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C: Chemical and Catalytic Reactivity at Interfaces

Chemical Equilibrium-Based Mechanism for the Electrochemical Reduction of DNA-Bound Methylene Blue Explains Double Redox Waves in Voltammetry
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  • J. D. Mahlum
    J. D. Mahlum
    Chemistry-Biology Interface Program, Zanvyl Krieger School of Arts & Sciences, Johns Hopkins University, Baltimore, Maryland 21218, United States
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    Orcidhttp://orcid.org/0000-0003-0220-3058
  • Miguel Aller Pellitero
    Miguel Aller Pellitero
    Department of Pharmacology and Molecular Sciences, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21202, United States
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    Orcidhttp://orcid.org/0000-0001-8739-2542
  • Netzahualcóyotl Arroyo-Currás*
    Netzahualcóyotl Arroyo-Currás
    Chemistry-Biology Interface Program, Zanvyl Krieger School of Arts & Sciences, Johns Hopkins University, Baltimore, Maryland 21218, United States
    Department of Pharmacology and Molecular Sciences, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21202, United States
    Department of Chemical and Biomolecular Engineering, Whiting School of Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United States
    Institute for Nanobiotechnology, Johns Hopkins University, Baltimore, Maryland 21218, United States
    *Email: [email protected]. Phone: 443-287-4798.
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    Orcidhttp://orcid.org/0000-0002-2740-6276
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The Journal of Physical Chemistry C

Cite this: J. Phys. Chem. C 2021, 125, 17, 9038–9049
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https://doi.org/10.1021/acs.jpcc.1c00336
Published April 21, 2021

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Abstract

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Methylene blue is widely used as a redox reporter in DNA-based electrochemical sensors and, in particular, it is the benchmark DNA-bound reporter used in electrochemical, aptamer-based sensors (E-ABs). Our group recently published an approach to interrogate E-ABs via cyclic voltammetry, which uses the cathodic to anodic peak-to-peak voltage separation (ΔEP) from methylene blue to report on the electron-transfer kinetics and binding state of these sensors. Although effective at scanning rates ≤10 V·s–1, the method is limited at faster scanning rates because cyclic voltammograms of methylene blue-modified, electrode-bound DNA present double faradaic waves that prevent the accurate estimation of ΔEP. These double waves have been observed in previous works, but their origin was unknown. In response, here we investigated the origin of these redox waves by developing a numerical model that incorporates methylene blue’s chemical equilibria in phosphate buffer to predict the shape and magnitude of cyclic voltammograms with 85% or better accuracy from single- and double-stranded DNA. Our model confirms that the peak splitting observed at scanning rates >10 V·s–1 originates from the protonation equilibrium of the radical intermediate species formed after methylene blue receives the first electron. Moreover, the model reveals a strong interaction between the proton transferred during the reduction of methylene blue and the chemical make of blocking self-assembled monolayers typically used in the fabrication of E-ABs. This interaction affects the apparent rate of the first electron-transfer step, accelerating or decelerating it depending on the hydrophobicity and polarity of the blocking monolayer. By expanding our understanding of the effect that monolayer chemistries have on methylene blue’s protonation rates and E-AB signaling, this work may serve the rational design of future sensors with tunable electron-transfer kinetics.

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Introduction

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Methylene blue is arguably one of the most used redox reporters in nucleic acid-based electrochemical sensors and is the benchmark reporter used in electrochemical, aptamer-based sensors (E-ABs). (1,2) This widespread use exists because, when bound to DNA, methylene blue and its reduced form, leucomethylene blue, are chemically stable and undergo an electrochemically reversible electron transfer. (3) Moreover, when co-tethered to the surface of gold electrodes with alkanethiol self-assembled monolayers (SAMs), methylene blue presents an ideal reduction potential far from background redox processes (e.g., the reduction of molecular oxygen and the surface oxidation of gold). (2,4) These features notwithstanding, the mechanism of methylene/leucomethylene blue electron transfer is complex. (5) First, it is pH-dependent, (6) making electron transfer susceptible to the acid–base chemistry of the medium. Furthermore, the voltammetric interrogation of the pair can generate multiple redox waves at fast voltage scanning rates. (7,8) These voltammetric features have been attributed to the occurrence of two different mechanisms of electron transfer (contact-mediated (9) vs through-DNA (10)). (7) Here, instead, we present a chemical equilibrium-based model that explains the origin of such features and demonstrates that they simply derive from the protonation equilibrium of an intermediate radical species.
The electrochemical reduction of methylene blue in aqueous solution (Figure 1A) involves a first electron-transfer step to produce a radical, (5) which undergoes a protonation reaction, followed by a second electron transfer to finally produce leucomethylene blue. Under conditions of molecular crowding, leucomethylene blue can self-associate to produce dimers (Figure 1B), (11−15) which appear in voltammograms at more negative reduction potentials relative to that of methylene blue. (13) It is expected that the electrochemical reduction of DNA-bound methylene blue follows the same electron-transfer mechanism as the solvated form. However, given the low surface density of methylene blue molecules on DNA-based sensors (typically in the order of a few picomolar (9)), self-stacking interactions are likely minimal. Thus, the voltammetric interrogation of such sensors often shows a single anodic/cathodic wave pair with peak-to-peak separation close to 0 mV at scanning rates ≤1 V s–1. (16,17)

Figure 1

Figure 1. Electrochemical reduction of methylene blue. This scheme uses the curved-arrow formalism to explain the electrochemical reduction of methylene blue according to the original mechanism proposed by Jean Chevalet. (13)

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In prior work, (18) we have demonstrated the potential advantages of employing cyclic voltammetry (CV) at scanning rates >1 V s–1 to measure the electron-transfer kinetics and signaling of methylene blue-modified, electrode-bound DNA aptamers. In our approach, the target concentration is reported via changes in the cathodic to anodic peak-to-peak voltage separation (ΔEP) of cyclic voltammograms. Because the magnitude of ΔEP is insensitive to variations in the number of aptamer probes on the electrode, ΔEP-interrogated E-ABs were resistant to drift and showed decreased batch-to-batch and day-to-day variability in sensor performance relative to interrogation based on other electrochemical methods. Moreover, ΔEP-based measurements could be performed every few hundred milliseconds and were, thus, competitive with other subsecond interrogation strategies such as chronoamperometry (19) and electrochemical impedance. (20) Unfortunately, the method fails at scanning rates faster than 10 V·s–1 because methylene blue’s faradaic waves start to split, (7,8) turning into two uneven redox waves.
To our knowledge, the faradaic splitting in methylene blue’s cyclic voltammograms has been reported twice before. In 2011, Yang and Lai showed that the voltammetric reduction peak of methylene blue in DNA duplexes separates into two waves at high scan rates (see Figure S7 in the Supporting Information of ref (8)). However, the authors did not provide a discussion regarding the origin of such splitting. One year later, Pheeney and Barton reported that, at fast scanning rates (5 V·s–1), cyclic voltammograms of methylene blue-modified DNA duplexes present two reductive peaks (see Figure 7 in ref (7)). The authors attributed the origin of each peak to two different mechanisms of electron transfer, one transferring electrons via a contact-mediated mechanism (9) and another transferring them through the DNA chain. (10) This interpretation seems unlikely since voltammetric peak splitting that occurs when using single-stranded DNA is the same as when using double-stranded DNA, irrespective of length or sequence. To clarify the origin of this peak splitting, here we use a numerical model to demonstrate that the protonation equilibrium of methylene blue’s intermediate radical is the source of the two voltammetric waves. By quantitatively comparing our simulated currents against experimental measurements, we show that this model can accurately predict voltammetric peak currents at scanning rates spanning 3 orders of magnitude and under different conditions of oligonucleotide length, pH, monolayer chemistry, and electron-transfer kinetics. In addition, the model reveals a strong interaction between the proton transferred during the first reduction step of methylene blue and the chemical make of blocking SAMs. This interaction affects the apparent rate of the first electron-transfer step, accelerating or decelerating it depending on the hydrophobicity and polarity of the blocking monolayer. The results herein expand our understanding of DNA-bound methylene blue’s electron-transfer properties and of how these are tuned by monolayer chemistries often used for the fabrication of DNA-based sensors.

Methods

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Chemicals and Materials

6-Mercaptohexanol, 1-hexanethiol, 1H,1H,2H,2H-perfluoro-1-hexanethiol, 1-mercaptohexanoic acid, and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich (St. Louis, MO). Phosphate-buffered saline (PBS, 11.9 mM HPO32–; 137 mM NaCl; 2.7 mM KCl; pH = 7.4), trace-metal-grade sulfuric acid (H2SO4), hydrochloric acid (HCl), and sodium hydroxide (NaOH) were purchased from Fisher Scientific (Waltham, MA). Tobramycin sulfate was purchased from GoldBio (St. Louis, MO). We prepared all aqueous solutions using deionized water from a Milli-Q Direct purification system with a resistivity of 18 MΩ. Gold working electrodes (PN: 002314, diameter: 1.6 mm) and coiled platinum wire counter-electrodes (PN: 012961) were obtained from ALS Inc. (Tokyo, Japan). Ag|AgCl (KCl-saturated) reference electrodes (PN: CHI111) were purchased from CH Instruments (Austin, TX). For polishing electrodes, 1200/P2500 silicon carbide grinding paper (PN: 36-08-1200) was bought from Buehler (Lake Bluff, IL); cloth pads (PN: MF-1040) and alumina slurry (PN: CF-1050) were purchased from BASi (West Lafayette, IN). Oligonucleotides, modified on the 5′ end with hexanethiol and on the 3′ end with methylene blue were purchased from and HPLC-purified by Sigma-Aldrich (Houston, TX). We used the DNA sequences reported in Table S1. To prepare DNA solutions, we first incubated 1 μL of 100 μM thiolated MB-modified DNA with 2 μL of 5 mM TCEP aqueous solution to reduce the disulfide bond. We then diluted the DNA with freshly prepared 1 mM thiol aqueous solution to a final concentration of either 100 nM, 500 nM, 1 μM, or 25 μM as determined via molecular absorbance measurements employing an Implen Nanophotometer NP80 (Westlake Village, CA). Using these solutions, we obtained DNA-packing densities on electrode surfaces of 1.93 ± 0.64 pmol·cm–2, 2.24 ± 0.58 pmol·cm–2, 2.66 ± 0.45 pmol·cm–2, and 4.61 ± 0.68 pmol·cm–2, respectively.

Electrode Preparation

Gold electrodes were polished for ∼2 min on 1200/P2500 silicon carbide grinding paper and subsequently for ∼4 min on a cloth pad with alumina slurry. After rinsing with water to remove polishing debris, they were then electrochemically cleaned in 0.5 M NaOH and 0.5 M H2SO4 following a previously reported protocol. (21) Briefly, (1) in 0.5 M NaOH, we scanned from −0.3 to −1.6 V versus Ag|AgCl 200 times at a scan rate of 0.5 V·s–1; (2) in 0.5 M H2SO4, we scanned from 0 to 1.6 V versus Ag|AgCl 200 times at a scan rate of 0.5 V·s–1. Once the electrodes were clean, we rinsed them with water and placed them immediately into 1 mM alkanethiol solutions, with or without a DNA construct listed in Table S1, in water to incubate overnight at 25 °C. We prepared sensors via co-incubation, as we reported before. (4) Post incubation, we rinsed the electrodes with water and placed them into a custom electrochemical cell containing 1× PBS. The cell was deaerated for 5 min by bubbling nitrogen gas into the solution prior to taking any electrochemical measurements. All measurements were performed under a N2 atmosphere. For pH experiments, we potentiometrically titrated the baseline PBS pH value (7.4) to pH = 4 using HCl and pH = 9 using NaOH. To prepare dsDNA surfaces, we incubated electrodes functionalized with the anchor strand in solutions containing a 2-fold higher concentration of the complement. We confirmed full hybridization on the electrode surface using frequency maps as previously reported by Dauphin-Ducharme and colleagues. (9)

Electrochemical Measurements

A CH Instruments Electrochemical Analyzer (CHI 1040C, Austin, TX) multichannel potentiostat and associated software was used for all CV measurements. We used a three-electrode cell configuration consisting of gold disk working, coiled platinum wire counter, and Ag|AgCl (saturated KCl) reference electrodes. All CV measurements were recorded in 1× PBS solution at scanning rates of either 1, 4, 10, 50, 100, or 150 V·s–1 after a 3 s quiet time, sampling current every millisecond.

Experimental CV Analysis

Each cyclic voltammogram condition used in our analysis had four replicates taken. Only one replicate was used to fit the simulated data because averaging voltammograms artificially decreased and broadened peak currents due to heterogeneity between sensor surfaces (Figure S1). To choose which replicate would be used, we compared them using the scatter plot function of Microsoft Excel. The replicate with the largest difference between charging current and Faradaic current was then used for further analysis. Because we did not include parameters to simulate charging current in our model, we subtracted the background currents from the chosen measurements. To find the background currents, we first collected data using electrodes created as described above but without methylene blue attached to the DNA as our background scans. We aligned voltammograms from non-methylene blue sensors with those from sensors using methylene blue and subtracted the two. The resulting background-subtracted data were used in all figures and is presented throughout as solid lines.

Computational Model

We employed COMSOL v5.4 to create a numerical model for a one-dimensional electrochemical cell in order to simulate cyclic voltammograms. The COMSOL file is provided as Supporting Information. Our model simulates both homogenous equilibrium reactions occurring between the buffer and methylene blue and heterogenous electron transfer occurring between methylene blue and the electrode surface. Parameters reported in the Supporting Information were determined by manually adjusting such parameters in the model until each simulation matched the background-subtracted experimental data with an accuracy of 85% or better as described below. Our numerical model (Figure 2) approaches the experimental setup illustrated in Figure 3A,B as a one-dimensional electrochemical cell. We previously reported a similar model for the determination of DNA-bound methylene blue electron-transfer rates via square wave voltammetry. (22) In our current model, we define two boundaries, one representing the surface of the working electrode, and the other representing the 3′ DNA terminus where methylene blue sits (Figure 2A). We set the separation between these two boundaries to 10 nm with a uniform mesh of 1% of the total cell length (0.1 nm/mesh element). This length includes the hexanethiol monolayer, 0.8 nm, (22,23) plus an approximated maximum persistence length for a 27-nt-long, monolayer-attached DNA strand (assuming double-stranded DNA, which rises approximately 3.4 nm every 10 nt, is the closest expanded-DNA conformation). (24) We postulate that the length of double-stranded DNA is a good approximation to the maximum length of our short oligonucleotides in the absence of literature consensus for the persistent length of single-stranded DNA. (25)

Figure 2

Figure 2. Description of the numerical model. (A) We created a one-dimensional model representing the DNA-functionalized electrodes of Figure 3A,B. (B) We simulate the voltammetric response of these electrodes by using a triangular voltage waveform with slope E·t–1, which corresponds to the voltage scanning rate. (C) In response to this varying potential, the experimental system (Figure 3A,B) produces a cyclic voltammogram (black trace) which varies in shape and magnitude according to the chemical equilibrium of the redox species considered. Our model accurately simulates the same voltammogram (red circles) via eqs 10–17, 19, and 20. The surface DNA concentration was 1.93 ± 0.64 pmol·cm–2 here and in all figures of the article, unless noted otherwise. All voltammetric measurements were performed in 1× PBS as defined in the Methods section, unless noted otherwise.

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Figure 3

Figure 3. Electrode-attached, methylene blue-modified DNA exhibits voltammetric features that change with increasing voltage scanning rate. (A) To illustrate these features, we employ DNA constructs of different lengths and secondary structures (Table S1), including one aptamer (illustrated here) that binds to the antibiotic tobramycin. We modify these constructs with a hexanethiol linker in the 5′ terminus and a methylene blue reporter at the 3′ terminus and co-deposit them onto gold electrodes along with 1-hexanethiol to form a SAM. (B) In the presence of tobramycin, the aptamer undergoes binding-induced conformational changes that accelerate electron transfer from methylene blue to the gold electrode, possibly by bringing methylene blue closer to the electrode surface. (C) When we interrogate aptamer-functionalized electrodes at scan rates ≤10 V·s–1 with surface coverage concentrations of 1.93 ± 0.64 pmol·cm–2 and either no tobramycin added or 1 mM of tobramycin, we observe no splitting of voltammetric waves. (D) However, at voltage scanning rates > 10 V·s–1, we observe splitting of the oxidation wave. Moreover, the addition of the target causes an inversion of behavior, showing peak splitting in the reduction but not in the oxidation wave. See the Methods section for additional experimental information.

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We based the reactions considered by our model on the mechanism proposed by Jean Chevalet (Figure 1) (13)
(1)
(2)
(3)
where O is MB+, R1 is MB•, R1H is LMB+•, R2H is LMB; kf1 and kf2 are the rates of reduction for the first and second electron transfers, respectively; kb1 and kb2 are the rates of oxidation for the first and second electron transfers, respectively; and kb,R1H and kf,R1H are the rate of protonation and the rate of deprotonation, respectively. We note that eqs 1–3 denote the general formula for an ErCrEr reaction scheme, (26) which involves a first electron transfer, followed by a homogeneous chemical reaction, and, finally, a second electron transfer. We define the reaction of methylene blue at the working electrode in our simulation (Figure 2A) by using Fick’s second law of diffusion to describe electrochemical flux
(4)
(5)
where C denotes concentration of the subscripted species, t is time, x is the distance from the electrode surface, J is flux, and D is the diffusion coefficient of the subscripted species. For simplicity, we assume D = 10–5 cm2·s–1 for all redox-active species (see Figure S2 for more details). Per eqs 1–3, the initial conditions for the redox-active species were CO = CO* (where CO* is the initial bulk concentration of species O) and CR1 = CR1H = CR2H = 0 M. Thus, the right-most, no-flux boundary conditions are set as follows
(6)
(7)
(8)
(9)
We set the fluxes (eqs 4 and 5) to obey the Butler–Volmer formalism of electron-transfer kinetics
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
where k10 and k20 are heterogeneous rate constants for the first and second electron transfers, respectively; α1 and α2 are the transfer coefficients for the first and second electron transfers, respectively; n is the number of electrons transferred, F is Faraday’s constant, R is the gas constant, T is temperature, E is the applied voltage, and E10 and E20 are the standard reduction potentials for the first and second electron transfers, respectively. Using eqs 10–17, we successfully model the electrochemical flux that occurs at the electrode boundary.
To evaluate pH effects related to the protonation step (eq 2), we incorporate all chemical equilibrium reactions participating in our experimental system, including protonation equilibria for phosphate-buffered saline (PBS, Table S2). We model PBS equilibria based on previously reported equilibrium constants and protonation rates. (27) To incorporate these equilibria into our flux calculations, we express eq 2 in its acid dissociation form
(18)
where CH+ is the concentration of protons. When the system is at equilibrium
(19)
(20)
where KR1H is the equilibrium constant for the proton dissociation of R1H. We set eq 20 as a domain variable so our model accounts for a proton equilibrium similar to our real-world experimental system.
Having defined the model physics, we next establish the triangular voltage waveform that CV uses (Figure 2B). To do so, we use the expression
(21)
where Ein is the initial voltage vs. Ag|AgCl (saturated KCl), Efin is the final voltage, sr is the scan rate, τ is the time required to sweep the voltage from Ein to Efin, and H1 and H2 define step functions: H1 jumps from 1 to 0 when χ1 = (τ – t)·t–1 ≤ 0, and H2 jumps from 0 to 1 when χ1 = (t – τ)·t–1 > 0. Thus, employing eqs 10–17 to account for electrochemical fluxes and eqs 18–21 to account for chemical equilibria and voltage perturbations, our model generates voltammetric currents that closely match the shape and magnitude of experimental voltammograms (Figure 2C).
To evaluate the accuracy of our numerical simulation, we modeled the effect the scanning rate has on the cyclic voltammograms of DNA-bound methylene blue. We perform this analysis by presenting a superposition of numerically simulated voltammograms over experimental, background-subtracted voltammograms measured at six different voltage scanning rates (Figure 4). To achieve a good fit, we adjusted the electron-transfer and proton dissociation parameters in eqs 1–3 and 10–13. By keeping such parameters within an order of magnitude or closer for all scanning rates (Table S3), we accurately simulate scanning-rate-dependent voltammetric features. To determine the accuracy of our model, we calculate error as the percent difference between simulated and experimental currents at each voltage point, using the formula
(22)

Figure 4

Figure 4. Modeling the electrochemical reduction of DNA-tethered methylene blue at different voltage scanning rates. We recorded these background-subtracted cyclic voltammograms on gold electrodes functionalized with a 26-base DNA aptamer modified at one end with methylene blue (Figure 2A). As a model system, we co-deposit 500 nM of an aptamer that binds to the antibiotic tobramycin with 1 mM 1-hexanethiol. We then obtain cyclic voltammograms (black traces) in 1× PBS solution at voltage scanning rates of (A) 1, (B) 4, (C) 10, (D) 55, (E) 100, and (F) 150 V·s–1. Using the parameters described in Table S3 in our numerical model, we generate simulated voltammograms (colored circles) closely matching the experimental ones. We only show 1 in every 8 simulated currents for clarity. Error bars report the true differential error between experimental and simulated voltammograms. The color indicates percent error, which we use to demonstrate that our numerical model correctly estimates the current magnitude of the different voltammetric peaks with an accuracy of 85% or better; that is, ≤15% error (Figure S3).

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To facilitate the visualization of relative error, we color-code the error bars in proper rainbow fashion to represent 0% (red) and 100% (violet) error (Figure 4). The error bar heights reported in each figure represent the real error between our model and the experimental data and are calculated by dividing P.D. by 100 and multiplying by the simulated data point. We stopped adjusting parameters when the peak magnitude for each simulation was 85% accurate or better (Figure S3). We observe that the largest error occurs in the flat regions of the voltammograms. However, this error arises because our numerical simulations report currents close to true zero, whereas the experimental voltammograms always have a nonzero current either because of noise or poor background correction in those regions.

Simulated CV Analysis

We exported the simulated voltammograms into a text file and mathematically compared them against experimental voltammograms in Microsoft Excel to determine the error. Using eq 22, we found the percent error. By dividing this by 100 and multiplying by the simulated value, we found the relative error. To visualize these errors, we used the built-in color function using the Rainbow color table. Error bars and simulated traces were colored according to the percent error where 0% error is red and 100% error is violet. Error bars represent the true error difference between simulated and experimental currents. All data were first analyzed in Microsoft Excel. Individual figure panels were graphed in Igor Pro v.8 and multipanel figures were assembled in Adobe Illustrator, Creative Cloud version.

Results and Discussion

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To analytically validate our numerical model, we use experimental voltammograms of the reduction of methylene blue covalently bound to single-stranded DNA of different lengths, sequences, and secondary structures and of double-stranded DNA (Table S1). To collect the experimental data, we modify these strands with a hexanethiol linker placed on the 5′ terminus and a linker functionalized with methylene blue on the 3′ terminus. (4) Then, we co-deposit the constructs with 1-hexanethiol onto the gold electrodes to form a SAM (Figure 3A). To better study the effect of electron-transfer kinetics on the voltammetric features of methylene blue, we include one aptamer sequence in our list of constructs (Table S1). In the presence of its target (the antibiotic tobramycin), this aptamer undergoes a binding-induced conformational change that accelerates the overall electron-transfer rate from methylene blue to the electrode (Figure 3B). (28,29) When we interrogate sensors functionalized with this aptamer by CV in PBS and at scanning rates ≤10 V·s–1, we observe faradaic waves with no visible splitting in either the reduction or oxidation peaks (Figure 3C). However, scanning the sensors at rates >10 V·s–1 causes a significant splitting of the oxidation wave (Figure 3D). Repeating the experiment in the presence of saturating concentrations of the aptamer’s target causes an inversion of the peak splitting behavior, with two waves becoming visible in the reduction scan but vanishing from the oxidation scan (Figure 3D). We explain this behavior by making the argument that such peak splitting is explained by the chemical equilibria involved in the voltammetric reduction of methylene blue.
The peak splitting we observe in the oxidation wave of voltammograms measured at >10 V·s–1 (Figure 4) originates from the protonation reaction of the methylene blue radical (Figure 1 and eq 2). Specifically, the acid dissociation constant (KR1H in eq 20) and protonation kinetics of this reaction determine how much the oxidation peak splits. To illustrate this effect, we performed numerical simulations holding all parameters constant in our model except for either KR1H (Figure S4) or kb,R1H (Figure S5). The resulting voltammograms clearly indicate that KR1H and kb,R1H strongly affect peak splitting. Fortunately, the acid dissociation constants for the methylene blue radical (pKa = 9.0) and leucomethylene blue (pKa = 5.8) have been reported before. (30) Thus, in our model, we assume the apparent acid dissociation constant for eq 2 to be approximately the average of the two known constants: pKa,R1H ∼ 7.4. Keeping this value constant in the model but modulating the protonation rate, we successfully fit all voltammograms reported in this work.
We can also use our model to discard the possibility that the peak splitting could be due to leucomethylene blue self-stacking (Figure 1B). To do this, we varied the concentration of the DNA aptamer used during the assembly of our monolayer. Increasing the DNA deposition concentration from 100 nM to 1 μM (packing densities of 1.93 ± 0.64, 2.24 ± 0.58, and 2.66 ± 0.45 pmol·cm–2, respectively) results in visually similar voltammograms (Figure 5A–C) that we accurately model (Figure S6 and Table S4) without including parameters to simulate leucomethylene blue dimerization. Employing the deposition concentrations used in the work by Pheeney and Barton (25 μM dsDNA solutions, Figure 5D), (7) we observe wave broadening and a 60 mV shift of the formal reduction potential in the negative direction but no additional voltammetric features. These results indicate that, at high electrode surface concentrations of methylene blue-modified DNA (4.61 ± 0.68 pmol·cm–2), stacking of neighboring methylene blue molecules may occur as suggested by the visible shift in reduction potential and wave broadening. However, this stacking does not seem to generate additional voltammetric features.

Figure 5

Figure 5. High deposition concentrations of methylene blue cause peak broadening and increased overpotentials but do not affect peak splitting. To show that higher packing density of DNA-bound methylene blue at the surface of an electrode does not affect peak splitting, we co-deposited 1-hexanethiol and either (A) 100 nM, (B) 500 nM, (C) 1 μM, or (D) 25 μM of the tobramycin aptamer (Table S1) onto gold electrodes with resulting surface coverages of (A) 1.93 ± 0.64, (B) 2.24 ± 0.58, (C) 2.66 ± 0.45,, and (D) 4.61 ± 0.68 pmol·cm–2. We then measured voltammograms at a scanning rate of 100 V·s–1 and fit the data with an accuracy of 85% or better (Figure S6) using the parameters found in Table S4. We speculate that the wave broadening and 60 mV shift in formal reduction potential observed in electrodes prepared from solutions containing 25 μM DNA relative to those prepared at 100 nM are due to leucomethylene blue self-stacking (Figure 1B).

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The splitting of methylene blue reduction or oxidation waves is also not caused by different mechanisms of electron transfer (contact-mediated (9) vs through-DNA (7,10)). We demonstrate this by presenting cyclic voltammograms that interrogate single-stranded DNA of different lengths and varying sequences and double-stranded DNA (Table S1 and Figure 6). Specifically, we include DNA strands composed only of deoxythymine, which lacks the possibility of forming double-stranded DNA (a requirement to enable through-DNA electron transfer). (31) Irrespective of sequence, all constructs show peak splitting behavior in the voltammograms. However, we observe less pronounced wave splitting with decreasing DNA length, which is an effect of increased electron-transfer kinetics of the system due to bringing methylene blue and the electrode surface closer together. Using our model and adjusting electron-transfer kinetics (Table S5), we accurately fit the shape of voltammmograms obtained for each of the DNA strands considered (Figure S7).

Figure 6

Figure 6. Voltammetric peak splitting is dependent on DNA secondary structure. We employed nucleotide sequences of (A) 10, (B) 20, (C) 26, (D) 37 nt, (E) tobramycin aptamer hybridized to a fully complementary strand, and (F) tobramycin aptamer in the presence of the target to test whether secondary structure affects peak splitting. Each of the constructs were co-deposited at 500 nM with 1 mM 1-hexanethiol onto gold electrodes and are modified with methylene blue at the distal terminus. All experiments were done in 1× PBS solution and experiments A–E were performed at 100 V·s–1, while experiment F was performed at 150 V·s–1. We report the theoretical secondary structures for each construct as generated by mfold software (Table S1). (34) Although all constructs exhibit peak splitting, we observe a reduction in this splitting with decreasing oligonucleotide length (A vs D). This is because, as the DNA shortens, the electron-transfer kinetics of the system increase as previously demonstrated. (35) We also observe a reversal in peak splitting behavior as a stable secondary structure is introduced (E for double-stranded DNA and F for a folded tobramycin-binding aptamer in the presence of 1 mM tobramycin). This is related to a change in protonation rate (Table S5) which suggests a change in hydrogen bonding interactions between methylene blue and the DNA. Error bars report the true differential error between experimental and simulated voltammograms. The color indicates percent error, which we use to demonstrate that each simulation fits the peak currents of background-subtracted voltammograms with an accuracy of 85% or better; that is, <15% error (Figure S7).

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The measurements using dsDNA (i.e., tobramycin-binding aptamer hybridized to a fully complementary strand) present a more pronounced peak splitting in the reduction scan (Figure 6E), in contrast to all ssDNA constructs, which present splitting in the reverse, oxidation scan. This inversion in electrochemical behavior may suggest a change in hydrogen bonding interactions between the DNA and methylene blue, a known DNA intercalator, (32,33) that affect the protonation reaction of the radical intermediate. We also observe this effect in ssDNA tobramycin-binding aptamers, which present significant self-complementarity when target-stabilized. To explore this, we challenged tobramycin-binding E-ABs with saturating tobramycin concentrations (1 mM) and fit the resulting voltammograms using our model (Figure 6F). When saturating concentrations of tobramycin are present, the overall electron-transfer kinetics of the system increase, as evidenced by a decrease in ΔEP visible between Figures 4F and 6F of ∼80 mV. The acceleration of the first electron-transfer step (from 261 to 354 s–1, Table S5) results in the disappearance of the original peak splitting in the oxidation wave (e.g., Figure 4F) and the appearance of new splitting in the reduction wave (Figure 6F). This is because although the first electron-transfer rate increases, the second electron transfer is still limited by the protonation rate, which in the case of the target-bound state has not significantly changed [4.0 × 107 m3·s–1·mol–1 for unbound vs 4.5 × 107 m3·s–1·mol–1 for bound aptamer (Table S5)]. These results demonstrate that peak splitting in methylene blue voltammetry does not arise from different mechanisms of electron transfer and underline the importance of understanding hydrogen bonding interactions between methylene blue and the DNA strand.
As one additional confirmation that our hypothesis is accurate and the observed peak splitting is caused by the rate-limiting protonation of the methylene blue radical (Figure 1 and eq 2), we tested peak splitting as a function of pH. The published literature indicates that methylene blue’s electron-transfer mechanism is itself pH-dependent. Specifically, several earlier reports (6,36,37) demonstrated that the formal reduction potential of methylene blue linearly becomes more negative with increasing pH, as expected from the Nernst equation. Yet, the slope of this line undergoes two marked transitions: one from −87 mV/pH (at pH < 5.4) to −58 mV/pH (at pH = 5.4–6.0) and a second transition to −28 mV/pH (at pH > 6.0). (6) These slope changes in the Nernstian curve are indirectly indicative of changes in the mechanism of electron transfer. Although such values are not transferable to our gold electrode system since they were measured on carbon fiber electrodes, they do indicate the mechanism of methylene blue reduction changes with pH.
Thus, we adapted our model to pH-induced changes in methylene blue’s reduction mechanism by assuming that the protonation reaction is no longer rate limiting at pH > 8.0 or pH < 6.0 (Figure 7). In other words, when the solution pH is far from methylene blue’s apparent pKa in either direction, the reaction mechanism changes from the ErCrEr scheme shown in eqs 1–3 to two serial and reversible electron transfers (ErEr)
(23)
(24)

Figure 7

Figure 7. Chemical equilibrium explains the changes in voltammetric features observed at different values of pH. To illustrate this point, we measured cyclic voltammograms on DNA-functionalized electrodes (tobramycin-binding aptamer) immersed in 1× PBS at pH = 4, 7, and 9. For the low and high pH values, we potentiometrically titrated the buffered solutions with HCl or sodium hydroxide, respectively. As shown here, our model accurately predicts the position and current magnitude of the main voltammetric features in background-subtracted voltammograms with greater than 85% accuracy at all values of pH (Figure S8). However, we note that at pH = 9 and pH = 4, the mechanism changes from an ErCrEr (eqs 1–3) reaction to an ErEr (eqs 23 and 24) scheme, where electrochemical reduction no longer requires protonation. The latter mechanism is included in our numerical model. We use the parameters reported in Table S6 to achieve these fits. Error bars report the true difference error between experimental and simulated voltammograms. The color indicates percent error.

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The validity of eqs 23 and 24 is supported by previously reported results from the work of Caram and colleagues (38) on voltammetric measurements of methylene blue performed in aprotic media in the presence and absence of proton donors (see, e.g., Scheme 2 in ref (38)). Using this assumption, we compared the voltammetric response of a new batch of DNA-functionalized electrodes at pH = 4.0 (Figure 7A), 7.0 (Figure 7B), and 9.0 (Figure 7C) versus the output of our model. Our simulated results successfully match the experimental voltammograms in peak current magnitude (Figure S8). Moreover, the E1/2 for each of the modeled voltammograms was within 50 mV of the values reported in the literature (Table S6). (6) These results demonstrate that pH-induced changes in methylene blue’s radical intermediate’s protonation successfully account for shape changes observed in experimental voltammograms in acidic, neutral, and alkaline solutions.
Having demonstrated the validity of our proton equilibrium hypothesis at various voltage scanning rates (Figure 4), deposition concentration (Figure 5), secondary structures (Table S1 and Figure 6), and pH values (Figure 7), we next explored how the hydrophobicity of alkanethiol monolayers affect the electrochemical behavior of DNA-bound methylene blue. We were motivated to pursue this study by prior results from our group demonstrating that the terminal chemistries of alkanethiols can dramatically affect the signaling behavior of electrochemical DNA biosensors (e.g., as in aptamer-based sensors, Figure 3). (4) For this purpose, we prepared a new batch of electrodes, this time functionalizing them with monolayers of either 6-mercaptohexanol (Figure 8A), 1-hexanethiol (Figure 8B), or a fluorinated analogue of 1-hexanethiol (Figure 8C), interrogated them at different voltage scanning rates (1, 10, and 100 V·s–1), and accurately simulated the voltammogram for each monolayer (Figure S9). The more hydrophilic monolayer of 6-mercaptohexanol (Figure 8A) produces background-subtracted voltammograms with sharper peaks and less peak splitting relative to monolayers made of 1-hexanethiol (Figure 8B) and the fluorinated monolayer (Figure 8C). In contrast, the fluorinated monolayer exhibits more peak splitting than either of the other two monolayers. The signal-to-noise level is worse in electrodes functionalized with 6-mercaptohexanol because they generally present larger capacitive currents (Figure S10) that contribute to hiding a fraction of methylene blue’s voltammetric waves. (4) Next, we used our numerical model to establish differences in fit parameters between the three monolayers (Table 1). Doing this, we determined that 6-mercaptohexanol increases the protonation rate of the methylene blue radical (i.e., increases kb,R1H in eq 2) nearly 2-fold relative to 1-hexanethiol, thereby decreasing the extent of peak splitting seen at, for example, 100 V·s–1. We also see a nearly 3-fold decrease in protonation rate for the fluorinated monolayer as compared to 1-hexanethiol (Table 1, kb,R1H). Aiming to investigate the effect of negatively charged monolayers, we also experimented on a monolayer formed from solutions of 6-mercaptohexanoic acid. Unfortunately, in our hands, this monolayer presents voltammetric features even in the absence of methylene blue-modified DNA (Figure S11). Thus, we did not pursue these experiments further. However, the results presented so far demonstrate that methylene blue protonation, and the interactions of this proton with monolayer end groups, can dramatically affect the redox chemistry of DNA-bound methylene blue.

Figure 8

Figure 8. Electrode-blocking SAMs affect the protonation rate of the leucomethylene blue radical. After co-depositing 100 nM of the tobramycin aptamer and 1 mM either (A) 6-mercaptohexanol, (B) 1-hexanethiol, or (C) fluorinated analogue of 1-hexanethiol onto gold electrodes, we collected cyclic voltammograms at 1, 10, and 100 V·s–1 that we model with an accuracy of 85% or greater (Figure S9). We adjusted the starting concentration of species O to account for the smaller background-corrected peak currents of 6-mercaptohexanol when compared to 1-hexanethiol, which are a product of the removal of the charging current (Table 1). (34) We observe less peak splitting occurring in 6-mercaptohexanol, which can be explained by its nearly 2-fold higher average apparent protonation rate as compared with 1-hexanethiol (Table 1). However, we observe greater peak splitting occurring for the fluorinated monolayer, which can be explained by the nearly 3-fold decrease in apparent protonation rate as compared with 1-hexanethiol.

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Table 1. Electrochemical and Equilibrium Parameters Corresponding to the Reduction of DNA-Bound Methylene Blue in the Presence of Different Monolayer Chemistries in Comparison to the Values Determined by Other Works
parameters6-mercaptohexanol1-hexanethiolfluorinated monolayerother works
k1° (s–1)124 ± 57417 ± 54143 ± 43k° = 80a
k2° (s–1)298 ± 208252 ± 196261 ± 197 
E1°′ (V)–0.30 ± 0.01–0.29 ± 0.00–0.31 ± 0.01 
E2°′ (V)–0.28 ± 0.02–0.29 ± 0.02–0.32 ± 0.01 
α10.57 ± 0.020.55 ± 0.000.49 ± 0.1 
α20.47 ± 0.120.41 ± 0.000.48 ± 0.06 
pKa7.00 ± 0.007.35 ± 0.037.30 ± 0.00∼7.4b
kb,R1H (m3 s–1 mol–1)5.01 × 107 ± 0.002.82 × 107 ± 0.000.97 × 107 ± 0.70 × 1072 × 108,c 4.5 × 108 ± 0.4 × 108,d
a

Reference (35).

b

Reference (30).

c

Reference (40).

d

Reference (39).

As demonstrated by the results presented in this work, methylene blue’s protonation equilibrium has a strong effect on E-AB signaling. For example, in prior work, we compared the signaling output of E-ABs functionalized with either 1-hexanethiol or 6-mercaptohexanol blocking monolayers. Doing so, we observed inverse signaling behavior between sensors functionalized with each of the two monolayers. (4) The results of this work (Table 1) now indicate that those differences in signaling are due, at least in part, to changes in proton interactions between methylene blue and monolayer end groups. The magnitudes of protonation rates reported in Table 1 are in the same order of those found by pulse photolysis from dilute solutions of methylene blue. (39,40) Thus, our numerical model represents an accurate, functional tool that will allow us to study and better understand electron transfer from DNA-bound methylene blue under various conditions of DNA sequence, blocking monolayer chemistries, and electrolyte pH. Note that Table 1 reports parameters used to fit voltammograms at pH = 7, corresponding to eqs 1–3. For equivalent parameters corresponding to eqs 23 and 24, we refer the reader to Table S6.

Conclusions

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We employed numerical simulations and CV to demonstrate that the voltammetric peak splitting observed in electrode-tethered, methylene blue-modified DNA at scan rates >10 V·s–1 originates from the protonation of a methylene blue radical. This peak splitting behavior limits the sensitivity and temporal resolution of ΔEP-based interrogation of E-ABs, which depends on the occurrence of single cathodic–anodic wave pairs. (18) However, by investigating the origin of this peak splitting behavior, we revealed that the rate of the electrochemical reduction of DNA-bound methylene blue can be a strong function of the extent of proton interactions with blocking monolayer end groups (Figure 8) and the DNA backbone (Figure 6E,F). These effects have been largely ignored in the field of E-ABs but could explain the dramatic changes in signaling behavior observed in sensors using varying aptamer secondary structures and monolayer chemistries; see, for example, ref (4).
Although we do not explore the position of methylene blue along the DNA chain in this work, it has been previously shown that internal placement of methylene blue causes a several-fold decrease in electron-transfer rates relative to identical constructs with the reporter at the 3′ terminus. (35) We now believe that this significant decrease in electron-transfer kinetics is caused by stronger hydrogen bonding interactions between methylene blue and the tail end of the DNA chain in internally modified oligonucleotides, which change the protonation rate of the radical intermediate. Therefore, a key result from this work is the increased understanding that methylene blue’s electron-transfer kinetics can be modulated by tuning the radical intermediate’s protonation rate, via either the positioning along the DNA chain, DNA secondary structure, and/or the chemical groups at the surface of blocking monolayer chemistries. We anticipate that similar effects are true for other redox reporters involving intermediate protonation steps, such as anthraquinone, nile blue, dabcyl, and others. (3)

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpcc.1c00336.

  • Experimental and simulated voltammograms under various conditions; numerical data tables derived from simulated voltammograms; DNA sequences used in this work (PDF)

  • COMSOL Model file (ZIP)

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Author Information

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  • Corresponding Author
    • Netzahualcóyotl Arroyo-Currás - Chemistry-Biology Interface Program, Zanvyl Krieger School of Arts & Sciences, Johns Hopkins University, Baltimore, Maryland 21218, United States;  Department of Pharmacology and Molecular Sciences, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21202, United States;  Department of Chemical and Biomolecular Engineering, Whiting School of Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United States;  Institute for Nanobiotechnology, Johns Hopkins University, Baltimore, Maryland 21218, United States;  Orcidhttp://orcid.org/0000-0002-2740-6276;  Email: [email protected]
  • Authors
    • J. D. Mahlum - Chemistry-Biology Interface Program, Zanvyl Krieger School of Arts & Sciences, Johns Hopkins University, Baltimore, Maryland 21218, United States;  Orcidhttp://orcid.org/0000-0003-0220-3058
    • Miguel Aller Pellitero - Department of Pharmacology and Molecular Sciences, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21202, United States;  Orcidhttp://orcid.org/0000-0001-8739-2542
  • Author Contributions

    J.D.M. built the numerical model and performed all simulations reported in this work. M.A.P. performed all experiments. N.A.C. collaboratively designed the experiments with J.D.M. and M.A.P. and supervised the development of the computational model. All authors contributed to the writing of this manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We thank Johns Hopkins University School of Medicine for the startup funds provided in support of this work. Moreover, we thank the NIH for providing support in the form of a T32 training grant (GM080189) to support the graduate education of J.D.M. as part of the Chemistry–Biology Interface Program at Johns Hopkins University.

References

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    In an effort to investigate DNA-mediated electron transfer involving ground-state reactants, the authors studied redox-active intercalators bound at discrete sites within the individual helixes of a DNA monolayer on gold. To investigate charge transduction through DNA as a function of distance, the authors have site-specifically cross-linked a redox-active intercalator, daunomycin (DM), into the DNA characterized. The site of intercalation was controlled in the duplexes by incorporating a single guanine-cytosine (GC) base step in otherwise adenine-thymine (AT) or inosine-cytosine (IC) sequences; as DM requires the N2 atom of guanine for covalent crosslinking, the intercalator is constrained to these positions. Moving the GC step along the duplex therefore provided a systematic variation in the location of the DM-binding site relative to the thiol-terminated linker. Crosslinking DM to the DNA in soln., and then depositing the labeled duplexes onto gold, afforded a series of films in which the intercalator was linked quant. at a known sepn. from the electrode surface. Here, long-range charge transport through DNA-modified films has been demonstrated in a series of structurally characterized assemblies.
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  11. 11
    Svetlicic, V.; Tomaic, J.; Zutic, V. A Kinetic-Study of Charge-Transfer and Phase-Transitions of the Methylene-Blue Leucomethylene Blue Couple Adsorbed at the Mercury Aqueous-Solution Interface. J. Electroanal. Chem. 1983, 146, 71– 92,  DOI: 10.1016/S0022-0728(83)80113-2
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    11
    A kinetic study of charge transfer and phase transitions of the methylene blue/leucomethylene blue couple adsorbed at the mercury/aqueous solution interface
    Svetlicic, Vesna; Tomaic, Jadranka; Zutic, Vera; Chevalet, Jean
    Journal of Electroanalytical Chemistry and Interfacial Electrochemistry (1983), 146 (1), 71-92CODEN: JEIEBC; ISSN:0022-0728.
    The interfacial behavior of a methylene blue (MB)/leucomethylene blue (LMB) redox couple was studied by using fast perturbation potentiostatic techniques at the dropping Hg electrode. The extent of adsorption and the structure of the adsorbed layer above the monolayer coverage were detd. by the presence of anions at the interface, anion hydration playing the important role. In 1M electrolytes (pH = 7.9) the tendency of MB dimers towards a stacking interaction in the aq. phase, as well as to an adsorption at the Hg/aq. soln. interface, increase in the sequence F- « NO3- < Cl- « ClO4-. Evidence is presented for a coadsorption of LMB and nitrate and well-defined phase transitions in the adsorbed film. There are 3 distinct potential-dependent phases of LMB at the interface: (1) flat mols. and/or smaller 2-dimensional aggregates at low LMB coverages; (2)2-dimensional cryst. layers of flat mols. after a monolayer of LMB is generated by redn. at pos. charges; (3) a compact LMB layer at the neg. charged electrode,after desorption of nitrate and a conformational change of LMB mols. The characteristics of the charge-transfer process are best interpreted in terms of 2 MB/LMB redox couples: the prewave corresponds to redox reactions of adsorbed species at the Hg surface, while the process at more neg. potentials corresponds to the MB/LMB couple at a chem. modified electrode-Hg, covered by a compact and conductive layer of solid LMB.
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  12. 12
    Svetličič, V.; Žutić, V.; Clavilier, J.; Chevalet, J. Supramolecular Phenomena in Organic Redox Films at Electrodes. J. Electroanal. Chem. Interfacial Electrochem. 1985, 195, 307– 319,  DOI: 10.1016/0022-0728(85)80051-6
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    12
    Supramolecular phenomena in organic redox films at electrodes. Part I. The methylene blue/leucomethylene blue redox couple at the platinum electrode
    Svetlicic, Vesna; Zutic, Vera; Clavilier, Jean; Chevalet, Jean
    Journal of Electroanalytical Chemistry and Interfacial Electrochemistry (1985), 195 (2), 307-19CODEN: JEIEBC; ISSN:0022-0728.
    It is possible to generate conductive multilayer structures by redn. of methylene blue cations at the interface: clean surface of a Pt electrode/1M aq. electrolyte. The characteristics of the multilayer ordered phase generated in fluoride and nitrate solns. are presented and the mechanism of a fast charge transfer between electrode/org. film and org./aq. soln. interfaces is discussed. The cond. of the film was interpreted by postulating formation of a mixed valence structure with the generation of a cation radical intermediate which is favored in the solid state at characteristic potentials.
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  13. 13
    Žutić, V.; Svetličić, V.; Clavilier, J.; Chevalet, J. Supramolecular Phenomena in Organic Redox Films at Electrodes. J. Electroanal. Chem. Interfacial Electrochem. 1987, 219, 183– 195,  DOI: 10.1016/0022-0728(87)85039-8
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    13
    Supramolecular phenomena in organic redox films at electrodes. Part II. The methylene blue/leucomethylene blue redox couple at the gold electrode
    Zutic, Vera; Svetlicic, Vesna; Clavilier, Jean; Chevalet, Jean
    Journal of Electroanalytical Chemistry and Interfacial Electrochemistry (1987), 219 (1-2), 183-95CODEN: JEIEBC; ISSN:0022-0728.
    Org. films formed by redn. of the methylene blue cation at the Au electrode/aq. electrolyte soln. interface were studied by purely electrochem. methods. The insol. redn. product, a mixed-valence salt of the cation radical and leucomethylene blue, forms 2 polymorphic conductive structures: one with metallic and the other with ionic cond. The supramol. organization in the electrogenerated films is detd. by the redox state and the structure of the Au/org. film interface.
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  14. 14
    Svetlicic, V.; Zutic, V.; Clavilier, J.; Chevalet, J. Organic Monolayer Formation at a Sulfur Modified Gold Electrode─the Methylene-Blue Leucomethylene Blue Redox Couple. J. Electroanal. Chem. Interfacial Electrochem. 1987, 233, 199– 210,  DOI: 10.1016/0022-0728(87)85016-7
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    14
    Organic monolayer formation at a sulfur modified gold electrode. The methylene blue/leucomethylene blue redox couple
    Svetlicic, Vesna; Zutic, Vera; Clavilier, Jean; Chevalet, Jean
    Journal of Electroanalytical Chemistry and Interfacial Electrochemistry (1987), 233 (1-2), 199-210CODEN: JEIEBC; ISSN:0022-0728.
    The electrochem. formation of a condensed methylene blue (MB+)/leucomethylene blue (LMB) monolayer at the S covered Au electrode (1 M NaF, KCl, or KNO3 soln., pH 7.9) is reported. The strong interaction between MB+ and adsorbed S in the monolayer was ascribed to a disulfide linkage between a S adatom and the S heteroatom of the perpendicularly oriented org. mol. This binding also resulted in a change in the elec. properties of the S monolayer, which increases the reversibility of the electron transfer in the 1st adsorbed MB monolayer. The stability of the reduced monolayer at the A-S/aq. electrolyte interface was affected strongly by the type of anion (F- > Cl- » NO3-).
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  15. 15
    Brdička, R. Measurements of Quantities Concerning the Adsorption of Certain Reducible Compounds or Their Reduction Products at the Dropping Mercury Electrode. Collect. Czech. Chem. Commun. 1947, 12, 522– 540,  DOI: 10.1135/cccc19470522
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    15
    Measurements of quantities concerning the adsorption of certain reducible compounds or their reduction products at the dropping-mercury electrode
    Brdicka, R.
    Collection of Czechoslovak Chemical Communications (1947), 12 (), 522-40CODEN: CCCCAK; ISSN:0010-0765.
    cf. C.A. 37, 5661.3,5. B. has generalized the adsorption theory previously proposed to explain the anomalous polarograms of certain reversible org. oxidation-reduction systems. Theoretically the polarigraphic curve should have the same shape as the corresponding potentiometric titration curve and the half-wave potential and normal oxidation-reduction potential should be the same. If one of the oxidation-reduction forms should be adsorbed on the Hg drops, the free energy change which occurs will shift the polarographic wave to more neg. potentials if only the oxidized form is adsorbed; and to more pos. values if only the reduced form is adsorbed. At low concns. of the oxidation-reduction system the Hg drops will be unsatd. and a single wave will appear. Above a certain concn. 2 waves appear: an "anomalous" wave and a normal wave. The height of the "anomalous" wave is independent of concn., since it corresponds to satn. of surface of the drops with adsorbed compd. It will follow the normal wave at more neg. values if the oxidized form only is adsorbed (e.g. phenosafranine), or precede the normal wave at more pos. potentials if the reduced form only is adsorbed (e.g. methylene blue and lactoflavin). The height of the "anomalous" or adsorption curve is a measure of the adsorption current from which can be evaluated the max. no. of adsorbed mols. per unit area of drop surface. From the difference between the half-wave potentials of the adsorption and diffusion currents the H.ovrddot.uckel adsorption coeff. can be calcd. If some plausible value is assumed for the vol. of the adsorbed mols. the free energy of adsorption can be detd. The adsorption current varies directly with the height of the Hg reservoir. Finally, if both oxidation-reduction forms are adsorbed equally, the polarogram should be normal. Data from previous articles are used to test the theory.
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  16. 16
    Bard, A. J.; Faulkner, L. R.; Leddy, J.; Zoski, C. G. Electrochemical Methods: Fundamentals and Applications; Wiley: New York, 1980; Vol. 2.
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  17. 17
    Bard, A. J.; Faulkner, L. R. Chapter 11; Section 11.7: Thin-layer electrochemistry. Electrochemical Methods: Fundamentals and Applications; Wiley: New York, 2001.
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  18. 18
    Pellitero, M. A.; Curtis, S. D.; Arroyo-Curras, N. Interrogation of Electrochemical Aptamer-Based Sensors Via Peak-to-Peak Separation in Cyclic Voltammetry Improves the Temporal Stability and Batch-to-Batch Variability in Biological Fluids. ACS Sens. 2021, 6, 1199
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    18
    Interrogation of Electrochemical Aptamer-Based Sensors via Peak-to-Peak Separation in Cyclic Voltammetry Improves the Temporal Stability and Batch-to-Batch Variability in Biological Fluids
    Pellitero, Miguel Aller; Curtis, Samuel D.; Arroyo-Curras, Netzahualcoyotl
    ACS Sensors (2021), 6 (3), 1199-1207CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
    Electrochem., aptamer-based (E-AB) sensors support continuous, real-time measurements of specific mol. targets in complex fluids such as undiluted serum. They achieve these measurements by using redox-reporter-modified, electrode-attached aptamers that undergo target binding-induced conformational changes which, in turn, change electron transfer between the reporter and the sensor surface. Traditionally, E-AB sensors are interrogated via pulse voltammetry to monitor binding-induced changes in transfer kinetics. While these pulse techniques are sensitive to changes in electron transfer, they also respond to progressive changes in the sensor surface driven by biofouling or monolayer desorption and, consequently, present a significant drift. Moreover, we have empirically obsd. that differential voltage pulsing can accelerate monolayer desorption from the sensor surface, presumably via field-induced actuation of aptamers. Here, in contrast, we demonstrate the potential advantages of employing cyclic voltammetry to measure electron-transfer changes directly. In our approach, the target concn. is reported via changes in the peak-to-peak sepn., ΔEP, of cyclic voltammograms. Because the magnitude of ΔEP is insensitive to variations in the no. of aptamer probes on the electrode, ΔEP-interrogated E-AB sensors are resistant to drift and show decreased batch-to-batch and day-to-day variability in sensor performance. Moreover, ΔEP-based measurements can also be performed in a few hundred milliseconds and are, thus, competitive with other subsecond interrogation strategies such as chronoamperometry but with the added benefit of retaining sensor capacitance information that can report on monolayer stability over time.
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  19. 19
    Arroyo-Currás, N.; Dauphin-Ducharme, P.; Ortega, G.; Ploense, K. L.; Kippin, T. E.; Plaxco, K. W. Subsecond-Resolved Molecular Measurements in the Living Body Using Chronoamperometrically Interrogated Aptamer-Based Sensors. ACS Sens. 2018, 3, 360– 366,  DOI: 10.1021/acssensors.7b00787
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    19
    Subsecond-Resolved Molecular Measurements in the Living Body Using Chronoamperometrically Interrogated Aptamer-Based Sensors
    Arroyo-Curras, Netzahualcoyotl; Dauphin-Ducharme, Philippe; Ortega, Gabriel; Ploense, Kyle L.; Kippin, Tod E.; Plaxco, Kevin W.
    ACS Sensors (2018), 3 (2), 360-366CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
    Electrochem., aptamer-based (E-AB) sensors support the continuous, real-time measurement of specific small mols. directly in situ in the living body over the course of many hours. They achieve this by employing binding-induced conformational changes to alter electron transfer from a redox-reporter-modified, electrode-attached aptamer. Previously the authors have used voltammetry (cyclic, a.c., and square wave) to monitor this binding-induced change in transfer kinetics indirectly. Here, however, the authors demonstrate the potential advantages of employing chronoamperometry to measure the change in kinetics directly. In this approach target concn. is reported via changes in the lifetime of the exponential current decay seen when the sensor is subjected to a potential step. Because the lifetime of this decay is independent of its amplitude (e.g., insensitive to variations in the no. of aptamer probes on the electrode), chronoamperometrically interrogated E-AB sensors are calibration-free and resistant to drift. Chronoamperometric measurements can also be performed in a few hundred milliseconds, improving the previous few-second time resoln. of E-AB sensing by an order of magnitude. To illustrate the potential value of the approach the authors demonstrate here the calibration-free measurement of the drug tobramycin in situ in the living body with 300 ms time resoln. and unprecedented, few-percent precision in the detn. of its pharmacokinetic phases.
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  20. 20
    Downs, A. M.; Gerson, J.; Ploense, K. L.; Plaxco, K. W.; Dauphin-Ducharme, P. Subsecond-Resolved Molecular Measurements Using Electrochemical Phase Interrogation of Aptamer-Based Sensors. Anal. Chem. 2020, 92, 14063– 14068,  DOI: 10.1021/acs.analchem.0c03109
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    20
    Subsecond-Resolved Molecular Measurements Using Electrochemical Phase Interrogation of Aptamer-Based Sensors
    Downs, Alex M.; Gerson, Julian; Ploense, Kyle L.; Plaxco, Kevin W.; Dauphin-Ducharme, Philippe
    Analytical Chemistry (Washington, DC, United States) (2020), 92 (20), 14063-14068CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Recent years have seen the development of a no. of biosensor architectures that rely on target binding-induced changes in the rate of electron transfer from an electrode-bound receptor. Most often, the interrogation of these sensors has relied on voltammetric methods, such as square-wave voltammetry, which limit their time resoln. to a few seconds. Here, we describe the use of an impedance-based approach, which we have termed electrochem. phase interrogation, as a means of collecting high time resoln. measurements with sensors in this class. Specifically, using changes in the electrochem. phase to monitor target binding in an electrochem.-aptamer based (EAB) sensor, we achieve subsecond temporal resoln. and multihour stability in measurements performed directly in undiluted whole blood. Electrochem. phase interrogation also offers improved insights into EAB sensors' signaling mechanism. By modeling the interfacial resistance and capacitance using equiv. circuits, we find that the only parameter that is altered by target binding is the charge-transfer resistance. This confirms previous claims that binding-induced changes in electron-transfer kinetics drive signaling in this class of sensors. Considering that a wide range of electrochem. biosensor architectures rely on this signaling mechanism, we believe that electrochem. phase interrogation may prove generalizable toward subsecond measurements of mol. targets.
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  21. 21
    Arroyo-Currás, N.; Scida, K.; Ploense, K. L.; Kippin, T. E.; Plaxco, K. W. High Surface Area Electrodes Generated Via Electrochemical Roughening Improve the Signaling of Electrochemical Aptamer-Based Biosensors. Anal. Chem. 2017, 89, 12185– 12191,  DOI: 10.1021/acs.analchem.7b02830
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    21
    High Surface Area Electrodes Generated via Electrochemical Roughening Improve the Signaling of Electrochemical Aptamer-Based Biosensors
    Arroyo-Curras, Netzahualcoyotl; Scida, Karen; Ploense, Kyle L.; Kippin, Tod E.; Plaxco, Kevin W.
    Analytical Chemistry (Washington, DC, United States) (2017), 89 (22), 12185-12191CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    The electrochem., aptamer-based (E-AB) sensor platform provides a modular approach to the continuous, real-time measurement of specific mol. targets (irresp. of their chem. reactivity) in situ in the living body. To achieve this, however, requires the fabrication of sensors small enough to insert into a vein, which, for the rat animal model the authors employ, entails devices less than 200 μm in diam. The limited surface area of these small devices leads, in turn, to low faradaic currents and poor signal-to-noise ratios when deployed in the complex, fluctuating environments found in vivo. In response the authors have developed an electrochem. roughening approach that enhances the signaling of small electrochem. sensors by increasing the microscopic surface area of gold electrodes, allowing in this case more redox-reporter-modified aptamers to be packed onto the surface, thus producing significantly improved signal-to-noise ratios. Unlike previous approaches to achieving microscopically rough gold surfaces, the method employs chronoamperometric pulsing in a 5 min etching process easily compatible with batch manufg. Using these high surface area electrodes, the authors demonstrate the ability of E-AB sensors to measure complete drug pharmacokinetic profiles in live rats with precision of better than 10% in the detn. of drug disposition parameters.
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  22. 22
    Dauphin-Ducharme, P.; Arroyo-Currás, N.; Kurnik, M.; Ortega, G.; Li, H.; Plaxco, K. W. Simulation-Based Approach to Determining Electron Transfer Rates Using Square-Wave Voltammetry. Langmuir 2017, 33, 4407– 4413,  DOI: 10.1021/acs.langmuir.7b00359
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    22
    Simulation-Based Approach to Determining Electron Transfer Rates Using Square-Wave Voltammetry
    Dauphin-Ducharme, Philippe; Arroyo-Curras, Netzahualcoyotl; Kurnik, Martin; Ortega, Gabriel; Li, Hui; Plaxco, Kevin W.
    Langmuir (2017), 33 (18), 4407-4413CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    The efficiency with which square-wave voltammetry differentiates faradaic and charging currents makes it a particularly sensitive electroanal. approach, as evidenced by its ability to measure nanomolar or even picomolar concns. of electroactive analytes. Because of the relative complexity of the potential sweep it uses, however, the extn. of detailed kinetic and mechanistic information from square-wave data remains challenging. In response, the authors demonstrate here a numerical approach by which square-wave data can be used to det. electron transfer rates. Specifically, the authors have developed a numerical approach in which the authors model the height and the shape of voltammograms collected over a range of square-wave frequencies and amplitudes to simulated voltammograms as functions of the heterogeneous rate const. and the electron transfer coeff. As validation of the approach, it was used to det. electron transfer kinetics in both freely diffusing and diffusionless surface-tethered species, obtaining electron transfer kinetics in all cases in good agreement with values derived using non-square-wave methods.
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  23. 23
    Lyshevski, S. E. Dekker Encyclopedia of Nanoscience and Nanotechnology; CRC Press, 2014.
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    There is no corresponding record for this reference.
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    Rothemund, P. W. K. Folding DNA to Create Nanoscale Shapes and Patterns. Nature 2006, 440, 297– 302,  DOI: 10.1038/nature04586
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    24
    Folding DNA to create nanoscale shapes and patterns
    Rothemund, Paul W. K.
    Nature (London, United Kingdom) (2006), 440 (7082), 297-302CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    'Bottom-up fabrication', which exploits the intrinsic properties of atoms and mols. to direct their self-organization, is widely used to make relatively simple nanostructures. A key goal for this approach is to create nanostructures of high complexity, matching that routinely achieved by 'top-down' methods. The self-assembly of DNA mols. provides an attractive route towards this goal. Here the author describe a simple method for folding long, single-stranded DNA mols. into arbitrary two-dimensional shapes. The design for a desired shape is made by raster-filling the shape with a 7-kilobase single-stranded scaffold and by choosing over 200 short oligonucleotide 'staple strands' to hold the scaffold in place. Once synthesized and mixed, the staple and scaffold strands self-assemble in a single step. The resulting DNA structures are roughly 100 nm in diam. and approx. desired shapes such as squares, disks and five-pointed stars with a spatial resoln. of 6 nm. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be programmed to bear complex patterns such as words and images on their surfaces. Finally, individual DNA structures can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles (which constitutes a 30-megadalton mol. complex).
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  25. 25
    Roth, E.; Glick Azaria, A.; Girshevitz, O.; Bitler, A.; Garini, Y. Measuring the Conformation and Persistence Length of Single-Stranded DNA Using a DNA Origami Structure. Nano Lett. 2018, 18, 6703– 6709,  DOI: 10.1021/acs.nanolett.8b02093
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    25
    Measuring the Conformation and Persistence Length of Single-Stranded DNA Using a DNA Origami Structure
    Roth, Efrat; Glick Azaria, Alex; Girshevitz, Olga; Bitler, Arkady; Garini, Yuval
    Nano Letters (2018), 18 (11), 6703-6709CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
    Measuring the mech. properties of single-stranded DNA (ssDNA) is a challenge that has been addressed by different methods lately. The short persistence length, fragile structure, and the appearance of stem loops complicate the measurement, and this leads to a large variability in the measured values. Here the authors describe an innovative method based on DNA origami for studying the biophys. properties of ssDNA. By synthesizing a DNA origami structure that consists of two rigid rods with an ssDNA segment between them, the authors developed a method to characterize the effective persistence length of a random-sequence ssDNA while allowing the formation of stem loops. The authors imaged the structure with an at. force microscope (AFM); the rigid rods provide a means for the exact identification of the ssDNA ends. This leads to an accurate detn. of the end-to-end distance of each ssDNA segment, and by fitting the measured distribution to the ideal chain polymer model the authors measured an effective persistence length of 1.98 ± 0.72 nm. This method enables one to measure short or long strands of ssDNA, and it can cope with the formation of stem loops that are often formed along ssDNA. The authors envision that this method can be used for measuring stem loops for detg. the effect of repetitive nucleotide sequences and environmental conditions on the mech. properties of ssDNA and the effect of interacting proteins with ssDNA. Further the method can be extended to nanoprobes for measuring the interactions of specific DNA sequences, because the DNA origami rods (or similar structures) can hold multiple fluorescent probes that can be easily detected.
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    Savéant, J.-M. Molecular Catalysis of Electrochemical Reactions. Mechanistic Aspects. Chem. Rev. 2008, 108, 2348– 2378,  DOI: 10.1021/cr068079z
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    26
    Molecular Catalysis of Electrochemical Reactions. Mechanistic Aspects
    Saveant, Jean-Michel
    Chemical Reviews (Washington, DC, United States) (2008), 108 (7), 2348-2378CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. The term "electrocatalysis" is traditionally used for reactions in which the electrode material-often, but not always, a metal-is chem. involved in the catalytic process. Although the chem. properties of the electrode material play an important role in governing the catalytic efficiency, geometric and crystallog. features, nature and no. of defects, may also be of paramount significance. The differences between the bulk properties of the metal and its surface properties are particularly important in this respect. It is therefore difficult, or even irrelevant, to analyze results and devise new catalytic systems on the basis of mol. concepts. Another approach to catalyzing electrochem. reactions is to use mols. as catalysts. "Mol. catalysis" thus defined may involve catalyst mols. either homogeneously dispersed in the soln. bathing the electrode or immobilized in a monolayer or multilayered coating deposited on the electrode surface.
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  27. 27
    Eden, A.; Scida, K.; Arroyo-Currás, N.; Eijkel, J. C. T.; Meinhart, C. D.; Pennathur, S. Modeling Faradaic Reactions and Electrokinetic Phenomena at a Nanochannel-Confined Bipolar Electrode. J. Phys. Chem. C 2019, 123, 5353– 5364,  DOI: 10.1021/acs.jpcc.8b10473
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    27
    Modeling Faradaic Reactions and Electrokinetic Phenomena at a Nanochannel-Confined Bipolar Electrode
    Eden, A.; Scida, K.; Arroyo-Curras, N.; Eijkel, J. C. T.; Meinhart, C. D.; Pennathur, S.
    Journal of Physical Chemistry C (2019), 123 (9), 5353-5364CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
    The authors present the most comprehensive 2-dimensional numerical model to date for a nanoconfined bipolar electrochem. system. By accounting for the compact Stern layer and resolving the diffuse part of the elec. double layer (EDL) at the BPE surface and channel walls, the authors' model captures the impact of surface polarization and ionic charge screening effects on the heterogeneous charge-transfer kinetics, as well as nonlinear electrokinetic transport phenomena such as induced-charge electroosmosis and concn. polarization. The authors employ the Poisson-Nernst-Planck and Stokes flow system of equations, unified with generalized Frumkin-Butler-Volmer reaction kinetics, to describe H2O electrolysis reactions and the resulting transport of ions and dissolved gases in the confined bipolar electrode (BPE) system. The authors' results demonstrate that under a sufficiently large applied elec. field, the rapid reaction kinetics on the authors' Pt BPE dynamically transition from charge-transfer limited to mass-transfer limited temporal regimes as regions depleted of redox species form and propagate outwards from the resp. BPE poles. This phenomenon was visualized exptl. with pH-sensitive fluorescein dye and showed excellent phenomenol. agreement with the authors' numerical calcns., providing a foundation for further understanding and developing bipolar electrochem. processes in confined geometries. The authors introduce two prospective applications arising from the authors' work: (1) a hybrid hydrodynamic/electrochem. peristaltic pump, and (2) deducing information about chem. kinetics through simulation.
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    Biosensors built using RNA aptamers show promise as tools for point-of-care medical diagnostics, but they remain vulnerable to nuclease degrdn. when deployed in clin. samples. To explore methods for protecting RNA-based biosensors from such degrdn. we have constructed and characterized an electrochem., aptamer-based sensor for the detection of aminoglycosidic antibiotics. We find that while this sensor achieves low micromolar detection limits and subminute equilibration times when challenged in buffer, it deteriorates rapidly when immersed directly in blood serum. In order to circumvent this problem, we have developed and tested sensors employing modified versions of the same aptamer. Our first effort to this end entailed the methylation of all of the 2'-hydroxyl groups outside of the aptamer's antibiotic binding pocket. However, while devices employing this modified aptamer are as sensitive as those employing an unmodified parent, the modification fails to confer greater stability when the sensor is challenged directly in blood serum. As a second potentially naive alternative, we replaced the RNA bases in the aptamer with their more degrdn.-resistant DNA equiv. Surprisingly and unlike control DNA-stem loops employing other sequences, this DNA aptamer retains the ability to bind aminoglycosides, albeit with poorer affinity than the parent RNA aptamer. Unfortunately, however, while sensors fabricated using this DNA aptamer are stable in blood serum, its lower affinity pushes their detection limits above the therapeutically relevant range. Finally, we find that ultrafiltration through a low-mol.-wt.-cutoff spin column rapidly and efficiently removes the relevant nucleases from serum samples spiked with gentamicin, allowing the convenient detection of this aminoglycoside at clin. relevant concns. using the original RNA-based sensor.
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    A review. The authors review some of the characteristics and mechanisms of DNA charge transport. Mechanisms discussed include transport through water, ions and phosphates; superexchange; localized hopping; and delocalized mechanisms.
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    The binding of the known DNA intercalators methylene blue and quinacrine with four sequence specific polynucleotides, viz. poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dT).poly(dA-dT) and poly(dA).poly(dT), have been compared using absorbance, fluorescence, competition dialysis and thermal melting and the thermodn. aspects of the interaction studied. In all the cases, non-cooperative binding phenomena obeying neighbor exclusion principle was obsd. though the affinity was remarkably higher for quinacrine and the nature of the binding was characterized to be true intercalation. The data on the salt dependence of binding derived from the plot of log K vs. log[Na+] revealed a slope of around 1.0, consistent with the values predicted by the theories for the binding of monovalent cations, and contained contributions from polyelectrolytic and non-polyelectrolytic forces. The bindings were characterized by strong stabilization of the polynucleotides against thermal strand sepn. in both optical melting as well as differential scanning calorimetry studies. The data analyzed from the thermal melting and isothermal titrn. calorimetry studies were in close proximity to those obtained from absorption spectral titrn. data. Isothermal titrn. calorimetry results revealed the bindings to poly(dG-dC).poly(dG-dC), poly(dG).poly(dC) and poly(dA-dT).poly(dA-dT) to be exothermic and favored by both neg. enthalpy and large favorable pos. entropy changes, while that to poly(dA).poly(dT) was endothermic and entropy driven. The heat capacity changes obtained from temp. dependence of enthalpy gave neg. values to all polynucleotides. New insights on the mol. aspects of interaction of these mols. to DNA have emerged from these studies.
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    We have used absorption and fluorimetric methods to study the interaction between methylene blue (MB) and calfthymus DNA. Based on Scatchard anal. of the exptl. data, we plotted the methylene blue-DNA binding curve. This curve consists of two linear sections, which indicates two types of interaction, for which we detd. the consts. K and the no. of binding sites n for binding of this ligand to DNA. Comparison of the data obtained with analogous values found for interaction between ethidium bromide and DNA allowed us to conclude that there are two modes of interaction between methylene blue and DNA: strong binding (semi-intercalation) and weak binding (electrostatic).
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    The abbreviated name, ' mfold web server', describes a no. of closely related software applications available on the World Wide Web (WWW) for the prediction of the secondary structure of single stranded nucleic acids. The objective of this web server is to provide easy access to RNA and DNA folding and hybridization software to the scientific community at large. By making use of universally available web GUIs (Graphical User Interfaces), the server circumvents the problem of portability of this software. Detailed output, in the form of structure plots with or without reliability information, single strand frequency plots and energy dot plots', are available for the folding of single sequences. A variety of bulk' servers give less information, but in a shorter time and for up to hundreds of sequences at once.
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    Journal of Physical Chemistry C (2018), 122 (37), 21441-21448CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
    A wide range of new devices aimed at in vivo mol. detection and point-of-care diagnostics rely on binding-induced changes in electron-transfer kinetics from an electrode-attached, redox-reporter-modified oligonucleotide as their signaling mechanism. In an effort to better characterize the mechanisms underlying these sensors, we have measured the electron-transfer kinetics assocd. with surface-attached, single-stranded DNAs modified with a methylene blue redox reporter either at the chain's distal end or at an internal chain position. We find that although the rate of electron transfer from a reporter placed either terminally or internally is independent of chain length for chains shorter than the length scale of methylene blue (and its linker), for longer chains it follows a power-law dependence on length of exponent approx. -2.2. Such behavior is consistent with a diffusion-controlled mechanism in which the diffusion of the DNA-bound reporter to the surface controls the rate of electron transfer. This said, the obsd. rates are, at 5-400 s-1, orders of magnitude slower than the intramol. dynamics of single-stranded oligonucleotides when free in soln. Likewise, the rates of transfer from reporters placed internally are several-fold slower than those seen for the equiv. terminally modified construct. We attribute these effects to electrostatic repulsion between the oligonucleotide and the electrode surface, which is neg. charged at the redox potential of methylene blue. Consistent with this, changing monolayer compn. so as to increase the neg. charge of the surface reduces the transfer rate still more without significantly altering its power-law chain length dependence. Simple theor. models and computer simulations performed in support of our exptl. studies find similar power-law dependencies, similar electrostatic slowing of the transfer rate, and similar rate differences between terminally an internally modified constructs.
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    The photoredn. of methylene blue by N-methyl-9-phenylacridan (ACH) is studied in acetonitrile by means of flash photolysis and quantum yields. In the first step, due to fast proton shift within the original electron transfer product protonated semi-methylene blue MBH•+ (I) and the deprotonated donor radical N-methyl-9-phenylacridanyl (AC) are formed with a rate const. of 2 × 108 M-1 s-1. In the radical pair a second electron is transferred very fast from AC to I with a rate const. ke2 ≈ 101- s-1 to form leuco-methylene blue and N-methyl-9-phenylacridinium salt (AC+). About 80% of the two-equiv redn. product, leuco-methylene blue, is formed within the first solvent cage during the flash. The max. quantum yields of photoredn. approach φisc of MB+ as expected for a two-equiv. redn. The out-of-cage reaction consists of the known disproportionation of I and its redn. by AC. From the decay kinetics kred = 3 × 109 M-1 s-1 and kdis = 8 × 108 M-1 s-1 are derived. The system sensitizes the dediazoniation of p-(dimethylamino)benzenediazonium tetrafluoroborate efficiently even at very low diazonium salt concns. (φ = 0.6).
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  • Abstract

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    Figure 1

    Figure 1. Electrochemical reduction of methylene blue. This scheme uses the curved-arrow formalism to explain the electrochemical reduction of methylene blue according to the original mechanism proposed by Jean Chevalet. (13)

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    Figure 2

    Figure 2. Description of the numerical model. (A) We created a one-dimensional model representing the DNA-functionalized electrodes of Figure 3A,B. (B) We simulate the voltammetric response of these electrodes by using a triangular voltage waveform with slope E·t–1, which corresponds to the voltage scanning rate. (C) In response to this varying potential, the experimental system (Figure 3A,B) produces a cyclic voltammogram (black trace) which varies in shape and magnitude according to the chemical equilibrium of the redox species considered. Our model accurately simulates the same voltammogram (red circles) via eqs 10–17, 19, and 20. The surface DNA concentration was 1.93 ± 0.64 pmol·cm–2 here and in all figures of the article, unless noted otherwise. All voltammetric measurements were performed in 1× PBS as defined in the Methods section, unless noted otherwise.

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    Figure 3

    Figure 3. Electrode-attached, methylene blue-modified DNA exhibits voltammetric features that change with increasing voltage scanning rate. (A) To illustrate these features, we employ DNA constructs of different lengths and secondary structures (Table S1), including one aptamer (illustrated here) that binds to the antibiotic tobramycin. We modify these constructs with a hexanethiol linker in the 5′ terminus and a methylene blue reporter at the 3′ terminus and co-deposit them onto gold electrodes along with 1-hexanethiol to form a SAM. (B) In the presence of tobramycin, the aptamer undergoes binding-induced conformational changes that accelerate electron transfer from methylene blue to the gold electrode, possibly by bringing methylene blue closer to the electrode surface. (C) When we interrogate aptamer-functionalized electrodes at scan rates ≤10 V·s–1 with surface coverage concentrations of 1.93 ± 0.64 pmol·cm–2 and either no tobramycin added or 1 mM of tobramycin, we observe no splitting of voltammetric waves. (D) However, at voltage scanning rates > 10 V·s–1, we observe splitting of the oxidation wave. Moreover, the addition of the target causes an inversion of behavior, showing peak splitting in the reduction but not in the oxidation wave. See the Methods section for additional experimental information.

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    Figure 4

    Figure 4. Modeling the electrochemical reduction of DNA-tethered methylene blue at different voltage scanning rates. We recorded these background-subtracted cyclic voltammograms on gold electrodes functionalized with a 26-base DNA aptamer modified at one end with methylene blue (Figure 2A). As a model system, we co-deposit 500 nM of an aptamer that binds to the antibiotic tobramycin with 1 mM 1-hexanethiol. We then obtain cyclic voltammograms (black traces) in 1× PBS solution at voltage scanning rates of (A) 1, (B) 4, (C) 10, (D) 55, (E) 100, and (F) 150 V·s–1. Using the parameters described in Table S3 in our numerical model, we generate simulated voltammograms (colored circles) closely matching the experimental ones. We only show 1 in every 8 simulated currents for clarity. Error bars report the true differential error between experimental and simulated voltammograms. The color indicates percent error, which we use to demonstrate that our numerical model correctly estimates the current magnitude of the different voltammetric peaks with an accuracy of 85% or better; that is, ≤15% error (Figure S3).

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    Figure 5

    Figure 5. High deposition concentrations of methylene blue cause peak broadening and increased overpotentials but do not affect peak splitting. To show that higher packing density of DNA-bound methylene blue at the surface of an electrode does not affect peak splitting, we co-deposited 1-hexanethiol and either (A) 100 nM, (B) 500 nM, (C) 1 μM, or (D) 25 μM of the tobramycin aptamer (Table S1) onto gold electrodes with resulting surface coverages of (A) 1.93 ± 0.64, (B) 2.24 ± 0.58, (C) 2.66 ± 0.45,, and (D) 4.61 ± 0.68 pmol·cm–2. We then measured voltammograms at a scanning rate of 100 V·s–1 and fit the data with an accuracy of 85% or better (Figure S6) using the parameters found in Table S4. We speculate that the wave broadening and 60 mV shift in formal reduction potential observed in electrodes prepared from solutions containing 25 μM DNA relative to those prepared at 100 nM are due to leucomethylene blue self-stacking (Figure 1B).

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    Figure 6

    Figure 6. Voltammetric peak splitting is dependent on DNA secondary structure. We employed nucleotide sequences of (A) 10, (B) 20, (C) 26, (D) 37 nt, (E) tobramycin aptamer hybridized to a fully complementary strand, and (F) tobramycin aptamer in the presence of the target to test whether secondary structure affects peak splitting. Each of the constructs were co-deposited at 500 nM with 1 mM 1-hexanethiol onto gold electrodes and are modified with methylene blue at the distal terminus. All experiments were done in 1× PBS solution and experiments A–E were performed at 100 V·s–1, while experiment F was performed at 150 V·s–1. We report the theoretical secondary structures for each construct as generated by mfold software (Table S1). (34) Although all constructs exhibit peak splitting, we observe a reduction in this splitting with decreasing oligonucleotide length (A vs D). This is because, as the DNA shortens, the electron-transfer kinetics of the system increase as previously demonstrated. (35) We also observe a reversal in peak splitting behavior as a stable secondary structure is introduced (E for double-stranded DNA and F for a folded tobramycin-binding aptamer in the presence of 1 mM tobramycin). This is related to a change in protonation rate (Table S5) which suggests a change in hydrogen bonding interactions between methylene blue and the DNA. Error bars report the true differential error between experimental and simulated voltammograms. The color indicates percent error, which we use to demonstrate that each simulation fits the peak currents of background-subtracted voltammograms with an accuracy of 85% or better; that is, <15% error (Figure S7).

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    Figure 7

    Figure 7. Chemical equilibrium explains the changes in voltammetric features observed at different values of pH. To illustrate this point, we measured cyclic voltammograms on DNA-functionalized electrodes (tobramycin-binding aptamer) immersed in 1× PBS at pH = 4, 7, and 9. For the low and high pH values, we potentiometrically titrated the buffered solutions with HCl or sodium hydroxide, respectively. As shown here, our model accurately predicts the position and current magnitude of the main voltammetric features in background-subtracted voltammograms with greater than 85% accuracy at all values of pH (Figure S8). However, we note that at pH = 9 and pH = 4, the mechanism changes from an ErCrEr (eqs 1–3) reaction to an ErEr (eqs 23 and 24) scheme, where electrochemical reduction no longer requires protonation. The latter mechanism is included in our numerical model. We use the parameters reported in Table S6 to achieve these fits. Error bars report the true difference error between experimental and simulated voltammograms. The color indicates percent error.

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    Figure 8

    Figure 8. Electrode-blocking SAMs affect the protonation rate of the leucomethylene blue radical. After co-depositing 100 nM of the tobramycin aptamer and 1 mM either (A) 6-mercaptohexanol, (B) 1-hexanethiol, or (C) fluorinated analogue of 1-hexanethiol onto gold electrodes, we collected cyclic voltammograms at 1, 10, and 100 V·s–1 that we model with an accuracy of 85% or greater (Figure S9). We adjusted the starting concentration of species O to account for the smaller background-corrected peak currents of 6-mercaptohexanol when compared to 1-hexanethiol, which are a product of the removal of the charging current (Table 1). (34) We observe less peak splitting occurring in 6-mercaptohexanol, which can be explained by its nearly 2-fold higher average apparent protonation rate as compared with 1-hexanethiol (Table 1). However, we observe greater peak splitting occurring for the fluorinated monolayer, which can be explained by the nearly 3-fold decrease in apparent protonation rate as compared with 1-hexanethiol.

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  • References

    This article references 40 other publications.

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      SciFinder Literature Search with the Terms DNA + Sensor + Methylene Blue Produced 897 Hits. Similar Searches Replacing Methylene Blue for Ferrocene and Anthraquinone Produced Only 543 and 85 Hits, Respectively; Chemical Abstracts Service: Columbus, OH (accessed 2020-06-15).
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      Survey of Redox-Active Moieties for Application in Multiplexed Electrochemical Biosensors
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      Analytical Chemistry (Washington, DC, United States) (2016), 88 (21), 10452-10458CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Recent years have seen the development of a large no. of electrochem. sandwich assays and reagentless biosensor architectures employing biomols. modified via the attachment of a redox-active "reporter.". Here the authors survey a large set of potential redox reporters to det. which exhibits the best long-duration stability in thiol-on-gold monolayer-based sensors and to identify reporter "sets" signaling at distinct, non-overlapping redox potentials in support of multiplexing and error correcting ratiometric or differential measurement approaches. Specifically, the authors have characterized the performance of more than a dozen potential reporters that are, first, redox active within the potential window over which thiol-on-gold monolayers are reasonably stable and, second, are available com. in forms that are readily conjugated to biomols. or can be converted into such forms in one or two simple synthetic steps. To test each of these reporters the authors conjugated it to one terminus of a single-stranded DNA "probe" that was attached by its other terminus via a six-carbon thiol to a gold electrode to form an "E-DNA" sensor responsive to its complementary DNA target. The authors then measured the signaling properties of each sensor as well as its stability against repeated voltammetric scans and against deployment in and reuse from blood serum. Doing so the performance of methylene blue-based, thiol-on-gold sensors is unmatched; the near-quant. stability of such sensors against repeated scanning in even very complex sample matrixes is unparalleled. While more modest, the stability of sensors employing a handful of other reporters, including anthraquinone, Nile blue, and ferrocene, is reasonable. The authors' work thus serves as both to highlight the exceptional properties of methylene blue as a redox reporter in such applications and as a cautionary tale; the authors wish to help other researchers avoid fruitless efforts to employ the many, seemingly promising and yet ultimately inadequate reporters the authors have studied. Finally, the authors hope that their work also serves as an illustration of the pressing need for the further development of useful redox reporters.
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      Shaver, A.; Curtis, S. D.; Arroyo-Currás, N. Alkanethiol Monolayer End Groups Affect the Long-Term Operational Stability and Signaling of Electrochemical, Aptamer-Based Sensors in Biological Fluids. ACS Appl. Mater. Interfaces 2020, 12, 11214– 11223,  DOI: 10.1021/acsami.9b22385
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      Alkanethiol Monolayer End Groups Affect the Long-Term Operational Stability and Signaling of Electrochemical, Aptamer-Based Sensors in Biological Fluids
      Shaver, Alexander; Curtis, Samuel D.; Arroyo-Curras, Netzahualcoyotl
      ACS Applied Materials & Interfaces (2020), 12 (9), 11214-11223CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
      Electrochem. aptamer-based (E-AB) sensors achieve highly precise measurements of specific mol. targets in untreated biol. fluids. This unique ability, together with their measurement frequency of seconds or faster, has enabled the real-time monitoring of drug pharmacokinetics in live animals with unprecedented temporal resoln. However, one important weakness of E-AB sensors is that their bioelectronic interface degrades upon continuous electrochem. interrogation-a process typically seen as a drop in faradaic and an increase in charging currents over time. This progressive degrdn. limits their in vivo operational life to 12 h at best, a period that is much shorter than the elimination half-life of the vast majority of drugs in humans. Thus, there is a crit. need to develop novel E-AB interfaces that resist continuous electrochem. interrogation in biol. fluids for prolonged periods. In response, our group is pursuing the development of better packed, more stable self-assembled monolayers (SAMs) to improve the signaling and extend the operational life of in vivo E-AB sensors from hours to days. By invoking hydrophobicity arguments, we have created SAMs that do not desorb from the electrode surface in aq. physiol. solns. and biol. fluids. These SAMs, formed from 1-hexanethiol solns., decrease the voltammetric charging currents of E-AB sensors by 3-fold relative to std. monolayers of 6-mercapto-1-hexanol, increase the total faradaic current, and alter the electron transfer kinetics of the platform. Moreover, the stability of our new SAMs enables uninterrupted, continuous E-AB interrogation for several days in biol. fluids, like undiluted serum, at a physiol. temp. of 37°C.
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      Mechanisms of methylene blue electrode processes studied by in situ electron paramagnetic resonance and ultraviolet-visible spectroelectrochemistry
      Zhan, Ruiyun; Song, Shihua; Liu, Yayan; Dong, Shaojun
      Journal of the Chemical Society, Faraday Transactions (1990), 86 (18), 3125-7CODEN: JCFTEV; ISSN:0956-5000.
      The redox process of methylene blue was studied using thin-layer cyclic voltammetry, UV-VIS spectroelectrochem. techniques and in-situ EPR. The in-situ EPR measurements provide direct evidence for the formation of a methylene blue cation radical. The electrode mechanism for methylene blue is discussed.
      >> More from SciFinder ®
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      Ju, H.; Zhou, J.; Cai, C.; Chen, H. The Electrochemical Behavior of Methylene Blue at a Microcylinder Carbon Fiber Electrode. Electroanalysis 1995, 7, 1165– 1170,  DOI: 10.1002/elan.1140071213
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      The electrochemical behavior of methylene blue at a microcylinder carbon fiber electrode
      Ju, Huangxian; Zhou, Jun; Cai, Chenxin; Chen, Hongyuan
      Electroanalysis (1995), 7 (12), 1165-70CODEN: ELANEU; ISSN:1040-0397. (VCH)
      The electrochem. behavior of methylene blue (MB) at a carbon fiber microcylinder electrode, was studied by cyclic voltammetry. The charge transfer coeff. α of the electrode reaction with two-electron transfer is 0.5, and the no. of H+ participating in the electrode process is 3 at pH 2.2-5.4, 2 at pH 5.4-6.0 and 1 at pH 6.0-10.7, resp. The std. electron transfer rate const. k°', and std. formal potential E°' of MB at various pH were detd. by using the carbon fiber microcylinder electrode. The electrode reaction mechanism of methylene blue at various pH is proposed. The adsorbability of MB at the electrode is discussed and explored by cyclic voltammetry and chronocoulometric technique.
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      Pheeney, C. G.; Barton, J. K. DNA Electrochemistry with Tethered Methylene Blue. Langmuir 2012, 28, 7063– 7070,  DOI: 10.1021/la300566x
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      DNA Electrochemistry with Tethered Methylene Blue
      Pheeney, Catrina G.; Barton, Jacqueline K.
      Langmuir (2012), 28 (17), 7063-7070CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      Methylene blue (MB'), covalently attached to DNA through a flexible C12 alkyl linker, provides a sensitive redox reporter in DNA electrochem. measurements. Tethered, intercalated MB' is reduced through DNA-mediated charge transport; the incorporation of a single base mismatch at position 3, 10, or 14 of a 17-mer causes an attenuation of the signal to 62 ± 3% of the well-matched DNA, irresp. of position in the duplex. The redox signal intensity for MB'-DNA is found to be least 3-fold larger than that of Nile blue (NB)-DNA, indicating that MB' is even more strongly coupled to the π-stack. The signal attenuation due to an intervening mismatch does, however, depend on DNA film d. and the backfilling agent used to passivate the surface. These results highlight two mechanisms for redn. of MB' on the DNA-modified electrode: redn. mediated by the DNA base pair stack and direct surface redn. of MB' at the electrode. These two mechanisms are distinguished by their rates of electron transfer that differ by 20-fold. The extent of direct redn. at the surface can be controlled by assembly and buffer conditions.
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      Yang, W.; Lai, R. Y. Comparison of the Stem-Loop and Linear Probe-Based Electrochemical DNA Sensors by Alternating Current Voltammetry and Cyclic Voltammetry. Langmuir 2011, 27, 14669– 14677,  DOI: 10.1021/la203015v
      8
      Comparison of the stem-loop and linear probe-based electrochemical DNA sensors by alternating current voltammetry and cyclic voltammetry
      Yang, Weiwei; Lai, Rebecca Y.
      Langmuir (2011), 27 (23), 14669-14677CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      Here we systematically characterized the sensor performance of the stem-loop probe (SLP) and linear probe (LP) electrochem. DNA sensors using a.c. voltammetry (ACV) and cyclic voltammetry (CV), with the goal of generating the set of operational criteria that best suits each sensor architecture, in addn. to elucidating the signaling mechanism behind these sensors. Although the LP sensor shows slightly better % signal suppression (SS) upon hybridization with the perfect match target at 10 Hz, our frequency-dependent study suggests that it shows optimal % SS only in a very limited AC frequency range. Similar results are obsd. in CV studies in which the LP sensor, when compared to the SLP sensor, displays a narrower range of voltammetric scan rates where the optimal % SS can be achieved. More importantly, the difference between the two sensors' performance is particularly pronounced if the change in integrated charge (Q) upon target hybridization, rather than the peak current (I), is measured in CV. The temp.-dependent study further highlights the differences between the two sensors, where the LP sensor, owing to the flexible linear probe architecture, is more readily perturbed by temp. changes. Both SLP and LP sensors, however, show a loss of % SS when operated at elevated temps., despite the significant improvement in the hybridization kinetics. In conjunction with the ACV, CV, and temp.-dependent studies, the electron-transfer kinetics study provides further evidence in support of the proposed signaling mechanism of these two sensors, in which the SLP sensor's signaling efficiency and sensor performance is directly linked to the hybridization-induced conformational change in the redox-labeled probe, whereas the performance of the LP sensor relies on the hybridization-induced change in probe dynamics.
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      Dauphin-Ducharme, P.; Arroyo-Currás, N.; Plaxco, K. W. High-Precision Electrochemical Measurements of the Guanine-, Mismatch-, and Length-Dependence of Electron Transfer from Electrode-Bound DNA Are Consistent with a Contact-Mediated Mechanism. J. Am. Chem. Soc. 2019, 141, 1304– 1311,  DOI: 10.1021/jacs.8b11341
      9
      High-Precision Electrochemical Measurements of the Guanine-, Mismatch-, and Length-Dependence of Electron Transfer from Electrode-Bound DNA Are Consistent with a Contact-Mediated Mechanism
      Dauphin-Ducharme, Philippe; Arroyo-Curras, Netzahualcoyotl; Plaxco, Kevin W.
      Journal of the American Chemical Society (2019), 141 (3), 1304-1311CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Despite 25 years' effort, serious questions remain regarding the mechanism(s) underlying electron transfer through (or from) electrode-bound double-stranded DNA. In part this is because a control expt. regarding the putatively crit. role of guanine bases in the most widely proposed transport mechanism (hopping from guanine to guanine through the π-stack) appears to be lacking from the prior literature. In response, the authors have employed chronoamperometry, which allows for high-precision detn. of electron transfer rates, to characterize transfer to a redox reporter appended onto electrode-bound DNA duplexes. Specifically, the authors have measured the effects of guanines and base mismatches on the electron transfer rate assocd. with such constructs. Upon doing so, the authors find that, counter to prior reports, the transfer rate is, to within relatively tight exptl. confidence intervals, unaffected by either. Parallel studies of the dependence of the electron transfer rate on the length of the DNA suggest that transfer from this system obeys a "collision" mechanism in which the redox reporter phys. contacts the electrode surface prior to the exchange of electrons.
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      Kelley, S. O.; Jackson, N. M.; Hill, M. G.; Barton, J. K. Long-Range Electron Transfer through DNA Films. Angew. Chem., Int. Ed. 1999, 38, 941– 945,  DOI: 10.1002/(sici)1521-3773(19990401)38:7<941::aid-anie941>3.0.co;2-7
      10
      Long-range electron transfer through DNA films
      Kelley, Shana O.; Jackson, Nicole M.; Hill, Michael G.; Barton, Jacqueline K.
      Angewandte Chemie, International Edition (1999), 38 (7), 941-945CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH)
      In an effort to investigate DNA-mediated electron transfer involving ground-state reactants, the authors studied redox-active intercalators bound at discrete sites within the individual helixes of a DNA monolayer on gold. To investigate charge transduction through DNA as a function of distance, the authors have site-specifically cross-linked a redox-active intercalator, daunomycin (DM), into the DNA characterized. The site of intercalation was controlled in the duplexes by incorporating a single guanine-cytosine (GC) base step in otherwise adenine-thymine (AT) or inosine-cytosine (IC) sequences; as DM requires the N2 atom of guanine for covalent crosslinking, the intercalator is constrained to these positions. Moving the GC step along the duplex therefore provided a systematic variation in the location of the DM-binding site relative to the thiol-terminated linker. Crosslinking DM to the DNA in soln., and then depositing the labeled duplexes onto gold, afforded a series of films in which the intercalator was linked quant. at a known sepn. from the electrode surface. Here, long-range charge transport through DNA-modified films has been demonstrated in a series of structurally characterized assemblies.
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    11. 11
      Svetlicic, V.; Tomaic, J.; Zutic, V. A Kinetic-Study of Charge-Transfer and Phase-Transitions of the Methylene-Blue Leucomethylene Blue Couple Adsorbed at the Mercury Aqueous-Solution Interface. J. Electroanal. Chem. 1983, 146, 71– 92,  DOI: 10.1016/S0022-0728(83)80113-2
      11
      A kinetic study of charge transfer and phase transitions of the methylene blue/leucomethylene blue couple adsorbed at the mercury/aqueous solution interface
      Svetlicic, Vesna; Tomaic, Jadranka; Zutic, Vera; Chevalet, Jean
      Journal of Electroanalytical Chemistry and Interfacial Electrochemistry (1983), 146 (1), 71-92CODEN: JEIEBC; ISSN:0022-0728.
      The interfacial behavior of a methylene blue (MB)/leucomethylene blue (LMB) redox couple was studied by using fast perturbation potentiostatic techniques at the dropping Hg electrode. The extent of adsorption and the structure of the adsorbed layer above the monolayer coverage were detd. by the presence of anions at the interface, anion hydration playing the important role. In 1M electrolytes (pH = 7.9) the tendency of MB dimers towards a stacking interaction in the aq. phase, as well as to an adsorption at the Hg/aq. soln. interface, increase in the sequence F- « NO3- < Cl- « ClO4-. Evidence is presented for a coadsorption of LMB and nitrate and well-defined phase transitions in the adsorbed film. There are 3 distinct potential-dependent phases of LMB at the interface: (1) flat mols. and/or smaller 2-dimensional aggregates at low LMB coverages; (2)2-dimensional cryst. layers of flat mols. after a monolayer of LMB is generated by redn. at pos. charges; (3) a compact LMB layer at the neg. charged electrode,after desorption of nitrate and a conformational change of LMB mols. The characteristics of the charge-transfer process are best interpreted in terms of 2 MB/LMB redox couples: the prewave corresponds to redox reactions of adsorbed species at the Hg surface, while the process at more neg. potentials corresponds to the MB/LMB couple at a chem. modified electrode-Hg, covered by a compact and conductive layer of solid LMB.
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      Svetličič, V.; Žutić, V.; Clavilier, J.; Chevalet, J. Supramolecular Phenomena in Organic Redox Films at Electrodes. J. Electroanal. Chem. Interfacial Electrochem. 1985, 195, 307– 319,  DOI: 10.1016/0022-0728(85)80051-6
      12
      Supramolecular phenomena in organic redox films at electrodes. Part I. The methylene blue/leucomethylene blue redox couple at the platinum electrode
      Svetlicic, Vesna; Zutic, Vera; Clavilier, Jean; Chevalet, Jean
      Journal of Electroanalytical Chemistry and Interfacial Electrochemistry (1985), 195 (2), 307-19CODEN: JEIEBC; ISSN:0022-0728.
      It is possible to generate conductive multilayer structures by redn. of methylene blue cations at the interface: clean surface of a Pt electrode/1M aq. electrolyte. The characteristics of the multilayer ordered phase generated in fluoride and nitrate solns. are presented and the mechanism of a fast charge transfer between electrode/org. film and org./aq. soln. interfaces is discussed. The cond. of the film was interpreted by postulating formation of a mixed valence structure with the generation of a cation radical intermediate which is favored in the solid state at characteristic potentials.
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    13. 13
      Žutić, V.; Svetličić, V.; Clavilier, J.; Chevalet, J. Supramolecular Phenomena in Organic Redox Films at Electrodes. J. Electroanal. Chem. Interfacial Electrochem. 1987, 219, 183– 195,  DOI: 10.1016/0022-0728(87)85039-8
      13
      Supramolecular phenomena in organic redox films at electrodes. Part II. The methylene blue/leucomethylene blue redox couple at the gold electrode
      Zutic, Vera; Svetlicic, Vesna; Clavilier, Jean; Chevalet, Jean
      Journal of Electroanalytical Chemistry and Interfacial Electrochemistry (1987), 219 (1-2), 183-95CODEN: JEIEBC; ISSN:0022-0728.
      Org. films formed by redn. of the methylene blue cation at the Au electrode/aq. electrolyte soln. interface were studied by purely electrochem. methods. The insol. redn. product, a mixed-valence salt of the cation radical and leucomethylene blue, forms 2 polymorphic conductive structures: one with metallic and the other with ionic cond. The supramol. organization in the electrogenerated films is detd. by the redox state and the structure of the Au/org. film interface.
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    14. 14
      Svetlicic, V.; Zutic, V.; Clavilier, J.; Chevalet, J. Organic Monolayer Formation at a Sulfur Modified Gold Electrode─the Methylene-Blue Leucomethylene Blue Redox Couple. J. Electroanal. Chem. Interfacial Electrochem. 1987, 233, 199– 210,  DOI: 10.1016/0022-0728(87)85016-7
      14
      Organic monolayer formation at a sulfur modified gold electrode. The methylene blue/leucomethylene blue redox couple
      Svetlicic, Vesna; Zutic, Vera; Clavilier, Jean; Chevalet, Jean
      Journal of Electroanalytical Chemistry and Interfacial Electrochemistry (1987), 233 (1-2), 199-210CODEN: JEIEBC; ISSN:0022-0728.
      The electrochem. formation of a condensed methylene blue (MB+)/leucomethylene blue (LMB) monolayer at the S covered Au electrode (1 M NaF, KCl, or KNO3 soln., pH 7.9) is reported. The strong interaction between MB+ and adsorbed S in the monolayer was ascribed to a disulfide linkage between a S adatom and the S heteroatom of the perpendicularly oriented org. mol. This binding also resulted in a change in the elec. properties of the S monolayer, which increases the reversibility of the electron transfer in the 1st adsorbed MB monolayer. The stability of the reduced monolayer at the A-S/aq. electrolyte interface was affected strongly by the type of anion (F- > Cl- » NO3-).
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    15. 15
      Brdička, R. Measurements of Quantities Concerning the Adsorption of Certain Reducible Compounds or Their Reduction Products at the Dropping Mercury Electrode. Collect. Czech. Chem. Commun. 1947, 12, 522– 540,  DOI: 10.1135/cccc19470522
      15
      Measurements of quantities concerning the adsorption of certain reducible compounds or their reduction products at the dropping-mercury electrode
      Brdicka, R.
      Collection of Czechoslovak Chemical Communications (1947), 12 (), 522-40CODEN: CCCCAK; ISSN:0010-0765.
      cf. C.A. 37, 5661.3,5. B. has generalized the adsorption theory previously proposed to explain the anomalous polarograms of certain reversible org. oxidation-reduction systems. Theoretically the polarigraphic curve should have the same shape as the corresponding potentiometric titration curve and the half-wave potential and normal oxidation-reduction potential should be the same. If one of the oxidation-reduction forms should be adsorbed on the Hg drops, the free energy change which occurs will shift the polarographic wave to more neg. potentials if only the oxidized form is adsorbed; and to more pos. values if only the reduced form is adsorbed. At low concns. of the oxidation-reduction system the Hg drops will be unsatd. and a single wave will appear. Above a certain concn. 2 waves appear: an "anomalous" wave and a normal wave. The height of the "anomalous" wave is independent of concn., since it corresponds to satn. of surface of the drops with adsorbed compd. It will follow the normal wave at more neg. values if the oxidized form only is adsorbed (e.g. phenosafranine), or precede the normal wave at more pos. potentials if the reduced form only is adsorbed (e.g. methylene blue and lactoflavin). The height of the "anomalous" or adsorption curve is a measure of the adsorption current from which can be evaluated the max. no. of adsorbed mols. per unit area of drop surface. From the difference between the half-wave potentials of the adsorption and diffusion currents the H.ovrddot.uckel adsorption coeff. can be calcd. If some plausible value is assumed for the vol. of the adsorbed mols. the free energy of adsorption can be detd. The adsorption current varies directly with the height of the Hg reservoir. Finally, if both oxidation-reduction forms are adsorbed equally, the polarogram should be normal. Data from previous articles are used to test the theory.
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    16. 16
      Bard, A. J.; Faulkner, L. R.; Leddy, J.; Zoski, C. G. Electrochemical Methods: Fundamentals and Applications; Wiley: New York, 1980; Vol. 2.
      There is no corresponding record for this reference.
    17. 17
      Bard, A. J.; Faulkner, L. R. Chapter 11; Section 11.7: Thin-layer electrochemistry. Electrochemical Methods: Fundamentals and Applications; Wiley: New York, 2001.
      There is no corresponding record for this reference.
    18. 18
      Pellitero, M. A.; Curtis, S. D.; Arroyo-Curras, N. Interrogation of Electrochemical Aptamer-Based Sensors Via Peak-to-Peak Separation in Cyclic Voltammetry Improves the Temporal Stability and Batch-to-Batch Variability in Biological Fluids. ACS Sens. 2021, 6, 1199
      18
      Interrogation of Electrochemical Aptamer-Based Sensors via Peak-to-Peak Separation in Cyclic Voltammetry Improves the Temporal Stability and Batch-to-Batch Variability in Biological Fluids
      Pellitero, Miguel Aller; Curtis, Samuel D.; Arroyo-Curras, Netzahualcoyotl
      ACS Sensors (2021), 6 (3), 1199-1207CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
      Electrochem., aptamer-based (E-AB) sensors support continuous, real-time measurements of specific mol. targets in complex fluids such as undiluted serum. They achieve these measurements by using redox-reporter-modified, electrode-attached aptamers that undergo target binding-induced conformational changes which, in turn, change electron transfer between the reporter and the sensor surface. Traditionally, E-AB sensors are interrogated via pulse voltammetry to monitor binding-induced changes in transfer kinetics. While these pulse techniques are sensitive to changes in electron transfer, they also respond to progressive changes in the sensor surface driven by biofouling or monolayer desorption and, consequently, present a significant drift. Moreover, we have empirically obsd. that differential voltage pulsing can accelerate monolayer desorption from the sensor surface, presumably via field-induced actuation of aptamers. Here, in contrast, we demonstrate the potential advantages of employing cyclic voltammetry to measure electron-transfer changes directly. In our approach, the target concn. is reported via changes in the peak-to-peak sepn., ΔEP, of cyclic voltammograms. Because the magnitude of ΔEP is insensitive to variations in the no. of aptamer probes on the electrode, ΔEP-interrogated E-AB sensors are resistant to drift and show decreased batch-to-batch and day-to-day variability in sensor performance. Moreover, ΔEP-based measurements can also be performed in a few hundred milliseconds and are, thus, competitive with other subsecond interrogation strategies such as chronoamperometry but with the added benefit of retaining sensor capacitance information that can report on monolayer stability over time.
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    19. 19
      Arroyo-Currás, N.; Dauphin-Ducharme, P.; Ortega, G.; Ploense, K. L.; Kippin, T. E.; Plaxco, K. W. Subsecond-Resolved Molecular Measurements in the Living Body Using Chronoamperometrically Interrogated Aptamer-Based Sensors. ACS Sens. 2018, 3, 360– 366,  DOI: 10.1021/acssensors.7b00787
      19
      Subsecond-Resolved Molecular Measurements in the Living Body Using Chronoamperometrically Interrogated Aptamer-Based Sensors
      Arroyo-Curras, Netzahualcoyotl; Dauphin-Ducharme, Philippe; Ortega, Gabriel; Ploense, Kyle L.; Kippin, Tod E.; Plaxco, Kevin W.
      ACS Sensors (2018), 3 (2), 360-366CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
      Electrochem., aptamer-based (E-AB) sensors support the continuous, real-time measurement of specific small mols. directly in situ in the living body over the course of many hours. They achieve this by employing binding-induced conformational changes to alter electron transfer from a redox-reporter-modified, electrode-attached aptamer. Previously the authors have used voltammetry (cyclic, a.c., and square wave) to monitor this binding-induced change in transfer kinetics indirectly. Here, however, the authors demonstrate the potential advantages of employing chronoamperometry to measure the change in kinetics directly. In this approach target concn. is reported via changes in the lifetime of the exponential current decay seen when the sensor is subjected to a potential step. Because the lifetime of this decay is independent of its amplitude (e.g., insensitive to variations in the no. of aptamer probes on the electrode), chronoamperometrically interrogated E-AB sensors are calibration-free and resistant to drift. Chronoamperometric measurements can also be performed in a few hundred milliseconds, improving the previous few-second time resoln. of E-AB sensing by an order of magnitude. To illustrate the potential value of the approach the authors demonstrate here the calibration-free measurement of the drug tobramycin in situ in the living body with 300 ms time resoln. and unprecedented, few-percent precision in the detn. of its pharmacokinetic phases.
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    20. 20
      Downs, A. M.; Gerson, J.; Ploense, K. L.; Plaxco, K. W.; Dauphin-Ducharme, P. Subsecond-Resolved Molecular Measurements Using Electrochemical Phase Interrogation of Aptamer-Based Sensors. Anal. Chem. 2020, 92, 14063– 14068,  DOI: 10.1021/acs.analchem.0c03109
      20
      Subsecond-Resolved Molecular Measurements Using Electrochemical Phase Interrogation of Aptamer-Based Sensors
      Downs, Alex M.; Gerson, Julian; Ploense, Kyle L.; Plaxco, Kevin W.; Dauphin-Ducharme, Philippe
      Analytical Chemistry (Washington, DC, United States) (2020), 92 (20), 14063-14068CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Recent years have seen the development of a no. of biosensor architectures that rely on target binding-induced changes in the rate of electron transfer from an electrode-bound receptor. Most often, the interrogation of these sensors has relied on voltammetric methods, such as square-wave voltammetry, which limit their time resoln. to a few seconds. Here, we describe the use of an impedance-based approach, which we have termed electrochem. phase interrogation, as a means of collecting high time resoln. measurements with sensors in this class. Specifically, using changes in the electrochem. phase to monitor target binding in an electrochem.-aptamer based (EAB) sensor, we achieve subsecond temporal resoln. and multihour stability in measurements performed directly in undiluted whole blood. Electrochem. phase interrogation also offers improved insights into EAB sensors' signaling mechanism. By modeling the interfacial resistance and capacitance using equiv. circuits, we find that the only parameter that is altered by target binding is the charge-transfer resistance. This confirms previous claims that binding-induced changes in electron-transfer kinetics drive signaling in this class of sensors. Considering that a wide range of electrochem. biosensor architectures rely on this signaling mechanism, we believe that electrochem. phase interrogation may prove generalizable toward subsecond measurements of mol. targets.
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    21. 21
      Arroyo-Currás, N.; Scida, K.; Ploense, K. L.; Kippin, T. E.; Plaxco, K. W. High Surface Area Electrodes Generated Via Electrochemical Roughening Improve the Signaling of Electrochemical Aptamer-Based Biosensors. Anal. Chem. 2017, 89, 12185– 12191,  DOI: 10.1021/acs.analchem.7b02830
      21
      High Surface Area Electrodes Generated via Electrochemical Roughening Improve the Signaling of Electrochemical Aptamer-Based Biosensors
      Arroyo-Curras, Netzahualcoyotl; Scida, Karen; Ploense, Kyle L.; Kippin, Tod E.; Plaxco, Kevin W.
      Analytical Chemistry (Washington, DC, United States) (2017), 89 (22), 12185-12191CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      The electrochem., aptamer-based (E-AB) sensor platform provides a modular approach to the continuous, real-time measurement of specific mol. targets (irresp. of their chem. reactivity) in situ in the living body. To achieve this, however, requires the fabrication of sensors small enough to insert into a vein, which, for the rat animal model the authors employ, entails devices less than 200 μm in diam. The limited surface area of these small devices leads, in turn, to low faradaic currents and poor signal-to-noise ratios when deployed in the complex, fluctuating environments found in vivo. In response the authors have developed an electrochem. roughening approach that enhances the signaling of small electrochem. sensors by increasing the microscopic surface area of gold electrodes, allowing in this case more redox-reporter-modified aptamers to be packed onto the surface, thus producing significantly improved signal-to-noise ratios. Unlike previous approaches to achieving microscopically rough gold surfaces, the method employs chronoamperometric pulsing in a 5 min etching process easily compatible with batch manufg. Using these high surface area electrodes, the authors demonstrate the ability of E-AB sensors to measure complete drug pharmacokinetic profiles in live rats with precision of better than 10% in the detn. of drug disposition parameters.
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    22. 22
      Dauphin-Ducharme, P.; Arroyo-Currás, N.; Kurnik, M.; Ortega, G.; Li, H.; Plaxco, K. W. Simulation-Based Approach to Determining Electron Transfer Rates Using Square-Wave Voltammetry. Langmuir 2017, 33, 4407– 4413,  DOI: 10.1021/acs.langmuir.7b00359
      22
      Simulation-Based Approach to Determining Electron Transfer Rates Using Square-Wave Voltammetry
      Dauphin-Ducharme, Philippe; Arroyo-Curras, Netzahualcoyotl; Kurnik, Martin; Ortega, Gabriel; Li, Hui; Plaxco, Kevin W.
      Langmuir (2017), 33 (18), 4407-4413CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      The efficiency with which square-wave voltammetry differentiates faradaic and charging currents makes it a particularly sensitive electroanal. approach, as evidenced by its ability to measure nanomolar or even picomolar concns. of electroactive analytes. Because of the relative complexity of the potential sweep it uses, however, the extn. of detailed kinetic and mechanistic information from square-wave data remains challenging. In response, the authors demonstrate here a numerical approach by which square-wave data can be used to det. electron transfer rates. Specifically, the authors have developed a numerical approach in which the authors model the height and the shape of voltammograms collected over a range of square-wave frequencies and amplitudes to simulated voltammograms as functions of the heterogeneous rate const. and the electron transfer coeff. As validation of the approach, it was used to det. electron transfer kinetics in both freely diffusing and diffusionless surface-tethered species, obtaining electron transfer kinetics in all cases in good agreement with values derived using non-square-wave methods.
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    23. 23
      Lyshevski, S. E. Dekker Encyclopedia of Nanoscience and Nanotechnology; CRC Press, 2014.
      There is no corresponding record for this reference.
    24. 24
      Rothemund, P. W. K. Folding DNA to Create Nanoscale Shapes and Patterns. Nature 2006, 440, 297– 302,  DOI: 10.1038/nature04586
      24
      Folding DNA to create nanoscale shapes and patterns
      Rothemund, Paul W. K.
      Nature (London, United Kingdom) (2006), 440 (7082), 297-302CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      'Bottom-up fabrication', which exploits the intrinsic properties of atoms and mols. to direct their self-organization, is widely used to make relatively simple nanostructures. A key goal for this approach is to create nanostructures of high complexity, matching that routinely achieved by 'top-down' methods. The self-assembly of DNA mols. provides an attractive route towards this goal. Here the author describe a simple method for folding long, single-stranded DNA mols. into arbitrary two-dimensional shapes. The design for a desired shape is made by raster-filling the shape with a 7-kilobase single-stranded scaffold and by choosing over 200 short oligonucleotide 'staple strands' to hold the scaffold in place. Once synthesized and mixed, the staple and scaffold strands self-assemble in a single step. The resulting DNA structures are roughly 100 nm in diam. and approx. desired shapes such as squares, disks and five-pointed stars with a spatial resoln. of 6 nm. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be programmed to bear complex patterns such as words and images on their surfaces. Finally, individual DNA structures can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles (which constitutes a 30-megadalton mol. complex).
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    25. 25
      Roth, E.; Glick Azaria, A.; Girshevitz, O.; Bitler, A.; Garini, Y. Measuring the Conformation and Persistence Length of Single-Stranded DNA Using a DNA Origami Structure. Nano Lett. 2018, 18, 6703– 6709,  DOI: 10.1021/acs.nanolett.8b02093
      25
      Measuring the Conformation and Persistence Length of Single-Stranded DNA Using a DNA Origami Structure
      Roth, Efrat; Glick Azaria, Alex; Girshevitz, Olga; Bitler, Arkady; Garini, Yuval
      Nano Letters (2018), 18 (11), 6703-6709CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      Measuring the mech. properties of single-stranded DNA (ssDNA) is a challenge that has been addressed by different methods lately. The short persistence length, fragile structure, and the appearance of stem loops complicate the measurement, and this leads to a large variability in the measured values. Here the authors describe an innovative method based on DNA origami for studying the biophys. properties of ssDNA. By synthesizing a DNA origami structure that consists of two rigid rods with an ssDNA segment between them, the authors developed a method to characterize the effective persistence length of a random-sequence ssDNA while allowing the formation of stem loops. The authors imaged the structure with an at. force microscope (AFM); the rigid rods provide a means for the exact identification of the ssDNA ends. This leads to an accurate detn. of the end-to-end distance of each ssDNA segment, and by fitting the measured distribution to the ideal chain polymer model the authors measured an effective persistence length of 1.98 ± 0.72 nm. This method enables one to measure short or long strands of ssDNA, and it can cope with the formation of stem loops that are often formed along ssDNA. The authors envision that this method can be used for measuring stem loops for detg. the effect of repetitive nucleotide sequences and environmental conditions on the mech. properties of ssDNA and the effect of interacting proteins with ssDNA. Further the method can be extended to nanoprobes for measuring the interactions of specific DNA sequences, because the DNA origami rods (or similar structures) can hold multiple fluorescent probes that can be easily detected.
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    26. 26
      Savéant, J.-M. Molecular Catalysis of Electrochemical Reactions. Mechanistic Aspects. Chem. Rev. 2008, 108, 2348– 2378,  DOI: 10.1021/cr068079z
      26
      Molecular Catalysis of Electrochemical Reactions. Mechanistic Aspects
      Saveant, Jean-Michel
      Chemical Reviews (Washington, DC, United States) (2008), 108 (7), 2348-2378CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. The term "electrocatalysis" is traditionally used for reactions in which the electrode material-often, but not always, a metal-is chem. involved in the catalytic process. Although the chem. properties of the electrode material play an important role in governing the catalytic efficiency, geometric and crystallog. features, nature and no. of defects, may also be of paramount significance. The differences between the bulk properties of the metal and its surface properties are particularly important in this respect. It is therefore difficult, or even irrelevant, to analyze results and devise new catalytic systems on the basis of mol. concepts. Another approach to catalyzing electrochem. reactions is to use mols. as catalysts. "Mol. catalysis" thus defined may involve catalyst mols. either homogeneously dispersed in the soln. bathing the electrode or immobilized in a monolayer or multilayered coating deposited on the electrode surface.
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    27. 27
      Eden, A.; Scida, K.; Arroyo-Currás, N.; Eijkel, J. C. T.; Meinhart, C. D.; Pennathur, S. Modeling Faradaic Reactions and Electrokinetic Phenomena at a Nanochannel-Confined Bipolar Electrode. J. Phys. Chem. C 2019, 123, 5353– 5364,  DOI: 10.1021/acs.jpcc.8b10473
      27
      Modeling Faradaic Reactions and Electrokinetic Phenomena at a Nanochannel-Confined Bipolar Electrode
      Eden, A.; Scida, K.; Arroyo-Curras, N.; Eijkel, J. C. T.; Meinhart, C. D.; Pennathur, S.
      Journal of Physical Chemistry C (2019), 123 (9), 5353-5364CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
      The authors present the most comprehensive 2-dimensional numerical model to date for a nanoconfined bipolar electrochem. system. By accounting for the compact Stern layer and resolving the diffuse part of the elec. double layer (EDL) at the BPE surface and channel walls, the authors' model captures the impact of surface polarization and ionic charge screening effects on the heterogeneous charge-transfer kinetics, as well as nonlinear electrokinetic transport phenomena such as induced-charge electroosmosis and concn. polarization. The authors employ the Poisson-Nernst-Planck and Stokes flow system of equations, unified with generalized Frumkin-Butler-Volmer reaction kinetics, to describe H2O electrolysis reactions and the resulting transport of ions and dissolved gases in the confined bipolar electrode (BPE) system. The authors' results demonstrate that under a sufficiently large applied elec. field, the rapid reaction kinetics on the authors' Pt BPE dynamically transition from charge-transfer limited to mass-transfer limited temporal regimes as regions depleted of redox species form and propagate outwards from the resp. BPE poles. This phenomenon was visualized exptl. with pH-sensitive fluorescein dye and showed excellent phenomenol. agreement with the authors' numerical calcns., providing a foundation for further understanding and developing bipolar electrochem. processes in confined geometries. The authors introduce two prospective applications arising from the authors' work: (1) a hybrid hydrodynamic/electrochem. peristaltic pump, and (2) deducing information about chem. kinetics through simulation.
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    28. 28
      Xiao, Y.; Lubin, A. A.; Heeger, A. J.; Plaxco, K. W. Label-Free Electronic Detection of Thrombin in Blood Serum by Using an Aptamer-Based Sensor. Angew. Chem., Int. Ed. Engl. 2005, 44, 5456– 5459,  DOI: 10.1002/anie.200500989
      28
      Label-free electronic detection of thrombin in blood serum by using an aptamer-based sensor
      Xiao Yi; Lubin Arica A; Heeger Alan J; Plaxco Kevin W
      Angewandte Chemie (International ed. in English) (2005), 44 (34), 5456-9 ISSN:1433-7851.
      There is no expanded citation for this reference.
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    29. 29
      Rowe, A. A.; Miller, E. A.; Plaxco, K. W. Reagentless Measurement of Aminoglycoside Antibiotics in Blood Serum Via an Electrochemical, Ribonucleic Acid Aptamer-Based Biosensor. Anal. Chem. 2010, 82, 7090– 7095,  DOI: 10.1021/ac101491d
      29
      Reagentless Measurement of Aminoglycoside Antibiotics in Blood Serum via an Electrochemical, Ribonucleic Acid Aptamer-Based Biosensor
      Rowe, Aaron A.; Miller, Erin A.; Plaxco, Kevin W.
      Analytical Chemistry (Washington, DC, United States) (2010), 82 (17), 7090-7095CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Biosensors built using RNA aptamers show promise as tools for point-of-care medical diagnostics, but they remain vulnerable to nuclease degrdn. when deployed in clin. samples. To explore methods for protecting RNA-based biosensors from such degrdn. we have constructed and characterized an electrochem., aptamer-based sensor for the detection of aminoglycosidic antibiotics. We find that while this sensor achieves low micromolar detection limits and subminute equilibration times when challenged in buffer, it deteriorates rapidly when immersed directly in blood serum. In order to circumvent this problem, we have developed and tested sensors employing modified versions of the same aptamer. Our first effort to this end entailed the methylation of all of the 2'-hydroxyl groups outside of the aptamer's antibiotic binding pocket. However, while devices employing this modified aptamer are as sensitive as those employing an unmodified parent, the modification fails to confer greater stability when the sensor is challenged directly in blood serum. As a second potentially naive alternative, we replaced the RNA bases in the aptamer with their more degrdn.-resistant DNA equiv. Surprisingly and unlike control DNA-stem loops employing other sequences, this DNA aptamer retains the ability to bind aminoglycosides, albeit with poorer affinity than the parent RNA aptamer. Unfortunately, however, while sensors fabricated using this DNA aptamer are stable in blood serum, its lower affinity pushes their detection limits above the therapeutically relevant range. Finally, we find that ultrafiltration through a low-mol.-wt.-cutoff spin column rapidly and efficiently removes the relevant nucleases from serum samples spiked with gentamicin, allowing the convenient detection of this aminoglycoside at clin. relevant concns. using the original RNA-based sensor.
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    30. 30
      Impert, O.; Katafias, A.; Kita, P.; Mills, A.; Pietkiewicz-Graczyk, A.; Wrzeszcz, G. Kinetics and Mechanism of a Fast Leuco-Methylene Blue Oxidation by Copper(II)-Halide Species in Acidic Aqueous Media. Dalton Trans. 2003, 3, 348– 353,  DOI: 10.1039/b205786g
      There is no corresponding record for this reference.
    31. 31
      Genereux, J. C.; Barton, J. K. Mechanisms for DNA Charge Transport. Chem. Rev. 2010, 110, 1642– 1662,  DOI: 10.1021/cr900228f
      31
      Mechanisms for DNA Charge Transport
      Genereux, Joseph C.; Barton, Jacqueline K.
      Chemical Reviews (Washington, DC, United States) (2010), 110 (3), 1642-1662CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. The authors review some of the characteristics and mechanisms of DNA charge transport. Mechanisms discussed include transport through water, ions and phosphates; superexchange; localized hopping; and delocalized mechanisms.
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    32. 32
      Hossain, M.; Suresh Kumar, G. DNA Intercalation of Methylene Blue and Quinacrine: New Insights into Base and Sequence Specificity from Structural and Thermodynamic Studies with Polynucleotides. Mol. Biosyst. 2009, 5, 1311– 1322,  DOI: 10.1039/b909563b
      32
      DNA intercalation of methylene blue and quinacrine: new insights into base and sequence specificity from structural and thermodynamic studies with polynucleotides
      Hossain, Maidul; Suresh Kumar, Gopinatha
      Molecular BioSystems (2009), 5 (11), 1311-1322CODEN: MBOIBW; ISSN:1742-206X. (Royal Society of Chemistry)
      The binding of the known DNA intercalators methylene blue and quinacrine with four sequence specific polynucleotides, viz. poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dT).poly(dA-dT) and poly(dA).poly(dT), have been compared using absorbance, fluorescence, competition dialysis and thermal melting and the thermodn. aspects of the interaction studied. In all the cases, non-cooperative binding phenomena obeying neighbor exclusion principle was obsd. though the affinity was remarkably higher for quinacrine and the nature of the binding was characterized to be true intercalation. The data on the salt dependence of binding derived from the plot of log K vs. log[Na+] revealed a slope of around 1.0, consistent with the values predicted by the theories for the binding of monovalent cations, and contained contributions from polyelectrolytic and non-polyelectrolytic forces. The bindings were characterized by strong stabilization of the polynucleotides against thermal strand sepn. in both optical melting as well as differential scanning calorimetry studies. The data analyzed from the thermal melting and isothermal titrn. calorimetry studies were in close proximity to those obtained from absorption spectral titrn. data. Isothermal titrn. calorimetry results revealed the bindings to poly(dG-dC).poly(dG-dC), poly(dG).poly(dC) and poly(dA-dT).poly(dA-dT) to be exothermic and favored by both neg. enthalpy and large favorable pos. entropy changes, while that to poly(dA).poly(dT) was endothermic and entropy driven. The heat capacity changes obtained from temp. dependence of enthalpy gave neg. values to all polynucleotides. New insights on the mol. aspects of interaction of these mols. to DNA have emerged from these studies.
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    33. 33
      Vardevanyan, P. O.; Antonyan, A. P.; Parsadanyan, M. A.; Shahinyan, M. A.; Hambardzumyan, L. A. Mechanisms for Binding between Methylene Blue and DNA. J. Appl. Spectrosc. 2013, 80, 595– 599,  DOI: 10.1007/s10812-013-9811-7
      33
      Mechanisms for Binding between Methylene Blue and DNA
      Vardevanyan, P. O.; Antonyan, A. P.; Parsadanyan, M. A.; Shahinyan, M. A.; Hambardzumyan, L. A.
      Journal of Applied Spectroscopy (2013), 80 (4), 595-599CODEN: JASYAP; ISSN:0021-9037. (Springer)
      We have used absorption and fluorimetric methods to study the interaction between methylene blue (MB) and calfthymus DNA. Based on Scatchard anal. of the exptl. data, we plotted the methylene blue-DNA binding curve. This curve consists of two linear sections, which indicates two types of interaction, for which we detd. the consts. K and the no. of binding sites n for binding of this ligand to DNA. Comparison of the data obtained with analogous values found for interaction between ethidium bromide and DNA allowed us to conclude that there are two modes of interaction between methylene blue and DNA: strong binding (semi-intercalation) and weak binding (electrostatic).
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    34. 34
      Zuker, M. Mfold Web Server for Nucleic Acid Folding and Hybridization Prediction. Nucleic Acids Res. 2003, 31, 3406– 3415,  DOI: 10.1093/nar/gkg595
      34
      Mfold web server for nucleic acid folding and hybridization prediction
      Zuker, Michael
      Nucleic Acids Research (2003), 31 (13), 3406-3415CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      The abbreviated name, ' mfold web server', describes a no. of closely related software applications available on the World Wide Web (WWW) for the prediction of the secondary structure of single stranded nucleic acids. The objective of this web server is to provide easy access to RNA and DNA folding and hybridization software to the scientific community at large. By making use of universally available web GUIs (Graphical User Interfaces), the server circumvents the problem of portability of this software. Detailed output, in the form of structure plots with or without reliability information, single strand frequency plots and energy dot plots', are available for the folding of single sequences. A variety of bulk' servers give less information, but in a shorter time and for up to hundreds of sequences at once.
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    35. 35
      Dauphin-Ducharme, P.; Arroyo-Currás, N.; Adhikari, R.; Somerson, J.; Ortega, G.; Makarov, D. E.; Plaxco, K. W. Chain Dynamics Limit Electron Transfer from Electrode-Bound, Single-Stranded Oligonucleotides. J. Phys. Chem. C 2018, 122, 21441– 21448,  DOI: 10.1021/acs.jpcc.8b06111
      35
      Chain Dynamics Limit Electron Transfer from Electrode-Bound, Single-Stranded Oligonucleotides
      Dauphin-Ducharme, Philippe; Arroyo-Curras, Netzahualcoyotl; Adhikari, Ramesh; Somerson, Jacob; Ortega, Gabriel; Makarov, Dmitrii E.; Plaxco, Kevin W.
      Journal of Physical Chemistry C (2018), 122 (37), 21441-21448CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
      A wide range of new devices aimed at in vivo mol. detection and point-of-care diagnostics rely on binding-induced changes in electron-transfer kinetics from an electrode-attached, redox-reporter-modified oligonucleotide as their signaling mechanism. In an effort to better characterize the mechanisms underlying these sensors, we have measured the electron-transfer kinetics assocd. with surface-attached, single-stranded DNAs modified with a methylene blue redox reporter either at the chain's distal end or at an internal chain position. We find that although the rate of electron transfer from a reporter placed either terminally or internally is independent of chain length for chains shorter than the length scale of methylene blue (and its linker), for longer chains it follows a power-law dependence on length of exponent approx. -2.2. Such behavior is consistent with a diffusion-controlled mechanism in which the diffusion of the DNA-bound reporter to the surface controls the rate of electron transfer. This said, the obsd. rates are, at 5-400 s-1, orders of magnitude slower than the intramol. dynamics of single-stranded oligonucleotides when free in soln. Likewise, the rates of transfer from reporters placed internally are several-fold slower than those seen for the equiv. terminally modified construct. We attribute these effects to electrostatic repulsion between the oligonucleotide and the electrode surface, which is neg. charged at the redox potential of methylene blue. Consistent with this, changing monolayer compn. so as to increase the neg. charge of the surface reduces the transfer rate still more without significantly altering its power-law chain length dependence. Simple theor. models and computer simulations performed in support of our exptl. studies find similar power-law dependencies, similar electrostatic slowing of the transfer rate, and similar rate differences between terminally an internally modified constructs.
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    36. 36
      Lu, Z.; Dong, S. Preparation and Electrochemical-Behavior of a Methylene Blue-Modified Electrode Based on a Nafion Polymer Film. J. Chem. Soc., Faraday Trans. 1988, 84, 2979– 2986,  DOI: 10.1039/f19888402979
      36
      Preparation and electrochemical behavior of a methylene blue-modified electrode based on a Nafion polymer film
      Lu, Ziling; Dong, Shaojun
      Journal of the Chemical Society, Faraday Transactions 1: Physical Chemistry in Condensed Phases (1988), 84 (9), 2979-86CODEN: JCFTAR; ISSN:0300-9599.
      A methylene blue (MB) chem. modified electrode was prepd. by incorporating MB mols. into a Nafion film on a glassy C surface. The electrochem. behavior of the MB-modified polymer film electrode is discussed in detail. The electrode reaction of MB bound to the polymer film shows a reversible, 2-electron transfer process with good stability and reproducibility. The equation of E1/2 vs. pH was deduced theor. and was proved to be reasonable exptl. by the effect of soln. pH on the MB-modified polymer-film electrode. The influence of supporting electrolytes on the electrode is discussed.
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    37. 37
      Guadalupe, A. R.; Liu, K. E.; Abruña, H. D. Transport-Properties of Cationic Dyes in Nafion Films - Unusually High Diffusion-Coefficients and Aggregation Effects. Electrochim. Acta 1991, 36, 881– 887,  DOI: 10.1016/0013-4686(91)85289-j
      37
      Transport properties of cationic dyes in Nafion films: unusually high diffusion coefficients and aggregation effects
      Guadalupe, A. R.; Liu, K. E.; Abruna, H. D.
      Electrochimica Acta (1991), 36 (5-6), 881-7CODEN: ELCAAV; ISSN:0013-4686.
      Studies on the transport properties of the cationic dyes methylene blue, thionine, safranine and meldola blue in Nafion-coated electrodes are reported. Diffusion coeffs. for the reduced and oxidized forms of the dyes are unusually high (up to 9 × 10-7 cm2 s-1) at low dye concns. in the film and show a dramatic decrease with increasing concn. These results are interpreted in terms of recently proposed models for transport through Nafion as well as to the formation of dye dimers and/or higher aggregates in the Nafion film. This is demonstrated for methylene blue by spectrophotometric expts. where the dye was incorporated in Nafion films at different concns.
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    38. 38
      Caram, J. A.; Suárez, J. F. M.; Gennaro, A. M.; Mirífico, M. V. Electrochemical Behaviour of Methylene Blue in Non-Aqueous Solvents. Electrochim. Acta 2015, 164, 353– 363,  DOI: 10.1016/j.electacta.2015.01.196
      38
      Electrochemical behavior of methylene blue in non-aqueous solvents
      Caram, J. A.; Suarez, J. F. Martinez; Gennaro, A. M.; Mirifico, M. V.
      Electrochimica Acta (2015), 164 (), 353-363CODEN: ELCAAV; ISSN:0013-4686. (Elsevier Ltd.)
      The electrochem. behavior of methylene blue in soln. of nonaq. solvents with different supporting electrolytes was studied by cyclic voltammetry. Dye electro-redn. presents two well-defined processes of monoelectronic charge transfer yielding a free radical in the 1st process and an anion in the 2nd electron transfer. Free radical and anion are long living species in some of the studied media. Effects of supporting electrolyte and solvent on the peak potentials, the peak current functions and the reversibility of the charge transfer processes are reported. A dissocn. equil. of the dye in soln. of nonaq. solvents and the acid or base added det. markedly the electrochem. responses. In the particular cases of KOH/DMF or EDA basic media the chem. formation of the stable methylene blue radical was detected and it was characterized by EPR spectroscopy. A general reaction scheme is proposed.
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    39. 39
      Ohno, T.; Osif, T. L.; Lichtin, N. N. A Previously Unreported Intense Absorption Band and the Pk, of Protonated Triplet Methylene Blue. Photochem. Photobiol. 1979, 30, 541– 546,  DOI: 10.1111/j.1751-1097.1979.tb07178.x
      39
      A previously unreported intense absorption band and the pKa of protonated triplet methylene blue
      Ohno, T.; Osif, T. L.; Lichtin, N. N.
      Photochemistry and Photobiology (1979), 30 (5), 541-6CODEN: PHCBAP; ISSN:0031-8655.
      Methylene blue (L) was converted to its protonated triplet state by excitation with a Q-switched giant ruby laser, and the absorption spectrum was measured by kinetic spectrophotometry. Previously reported triplet-triplet absorption in the violet in acidic and alk. solns. and in the near IR in alk. soln. was confirmed. Long-wavelength triplet-triplet absorption in acidic soln. was found. Observation of a pH-independent isosbestic point at ∼720 nm confirmed that the long-wavelength absorptions were due to different protonated states of the same species. The pKa of the conjugate acid LH2+ was detd. from the dependence on pH of absorption and from the kinetics of decay of triplet absorption. The specific rate of protonation of L+ by H2PO4- ions was also measured.
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    40. 40
      Becker, H. G. O.; Kohrs, K. Photoreduction of Methyleneblue by the Two-Equivalent Electron Donor N-Methyl-9-Phenylacridane and the Use of the System for the Spectrally Sensitized Dediazoniation of P-N,N-Dimethylamino Benzenediazonium Tetrafluoroborate. J. Prakt. Chem. 1990, 332, 651– 657,  DOI: 10.1002/prac.19903320510
      40
      Photoreduction of methylene blue by the two-equivalent electron donor N-methyl-9-phenylacridan and the use of the system for the spectrally sensitized dediazoniation of p-(dimethylamino)benzenediazonium tetrafluoroborate
      Becker, H. G. O.; Kohrs, K.
      Journal fuer Praktische Chemie (Leipzig) (1990), 332 (5), 651-7CODEN: JPCEAO; ISSN:0021-8383.
      The photoredn. of methylene blue by N-methyl-9-phenylacridan (ACH) is studied in acetonitrile by means of flash photolysis and quantum yields. In the first step, due to fast proton shift within the original electron transfer product protonated semi-methylene blue MBH•+ (I) and the deprotonated donor radical N-methyl-9-phenylacridanyl (AC) are formed with a rate const. of 2 × 108 M-1 s-1. In the radical pair a second electron is transferred very fast from AC to I with a rate const. ke2 ≈ 101- s-1 to form leuco-methylene blue and N-methyl-9-phenylacridinium salt (AC+). About 80% of the two-equiv redn. product, leuco-methylene blue, is formed within the first solvent cage during the flash. The max. quantum yields of photoredn. approach φisc of MB+ as expected for a two-equiv. redn. The out-of-cage reaction consists of the known disproportionation of I and its redn. by AC. From the decay kinetics kred = 3 × 109 M-1 s-1 and kdis = 8 × 108 M-1 s-1 are derived. The system sensitizes the dediazoniation of p-(dimethylamino)benzenediazonium tetrafluoroborate efficiently even at very low diazonium salt concns. (φ = 0.6).
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpcc.1c00336.

    • Experimental and simulated voltammograms under various conditions; numerical data tables derived from simulated voltammograms; DNA sequences used in this work (PDF)

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