Research Approaches
In order to make diagnostic tools accessible, certain characteristics need to be considered, including reliability, simplicity, usability, cost effectiveness, and portability. Addressing these criteria is essential to meet the diverse needs of global populations, particularly in under-resourced areas.
Currently, we are advancing our objectives through three primary technical approaches: 1) Digital immuno-sensing; 2) Microfluidic based assay automation; 3) CRISPR molecular switch. These innovative approaches are geared towards transforming the accessibility of diagnostic solutions, making them more adaptable to various settings without compromising on performance.
Digital immuno-sensing
The beginning of the 21st century has witnessed significant advances in pursuit of ultra-high sensitivity for protein biomarker detections in the research settings. In 2010, the single-molecule enzyme-linked immunosorbent assay (digital ELISA) by Rissin et al. first introduced the revolutionary “digital immuno-sensing” concept into the field of protein detection. In digital ELISA, individual protein molecules were directly counted via the discrete fluorescent digital signals, achieving PCR-like sensitivity for protein detection. Although sensors in digital assays only need to distinguish between positive and negative signals, digital ELISA mainly relies on fluorescent labels and requires sophisticated and nonportable laboratory based high-resolution fluorescence microscopy system.
Compared with fluorescence, direct visualization as a readout method can make digital immuno-sensing more accessible by simplifying the system and reducing the cost, since no extra optical system is needed to filter excitation and emission light.
We aim to explore new signaling strategies, involving colorimetric or morphology signals, for immuno-sensing: keep its ultrahigh sensitivity yet make it more accessible, simpler and more affordable. One of our initial approach was to integrate digital immuno-sensing with immobilized-microbubbling, one distinguishable physical transformation process involving quick volume amplification with minimum mass increase. We envisioned microbubbling as an ideal “bridge” to connect the “invisible” nano-world to the “visible” micro-world.
Microfluidic based assay automation
Microfluidic sensing platfor that can integrate multiple functionalities on a single device are with several advantages over conventional immunodiagnostics methods, including automation, portability, faster detection, portability, sample volume reduction, and cost reduction, making microfludic-based assays a well-established component for accessible diagnosis. Central to microfluidic assays is the control of sequential flow of assay reagents flow within microconduits. Most traditional microfluidic systems depend on complicated, nonportable and expensive peripheral equipment to effect and control the flow of assay reagents. Another notorious limitation of microfluidics in general is the nature of steady laminar flow in microchannels, where mass transfer is dominated by the diffusivity of reagents not by turbulent transport at the macroscale. To address these issues, we are innovating all-in-one automated handheld microfluidic systems with programmable sequential oscillatory flow control. In these systems, fluids are subjected to a back-and-forth motion by utilizing a periodically changed pressure using mini peristaltic pump, which significantly increases the effective travel footprints. These periodic oscillatory flows effectively increased the mixing and binding performance between the target molecules in the sample solution and surface functionalized with binding ligands via the repeated turbulent transport at the connected macroscale reservoirs and increased molecular collision induced by the periodic oscillatory flows.
CRISPR switch
(allosteric switch)
Recently, the clustered regularly interspaced short
palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has attracted considerable scientific interest. In addition to its application in gene editing and cancer therapy, CRISPR/Cas technology has also been used in molecular diagnosis, involving the selective activation of the nuclease activity of Cas-associated proteins under the direction of a guide RNA to target nucleic acids. For example, groundbreaking
CRISPR/Cas biosensing methods, including SHERLOCK
(specific high-sensitivity enzymatic reporter unlocking) and DETECTR (DNA endonuclease-targeted CRISPR trans reporter), have successfully achieved sensitive pathogen detection and genotyping. However, most current CRISPR DX systems only detect nucleic acids. We aim to leverage the power allosteric switch properties of CRISPR simplify the setup and increase the accessibility for the detection of a variety of biomarkers including non-nucleic acid biomolecules.