Electrochemical Biosensing Techniques for Detection of NADH, Estradiol and Choline
- Author(s): FENG, LILI
- Advisor(s): Monbouquette, Harold G
- et al.
This thesis primarily focuses on optimizing electrode behavior for sensing NADH, estradiol and a metabolite of a common neurotransmitter, choline. NADH is a crucial coenzyme involved in cellular energy metabolism and is one of the products of enzyme-catalyzed oxidation of estradiol that is used in its sensing. The sensing of estradiol is important for the prediction and prevention of ovarian hyperstimulation syndrome. Most current electrooxidation methods for detection of NADH rely on glassy carbon electrodes so as to avoid the electrode fouling observed with platinum and other noble metal electrodes. The modification of platinum electrodes with an acid-treated, carbon nanotube film was applied in this work to eliminate the electrode fouling effect and to increase the electrode sensitivity. A detection limit as low as 0.17 μM and an average sensitivity of 370 mA M−1 cm−2 were achieved. Also, a reduced potential could be used at the sensing electrode due to the electrocatalytic properties of the carbon nanotube coating, thus less interference from electroactive species in a sample was observed.
These promising NADH sensing results suggested the feasibility of an electrochemical enzyme-multiplied immunoassay technique (EMIT) for estradiol detection. EMIT is a fast and convenient homogeneous immunoassay technology that can be practiced without much of the bulky and expensive instrumentation and tedious washing steps associated with the competing ELISA approach. EMIT relies on antibody-induced inhibition of an analyte-labeled reporter enzyme. Free analyte in solution competes for binding antibody thereby reducing reporter enzyme inhibition and resulting in increased signal with increased analyte concentration. The reporter enzyme, glucose-6-phosphate dehydrogenase, catalyzes the oxidation of glucose-6-phosphate and transfer of electrons to the β-nicotinamide adenine dinucleotide (NAD+) coenzyme to give NADH. The NADH produced has historically been detected spectrophotometrically, however our electrochemical NADH detection technology can also be used to follow reporter enzyme activity by constant potential amperometry. Electrochemical EMIT shows a linear logit-log range from 3 nM to 30 μM stradiol and exhibits a background signal that is about two times better than with spectrophotometric EMIT.
The second major project of this thesis research was focused on sensing an important neurochemical, choline, with a microbiosensor on a microelectrode array microprobe. Choline is produced in the brain when the neurotransmitter, acetylcholine, is rapidly hydrolyzed. In fact, choline may serve as an effective surrogate for acetylcholine since it is turned over so rapidly to choline and acetate. Thus, there is great interest among the neuroscience community in a sensitive and fast choline sensor for near real time in vivo recording. The choline biosensor fabrication process first entails the electropolymerization of a permselective poly-m-phenylenediamine (PPD) film on the electrode surface to provide good selectivity against electroactive interferents, such as ascorbic acid (AA) and dopamine (DA). Subsequently, Nafion dip coating is performed to improve rejection of AA due to instability of the PPD film. However, the Nafion dip coating step can be omitted if the choline sensors are used in short term (i.e., acute) applications in vivo as opposed to chronic studies. Finally, choline oxidase is mixed with bovine serum albumin (BSA) and glutaraldehye (GAH) in aqueous solution to give the crosslinking mixture for enzyme immobilization on the coated microelectrodes. Twelve layers of this enzyme mixture are loaded manually on two of the four-microelectrode sites on a microprobe, and the other two sites are used as controls. A high sensitivity of 390 μA∙mM-1∙cm-2 with a corresponding detection limit of 0.16 μM was achieved, which is good enough for applications in vivo. The choline sensors fabricated with the PPD coating exhibited among the highest sensitivities and lowest detection limits described in the literature to date.
The incorporation of an on-probe iridum oxide (IrOx) reference electrode was also investigated to reduce the background noise and to eliminate the necessity of a separate reference electrode. However, reference electrode with only IrOx sometimes has an issue of sudden current drop and is not stable. Platinum black was employed to increase the effective electrode surface area, which improved the IrOx reference coating. A very high sensitivity of 440 μA mM-1 cm-2 with a corresponding low detection limit of 0.04 μM was achieved with a linear range up to156 μM. However, platinum black plating solution has cytotoxic component lead, which is not good for the in vivo use. A novel nanomaterial called “platinum grass” (Pt-grass) was then used to replace platinum black as the reference electrode pre-coating material. A sensitivity of 310 μA mM-1 cm-2 and detection limit 0.07 μM were gained, which are ideal for the in vivo purpose.
Pt-grass was also used in modifying working electrode to reduce overpotential. Because of the background noise brought by the rough surface of Pt-grass on working electrode, the sensitivity and detection limit were affected, which were 196 μA mM-1 cm-2 and 2 μM, respectively. However, the electrocatalytic properties of Pt-grass could enable an operation of the choline biosensor at a lower working potential of 0.45 V, 0.25 V lower than that typically used, which resulted in improved selectivity. The Pt-grass all-in-one sensor, with Pt-grass on both working and reference electrode, has a sensitivity of 123 μA mM-1 cm-2 and detection limit of 3 μM under an applied potential 0.35 V. The all-in-one choline sensors are also quite promising for the in vivo usage since it can both minimize the electrode size and reduce the overpotential.