Comparison of Illumination Methods for Flow-Through Optofluidic Biosensors.

Optofluidic biosensors have become an important medical diagnostic tool because they allow for rapid, high-sensitivity testing of small samples compared to standard lab testing. For these devices, the practicality of use in a medical setting depends heavily on both the sensitivity of the device and the ease of alignment of passive chips to a light source. This paper uses a model previously validated by comparison to physical devices to compare alignment, power loss, and signal quality for windowed, laser line, and laser spot methods of top-down illumination.

FPGA Integrated Optofluidic Biosensor for Real-Time Single Biomarker Analysis.

Integrated optofluidic biosensors can fill the need for sensitive, amplification-free, multiplex single molecule detection which is relevant for containing the spread of infectious diseases such as COVID-19. Here, we demonstrate a rapid sample-to-answer scheme that uses a field programmable gate array (FPGA) to enable live monitoring of single particle fluorescence analysis on an optofluidic chip. Fluorescent nanobeads flowing through a micro channel are detected with 99% accuracy and particle concentrations in clinically relevant ranges from 3.4×104 to 3.4 × 106/ml are determined within seconds to a few minutes without the need for post-experiment data extraction and analysis. In addition, other extract salient experimental parameters such as dynamic flow rate changes can be monitored in real time. The sensor is validated with real-time fluorescence detection of single bacterial plasmid DNA at attomolar concentrations, showing excellent promise for implementation as a point of care (POC) diagnostic tool.

Optical trapping assisted label-free and amplification-free detection of SARS-CoV-2 RNAs with an optofluidic nanopore sensor.

Ultrasensitive, versatile sensors for molecular biomarkers are a critical component of disease diagnostics and personalized medicine as the COVID-19 pandemic has revealed in dramatic fashion. Integrated electrical nanopore sensors can fill this need via label-free, direct detection of individual biomolecules, but a fully functional device for clinical sample analysis has yet to be developed. Here, we report amplification-free detection of SARS-CoV-2 RNAs with single molecule sensitivity from clinical nasopharyngeal swab samples on an electro-optofluidic chip. The device relies on optically assisted delivery of target carrying microbeads to the nanopore for single RNA detection after release. A sensing rate enhancement of over 2,000x with favorable scaling towards lower concentrations is demonstrated. The combination of target specificity, chip-scale integration and rapid detection ensures the practicality of this approach for COVID-19 diagnosis over the entire clinically relevant concentration range from 104-109 copies/mL.

Performance Comparison of Flow-Through Optofluidic Biosensor Designs.

Optofluidic flow-through biosensors are being developed for single particle detection, particularly as a tool for pathogen diagnosis. The sensitivity of the biosensor chip depends on design parameters, illumination format (side vs. top), and flow configuration (parabolic, two- and three-dimensional hydrodynamic focused (2DHF and 3DHF)). We study the signal differences between various combinations of these design aspects. Our model is validated against a sample of physical devices. We find that side-illumination with 3DHF produces the strongest and consistent signal, but parabolic flow devices process a sample volume more quickly. Practical matters of optical alignment are also discussed, which may affect design choice.

Optofluidic multiplex detection of single SARS-CoV-2 and influenza A antigens using a novel bright fluorescent probe assay.

The urgency for the development of a sensitive, specific, and rapid point-of-care diagnostic test has deepened during the ongoing COVID-19 pandemic. Here, we introduce an ultrasensitive chip-based antigen test with single protein biomarker sensitivity for the differentiated detection of both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A antigens in nasopharyngeal swab samples at diagnostically relevant concentrations. The single-antigen assay is enabled by synthesizing a brightly fluorescent reporter probe, which is incorporated into a bead-based solid-phase extraction assay centered on an antibody sandwich protocol for the capture of target antigens. After optimization of the probe release for detection using ultraviolet light, the full assay is validated with both SARS-CoV-2 and influenza A antigens from clinical nasopharyngeal swab samples (PCR-negative spiked with target antigens). Spectrally multiplexed detection of both targets is implemented by multispot excitation on a multimode interference waveguide platform, and detection at 30 ng/mL with single-antigen sensitivity is reported.