We report the synthesis and application of three new antifouling diluents for the fabrication of an E-PB HIV sensor. Among the three thiolated antifouling diluents used in this study, the methoxy-terminated diluent (C6-MEG) is the most effective in alleviating both nonspecific binding and adsorption of matrix contaminants onto the sensor surface, especially when compared to the mannose- (C6-MAN) and ethylene-glycol-terminated (C6-EG) diluents. The sensor fabricated with C6-MEG has a specificity factor (∼13.5) substantially higher than the sensor passivated with only 6-mercapto-1-hexanol (∼1.5). It is functional even when employed directly in 25% serum, an achievement that has not been observed with this class of E-PB sensors. More importantly, incorporation of these antifouling diluents has negligible impact on other important sensor properties such as sensitivity and binding kinetics. This sensor passivation strategy is versatile and can potentially be used with other E-PB sensors, as well as surface-based sensors that utilize thiol-gold self-assembled monolayer chemistry.

We have developed a simple approach to minimize both nonspecific binding and nonspecific adsorption of random targets and matrix contaminants on self-assembled monolayer-based biosensors. The effectiveness of this approach has been verified using an electrochemical peptide-based (E-PB) sensor as a model system.1 The peptide recognition sequence used to fabricated this E-PB sensor is an immunodominant epitope from the HIV-1 p24 antigen ((n)QGPKEPFRDYVDRFYKTLRAE(c)).2 The sensor itself consists of a thiolated peptide labeled with methylene blue (DRY-MB), mercapto-6-hexan-1-ol (C6-OH), and one type of antifouling molecules. The three new anti-fouling amphiphiles used in this study are HS-(CH2)6-α-mannose (C6-MAN), HS-(CH2)6-O-(CH2)2-OH (C6-EG), and HS-(CH2)6-O-(CH2)2-O-(CH2)3 (C6-MEG). As shown in Scheme 1, in the absence of the target, the MB redox label on the probe is relatively flexible, resulting in high MB current. However, in the presence of the target (anti-p24 antibody), the peptide probe will bind to the target and the observed MB current will decrease substantially. While the previously reported sensors have demonstrated good sensitivity and specificity, their ability to function well in a complex medium has not been verified. To determine the specificity of the sensor, we first added random human antibodies (Wrong Abs) to the Phys2 buffer solution, followed by the addition of equimolar amounts of the target antibody (Ab). The sensors fabricated without any antifouling molecules showed ~20 % signal suppression (%SS) for the Wrong Abs and ~40 %SS for the target Ab. For sensors that incorporated one of the three antifouling molecules, the %SS was less than 5 % for the Wrong Abs and greater than 40 % for the target Ab. These results suggest that the incorporation of the antifouling molecules not only minimizes the response of the Wrong Abs, it also enhances the response of the target Ab. Furthermore, we noted that the extent of nonspecific interactions is highly dependent on the peptide probe sequence, in which peptide probes with more arginine and aspartic acid residues are more susceptible to this problem.3 Last, to determine sensor selectivity, we interrogated the sensors in a Phys2 buffer spiked with either human serum or synthetic human saliva. The sensors are fully capable of recognizing the target Ab in these complex matrices; this level of selectivity has not been reported for this class of E-PB sensors. References [1] a.) J.Y. Gerasimov, R.Y. Lai, Chem. Commun. 2011, 47, 8688-8690. b.) J.Y. Gerasimov, R.Y. Lai, Chem. Commun. 2010, 46, 395-397. c.) R.J. White, H.M. Kallewaard, W. Hsieh, A.S. Patterson, J.B. Kasehagen, K.J. Cash, T. Uzawa, H. Tom Soh, K.W. Plaxco, Anal. Chem., 2012, 84, 1098-1103. d) R. Partovi-Nia,S. Beheshti, Z. Qin, H.S. Mandal, Y.-T. Long, H.H. Girault, H.-B. Kraatz, Langmuir 2012, 28, 6377-6385. e.)A.K. Nowinski, F. Sun, A.D. White, A.J. Keefe, S. Jiang, J. Am. Chem. Soc. 2012, 134, 6000-6005. [2] a.) N. Frahm, C. Linde, C. Brander, HIV Molecular Immunology 2006, Los Alamos National Laboratory, Los Alamos, NM, 2006, pp. 3–28.b.) G. Tonarelli, J. Lottersberger, J.L. Salvetti, S. Jacchieri, R.A. Silva-Lucca, L.M. Beltramini, Lett. Pept. Sci., 2000, 7, 217–224. [3] a.) S.P. Massia, J.A. Hubbell, Anal. Biochem. 1980, 187, 292-301. b.) S.N. Khilko, M. Corr, L.F. Boyd, A. Lees, J.K. Inman, D.H. Margulies, J. Biol. Chem. 1993, 268, 15425-15434. Acknowledgements This work was supported by the National Science Foundation (CHE-0955439) and Nebraska EPSCoR (EPS-1004094).