Porous Laser-Scribed Graphene Electrodes Modified with Zwitterionic Moieties: A Strategy for Antibiofouling and Low-Impedance Interfaces

Laser-scribed graphene electrodes (LSGEs) are promising platforms for the development of electrochemical biosensors for point-of-care settings and continuous monitoring and wearable applications. However, the frequent occurrence of biofouling drastically reduces the sensitivity and selectivity of these devices, hampering their sensing performance. Herein, we describe a versatile, low-impedance, and robust antibiofouling interface based on sulfobetaine-zwitterionic moieties. The interface induces the formation of a hydration layer and exerts electrostatic repulsion, protecting the electrode surface from the nonspecific adsorption of various biofouling agents. We demonstrate through electrochemical and microscopy techniques that the modified electrode exhibits outstanding antifouling properties, preserving more than 90% of the original signal after 24 h of exposure to bovine serum albumin protein, HeLa cells, and Escherichia coli bacteria. The promising performance of this antifouling strategy suggests that it is a viable option for prolonging the lifetime of LSGEs-based sensors when operating on complex biological systems.


ZW Characterization
ZW was characterized by FT-IR, 1 H NMR, and mass spectroscopy.As can be seen in Figure S 1a, the IR spectrum of the zwitterionic compound presents the O-H bond stretching broad band at around 3500 cm -1 related to water molecules adsorbed to this hydroscopic compound.The following bands are related to asymmetric and symmetric stretching modes of N-H and C-H bonds.
C-H bond bending mode and also C-N + bond stretching mode are associated with weak bands observed at around 1400 cm -1 .The S=O stretching modes in the sulfonate group are associated with the strong bands observed at 1207 and 1151 cm -1 , while the S-O stretching mode is depicted by the band at 1028 cm -1 .The C-N stretching mode in primary aliphatic amines is normally observed in the 1250-1020 cm -1 range and therefore overlaps with sulfonate group vibrational modes.Finally, the low-intensity bands around 700 cm -1 are associated with aliphatic skeleton vibrations.C-S stretching mode band is expected to lower than 600 cm -1 and therefore is out of the acquired IR spectrum range.

Optimization of the ZW functionalization
Two approaches were tested in order to optimize the concentrations of the solutions and obtain the highest antifouling effect.Initially, the concentration of the zwitterionic solution was varied using a geometric progression of (0, 0.1, 0.2, and 0.4M), while maintaining the carbodiimide:sulfosuccinimide solution constant at (0.1 M).However, the system was not fully saturated and the antifouling effect was not ideal.Therefore, the concentration of the carbodiimide:sulfosuccinimide solution was increased using a geometric progression of (0, 0.25, 0.5, and 1 M) with the zwitterionic solution set at a maximum of (2 M) in order to couple all the free carboxyl (-OOH) with the primary amine (-NH2) species.

Long-term extrapolation
The long-term behavior of LSGE and modified LSGE/ZW was extrapolated using logarithmic functions of time, with the form of () =  +  × ( + 1).These functions are fitted on the measurements of the current density for each group of electrodes and for each concentration of albumin solution as depicted in Figure S10.An F-test was used to analyze the goodness of the fit.S1b, the coefficients of determination R 2 are close to 1, the F-values are large, and the p-values are significantly less than the standard threshold 0.05, which indicates that this model has remarkable statistical significance.Therefore, our logarithmic assumption can be used reliably to estimate the current density values of the electrodes at future times.As can be seen in Table S1a, projections of the current density values after 30 days of albumin solution exposure of bare LSGE show a reduction in the current response of ~69.8%, ~64%, and ~64%, for 10 mg mL -1 , 30 mg mL -1 , and 50 mg mL -1 , respectively.In contrast, LSGE/ZW exhibits a reduction of ~27%, ~28%, and ~34%, for the different concentrations of albumin solution.

Investigation of the influence of surface charge on antifouling performance
To investigate the influence of protein charge on the ZW performance, LSGE and LSGE/ZW electrodes were immersed in both 30 mg mL -1 of myoglobin and lysozyme.The current response was measured before and after immersion to estimate the degree of biofouling after 24 hours.We observed that the LSGEs' current response was reduced by ~60% after immersion in the myoglobin solution, whereas LSGE/ZWs' response only decreased by ~28%.Myoglobin is composed of one short peptide chain with 153 residues and a heme group buried in a central hydrophobic pocket.The isoelectric point of myoglobin is at pH 7.0 and the immersion testing was conducted using 0.01 M PBS (pH 7.40) as the background.As such, the protein could be regarded as neutrally charged, thereby nullifying the effect of charge electrostatic repulsion and allowing it to adhere to the surface.Previously, at room temperature, myoglobin has been demonstrated to unfold at slightly basic pH levels, which releases the heme group to interact with the aqueous phase. 1 Consequently, this explains the higher reduction in the current response experienced by LSGE/ZW when immersed in myoglobin relative to that experienced when immersing in albumin.Contrastingly, the influence of electrostatic repulsion is highlighted in the lysozyme findings.The bare LSGEs and the LSGE/ZW experienced slight drops of ~8% and ~6%, respectively, after immersion in a lysozyme-containing solution.As the aggregate charge of lysozymes is positive at the test pH of 7.4, we suspect that the larger degree of electrostatic repulsion played a role in preserving the current response in both electrodes.Furthermore, lysozymes have four native disulfide bonds and a number of interhelical interactions that stabilize the protein. 2,3This stabilization mitigates unfolding at test conditions.This restricts the specific surface area of the protein which in turn limits the site of non-specific adhesion further reducing the potential for adherence of the protein onto the surface of the electrodes.LSGE/ZW (blue plot line, plot column on the right) before and after (red plot lines) 24 hours of immersion in 10 mg mL -1 (top row), 30 mg mL -1 (middle row), and 50 mg mL -1 (bottom row) of albumin.The DPV test was performed using ferri/ferrocyanide as a redox probe.

S-15
H NMR signals were analyzed and schematically associated to zwitterionic compound structure by the letters A to G: methyl groups (-CH3) 6HA at 1.255 ppm ( 3 JAG = 7.3 Hz), amino group (-NH2) 2HD at 2.680 ppm ( 3 JDE = 7.6 Hz), and methylene groups (-CH2-) 2HB at 1.896 ppm, 2HC at 2.002 ppm ( 3 JBC = 7.5 Hz), 2HE at 2.893 ppm ( 3 JEH = 7.5 Hz), 2HF at 2.960 ppm ( 3 JFC = 7.5 Hz), 4HG at 3.162 ppm and 2HH at 3.241 ppm as depicted in Figure S 1b.Finally, the mass spectrum of the compound 3-((2-aminoethyl)diethylammonium) propane-1sulfonate (here referred to as ZW) is shown in Figure S 1c.The primary ion corresponding to the compound ZW is the peak at m/z 239.1428, which represents a protonated [ZW+H] + ion of the compound.As shown in the inset table in Figure S 1c, the calculated mass error is -1.6 ppm and the topmost proposed formula by the Bruker Data Analysis Software matches that of the protonated species of ZW (C9H23N2O3S).The lower Sigma score for this formula shows that the observed ion has the closest isotopic pattern to the theoretical formula of protonated species of ZW among the possible formulas.The higher intensity signal at m/z 261.1247 corresponds to the sodiated species of ZW [ZW+Na] + (i.e.C9H22N2O3SNa).Signals at around m/z 117 correspond to the N,N-diethylethylenediamine starting amine respective positive ions, and higher m/z values signals correspond to ZW positive charged aggregates.

Figure
Figure S1.(a) FT-IR (b) 1 H NMR (c) mass spectroscopy of the ZW compound.

Figure S9 .Figure S10 .
Figure S9.A bar plot of the calculated remaining current sensitivities before and after immersion in 10 mg mL -1 , 30 mg mL -1 , and 50 mg mL -1 of albumin for bare LSGE (red) and

Figure S12 .Figure S14 .
Figure S12.(a, b) Raw image obtained from the SEM contrast-enhanced to normalize the

Table S2 :
Anti-biofouling strategies applied to carbon-based electrodes with performance benchmark comparisons.