Biosensor technology has high specificity and sensitivity, and is widely used in the online analysis and detection of complex systems. It has good development prospects in fields such as clinical diagnosis, analytical chemistry, food testing, pharmaceutical analysis, and chemical engineering.
Biosensor technology is a branch of analytical biology, permeating multiple disciplines such as analytical chemistry, biology, life sciences, and physics. Biosensors mainly consist of two parts: a recognition system and a signal conversion system.
The ability of a specific substance to react specifically with a sensor through a recognition system is key to the sensor's recognizability, highlighting its specificity for detecting the substance. Substances detected by recognition systems can include proteins, enzymes, antigens/antibodies, DNA, nucleic acids, biomembranes, cells, tissues, microorganisms, and other materials. Based on the type of material being recognized, biosensors can be categorized into enzyme sensors, immunosensors, cell sensors, etc. Another part of the system, the signal conversion system, converts the specific reaction between the substance and the recognition system into recognizable information (such as light, heat, or electrical information), amplifies it, and outputs it. Based on the signal conversion method, sensors can be further classified into photobiosensors, electrochemical biosensors, etc.
Due to the advantages of electrical signals, such as fast response speed, ease of conversion and acquisition, and simple and intuitive data analysis, electrochemical biosensors have become the earliest developed, most researched, and most widely used sensors. Electrochemical biosensors primarily use electrodes as information conversion materials to convert specific reaction processes of substances into electrical signals, and the magnitude of these electrical signals indirectly represents the concentration of reactants. Among these, the development of enzyme electrodes is the most representative in the field of biosensors.
Enzyme electrode sensor
Enzyme electrodes are the most widely studied biosensors, primarily due to the high sensitivity, specificity, simplicity of instrumentation, and rapid response of enzymes. Enzyme electrode biosensors utilize biological enzymes as recognition units, immobilizing the enzyme on a modified electrode surface. When a specific substance corresponding to the enzyme is present in the substrate, it catalyzes its oxidation. This reaction generates electron exchange on the electrode surface, and the change in current reflects the chemical reaction, thus indicating the change in substrate concentration. However, biological enzymes typically have one or more redox active sites composed of metal ions, most of which are deeply embedded within the protein peptide chain, making direct electron exchange between the enzyme's active site and the electrode surface difficult.
To address the charge transfer problem between the active site of an enzyme and the electrode, the development of bio-enzyme electrochemical sensors has mainly gone through three stages.
The first-stage enzyme electrode uses oxygen as an electron acceptor. Taking a glucose oxidase sensor as an example, the reaction process is as shown in (1) and (2). GOx(FAD) oxidized glucose oxidizes glucose into glucono-delta-lactone, while the reduced enzyme GOx(FADH2) reduces oxygen in the solution to hydrogen peroxide. The glucose concentration is indirectly determined by measuring the change in the concentration of oxygen or hydrogen peroxide during the reaction. However, the sensor in this stage is highly susceptible to the influence of oxygen in the environment and has poor anti-interference ability.
GOx(FAD)+glucose→GOx(FADH2)+glucolactone(1)
GOx(FADH2)+O2→GOx(FAD)+H2O2(2)
The second-stage sensor adds a mediator layer for electron transfer between the enzyme and the electrode, replacing oxygen as the electron acceptor and overcoming the problem of interference. A mediator material capable of rapid redox reactions is used as an intermediate for electron transfer between the enzyme's active site and the electrode surface, as shown in (3), (4), and (5). The oxidized enzyme oxidizes the substrate to a reduced state, while the process of reducing and oxidizing the mediator material transfers the reaction charge to the electrode surface, and the amount of charge represents the substrate concentration. However, the mediator material is prone to diffusion, which places higher demands on its immobilization.
GOx(FAD)+glucose→GOx(FADH2)+glucolactone(3)
GOx(FADH2)+2Medox+2e-→GOx(FAD)+2Medred+2H+(4)
2Medred→2Medox+2e-(5)
The third-stage enzyme electrode sensor does not require oxygen or a mediator as an electron acceptor. Instead, it uses chemical methods to open the peptide chain of the biological enzyme protein to expose the enzyme's active site or to perform special treatment on the electrode surface to immobilize the biological enzyme on the electrode surface. While catalyzing the oxidation of reactants, it directly exchanges charge with the electrode, as shown in (6) and (7). However, due to the inherent properties of the biological enzyme, the electron transport efficiency is still limited.
GOx(FAD)+glucose→GOx(FADH2)+glucolactone(6)
GOx(FADH2)+2e-→GOx(FAD)+2H+(7)
Enzyme-free electrode sensor
In enzyme electrode sensors, enzyme activity is a key factor determining the sensor's stability and sensitivity. However, enzymes are prone to denaturation and inactivation during immobilization, and their activity is easily affected by environmental factors such as humidity, temperature, and chemical conditions. Furthermore, enzyme leakage may occur during immobilization, and some biological enzymes are expensive. Therefore, a method has been proposed that utilize metals, metal oxides, alloys, etc., with multiple oxidation states as catalytic materials to replace biological enzymes immobilized on the electrode surface for the catalytic oxidation of analytes. Two oxidation mechanisms are widely accepted theories regarding the catalytic oxidation of substances on the electrode surface.
Taking the oxidation of glucose on the electrode surface as an example, the first theory is the adjacent site adsorption theory, which states that when glucose adsorbed on the electrode surface is oxidized, the CH bond on the hemiacetal carbon in the glucose molecule breaks, and the hydrogen atom and the hemiacetal carbon simultaneously form chemical bonds on the electrode surface, as shown in Figure 1.
Figure 1. Schematic diagram of adsorption at adjacent sites
The second theory is the intermediate oxidation theory. When metal atoms are adsorbed by glucose molecules, they form a metal ion membrane. The metal ions oxidize the adsorbed glucose molecules into gluconeuronic acid. The ion membrane is then reduced to metal atoms on the electrode surface, thereby achieving charge exchange, as shown in Figure 2.
Figure 2 Schematic diagram of intermediate oxidation
Currently, there is considerable research on enzyme-free sensors, with glucose enzyme-free sensors being the most extensively studied. Examples include enzyme-free sensors using noble metals Pt, Au, and Pd as glucose catalytic materials for electrode fabrication; transition metals Ni and Cu, and their oxides, modified to create sensor electrodes; and electrodes made from hybrid metals or oxides. While enzyme-free sensors are unaffected by enzyme activity, they also present some challenges. For instance, noble metals Pt and Au are expensive; although they exhibit good catalytic activity for glucose, Cl- ions in solution readily adsorb onto the electrode surface; Pd nanoparticles are prone to polymerization; and while transition metals Ni and Cu, and their oxides, possess high sensitivity, they suffer from a narrow linear detection range for glucose. Enzyme-free sensors are highly susceptible to chemical influences and have stringent requirements for the detection environment; therefore, detection is typically performed in buffer solutions.
