Enzyme-Based Electrochemical Biosensors

By Kal Kaur

Introduction
Enzyme-based Electrochemical Biosensors
-    Enzyme Immobilization
Electrical Communication
-    Artificial Redox Mediators
-    Use of a Natural Substrate
-    Transfer of Electrons between the Enzyme and the Electron
Glucose Biosensors
References

Introduction

Electrochemical sensors involve a transduction element that is encapsulated by a biological recognition layer. The recognition layer interacts with the target analyte and forces a chemical reaction to occur, a process that is interpreted by the transducing element into an electrical wave.

Since as early as 2005, analytical research has become more involved in the application of electrochemical sensors in industries ranging from clinical to industrial, environmental, and agricultural analysis. The bio-catalytic sensor (i.e., use of an enzyme or cell as an immobilized component) is a common type of electrochemical sensor and will be discussed in detail for the scope of this article.

Enzyme-based Electrochemical Biosensors

Enzyme Immobilization

Enzymes help catalyze a chemical reaction in cellular homeostasis. With relation to enzyme-based electrochemical sensors, there is a coupling reaction between an enzyme and an electrode. In a biological cell, enzyme catalysis normally involves thermal energy that forces the enzyme and substrate to bind via van der Walls forces. During the catalytic reaction, the enzyme reduces the activation energy making it easier for reactant molecules to form a product with the enzyme. However, with enzyme electrodes, the enzyme binds to the electrode surface by a number of processes: covalent cross-linking, entrapment with a gel layer on the electrode surface, encapsulation using low thermal energy, or absorption onto a graphite surface – all such methods are capable of immobilizing an enzyme to an electrode surface (Figure 1). 

Figure 1. Methods to bind an enzyme to the surface of an electrode. Source: Wang, J. (2006). Analytical Electrochemistry. Canada: John Wiley and Sons, Inc.

Electrical Communication

The next step in understanding electrochemical enzyme-based biosensors is being able to create an electrical communication between the enzyme active site and the electrode. Three types of enzyme-electrode reactions can allow for this:

  • Artificial Redox Mediators

During this reaction, the enzyme either generates or consumes a redox-active compound (biologically active charge carriers) during conversion of the target analyte. The active compound is oxidized by the enzyme, which generates electrons that charge the transducing element at the electrode surface to generate an electrical potential that corresponds to the amount of analyte oxidised (Figure 2). Application of a mediated system, whereby molecules are used to shift electrons between the redox reaction point of the enzyme and the electrode, requires less operating voltage to power the catalytic reaction. The most common type of redox mediators includes ferrocene and its derivatives based on their large potential to power a redox reaction.

Figure 2. Elextrical communication via application of artificial redox mediators. Source: Enzyme Electrodes. PowerPoint Presentation. Biosensors and Bioelectronics (2008/2009). Walter Schottky Institut Center for Nanotechnology and Nanomaterials.

  • Use of a Natural Substrate

One of the most common types of electrochemical bio-sensing reactions involves natural substrates that are oxidized to transfer electrons to molecular oxygen (O2) resulting in the production of hydrogen peroxide (H2O2). Both oxygen and H2O2 are electrochemically active and can transfer electrons into the neighbouring electrode to generate an electrical current. Though this method is known for being simple compared to the use of artificial redox mediators, it does carry its disadvantages, such as a slow response rate, problems with miniaturization, and poor validity. Oxidation of O2 to H2O2 does require a high voltage and this is probably one of the biggest drawbacks to this method.

  • Transfer of Electrons between the Enzyme and the Electron
Research efforts are moving towards looking at direct electrical communication between the electrode and the enzyme component. One particular enzyme capable of performing direct electron transfer is the horseradish peroxide enzyme. The enzyme works by catalyzing the reduction of H2O2. This enzyme does not work alone to catalyze the reduction of H2O2; the enzyme works as a complex system with an oxidase enzyme. The peroxidase electrode linked to the peroxidase enzyme works at a low voltage, which prevents interference of other electroactive species in this catalytic reaction.

Glucose Biosensors

The glucose biosensor (based on probes manufactured by YSI Inc.) is a great example of an electrochemical biosensor. The device has a platinum cathode and silver anode electrode, both responsible for carrying an electrical current. At the front end of the biosensor there are four layers that enhance the enzyme reaction. The glucose passes through the O-ring layer and the first polycarbonate membrane to the third membrane layer where the glucose is oxidized to H2O2 and gluconic acid. The H2O2 passes through the cellulose acetate membrane to the platinum cathode where it is converted into O2, a hydrogen molecule, and 2 electrons. The third reaction involves combination of silver nitrate with an electron on the silver anode, a catalytic reaction that generates silver and chlorine. The hydrogen and O2 molecules react on the silver anode at the opposite end of the biosensor, which releases electrons to power an electrical current.

Research is now focusing more on in-vivo and in-vitro electrochemical biosensors for investigating a wider range of physiological compounds. Based on the drawbacks to some of the methods to electrochemical sensing, efforts still need to focus on making these sensors more sensitive and reliable.

References

  • Newman J.D., Setford S.J. Enzymatic Biosensors. Molecular Biotechnology 2006;32:249.
  • Wang, J. (2006). Analytical Electrochemistry. Canada: John Wiley and Sons, Inc.
  • Wollenberger U. Electrochemical Biosensors – Ways to Improve Sensor Performance. Biotechnology and Genetic Engineering Reviews 1995;13: 237–266.
  • The Enzyme Electrode
  • Wang J. Glucose Biosensors: 40 Years of Advances and Challenges. Electroanalysis 2001;13(12):983–988.

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