Electrosynthesis of Surface-bound Hydroxylamine Linkers for Antibody Immobilization
Boone M. Prentice, Advisor Melissa C. Rhoten
There is an increasing need for fast, accurate, repeatable and economical methods for the analysis of biohazards and environmental toxins in air and water. Immunoassays have traditionally been used to detect and quantify toxins, but suffer from long analysis times, one-time use, automation challenges, and quantification variability. The use of polarized liquid crystal materials for antibody immobilization is a novel approach in immunosensor design. In this work 4-(4-nitro-phenylethynyl)-benzenethiol has been synthesized and allowed to self-assemble at gold quartz crystal microbalance (QCM) electrodes. Prior to covalent attachment of the antibody of interest, the nitro group must be reduced to a hydroxylamine. This is accomplished electrochemically and is the focus of this poster. Results for the electroreduction of the nitro group of 4-nitrothiophenol to the hydroxylamine is also presented to demonstrate the feasibility of this approach. The electroreduction at gold QCM electrodes is also compared to results obtained at molybdenum electrodes.
Electrode Modification Procedure
Capacitance measurements were conducted at each gold QCM electrode prior to cleaning. Three cyclic voltammograms were recorded in 4.4 mM phosphate buffer (pH 7.4) from 0.1 V to 0.4 V [versus Ag/AgCl (1 M KCl)] at a scan rate of 0.1 V/s. The electrode was then washed with doubly deionized water.
Each electrode was cleaned in a KCl (0.01 M)/H2SO4 (0.1 M) solution under the following voltammetric conditions: scans 1-3: 0.2 V ↔ 0.9 V, scans 4-6: 0.2 V ↔ 1.2 V, scans 7-9: 0.2 V ↔ 1.35 V, scan rate = 0.1 V/s, reference electrode = Ag/AgCl (1 M KCl). The electrodes were again washed with doubly deionized water.
Post-cleaning capacitance measurements were recorded at each electrode in 4.4 mM phosphate buffer (pH 7.4) from 0.1 V to 0.4 V (versus Ag/AgCl (1 M)) at a scan rate of 0.1 V/s.
Each electrode was washed with copious amounts of doubly deionized water, ethanol, water, and ethanol prior to the addition of the freshly-prepared thiol solution (50 mM) of interest (in dichloromethane). Self-assembly was allowed to proceed for 48 hours.
The 4-nitrothiophenol system
After modification with 4-NTP, the electrode is poised at a potential of -400 mV vs. Ag/AgCl (1 M KCl) for 15-60 minutes.
Application of the initial potential reduces the nitro group to the hydroxylamine.
CVs are taken at 15 minute intervals between -400 mV and 0 mV.
Oxidation of the hydroxylamine produces the nitroso group followed by re-reduction back to the hydroxylamine.
PDP (pyridoxal phosphate) is an aqueous molecule that serves as an antibody model system (i.e., it contains an aldehyde group that is capable of reaction with the hydroxylamine). The 4-NTP modified electrode is exposed to PDP (50 mM) while being held at -400 mV for various lengths of time.
Initially it was proposed that the diminished current response over time was due to binding of the PDP to the surface-bound hydroxylamine.
The 4-NTP electrode was initially held at -400 mV for 35 minutes followed by interrogation with CV at open circuit. Scans were taken between -400 mV and 0 mV over the course of 6 hours revealing a large decrease in current response for the hydroxylamine/nitroso redox couple. A potential of -400 mV was reapplied for 35 minutes after this 6 hour time period, but no significant increase in current response was seen. This could indicate that a non-electroactive species is being produced over time which may reduce the number of hydroxylamine moieties for reaction with PDP (or an antibody).
The 4-(4-nitro-phenylethynyl)-benzenethiol system
This molecule is capable of producing the same electrochemistry as 4-NTP (i.e., reduction of the nitro group to the hydroxylamine).
CVs shown above represent the oxidation of the hydroxylamine to the nitroso compound over time (i.e., electrode is held at -400 mV for 0-120 minutes).
We postulate that this molecule could be used to affix antibodies to the gold surface and facilitate shorter surface regeneration time because of the molecule's highly conductive nature.
- Dr. Fred Hawkridge, VCU
- Dr. Michael Leopold, University of Richmond
- Longwood Citizen Scholars Program