Multi-Channel Immunoassay System with Electrochemical Detection Toward Point-of-Care Diagnostics Using Magnetic Nanoparticles

The electrochemical biosensors are widely used in point-of-care test (POCT) applications because of their simple measurement methods and their ease of implementation in small diagnostic devices. Most electrochemical biosensors perform sample analysis one by one, and there are limitations to using electrochemical immunoassays as multiple reactions and multiple assays required in practical POCT applications. Here, we developed a multi-channel electrochemical immunoassay system and simultaneously assayed one set of electrochemical ELISA. We quantitatively analyzed with electrocmafhemical measurements using multiple sensor arrays. In the immune reaction, we used 8 microwell array in 96-well plate (8 by 12) using magnetic nanoparticles (MNPs). As a model system, the immune reaction of the sandwich method of detecting matrix metalloproteinase-9 (MMP-9) as a bladder cancer marker using MNPs coated with streptavidin was performed. Matrix metalloproteinases play a major role in the invasion and metastasis of cancer cells, and MMP-9 is produced in both normal and cancer cells. MMP-9 is known to be closely associated with malignancy of tumor cells. In the experiment, the 96-well plate and multi-channel immunoassay system-assay were used. To immobilize biotinylated MMP-9 on streptavidin-coated MNPs, 15 μL of a 1% PBST buffer solution containing 10 µg/ml biotinylated MMP-9 was incubated onto the streptavidin-coated MNPs, the incubated state was maintained for 30 min at 25 °C, and the MNPs were washed three times with rinsing buffer. Subsequently, 15 μL of a 1% BSA buffer solution was incubated onto the biotinylated MMP-9 modified MNPs, then incubated state was maintained for overnight at 4 °C, and the electrodes were washed three times with rinsing buffer. The resulting MNPs were stored at 4 °C before their use. The storage solution should use 1% BSA. For the binding of the target protein to the MNPs, 150 μL of 1 % PBST buffer solutions containing different concentrations of MMP-9 were incubated onto the MNPs, the incubated state was maintained for 30 min at 25 °C, and MNPs were washed three times with rinsing buffer. Afterward, 150 μL of a 1%BSA buffer solution containing 1 µg/ml enzyme alkaline phosphatase-conjugated MMP-9 were incubated on the target-treated MNPs, and the incubated state was maintained for 30 min at 25 °C, followed by washing three times with rinsing buffer. All reactions at 25 °C were made with a shaker. After completion of the immune reaction, the MNPs were performed by electrochemical measuring using a multi-array instrument after the reaction in a substrate solution. 100 μL of a tris-buffer solution containing 1mM ascorbic acid 2-phosphate (AAP) were incubated on the prepared MNPs, and the incubated state was maintained for 10 min at 25 °C, and the MNPs were then removed. In the solution, the reaction between AAP and ALP no longer occurs, and only AAP which does not react with ascorbic acid (AA), which is completed, is present. Here AA and AAP have electrochemically different physical properties and can only detect oxidation of AA in a specific voltage range. The results of the immune reaction were measured using a multichannel immunoassay system-detection developed to measure electrochemical signals. The electrochemical sensor modules are only exposed to the complete solution of the substrate reaction and can be reused after a wash, which will be useful in practical applications. A multichannel electrochemical sensor array was added to the reaction solution and subjected to chronocoulometry (CC). As a result, the measuring range is 10 pM to 10 nM, and the detection limit is 0.35 pM. In other words, the detection limit of 0.322 ng / mL and the measurement range of 0.92 - 920 ng/ ml have similar sensitivity and higher assay range as commercialized MMP-9 ELISA kit All reactions related to immune reaction were made possible by multiple reactions in 1 set unit using MNPs. Besides, by separating the signal measurement surface and the immune reaction surface, the enzyme reaction can be terminated at the same time. It is important to terminate the enzymatic reaction at the same time because it is difficult to control the reaction time in an one-by-one electrochemical immunoassay. Thereby, the developed system can provide the convenience and simplicity of the electrochemical immunoassay. Previous studies on bladder cancer in our group confirmed that the diagnostic accuracy of two or three multiple makers including MMP-9 was significantly higher than the single marker of MMP-9. The results suggest the necessity of diagnosis using various biomarkers and the importance and the advantage of a multi-channel immunoassay system as well. A multi-channel immune sensor system was developed to measure a wide range of concentrations, from low to high concentration of target markers, and to perform from 1 set of immune reactions to measurements. This work was financially supported by the research funds of the Bio & Medical Technology Development Program of the National Research Foundation (NRF-2015M3A9E2028480) and the research funds (Grant No. 10077648) of the Ministry of trade, Industry and Energy, Korea. Streptavidin coated magnetic nanoparticles were provided by Amolifescience, Co. Ltd, Korea. Figure 1