Sequential Protein Capture in Multiplex Single Molecule Arrays: A Strategy for Eliminating Assay Cross-Reactivity.

Measurements of multiple biomolecules within the same biological sample are important for many clinical applications to enable accurate disease diagnosis or classification. These disease-related biomarkers often exist at very low levels in biological fluids, necessitating ultrasensitive measurement methods. Single-molecule arrays (Simoa), a bead-based digital enzyme-linked immunosorbent assay, is the current state of the art for ultrasensitive protein detection and can detect sub-femtomolar protein concentrations, but its ability to achieve high-order multiplexing without cross-reactivity remains a challenge. Here, a sequential protein capture approach for multiplex Simoa assays is implemented to eliminate cross-reactivity between binding reagents by sequentially capturing each protein analyte and then incubating each capture bead with only its corresponding detection antibody. This strategy not only reduces cross-reactivity to background levels and significantly improves measurement accuracies, but also enables higher-order multiplexing. As a proof of concept, the sequential multiplex Simoa assay is used to measure five different cytokines in plasma samples from Coronavirus Disease 2019 (COVID-19) patients. The ultrasensitive sequential multiplex Simoa assays will enable the simultaneous measurements of multiple low-abundance analytes in a time- and cost-effective manner and will prove especially critical in many cases where sample volumes are limited.

The Primarily Undergraduate Nanomaterials Cooperative: A New Model for Supporting Collaborative Research at Small Institutions on a National Scale.

The Primarily Undergraduate Nanomaterials Cooperative (PUNC) is an organization for research-active faculty studying nanomaterials at Primarily Undergraduate Institutions (PUIs), where undergraduate teaching and research go hand-in-hand. In this perspective, we outline the differences in maintaining an active research group at a PUI compared to an R1 institution. We also discuss the work of PUNC, which focuses on community building, instrument sharing, and facilitating new collaborations. Currently consisting of 37 members from across the United States, PUNC has created an online community consisting of its Web site (nanocooperative.org), a weekly online summer group meeting program for faculty and students, and a Discord server for informal conversations. Additionally, in-person symposia at ACS conferences and PUNC-specific conferences are planned for the future. It is our hope that in the years to come PUNC will be seen as a model organization for community building and research support at primarily undergraduate institutions.

Simplified Digital Enzyme-Linked Immunosorbent Assay Using Tyramide Signal Amplification and Fibrin Hydrogels.

Many protein biomarkers occur at very low concentrations in biofluids like blood and saliva, and ultrasensitive detection methods are required in order to measure them. Approaches such as digital enzyme-linked immunosorbent assays (ELISA) and single molecule arrays (Simoa) have been developed to accurately quantitate protein concentrations as low as attomolar levels. Although these techniques are being implemented in research and clinical laboratories to develop ultrasensitive clinical diagnostic assays, the size and cost of the instruments required to run these digital assays have precluded them from being implemented into point-of-care diagnostic formats. Here, we report the development of a simplified digital ELISA format that is more amenable to point-of-care technologies, referred to as catalyzed reporter deposition digital ELISA (CARD-dELISA). On-bead signal generation using the CARD tyramide signal amplification technique is combined with bead immobilization in fibrin hydrogels for single molecule counting in a simplified workflow format. CARD-dELISA allows for ultrasensitive protein detection (IL-6: ∼1 fM) with a dynamic range similar to the conventional Simoa assay. We use CARD-dELISA to measure IL-6 in saliva samples and show good agreement with conventional Simoa.

Ultra-Sensitive Serial Profiling of SARS-CoV-2 Antigens and Antibodies in Plasma to Understand Disease Progression in COVID-19 Patients with Severe Disease.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 21 million people worldwide since August 16, 2020. Compared to PCR and serology tests, SARS-CoV-2 antigen assays are underdeveloped, despite their potential to identify active infection and monitor disease progression. METHODS: We used Single Molecule Array (Simoa) assays to quantitatively detect SARS-CoV-2 spike, S1 subunit, and nucleocapsid antigens in the plasma of patients with coronavirus disease (COVID-19). We studied plasma from 64 patients who were COVID-19 positive, 17 who were COVID-19 negative, and 34 prepandemic patients. Combined with Simoa anti-SARS-CoV-2 serological assays, we quantified changes in 31 SARS-CoV-2 biomarkers in 272 longitudinal plasma samples obtained for 39 patients with COVID-19. Data were analyzed by hierarchical clustering and were compared to longitudinal RT-PCR test results and clinical outcomes. RESULTS: SARS-CoV-2 S1 and N antigens were detectable in 41 out of 64 COVID-19 positive patients. In these patients, full antigen clearance in plasma was observed a mean ± 95% CI of 5 ± 1 days after seroconversion and nasopharyngeal RT-PCR tests reported positive results for 15 ± 5 days after viral-antigen clearance. Correlation between patients with high concentrations of S1 antigen and ICU admission (77%) and time to intubation (within 1 day) was statistically significant. CONCLUSIONS: The reported SARS-CoV-2 Simoa antigen assay is the first to detect viral antigens in the plasma of patients who were COVID-19 positive to date. These data show that SARS-CoV-2 viral antigens in the blood are associated with disease progression, such as respiratory failure, in COVID-19 cases with severe disease.

Ultrasensitive high-resolution profiling of early seroconversion in patients with COVID-19.

Sensitive assays are essential for the accurate identification of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we report a multiplexed assay for the fluorescence-based detection of seroconversion in infected individuals from less than 1 µl of blood, and as early as the day of the first positive nucleic acid test after symptom onset. The assay uses dye-encoded antigen-coated beads to quantify the levels of immunoglobulin G (IgG), IgM and IgA antibodies against four SARS-CoV-2 antigens. A logistic regression model trained using samples collected during the pandemic and samples collected from healthy individuals and patients with respiratory infections before the first outbreak of coronavirus disease 2019 (COVID-19) was 99% accurate in the detection of seroconversion in a blinded validation cohort of samples collected before the pandemic and from patients with COVID-19 five or more days after a positive nasopharyngeal test by PCR with reverse transcription. The high-throughput serological profiling of patients with COVID-19 allows for the interrogation of interactions between antibody isotypes and viral proteins, and should help us to understand the heterogeneity of clinical presentations.

Ultra-Sensitive High-Resolution Profiling of Anti-SARS-CoV-2 Antibodies for Detecting Early Seroconversion in COVID-19 Patients.

The COVID-19 pandemic continues to infect millions of people worldwide. In order to curb its spread and reduce morbidity and mortality, it is essential to develop sensitive and quantitative methods that identify infected individuals and enable accurate population-wide screening of both past and present infection. Here we show that Single Molecule Array assays detect seroconversion in COVID-19 patients as soon as one day after symptom onset using less than a microliter of blood. This multiplexed assay format allows us to quantitate IgG, IgM and IgA immunoglobulins against four SARS-CoV-2 targets, thereby interrogating 12 antibody isotype-viral protein interactions to give a high resolution profile of the immune response. Using a cohort of samples collected prior to the outbreak as well as samples collected during the pandemic, we demonstrate a sensitivity of 86% and a specificity of 100% during the first week of infection, and 100% sensitivity and specificity thereafter. This assay should become the gold standard for COVID19 serological profiling and will be a valuable tool for answering important questions about the heterogeneity of clinical presentation seen in the ongoing pandemic.

Single-molecule measurements in microwells for clinical applications.

The ability to detect and analyze proteins, nucleic acids, and other biomolecules is critical for clinical diagnostics and for understanding the underlying mechanisms of disease. Current detection methods in clinical and research laboratories rely upon bulk measurement techniques such as immunoassays, polymerase chain reaction, and mass spectrometry to detect these biomarkers. However, many potentially useful protein or nucleic acid biomarkers in blood, saliva, or other biofluids exist at concentrations well below the detection limits of current methods, necessitating the development of more sensitive technologies. Single-molecule measurements are poised to address this challenge, vastly improving sensitivity for detecting low abundance biomarkers and rare events within a population. Microwell arrays have emerged as a powerful tool for single-molecule measurements, enabling ultrasensitive detection of disease-relevant biomolecules in easily accessible biofluids. This review discusses the development, fundamentals, and clinical applications of microwell-based single-molecule methods, as well as challenges and future directions for translating these methods to the clinic.

Characterizing Single Polymeric and Protein Nanoparticles with Surface Plasmon Resonance Imaging Measurements.

Near-infrared surface plasmon resonance imaging (SPRI) microscopy is used to detect and characterize the adsorption of single polymeric and protein nanoparticles (PPNPs) onto chemically modified gold thin films in real time. The single-nanoparticle SPRI responses, Δ%RNP, from several hundred adsorbed nanoparticles are collected in a single SPRI adsorption measurement. Analysis of Δ%RNP frequency distribution histograms is used to provide information on the size, material content, and interparticle interactions of the PPNPs. Examples include the measurement of log-normal Δ%RNP distributions for mixtures of polystyrene nanoparticles, the quantitation of bioaffinity uptake into and aggregation of porous NIPAm-based (N-isopropylacrylamide) hydrogel nanoparticles specifically engineered to bind peptides and proteins, and the characterization of the negative single-nanoparticle SPRI response and log-normal Δ%RNP distributions obtained for three different types of genetically encoded gas-filled protein nanostructures derived from bacteria.

Fabrication of PEDOT Nanocone Arrays with Electrochemically Modulated Broadband Antireflective Properties.

Ordered nanocone arrays of the electroactive polymer poly(3,4-ethylenedioxythiophene) (PEDOT) were fabricated by the simultaneous oxygen plasma etching of an electrodeposited PEDOT thin film coated with a hexagonally closed packed polystyrene bead monolayer. PEDOT nanocone arrays with an intercone spacing of 200 nm and an average nanocone height of 350 nm exhibited a low broadband reflectivity of <1.5% from 550 to 800 nm. Electrochemical modulation of the oxidation state of the PEDOT nanocone array film was used to change both its ex situ absorption spectrum (electrochromism) and reflection spectrum (electroreflectivity). The sign of the PEDOT nanocone array electroreflectivity was opposite to that observed from unmodified PEDOT thin films; this significant difference is attributed to the unique optical behavior of nanostructured surfaces with an interfacial layer that contains a graded mix of air and highly absorptive nanocones. The combined electrochromic and electroreflective behavior of the antireflective PEDOT nanocone array films should find promising applications in solar energy cells, sensors and other optical devices.