Lab-on-a-chip immunoassay for multiple antibodies using microsphere light scattering and quantum dot emission

Detection of multiple biomarkers has recently received great interest from the biosensors community. These diagnostic methods must be rapid, specific, sensitive, and cost-effective. In 2006, our group demonstrated a successful lab-on-a-chip immunoassay using microsphere light scattering, which is essentially a one-step, automated protocol, on a reusable chip. In the past, this had been difficult due to the limitations of microfluidic mixing and false-positive readings of particle immunoassays in a chip environment. In this current study, we conjugated quantum dots (QDs) onto microspheres to enable multiplex assays as well as to enhance the limit of detection (LOD). We named this configuration "nano-on-micro" or "NOM." Upon radiation with UVlight (380 nm), a stronger light scattering signal is observed with NOMs than QDs or microspheres alone. Additionally, NOMs are easier to handle than QDs. Since QDs also provide fluorescent emission, we are able to utilize an increase in light scattering for detecting antigen-antibody reaction and a decrease in QD emission to identify which antibody (or antigen) is present. Two types of NOM combinations were used. One batch of microspheres was coated with QDs emitting at 655 nm and mouse IgG (mlgG); the other with QDs emitting at 605 nm and bovine serum albumin (BSA). A mixture of these two NOMs was used to identify either anti-mlgG or anti-BSA. NOM particles and target solutions were mixed in a microfluidic device and on-chip detection was performed using proximity optical fibers. Forward light scattering at 380 nm was collected. With the positive target, the scattering signal was increased. The LOD was 25 ng ml^-1 (165 pM) with p<0.05. Fluorescent emission (655 or 605 nm) was simultaneously collected. With the positive target, the emission signal was attenuated. Therefore, we were able to detect two different antibodies simultaneously with two different detection protocols. We believe this NOM bioassay has the ability to screen for and detect multiple antibodies with minimal sample processing and handling.