Biology subjects

Ng, C. F.

Publications and source records attributed to Ng, C. F..

4 recordsLinked to original sources

bioRxiv2026

Bead-based RNA Detection with Cas13a: Multi-scale Design and Validation

CRISPR-based assays offer sensitive and specific nucleic acid detection for a broad range of diagnostic and surveillance applications. While standard solution-based assays are simple, they typically lack the ability to detect multiple targets individually without the need for more complex instrumentation and multi-step workflows. Surface-based strategies offer greater potential for multiplexing since the identity of a target can be associated with a location on a slide or a specific bead, as in the case of DNA arrays or Luminex bead assays. However, the design principles for optimizing surface-based CRISPR assays have yet to be developed. Here we present a multi-scale analytical framework integrating nanoscale enzyme kinetics, microscale diffusion constraints, and macroscale assay parameters that we use to develop a bead-based Cas13a assay where the bead becomes fluorescent when the target RNA for that bead is detected. The assay, which we call SurfCas, uses surface-bound guide RNAs and reporter RNAs to detect one or more soluble target RNAs in a one-pot assay. We show that SurfCas has a sensitivity approaching bulk reactions, and we demonstrate multiplexed detection of target RNAs in a complex biological matrix. The design principles we establish for surface-based CRISPR assays offer guidance on how to further improve SurfCas sensitivity and scale multiplexed detection of target RNAs in a single reaction.

bioengineering↗

bioRxiv2026

LUCas: Light-Uncaged Cas13a using photocleavable interfering guide RNAs

CRISPR diagnostics have emerged as powerful tools for detecting infectious diseases, with the RNA endonuclease Cas13a enabling sensitive and specific, amplification-free RNA detection through collateral trans-cleavage of fluorescent reporters. However, background cleavage from unbound enzyme, contaminating nucleases, and unsynchronized initiation of reactions limits assay sensitivity and interpretability. A strategy to precisely control the onset of Cas13a catalytic activity, essentially a molecular "starting gun", would address these challenges and expand assay design space. Here, we introduce Light-Uncaged Cas13a (LUCas), a light controllable system that directly gates Cas13a using a photocleavable interfering guide RNA (pc-igRNA) that suppresses trans-cleavage activity even in the presence of target RNA. Brief UV illumination releases this suppression, restoring full activity. Quantitative kinetic analysis reveals an approximately 100-fold suppression of trans-cleavage activity prior to photo-activation. Importantly, LUCas also suppresses target-independent background activity, enabling a predictive, background-limited determination of assay sensitivity. Using measured kinetic parameters, we predict and experimentally validate the limit-of-detection of the LUCas system. Finally, we demonstrate a multiplexed detection strategy termed "temporal barcoding," which enables quantitative detection of viral co-infections in a single bulk reaction. Together, these results establish LUCas as a general framework for mechanistically informed, light-based control of Cas13a activity.

bioengineering↗

bioRxiv2019

A robotic platform for fluidically-linked human body-on-chips experimentation

Here we describe of an Interrogator instrument that uses liquid-handling robotics, a custom software package, and an integrated mobile microscope to enable automated culture, perfusion, medium addition, fluidic linking, sample collection, and in situ microscopic imaging of up to 10 Organ Chips inside a standard tissue culture incubator. The automated Interrogator platform maintained the viability and organ-specific functions of 8 different vascularized, 2-channel, Organ Chips (intestine, liver, kidney, heart, lung, skin, blood-brain barrier (BBB), and brain) for 3 weeks in culture when fluidically coupled through their endothelium-lined vascular channels using a common blood substitute medium. When an inulin tracer was perfused through the multi-organ Human Body-on-Chips (HuBoC) fluidic network, quantitative distributions of this tracer could be accurately predicted using a physiologically-based multi-compartmental reduced order (MCRO) in silico model of the experimental system derived from first principles. This automated culture platform enables non-invasive imaging of cells within human Organ Chips and repeated sampling of both the vascular and interstitial compartments without compromising fluidic coupling, which should facilitate future HuBoc studies and pharmacokinetics (PK) analysis in vitro.

bioengineering↗

bioRxiv2018

Human bone marrow disorders recapitulated in vitro using organ chip technology

Understanding human bone marrow (BM) pathophysiology in the context of myelotoxic stress induced by drugs, radiation, or genetic mutations is of critical importance in clinical medicine. However, study of these dynamic cellular responses is hampered by the inaccessibility of living BM in vivo. Here, we describe a vascularized human Bone Marrow-on-a-Chip (BM Chip) microfluidic culture device for modeling bone marrow function and disease states. The BM Chip is comprised of a fluidic channel filled with a fibrin gel in which patient-derived CD34+ cells and bone marrow-derived stromal cells (BMSCs) are co-cultured, which is separated by a porous membrane from a parallel fluidic channel lined by human vascular endothelium. When perfused with culture medium through the vascular channel, the BM Chip maintains human CD34+ cells and supports differentiation and maturation of multiple blood cell lineages over 1 month in culture. Moreover, it recapitulates human myeloerythroid injury responses to drugs and gamma radiation exposure, as well as key hematopoietic abnormalities found in patients with the genetic disorder, Shwachman-Diamond Syndrome (SDS). These data establish the BM Chip as a new human in vitro model with broad potential utility for studies of BM dysfunction.

bioengineering↗