Four-Channel Photothermal Plate Reader for High-Throughput Nanoparticle-Amplified Immunoassay

We enhanced the sample throughput of microplate-based photothermal detection by using a semicylindrical prism to expand a point laser source to a long beam for illuminating multiple wells. Coupled with four epoxy-coated thermocouples in alignment with wells on a 96-well microplate, four parallel immunoassays of C-reaction protein (CRP) with antibody-conjugated gold nanoparticles can be simultaneously performed. The sample throughput is further increased by mounting the Styrofoam-enclosed microplate onto a translational/elevator stage so that immunoassays and thermocouple rinse/drying cycles can be implemented in a programmed fashion. The automated assay with three rinse/drying cycles takes only 34.5 min for four samples or 8.62 min/sample, whereas the manual mode with a single thermocouple and a point light source requires at least 66 min for just one sample. With careful calibration of the energy distribution of the expanded laser beam and controllable immersion of the thermocouples, excellent well-to-well (RSD = 1.3%) and cycle-to-cycle (RSD = 4.0%) reproducibility can be attained. The temperature changes can be correlated with the CRP concentration by the Langmuir isotherm, and the low limit of detection, 0.52 ng/mL or 4.33 pM, is well below the plasma CRP levels of both healthy people (<5 μg/mL) and patients (10-500 μg/mL). The serum CRP concentrations quantified by our plate reader are in excellent agreement with the immunoturbidimetric results, demonstrating that this cost-effective, robust, and high-throughput mode for microplate-based immunoassays is amenable to detecting biomarkers in many clinical samples.