Environmentally Resilient Microfluidic Point-of-Care Immunoassay Enables Rapid Diagnosis of Talaromycosis.

Point-of-care tests (POCTs) are increasingly being used in field settings, particularly outdoors. The performance of current POCTs─most commonly the lateral flow immunoassay─can be adversely affected by ambient temperature and humidity. We developed a self-contained immunoassay platform─the D4 POCT─that can be conducted at the POC by integrating all reagents in a capillary-driven passive microfluidic cassette that minimizes user intervention. The assay can be imaged and analyzed on a portable fluorescence reader─the D4Scope─and provide quantitative outputs. Here, we systematically investigated the resilience of our D4 POCT to varied temperature and humidity and to physiologically diverse human whole blood samples that span a wide range of physiological hematocrit (30-65%). For all conditions, we showed that the platform maintained high sensitivity (0.05-0.41 ng/mL limits of detection). The platform also demonstrated good accuracy in reporting true analyte concentration across environmental extremes when compared to the manually operated format of the same test to detect a model analyte─ovalbumin. Additionally, we engineered an improved version of the microfluidic cassette that improved the ease-of-use of the device and shortened the time-to-result. We implemented this new cassette to create a rapid diagnostic test to detect talaromycosis infection in patients with advanced HIV disease at the POC, demonstrating comparable sensitivity and specificity to the laboratory test for the disease.

Engineering Innovative Interfaces for Point-of-Care Diagnostics.

The ongoing Coronavirus disease 2019 (COVID-19) pandemic illustrates the need for sensitive and reliable tools to diagnose and monitor diseases. Traditional diagnostic approaches rely on centralized laboratory tests that result in long wait times to results and reduce the number of tests that can be given. Point-of-care tests (POCTs) are a group of technologies that miniaturize clinical assays into portable form factors that can be run both in clinical areas --in place of traditional tests-- and outside of traditional clinical settings --to enable new testing paradigms. Hallmark examples of POCTs are the pregnancy test lateral flow assay and the blood glucose meter. Other uses for POCTs include diagnostic assays for diseases like COVID-19, HIV, and malaria but despite some successes, there are still unsolved challenges for fully translating these lower cost and more versatile solutions. To overcome these challenges, researchers have exploited innovations in colloid and interface science to develop various designs of POCTs for clinical applications. Herein, we provide a review of recent advancements in lateral flow assays, other paper based POCTs, protein microarray assays, microbead flow assays, and nucleic acid amplification assays. Features that are desirable to integrate into future POCTs, including simplified sample collection, end-to-end connectivity, and machine learning, are also discussed in this review.

COVID-19 Diagnosis and SARS-CoV-2 Strain Identification by a Rapid, Multiplexed, Point-of-Care Antibody Microarray.

Antigen tests to detect SARS-CoV-2 have emerged as a promising rapid diagnostic method for COVID-19, but they are unable to differentiate between variants of concern (VOCs). Here, we report a rapid point-of-care test (POC-T), termed CoVariant-SPOT, that uses a set of antibodies that are either tolerant or intolerant to spike protein mutations to identify the likely SARS-CoV-2 strain concurrent with COVID-19 diagnosis using antibodies targeting the nucleocapsid protein. All reagents are incorporated into a portable, multiplexed, and sensitive diagnostic platform built upon a nonfouling polymer brush. To validate CoVariant-SPOT, we tested recombinant SARS-CoV-2 proteins, inactivated viruses, and nasopharyngeal swab samples from COVID-19 positive and negative individuals and showed that CoVariant-SPOT can readily distinguish between two VOCs: Delta and Omicron. We believe that CoVariant-SPOT can serve as a valuable adjunct to next-generation sequencing to rapidly identify variants using a scalable and deployable POC-T, thereby enhancing community surveillance efforts worldwide and informing treatment selection.

Technologies for Frugal and Sensitive Point-of-Care Immunoassays.

Immunoassays are a powerful tool for sensitive and quantitative analysis of a wide range of biomolecular analytes in the clinic and in research laboratories. However, enzyme-linked immunosorbent assay (ELISA)-the gold-standard assay-requires significant user intervention, time, and clinical resources, making its deployment at the point-of-care (POC) impractical. Researchers have made great strides toward democratizing access to clinical quality immunoassays at the POC and at an affordable price. In this review, we first summarize the commercially available options that offer high performance, albeit at high cost. Next, we describe strategies for the development of frugal POC assays that repurpose consumer electronics and smartphones for the quantitative detection of analytes. Finally, we discuss innovative assay formats that enable highly sensitive analysis in the field with simple instrumentation.

Smartphone Enabled Point-of-Care Detection of Serum Biomarkers.

Sandwich immunoassays are the gold standard for detection of protein analytes. Here, we describe an ultrasensitive point-of-care sandwich immunoassay platform for the detection of biomarkers directly from blood or serum using a custom-built smartphone detector. Testing undiluted blood or serum is challenging due to the complexity of the matrix. Proteins nonspecifically adsorb to and cells often adhere to the assay surface, which can drastically impact the analytical sensitivity of the assay. To address this problem, our assay is built upon a "nonfouling" polymer brush "grafted from" a glass slide, which eliminates nearly all nonspecific binding and therefore increases the signal-to-noise ratio and greatly improves the analytical performance of the test. The two components required to perform a sandwich immunoassay are inkjet-printed directly onto the surface: (1) "stable" capture antibodies that remain entrapped in the brush even after exposure to a liquid sample and (2) fluorescently labeled "soluble" detection antibodies that dissolve upon exposure to a liquid sample. The polymer brush provides hydration to the antibodies, allowing them to remain stable and active over prolonged periods of time. When a liquid sample containing a biomarker of interest is dispensed onto the chip, the detection antibodies dissolve and diffuse to the stable capture spots forming a complex that sandwiches the analyte and that has a fluorescence intensity proportional to the concentration of the biomarker in solution, which can be measured using a custom-built smartphone detector. As multiple capture antibodies can be printed as discrete capture spots, the assay can be easily multiplexed without the need for multiple fluorophores. This chip and detector platform can be utilized for the point-of-care detection of low-abundance biomarkers directly from blood or serum in low-resource settings.

Rapid test to assess the escape of SARS-CoV-2 variants of concern.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are concerning in the ongoing coronavirus disease 2019 (COVID-19) pandemic. Here, we developed a rapid test, termed CoVariant-SCAN, that detects neutralizing antibodies (nAbs) capable of blocking interactions between the angiotensin-converting enzyme 2 receptor and the spike protein of wild-type (WT) SARS-CoV-2 and three other variants: B.1.1.7, B.1.351, and P.1. Using CoVariant-SCAN, we assessed neutralization/blocking of monoclonal antibodies and plasma from COVID-19­positive and vaccinated individuals. For several monoclonal antibodies and most plasma samples, neutralization against B.1.351 and P.1 variants is diminished relative to WT, while B.1.1.7 is largely cross-neutralized. We also showed that we can rapidly adapt the platform to detect nAbs against an additional variant­B.1.617.2 (Delta)­without reengineering or reoptimizing the assay. Results using CoVariant-SCAN are consistent with live virus neutralization assays and demonstrate that this easy-to-deploy test could be used to rapidly assess nAb response against multiple SARS-CoV-2 variants.

Cellphone enabled point-of-care assessment of breast tumor cytology and molecular HER2 expression from fine-needle aspirates.

Management of breast cancer in limited-resource settings is hindered by a lack of low-cost, logistically sustainable approaches toward molecular and cellular diagnostic pathology services that are needed to guide therapy. To address these limitations, we have developed a multimodal cellphone-based platform-the EpiView-D4-that can evaluate both cellular morphology and molecular expression of clinically relevant biomarkers directly from fine-needle aspiration (FNA) of breast tissue specimens within 1 h. The EpiView-D4 is comprised of two components: (1) an immunodiagnostic chip built upon a "non-fouling" polymer brush-coating (the "D4") which quantifies expression of protein biomarkers directly from crude cell lysates, and (2) a custom cellphone-based optical microscope ("EpiView") designed for imaging cytology preparations and D4 assay readout. As a proof-of-concept, we used the EpiView-D4 for assessment of human epidermal growth factor receptor-2 (HER2) expression and validated the performance using cancer cell lines, animal models, and human tissue specimens. We found that FNA cytology specimens (prepared in less than 5 min with rapid staining kits) imaged by the EpiView-D4 were adequate for assessment of lesional cellularity and tumor content. We also found our device could reliably distinguish between HER2 expression levels across multiple different cell lines and animal xenografts. In a pilot study with human tissue (n = 19), we were able to accurately categorize HER2-negative and HER2-positve tumors from FNA specimens. Taken together, the EpiView-D4 offers a promising alternative to invasive-and often unavailable-pathology services and may enable the democratization of effective breast cancer management in limited-resource settings.

Multiplexed, quantitative serological profiling of COVID-19 from blood by a point-of-care test.

Highly sensitive, specific, and point-of-care (POC) serological assays are an essential tool to manage coronavirus disease 2019 (COVID-19). Here, we report on a microfluidic POC test that can profile the antibody response against multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens-spike S1 (S1), nucleocapsid (N), and the receptor binding domain (RBD)-simultaneously from 60 µl of blood, plasma, or serum. We assessed the levels of antibodies in plasma samples from 31 individuals (with longitudinal sampling) with severe COVID-19, 41 healthy individuals, and 18 individuals with seasonal coronavirus infections. This POC assay achieved high sensitivity and specificity, tracked seroconversion, and showed good concordance with a live virus microneutralization assay. We can also detect a prognostic biomarker of severity, IP-10 (interferon-γ-induced protein 10), on the same chip. Because our test requires minimal user intervention and is read by a handheld detector, it can be globally deployed to combat COVID-19.

Multiplexed, quantitative serological profiling of COVID-19 from a drop of blood by a point-of-care test.

Highly sensitive, specific, and point-of-care (POC) serological assays are an essential tool to manage the COVID-19 pandemic. Here, we report on a microfluidic, multiplexed POC test that can profile the antibody response against multiple SARS-CoV-2 antigens - Spike S1 (S1), Nucleocapsid (N), and the receptor binding domain (RBD) - simultaneously from a 60 microliter drop of blood, plasma, or serum. We assessed the levels of anti-SARS-CoV-2 antibodies in plasma samples from 19 individuals (at multiple time points) with COVID-19 that required admission to the intensive care unit and from 10 healthy individuals. This POC assay shows good concordance with a live virus microneutralization assay, achieved high sensitivity (100%) and specificity (100%), and successfully tracked the longitudinal evolution of the antibody response in infected individuals. We also demonstrated that we can detect a chemokine, IP-10, on the same chip, which may provide prognostic insight into patient outcomes. Because our test requires minimal user intervention and is read by a handheld detector, it can be globally deployed in the fight against COVID-19 by democratizing access to laboratory quality tests.

In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design.

The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l-1. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.

In Pursuit of Zero 2.0: Recent Developments in Nonfouling Polymer Brushes for Immunoassays.

"Nonfouling" polymer brush surfaces can greatly improve the performance of in vitro diagnostic (IVD) assays due to the reduction of nonspecific protein adsorption and consequent improvement of signal-to-noise ratios. The development of synthetic polymer brush architectures that suppress adventitious protein adsorption is reviewed, and their integration into surface plasmon resonance and fluorescent sandwich immunoassay formats is discussed. Also, highlighted is a novel, self-contained immunoassay platform (the D4 assay) that transforms time-consuming laboratory-based assays into a user-friendly and point-of-care format with a sensitivity and specificity comparable or better than standard enzyme-linked immunosorbent assay (ELISA) directly from unprocessed samples. These advancements clearly demonstrate the utility of nonfouling polymer brushes as a substrate for ultrasensitive and robust diagnostic assays that may be suitable for clinical testing, in field and laboratory settings.

Architectural Modification of Conformal PEG-Bottlebrush Coatings Minimizes Anti-PEG Antigenicity While Preserving Stealth Properties.

Poly(ethylene glycol) (PEG), a linear polymer known for its "stealth" properties, is commonly used to passivate the surface of biomedical implants and devices, and it is conjugated to biologic drugs to improve their pharmacokinetics. However, its antigenicity is a growing concern. Here, the antigenicity of PEG is investigated when assembled in a poly(oligoethylene glycol) methacrylate (POEGMA) "bottlebrush" configuration on a planar surface. Using ethylene glycol (EG) repeat lengths of the POEGMA sidechains as a tunable parameter for optimization, POEGMA brushes with sidechain lengths of two and three EG repeats are identified as the optimal polymer architecture to minimize binding of anti-PEG antibodies (APAs), while retaining resistance to nonspecific binding by bovine serum albumin and cultured cells. Binding of backbone- versus endgroup-selective APAs to POEGMA brushes is further investigated, and finally the antigenicity of POEGMA coatings is assessed against APA-positive clinical plasma samples. These results are applied toward fabricating immunoassays on POEGMA surfaces with minimal reactivity toward APAs while retaining a low limit-of-detection for the analyte. Taken together, these results offer useful design concepts to reduce the antigenicity of polymer brush-based surface coatings used in applications involving human or animal matrices.

Controlling cell-free metabolism through physiochemical perturbations.

Building biosynthetic pathways and engineering metabolic reactions in cells can be time-consuming due to complexities in cellular metabolism. These complexities often convolute the combinatorial testing of biosynthetic pathway designs needed to define an optimal biosynthetic system. To simplify the optimization of biosynthetic systems, we recently reported a new cell-free framework for pathway construction and testing. In this framework, multiple crude-cell extracts are selectively enriched with individual pathway enzymes, which are then mixed to construct full biosynthetic pathways on the time scale of a day. This rapid approach to building pathways aids in the study of metabolic pathway performance by providing a unique freedom of design to modify and control biological systems for both fundamental and applied biotechnology. The goal of this work was to demonstrate the ability to probe biosynthetic pathway performance in our cell-free framework by perturbing physiochemical conditions, using n-butanol synthesis as a model. We carried out three unique case studies. First, we demonstrated the power of our cell-free approach to maximize biosynthesis yields by mapping physiochemical landscapes using a robotic liquid-handler. This allowed us to determine that NAD and CoA are the most important factors that govern cell-free n-butanol metabolism. Second, we compared metabolic profile differences between two different approaches for building pathways from enriched lysates, heterologous expression and cell-free protein synthesis. We discover that phosphate from PEP utilization, along with other physiochemical reagents, during cell-free protein synthesis-coupled, crude-lysate metabolic system operation inhibits optimal cell-free n-butanol metabolism. Third, we show that non-phosphorylated secondary energy substrates can be used to fuel cell-free protein synthesis and n-butanol biosynthesis. Taken together, our work highlights the ease of using cell-free systems to explore physiochemical perturbations and suggests the need for a more controllable, multi-step, separated cell-free framework for future pathway prototyping and enzyme discovery efforts.