ACS Publications. Most Trusted. Most Cited. Most Read
Amplification-Free Attomolar Detection of Short Nucleic Acids with Upconversion Luminescence: Eliminating Nonspecific Binding by Hybridization Complex Transfer
More

OR SEARCH CITATIONS

My Activity
Publications

Figure 1Loading Img
Download Hi-Res ImageDownload to MS-PowerPointCite This:Anal. Chem. 2025, 97, 3, 1775-1782
  • Open Access
Article

Amplification-Free Attomolar Detection of Short Nucleic Acids with Upconversion Luminescence: Eliminating Nonspecific Binding by Hybridization Complex Transfer
Click to copy article linkArticle link copied!

Open PDFSupporting Information (1)

Analytical Chemistry

Cite this: Anal. Chem. 2025, 97, 3, 1775–1782
Click to copy citationCitation copied!
https://doi.org/10.1021/acs.analchem.4c05401
Published January 12, 2025

Copyright © 2025 The Authors. Published by American Chemical Society. This publication is licensed under

CC-BY-NC-ND 4.0 .

Abstract

Click to copy section linkSection link copied!
High Resolution Image
Download MS PowerPoint Slide

The anti-Stokes emission of photon upconversion nanoparticles (UCNPs) facilitates their use as labels for ultrasensitive detection in biological samples as infrared excitation does not induce autofluorescence at visible wavelengths. The detection of extremely low-abundance analytes, however, remains challenging as it is impossible to completely avoid nonspecific binding of label conjugates. To overcome this limitation, we developed a novel hybridization complex transfer technique using UCNP labels to detect short nucleic acids directly without target amplification. The assay involves capturing the target–label complexes on an initial solid phase, then using releasing oligonucleotides to specifically elute only the target–UCNP complexes and recapturing them on another solid phase. The nonspecifically adsorbed labels remain on the first solid phase, enabling background-free, ultrasensitive detection. When magnetic microparticles were used as the first solid phase in a sample volume of 120 μL, the assay achieved a limit of detection (LOD) of 310 aM, a 27-fold improvement over the reference assay without transfer. Moreover, the additional target-specific steps introduced in the complex transfer procedure improved the sequence specificity of the complex transfer assay compared with the reference assay. The suitability for clinical analysis was confirmed using spiked plasma samples, resulting in an LOD of 190 aM. By increasing the sample volume to 600 μL and using magnetic preconcentration, the LOD was improved to 46 aM. These results highlight the importance of background elimination in achieving ultralow LODs for the analysis of low-abundance biomarkers.

This publication is licensed under

CC-BY-NC-ND 4.0 .
  • cc licence
  • by licence
  • nc licence
  • nd licence
Copyright © 2025 The Authors. Published by American Chemical Society

Subjects

what are subjects

Introduction

Click to copy section linkSection link copied!
Nucleic acid testing is an essential part of modern diagnostics, especially in the diagnostics of infectious diseases and cancer. A major part of nucleic acid testing is done by using quantitative polymerase chain reaction and other target amplification-based methods, as they generally reach the lowest limits of detection (LOD). (1) However, amplification-based methods are prone to false results upon contamination and amplification biases. Additionally, the quantitative detection of short targets, such as microRNAs (miRNA), is challenging, as the hybridization of short primers is not always efficient and reliable. (2) Furthermore, amplification-based methods typically require isolation of nucleic acids from the sample matrix to remove enzyme inhibitors, introducing an additional step and potential source of bias to the workflow. (3,4) On the other hand, amplification-free methods, such as Northern blot, in situ hybridization, next-generation sequencing, and microarrays, are designed to detect the target sequence directly. (5−7) However, their sensitivity is often not sufficient for diagnostic use.
Photon-upconversion nanoparticles (UCNPs) are a group of nanomaterials capable of a unique type of luminescence emission, anti-Stokes photoluminescence, (8) rendering them highly attractive labels for bioaffinity assays. The lanthanide-doped inorganic UCNPs (e.g., hexagonal NaYF4:Yb3+, Er3+) can produce visible emission under infrared excitation due to the ladder-like long-lived energy states of the lanthanides and the capability to stack the energy of two or more sequentially absorbed photons. (9) As a result of the anti-Stokes emission, the autofluorescence of the biological sample matrix and solid-phase material can be completely eliminated. Therefore, biomarker detection and imaging can be carried out even in complex biological matrices without optical background interference. (10) Photostability and resistance to photobleaching under continuous excitation are other advantages of UCNPs over conventional labels, such as organic fluorophores. (11)
Due to their excellent properties as labels, UCNPs have also been used for nucleic acid detection. For example, Guan et al. (12) reported the amplification of miRNA using CRISPR/Cas13-based enzymes, which was then detected by magnetic UCNPs on a biosensor platform, achieving a limit of detection (LOD) of 83 fM. Wu et al. (13) detected two miRNA biomarkers for breast cancer simultaneously using green- and blue-emitting UCNPs as Förster resonance energy transfer (FRET) donors and MoS2 nanosheets as FRET quenchers. The presence of miRNAs in the sample prevented FRET, resulting in LODs of 0.17 nM for miRNA-593 and 0.25 nM for miRNA-155. Chen et al. (14) employed UCNPs in a multimode lateral flow assay to detect miRNA via colorimetry, luminescence, and surface-enhanced Raman spectroscopy. This platform allowed miRNA detection in the concentration range from 1 fM to 2 nM. Wang et al. (15) reported the detection of circulating tumor DNA (ctDNA) based on FRET between UCNP-based donor probes and gold nanocages acting as energy acceptors, reaching an LOD of 6.3 pM.
While UCNPs are detectable without optical background interference, the background signal generated by nonspecific binding of label conjugates becomes the sensitivity-limiting factor. Variation of the nonspecific binding in the assays can also contribute to the inaccuracy, ultimately leading to falsely negative or positive results. (16,17) There are several ways to reduce the amount of nonspecifically bound labels, for example, using more effective washing procedures, thorough optimization of the composition and amount of the assay components, and efficient blocking of the detection surface. (18−20) However, excessive washing can decrease assay sensitivity by washing off even a substantial part of the specifically bound labels. (18) Target-dependent covalent ligation has been used to enable more stringent washing. (21) Another way of increasing the signal-to-background ratio is by promoting specific interactions. For example, the rate of specific hybridization can be increased by concentrating the target oligonucleotides by addition of crowding agents, such as poly(ethylene glycol). (22)
Another well-known way to improve the analytical sensitivity of bioaffinity assays is the digital readout, based on counting single labels bound to the solid-phase surface. (24) However, if the analytical sensitivity is limited by nonspecific binding, the digital readout does not necessarily improve the assay performance. (23) An alternative approach to increase the assay sensitivity is reducing the capture surface area to maximize the density of the specific binding, thus increasing the signal-to-background ratio by the so-called ambient analyte assays. (25)
For bioanalytical applications, surface modification is required to render UCNPs dispersible in aqueous solutions and enable conjugation with biomolecules. Surface coating with polymers, such as poly(ethylene glycol) or poly(acrylic acid), yields a protective layer and prevents the nonspecific binding of assay components to the particle surface. (23,26) Different surface modifications and conjugated biomolecules have different tendencies for nonspecific interactions with other biomolecules and biomolecule-coated surfaces. (23,27−29) Thus, each particular combination of surface chemistry and molecular recognition elements in the assay may require separate optimization.
Despite efforts to minimize nonspecific binding, it has been practically impossible to eliminate it completely. However, we introduce an assay principle for the detection of short nucleic acid sequences that overcomes this limitation by utilizing the hybridization complex transfer technique. The assay uses two consecutive target-specific capture steps, and the elimination of the nonspecific background signal is based on the specific release of the target sequences from the first capture probes by toehold-mediated strand displacement (30) with releasing oligonucleotides. As a result, only the target–UCNP complexes are released from the first capture probes and recaptured onto the second solid phase, while nonspecifically bound UCNPs remain on the first solid phase. Therefore, only specifically target-bound UCNPs are detected on the second solid phase, resulting in complete elimination of the assay background and ultrasensitive detection of the target. This type of complex transfer assay has not been reported before for the detection of nucleic acids. The assay principle is an improved version of the immunocomplex transfer assay originally developed by Kohno et al. (31,32) In their assay, the entire two-site immunocomplex, including the capture antibody, was released and recaptured. Therefore, the unoccupied capture antibodies were transferred, too. With nucleic acids, it is easier to release only the target–label complexes without releasing capture probes. This enables the use of a large surface area in the first capture step to enhance the binding kinetics, followed by the second capture step onto a smaller surface area to maximize the signal intensity on the readout area. (33) By using magnetic beads (MBs) as the solid phase, this also enables the preconcentration of the target from a larger sample volume, further improving the sensitivity of the assay. We used synthetic DNA oligonucleotide corresponding to the miR-20a sequence (DNA-miR-20a) as a model target in assay development to demonstrate the potential of the complex transfer assay technique while excluding possible stability issues of RNA. (34)

Experimental Section

Click to copy section linkSection link copied!

Chemicals and Materials

Synthetic oligonucleotides (sequences listed in Table S1, Supporting Information) were obtained from Biomers (Germany). Kaivogen buffer solution (assay buffer), Kaivogen wash buffer, and white streptavidin-coated microtiter plates (MTP) were purchased from Uniogen Oy (Finland). Pierce 1 μm streptavidin MBs and Lumi Lockwell White Maxisorp 96-well MTPs were purchased from Thermo Fisher (USA). Streptavidin was obtained from IBA Life Sciences (Germany). Bovine serum albumin was obtained from Bioreba (Switzerland). Hybridization buffer and elution buffer were prepared by adding NaCl and KF to the assay buffer, resulting in 3.6 M NaCl and 5 mM KF in the hybridization buffer and 1 M NaCl and 1 mM KF in the elution buffer. Hybridization wash buffer was prepared by adding 0.3 M NaCl, 0.1% (w/v) Tween 20, and 1 mM KF to the wash buffer.
Information on the oligonucleotide design, protocols for synthesis, and surface modification of UCNPs (NaYF4:Yb3+, Er3+) and their conjugation with tracer probes, as well as characterization of UCNPs and UCNP-tracer probe conjugate with transmission electron microscopy and dynamic light scattering (Figure S1), and information on the plasma pool collection and in-house coating of MTPs with streptavidin are provided in the Supporting Information.

Hybridization Complex Transfer Assay with Microtiter Wells as the First Capture Surface

MTPs for the first and second capture steps were coated with capture probes just before use. The wells of commercial streptavidin-coated 96-well MTPs were prewashed with the wash buffer and coated with the biotinylated first capture probe by adding 150 μL of 50 nM probe dilution in assay buffer to each well and incubating the plate at room temperature under slow shaking for 30 min. For the second capture step, the biotinylated second capture probes were diluted to a final concentration of 50 nM in assay buffer supplemented with 5% ethanol, heated to 80 °C for 5 min to denature probe dimers, and cooled at room temperature. The in-house coated streptavidin MTP was prewashed with the wash buffer, and the biotinylated second capture probe was immobilized onto the bottom of the wells by incubating 25 μL of the probe per well for 30 min without shaking. The unbound probes were removed by washing the wells once with a wash buffer.
Calibrators were prepared by diluting target DNA oligonucleotides in 50 mM Tris-HCl, pH 7.75, 0.9% NaCl, 0.05% NaN3 containing 7.5% bovine serum albumin. UCNP–DNA probe conjugates were diluted to a concentration of 75 μg/mL in hybridization buffer and bath-sonicated for 3 min. The UCNP conjugate dilution was mixed with samples or calibrators in a 20:80 volume ratio (resulting in 15 μg/mL UCNP conjugates, 0.85 M NaCl, and 1 mM KF in the hybridization reaction). The UCNP–target complexes were formed by incubating the hybridization reactions for 30 min at room temperature under slow rotation, after which 150 μL of hybridization reaction mixtures (corresponding to 30 μL of the UCNP conjugates in hybridization buffer and 120 μL of the sample) was pipetted to the first capture probe-coated wells. The complexes were captured by incubating the plate at room temperature under slow shaking for 1 h. The wells were washed four times with hybridization wash buffer, and 5 pmol releasing oligonucleotide was added to each well in 150 μL of elution buffer and incubated at room temperature under slow shaking for 45 min. The released target–UCNP complexes were transferred to the second capture wells (140 μL/well) and incubated for 90 min at room temperature under slow shaking. The wells were washed four times with the hybridization wash buffer and left to dry. Finally, the upconversion luminescence (UCL) was measured.

Hybridization Complex Transfer Assay Using MBs as the First Capture Surface

Complex transfer assay with MBs as the first capture surface was carried out as described for the MTP wells, with some modifications. Streptavidin-modified MBs were prewashed twice with wash buffer and coated with the biotinylated first capture probes (1 mg/mL beads, 100 nM first capture probes in assay buffer) for 60 min under 1200 rpm shaking. The capture probe-coated MBs were washed 3 times with wash buffer to remove unbound probes. Calibrators were prepared by diluting target DNA oligonucleotides in the assay buffer. The volume of samples or calibrators was either 120 or 600 μL per reaction, and the volume of the UCNP dilution was scaled accordingly (i.e., 30 or 150 μL per reaction, respectively). The UCNP–target complexes were formed as described for the MTP assay. To capture the complexes, a final concentration of 0.17 mg/mL MBs was added to the UCNP–target mixtures, and the reaction mixture was incubated in microtubes under 1200 rpm orbital shaking for 60 min at room temperature. Afterward, the MBs were washed four times with hybridization wash buffer, and the target–UCNP complexes were released by adding 0.1 nmol of releasing oligonucleotide per 1 mg of MBs in 40 μL/reaction in the elution buffer. The strand displacement reaction mixture was incubated for 30 min under 1200 rpm shaking. The MBs were removed from the reactions with a magnet, and 40 μL of the supernatant was transferred into each second capture well, prepared as described for the MTP assay. Thereafter, the wells were incubated for 30 min at room temperature under slow shaking to capture the UCNP–target complexes. After the wells were washed and dried, the UCL was measured.

UCL Readout and Data Evaluation

UCL at 540 nm was measured as an average of a 3 × 3 point raster (point-to-point distance 1.5 mm) measurement of 2 s readouts from the bottom of a microtiter well using a modified Chameleon plate reader (Hidex Oy, Finland) equipped with a 980 nm laser excitation source. (35) The standard curves were fitted with a 4-parameter logistic function in Origin 2016, and the LODs were calculated by adding three times the standard deviation of the zero calibrator to the average signal of the zero calibrator, followed by determining the corresponding concentration from the regression curve. The data evaluation and LOD determination are described in detail in the Supporting Information.

Results and Discussion

Click to copy section linkSection link copied!
The principle of the hybridization complex transfer assay is illustrated in Figure 1. The sample containing the target or calibrator DNA is first mixed with a hybridization buffer containing UCNPs conjugated with hairpin-structured DNA probes to form target–UCNP complexes (step I). The hairpin structures stabilize the hybridization via base-stacking interactions between adjacent bases. (36) The complexes are then captured using biotin-modified capture DNA probes immobilized on the first capture surface of either streptavidin-coated MTP wells or streptavidin-coated MBs (step II). After washing off the unbound reagents, releasing DNA oligonucleotides are added. The releasing oligonucleotides were designed to hybridize with the capture probes more strongly than the target, thus displacing the target–UCNP complexes via a toehold-mediated strand displacement reaction. The capture probes contain a toehold domain, which is located next to the target binding domain and is complementary to the releasing oligonucleotide. The partial hybridization of the releasing oligonucleotide to the toehold domain in the capture probe initiates the strand displacement reaction (step III). The releasing oligonucleotide then displaces the target sequence via branch migration and releases the target–UCNP complex into the solution. In contrast, UCNPs nonspecifically bound to the first capture surface are not released (step IV). The solution containing the released target–UCNP complexes is transferred to a fresh streptavidin-coated MTP modified with a biotinylated, hairpin-structured, and target-specific capture probe (step V). The target–UCNP complexes bind to the second capture probe, and after washing and drying, the UCL at 540 nm is measured from the bottom of the well upon 980 nm laser excitation (step VI).

Figure 1

Figure 1. Principle of the hybridization complex transfer assay utilizing MBs as the first capture surface. (I) The sample is mixed with the UCNP–DNA probe conjugates to form target–UCNP complexes. (II) The complexes are captured with DNA probes immobilized on the surface of MBs. (III) The beads are washed, and releasing oligonucleotides are added. Releasing oligonucleotides hybridize with the capture probes, displacing the target via toehold-mediated strand displacement. (IV) Target–UCNP complexes are released into the solution, and (V) the solution is transferred into microtiter plate wells coated with another capture probe. (VI) The target–UCNP complexes are collected with the capture probes, the wells are washed and dried, and UCL is measured. In the reference assay (R), the complexes formed in the step (I) are directly captured onto wells coated with the second capture probes.

High Resolution Image
Download MS PowerPoint Slide
The assay was first optimized using an MTP as a solid phase for both capture steps to find the optimal conditions for the hybridization of the target with capture probes as well as for releasing the target complex. The first capture of the target from the sample was carried out in commercial MTP wells completely coated with streptavidin. The second capture step was carried out in MTP where streptavidin was present only on the bottom of the wells to concentrate the UCNP–target complexes on the scanned area of the well to maximize the signal intensities. Figure S2 in the Supporting Information shows that 13 nucleotides were an optimal length for the sequence in the releasing oligonucleotide binding to the toehold domain in the first capture probe. Figure S3 shows that the optimal elution buffer contained a minimum of 0.7 M NaCl. The complex transfer assay was performed in a sample volume of 120 μL per MTP well. In a reference assay, the UCNP–target complexes were formed in the same volume as in the complex transfer assay, but the complexes were captured directly onto the MTP solid phase used in the second capture step, i.e., without the first capture and complex transfer steps.
The standard curves of the hybridization complex transfer assay and the reference assay are presented in Figure 2. The complex transfer assay yielded an LOD of 0.66 fM, about 9-fold lower than the LOD of 5.7 fM obtained by the reference assay. Although the signal responses in the linear range obtained by the complex transfer assay were 48–56% lower compared to the reference assay, the background signal originating from nonspecifically bound labels (i.e., the signal of the zero calibrators after subtracting the instrument background) was reduced by more than 99%, thus being much more important to obtain the lowest possible LOD than the overall luminescence signal. The signal of zero calibrators (595 ± 61 counts) was at the level of the instrumental background (555 ± 23 counts), which was now the limiting factor of the sensitivity of the complex transfer assay together with the binding and detectability of the labels (37) instead of the nonspecific binding. This confirmed the assumption that complex transfer and subsequent elimination of the background caused by nonspecifically bound labels would allow for achieving lower LODs.

Figure 2

Figure 2. Standard curves of the complex transfer assay (red, circles) and the reference assay without complex transfer (blue, squares). Both captures of the complex transfer assay were carried out in an MTP. Dashed lines indicate the LODs, and the dotted line shows the instrument background. Error bars represent the standard deviations of three replicates (or six replicates in case of zero calibrators).

High Resolution Image
Download MS PowerPoint Slide
To further improve the performance of the complex transfer assay, MTP was replaced by streptavidin-coated magnetic MBs as the first capture surface. The advantage of using MBs as a solid phase is their large surface area and the possibility of increasing the ratio of the capture surface area to the sample volume, thus increasing the concentration of capture probes and subsequently improving the capture kinetics. For the first capture step, the MBs were incubated for 1 h in a mixture containing 120 μL of the sample and 30 μL of UCNPs in the hybridization buffer. The binding kinetics of the UCNP–target complexes depended on the MB concentration, and the binding was slower when the MB concentration was reduced to 50% (Figure S4 in the Supporting Information).
In order to obtain the best possible performance of the complex transfer assay utilizing the MBs as the first capture surface, the UCNP label concentration, releasing oligonucleotide concentration, incubation times of the first capture on MBs and second capture on MTP, and the elution of the complexes from MBs were optimized (Figures S5 and S6 in the Supporting Information). Under optimal conditions, even though the signal responses in the complex transfer assay were 40–60% lower compared to the reference assay (Figure 3A), the hybridization complex transfer assay achieved an LOD of 0.31 fM (corresponding to approximately 22,000 target DNA molecules), improving the LOD by a factor of 27, compared to the reference assay with an LOD of 8.3 fM (corresponding to 600,000 target DNA molecules).

Figure 3

Figure 3. Standard curves of the complex transfer assay for the detection of DNA-miR-20a utilizing the MBs as the first capture surface (red circles) and the reference assay with direct capture on the second capture surface (blue squares) by using (A) 120 μL of sample volume per reaction with the assay buffer as the sample matrix, (B) 120 μL sample volume per reaction with EDTA plasma pool as a sample matrix, and (C) preconcentration from 600 μL of sample volume per reaction with the assay buffer as the sample matrix. Dashed lines indicate the LODs, and dotted lines show the instrument background. Error bars represent the standard deviations of three replicates (or eight replicates in case of zero calibrators).

High Resolution Image
Download MS PowerPoint Slide
To demonstrate that the complex transfer assay is suitable for detecting the target directly from plasma samples, EDTA plasma pool aliquots were spiked with known target concentrations and analyzed by the complex transfer assay, utilizing MBs as the first capture surface and by the reference assay. The LOD in EDTA plasma was 0.19 fM (14,000 molecules) for the hybridization complex transfer assay and 4.0 fM (290,000 molecules) for the reference assay (Figure 3B). The assay was able to detect similar target concentrations in undiluted plasma as in the buffer, and the LOD was 4 orders of magnitude lower than the reported miR-20a concentration in plasma. (38) This LOD indicates that the target can be detected directly in plasma without additional sample pretreatment. Generally, circulating miRNA concentrations are in the atto- to picomolar range. (39,40) Thus, the assay is likely to be almost universally applicable for the detection of miRNA by simply changing the sequences of the capture and tracer probes.
Using magnetic MBs as the solid phase in the first capture step has the additional benefit of enabling the collection of target molecules from a relatively large sample volume and concentrating them by doing the strand displacement reaction in a significantly smaller volume. The preconcentration step enables the detection of even lower target concentrations in the sample. Therefore, a 5-fold increase in the initial sample volume (600 μL) was used (Figure 3C), resulting in a 6.7-fold improvement in the LOD (46 aM, corresponding to 17,000 molecules). Compared to the reference assay in Figure 3A without complex transfer and preconcentration, the improvement was 180-fold.
As the sensitivity of the complex transfer assay was limited by the instrumental background noise and the detectability of the label, it is expected that the LOD could be further reduced by using larger UCNPs to increase their brightness or by changing the detection method. For example, only a small number of UCNPs bound to the surface are sufficient for a digital readout. (23,41) Additionally, the detectability of the signal could be improved by reducing the capture surface area in the second capture step to increase the label density. (25,42)
Some miRNAs differ by only a single nucleotide, making the ability to discriminate between minor sequence variations an essential feature of miRNA assays. To generate a signal in the complex transfer assay, the noncomplementary sequence has to cross-react with two capture probes sequentially, whereas in the reference assay, binding to one capture probe is enough. Therefore, the complex transfer assay was expected to have a lower cross-reactivity than the reference one. The cross-reactivities of MB-based complex transfer assay and reference assay were studied by using 0.5 pM DNA oligonucleotides, which contain minor variations in the sequence of the complementary target (Table S1). The luminescence signals were normalized to the complementary target signal; the normalized signal responses of each sequence are presented in Figure 4. The sequence with only a single nucleotide difference from the complementary target resulted in 1.8% and 19% cross-reactivity in complex transfer and reference assay, respectively. The sequence containing two mismatched nucleotides (DNA analogue of miR-20b) had 0.4% and 2.6% cross-reactivity in complex transfer and reference assay, respectively. Additionally, to study whether highly homologous sequences interfere with the quantitation of the target, samples containing various combinations of DNA analogues of miR-20a and miR-20b were prepared and analyzed with the complex transfer assay, and even 10-fold excess of nontarget sequence did not interfere with the quantification of the target (Figure S7, Supporting Information). These results support the hypothesis that the complex transfer procedure significantly improves the assay specificity.

Figure 4

Figure 4. Cross-reactivity of the complex transfer assay (red) and the reference assay (blue) with sequences containing minor target sequence variations (0.5 pM; complete sequences are provided in Table S1). The luminescence signal responses are presented as percentage of the signal of the complementary target (DNA-miR-20a). The error bars represent the standard deviations of three replicate wells.

High Resolution Image
Download MS PowerPoint Slide
In our previous work, we developed a conventional sandwich hybridization assay on an MTP for the detection of the same sequence as in this work. (43) The direct assay principle was similar to that of the reference assay presented in this work. However, relatively long incubation times were necessary to compensate for the low UCNP label concentration employed to minimize nonspecific binding and, subsequently, the background signal. With a total assay time of approximately 4 h, the direct assay achieved an LOD of 0.73 fM. Although the complex transfer procedure in this work requires additional steps, it also allows for using 15-fold higher label concentration to improve binding kinetics without increasing the background signal, and the total assay time was decreased by approximately 1 h while achieving 16 times lower LOD.
Watanabe and Hashida (44) developed immunocomplex transfer assays with enzyme labels for three cytokines and compared them with similar assays without immunocomplex transfer. In their assay for TNF-α, the complex transfer decreased the specific signal by 65–71%, but due to the reduced nonspecific binding, the LOD improved 100-fold to 0.03 pg/mL, which corresponds to 1.7 fM monomeric TNF-α. Morrissey et al. (45) utilized a partly similar approach to improve the LOD of a radiolabel hybridization assay. Their approach used poly(dA)-tailed capture probes to enable the reversible capture of the target–probe complexes. The complexes were captured with polyT probes and eluted by the addition of a chaotropic salt. By repeating the capture and elution cycle three times, a 1000-fold improvement in the LOD and a detection limit of 40,000–100,000 molecules were achieved. In comparison, our complex transfer assay achieved an LOD of approximately 14,000 DNA target molecules.
Masterson et al. (46) reported localized surface plasmon resonance nanoplasmonic quantitation of microRNA-10b and microRNA-let7a, which are related to pancreatic ductal adenocarcinoma. They achieved an LOD of approximately 130 aM for both targets. Compared to the complex transfer assay reported here, their assay was carried out only in 10% plasma, and required overnight incubation. Ramshani et al. (47) developed a microfluidic sensor based on the electrokinetic membrane for free and extracellular vesicle-encapsulated microRNA miR-21 detection. This assay required only 20 μL of the sample, and the assay time was only 30 min, but the LOD of 1 pM was 4 orders of magnitude higher compared to our complex transfer assay. Majd et al. (48) developed a label-free hybridization assay using a molybdenum disulfide field-effect transistor biosensor for the detection of miRNA-155 as a breast cancer biomarker. They achieved LOD values of 0.03 fM in buffer and 0.1 fM in spiked human serum. However, the serum samples were 10-fold diluted with buffer before analysis, so the target concentration would have to be 10-fold higher in the untreated serum to be detectable. Wegman et al. (49) reported amplification-free analysis of multiple miRNAs by capillary electrophoresis with isotachophoresis preconcentration. Although the advantage of their approach is the possibility to detect several miRNAs in one experiment, their LOD was only 1 pM, which is about 4 orders of magnitude higher than the one obtained with our complex transfer assay.

Conclusions

Click to copy section linkSection link copied!
A novel complex transfer technique was developed to eliminate the background signal from nonspecifically bound UCNPs in heterogeneous hybridization assays. The functionality of the method was demonstrated in the hybridization assay for the DNA analogue of miR-20a sequence as a model target, but the assay can be easily modified for the ultrasensitive detection of other nucleic acid biomarkers by changing the probe sequences. First, both capture steps were carried out in the MTP, achieving an LOD of 0.66 fM, which was 8.7 times lower compared to the LOD of the reference assay without the complex transfer. These results indicate that despite the unique optical properties of UCNPs, the nonspecific adsorption of labels to the detection surface is the biggest obstacle in reaching ultralow LODs. This limitation was overcome by our complex transfer approach, which eliminated the background and thus improved the LOD. Moreover, the additional steps combined with the MBs as the first detection surface enabled preconcentration of the target, which can be exploited to further improve the sensitivity. Using MBs as the first capture surface, LODs of 0.31 and 0.19 fM were achieved for buffer and EDTA plasma, respectively. These represent 27-fold and 21-fold improvements compared to the reference assay. A slightly lower LOD in the plasma proves the ability of the assay to be sensitive for the detection of nucleic acids in clinical samples without matrix interference. Additionally, using MBs as the first capture surface allowed sample preconcentration from a larger volume, resulting in an LOD of 46 aM. Furthermore, using two target-specific capture steps in the complex transfer assay improved discrimination between highly homologous sequences compared to the reference assay involving only a single capture step. The results indicate that the complex transfer assay is suitable for the direct detection of low-abundance circulating miRNA biomarkers, making it a powerful tool for cancer screening and early stage diagnostics.

Supporting Information

Click to copy section linkSection link copied!

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.4c05401.

  • Information on oligonucleotide design and sequences of oligonucleotides, synthesis and surface modification of UCNP labels and their conjugation with oligonucleotide probes; information about plasma pool collection and in-house coating of MTPs with streptavidin; characterization of UCNP labels; and results from the optimization of the assay performance (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

Click to copy section linkSection link copied!
  • Corresponding Author
  • Authors
    • Jakub Máčala - Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
    • Satu Lahtinen - Department of Life Technologies/Biotechnology, Faculty of Technology, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland;  Orcidhttps://orcid.org/0000-0003-2816-7809
    • Hans H. Gorris - Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic;  Orcidhttps://orcid.org/0000-0003-1148-4293
    • Petr Skládal - Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic;  Orcidhttps://orcid.org/0000-0002-3868-5725
    • Zdeněk Farka - Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic;  Orcidhttps://orcid.org/0000-0002-6842-7081
    • Tero Soukka - Department of Life Technologies/Biotechnology, Faculty of Technology, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland;  Orcidhttps://orcid.org/0000-0002-1144-6724
  • Author Contributions

    J.M. and S.K. contributed equally to this work.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

Click to copy section linkSection link copied!

The authors thank Jaana Rosenberg from the University of Turku for synthesizing the UCNPs and Julian Brandmeier from the University of Regensburg for the surface modification of UCNPs. The work was supported by grant GA22-27580S from the Czech Science Foundation.

References

Click to copy section linkSection link copied!

This article references 49 other publications.

  1. 1
    Li, M.; Yin, F.; Song, L.; Mao, X.; Li, F.; Fan, C.; Zuo, X.; Xia, Q. Nucleic Acid Tests for Clinical Translation. Chem. Rev. 2021, 121 (17), 10469– 10558,  DOI: 10.1021/acs.chemrev.1c00241
    Google Scholar
    1
    Nucleic Acid Tests for Clinical Translation
    Li, Min; Yin, Fangfei; Song, Lu; Mao, Xiuhai; Li, Fan; Fan, Chunhai; Zuo, Xiaolei; Xia, Qiang
    Chemical Reviews (Washington, DC, United States) (2021), 121 (17), 10469-10558CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. Nucleic acids, including DNA (DNA) and RNA (RNA), are natural biopolymers composed of nucleotides that store, transmit, and express genetic information. Overexpressed or underexpressed as well as mutated nucleic acids have been implicated in many diseases. Therefore, nucleic acid tests (NATs) are extremely important. Inspired by intracellular DNA replication and RNA transcription, in vitro NATs have been extensively developed to improve the detection specificity, sensitivity, and simplicity. The principles of NATs can be in general classified into three categories: nucleic acid hybridization, thermal-cycle or isothermal amplification, and signal amplification. Driven by pressing needs in clin. diagnosis and prevention of infectious diseases, NATs have evolved to be a rapidly advancing field. During the past ten years, an explosive increase of research interest in both basic research and clin. translation has been witnessed. In this review, we aim to provide comprehensive coverage of the progress to analyze nucleic acids, use nucleic acids as recognition probes, construct detection devices based on nucleic acids, and utilize nucleic acids in clin. diagnosis and other important fields. We also discuss the new frontiers in the field and the challenges to be addressed.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsFaku7zF&md5=8f3ce9510fb68c111ff7c95f432979e6
  2. 2
    Valihrach, L.; Androvic, P.; Kubista, M. Circulating MiRNA Analysis for Cancer Diagnostics and Therapy. Mol. Aspects Med. 2020, 72, 100825,  DOI: 10.1016/j.mam.2019.10.002
    Google Scholar
    There is no corresponding record for this reference.
  3. 3
    Qian, S.; Chen, Y.; Xu, X.; Peng, C.; Wang, X.; Wu, H.; Liu, Y.; Zhong, X.; Xu, J.; Wu, J. Advances in Amplification-Free Detection of Nucleic Acid: CRISPR/Cas System as a Powerful Tool. Anal. Biochem. 2022, 643, 114593,  DOI: 10.1016/j.ab.2022.114593
    Google Scholar
    3
    Advances in amplification-free detection of nucleic acid: CRISPR/Cas system as a powerful tool
    Qian, Siwenjie; Chen, Yanju; Xu, Xiaoli; Peng, Cheng; Wang, Xiaofu; Wu, Hui; Liu, Yang; Zhong, Xiaoping; Xu, Junfeng; Wu, Jian
    Analytical Biochemistry (2022), 643 (), 114593CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
    Amplification technologies such as polymerase chain reaction (PCR) play an important role in nucleic acid detection. However, they require bulky and sophisticated thermal cycling instrument, as well as are prone to get false-pos. results due to amplicon contamination. Currently, CRISPR/Cas system has become an increasingly popular diagnostic tool for nucleic acid with the discovery of its trans-cleavage activity which can degrade single-stranded DNA or RNA at a very high turnover rate. This inherent signal amplification capability allows CRISPR/Cas system to detect unamplified nucleic acids. Here, we reviewed the recent advances of CRISPR-based amplification-free methods for nucleic acid detection. With the assistance of various signal enhancement strategies, the detection sensitivity could be comparable to that of amplification-based methods. We then presented the pros and cons of these methods. And the subsistent challenges including sample prepn., off-target effect, sequences limit, quant. and multiplex detection were further discussed in this review. It is probable for CRISPR-powered detection methods to pave the road for rapid, cheap, highly sensitive and specific on-site detection without amplification.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjslSht7s%253D&md5=64bee4939693c4f1f70399445975d0e3
  4. 4
    Kim, D.-J.; Linnstaedt, S.; Palma, J.; Park, J. C.; Ntrivalas, E.; Kwak-Kim, J. Y. H.; Gilman-Sachs, A.; Beaman, K.; Hastings, M. L.; Martin, J. N.; Duelli, D. M. Plasma Components Affect Accuracy of Circulating Cancer-Related MicroRNA Quantitation. J. Mol. Diagn. 2012, 14 (1), 71– 80,  DOI: 10.1016/j.jmoldx.2011.09.002
    Google Scholar
    4
    Plasma components affect accuracy of circulating cancer-related microRNA quantitation
    Kim, Dong-Ja; Linnstaedt, Sarah; Palma, Jaime; Park, Joon Cheol; Ntrivalas, Evangelos; Kwak-Kim, Joanne Y. H.; Gilman-Sachs, Alice; Beaman, Kenneth; Hastings, Michelle L.; Martin, Jeffrey N.; Duelli, Dominik M.
    Journal of Molecular Diagnostics (2012), 14 (1), 71-80CODEN: JMDIFP; ISSN:1525-1578. (Elsevier)
    Circulating microRNAs (miRNAs) have emerged as candidate biomarkers of various diseases and conditions including malignancy and pregnancy. This approach requires sensitive and accurate quantitation of miRNA concns. in body fluids. Herein we report that enzyme-based miRNA quantitation, which is currently the mainstream approach for identifying differences in miRNA abundance among samples, is skewed by endogenous serum factors that co-purify with miRNAs and anticoagulant agents used during collection. Of importance, different miRNAs were affected to varying extent among patient samples. By developing measures to overcome these interfering activities, we increased the accuracy, and improved the sensitivity of miRNA detection up to 30-fold. Overall, the present study outlines key factors that prevent accurate miRNA quantitation in body fluids and provides approaches that enable faithful quantitation of miRNA abundance in body fluids.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVCrtb0%253D&md5=176c4ffc5f7ef4052523ae2b413b6fe9
  5. 5
    Koshiol, J.; Wang, E.; Zhao, Y.; Marincola, F.; Landi, M. T. Strengths and Limitations of Laboratory Procedures for MicroRNA Detection. Cancer Epidemiol. Biomarkers Prev. 2010, 19 (4), 907– 911,  DOI: 10.1158/1055-9965.EPI-10-0071
    Google Scholar
    There is no corresponding record for this reference.
  6. 6
    Li, W.; Ruan, K. MicroRNA Detection by Microarray. Anal. Bioanal. Chem. 2009, 394 (4), 1117– 1124,  DOI: 10.1007/s00216-008-2570-2
    Google Scholar
    6
    MicroRNA detection by microarray
    Li, Wei; Ruan, Kangcheng
    Analytical and Bioanalytical Chemistry (2009), 394 (4), 1117-1124CODEN: ABCNBP; ISSN:1618-2642. (Springer)
    A review. MicroRNAs (miRNAs) are a class of small noncoding RNAs ∼22 nt in length that regulate gene expression and play fundamental roles in multiple biol. processes, including cell differentiation, proliferation and apoptosis as well as disease processes. The study of miRNA has thus become a rapidly emerging field in life science. The detection of miRNA expression is a very important first step in miRNA exploration. Several methodologies, including cloning, northern blotting, real-time RT-PCR, microRNA arrays and ISH (in situ hybridization), have been developed and applied successfully in miRNA profiling. This review discusses the main existing microRNA detection technologies, while emphasizing microRNA arrays.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXktVamsw%253D%253D&md5=e8b0d1482009f44537cb50a763d20024
  7. 7
    Koscianska, E.; Starega-Roslan, J.; Sznajder, L. J.; Olejniczak, M.; Galka-Marciniak, P.; Krzyzosiak, W. J. Northern Blotting Analysis of MicroRNAs, Their Precursors and RNA Interference Triggers. BMC Mol. Biol. 2011, 12 (1), 14,  DOI: 10.1186/1471-2199-12-14
    Google Scholar
    7
    Northern blotting analysis of microRNAs, their precursors and RNA interference triggers
    Koscianska, Edyta; Starega-Roslan, Julia; Sznajder, Lukasz J.; Olejniczak, Marta; Galka-Marciniak, Paulina; Krzyzosiak, Wlodzimierz J.
    BMC Molecular Biology (2011), 12 (), 14CODEN: BMBMC4; ISSN:1471-2199. (BioMed Central Ltd.)
    Background: Numerous microRNAs (miRNAs) have heterogeneous ends resulting from imprecise cleavages by processing nucleases and from various non-templated nucleotide addns. The scale of miRNA end-heterogeneity is best shown by deep sequencing data revealing not only the major miRNA variants but also those that occur in only minute amts. and are unlikely to be of functional importance. All RNA interference (RNAi) technol. reagents that are expressed and processed in cells are also exposed to the same machinery generating end-heterogeneity of the released short interfering RNAs (siRNAs) or miRNA mimetics. Results: In this study we have analyzed endogenous and exogenous RNAs in the range of 20-70 nt by high-resoln. northern blotting. We have validated the results obtained with northern blotting by comparing them with data derived from miRNA deep sequencing; therefore we have demonstrated the usefulness of the northern blotting technique in the investigation of miRNA biogenesis, as well as in the characterization of RNAi technol. reagents. Conclusions: The conventional northern blotting enhanced to high resoln. may be a useful adjunct to other miRNA discovery, detection and characterization methods. It provides quant. data on distribution of major length variants of abundant endogenous miRNAs, as well as on length heterogeneity of RNAi technol. reagents expressed in cells.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXltVWqs7c%253D&md5=818f990c182f607d854b33c2793beb2b
  8. 8
    Liang, G.; Wang, H.; Shi, H.; Wang, H.; Zhu, M.; Jing, A.; Li, J.; Li, G. Recent Progress in the Development of Upconversion Nanomaterials in Bioimaging and Disease Treatment. J. Nanobiotechnol. 2020, 18 (1), 154,  DOI: 10.1186/s12951-020-00713-3
    Google Scholar
    8
    Recent progress in the development of upconversion nanomaterials in bioimaging and disease treatment
    Liang Gaofeng; Wang Haojie; Zhu Mengxi; Shi Hao; Jing Aihua; Li Jinghua; Li Guangda; Wang Haitao
    Journal of nanobiotechnology (2020), 18 (1), 154 ISSN:.
    Multifunctional lanthanide-based upconversion nanoparticles (UCNPs), which feature efficiently convert low-energy photons into high-energy photons, have attracted considerable attention in the domain of materials science and biomedical applications. Due to their unique photophysical properties, including light-emitting stability, excellent upconversion luminescence efficiency, low autofluorescence, and high detection sensitivity, and high penetration depth in samples, UCNPs have been widely applied in biomedical applications, such as biosensing, imaging and theranostics. In this review, we briefly introduced the major components of UCNPs and the luminescence mechanism. Then, we compared several common design synthesis strategies and presented their advantages and disadvantages. Several examples of the functionalization of UCNPs were given. Next, we detailed their biological applications in bioimaging and disease treatment, particularly drug delivery and photodynamic therapy, including antibacterial photodynamic therapy. Finally, the future practical applications in materials science and biomedical fields, as well as the remaining challenges to UCNPs application, were described. This review provides useful practical information and insights for the research on and application of UCNPs in the field of cancer.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3s7nvFehug%253D%253D&md5=74d17e757c999be9fa5661306657845d
  9. 9
    Wen, S.; Zhou, J.; Zheng, K.; Bednarkiewicz, A.; Liu, X.; Jin, D. Advances in Highly Doped Upconversion Nanoparticles. Nat. Commun. 2018, 9 (1), 2415,  DOI: 10.1038/s41467-018-04813-5
    Google Scholar
    9
    Advances in highly doped upconversion nanoparticles
    Wen Shihui; Zhou Jiajia; Jin Dayong; Zheng Kezhi; Liu Xiaogang; Bednarkiewicz Artur; Bednarkiewicz Artur
    Nature communications (2018), 9 (1), 2415 ISSN:.
    Lanthanide-doped upconversion nanoparticles (UCNPs) are capable of converting near-infra-red excitation into visible and ultraviolet emission. Their unique optical properties have advanced a broad range of applications, such as fluorescent microscopy, deep-tissue bioimaging, nanomedicine, optogenetics, security labelling and volumetric display. However, the constraint of concentration quenching on upconversion luminescence has hampered the nanoscience community to develop bright UCNPs with a large number of dopants. This review surveys recent advances in developing highly doped UCNPs, highlights the strategies that bypass the concentration quenching effect, and discusses new optical properties as well as emerging applications enabled by these nanoparticles.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1Mbps1Cluw%253D%253D&md5=615b19adc979238a82e23c423ae5af05
  10. 10
    Hlaváček, A.; Farka, Z.; Mickert, M. J.; Kostiv, U.; Brandmeier, J. C.; Horák, D.; Skládal, P.; Foret, F.; Gorris, H. H. Bioconjugates of Photon-Upconversion Nanoparticles for Cancer Biomarker Detection and Imaging. Nat. Protoc. 2022, 17 (4), 1028– 1072,  DOI: 10.1038/s41596-021-00670-7
    Google Scholar
    10
    Bioconjugates of photon-upconversion nanoparticles for cancer biomarker detection and imaging
    Hlavacek, Antonin; Farka, Zdenek; Mickert, Matthias J.; Kostiv, Uliana; Brandmeier, Julian C.; Horak, Daniel; Skladal, Petr; Foret, Frantisek; Gorris, Hans H.
    Nature Protocols (2022), 17 (4), 1028-1072CODEN: NPARDW; ISSN:1750-2799. (Nature Portfolio)
    The detection of cancer biomarkers in histol. samples and blood is of paramount importance for clin. diagnosis. Current methods are limited in terms of sensitivity, hindering early detection of disease. We have overcome the shortcomings of currently available staining and fluorescence labeling methods by taking an integrative approach to establish photon-upconversion nanoparticles (UCNP) as a powerful platform for cancer detection. These nanoparticles are readily synthesized in different sizes to yield efficient and tunable short-wavelength light emission under near-IR excitation, which eliminates optical background interference of the specimen. Here we present a protocol for the synthesis of UCNPs by high-temp. co-pptn. or seed-mediated growth by thermal decompn., surface modification by silica or poly(ethylene glycol) that renders the particles resistant to nonspecific binding, and the conjugation of streptavidin or antibodies for biol. detection. To detect blood-based biomarkers, we present an upconversion-linked immunosorbent assay for the analog and digital detection of the cancer marker prostate-specific antigen. When applied to immunocytochem. anal., UCNPs enable the detection of the breast cancer marker human epidermal growth factor receptor 2 with a signal-to-background ratio 50-fold higher than conventional fluorescent labels. UCNP synthesis takes 4.5 d, the prepn. of the antibody-silica-UCNP conjugate takes 3 d, the streptavidin-poly(ethylene glycol)-UCNP conjugate takes 2-3 wk, upconversion-linked immunosorbent assay takes 2-4 d and immunocytochem. takes 8-10 h. The procedures can be performed after std. lab. training in nanomaterials research.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xktlyrt7k%253D&md5=f624bb3e5e2b1fef2b6f41aae0773c20
  11. 11
    Wu, S.; Han, G.; Milliron, D. J.; Aloni, S.; Altoe, V.; Talapin, D. V.; Cohen, B. E.; Schuck, P. J. Non-Blinking and Photostable Upconverted Luminescence from Single Lanthanide-Doped Nanocrystals. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (27), 10917– 10921,  DOI: 10.1073/pnas.0904792106
    Google Scholar
    11
    Non-blinking and photostable up-converted luminescence from single lanthanide-doped nanocrystals
    Wu, Shiwei; Han, Gang; Milliron, Delia J.; Aloni, Shaul; Altoe, Virginia; Talapin, Dmitri V.; Cohen, Bruce E.; Schuck, P. James
    Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (27), 10917-10921, S10917/1-S10917/9CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The development of probes for single-mol. imaging has dramatically facilitated the study of individual mols. in cells and other complex environments. Single-mol. probes ideally exhibit good brightness, uninterrupted emission, resistance to photobleaching, and minimal spectral overlap with cellular autofluorescence. However, most single-mol. probes are imperfect in several of these aspects, and none have been shown to possess all of these characteristics. Here the authors show that individual lanthanide-doped up-converting nanoparticles (UCNPs) - specifically, hexagonal phase NaYF4 (β-NaYF4) nanocrystals with multiple Yb3+ and Er3+ dopants - emit bright anti-Stokes visible up-converted luminescence with exceptional photostability when excited by a 980-nm continuous wave laser. Individual UCNPs exhibit no on/off emission behavior, or "blinking," down to the millisecond time-scale, and no loss of intensity following an hour of continuous excitation. Amphiphilic polymer coatings permit the transfer of hydrophobic UCNPs into water, resulting in individual water-sol. nanoparticles with undiminished photophys. characteristics. These UCNPs are endocytosed by cells and show strong up-converted luminescence, with no measurable anti-Stokes background autofluorescence, suggesting that UCNPs are ideally suited for single-mol. imaging expts.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXptVSntLc%253D&md5=d7e1f8475c88639148ffa7626c0825e3
  12. 12
    Guan, L.; Peng, J.; Liu, T.; Huang, S.; Yang, Y.; Wang, X.; Hao, X. Ultrasensitive MiRNA Detection Based on Magnetic Upconversion Nanoparticle Enhancement and CRISPR/Cas13a-Driven Signal Amplification. Anal. Chem. 2023, 95 (48), 17708– 17715,  DOI: 10.1021/acs.analchem.3c03554
    Google Scholar
    There is no corresponding record for this reference.
  13. 13
    Wu, X.; Li, Y.; Yang, M. Y.; Mao, C. B. Simultaneous Ultrasensitive Detection of Two Breast Cancer MicroRNA Biomarkers by Using a Dual Nanoparticle/Nanosheet Fluorescence Resonance Energy Transfer Sensor. Mater. Today Adv. 2021, 12, 100163,  DOI: 10.1016/j.mtadv.2021.100163
    Google Scholar
    There is no corresponding record for this reference.
  14. 14
    Chen, C.; Hu, S.; Tian, L.; Qi, M.; Chang, Z.; Li, L.; Wang, L.; Dong, B. A Versatile Upconversion-Based Multimode Lateral Flow Platform for Rapid and Ultrasensitive Detection of MicroRNA towards Health Monitoring. Biosens. Bioelectron. 2024, 252, 116135,  DOI: 10.1016/j.bios.2024.116135
    Google Scholar
    There is no corresponding record for this reference.
  15. 15
    Wang, J.; Hua, G.; Li, L.; Li, D.; Wang, F.; Wu, J.; Ye, Z.; Zhou, X.; Ye, S.; Yang, J.; Zhang, X.; Ren, L. Upconversion Nanoparticle and Gold Nanocage Satellite Assemblies for Sensitive CtDNA Detection in Serum. Analyst 2020, 145 (16), 5553– 5562,  DOI: 10.1039/D0AN00701C
    Google Scholar
    There is no corresponding record for this reference.
  16. 16
    Güven, E.; Duus, K.; Lydolph, M. C.; Jørgensen, C. S.; Laursen, I.; Houen, G. Non-Specific Binding in Solid Phase Immunoassays for Autoantibodies Correlates with Inflammation Markers. J. Immunol. Methods 2014, 403 (1–2), 26– 36,  DOI: 10.1016/j.jim.2013.11.014
    Google Scholar
    There is no corresponding record for this reference.
  17. 17
    Lahtinen, S.; Lyytikäinen, A.; Sirkka, N.; Päkkilä, H.; Soukka, T. Improving the Sensitivity of Immunoassays by Reducing Non-Specific Binding of Poly(Acrylic Acid) Coated Upconverting Nanoparticles by Adding Free Poly(Acrylic Acid). Microchim. Acta 2018, 185 (4), 220,  DOI: 10.1007/s00604-018-2756-z
    Google Scholar
    There is no corresponding record for this reference.
  18. 18
    Hariri, A. A.; Newman, S. S.; Tan, S.; Mamerow, D.; Adams, A. M.; Maganzini, N.; Zhong, B. L.; Eisenstein, M.; Dunn, A. R.; Soh, H. T. Improved Immunoassay Sensitivity and Specificity Using Single-Molecule Colocalization. Nat. Commun. 2022, 13 (1), 5359,  DOI: 10.1038/s41467-022-32796-x
    Google Scholar
    18
    Improved immunoassay sensitivity and specificity using single-molecule colocalization
    Hariri, Amani A.; Newman, Sharon S.; Tan, Steven; Mamerow, Dan; Adams, Alexandra M.; Maganzini, Nicolo; Zhong, Brian L.; Eisenstein, Michael; Dunn, Alexander R.; Soh, H. Tom
    Nature Communications (2022), 13 (1), 5359CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)
    Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern mol. detection, but the technique still faces notable challenges. One of the biggest problems is discriminating true signal generated by target mols. vs. non-specific background. Here, we developed a Single-Mol. Colocalization Assay (SiMCA) that overcomes this problem by employing total internal reflection fluorescence microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized signals, we can eliminate the effects of background produced by non-specific binding of detection antibodies. Using TNF-α, we show that SiMCA achieves a three-fold lower limit of detection compared to conventional single-color assays and exhibits consistent performance for assays performed in complex specimens such as serum and blood. Our results help define the pernicious effects of non-specific background in immunoassays and demonstrate the diagnostic gains that can be achieved by eliminating those effects.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitlKrsLzJ&md5=6619c42f5f7dfd10214116d5e36113db
  19. 19
    Buchwalow, I.; Samoilova, V.; Boecker, W.; Tiemann, M. Non-Specific Binding of Antibodies in Immunohistochemistry: Fallacies and Facts. Sci. Rep. 2011, 1 (1), 28,  DOI: 10.1038/srep00028
    Google Scholar
    There is no corresponding record for this reference.
  20. 20
    Wauthier, L.; Plebani, M.; Favresse, J. Interferences in Immunoassays: Review and Practical Algorithm. Clin. Chem. Lab. Med. 2022, 60 (6), 808– 820,  DOI: 10.1515/cclm-2021-1288
    Google Scholar
    There is no corresponding record for this reference.
  21. 21
    Mendez-Gonzalez, D.; Lahtinen, S.; Laurenti, M.; López-Cabarcos, E.; Rubio-Retama, J.; Soukka, T. Photochemical Ligation to Ultrasensitive DNA Detection with Upconverting Nanoparticles. Anal. Chem. 2018, 90 (22), 13385– 13392,  DOI: 10.1021/acs.analchem.8b03106
    Google Scholar
    21
    Photochemical Ligation to Ultrasensitive DNA Detection with Upconverting Nanoparticles
    Mendez-Gonzalez, Diego; Lahtinen, Satu; Laurenti, Marco; Lopez-Cabarcos, Enrique; Rubio-Retama, Jorge; Soukka, Tero
    Analytical Chemistry (Washington, DC, United States) (2018), 90 (22), 13385-13392CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    In this work, we explore a photochem. ligation reaction to covalently modify oligonucleotide-conjugated upconverting nanoparticles (UCNPs) in the presence of a specific target DNA sequence. The target sequence acts as a hybridization template, bringing together a biotinylated photoactivatable oligonucleotide probe and the oligonucleotide probe that is attached to UCNPs. The illumination of the UCNPs by NIR light to generate UV emission internally or illuminating the photoactivatable probe directly by an external UV light promotes the photochem. ligation reaction, yielding covalently biotin functionalized UCNPs that can be selectively captured in streptavidin-coated microwells. Following this strategy, we developed a DNA sensor with a limit of detection of 1 × 10-18 mol per well (20 fM). In addn., we demonstrate the possibility to create UCNP patterns on the surface of solid supports upon NIR illumination that are selectively formed under the presence of the target oligonucleotide.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvFKnu7vE&md5=18b56c80456a7e0c1de9caaf95fc830d
  22. 22
    Baltierra-Jasso, L. E.; Morten, M. J.; Laflör, L.; Quinn, S. D.; Magennis, S. W. Crowding-Induced Hybridization of Single DNA Hairpins. J. Am. Chem. Soc. 2015, 137 (51), 16020– 16023,  DOI: 10.1021/jacs.5b11829
    Google Scholar
    22
    Crowding-Induced Hybridization of Single DNA Hairpins
    Baltierra-Jasso, Laura E.; Morten, Michael J.; Laflor, Linda; Quinn, Steven D.; Magennis, Steven W.
    Journal of the American Chemical Society (2015), 137 (51), 16020-16023CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    It is clear that a crowded environment influences the structure, dynamics, and interactions of biol. mols., but the complexity of this phenomenon demands the development of new exptl. and theor. approaches. Here we use two complementary single-mol. FRET techniques to show that the kinetics of DNA base pairing and unpairing, which are fundamental to both the biol. role of DNA and its technol. applications, are strongly modulated by a crowded environment. We directly obsd. single DNA hairpins, which are excellent model systems for studying hybridization, either freely diffusing in soln. or immobilized on a surface under crowding conditions. The hairpins followed two-state folding dynamics with a closing rate increasing by 4-fold and the opening rate decreasing 2-fold, for only modest concns. of crowder [10% (wt./wt.) polyethylene glycol (PEG)]. These expts. serve both to unambiguously highlight the impact of a crowded environment on a fundamental biol. process, DNA base pairing, and to illustrate the benefits of single-mol. approaches to probing the structure and dynamics of complex biomol. systems.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvF2ltL7N&md5=41090b083fa05da3dffcf7e608be31b4
  23. 23
    Brandmeier, J. C.; Raiko, K.; Farka, Z.; Peltomaa, R.; Mickert, M. J.; Hlaváček, A.; Skládal, P.; Soukka, T.; Gorris, H. H. Effect of Particle Size and Surface Chemistry of Photon-Upconversion Nanoparticles on Analog and Digital Immunoassays for Cardiac Troponin. Adv. Healthcare Mater. 2021, 10 (18), 2100506,  DOI: 10.1002/adhm.202100506
    Google Scholar
    There is no corresponding record for this reference.
  24. 24
    Chang, L.; Rissin, D. M.; Fournier, D. R.; Piech, T.; Patel, P. P.; Wilson, D. H.; Duffy, D. C. Single Molecule Enzyme-Linked Immunosorbent Assays: Theoretical Considerations. J. Immunol. Methods 2012, 378 (1–2), 102– 115,  DOI: 10.1016/j.jim.2012.02.011
    Google Scholar
    24
    Single molecule enzyme-linked immunosorbent assays: Theoretical considerations
    Chang, Lei; Rissin, David M.; Fournier, David R.; Piech, Tomasz; Patel, Purvish P.; Wilson, David H.; Duffy, David C.
    Journal of Immunological Methods (2012), 378 (1-2), 102-115CODEN: JIMMBG; ISSN:0022-1759. (Elsevier B.V.)
    We have developed a highly sensitive immunoassay-called digital ELISA-that is based on the detection of single enzyme-linked immunocomplexes on beads that are sealed in arrays of femtoliter wells. Digital ELISA was designed to be highly efficient in the capturing of target proteins, labeling of these proteins, and their detection in single mol. arrays (SiMoA); in essence, the goal of the assay is to "capture every mol., detect every mol.". Here we provide the theor. basis for the design of this assay derived from simple equations based on bimol. interactions. Using these equations and knowledge of the concns. of reagents, the times of interactions, and the on- and off-rates of the mol. interactions for each step of the assay, it is possible to predict the no. of immunocomplexes that are formed and detected by SiMoA. The unique ability of SiMoA to count single immunocomplexes and det. an av. no. of enzymes per bead (AEB), makes it possible to directly compare the no. of mols. detected exptl. to those predicted by theory. These predictions compare favorably to exptl. data generated for a digital ELISA for prostate specific antigen (PSA). The digital ELISA process is efficient across a range of antibody affinities (KD ~ 10-11-10-9 M), and antibodies with high on-rates (kon > 105 M-1 s-1) are predicted to perform best. The high efficiency of digital ELISA and sensitivity of SiMoA to enzyme label also makes it possible to reduce the concn. of labeling reagent, reduce backgrounds, and increasing the specificity of the approach. Strategies for dealing with the dissocn. of antibody complexes over time that can affect the signals in an assay are also described.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XjsFOhsLc%253D&md5=7657279ca929d26c6c245ba0bd3d0009
  25. 25
    Ekins, R. P.; Chu, F. W. Multianalyte Microspot Immunoassay--Microanalytical “Compact Disk” of the Future. Clin. Chem. 1991, 37 (11), 1955– 1967,  DOI: 10.1093/clinchem/37.11.1955
    Google Scholar
    25
    Multianalyte microspot immunoassay-microanalytical "compact disk" of the future
    Ekins, R. P.; Chu, F. W.
    Clinical Chemistry (Washington, DC, United States) (1991), 37 (11), 1955-67CODEN: CLCHAU; ISSN:0009-9147.
    Throughout the 1970s, controversy centered both on immunoassay sensitivity per se and on the relative sensitivities of labeled antibody (Ab) and labeled analyte methods. Theor. studies revealed that RIA sensitivities could be surpassed only by the use of very high-specific-activity nonisotopic labels in noncompetitive designs, preferably with monoclonal antibodies. The time-resolved fluorescence methodol. known as DELFIA represented the first com. ultrasensitive nonisotopic technique based on these theor. insights, the same concepts being subsequently adopted in comparable methodologies relying on the use of chemiluminescent and enzyme labels. However, high-specific-activity labels also permit the development of multianalyte immunoassay systems combining ultrasensitivity with the simultaneous measurement of tens, hundreds, or thousands of analytes in a small biol. sample. This possibility relies on simple, albeit hitherto-unexploited, physiochem. concepts. The first is that all immunoassays rely on the measurement of Ab occupancy by analyte. The second is that, provided the Ab concn. used is vanishingly small, fractional Ab occupancy is independent of both Ab concn. and sample vol. This leads to the notion of ratiometric immunoassay, involving measurement of the ratio of signals (e.g., fluorescent Ab) deposited as a microspot on a solid support, the second (a developing Ab) directed against either occupied or unoccupied binding sites of the sensor Ab. The authors' preliminary studies of this approach have relied on a dual-channel scanning-laser confocal microscope, permitting microspots of area 100 μm2 or less to be analyzed, and implying that an array of 106 Ab-contg. microspots, each directed against a different analyte, could in principle, be accommodated on an area of 1 cm2. Although measurement of such analyte nos. is unlikely ever to be required, the ability to analyze biol. fluids for a wide spectrum of analytes is likely to transform immunodiagnostics in the next decade.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38Xjslekuw%253D%253D&md5=56792c2461f50069cb11ece83e5a68a5
  26. 26
    Shapoval, O.; Brandmeier, J. C.; Nahorniak, M.; Oleksa, V.; Makhneva, E.; Gorris, H. H.; Farka, Z.; Horák, D. PMVEMA-Coated Upconverting Nanoparticles for Upconversion-Linked Immunoassay of Cardiac Troponin. Talanta 2022, 244, 123400,  DOI: 10.1016/j.talanta.2022.123400
    Google Scholar
    There is no corresponding record for this reference.
  27. 27
    Nsubuga, A.; Sgarzi, M.; Zarschler, K.; Kubeil, M.; Hübner, R.; Steudtner, R.; Graham, B.; Joshi, T.; Stephan, H. Facile Preparation of Multifunctionalisable ‘Stealth’ Upconverting Nanoparticles for Biomedical Applications. Dalton Trans. 2018, 47 (26), 8595– 8604,  DOI: 10.1039/C8DT00241J
    Google Scholar
    There is no corresponding record for this reference.
  28. 28
    Raiko, K.; Lyytikäinen, A.; Ekman, M.; Nokelainen, A.; Lahtinen, S.; Soukka, T. Supersensitive Photon Upconversion Based Immunoassay for Detection of Cardiac Troponin I in Human Plasma. Clin. Chim. Acta 2021, 523, 380– 385,  DOI: 10.1016/j.cca.2021.10.023
    Google Scholar
    There is no corresponding record for this reference.
  29. 29
    Chen, H.; Wang, L.; Yeh, J.; Wu, X.; Cao, Z.; Wang, Y. A.; Zhang, M.; Yang, L.; Mao, H. Reducing Non-Specific Binding and Uptake of Nanoparticles and Improving Cell Targeting with an Antifouling PEO-b-PγMPS Copolymer Coating. Biomaterials 2010, 31 (20), 5397– 5407,  DOI: 10.1016/j.biomaterials.2010.03.036
    Google Scholar
    There is no corresponding record for this reference.
  30. 30
    Weng, Z.; Yu, H.; Luo, W.; Guo, Y.; Liu, Q.; Zhang, L.; Zhang, Z.; Wang, T.; Dai, L.; Zhou, X.; Han, X.; Wang, L.; Li, J.; Yang, Y.; Xie, G. Cooperative Branch Migration: A Mechanism for Flexible Control of DNA Strand Displacement. ACS Nano 2022, 16 (2), 3135– 3144,  DOI: 10.1021/acsnano.1c10797
    Google Scholar
    30
    Cooperative Branch Migration: A Mechanism for Flexible Control of DNA Strand Displacement
    Weng, Zhi; Yu, Hongyan; Luo, Wang; Guo, Yongcan; Liu, Qian; Zhang, Li; Zhang, Zhang; Wang, Ting; Dai, Ling; Zhou, Xi; Han, Xiaole; Wang, Luojia; Li, Junjie; Yang, Yujun; Xie, Guoming
    ACS Nano (2022), 16 (2), 3135-3144CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    DNA strand displacement plays an essential role in the field of dynamic DNA nanotechnol. However, flexible regulation of strand displacement remains a significant challenge. Most previous regulatory tools focused on controllable activation of toehold and thus limited the design flexibility. Here, we introduce a regulatory tool termed cooperative branch migration (CBM), through which DNA strand displacement can be controlled by regulating the complementarity of branch migration domains. CBM shows perfect compatibility with the majority of existing regulatory tools, and when combined with forked toehold, it permits continuous fine-tuning of the strand displacement rate spanning 5 orders of magnitude. CBM manifests multifunctional regulation ability, including rate fine-tuning, continuous dynamic regulation, reaction resetting, and selective activation. To exemplify the powerful function, we also constructed a nested if-function signal processing system on the basis of cascading CBM reactions. We believe that the proposed regulatory strategy would effectively enrich the DNA strand displacement toolbox and ultimately promote the construction of DNA machines of higher complexity in nucleic acid research and biomedical applications.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xis1ykurw%253D&md5=7a93cf13c2424daa710c580db80b9382
  31. 31
    Kohno, T.; Ishikawa, E.; Mitsukawa, T.; Matsukura, S. Novel Enzyme Immunoassay (Immune Complex Transfer Enzyme Immunoassay) for Anti-thyroglobulin IgG in Human Serum. J. Clin. Lab. Anal. 1988, 2 (4), 209– 214,  DOI: 10.1002/jcla.1860020406
    Google Scholar
    There is no corresponding record for this reference.
  32. 32
    Kohno, T.; Mitsukawa, T.; Matsukura, S.; Tsunetoshi, Y.; Ishikawa, E. More Sensitive and Simpler Immune Complex Transfer Enzyme Immunoassay for Antithyroglobulin Igg in Serum. J. Clin. Lab. Anal. 1989, 3 (3), 163– 168,  DOI: 10.1002/jcla.1860030306
    Google Scholar
    There is no corresponding record for this reference.
  33. 33
    Gorris, H. H.; Soukka, T. What Digital Immunoassays Can Learn from Ambient Analyte Theory: A Perspective. Anal. Chem. 2022, 94 (16), 6073– 6083,  DOI: 10.1021/acs.analchem.1c05591
    Google Scholar
    33
    What Digital Immunoassays Can Learn from Ambient Analyte Theory: A Perspective
    Gorris, Hans H.; Soukka, Tero
    Analytical Chemistry (Washington, DC, United States) (2022), 94 (16), 6073-6083CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    A review. Immunoassays are important tools for clin. diagnosis as well as environmental and food anal. because they enable highly sensitive and quant. measurements of analyte concns. In the 1980s, Roger Ekins suggested to improve the sensitivity of immunoassays by employing microspot assays, which are carried out under ambient analyte conditions and do not change the bulk analyte concn. of a sample during a measurement. More recently, the measurement of single analyte mols. has addnl. attracted wide research interest. Although the ability to detect a single analyte mol. is not synonymous with the highest anal. sensitivity, single-mol. detection makes new routes accessible to avoiding background noise. This perspective follows the development of solid-phase immunoassays from the design of label techniques to single-mol. (digital) assays against the backdrop of Ekins's fundamental work on immunoassay theory. The essential aspects of both ambient analyte and digital assay approaches are presented as a guideline to finding a balance between the speed, sensitivity, and precision of immunoassays.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xpt1ynt7Y%253D&md5=fc3c8d44561d32aa1bd51f7d64afc196
  34. 34
    Chheda, U.; Pradeepan, S.; Esposito, E.; Strezsak, S.; Fernandez-Delgado, O.; Kranz, J. Factors Affecting Stability of RNA – Temperature, Length, Concentration, PH, and Buffering Species. J. Pharm. Sci. 2024, 113 (2), 377– 385,  DOI: 10.1016/j.xphs.2023.11.023
    Google Scholar
    There is no corresponding record for this reference.
  35. 35
    Soukka, T.; Kuningas, K.; Rantanen, T.; Haaslahti, V.; Lövgren, T. Photochemical Characterization of Up-Converting Inorganic Lanthanide Phosphors as Potential Labels. J. Fluoresc. 2005, 15 (4), 513– 528,  DOI: 10.1007/s10895-005-2825-7
    Google Scholar
    35
    Photochemical characterization of up-converting inorganic lanthanide phosphors as potential labels
    Soukka, Tero; Kuningas, Katri; Rantanen, Terhi; Haaslahti, Ville; Loevgren, Timo
    Journal of Fluorescence (2005), 15 (4), 513-528CODEN: JOFLEN; ISSN:1053-0509. (Springer Science+Business Media, Inc.)
    The authors have characterized com. available up-converting inorg. lanthanide phosphors for their rare earth compn. and photoluminescence properties under IR laser diode excitation. These up-converting phosphors, in contrast to proprietary materials reported earlier, are readily available to be utilized as particulate reporters in various ligand binding assays after grinding to submicron particle size. The laser power d. required at 980 nm to generate anti-Stokes photoluminescence from these particulate reporters is significantly lower than required for two-photon excitation. The narrow photoluminescence emission bands at 520-550 nm and at 650-670 nm are at shorter wavelengths and thus totally discriminated from autofluorescence and scattered excitation light even without temporal resoln. Transparent soln. of colloidal bead-milled up-converting phosphor nanoparticles provides intense green emission visible to the human eye under illumination by an IR laser pointer. In this article, the authors show that the unique photoluminescence properties of the up-converting phosphors and the inexpensive measurement configuration, which is adequate for their sensitive detection, render the up-conversion an attractive alternative to the UV-excited time-resolved fluorescence of down-converting lanthanide compds. widely employed in biomedical research and diagnostics.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtVSqu7%252FM&md5=46cf461e1e56dd0615e44e4ad0b43124
  36. 36
    Yakovchuk, P.; Protozanova, E.; Frank-Kamenetskii, M. D. Base-Stacking and Base-Pairing Contributions into Thermal Stability of the DNA Double Helix. Nucleic Acids Res. 2006, 34 (2), 564– 574,  DOI: 10.1093/nar/gkj454
    Google Scholar
    36
    Base-stacking and base-pairing contributions into thermal stability of the DNA double helix
    Yakovchuk, Peter; Protozanova, Ekaterina; Frank-Kamenetskii, Maxim D.
    Nucleic Acids Research (2006), 34 (2), 564-574CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    Two factors are mainly responsible for the stability of the DNA double helix: base pairing between complementary strands and stacking between adjacent bases. By studying DNA mols. with solitary nicks and gaps we measure temp. and salt dependence of the stacking free energy of the DNA double helix. For the first time, DNA stacking parameters are obtained directly (without extrapolation) for temps. from below room temp. to close to melting temp. We also obtain DNA stacking parameters for different salt concns. ranging from 15 to 100 mM Na+. From stacking parameters of individual contacts, we calc. base-stacking contribution to the stability of A•T- and G•C-contg. DNA polymers. We find that temp. and salt dependences of the stacking term fully det. the temp. and the salt dependence of DNA stability parameters. For all temps. and salt concns. employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A•T pairing is always destabilizing and G•C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtlOnuro%253D&md5=2c72728f600373744c60a81684a64409
  37. 37
    Ekins, R.; Chu, F.; Micallef, J. High Specific Activity Chemiluminescent and Fluorescent Markers: Their Potential Application to High Sensitivity and ‘Multi-analyte’ Immunoassays. J. Biolumin. Chemilumin. 1989, 4 (1), 59– 78,  DOI: 10.1002/bio.1170040113
    Google Scholar
    There is no corresponding record for this reference.
  38. 38
    Zhou, X.; Zhu, W.; Li, H.; Wen, W.; Cheng, W.; Wang, F.; Wu, Y.; Qi, L.; Fan, Y.; Chen, Y.; Ding, Y.; Xu, J.; Qian, J.; Huang, Z.; Wang, T.; Zhu, D.; Shu, Y.; Liu, P. Diagnostic Value of a Plasma MicroRNA Signature in Gastric Cancer: A MicroRNA Expression Analysis. Sci. Rep. 2015, 5 (1), 11251,  DOI: 10.1038/srep11251
    Google Scholar
    There is no corresponding record for this reference.
  39. 39
    Ferracin, M.; Lupini, L.; Salamon, I.; Saccenti, E.; Zanzi, M. V.; Rocchi, A.; Da Ros, L.; Zagatti, B.; Musa, G.; Bassi, C.; Mangolini, A.; Cavallesco, G.; Frassoldati, A.; Volpato, S.; Carcoforo, P.; Hollingsworth, A. B.; Negrini, M. Absolute Quantification of Cell-Free MicroRNAs in Cancer Patients. Oncotarget 2015, 6 (16), 14545– 14555,  DOI: 10.18632/oncotarget.3859
    Google Scholar
    There is no corresponding record for this reference.
  40. 40
    Mitchell, P. S.; Parkin, R. K.; Kroh, E. M.; Fritz, B. R.; Wyman, S. K.; Pogosova-Agadjanyan, E. L.; Peterson, A.; Noteboom, J.; O’Briant, K. C.; Allen, A.; Lin, D. W.; Urban, N.; Drescher, C. W.; Knudsen, B. S.; Stirewalt, D. L.; Gentleman, R.; Vessella, R. L.; Nelson, P. S.; Martin, D. B.; Tewari, M. Circulating MicroRNAs as Stable Blood-Based Markers for Cancer Detection. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (30), 10513– 10518,  DOI: 10.1073/pnas.0804549105
    Google Scholar
    40
    Circulating microRNAs as stable blood-based markers for cancer detection
    Mitchell, Patrick S.; Parkin, Rachael K.; Kroh, Evan M.; Fritz, Brian R.; Wyman, Stacia K.; Pogosova-Agadjanyan, Era L.; Peterson, Amelia; Noteboom, Jennifer; O'Briant, Kathy C.; Allen, April; Lin, Daniel W.; Urban, Nicole; Drescher, Charles W.; Knudsen, Beatrice S.; Stirewalt, Derek L.; Gentleman, Robert; Vessella, Robert L.; Nelson, Peter S.; Martin, Daniel B.; Tewari, Muneesh
    Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (30), 10513-10518CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small (≈22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. The authors show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. MiRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. These results establish the measurement of tumor-derived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXpsVCltL0%253D&md5=c802fd49d4fae2e536c03abfd7a34996
  41. 41
    Farka, Z.; Mickert, M. J.; Hlaváček, A.; Skládal, P.; Gorris, H. H. Single Molecule Upconversion-Linked Immunosorbent Assay with Extended Dynamic Range for the Sensitive Detection of Diagnostic Biomarkers. Anal. Chem. 2017, 89 (21), 11825– 11830,  DOI: 10.1021/acs.analchem.7b03542
    Google Scholar
    41
    Single Molecule Upconversion-Linked Immunosorbent Assay with Extended Dynamic Range for the Sensitive Detection of Diagnostic Biomarkers
    Farka, Zdenek; Mickert, Matthias J.; Hlavacek, Antonin; Skladal, Petr; Gorris, Hans H.
    Analytical Chemistry (Washington, DC, United States) (2017), 89 (21), 11825-11830CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    The ability to detect disease markers at the single mol. level promises the ultimate sensitivity in clin. diagnosis. Fluorescence-based single-mol. anal., however, is limited by matrix interference and can only probe a very small detection vol., which is typically not suitable for real world anal. applications. The authors have developed a microtiter plate immunoassay for counting single mols. of the cancer marker prostate specific antigen (PSA) using photon-upconversion nanoparticles (UCNPs) as labels that can be detected without background fluorescence. Individual sandwich immunocomplexes consisting of (1) an anti-PSA antibody immobilized to the surface of a microtiter well, (2) PSA, and (3) an anti-PSA antibody-UCNP conjugate were counted under a wide-field epifluorescence microscope equipped with a 980 nm laser excitation source. The single-mol. (digital) upconversion-linked immunosorbent assay (ULISA) reaches a limit of detection of 1.2 pg mL-1 (42 fM) PSA in 25% blood serum, which is about ten times more sensitive than com. ELISAs and covers a dynamic range of three orders of magnitude. This upconversion detection mode has the potential to pave the way for a new generation of digital immunoassays.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFOjtrfP&md5=d6c0a2d5ab2eb655077152c109e0e027
  42. 42
    Christopoulos, T. K.; Lianidou, E. S.; Diamandis, E. P. Ultrasensitive Time-Resolved Fluorescence Method for α-Fetoprotein. Clin. Chem. 1990, 36 (8), 1497– 1502,  DOI: 10.1093/clinchem/36.8.1497
    Google Scholar
    There is no corresponding record for this reference.
  43. 43
    Kuusinen, S.; Lahtinen, S.; Soukka, T. Upconversion Luminescence Based Direct Hybridization Assay to Detect Subfemtomolar MiR-20 a DNA Analogue in Plasma. Anal. Sens. 2024, 4 (4), e202400005  DOI: 10.1002/anse.202400005
    Google Scholar
    There is no corresponding record for this reference.
  44. 44
    Watanabe, T.; Hashida, S. The Immune Complex Transfer Enzyme Immunoassay: Mechanism of Improved Sensitivity Compared with Conventional Sandwich Enzyme Immunoassay. J. Immunol. Methods 2018, 459, 76– 80,  DOI: 10.1016/j.jim.2018.05.010
    Google Scholar
    There is no corresponding record for this reference.
  45. 45
    Morrissey, D. V.; Lombardo, M.; Eldredge, J. K.; Kearney, K. R.; Groody, E. P.; Collins, M. L. Nucleic Acid Hybridization Assays Employing DA-Tailed Capture Probes. Anal. Biochem. 1989, 181 (2), 345– 359,  DOI: 10.1016/0003-2697(89)90255-8
    Google Scholar
    There is no corresponding record for this reference.
  46. 46
    Masterson, A. N.; Chowdhury, N. N.; Fang, Y.; Yip-Schneider, M. T.; Hati, S.; Gupta, P.; Cao, S.; Wu, H.; Schmidt, C. M.; Fishel, M. L.; Sardar, R. Amplification-Free, High-Throughput Nanoplasmonic Quantification of Circulating MicroRNAs in Unprocessed Plasma Microsamples for Earlier Pancreatic Cancer Detection. ACS Sens. 2023, 8 (3), 1085– 1100,  DOI: 10.1021/acssensors.2c02105
    Google Scholar
    There is no corresponding record for this reference.
  47. 47
    Ramshani, Z.; Zhang, C.; Richards, K.; Chen, L.; Xu, G.; Stiles, B. L.; Hill, R.; Senapati, S.; Go, D. B.; Chang, H.-C. Extracellular Vesicle MicroRNA Quantification from Plasma Using an Integrated Microfluidic Device. Commun. Biol. 2019, 2 (1), 189,  DOI: 10.1038/s42003-019-0435-1
    Google Scholar
    47
    Extracellular vesicle microRNA quantification from plasma using an integrated microfluidic device
    Ramshani Zeinab; Zhang Chenguang; Senapati Satyajyoti; Go David B; Chang Hsueh-Chia; Ramshani Zeinab; Zhang Chenguang; Senapati Satyajyoti; Chang Hsueh-Chia; Ramshani Zeinab; Richards Katherine; Senapati Satyajyoti; Chang Hsueh-Chia; Richards Katherine; Chen Lulu; Stiles Bangyan L; Xu Geyang; Hill Reginald; Hill Reginald; Go David B; Chang Hsueh-Chia
    Communications biology (2019), 2 (), 189 ISSN:.
    Extracellular vesicles (EV) containing microRNAs (miRNAs) have tremendous potential as biomarkers for the early detection of disease. Here, we present a simple and rapid PCR-free integrated microfluidics platform capable of absolute quantification (<10% uncertainty) of both free-floating miRNAs and EV-miRNAs in plasma with 1 pM detection sensitivity. The assay time is only 30 minutes as opposed to 13 h and requires only ~20 μL of sample as oppose to 1 mL for conventional RT-qPCR techniques. The platform integrates a surface acoustic wave (SAW) EV lysing microfluidic chip with a concentration and sensing microfluidic chip incorporating an electrokinetic membrane sensor that is based on non-equilibrium ionic currents. Unlike conventional RT-qPCR methods, this technology does not require EV extraction, RNA purification, reverse transcription, or amplification. This platform can be easily extended for other RNA and DNA targets of interest, thus providing a viable screening tool for early disease diagnosis, prognosis, and monitoring of therapeutic response.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3M7pt1CksA%253D%253D&md5=2b1721ce83d533ad89c98b76e55bf780
  48. 48
    Majd, S. M.; Salimi, A.; Ghasemi, F. An Ultrasensitive Detection of MiRNA-155 in Breast Cancer via Direct Hybridization Assay Using Two-Dimensional Molybdenum Disulfide Field-Effect Transistor Biosensor. Biosens. Bioelectron. 2018, 105, 6– 13,  DOI: 10.1016/j.bios.2018.01.009
    Google Scholar
    48
    An ultrasensitive detection of miRNA-155 in breast cancer via direct hybridization assay using two-dimensional molybdenum disulfide field-effect transistor biosensor
    Majd, Samira Mansouri; Salimi, Abdollah; Ghasemi, Foad
    Biosensors & Bioelectronics (2018), 105 (), 6-13CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
    MicroRNAs (miRNAs), crit. biomarkers of acute and chronic diseases, play key regulatory roles in many biol. processes. As a result, robust assay platforms to enable an accurate and efficient detection of low-level miRNAs in complex biol. samples are of great significance. In this work, a label-free and direct hybridization assay using molybdenum disulfide (MoS2) field-effect transistor (FET) biosensor has been developed for ultrasensitive detection of miRNA-155 as a breast cancer biomarker in human serum and cell-line samples. MoS2, the novel 2D layered material with excellent phys. and chem. properties, was prepd. through sequential solvent exchange method and was used as an active channel material. MoS2 was comprehensively characterized by spectroscopic and microscopic methods and it was applied for fabrication of FET device by drop-casting MoS2 flacks suspension onto the FET surface. MoS2 FET device showed a relatively low subthreshold swing of 48.10 mV/decade and a high mobility of 1.98 × 103 cm2 V-1 s-1. Subsequently, probe miRNA-155 strands were immobilized on the surface of the MoS2 FET device. Under optimized conditions detection limit of 0.03 fM and concn. range 0.1 fM to 10 nM were achieved. The developed biosensor not only was capable to identification of fully matched vs. one-base mismatch miRNA-155 sequence, but also it could detect target miRNA-155 in spiked real human serum and exts. from human breast cancer cell-line samples. This approach paves a way for label-free, early detection of miRNA as a biomarker in cancer diagnostics with very high sensitivity and good specificity, thus offering a significant potential for clin. application.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXnvVKqtw%253D%253D&md5=ecd8c227ae2e8e140ff1f414e92b58bc
  49. 49
    Wegman, D. W.; Ghasemi, F.; Khorshidi, A.; Yang, B. B.; Liu, S. K.; Yousef, G. M.; Krylov, S. N. Highly-Sensitive Amplification-Free Analysis of Multiple MiRNAs by Capillary Electrophoresis. Anal. Chem. 2015, 87 (2), 1404– 1410,  DOI: 10.1021/ac504406s
    Google Scholar
    There is no corresponding record for this reference.

Cited By

Click to copy section linkSection link copied!

This article has not yet been cited by other publications.

Download PDF
Go to Analytical Chemistry
Get e-Alerts
Get e-Alerts

Analytical Chemistry

Cite this: Anal. Chem. 2025, 97, 3, 1775–1782
Click to copy citationCitation copied!
https://doi.org/10.1021/acs.analchem.4c05401
Published January 12, 2025

Copyright © 2025 The Authors. Published by American Chemical Society. This publication is licensed under

CC-BY-NC-ND 4.0 .

Article Views

890

Altmetric

-

Citations

-
Learn about these metrics

Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.

Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.

The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.

  • Abstract

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 1

    Figure 1. Principle of the hybridization complex transfer assay utilizing MBs as the first capture surface. (I) The sample is mixed with the UCNP–DNA probe conjugates to form target–UCNP complexes. (II) The complexes are captured with DNA probes immobilized on the surface of MBs. (III) The beads are washed, and releasing oligonucleotides are added. Releasing oligonucleotides hybridize with the capture probes, displacing the target via toehold-mediated strand displacement. (IV) Target–UCNP complexes are released into the solution, and (V) the solution is transferred into microtiter plate wells coated with another capture probe. (VI) The target–UCNP complexes are collected with the capture probes, the wells are washed and dried, and UCL is measured. In the reference assay (R), the complexes formed in the step (I) are directly captured onto wells coated with the second capture probes.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 2

    Figure 2. Standard curves of the complex transfer assay (red, circles) and the reference assay without complex transfer (blue, squares). Both captures of the complex transfer assay were carried out in an MTP. Dashed lines indicate the LODs, and the dotted line shows the instrument background. Error bars represent the standard deviations of three replicates (or six replicates in case of zero calibrators).

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 3

    Figure 3. Standard curves of the complex transfer assay for the detection of DNA-miR-20a utilizing the MBs as the first capture surface (red circles) and the reference assay with direct capture on the second capture surface (blue squares) by using (A) 120 μL of sample volume per reaction with the assay buffer as the sample matrix, (B) 120 μL sample volume per reaction with EDTA plasma pool as a sample matrix, and (C) preconcentration from 600 μL of sample volume per reaction with the assay buffer as the sample matrix. Dashed lines indicate the LODs, and dotted lines show the instrument background. Error bars represent the standard deviations of three replicates (or eight replicates in case of zero calibrators).

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 4

    Figure 4. Cross-reactivity of the complex transfer assay (red) and the reference assay (blue) with sequences containing minor target sequence variations (0.5 pM; complete sequences are provided in Table S1). The luminescence signal responses are presented as percentage of the signal of the complementary target (DNA-miR-20a). The error bars represent the standard deviations of three replicate wells.

    High Resolution Image
    Download MS PowerPoint Slide

  • References

    This article references 49 other publications.

    1. 1
      Li, M.; Yin, F.; Song, L.; Mao, X.; Li, F.; Fan, C.; Zuo, X.; Xia, Q. Nucleic Acid Tests for Clinical Translation. Chem. Rev. 2021, 121 (17), 10469– 10558,  DOI: 10.1021/acs.chemrev.1c00241
      1
      Nucleic Acid Tests for Clinical Translation
      Li, Min; Yin, Fangfei; Song, Lu; Mao, Xiuhai; Li, Fan; Fan, Chunhai; Zuo, Xiaolei; Xia, Qiang
      Chemical Reviews (Washington, DC, United States) (2021), 121 (17), 10469-10558CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. Nucleic acids, including DNA (DNA) and RNA (RNA), are natural biopolymers composed of nucleotides that store, transmit, and express genetic information. Overexpressed or underexpressed as well as mutated nucleic acids have been implicated in many diseases. Therefore, nucleic acid tests (NATs) are extremely important. Inspired by intracellular DNA replication and RNA transcription, in vitro NATs have been extensively developed to improve the detection specificity, sensitivity, and simplicity. The principles of NATs can be in general classified into three categories: nucleic acid hybridization, thermal-cycle or isothermal amplification, and signal amplification. Driven by pressing needs in clin. diagnosis and prevention of infectious diseases, NATs have evolved to be a rapidly advancing field. During the past ten years, an explosive increase of research interest in both basic research and clin. translation has been witnessed. In this review, we aim to provide comprehensive coverage of the progress to analyze nucleic acids, use nucleic acids as recognition probes, construct detection devices based on nucleic acids, and utilize nucleic acids in clin. diagnosis and other important fields. We also discuss the new frontiers in the field and the challenges to be addressed.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsFaku7zF&md5=8f3ce9510fb68c111ff7c95f432979e6
    2. 2
      Valihrach, L.; Androvic, P.; Kubista, M. Circulating MiRNA Analysis for Cancer Diagnostics and Therapy. Mol. Aspects Med. 2020, 72, 100825,  DOI: 10.1016/j.mam.2019.10.002
      There is no corresponding record for this reference.
    3. 3
      Qian, S.; Chen, Y.; Xu, X.; Peng, C.; Wang, X.; Wu, H.; Liu, Y.; Zhong, X.; Xu, J.; Wu, J. Advances in Amplification-Free Detection of Nucleic Acid: CRISPR/Cas System as a Powerful Tool. Anal. Biochem. 2022, 643, 114593,  DOI: 10.1016/j.ab.2022.114593
      3
      Advances in amplification-free detection of nucleic acid: CRISPR/Cas system as a powerful tool
      Qian, Siwenjie; Chen, Yanju; Xu, Xiaoli; Peng, Cheng; Wang, Xiaofu; Wu, Hui; Liu, Yang; Zhong, Xiaoping; Xu, Junfeng; Wu, Jian
      Analytical Biochemistry (2022), 643 (), 114593CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)
      Amplification technologies such as polymerase chain reaction (PCR) play an important role in nucleic acid detection. However, they require bulky and sophisticated thermal cycling instrument, as well as are prone to get false-pos. results due to amplicon contamination. Currently, CRISPR/Cas system has become an increasingly popular diagnostic tool for nucleic acid with the discovery of its trans-cleavage activity which can degrade single-stranded DNA or RNA at a very high turnover rate. This inherent signal amplification capability allows CRISPR/Cas system to detect unamplified nucleic acids. Here, we reviewed the recent advances of CRISPR-based amplification-free methods for nucleic acid detection. With the assistance of various signal enhancement strategies, the detection sensitivity could be comparable to that of amplification-based methods. We then presented the pros and cons of these methods. And the subsistent challenges including sample prepn., off-target effect, sequences limit, quant. and multiplex detection were further discussed in this review. It is probable for CRISPR-powered detection methods to pave the road for rapid, cheap, highly sensitive and specific on-site detection without amplification.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjslSht7s%253D&md5=64bee4939693c4f1f70399445975d0e3
    4. 4
      Kim, D.-J.; Linnstaedt, S.; Palma, J.; Park, J. C.; Ntrivalas, E.; Kwak-Kim, J. Y. H.; Gilman-Sachs, A.; Beaman, K.; Hastings, M. L.; Martin, J. N.; Duelli, D. M. Plasma Components Affect Accuracy of Circulating Cancer-Related MicroRNA Quantitation. J. Mol. Diagn. 2012, 14 (1), 71– 80,  DOI: 10.1016/j.jmoldx.2011.09.002
      4
      Plasma components affect accuracy of circulating cancer-related microRNA quantitation
      Kim, Dong-Ja; Linnstaedt, Sarah; Palma, Jaime; Park, Joon Cheol; Ntrivalas, Evangelos; Kwak-Kim, Joanne Y. H.; Gilman-Sachs, Alice; Beaman, Kenneth; Hastings, Michelle L.; Martin, Jeffrey N.; Duelli, Dominik M.
      Journal of Molecular Diagnostics (2012), 14 (1), 71-80CODEN: JMDIFP; ISSN:1525-1578. (Elsevier)
      Circulating microRNAs (miRNAs) have emerged as candidate biomarkers of various diseases and conditions including malignancy and pregnancy. This approach requires sensitive and accurate quantitation of miRNA concns. in body fluids. Herein we report that enzyme-based miRNA quantitation, which is currently the mainstream approach for identifying differences in miRNA abundance among samples, is skewed by endogenous serum factors that co-purify with miRNAs and anticoagulant agents used during collection. Of importance, different miRNAs were affected to varying extent among patient samples. By developing measures to overcome these interfering activities, we increased the accuracy, and improved the sensitivity of miRNA detection up to 30-fold. Overall, the present study outlines key factors that prevent accurate miRNA quantitation in body fluids and provides approaches that enable faithful quantitation of miRNA abundance in body fluids.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVCrtb0%253D&md5=176c4ffc5f7ef4052523ae2b413b6fe9
    5. 5
      Koshiol, J.; Wang, E.; Zhao, Y.; Marincola, F.; Landi, M. T. Strengths and Limitations of Laboratory Procedures for MicroRNA Detection. Cancer Epidemiol. Biomarkers Prev. 2010, 19 (4), 907– 911,  DOI: 10.1158/1055-9965.EPI-10-0071
      There is no corresponding record for this reference.
    6. 6
      Li, W.; Ruan, K. MicroRNA Detection by Microarray. Anal. Bioanal. Chem. 2009, 394 (4), 1117– 1124,  DOI: 10.1007/s00216-008-2570-2
      6
      MicroRNA detection by microarray
      Li, Wei; Ruan, Kangcheng
      Analytical and Bioanalytical Chemistry (2009), 394 (4), 1117-1124CODEN: ABCNBP; ISSN:1618-2642. (Springer)
      A review. MicroRNAs (miRNAs) are a class of small noncoding RNAs ∼22 nt in length that regulate gene expression and play fundamental roles in multiple biol. processes, including cell differentiation, proliferation and apoptosis as well as disease processes. The study of miRNA has thus become a rapidly emerging field in life science. The detection of miRNA expression is a very important first step in miRNA exploration. Several methodologies, including cloning, northern blotting, real-time RT-PCR, microRNA arrays and ISH (in situ hybridization), have been developed and applied successfully in miRNA profiling. This review discusses the main existing microRNA detection technologies, while emphasizing microRNA arrays.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXktVamsw%253D%253D&md5=e8b0d1482009f44537cb50a763d20024
    7. 7
      Koscianska, E.; Starega-Roslan, J.; Sznajder, L. J.; Olejniczak, M.; Galka-Marciniak, P.; Krzyzosiak, W. J. Northern Blotting Analysis of MicroRNAs, Their Precursors and RNA Interference Triggers. BMC Mol. Biol. 2011, 12 (1), 14,  DOI: 10.1186/1471-2199-12-14
      7
      Northern blotting analysis of microRNAs, their precursors and RNA interference triggers
      Koscianska, Edyta; Starega-Roslan, Julia; Sznajder, Lukasz J.; Olejniczak, Marta; Galka-Marciniak, Paulina; Krzyzosiak, Wlodzimierz J.
      BMC Molecular Biology (2011), 12 (), 14CODEN: BMBMC4; ISSN:1471-2199. (BioMed Central Ltd.)
      Background: Numerous microRNAs (miRNAs) have heterogeneous ends resulting from imprecise cleavages by processing nucleases and from various non-templated nucleotide addns. The scale of miRNA end-heterogeneity is best shown by deep sequencing data revealing not only the major miRNA variants but also those that occur in only minute amts. and are unlikely to be of functional importance. All RNA interference (RNAi) technol. reagents that are expressed and processed in cells are also exposed to the same machinery generating end-heterogeneity of the released short interfering RNAs (siRNAs) or miRNA mimetics. Results: In this study we have analyzed endogenous and exogenous RNAs in the range of 20-70 nt by high-resoln. northern blotting. We have validated the results obtained with northern blotting by comparing them with data derived from miRNA deep sequencing; therefore we have demonstrated the usefulness of the northern blotting technique in the investigation of miRNA biogenesis, as well as in the characterization of RNAi technol. reagents. Conclusions: The conventional northern blotting enhanced to high resoln. may be a useful adjunct to other miRNA discovery, detection and characterization methods. It provides quant. data on distribution of major length variants of abundant endogenous miRNAs, as well as on length heterogeneity of RNAi technol. reagents expressed in cells.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXltVWqs7c%253D&md5=818f990c182f607d854b33c2793beb2b
    8. 8
      Liang, G.; Wang, H.; Shi, H.; Wang, H.; Zhu, M.; Jing, A.; Li, J.; Li, G. Recent Progress in the Development of Upconversion Nanomaterials in Bioimaging and Disease Treatment. J. Nanobiotechnol. 2020, 18 (1), 154,  DOI: 10.1186/s12951-020-00713-3
      8
      Recent progress in the development of upconversion nanomaterials in bioimaging and disease treatment
      Liang Gaofeng; Wang Haojie; Zhu Mengxi; Shi Hao; Jing Aihua; Li Jinghua; Li Guangda; Wang Haitao
      Journal of nanobiotechnology (2020), 18 (1), 154 ISSN:.
      Multifunctional lanthanide-based upconversion nanoparticles (UCNPs), which feature efficiently convert low-energy photons into high-energy photons, have attracted considerable attention in the domain of materials science and biomedical applications. Due to their unique photophysical properties, including light-emitting stability, excellent upconversion luminescence efficiency, low autofluorescence, and high detection sensitivity, and high penetration depth in samples, UCNPs have been widely applied in biomedical applications, such as biosensing, imaging and theranostics. In this review, we briefly introduced the major components of UCNPs and the luminescence mechanism. Then, we compared several common design synthesis strategies and presented their advantages and disadvantages. Several examples of the functionalization of UCNPs were given. Next, we detailed their biological applications in bioimaging and disease treatment, particularly drug delivery and photodynamic therapy, including antibacterial photodynamic therapy. Finally, the future practical applications in materials science and biomedical fields, as well as the remaining challenges to UCNPs application, were described. This review provides useful practical information and insights for the research on and application of UCNPs in the field of cancer.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3s7nvFehug%253D%253D&md5=74d17e757c999be9fa5661306657845d
    9. 9
      Wen, S.; Zhou, J.; Zheng, K.; Bednarkiewicz, A.; Liu, X.; Jin, D. Advances in Highly Doped Upconversion Nanoparticles. Nat. Commun. 2018, 9 (1), 2415,  DOI: 10.1038/s41467-018-04813-5
      9
      Advances in highly doped upconversion nanoparticles
      Wen Shihui; Zhou Jiajia; Jin Dayong; Zheng Kezhi; Liu Xiaogang; Bednarkiewicz Artur; Bednarkiewicz Artur
      Nature communications (2018), 9 (1), 2415 ISSN:.
      Lanthanide-doped upconversion nanoparticles (UCNPs) are capable of converting near-infra-red excitation into visible and ultraviolet emission. Their unique optical properties have advanced a broad range of applications, such as fluorescent microscopy, deep-tissue bioimaging, nanomedicine, optogenetics, security labelling and volumetric display. However, the constraint of concentration quenching on upconversion luminescence has hampered the nanoscience community to develop bright UCNPs with a large number of dopants. This review surveys recent advances in developing highly doped UCNPs, highlights the strategies that bypass the concentration quenching effect, and discusses new optical properties as well as emerging applications enabled by these nanoparticles.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1Mbps1Cluw%253D%253D&md5=615b19adc979238a82e23c423ae5af05
    10. 10
      Hlaváček, A.; Farka, Z.; Mickert, M. J.; Kostiv, U.; Brandmeier, J. C.; Horák, D.; Skládal, P.; Foret, F.; Gorris, H. H. Bioconjugates of Photon-Upconversion Nanoparticles for Cancer Biomarker Detection and Imaging. Nat. Protoc. 2022, 17 (4), 1028– 1072,  DOI: 10.1038/s41596-021-00670-7
      10
      Bioconjugates of photon-upconversion nanoparticles for cancer biomarker detection and imaging
      Hlavacek, Antonin; Farka, Zdenek; Mickert, Matthias J.; Kostiv, Uliana; Brandmeier, Julian C.; Horak, Daniel; Skladal, Petr; Foret, Frantisek; Gorris, Hans H.
      Nature Protocols (2022), 17 (4), 1028-1072CODEN: NPARDW; ISSN:1750-2799. (Nature Portfolio)
      The detection of cancer biomarkers in histol. samples and blood is of paramount importance for clin. diagnosis. Current methods are limited in terms of sensitivity, hindering early detection of disease. We have overcome the shortcomings of currently available staining and fluorescence labeling methods by taking an integrative approach to establish photon-upconversion nanoparticles (UCNP) as a powerful platform for cancer detection. These nanoparticles are readily synthesized in different sizes to yield efficient and tunable short-wavelength light emission under near-IR excitation, which eliminates optical background interference of the specimen. Here we present a protocol for the synthesis of UCNPs by high-temp. co-pptn. or seed-mediated growth by thermal decompn., surface modification by silica or poly(ethylene glycol) that renders the particles resistant to nonspecific binding, and the conjugation of streptavidin or antibodies for biol. detection. To detect blood-based biomarkers, we present an upconversion-linked immunosorbent assay for the analog and digital detection of the cancer marker prostate-specific antigen. When applied to immunocytochem. anal., UCNPs enable the detection of the breast cancer marker human epidermal growth factor receptor 2 with a signal-to-background ratio 50-fold higher than conventional fluorescent labels. UCNP synthesis takes 4.5 d, the prepn. of the antibody-silica-UCNP conjugate takes 3 d, the streptavidin-poly(ethylene glycol)-UCNP conjugate takes 2-3 wk, upconversion-linked immunosorbent assay takes 2-4 d and immunocytochem. takes 8-10 h. The procedures can be performed after std. lab. training in nanomaterials research.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xktlyrt7k%253D&md5=f624bb3e5e2b1fef2b6f41aae0773c20
    11. 11
      Wu, S.; Han, G.; Milliron, D. J.; Aloni, S.; Altoe, V.; Talapin, D. V.; Cohen, B. E.; Schuck, P. J. Non-Blinking and Photostable Upconverted Luminescence from Single Lanthanide-Doped Nanocrystals. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (27), 10917– 10921,  DOI: 10.1073/pnas.0904792106
      11
      Non-blinking and photostable up-converted luminescence from single lanthanide-doped nanocrystals
      Wu, Shiwei; Han, Gang; Milliron, Delia J.; Aloni, Shaul; Altoe, Virginia; Talapin, Dmitri V.; Cohen, Bruce E.; Schuck, P. James
      Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (27), 10917-10921, S10917/1-S10917/9CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The development of probes for single-mol. imaging has dramatically facilitated the study of individual mols. in cells and other complex environments. Single-mol. probes ideally exhibit good brightness, uninterrupted emission, resistance to photobleaching, and minimal spectral overlap with cellular autofluorescence. However, most single-mol. probes are imperfect in several of these aspects, and none have been shown to possess all of these characteristics. Here the authors show that individual lanthanide-doped up-converting nanoparticles (UCNPs) - specifically, hexagonal phase NaYF4 (β-NaYF4) nanocrystals with multiple Yb3+ and Er3+ dopants - emit bright anti-Stokes visible up-converted luminescence with exceptional photostability when excited by a 980-nm continuous wave laser. Individual UCNPs exhibit no on/off emission behavior, or "blinking," down to the millisecond time-scale, and no loss of intensity following an hour of continuous excitation. Amphiphilic polymer coatings permit the transfer of hydrophobic UCNPs into water, resulting in individual water-sol. nanoparticles with undiminished photophys. characteristics. These UCNPs are endocytosed by cells and show strong up-converted luminescence, with no measurable anti-Stokes background autofluorescence, suggesting that UCNPs are ideally suited for single-mol. imaging expts.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXptVSntLc%253D&md5=d7e1f8475c88639148ffa7626c0825e3
    12. 12
      Guan, L.; Peng, J.; Liu, T.; Huang, S.; Yang, Y.; Wang, X.; Hao, X. Ultrasensitive MiRNA Detection Based on Magnetic Upconversion Nanoparticle Enhancement and CRISPR/Cas13a-Driven Signal Amplification. Anal. Chem. 2023, 95 (48), 17708– 17715,  DOI: 10.1021/acs.analchem.3c03554
      There is no corresponding record for this reference.
    13. 13
      Wu, X.; Li, Y.; Yang, M. Y.; Mao, C. B. Simultaneous Ultrasensitive Detection of Two Breast Cancer MicroRNA Biomarkers by Using a Dual Nanoparticle/Nanosheet Fluorescence Resonance Energy Transfer Sensor. Mater. Today Adv. 2021, 12, 100163,  DOI: 10.1016/j.mtadv.2021.100163
      There is no corresponding record for this reference.
    14. 14
      Chen, C.; Hu, S.; Tian, L.; Qi, M.; Chang, Z.; Li, L.; Wang, L.; Dong, B. A Versatile Upconversion-Based Multimode Lateral Flow Platform for Rapid and Ultrasensitive Detection of MicroRNA towards Health Monitoring. Biosens. Bioelectron. 2024, 252, 116135,  DOI: 10.1016/j.bios.2024.116135
      There is no corresponding record for this reference.
    15. 15
      Wang, J.; Hua, G.; Li, L.; Li, D.; Wang, F.; Wu, J.; Ye, Z.; Zhou, X.; Ye, S.; Yang, J.; Zhang, X.; Ren, L. Upconversion Nanoparticle and Gold Nanocage Satellite Assemblies for Sensitive CtDNA Detection in Serum. Analyst 2020, 145 (16), 5553– 5562,  DOI: 10.1039/D0AN00701C
      There is no corresponding record for this reference.
    16. 16
      Güven, E.; Duus, K.; Lydolph, M. C.; Jørgensen, C. S.; Laursen, I.; Houen, G. Non-Specific Binding in Solid Phase Immunoassays for Autoantibodies Correlates with Inflammation Markers. J. Immunol. Methods 2014, 403 (1–2), 26– 36,  DOI: 10.1016/j.jim.2013.11.014
      There is no corresponding record for this reference.
    17. 17
      Lahtinen, S.; Lyytikäinen, A.; Sirkka, N.; Päkkilä, H.; Soukka, T. Improving the Sensitivity of Immunoassays by Reducing Non-Specific Binding of Poly(Acrylic Acid) Coated Upconverting Nanoparticles by Adding Free Poly(Acrylic Acid). Microchim. Acta 2018, 185 (4), 220,  DOI: 10.1007/s00604-018-2756-z
      There is no corresponding record for this reference.
    18. 18
      Hariri, A. A.; Newman, S. S.; Tan, S.; Mamerow, D.; Adams, A. M.; Maganzini, N.; Zhong, B. L.; Eisenstein, M.; Dunn, A. R.; Soh, H. T. Improved Immunoassay Sensitivity and Specificity Using Single-Molecule Colocalization. Nat. Commun. 2022, 13 (1), 5359,  DOI: 10.1038/s41467-022-32796-x
      18
      Improved immunoassay sensitivity and specificity using single-molecule colocalization
      Hariri, Amani A.; Newman, Sharon S.; Tan, Steven; Mamerow, Dan; Adams, Alexandra M.; Maganzini, Nicolo; Zhong, Brian L.; Eisenstein, Michael; Dunn, Alexander R.; Soh, H. Tom
      Nature Communications (2022), 13 (1), 5359CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)
      Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern mol. detection, but the technique still faces notable challenges. One of the biggest problems is discriminating true signal generated by target mols. vs. non-specific background. Here, we developed a Single-Mol. Colocalization Assay (SiMCA) that overcomes this problem by employing total internal reflection fluorescence microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized signals, we can eliminate the effects of background produced by non-specific binding of detection antibodies. Using TNF-α, we show that SiMCA achieves a three-fold lower limit of detection compared to conventional single-color assays and exhibits consistent performance for assays performed in complex specimens such as serum and blood. Our results help define the pernicious effects of non-specific background in immunoassays and demonstrate the diagnostic gains that can be achieved by eliminating those effects.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XitlKrsLzJ&md5=6619c42f5f7dfd10214116d5e36113db
    19. 19
      Buchwalow, I.; Samoilova, V.; Boecker, W.; Tiemann, M. Non-Specific Binding of Antibodies in Immunohistochemistry: Fallacies and Facts. Sci. Rep. 2011, 1 (1), 28,  DOI: 10.1038/srep00028
      There is no corresponding record for this reference.
    20. 20
      Wauthier, L.; Plebani, M.; Favresse, J. Interferences in Immunoassays: Review and Practical Algorithm. Clin. Chem. Lab. Med. 2022, 60 (6), 808– 820,  DOI: 10.1515/cclm-2021-1288
      There is no corresponding record for this reference.
    21. 21
      Mendez-Gonzalez, D.; Lahtinen, S.; Laurenti, M.; López-Cabarcos, E.; Rubio-Retama, J.; Soukka, T. Photochemical Ligation to Ultrasensitive DNA Detection with Upconverting Nanoparticles. Anal. Chem. 2018, 90 (22), 13385– 13392,  DOI: 10.1021/acs.analchem.8b03106
      21
      Photochemical Ligation to Ultrasensitive DNA Detection with Upconverting Nanoparticles
      Mendez-Gonzalez, Diego; Lahtinen, Satu; Laurenti, Marco; Lopez-Cabarcos, Enrique; Rubio-Retama, Jorge; Soukka, Tero
      Analytical Chemistry (Washington, DC, United States) (2018), 90 (22), 13385-13392CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      In this work, we explore a photochem. ligation reaction to covalently modify oligonucleotide-conjugated upconverting nanoparticles (UCNPs) in the presence of a specific target DNA sequence. The target sequence acts as a hybridization template, bringing together a biotinylated photoactivatable oligonucleotide probe and the oligonucleotide probe that is attached to UCNPs. The illumination of the UCNPs by NIR light to generate UV emission internally or illuminating the photoactivatable probe directly by an external UV light promotes the photochem. ligation reaction, yielding covalently biotin functionalized UCNPs that can be selectively captured in streptavidin-coated microwells. Following this strategy, we developed a DNA sensor with a limit of detection of 1 × 10-18 mol per well (20 fM). In addn., we demonstrate the possibility to create UCNP patterns on the surface of solid supports upon NIR illumination that are selectively formed under the presence of the target oligonucleotide.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhvFKnu7vE&md5=18b56c80456a7e0c1de9caaf95fc830d
    22. 22
      Baltierra-Jasso, L. E.; Morten, M. J.; Laflör, L.; Quinn, S. D.; Magennis, S. W. Crowding-Induced Hybridization of Single DNA Hairpins. J. Am. Chem. Soc. 2015, 137 (51), 16020– 16023,  DOI: 10.1021/jacs.5b11829
      22
      Crowding-Induced Hybridization of Single DNA Hairpins
      Baltierra-Jasso, Laura E.; Morten, Michael J.; Laflor, Linda; Quinn, Steven D.; Magennis, Steven W.
      Journal of the American Chemical Society (2015), 137 (51), 16020-16023CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      It is clear that a crowded environment influences the structure, dynamics, and interactions of biol. mols., but the complexity of this phenomenon demands the development of new exptl. and theor. approaches. Here we use two complementary single-mol. FRET techniques to show that the kinetics of DNA base pairing and unpairing, which are fundamental to both the biol. role of DNA and its technol. applications, are strongly modulated by a crowded environment. We directly obsd. single DNA hairpins, which are excellent model systems for studying hybridization, either freely diffusing in soln. or immobilized on a surface under crowding conditions. The hairpins followed two-state folding dynamics with a closing rate increasing by 4-fold and the opening rate decreasing 2-fold, for only modest concns. of crowder [10% (wt./wt.) polyethylene glycol (PEG)]. These expts. serve both to unambiguously highlight the impact of a crowded environment on a fundamental biol. process, DNA base pairing, and to illustrate the benefits of single-mol. approaches to probing the structure and dynamics of complex biomol. systems.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvF2ltL7N&md5=41090b083fa05da3dffcf7e608be31b4
    23. 23
      Brandmeier, J. C.; Raiko, K.; Farka, Z.; Peltomaa, R.; Mickert, M. J.; Hlaváček, A.; Skládal, P.; Soukka, T.; Gorris, H. H. Effect of Particle Size and Surface Chemistry of Photon-Upconversion Nanoparticles on Analog and Digital Immunoassays for Cardiac Troponin. Adv. Healthcare Mater. 2021, 10 (18), 2100506,  DOI: 10.1002/adhm.202100506
      There is no corresponding record for this reference.
    24. 24
      Chang, L.; Rissin, D. M.; Fournier, D. R.; Piech, T.; Patel, P. P.; Wilson, D. H.; Duffy, D. C. Single Molecule Enzyme-Linked Immunosorbent Assays: Theoretical Considerations. J. Immunol. Methods 2012, 378 (1–2), 102– 115,  DOI: 10.1016/j.jim.2012.02.011
      24
      Single molecule enzyme-linked immunosorbent assays: Theoretical considerations
      Chang, Lei; Rissin, David M.; Fournier, David R.; Piech, Tomasz; Patel, Purvish P.; Wilson, David H.; Duffy, David C.
      Journal of Immunological Methods (2012), 378 (1-2), 102-115CODEN: JIMMBG; ISSN:0022-1759. (Elsevier B.V.)
      We have developed a highly sensitive immunoassay-called digital ELISA-that is based on the detection of single enzyme-linked immunocomplexes on beads that are sealed in arrays of femtoliter wells. Digital ELISA was designed to be highly efficient in the capturing of target proteins, labeling of these proteins, and their detection in single mol. arrays (SiMoA); in essence, the goal of the assay is to "capture every mol., detect every mol.". Here we provide the theor. basis for the design of this assay derived from simple equations based on bimol. interactions. Using these equations and knowledge of the concns. of reagents, the times of interactions, and the on- and off-rates of the mol. interactions for each step of the assay, it is possible to predict the no. of immunocomplexes that are formed and detected by SiMoA. The unique ability of SiMoA to count single immunocomplexes and det. an av. no. of enzymes per bead (AEB), makes it possible to directly compare the no. of mols. detected exptl. to those predicted by theory. These predictions compare favorably to exptl. data generated for a digital ELISA for prostate specific antigen (PSA). The digital ELISA process is efficient across a range of antibody affinities (KD ~ 10-11-10-9 M), and antibodies with high on-rates (kon > 105 M-1 s-1) are predicted to perform best. The high efficiency of digital ELISA and sensitivity of SiMoA to enzyme label also makes it possible to reduce the concn. of labeling reagent, reduce backgrounds, and increasing the specificity of the approach. Strategies for dealing with the dissocn. of antibody complexes over time that can affect the signals in an assay are also described.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XjsFOhsLc%253D&md5=7657279ca929d26c6c245ba0bd3d0009
    25. 25
      Ekins, R. P.; Chu, F. W. Multianalyte Microspot Immunoassay--Microanalytical “Compact Disk” of the Future. Clin. Chem. 1991, 37 (11), 1955– 1967,  DOI: 10.1093/clinchem/37.11.1955
      25
      Multianalyte microspot immunoassay-microanalytical "compact disk" of the future
      Ekins, R. P.; Chu, F. W.
      Clinical Chemistry (Washington, DC, United States) (1991), 37 (11), 1955-67CODEN: CLCHAU; ISSN:0009-9147.
      Throughout the 1970s, controversy centered both on immunoassay sensitivity per se and on the relative sensitivities of labeled antibody (Ab) and labeled analyte methods. Theor. studies revealed that RIA sensitivities could be surpassed only by the use of very high-specific-activity nonisotopic labels in noncompetitive designs, preferably with monoclonal antibodies. The time-resolved fluorescence methodol. known as DELFIA represented the first com. ultrasensitive nonisotopic technique based on these theor. insights, the same concepts being subsequently adopted in comparable methodologies relying on the use of chemiluminescent and enzyme labels. However, high-specific-activity labels also permit the development of multianalyte immunoassay systems combining ultrasensitivity with the simultaneous measurement of tens, hundreds, or thousands of analytes in a small biol. sample. This possibility relies on simple, albeit hitherto-unexploited, physiochem. concepts. The first is that all immunoassays rely on the measurement of Ab occupancy by analyte. The second is that, provided the Ab concn. used is vanishingly small, fractional Ab occupancy is independent of both Ab concn. and sample vol. This leads to the notion of ratiometric immunoassay, involving measurement of the ratio of signals (e.g., fluorescent Ab) deposited as a microspot on a solid support, the second (a developing Ab) directed against either occupied or unoccupied binding sites of the sensor Ab. The authors' preliminary studies of this approach have relied on a dual-channel scanning-laser confocal microscope, permitting microspots of area 100 μm2 or less to be analyzed, and implying that an array of 106 Ab-contg. microspots, each directed against a different analyte, could in principle, be accommodated on an area of 1 cm2. Although measurement of such analyte nos. is unlikely ever to be required, the ability to analyze biol. fluids for a wide spectrum of analytes is likely to transform immunodiagnostics in the next decade.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38Xjslekuw%253D%253D&md5=56792c2461f50069cb11ece83e5a68a5
    26. 26
      Shapoval, O.; Brandmeier, J. C.; Nahorniak, M.; Oleksa, V.; Makhneva, E.; Gorris, H. H.; Farka, Z.; Horák, D. PMVEMA-Coated Upconverting Nanoparticles for Upconversion-Linked Immunoassay of Cardiac Troponin. Talanta 2022, 244, 123400,  DOI: 10.1016/j.talanta.2022.123400
      There is no corresponding record for this reference.
    27. 27
      Nsubuga, A.; Sgarzi, M.; Zarschler, K.; Kubeil, M.; Hübner, R.; Steudtner, R.; Graham, B.; Joshi, T.; Stephan, H. Facile Preparation of Multifunctionalisable ‘Stealth’ Upconverting Nanoparticles for Biomedical Applications. Dalton Trans. 2018, 47 (26), 8595– 8604,  DOI: 10.1039/C8DT00241J
      There is no corresponding record for this reference.
    28. 28
      Raiko, K.; Lyytikäinen, A.; Ekman, M.; Nokelainen, A.; Lahtinen, S.; Soukka, T. Supersensitive Photon Upconversion Based Immunoassay for Detection of Cardiac Troponin I in Human Plasma. Clin. Chim. Acta 2021, 523, 380– 385,  DOI: 10.1016/j.cca.2021.10.023
      There is no corresponding record for this reference.
    29. 29
      Chen, H.; Wang, L.; Yeh, J.; Wu, X.; Cao, Z.; Wang, Y. A.; Zhang, M.; Yang, L.; Mao, H. Reducing Non-Specific Binding and Uptake of Nanoparticles and Improving Cell Targeting with an Antifouling PEO-b-PγMPS Copolymer Coating. Biomaterials 2010, 31 (20), 5397– 5407,  DOI: 10.1016/j.biomaterials.2010.03.036
      There is no corresponding record for this reference.
    30. 30
      Weng, Z.; Yu, H.; Luo, W.; Guo, Y.; Liu, Q.; Zhang, L.; Zhang, Z.; Wang, T.; Dai, L.; Zhou, X.; Han, X.; Wang, L.; Li, J.; Yang, Y.; Xie, G. Cooperative Branch Migration: A Mechanism for Flexible Control of DNA Strand Displacement. ACS Nano 2022, 16 (2), 3135– 3144,  DOI: 10.1021/acsnano.1c10797
      30
      Cooperative Branch Migration: A Mechanism for Flexible Control of DNA Strand Displacement
      Weng, Zhi; Yu, Hongyan; Luo, Wang; Guo, Yongcan; Liu, Qian; Zhang, Li; Zhang, Zhang; Wang, Ting; Dai, Ling; Zhou, Xi; Han, Xiaole; Wang, Luojia; Li, Junjie; Yang, Yujun; Xie, Guoming
      ACS Nano (2022), 16 (2), 3135-3144CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      DNA strand displacement plays an essential role in the field of dynamic DNA nanotechnol. However, flexible regulation of strand displacement remains a significant challenge. Most previous regulatory tools focused on controllable activation of toehold and thus limited the design flexibility. Here, we introduce a regulatory tool termed cooperative branch migration (CBM), through which DNA strand displacement can be controlled by regulating the complementarity of branch migration domains. CBM shows perfect compatibility with the majority of existing regulatory tools, and when combined with forked toehold, it permits continuous fine-tuning of the strand displacement rate spanning 5 orders of magnitude. CBM manifests multifunctional regulation ability, including rate fine-tuning, continuous dynamic regulation, reaction resetting, and selective activation. To exemplify the powerful function, we also constructed a nested if-function signal processing system on the basis of cascading CBM reactions. We believe that the proposed regulatory strategy would effectively enrich the DNA strand displacement toolbox and ultimately promote the construction of DNA machines of higher complexity in nucleic acid research and biomedical applications.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xis1ykurw%253D&md5=7a93cf13c2424daa710c580db80b9382
    31. 31
      Kohno, T.; Ishikawa, E.; Mitsukawa, T.; Matsukura, S. Novel Enzyme Immunoassay (Immune Complex Transfer Enzyme Immunoassay) for Anti-thyroglobulin IgG in Human Serum. J. Clin. Lab. Anal. 1988, 2 (4), 209– 214,  DOI: 10.1002/jcla.1860020406
      There is no corresponding record for this reference.
    32. 32
      Kohno, T.; Mitsukawa, T.; Matsukura, S.; Tsunetoshi, Y.; Ishikawa, E. More Sensitive and Simpler Immune Complex Transfer Enzyme Immunoassay for Antithyroglobulin Igg in Serum. J. Clin. Lab. Anal. 1989, 3 (3), 163– 168,  DOI: 10.1002/jcla.1860030306
      There is no corresponding record for this reference.
    33. 33
      Gorris, H. H.; Soukka, T. What Digital Immunoassays Can Learn from Ambient Analyte Theory: A Perspective. Anal. Chem. 2022, 94 (16), 6073– 6083,  DOI: 10.1021/acs.analchem.1c05591
      33
      What Digital Immunoassays Can Learn from Ambient Analyte Theory: A Perspective
      Gorris, Hans H.; Soukka, Tero
      Analytical Chemistry (Washington, DC, United States) (2022), 94 (16), 6073-6083CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      A review. Immunoassays are important tools for clin. diagnosis as well as environmental and food anal. because they enable highly sensitive and quant. measurements of analyte concns. In the 1980s, Roger Ekins suggested to improve the sensitivity of immunoassays by employing microspot assays, which are carried out under ambient analyte conditions and do not change the bulk analyte concn. of a sample during a measurement. More recently, the measurement of single analyte mols. has addnl. attracted wide research interest. Although the ability to detect a single analyte mol. is not synonymous with the highest anal. sensitivity, single-mol. detection makes new routes accessible to avoiding background noise. This perspective follows the development of solid-phase immunoassays from the design of label techniques to single-mol. (digital) assays against the backdrop of Ekins's fundamental work on immunoassay theory. The essential aspects of both ambient analyte and digital assay approaches are presented as a guideline to finding a balance between the speed, sensitivity, and precision of immunoassays.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xpt1ynt7Y%253D&md5=fc3c8d44561d32aa1bd51f7d64afc196
    34. 34
      Chheda, U.; Pradeepan, S.; Esposito, E.; Strezsak, S.; Fernandez-Delgado, O.; Kranz, J. Factors Affecting Stability of RNA – Temperature, Length, Concentration, PH, and Buffering Species. J. Pharm. Sci. 2024, 113 (2), 377– 385,  DOI: 10.1016/j.xphs.2023.11.023
      There is no corresponding record for this reference.
    35. 35
      Soukka, T.; Kuningas, K.; Rantanen, T.; Haaslahti, V.; Lövgren, T. Photochemical Characterization of Up-Converting Inorganic Lanthanide Phosphors as Potential Labels. J. Fluoresc. 2005, 15 (4), 513– 528,  DOI: 10.1007/s10895-005-2825-7
      35
      Photochemical characterization of up-converting inorganic lanthanide phosphors as potential labels
      Soukka, Tero; Kuningas, Katri; Rantanen, Terhi; Haaslahti, Ville; Loevgren, Timo
      Journal of Fluorescence (2005), 15 (4), 513-528CODEN: JOFLEN; ISSN:1053-0509. (Springer Science+Business Media, Inc.)
      The authors have characterized com. available up-converting inorg. lanthanide phosphors for their rare earth compn. and photoluminescence properties under IR laser diode excitation. These up-converting phosphors, in contrast to proprietary materials reported earlier, are readily available to be utilized as particulate reporters in various ligand binding assays after grinding to submicron particle size. The laser power d. required at 980 nm to generate anti-Stokes photoluminescence from these particulate reporters is significantly lower than required for two-photon excitation. The narrow photoluminescence emission bands at 520-550 nm and at 650-670 nm are at shorter wavelengths and thus totally discriminated from autofluorescence and scattered excitation light even without temporal resoln. Transparent soln. of colloidal bead-milled up-converting phosphor nanoparticles provides intense green emission visible to the human eye under illumination by an IR laser pointer. In this article, the authors show that the unique photoluminescence properties of the up-converting phosphors and the inexpensive measurement configuration, which is adequate for their sensitive detection, render the up-conversion an attractive alternative to the UV-excited time-resolved fluorescence of down-converting lanthanide compds. widely employed in biomedical research and diagnostics.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtVSqu7%252FM&md5=46cf461e1e56dd0615e44e4ad0b43124
    36. 36
      Yakovchuk, P.; Protozanova, E.; Frank-Kamenetskii, M. D. Base-Stacking and Base-Pairing Contributions into Thermal Stability of the DNA Double Helix. Nucleic Acids Res. 2006, 34 (2), 564– 574,  DOI: 10.1093/nar/gkj454
      36
      Base-stacking and base-pairing contributions into thermal stability of the DNA double helix
      Yakovchuk, Peter; Protozanova, Ekaterina; Frank-Kamenetskii, Maxim D.
      Nucleic Acids Research (2006), 34 (2), 564-574CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      Two factors are mainly responsible for the stability of the DNA double helix: base pairing between complementary strands and stacking between adjacent bases. By studying DNA mols. with solitary nicks and gaps we measure temp. and salt dependence of the stacking free energy of the DNA double helix. For the first time, DNA stacking parameters are obtained directly (without extrapolation) for temps. from below room temp. to close to melting temp. We also obtain DNA stacking parameters for different salt concns. ranging from 15 to 100 mM Na+. From stacking parameters of individual contacts, we calc. base-stacking contribution to the stability of A•T- and G•C-contg. DNA polymers. We find that temp. and salt dependences of the stacking term fully det. the temp. and the salt dependence of DNA stability parameters. For all temps. and salt concns. employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A•T pairing is always destabilizing and G•C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtlOnuro%253D&md5=2c72728f600373744c60a81684a64409
    37. 37
      Ekins, R.; Chu, F.; Micallef, J. High Specific Activity Chemiluminescent and Fluorescent Markers: Their Potential Application to High Sensitivity and ‘Multi-analyte’ Immunoassays. J. Biolumin. Chemilumin. 1989, 4 (1), 59– 78,  DOI: 10.1002/bio.1170040113
      There is no corresponding record for this reference.
    38. 38
      Zhou, X.; Zhu, W.; Li, H.; Wen, W.; Cheng, W.; Wang, F.; Wu, Y.; Qi, L.; Fan, Y.; Chen, Y.; Ding, Y.; Xu, J.; Qian, J.; Huang, Z.; Wang, T.; Zhu, D.; Shu, Y.; Liu, P. Diagnostic Value of a Plasma MicroRNA Signature in Gastric Cancer: A MicroRNA Expression Analysis. Sci. Rep. 2015, 5 (1), 11251,  DOI: 10.1038/srep11251
      There is no corresponding record for this reference.
    39. 39
      Ferracin, M.; Lupini, L.; Salamon, I.; Saccenti, E.; Zanzi, M. V.; Rocchi, A.; Da Ros, L.; Zagatti, B.; Musa, G.; Bassi, C.; Mangolini, A.; Cavallesco, G.; Frassoldati, A.; Volpato, S.; Carcoforo, P.; Hollingsworth, A. B.; Negrini, M. Absolute Quantification of Cell-Free MicroRNAs in Cancer Patients. Oncotarget 2015, 6 (16), 14545– 14555,  DOI: 10.18632/oncotarget.3859
      There is no corresponding record for this reference.
    40. 40
      Mitchell, P. S.; Parkin, R. K.; Kroh, E. M.; Fritz, B. R.; Wyman, S. K.; Pogosova-Agadjanyan, E. L.; Peterson, A.; Noteboom, J.; O’Briant, K. C.; Allen, A.; Lin, D. W.; Urban, N.; Drescher, C. W.; Knudsen, B. S.; Stirewalt, D. L.; Gentleman, R.; Vessella, R. L.; Nelson, P. S.; Martin, D. B.; Tewari, M. Circulating MicroRNAs as Stable Blood-Based Markers for Cancer Detection. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (30), 10513– 10518,  DOI: 10.1073/pnas.0804549105
      40
      Circulating microRNAs as stable blood-based markers for cancer detection
      Mitchell, Patrick S.; Parkin, Rachael K.; Kroh, Evan M.; Fritz, Brian R.; Wyman, Stacia K.; Pogosova-Agadjanyan, Era L.; Peterson, Amelia; Noteboom, Jennifer; O'Briant, Kathy C.; Allen, April; Lin, Daniel W.; Urban, Nicole; Drescher, Charles W.; Knudsen, Beatrice S.; Stirewalt, Derek L.; Gentleman, Robert; Vessella, Robert L.; Nelson, Peter S.; Martin, Daniel B.; Tewari, Muneesh
      Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (30), 10513-10518CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small (≈22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. The authors show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. MiRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. These results establish the measurement of tumor-derived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXpsVCltL0%253D&md5=c802fd49d4fae2e536c03abfd7a34996
    41. 41
      Farka, Z.; Mickert, M. J.; Hlaváček, A.; Skládal, P.; Gorris, H. H. Single Molecule Upconversion-Linked Immunosorbent Assay with Extended Dynamic Range for the Sensitive Detection of Diagnostic Biomarkers. Anal. Chem. 2017, 89 (21), 11825– 11830,  DOI: 10.1021/acs.analchem.7b03542
      41
      Single Molecule Upconversion-Linked Immunosorbent Assay with Extended Dynamic Range for the Sensitive Detection of Diagnostic Biomarkers
      Farka, Zdenek; Mickert, Matthias J.; Hlavacek, Antonin; Skladal, Petr; Gorris, Hans H.
      Analytical Chemistry (Washington, DC, United States) (2017), 89 (21), 11825-11830CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      The ability to detect disease markers at the single mol. level promises the ultimate sensitivity in clin. diagnosis. Fluorescence-based single-mol. anal., however, is limited by matrix interference and can only probe a very small detection vol., which is typically not suitable for real world anal. applications. The authors have developed a microtiter plate immunoassay for counting single mols. of the cancer marker prostate specific antigen (PSA) using photon-upconversion nanoparticles (UCNPs) as labels that can be detected without background fluorescence. Individual sandwich immunocomplexes consisting of (1) an anti-PSA antibody immobilized to the surface of a microtiter well, (2) PSA, and (3) an anti-PSA antibody-UCNP conjugate were counted under a wide-field epifluorescence microscope equipped with a 980 nm laser excitation source. The single-mol. (digital) upconversion-linked immunosorbent assay (ULISA) reaches a limit of detection of 1.2 pg mL-1 (42 fM) PSA in 25% blood serum, which is about ten times more sensitive than com. ELISAs and covers a dynamic range of three orders of magnitude. This upconversion detection mode has the potential to pave the way for a new generation of digital immunoassays.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFOjtrfP&md5=d6c0a2d5ab2eb655077152c109e0e027
    42. 42
      Christopoulos, T. K.; Lianidou, E. S.; Diamandis, E. P. Ultrasensitive Time-Resolved Fluorescence Method for α-Fetoprotein. Clin. Chem. 1990, 36 (8), 1497– 1502,  DOI: 10.1093/clinchem/36.8.1497
      There is no corresponding record for this reference.
    43. 43
      Kuusinen, S.; Lahtinen, S.; Soukka, T. Upconversion Luminescence Based Direct Hybridization Assay to Detect Subfemtomolar MiR-20 a DNA Analogue in Plasma. Anal. Sens. 2024, 4 (4), e202400005  DOI: 10.1002/anse.202400005
      There is no corresponding record for this reference.
    44. 44
      Watanabe, T.; Hashida, S. The Immune Complex Transfer Enzyme Immunoassay: Mechanism of Improved Sensitivity Compared with Conventional Sandwich Enzyme Immunoassay. J. Immunol. Methods 2018, 459, 76– 80,  DOI: 10.1016/j.jim.2018.05.010
      There is no corresponding record for this reference.
    45. 45
      Morrissey, D. V.; Lombardo, M.; Eldredge, J. K.; Kearney, K. R.; Groody, E. P.; Collins, M. L. Nucleic Acid Hybridization Assays Employing DA-Tailed Capture Probes. Anal. Biochem. 1989, 181 (2), 345– 359,  DOI: 10.1016/0003-2697(89)90255-8
      There is no corresponding record for this reference.
    46. 46
      Masterson, A. N.; Chowdhury, N. N.; Fang, Y.; Yip-Schneider, M. T.; Hati, S.; Gupta, P.; Cao, S.; Wu, H.; Schmidt, C. M.; Fishel, M. L.; Sardar, R. Amplification-Free, High-Throughput Nanoplasmonic Quantification of Circulating MicroRNAs in Unprocessed Plasma Microsamples for Earlier Pancreatic Cancer Detection. ACS Sens. 2023, 8 (3), 1085– 1100,  DOI: 10.1021/acssensors.2c02105
      There is no corresponding record for this reference.
    47. 47
      Ramshani, Z.; Zhang, C.; Richards, K.; Chen, L.; Xu, G.; Stiles, B. L.; Hill, R.; Senapati, S.; Go, D. B.; Chang, H.-C. Extracellular Vesicle MicroRNA Quantification from Plasma Using an Integrated Microfluidic Device. Commun. Biol. 2019, 2 (1), 189,  DOI: 10.1038/s42003-019-0435-1
      47
      Extracellular vesicle microRNA quantification from plasma using an integrated microfluidic device
      Ramshani Zeinab; Zhang Chenguang; Senapati Satyajyoti; Go David B; Chang Hsueh-Chia; Ramshani Zeinab; Zhang Chenguang; Senapati Satyajyoti; Chang Hsueh-Chia; Ramshani Zeinab; Richards Katherine; Senapati Satyajyoti; Chang Hsueh-Chia; Richards Katherine; Chen Lulu; Stiles Bangyan L; Xu Geyang; Hill Reginald; Hill Reginald; Go David B; Chang Hsueh-Chia
      Communications biology (2019), 2 (), 189 ISSN:.
      Extracellular vesicles (EV) containing microRNAs (miRNAs) have tremendous potential as biomarkers for the early detection of disease. Here, we present a simple and rapid PCR-free integrated microfluidics platform capable of absolute quantification (<10% uncertainty) of both free-floating miRNAs and EV-miRNAs in plasma with 1 pM detection sensitivity. The assay time is only 30 minutes as opposed to 13 h and requires only ~20 μL of sample as oppose to 1 mL for conventional RT-qPCR techniques. The platform integrates a surface acoustic wave (SAW) EV lysing microfluidic chip with a concentration and sensing microfluidic chip incorporating an electrokinetic membrane sensor that is based on non-equilibrium ionic currents. Unlike conventional RT-qPCR methods, this technology does not require EV extraction, RNA purification, reverse transcription, or amplification. This platform can be easily extended for other RNA and DNA targets of interest, thus providing a viable screening tool for early disease diagnosis, prognosis, and monitoring of therapeutic response.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3M7pt1CksA%253D%253D&md5=2b1721ce83d533ad89c98b76e55bf780
    48. 48
      Majd, S. M.; Salimi, A.; Ghasemi, F. An Ultrasensitive Detection of MiRNA-155 in Breast Cancer via Direct Hybridization Assay Using Two-Dimensional Molybdenum Disulfide Field-Effect Transistor Biosensor. Biosens. Bioelectron. 2018, 105, 6– 13,  DOI: 10.1016/j.bios.2018.01.009
      48
      An ultrasensitive detection of miRNA-155 in breast cancer via direct hybridization assay using two-dimensional molybdenum disulfide field-effect transistor biosensor
      Majd, Samira Mansouri; Salimi, Abdollah; Ghasemi, Foad
      Biosensors & Bioelectronics (2018), 105 (), 6-13CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
      MicroRNAs (miRNAs), crit. biomarkers of acute and chronic diseases, play key regulatory roles in many biol. processes. As a result, robust assay platforms to enable an accurate and efficient detection of low-level miRNAs in complex biol. samples are of great significance. In this work, a label-free and direct hybridization assay using molybdenum disulfide (MoS2) field-effect transistor (FET) biosensor has been developed for ultrasensitive detection of miRNA-155 as a breast cancer biomarker in human serum and cell-line samples. MoS2, the novel 2D layered material with excellent phys. and chem. properties, was prepd. through sequential solvent exchange method and was used as an active channel material. MoS2 was comprehensively characterized by spectroscopic and microscopic methods and it was applied for fabrication of FET device by drop-casting MoS2 flacks suspension onto the FET surface. MoS2 FET device showed a relatively low subthreshold swing of 48.10 mV/decade and a high mobility of 1.98 × 103 cm2 V-1 s-1. Subsequently, probe miRNA-155 strands were immobilized on the surface of the MoS2 FET device. Under optimized conditions detection limit of 0.03 fM and concn. range 0.1 fM to 10 nM were achieved. The developed biosensor not only was capable to identification of fully matched vs. one-base mismatch miRNA-155 sequence, but also it could detect target miRNA-155 in spiked real human serum and exts. from human breast cancer cell-line samples. This approach paves a way for label-free, early detection of miRNA as a biomarker in cancer diagnostics with very high sensitivity and good specificity, thus offering a significant potential for clin. application.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXnvVKqtw%253D%253D&md5=ecd8c227ae2e8e140ff1f414e92b58bc
    49. 49
      Wegman, D. W.; Ghasemi, F.; Khorshidi, A.; Yang, B. B.; Liu, S. K.; Yousef, G. M.; Krylov, S. N. Highly-Sensitive Amplification-Free Analysis of Multiple MiRNAs by Capillary Electrophoresis. Anal. Chem. 2015, 87 (2), 1404– 1410,  DOI: 10.1021/ac504406s
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.4c05401.

    • Information on oligonucleotide design and sequences of oligonucleotides, synthesis and surface modification of UCNP labels and their conjugation with oligonucleotide probes; information about plasma pool collection and in-house coating of MTPs with streptavidin; characterization of UCNP labels; and results from the optimization of the assay performance (PDF)


    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.