A single-step electrochemical immunochromatography has been developed: the device was based on two pieces of nitrocellulose membrane, a sample pad with anti-mouse IgG antibody labeled with glucose oxidase (GOx-labeled antibody), a conjugate pad with glucose, and a Pt working electrode. Either antibody or antigen was immobilized on the membrane. The addition of a solution containing mouse IgG, a model target, allows for the dissolution of GOx-labeled antibody in the sample pad to form an immunocomplex. The produced immunocomplex was automatically separated by capturing to the antibody immobilized on the membrane with the sandwich structure or by passing through the membrane modified with an antigen for the competitive reaction. The separated GOx label arrived at the conjugate pad with glucose to undergo the enzyme reaction. Hydrogen peroxide generated by this reaction was detected at the Pt electrode prepared on the second nitrocellulose membrane downstream from the conjugate pad. The results demonstrated that the designed immunochromatography can be applied to quantitative detection with a single-step procedure, because both the GOxlabeled antibody for revealing the immunoreactions and the substrate for the enzyme reaction were prepared in the device. Moreover, the initial concentration of the GOx-labeled antibody permitted control of the detectable concentration for mouse IgG.

Immunochromatography is a well-established technique and widely used as a practical kit for detection of various diagnoses for the initial screening of medical diagnoses. When a solution containing target analytes is added at the end of a membrane, the solution spontaneously migrates toward the other end of the membrane by capillary force. This automatic migration of the solution is combined with the capturing of analytes by the immunorecognition events. The target moleculses in the solutions flowing in the membrane arrived at the region immobilized with antibody and captured to form immunocomplexes. The antibody conjugated with the signal source is also migrated with target molecules to concentrate the signal source. The concentrated signal source can be detected by colorimetric or fluorescent measurements within 10 - 20 min. Moreover, the unreacted signal source was spontaneously removed to downstream in the membrane strips, and automatically discriminated from the captured molecules. Thus, the immunochromatography is a simple and rapid immunosensing system. A visual colorimetric measurement by necked eye is commonly utilized for the decision of positivity or negativity, but is of low sensitivity, and thus insufficient for quantitative analysis. Complete dark box and precisely arranged optical system are required to utilize the fluorescence detection for quantitative analysis. In this presentation, the electrochemical detection system was incorporated in lateral flow immunochromatographic assays for the quantitative and automatic determination of target molecules. The working electrode was placed at the downstream from the region immobilized with antibody. The hydrogen peroxide (redox species) generated by the labeled enzyme captured on the antibody immobilized region can flow in the downstream to arrive the electrode and be detected by the amperometry. The device for electrochemical immunochromatography consisted of a test strip (nitrocellulose membrane) with the antibody immobilization area and absorbent pad and acrylic plates incorporating Pt disk electrode (50 m) and poles for supporting the membranes. The silver wire was inserted through another plate to use as a pseudo-reference/counter electrode. Cyclic voltammetry of Fe(CN)6 4− flowing in the test strip was measured by using the device for immunochromatography. The steady-state oxidation current was observed at a region of positive potential due to the continuous supply of solution flow containing Fe(CN)6 4−. In addition, the oxidation current obtained without flow in the test strip is smaller than that obtained with flow. The redox species flowing in the test strip can be detected with the present system. Amperometry was also performed in the test strip with flow at different concentrations of Fe(CN)6 4−. The obtained steady-state current increased linearly with increasing the concentration of Fe(CN)6 4− in the flowing solution. Therefore, we would determine the concentration of redox species flowing in the test strip. Hydrogen peroxide generated by the reaction of labeled enzyme (glucose oxidase, GOx) was detected to demonstrate the determination of target molecules. The solution containing different concentrations of target analyte, mouse IgG was applied to the end of the test strip. When mouse IgG and GOx-labeled antibody arrived at the region immobilized with antibody, sandwich type of immunocomplex was formed at the region. The addition of the solution containing glucose that is the substrate of the labeled enzyme allows to producing hydrogen peroxide to detect captured mouse IgG and removing unreacted labeled enzyme to downstream. When glucose was added, the oxidation current increased gradually and reached at almost a steady-state. The current response increased with increasing concentration of mouse IgG. Thus, amount of the captured mouse IgG can be determined. The total measurement time required with the present system is around 20 min, which is equivalent to conventional immunochromatographic formats.Therefore, we can incorporate electrochemical quantitation in a simple immunosensing system for immunochromatography.