For antibody based biosensors calibration is complicated

For antibody-based biosensors, calibration is complicated by the strength and irreversibility of antibody-antigen binding. One Micafungin of exposure to a chaotropic agent and antibody refolding allows calibration of impedance biosensors, whereas multiple cycles allow regeneration of impedance biosensors. Antibody-based ELISA tests typically allow 50–60cycles of regeneration, most commonly using strong acids or bases as chaotropic agents [7]. For impedance biosensors, high or low pH solutions may not be compatible with the chemistries employed for protein immobilization, and are particularly problematic for integration into ULSI or MEMS devices. Here several mild chaotropic agents are tested for antibody regeneration of impedance biosensors with the antibody to peanut protein Ara h 1 covalently immobilized onto Au and degenerate Si electrodes. Biosensors for detection of peanut proteins, and other food allergens, have recently attracted significant interest [8–11]. Peanuts are a particularly problematic food allergen due to the prevalence of peanuts in a wide variety of food products, the high sensitivity of some individuals, and the stability of some allergenic peanut proteins during food manufacturing and human digestion [8–11]. Au-thiol self-assembly chemistry has been most commonly employed for antibody immobilization onto a conductive electrode [12]. However, depending on storage conditions, the shelf life of such sensors has been reported to be limited to days to weeks [13]. For these reasons, other substrate materials such as C, Si, Pt, Ti, and ITO have also been tested for impedance biosensors [14–23]. We recently reported degenerate (highly doped) Si as an alternative electrode material to Au, and demonstrated impedance detection of an allergenic food protein, peanut protein Ara h 1 [24,25]. Advantages of degenerate Si as a sensor electrode include the greater strength of Si–C covalent bonds relative to Au–S, simpler equivalent circuit relative to n-type or p-type Si, easier surface preparation relative to C, widespread availability of Si wafers, and ease of incorporation into ULSI devices [24]. Here antibody regeneration atop degenerate Si is successfully reported for a 30-day trial using 0.2M KSCN and 10mM HF as the chaotropic reagent, and compared to antibody regeneration atop Au electrodes.
Experimental
Results and discussion
Conclusions Antibody regeneration was tested using several reagents for the mouse monoclonal antibody to peanut protein Ara h 1 covalently immobilized atop by degenerate (highly doped) Si and Au electrodes. Only 200mM KSCN is effective for antibody regeneration and detection of Ara h 1 atop Au electrodes, allowing 15days of sensor usage after daily antibody unfolding and refolding. On the other hand, 30days of sensor usage were possible for degenerate Si using the same denaturing agent, but with addition of 10mM HF. For degenerate Si, cyclic voltammetry in the presence of 5.0mM K3Fe(CN)6/K4Fe(CN)6 at pH7.3 showed that oxidation/reduction peaks are only observed in the presence of 10mM HF, demonstrating that this reagent is necessary to prevent Si oxidation. Although the impedance spectrum for detection of Ara h 1 gradually degrades during these multi-day regeneration trials, experiments performed within one day illustrate that sensor electrodes can be calibrated on the day of use to within about 2%.
Acknowledgements The authors gratefully acknowledge the support of this research by National Science Foundation grant # 1342618.
Introduction Electrochemiluminescence(ECL) detection is one of the prevailing electrochromism approaches to test biotin or chemicals [1]. It involves electron transfer between electrochemically generated radical ions in solution to produce excited species that emit light. When voltage is exerted between the working electrode and the counter electrode, elements or radicals can be exited to metastable potentials, which would be capable to transmit photon when moving back to stable valance [2]. ECL signals are unique to specific substances since wavelength and luminescence intensities are distinct [3]. In addition, the voltage of photo multiplier tube (PMT) also contributes to ECL signals [4]. Combined with electrochemical (EC) detection, we can forecast the concentration range and the substance type.