Enteric and diarrheal diseases are a major cause of childhood illness and death in countries with developing economies. Each year, more than half of a million children under the age of five die from these diseases. We have developed a portable, microfluidic platform capable of simultaneous, multiplexed detection of several of the bacterial pathogens that cause these diseases. This platform can perform fast, sensitive immunoassays directly from relevant, complex clinical matrices such as stool without extensive sample cleanup or preparation. Using only 1 µL of sample per assay, we demonstrate simultaneous multiplexed detection of four bacterial pathogens implicated in diarrheal and enteric diseases in less than 20 min.

The challenges of diagnosing infectious disease, especially in the developing world, and the shortcomings of available instrumentation have exposed the need for portable, easy-to-use diagnostic tools capable of detecting the wide range of causative microbes while operating in low resource settings. We present a centrifugal microfluidic platform that combines ultrasensitive immunoassay and isothermal amplification-based screening for the orthogonal detection of both protein and nucleic acid targets at the point-of-care. A disposable disc with automatic aliquoting inlets is paired with a non-contact heating system and precise rotary control system to yield an easy-to-use, field-deployable platform with versatile screening capabilities. The detection of three enterotoxins (cholera toxin, Staphylococcal enterotoxin B, and Shiga-like toxin 1) and three enteric bacteria (C. jejuni, E. coli, and S. typhimurium) were performed independently and shown to be highly sensitive (limit of detection = 1.35–5.50 ng/mL for immunoassays and 1–30 cells for isothermal amplification), highly exclusive in the presence of non-specific targets, and capable of handling a complex sample matrix like stool. The full panel of toxins and bacteria were reliably detected simultaneously on a single disc at clinically relevant sample concentrations in less than an hour. The ability of our technology to detect multiple analyte types in parallel at the point-of-care can serve a variety of needs, from routine patient care to outbreak triage, in a variety of settings to reduce disease impact and expedite effective treatment.

Here, we describe the isolation of 18 unique anti SARS-CoV-2 human single-chain antibodies from an antibody library derived from healthy donors. The selection used a combination of phage and yeast display technologies and included counter-selection strategies meant to direct the selection of the receptor-binding motif (RBM) of SARS-CoV-2 spike protein’s receptor binding domain (RBD2). Selected antibodies were characterized in various formats including IgG, using flow cytometry, ELISA, high throughput SPR, and fluorescence microscopy. We report antibodies’ RBD2 recognition specificity, binding affinity, and epitope diversity, as well as ability to block RBD2 binding to the human receptor angiotensin-converting enzyme 2 (ACE2) and to neutralize authentic SARS-CoV-2 virus infection in vitro. We present evidence supporting that: 1) most of our antibodies (16 out of 18) selectively recognize RBD2; 2) the best performing 8 antibodies target eight different epitopes of RBD2; 3) one of the pairs tested in sandwich assays detects RBD2 with sub-picomolar sensitivity; and 4) two antibody pairs inhibit SARS-CoV-2 infection at low nanomolar half neutralization titers. Based on these results, we conclude that our antibodies have high potential for therapeutic and diagnostic applications. Importantly, our results indicate that readily available non immune (naïve) antibody libraries obtained from healthy donors can be used to select high-quality monoclonal antibodies, bypassing the need for blood of infected patients, and offering a widely accessible and low-cost alternative to more sophisticated and expensive antibody selection approaches (e.g. single B cell analysis and natural evolution in humanized mice).

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