Sensitive, Single-Pot, Two-Stage Assay for Hepatitis Viruses.

When left untreated, hepatitis B virus (HBV) and hepatitis C virus (HCV) infections may cause severe illnesses. Since these infections remain asymptomatic for many years, routine screening of populations at risk is critical for therapy initiation. The current standard of care mandates a screening antibody test for HCV, followed by a confirmatory laboratory-based molecular test and treatment. Multiple visits to the clinic are inconvenient, and many patients fail to follow up. To address this challenge, we have developed sensitive, two-stage, isothermal molecular (Penn-RAMP) point-of-care tests to enable test and treat strategy. Penn-RAMP's first stage is comprised of recombinase polymerase amplification (RPA), while its second stage is comprised of loop-mediated isothermal amplification (LAMP). Penn-RAMP is more sensitive than LAMP or RPA alone. We designed a custom pre-LAMP buffer to maximize the volume of RPA products that can be added to the LAMP reaction mix without inhibition and forward and backward primers. Penn-RAMP was implemented in a single pot comprised of two compartments separated by a thermally removable barrier. RAMP's first stage is carried out above the barrier at the RPA incubation temperature. When the pot is heated to the LAMP incubation temperature, the barrier melts away, and the RPA reaction volume mixes with the pre-LAMP buffer, facilitating second-stage amplification. This entire process can be carried out with minimal instrumentation. Our HBV and HCV tests detect, respectively, as few as 10 and 25 virions within 30 min. The viral load can be estimated based on signal threshold time.

Manually-Operated, Slider Cassette for Multiplexed Molecular Detection at the Point of Care.

Effective control of epidemics, individualized medicine, and new drugs with virologic response-dependent dose and timing require, among other things, simple, inexpensive, multiplexed molecular detection platforms suitable for point of care and home use. Herein, we describe our progress towards developing such a platform that includes sample lysis, nucleic acid isolation, concentration, purification, and amplification. Our diagnostic device comprises a sliding component that houses the nucleic acid isolation membrane and a housing containing three amplification reaction chambers with dry stored reagents, blisters with buffers and wash solutions, and absorption pads to facilitate capillarity pull and waste storage. After sample introduction, the user slides the slider within the housing from one station to another to carry out various unit operations. The slider motion induces blisters to discharge their contents, effectuating washes, and eventual elution of captured nucleic acids into reaction chambers. The slider cassette mates with a processor that incubates isothermal amplification but can also be made to operate instrumentation-free. We demonstrate our cassette's utility for the co-detection of the human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV). These three blood-borne pathogens co-infect many people worldwide with severe personal and public health consequences.

Rapid single-molecule digital detection of protein biomarkers for continuous monitoring of systemic immune disorders.

Digital protein assays have great potential to advance immunodiagnostics because of their single-molecule sensitivity, high precision, and robust measurements. However, translating digital protein assays to acute clinical care has been challenging because it requires deployment of these assays with a rapid turnaround. Herein, we present a technology platform for ultrafast digital protein biomarker detection by using single-molecule counting of immune-complex formation events at an early, pre-equilibrium state. This method, which we term "pre-equilibrium digital enzyme-linked immunosorbent assay" (PEdELISA), can quantify a multiplexed panel of protein biomarkers in 10 µL of serum within an unprecedented assay incubation time of 15 to 300 seconds over a 104 dynamic range. PEdELISA allowed us to perform rapid monitoring of protein biomarkers in patients manifesting post-chimeric antigen receptor T-cell therapy cytokine release syndrome, with ∼30-minute sample-to-answer time and a sub-picograms per mL limit of detection. The rapid, sensitive, and low-input volume biomarker quantification enabled by PEdELISA is broadly applicable to timely monitoring of acute disease, potentially enabling more personalized treatment.