Sensitive optical detection of clinically relevant biomarkers in affordable microfluidic devices: Overcoming substrate diffusion limitations

[Display omitted] •We unravelled the full potential of chromogenic substrates for optical detection biomarkers in microfluidic devices.•Commercial chromogenic substrate formulations are suboptimum tuned to meso-scale systems.•Optical detection of clinically relevant biomarkers is shown for a microfluidic device with optimized OPD:H2O2 formulation.•Assay times and lower limit of detection for PSA and IL-1β reduced by one order of magnitude. One of the biggest challenges in miniaturization of optical immunoassays is the short light path distance of microchannels/microcapillaries. Protein biomarkers are often presented in circulating blood in the picomolar-femtomolar range, requiring exceptional levels of sensitivity that cannot be met with traditional chromogenic substrates and without sophisticated, bulky detection systems. This study discloses an effective strategy for increasing the sensitivity and shorten the total test time for sandwich ELISAs in microfluidic devices optically interrogated, based on enhancing enzymatic amplification. We found that activity of Horseradish Peroxidase (HRP) in mesofluidic systems is highly limited by diffusion, therefore increasing the concentration of enzymatic substrate in these systems does not translate into an enhancement in enzymatic conversation. The opposite happens in microfluidic systems due to short diffusion distances, however increased concentration of the second enzymatic substrate, hydrogen peroxide (H2O2), leads to enzyme inhibition as herein reported. Consequently, we found that the molar ratio of o-phenylenediamine (OPD) to hydrogen peroxide from commercially substrate formulations is not suitable for miniaturized systems. Sandwich ELISA quantitation of a cancer biomarker PSA and human cytokine IL-1β in fluoropolymer microfluidic strips revealed over one order of magnitude increase in sensitivity and 10-fold decrease in incubation time by simply changing the molar ratio of OPD:H2O2 from 1:3 to 1:1 and increasing OPD concentration from 1 to 4mg/ml. This enhancement in enzymatic amplification offers finally the sensitivity required for optical interrogation of novel portable and affordable microfluidic devices with inexpensive and ubiquitous smartphones and flatbed scanners.