Adeno-associated virus genome quantification with amplification-free CRISPR-Cas12a.

Efficient manufacturing of recombinant Adeno-Associated Viral (rAAV) vectors to meet rising clinical demand remains a major hurdle. One of the most significant challenges is the generation of large amounts of empty capsids without the therapeutic genome. There is no standardized analytical method to accurately quantify the viral genes, and subsequently the empty-to-full ratio, making the manufacturing challenges even more complex. We propose the use of CRISPR diagnostics (CRISPR-Dx) as a robust and rapid approach to determine AAV genome titers. We designed and developed the CRISPR-AAV Evaluation (CRAAVE) assay to maximize sensitivity, minimize time-to-result, and provide a potentially universal design for quantifying multiple transgene constructs encapsidated within different AAV serotypes. We also demonstrate an on-chip CRAAVE assay with lyophilized reagents to minimize end user assay input. The CRAAVE assay was able to detect AAV titers as low as 7e7 vg/mL with high precision (<3% error) in quantifying unknown AAV titers when compared with conventional quantitative PCR (qPCR) method. The assay only requires 30 min of assay time, shortening the analytical workflow drastically. Our results suggest CRISPR-Dx could be a promising tool for efficient rAAV genome titer quantification and has the potential to revolutionize biomanufacturing process analytical technology (PAT).

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.

Ultrabright Fluorescence Readout of an Inkjet-Printed Immunoassay Using Plasmonic Nanogap Cavities.

Fluorescence-based microarrays are promising diagnostic tools due to their high throughput, small sample volume requirements, and multiplexing capabilities. However, their low fluorescence output has limited their implementation for in vitro diagnostics applications in point-of-care (POC) settings. Here, by integration of a sandwich immunoassay microarray within a plasmonic nanogap cavity, we demonstrate strongly enhanced fluorescence which is critical for readout by inexpensive POC detectors. The immunoassay consists of inkjet-printed antibodies on a polymer brush which is grown on a gold film. Colloidally synthesized silver nanocubes are placed on top and interact with the underlying gold film creating high local electromagnetic field enhancements. By varying the thickness of the brush from 5 to 20 nm, up to a 151-fold increase in fluorescence and 14-fold improvement in the limit-of-detection is observed for the cardiac biomarker B-type natriuretic peptide (BNP) compared to the unenhanced assay, paving the way for a new generation of POC clinical diagnostics.

Inkjet-printed point-of-care immunoassay on a nanoscale polymer brush enables subpicomolar detection of analytes in blood.

The ELISA is the mainstay for sensitive and quantitative detection of protein analytes. Despite its utility, ELISA is time-consuming, resource-intensive, and infrastructure-dependent, limiting its availability in resource-limited regions. Here, we describe a self-contained immunoassay platform (the "D4 assay") that converts the sandwich immunoassay into a point-of-care test (POCT). The D4 assay is fabricated by inkjet printing assay reagents as microarrays on nanoscale polymer brushes on glass chips, so that all reagents are "on-chip," and these chips show durable storage stability without cold storage. The D4 assay can interrogate multiple analytes from a drop of blood, is compatible with a smartphone detector, and displays analytical figures of merit that are comparable to standard laboratory-based ELISA in whole blood. These attributes of the D4 POCT have the potential to democratize access to high-performance immunoassays in resource-limited settings without sacrificing their performance.

Engineering the Surface Properties of a Zwitterionic Polymer Brush to Enable the Simple Fabrication of Inkjet-Printed Point-of-Care Immunoassays.

Motivated by the lack of adventitious protein adsorption on zwitterionic polymer brushes that promise low noise and hence high analytical sensitivity for surface-based immunoassays, we explored their use as a substrate for immunoassay fabrication by the inkjet printing of antibodies. We observed that a poly(sulfobetaine)methacrylate brush on glass is far too hydrophilic to enable the noncovalent immobilization of antibodies by inkjet printing. To circumvent this limitation, we developed a series of hybrid zwitterionic-cationic surface coatings with tunable surface wettability that are suitable for the inkjet printing of antibodies but also have low protein adsorption. We show that in a microarray format in which both the capture and detection antibodies are discretely printed as spots on these hybrid brushes, a point-of-care sandwich immunoassay can be carried out with an analytical sensitivity and dynamic range that is similar to or better than those of the same assay fabricated on a PEG-like brush. We also show that the hybrid polymer brushes do not bind anti-PEG antibodies that are ubiquitous in human blood, which can be a problem with immunoassays fabricated on PEG-like coatings.

Direct fluorescence detection of RNA on microarrays by surface-initiated enzymatic polymerization.

We report the first demonstration of surface-initiated enzymatic polymerization (SIEP) for the direct detection of RNA in a fluorescence microarray format. This new method incorporates multiple fluorophores into an RNA strand using the two-step sequential and complementary reactions catalyzed by yeast poly(A) polymerase (PaP) to incorporate deoxyadenosine triphosphate (dATP) at the 3'-OH of an RNA molecule, followed by terminal deoxynucleotidyl transferase (TdT) to catalyze the sequential addition of a mixture of natural and fluorescent deoxynucleotides (dNTPs) at the 3'-OH of an RNA-DNA hybrid. We found that the 3'-end of RNA can be efficiently converted into DNA (∼50% conversion) by polymerization of dATP using yeast PaP, and the short DNA strand appended to the end of the RNA by PaP acts as the initiator for the TdT-catalyzed polymerization of longer DNA strands from a mixture of natural and fluorescent dNTPs that contain up to ∼45 Cy3 fluorophores per 1 kb DNA. We obtained an ∼2 pM limit of detection (LOD) and a 3 log-linear dynamic range for hybridization of a short 21 base-long RNA target to an immobilized peptide nucleic acid probe, while fragmented mRNA targets from three different full length mRNA transcripts yielded a ∼10 pM LOD with a similar dynamic range in a microarray format.

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.

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.

PEG-Like Brush Polymer Conjugate of RNA Aptamer That Shows Reversible Anticoagulant Activity and Minimal Immune Response.

Ribonucleic acid (RNA) therapeutics are an emerging class of drugs. RNA aptamers are of significant therapeutic and clinical interest because their activity can be easily reversed in vivo-a useful feature that is difficult to achieve using other therapeutic modalities. Despite their therapeutic promise, RNA aptamers are limited by their poor blood circulation. The attachment of polyethylene glycol (PEG) to RNA aptamers addresses this limitation. However, an RNA aptamer-PEG conjugate that is a reversible anticoagulant fails in a clinical trial due to the reactivity of the conjugate with pre-existing PEG antibodies and has cast a pall over PEGylation of aptamers and other biologics, despite its long history of utility in drug delivery. Here, PEG antibody-reactivity of this RNA aptamer is eliminated by conjugating it to a next-generation PEG-like brush polymer-poly[(oligoethylene glycol) methyl ether methacrylate)] (POEGMA). The conjugate retained the drug's therapeutic action and the ability to be easily reversed. Importantly, this conjugate does not bind pre-existing PEG antibodies that are prevalent in humans and does not induce a humoral immune response against the polymer itself in mice. These findings suggest a path to rescuing the PEGylation of RNA therapeutics and vaccines from the deleterious side-effects of PEG.

Polyethylene Glycol-Like Brush Polymer Conjugate of a Protein Drug Does Not Induce an Antipolymer Immune Response and Has Enhanced Pharmacokinetics than Its Polyethylene Glycol Counterpart.

Protein therapeutics, except for antibodies, have a short plasma half-life and poor stability in circulation. Covalent coupling of polyethylene glycol (PEG) to protein drugs addresses this limitation. However, unlike previously thought, PEG is immunogenic. In addition to induced PEG antibodies, ≈70% of the US population has pre-existing anti-PEG antibodies. Both induced and preexisting anti-PEG antibodies result in accelerated drug clearance, reduced clinical efficacy, and severe hypersensitivity reactions that have limited the clinical utility of uricase, an enzyme drug for treatment for refractory gout that is decorated with a PEG corona. Here, the authors synthesize a poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) conjugate of uricase that decorates the protein with multiple polymer chains to create a corona to solve these problems. The resulting uricase-POEGMA is well-defined, has high bioactivity, and outperforms its PEG counterparts in its pharmacokinetics (PK). Furthermore, the conjugate does not induce anti-POEGMA antibodies and is not recognized by anti-PEG antibodies. These findings suggest that POEGMA conjugation may provide a solution to the immunogenicity and antigenicity limitations of PEG while improving upon its PK benefits. These results transcend uricase and can be applied to other PEGylated therapeutics and the broader class of biologics with suboptimal PK.

Amplified on-chip fluorescence detection of DNA hybridization by surface-initiated enzymatic polymerization.

We describe the incorporation of multiple fluorophores into a single stranded DNA (ssDNA) chain using terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase that catalyzes the sequential addition of deoxynucleotides (dNTPs) at the 3'-OH group of an oligonucleotide primer; we term this methodology surface initiated enzymatic polymerization (SIEP) of DNA. We found that long (>1 Kb) ssDNA homopolymer can be grown by SIEP, and that the length of the ssDNA product is determined by the monomer to oligonucleotide initiator ratio. We observed efficient initiation (≥50%) and narrow polydispersity of the extended product when fluorescently labeled nucleotides are incorporated. TdT's ability to incorporate fluorescent dNTPs into a ssDNA chain was characterized by examining the effect of the molar ratios of fluorescent dNTP to natural dNTP on the degree of fluorophore incorporation and the length of the polymerized DNA strand. These experiments allowed us to optimize the polymerization conditions to incorporate up to ~50 fluorescent Cy3-labeled dNTPs per kilobase into a ssDNA chain. With the goal of using TdT as an on-chip labeling method, we also quantified TdT mediated signal amplification on the surface by immobilizing ssDNA oligonucleotide initiators on a glass surface followed by SIEP of DNA. The incorporation of multiple fluorophores into the extended DNA chain by SIEP translated to a ~45 fold signal amplification compared to the incorporation of a single fluorophore. SIEP was then employed to detect hybridization of DNA, by the posthybridization, on-chip polymerization of fluorescently labeled ssDNA that was grown from the 3'-OH of target strands that hybridized to DNA probes that were printed on a surface. A dose-response curve for detection of DNA hybridization by SIEP was generated, with a ~1 pM limit of detection and a linear dynamic range of 2 logs.

Ultrasensitive point-of-care immunoassay for secreted glycoprotein detects Ebola infection earlier than PCR.

Ebola virus (EBOV) hemorrhagic fever outbreaks have been challenging to deter due to the lack of health care infrastructure in disease-endemic countries and a corresponding inability to diagnose and contain the disease at an early stage. EBOV vaccines and therapies have improved disease outcomes, but the advent of an affordable, easily accessed, mass-produced rapid diagnostic test (RDT) that matches the performance of more resource-intensive polymerase chain reaction (PCR) assays would be invaluable in containing future outbreaks. Here, we developed and demonstrated the performance of a new ultrasensitive point-of-care immunoassay, the EBOV D4 assay, which targets the secreted glycoprotein of EBOV. The EBOV D4 assay is 1000-fold more sensitive than the U.S. Food and Drug Administration-approved RDTs and detected EBOV infection earlier than PCR in a standard nonhuman primate model. The EBOV D4 assay is suitable for low-resource settings and may facilitate earlier detection, containment, and treatment during outbreaks of the disease.

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.

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.

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.

A versatile diffractive maskless lithography for single-shot and serial microfabrication.

We demonstrate a diffractive maskless lithographic system that is capable of rapidly performing both serial and single-shot micropatterning. Utilizing the diffractive properties of phase holograms displayed on a spatial light modulator, arbitrary intensity distributions were produced to form two and three dimensional micropatterns/structures in a variety of substrates. A straightforward graphical user interface was implemented to allow users to load templates and change patterning modes within the span of a few minutes. A minimum resolution of approximately 700 nm is demonstrated for both patterning modes, which compares favorably to the 232 nm resolution limit predicted by the Rayleigh criterion. The presented method is rapid and adaptable, allowing for the parallel fabrication of microstructures in photoresist as well as the fabrication of protein microstructures that retain functional activity.

Protein patterning by UV-induced photodegradation of poly(oligo(ethylene glycol) methacrylate) brushes.

The UV photodegradation of protein-resistant poly(oligo(ethylene glycol) methacrylate) (POEGMA) bottle-brush films, grown on silicon oxide by surface-initiated atom radical transfer polymerization, was studied using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Exposure to light with a wavelength of 244 nm caused a loss of polyether units from the brush structure and the creation of aldehyde groups that could be derivatized with amines. An increase was measured in the coefficient of friction of the photodegraded polymer brush compared to the native brush, attributed to the creation of a heterogeneous surface film, leading to increased energy dissipation through film deformation and the creation of new polar functional groups at the surface. Exposure of the films through a photomask yielded sharp, well-defined patterns. Analysis of topographical images showed that physical removal of material occurred during exposure, at a rate of 1.35 nm J(-1) cm(2). Using fluorescence microscopy, the adsorption of labeled proteins onto the exposed surfaces was studied. It was found that protein strongly adsorbed to exposed areas, while the masked regions retained their protein resistance. Exposure of the film to UV light from a scanning near-field optical microscope yielded submicrometer-scale patterns. These data indicate that a simple, rapid, one-step photoconversion of the poly(OEGMA) brush occurs that transforms it from a highly protein-resistant material to one that adsorbs protein and can covalently bind amine-containing molecules and that this photoconversion can be spatially addressed with high spatial resolution.

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.

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.

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.