A centrifugal fluidic immunoassay for ocular diagnostics with an enzymatically hydrolyzed fluorogenic substrate.

We present a novel "Lab-on-a-Disk" platform and demonstrate its capability for rapid and sensitive measurement of vascular endothelial growth factor (VEGF) intended for patients suffering from diabetic retinopathy (DR) and age-related macular degeneration (AMD). This approach combines sedimentation principles applied to microspheres under centrifugal force with signal amplification using an enzyme and a fluorogenic substrate for readout. The simple single channel per assay platform separates, washes and concentrates antibody-coated microspheres from excess label to produce a sensitive fluorogenic response proportional to the amount of VEGF in the sample. This platform has comparable sensitivity to conventional ELISA and can generate a readout within 16-18 min with no sample preparation beyond mixing assay reagents and loading on the disk. In the context of ocular diagnostics, this device has the potential to facilitate accurate dosing of anti-VEGF medications utilized to treat DR and AMD, as well as identify patients whose ocular VEGF levels are not elevated and who would therefore not benefit from standard anti-VEGF medications.

Microfluidic digital isoelectric fractionation for rapid multidimensional glycoprotein analysis.

Here we present an integrated microfluidic device for rapid and automated isolation and quantification of glycoprotein biomarkers directly from biological samples on a multidimensional analysis platform. In the first dimension, digital isoelectric fractionation (dIEF) uses discrete pH-specific membranes to separate proteins and their isoforms into precise bins in a highly flexible spatial arrangement on-chip. dIEF provides high sample preconcentration factors followed by immediate high-fidelity transfer of fractions for downstream analysis. We successfully fractionate isoforms of two potential glycoprotein cancer markers, fetuin and prostate-specific antigen (PSA), with 10 min run time, and results are compared qualitatively and quantitatively to conventional slab gel IEF. In the second dimension, functionalized monolithic columns are used to capture and detect targeted analytes from each fraction. We demonstrate rapid two-dimensional fractionation, immunocapture, and detection of C-reactive protein (CRP) spiked in human serum. This rapid, flexible, and automated approach is well-suited for glycoprotein biomarker research and verification studies and represents a practical avenue for glycoprotein isoform-based diagnostic testing.