Ultra-Low Level Sensing of Biogenic Analytes

 Goal: To develop an ultrasensitive, peroxide-based electrochemical detection method for biogenic analytes in cells and tissues.

Studies:

• Development of a low-level (<1 µM) sensing scheme to detect enzymatically generated H₂O₂ utilizing a H₂O₂-activated redox mediator and pulse voltammetry.
• Application of this sensing scheme in quantitative determination of target analytes (glucose, cholesterol, etc.) in tissue samples.
• Extraction of enzyme kinetic parameters (Michaelis-Menten constants, enzyme turnover rates) to aid in evaluation of biochemical systems, including three-dimensional enzyme scaffolds constructed via multiphoton excitation lithography (see Dr. Jason Shear’s research).

Scheme 1: Depiction of enzymatically generated H₂O₂ detection using a chemically activated redox mediator and either soluble or immobilized HRP

Figure 1: Demonstration of the S/N improvement attainable through use of pulse voltammetry for analysis of a 300 nM H₂O₂ sample. For comparison, a CV of the same sample is also shown.

Figure 2: (a) Calibration curves for H₂O₂ detection using our redox mediator/pulse voltammetry method. (b) Apparent Michaelis-Menten behavior observed at increasing concentrations of H₂O₂.

Significance: Biosensors developed for diagnostic analysis of target analytes (such as glucose and cholesterol) often depend on these analytes’ interaction with enzymes to catalytically generate hydrogen peroxide (H₂O₂), which is then electrochemically oxidized to determine analyte concentrations. However, H₂O₂ oxidation poses many disadvantages when used in biological environments, including the physiologically extreme potentials required for its oxidation and its complex reaction mechanism at biological pH values.

To avoid the problems inherent with direct H₂O₂ oxidation, we have developed a mediated, low-potential electrochemical method for bioanalyte detection (Scheme 1). In this scheme, horseradish peroxidase (HRP) that is either free in solution or immobilized at a substrate catalyzes the decomposition of enzymatically generated H₂O₂ to peroxy radicals, which are then reduced to water via oxidation of a mediator (med) to its redox-active form (med_ox). Med_ox is detected electrochemically at a GC electrode via a reversible, two-electron reduction step at -100 mV vs. Ag/AgCl. Because H₂O₂ oxidizes the mediator in a 1:1 ratio, the amount of free H₂O₂ created through enzyme-analyte interaction can be quantified at a lower, more biologically-friendly potential. We have further improved upon traditional peroxide-based electrochemical sensing methods by using pulse voltammetry³ to optimize sensing at ultralow concentrations (<1 µM) by eliminating background current contributions (Figure 1).⁴

Calibration curves obtained using our method, shown in Figure 2(a), are linear over five orders of magnitude (18 pM to 3 µM) and display significantly improved amperometric sensitivities compared to previously published electrochemical and optical peroxide detection methods.⁵ Our method is amenable to any detection scheme that uses enzymatically generated H₂O₂ for detection of a biogenic species. Future studies are focused on extending this scheme to quantitative analyte measurements in cells and tissues.

Related Publications:

(1)    Heller, A. Annual Review of Biomedical Engineering 1999, 1, 153-175.

(2)    Wilson, G. S.; Hu, Y. Chemical Reviews (Washington, D. C.) 2000, 100, 2693-2704.

(3)    Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications, 2nd ed.; John Wiley & Sons: Hoboken, NJ, 2001, pp 293-301.

(4)    Osteryoung, J.; O'Dea, J. J. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1986; Vol. 14, pp 209-308.

Students currently involved in this project: Jen Lyon