Detection of low-abundance biomarker lipocalin 1 for diabetic retinopathy using optoelectrokinetic bead-based immunosensing.

Early diagnosis of diabetic retinopathy (DR) is vital but challenging. DR is a common complication and a major cause of vision loss in patients with diabetes mellitus. Without appropriate medical intervention, visual impairment may become a great burden to our healthcare system. In clinical practice, the current diagnostic methods, such as fluorescence angiography and optical coherence tomography, remain constrained by non-quantitative examinations and individual ophthalmologists' experiences. Late diagnosis often prevents early treatment. To address the constraints on current diagnostics, this study developed an optoelectrokinetic bead-based immunosensing technique for detecting lipocalin 1 (LCN1), a DR biomarker. The concentration level of LCN1 in the tears of DR patients increases with DR severity. The immunoassay was dependent on the formation of sandwiched immunocomplexes on the particles. A secondary antibody labeled with dyes/quantum dots (QDs) was used to visualize the presence of the target antigens. Rapid electrokinetic patterning (REP), an optoelectrokinetic technique, was used to dynamically enhance the fluorescent signal by concentrating the modified particles. The limit of detection (LOD) of the technique could reach 110pg/mL. Only 1.5µL of a sample fluid was required for the measurement. Our results showed that highly sensitive and improved LOD is subjected to particle stacking, small particle size, and compact cluster. By labeling different particle sizes with dyes/QDs for LCN1 and TNF-α, we successfully used REP to detect the two DR biomarkers on the same platform. The development of an optoelectrokinetic bead-based immunosensing technique can provide new insights into diagnosing other low-abundance diseases in the future.

A bead-based fluorescence immunosensing technique enabled by the integration of Förster resonance energy transfer and optoelectrokinetic concentration.

Bead-based immunosensing has been growing as a promising technology in the point-of-care diagnostics due to great flexibility. For dilute samples, functionalized particles can be used to collect dispersed analytes and act as carriers for particle manipulation. To realize rapid and visual immunosensing, Förster resonance energy transfer (FRET) was used herein to ensure only the diabetic biomarker, lipocalin 1, to be detected. The measurement was made in an aqueous droplet sandwiched between two parallel plate electrodes. With an electric field and a focused laser beam applying on the microchip simultaneously, the immunocomplexes in the droplet were further concentrated to enhance the FRET fluorescent signal. The optoelectrokinetic technique, termed rapid electrokinetic patterning (REP), has been proven to be excellent in dynamic and programmable particle manipulation. Therefore, the detection can be complete within several tens of seconds. The lower detection limit of the REP-enabled bead-based diagnosis reached nearly 5 nM. The combinative use of FRET and the optoelectrokinetic technique for the bead-based immunosensing enables a rapid measure to diagnose early stage diseases and dilute analytes.