Enzyme-linked immunosorbent assay (ELISA) has been widely used for detection and quantification of biological agents (mainly proteins and polypeptides). Its high selectivity and sensitivity draw great attractions in clinical, food safety, and environmental applications. However, conventional ELISA involves a tedious and labor-intensive protocol due to its series of steps and long incubation times, which are mostly attributed to inefficient mass transport of the regents from bulk solution to solid surface.
Integrated Microfluidic platforms can easily provide high specific surface area, low reagent consumptions and short diffusion length. Such microdevices can significantly reduce the assay time and sample or reagents consumptions, enhance the reaction efficiency, and provide highly portable systems. Several developmental advances have been realized in miniaturization of capillary electrophoresis-based immunoassays and microchip-based ELISA. Unfortunately, each step of these ELISA chips was still carried out manually. In our laboratory, an integrated microfluidic device on a compact disk (CD) has been developed to automatically perform ELISA for rat IgG from hybridoma cell culture.
The design of our CD-like ELISA chip is shown in Figure 1a with 24 sets of ELISA on a 5” disk. One set is highlighted on Figure 1b and a photo of computer numerically controlled (CNC)-machined disc is shown in Figure 1c. The microfluidic platform combined several microfluidic functions (e.g., capillary valving, centrifugal pumping, and flow sequencing). Centrifugal and capillary forces are used to control the flow sequence of several different solutions involved in ELISA process. The capillary force will hold liquid from a small channel to an expanded area, while the centrifugal force may release the fluid from its reservoir when it is larger than the capillary force. A PC computer controls the rotation speed of the disk to achieve proper flow sequencing.
The whole ELISA process can be carried out automatically after preloading necessary reagents into corresponding reservoirs before test. At a low rotation speed, the antigen solution is released into the measurement site first. To enhance antigen binding and shorten the incubation time, the solid surface at the measurement site needs to be modified so that they have high protein affinity. After incubation, the washing solution is released to wash out the unbounded antigen to the waste reservoir. Then the blocking protein, the washing solution, the primary antibody, the washing solution, the conjugate solution, the washing solution, and finally the substrate are delivered sequentially to the measurement site when increasing rotation speeds. Figure 2 shows the calibration curves for the 96-well microtiter plate and the microchip. Results in the microchip have a similar signal as that of the microtiter plate within the same detection range. The detection limit is 5 mg/L (31nM) of the rat IgG (MW ~ 160,000). Since the concentration of the rat IgG from the hybridoma culture is typically in the range of 1 to 100 mg/L, this microfluidic platform is expected to be suitable for practical measurement.
Compared to 96-well microtiter plate, the consumption of reagents (antigen and antibody) and the assay time are reduced to about one tenth of that used in the 96-well microtiter plate; The total assay time was a little more than an hour, which is much less than that required for the microtiter plate. The assay conditions chosen currently are not fully optimized. The ultimate detection limit is determined by many factors such as the choice of the reagents and the immobilization of the first component on the solid surface, etc. Through improving the binding capability of the first reagent and optimizing the assay conditions, the reagent consumption can be further reduced to less than 1 ml for each step and each assay can be finished within half an hour.
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