ELISA (enzyme linked immunosorbant assay) methods are being developed on microfluidics in our lab. My projects involve improving sensitivity of ELISA using microfluidic platform. We fabricate and use glass based microfluidic chips. I have seen some papers which describe ELISA on PDMS chips. The glass chips are cheaper to fabricate, does not require sophisticated instrumentations. We use photolithography, chemical wet etching and room temperature bonding technology to fabricate glass microfluidic chips. I feel I am good at fabricating chips. I don't have any problems till now from the beginning on fabrication. Actually I learned this step quickly. After being able to fabricate chips, I started doing static ELISA. Here comes the problem. Its being two weeks I am not able to get expected data.
ELISA in microchannel involves different coating steps. First, channels are cleaned with sodium hydroxide to form negatively charged-hydrophilic glass surface. We use horse redish peroxide (HRPase) as enzyme which converts amplex red (fluorophore) to resorufin (a fluorescent anionic molecule). After completing appropriate ELISA steps, amplex red containing hydrogen peroxide is introduced into the channel and the fluorescent signal of resorufin molecules produced by enzymatic conversion of amplex red is monitored.
Fluorescent signal should correspond to the concentration of antigen in the sample. At this point in my experiments, the signal does not increase with the increase in the concentration of analyte. I repeated the same experiment 4 times and got the same result. I might be repeating the same stupid mistake again and again. There must be a systemic error which I am not able to figure out till now.
If you have any clue, advise plz let me know. I would be very great full.
Thank you for reading my experience with ELISA.