Photopolymerization-based signal amplification

Summary

Photopolymerization-based signal amplification (PBA) is one method to amplify detection signals from molecular recognition events in an immunoassay by utilizing a radical polymerization initiated by illumination of light. To contrast between a negative and a positive result, PBA is linked to a colorimetric method, thereby resulting in a change in color when a targeted analyte is detected, i.e., a positive signal. PBA is also used to quantify a concentration of the analyte by measuring intensity of the color.

Method

PBA is achieved by sequentially adding three kinds of solutions to a test strip and illuminating it with green light. First, a droplet of a patient’s sample is loaded on the test strip whose surface is covered with immobilized antibodies. If the sample has target antigens, they bind to the immobilized antibodies. (Figure 1a) Second, eosin-conjugated antibodies are added to the patient’s sample. This second antibody specifically binds with the bound antigens, thereby causing each bound antigen to be sandwiched between the first antibody and the eosin-conjugated antibody. (Figure 1b) After ten minutes, the droplet on the surface is rinsed away in order to make sure that only the sandwiched binding complexes are left on the surface before adding the third solution.

Lastly, a droplet of monomers -- PEGDA and N-vinyl pyrrolidone -- and phenolphthalein mixture is added to the test strip, and the droplet is illuminated with green visible light, by which eosin molecules become excited and produce radicals. (Figure 1c) As a result, propagation is caused and polymers are formed. Since phenolphthalein molecules are surrounded by the polymers and thus left on the surface even after another rinse, the test strip turns red when a base is added. (Figure 1d) On the other hand, if the patient’s sample does not include any targeted antigens, the sandwiched binding complexes on the surface will not be formed, which leads to no red color in the end.