The biosensory analytical technique has become one of the most important technologies in the 21st century. Biosensors are analytical systems that utilize the biosensory analytical technique, and consist of biological identification materials and various signal converters. Among them, an electrochemical biosensor is easy to operate and has outstanding sensitivity, and therefore is an excellent sensory element. In addition, in view of the specificity of different biological molecules, the problem of selectivity in most sensory elements can be overcome. Since a biosensor with an electrode strip, such as an enzymatic-electrochemical sensor, can provide an accurate result rapidly, it is widely used in detecting a large amount of samples in research and clinical studies. For example, the electrochemical blood sugar detecting systems sold in the market use an electrode coated with a glucose oxidase to measure the concentration of the glucose molecule. The development of enzyme-immobilized biosensors generally has three stages. The first stage is the utilization of a dissolved oxygen-detecting electrode in the measurement of the amount of the dissolved oxygen consumed during the catalytic process of the substance with the oxidative enzyme, so that the concentration of the substance can be indirectly obtained. Alternatively, the product having an electrochemical activity, such as hydrogen peroxide, produced during the enzymatic-catalyzing reaction can be detected. The second stage mainly lies in the addition of an electron transporter, which improves the efficiency of the transportation of electrons to the surface of the electrode. Furthermore, the electron transporter has the property of reversing the reduction/oxidation reactions so that it can receive the electrons produced from the enzymatic-catalyzing reaction and become the reduced form, and the oxidation reaction on the surface of the electrode can pass the electrons to the electrode so as to generate electric signals. Because of the low reduction/oxidation potential of the electron transporter, it can decrease the electric potential required for the detection and avoid the interference caused by the substances produced under the high electric potential condition. In the third stage, co-factors of enzymes are applied so as to decrease the resistance resulting from the transportation of electrons from the enzyme during the enzymatic-catalyzing oxidation or reduction reaction. Nicotinamide adenine dinucleotide (NADH) is the commonly used co-factor, and transports electrons to the electrode by way of the reversible oxidation/reduction process. A lot of research has demonstrated that the efficiency of electron transportation of the biosensor used at this stage is far higher than those used in the previous two stages and thus the sensor has a higher sensitivity. However, the disadvantages of the biosensor used at this stage are the complexities on the enzyme immobilization procedure and the poor stability under room temperature, and thus it is not suited for transportation and storage.
Because antibodies/antigens or complementary or partially complementary double-strand ribonucleic acids (RNA) or deoxyribonucleic acids (DNA) are biological molecules having high selectivity and affinity, they can be designed to detect different molecules. Researchers can immobilize the biological molecules having high selectivity and affinity on various types of sensors as the tag for the detection. The biological molecules include, but are not limited to antibodies, antigens, enzymes, nucleic acids, tissues or cells. For example, by the utilization of the mechanism similar to the conventional solid phase immunoassay, a combination of an electrochemical device and a selected and immobilized antibody can be used for the detection of the binding of the solid-phase molecules (e.g., the antibody) with the corresponding mobile-phase molecules (e.g., the antigen). In this combination, a converter in the sensor amplifies the detected electric signals so that a quantitative analysis can be conducted. Such combination is called an “electrochemical immunosensor.”
The enzyme-labeled electrochemical immunosensor is the most well-developed system in the art. A non-heterogeneous enzymatic immunoassay comprises two analytic methods, i.e., the competitive analysis and the sandwich analysis. A competitive analysis mainly comprises the steps of: (1) immobilizing an antibody, which is specific to the targeting antigen, on the surface of an electrode; (2) contacting the electrode with an enzyme-labeled targeting antigen and the antigen sample; (3) rinsing the electrode to remove the unbound antigen, (4) adding the substrate for the labeled enzyme to conduct the catalytic reaction and thus produce the electrochemical product; and (5) quantifying the amount of the targeting antigen in the sample by measuring the amount of said product. The electric signal obtained from the competitive analysis is in inverse proportion to the concentration of the targeting antigen. In contrast, the electrical signal obtained from the sandwich analysis is in direct proportion to the concentration of the targeting antigen. Compared to the traditional immunoassay, an electrochemical immunosensor can effectively decrease the operation cost for analyzing various samples presented in a small amount. However, when an electrochemical immunosensor is actually used, it is often found that the electric signal measured cannot be distinguished from the background noise because of the low concentration of the target. Thus, there is a need to develop an electrochemical immunosensor that has a biologically sensory electrode strip capable of amplifying the redox electrical signal and improving the accuracy of the result.
A wide number of species of the biological samples or molecules can be detected by the immunoassay. For example, Escherichia coli (E. coli) and Vibrio parahaemolyticus, which cause food poisoning, are the common research subjects for developing new immunoassays. There are a variety of pathogenic bacteria in a person's daily diet. Traditionally, in order to identify the species of a bacterium precisely, different cultural broths and selective media and further biochemical reaction tests are needed. Therefore, a traditional detecting method is more time-consuming and labor-intensive. Moreover, it cannot detect and identify new strains or mutants of the pathogenic bacteria. It is also a problem that urgently requires a solution.
U.S. Pat. No. 6,491,803, CN 1462880 A and CN 1462881 A pertain to the application of a nano-scaled material to a biochemically sensory electrode. However, these cases still require complicated preparation procedures. For the preparation of the test strip disclosed in U.S. Pat. No. 6,491,803 B1, all reaction substances, including nanometer metal particles, must be first admixed and then coated on the electrode by screen printing, and, in order to evenly coat the admixed substances, the conditions for screen printing are rather strict. In CN 1462880 A and CN 1462881 A, at least three layers of materials including a water-soluble polymer carrier (e.g., carboxymethyl cellulose), a modified nanometer carbon tube and an enzyme reaction layer (including an enzyme, an electron mediator, a stabilizer, a buffer, etc.) are sequentially coated and dried on the test strip. Therefore, the preparation processes disclosed in the two cases are complex. TW Patent No: 1276799 discloses a simplified process for the preparation of a biochemically sensory electrode.
In the combination of all the technologies described above, which include the utilization of the electron mediator, the identification process of the biological materials having affinity to each other, and the utilization of the nanometer materials on electrochemical measurement, an artisan cannot easily deduce the solution to the problems caused by the repetitious soaking and washing procedures conducted in the immuno-identification process, and to the problem of low signal/noise ratio in view of the low concentration of the immunoassay target. Therefore, in this technical field, there is a need to develop a technology to prepare an electrode strip without the complicated preparation processes conventionally used, and to obtain strong electric signals from the electrode strip. The present invention provides an applicable solution for this object.