Morphology Control of Self-Assembled Copper Coordination Polymers for Glucose Assays.

Low-cost analytical assays enable accessible detection of clinically and environmentally important analytes; however, common enzyme-based assays suffer from high production and storage costs. Catalytically active synthetic materials serve as replacements for natural enzymes, but development of cost-effective, highly efficient synthetic strategies remains a challenge. Here, we utilized a facile synthesis for copper bipyridine coordination polymers (CuBpyCPs) and investigated structure-function relationships to achieve optimal catalytic properties for a glucose assay. We demonstrated the manipulation of CuBpyCP morphology, resulting in nanoscale petal-like structures and microscale high-index faceted structures, and identified three pure crystal morphologies exhibiting a comparable catalytic activity (Km = 0.3-0.5 mM) to horseradish peroxidase.

Role of Molecular Modification and Protein Folding in the Nucleation and Growth of Protein-Metal-Organic Frameworks.

Metal-organic frameworks (MOFs) are a class of porous nanomaterials that have been extensively studied as enzyme immobilization substrates. During in situ immobilization, MOF nucleation is driven by biomolecules with low isoelectric points. Investigation of how biomolecules control MOF self-assembly mechanisms on the molecular level is key to designing nanomaterials with desired physical and chemical properties. Here, we demonstrate how molecular modifications of bovine serum albumin (BSA) with fluorescein isothiocyanate (FITC) can affect MOF crystal size, morphology, and encapsulation efficiency. Final crystal properties are characterized using scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), fluorescent microscopy, and fluorescence spectroscopy. To probe MOF self-assembly, in situ experiments were performed using cryogenic transmission electron microscopy (cryo-TEM) and X-ray diffraction (XRD). Biophysical characterization of BSA and FITC-BSA was performed using ζ potential, mass spectrometry, circular dichroism studies, fluorescence spectroscopy, and Fourier transform infrared (FTIR) spectroscopy. The combined data reveal that protein folding and stability within amorphous precursors are contributing factors in the rate, extent, and mechanism of crystallization. Thus, our results suggest molecular modifications as promising methods for fine-tuning protein@MOFs' nucleation and growth.

Single-molecule studies reveal method for tuning the heterogeneous activity of alkaline phosphatase.

Single-molecule-enzymology (SME) methods have enabled the observation of heterogeneous catalytic activities within a single enzyme population. Heterogeneous activity is hypothesized to originate from conformational changes in the enzyme that result from changes in the local environment leading to catalytically active substates. Here, we use SME to investigate the mechanisms of heterogeneous activity exhibited by tissue nonspecific alkaline phosphatase (TNSALP), which reveals two subpopulations with different catalytic activities. We show the effect of pH and temperature on the distribution of TNSALP activity and confirm the presence of two subpopulations attributed to half- and fully active TNSALP substates. We provide mechanistic insight about protein structure using molecular dynamic simulations and show pH- and temperature-dependent conformational transitions that corroborate experimentally observed changes in TNSALP activity. These results show the utility of SME to understand heterogeneous enzyme activity and demonstrate a simple approach using pH and temperature to tune catalytic activity within an enzyme population.

Severe Acute Respiratory Syndrome Coronavirus 2 Antigens as Targets of Antibody Responses.

Humoral immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during acute infection and convalescence has been widely studied since March 2020. In this review, the authors summarize literature on humoral responses to SARS-CoV-2 antigens with a focus on spike, nucleocapsid, and the receptor-binding domain as targets of antibody responses. They highlight serologic studies during acute SARS-CoV-2 infection and discuss the clinical relevance of antibody levels in COVID-19 progression. Antibody responses in pediatric COVID-19 patients are also reviewed. Finally, the authors discuss antibody responses during convalescence and their role in protection from SARS-CoV-2 reinfection.

Single-Molecule Enzymology for Diagnostics: Profiling Alkaline Phosphatase Activity in Clinical Samples.

Enzymes can be used as biomarkers for a variety of diseases. However, profiling enzyme activity in clinical samples is challenging due to the heterogeneity in enzyme activity, and the low abundance of the target enzyme in biofluids. Single-molecule methods can overcome these challenges by providing information on the distribution of enzyme activities in a sample. Here, we describe the concept of using the single-molecule enzymology (SME) method to analyze enzymatic activity in clinical samples. We present recent work focused on measuring alkaline phosphatase isotypes in serum samples using SME. Future work will involve improving and simplifying this technology, and applying it to other enzymes for diagnostics.

Circulating Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Vaccine Antigen Detected in the Plasma of mRNA-1273 Vaccine Recipients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins were measured in longitudinal plasma samples collected from 13 participants who received two doses of mRNA-1273 vaccine. Eleven of 13 participants showed detectable levels of SARS-CoV-2 protein as early as day 1 after first vaccine injection. Clearance of detectable SARS-CoV-2 protein correlated with production of immunoglobulin G (IgG) and immunoglobulin A (IgA).

Multisystem inflammatory syndrome in children is driven by zonulin-dependent loss of gut mucosal barrier.

BACKGROUNDWeeks after SARS-CoV-2 infection or exposure, some children develop a severe, life-threatening illness called multisystem inflammatory syndrome in children (MIS-C). Gastrointestinal (GI) symptoms are common in patients with MIS-C, and a severe hyperinflammatory response ensues with potential for cardiac complications. The cause of MIS-C has not been identified to date.METHODSHere, we analyzed biospecimens from 100 children: 19 with MIS-C, 26 with acute COVID-19, and 55 controls. Stools were assessed for SARS-CoV-2 by reverse transcription PCR (RT-PCR), and plasma was examined for markers of breakdown of mucosal barrier integrity, including zonulin. Ultrasensitive antigen detection was used to probe for SARS-CoV-2 antigenemia in plasma, and immune responses were characterized. As a proof of concept, we treated a patient with MIS-C with larazotide, a zonulin antagonist, and monitored the effect on antigenemia and the patient's clinical response.RESULTSWe showed that in children with MIS-C, a prolonged presence of SARS-CoV-2 in the GI tract led to the release of zonulin, a biomarker of intestinal permeability, with subsequent trafficking of SARS-CoV-2 antigens into the bloodstream, leading to hyperinflammation. The patient with MIS-C treated with larazotide had a coinciding decrease in plasma SARS-CoV-2 spike antigen levels and inflammatory markers and a resultant clinical improvement above that achieved with currently available treatments.CONCLUSIONThese mechanistic data on MIS-C pathogenesis provide insight into targets for diagnosing, treating, and preventing MIS-C, which are urgently needed for this increasingly common severe COVID-19-related disease in children.

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.

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 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 Detection of Enzymatic Activity Using Single Molecule Arrays.

Enzyme assays are important for many applications including clinical diagnostics, functional proteomics, and drug discovery. Current methods for enzymatic activity measurement often suffer from low analytical sensitivity. We developed an ultrasensitive method for the detection of enzymatic activity using Single Molecule Arrays (eSimoa). The eSimoa assay is accomplished by conjugating substrates to paramagnetic beads and measuring the conversion of substrates to products using single molecule analysis. We demonstrated the eSimoa method for the detection of protein kinases, telomerase, histone H3 methyltransferase SET7/9, and polypeptide N-acetylgalactosaminyltransferase with unprecedented sensitivity. In addition, we tested enzyme inhibition and performed theoretical calculations for the binding of inhibitor to its target enzyme and show the need for an ultrasensitive enzymatic assay to evaluate the potency of tight binding inhibitors. The eSimoa assay was successfully used to determine inhibition constants of both bosutinib and dasatinib. Due to the ultrasensitivity of this method, we also were able to measure the kinase activities at the single cell level. We show that the eSimoa assay is a simple, fast, and highly sensitive approach, which can be easily extended to detect a variety of other enzymes, providing a promising platform for enzyme-related fundamental research and inhibitor screening.

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.

Virus Bioresistor (VBR) for Detection of Bladder Cancer Marker DJ-1 in Urine at 10 pM in One Minute.

DJ-1, a 20.7 kDa protein, is overexpressed in people who have bladder cancer (BC). Its elevated concentration in urine allows it to serve as a marker for BC. However, no biosensor for the detection of DJ-1 has been demonstrated. Here, we describe a virus bioresistor (VBR) capable of detecting DJ-1 in urine at a concentration of 10 pM in 1 min. The VBR consists of a pair of millimeter-scale gold electrodes that measure the electrical impedance of an ultrathin (≈ 150-200 nm), two-layer polymeric channel. The top layer of this channel (90-105 nm in thickness) consists of an electrodeposited virus-PEDOT (PEDOT is poly(3,4-ethylenedioxythiophene)) composite containing embedded M13 virus particles that are engineered to recognize and bind to the target protein of interest, DJ-1. The bottom layer consists of spin-coated PEDOT-PSS (poly(styrenesulfonate)). Together, these two layers constitute a current divider. We demonstrate here that reducing the thickness of the bottom PEDOT-PSS layer increases its resistance and concentrates the resistance drop of the channel in the top virus-PEDOT layer, thereby increasing the sensitivity of the VBR and enabling the detection of DJ-1. Large signal amplitudes coupled with the inherent simplicity of the VBR sensor design result in high signal-to-noise (S/N > 100) and excellent sensor-to-sensor reproducibility characterized by coefficients of variation in the range of 3-7% across the DJ-1 binding curve down to a concentration of 30 pM, near the 10 pM limit of detection (LOD), encompassing four orders of magnitude in concentration.

Direct Observation of Amorphous Precursor Phases in the Nucleation of Protein-Metal-Organic Frameworks.

Protein-metal-organic frameworks (p-MOFs) are a prototypical example of how synthetic biological hybrid systems can be used to develop next-generation materials. Controlling p-MOF formation enables the design of hybrid materials with enhanced biological activity and high stability. However, such control is yet to be fully realized due to an insufficient understanding of the governing nucleation and growth mechanisms in p-MOF systems. The structural evolution of p-MOFs was probed by cryo-transmission electron microscopy, revealing nonclassical pathways via dissolution-recrystallization of highly hydrated amorphous particles and solid-state transformation of a protein-rich amorphous phase. On the basis of these data, we propose a general description of p-MOF crystallization which is best characterized by particle aggregation and colloidal theory for future synthetic strategies.

Rapid, Wet Chemical Fabrication of Radial Junction Electroluminescent Wires.

A wet chemical process involving two electrodeposition steps followed by a solution casting step, the "EESC" process, is described for the fabrication of electroluminescent, radial junction wires. EESC is demonstrated by assembling three well-studied nanocrystalline (or amorphous) materials: Au, CdSe, and poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS). The tri-layered device architecture produced by EESC minimizes the influence of an electrically resistive CdSe emitter layer by using a highly conductive gold nanowire that serves as both a current collector and a negative electrode. Hole injection, at a high barrier CdSe-PEDOT:PSS interface (ϕh ≈ 1.1 V), is facilitated by a contact area that is 1.9-4.7-fold larger than the complimentary gold-CdSe electron-injecting contact (ϕe ≈ 0.6 V), contributing to low-voltage thresholds (1.4-1.7 V) for electroluminescence (EL) emission. Au@CdSe@PEDOT:PSS wire EL emitters are 25 µm in length, amongst the longest so far demonstrated to our knowledge, but the EESC process is scalable to nanowires of any length, limited only by the length of the central gold nanowire that serves as a template for the fabrication process. Radial carrier transport within these multishell wires conforms to the back-to-back diode model.

An Impedance-Transduced Chemiresistor with a Porous Carbon Channel for Rapid, Nonenzymatic, Glucose Sensing.

A new type of chemiresistor, the impedance-transduced chemiresistor (ITCR), is described for the rapid analysis of glucose. The ITCR exploits porous, high surface area, fluorine-doped carbon nanofibers prepared by electrospinning of fluorinated polymer nanofibers followed by pyrolysis. These nanofibers are functionalized with a boronic acid receptor and stabilized by Nafion to form the ITCR channel for glucose detection. The recognition and binding of glucose by the ITCR is detected by measuring its electrical impedance at a single frequency. The analysis frequency is selected by measuring the signal-to-noise ( S/ N) for glucose detection across 5 orders of magnitude, evaluating both the imaginary and real components of the complex impedance. On the basis of this analysis, an optimal frequency of 13 kHz is selected for glucose detection, yielding an S/ N ratio of 60-100 for [glucose] = 5 mM using the change in the total impedance, Δ Z. The resulting ITCR glucose sensor shows a rapid analysis time (<8 s), low coefficient of variation for a series of sensors (<10%), an analysis range of 50 µM to 5 mM, and excellent specificity versus fructose, ascorbic acid, and uric acid. These metrics for the ITCR are obtained using a sample size as small as 5 µL.

The Virus Bioresistor: Wiring Virus Particles for the Direct, Label-Free Detection of Target Proteins.

The virus bioresistor (VBR) is a chemiresistor that directly transfers information from virus particles to an electrical circuit. Specifically, the VBR enables the label-free detection of a target protein that is recognized and bound by filamentous M13 virus particles, each with dimensions of 6 nm ( w) × 1 µm ( l), entrained in an ultrathin (∼250 nm) composite virus-polymer resistor. Signal produced by the specific binding of virus to target molecules is monitored using the electrical impedance of the VBR: The VBR presents a complex impedance that is modeled by an equivalent circuit containing just three circuit elements: a solution resistance ( Rsoln), a channel resistance ( RVBR), and an interfacial capacitance ( CVBR). The value of RVBR, measured across 5 orders of magnitude in frequency, is increased by the specific recognition and binding of a target protein to the virus particles in the resistor, producing a signal Δ RVBR. The VBR concept is demonstrated using a model system in which human serum albumin (HSA, 66 kDa) is detected in a phosphate buffer solution. The VBR cleanly discriminates between a change in the electrical resistance of the buffer, measured by Rsoln, and selective binding of HSA to virus particles, measured by RVBR. The Δ RVBR induced by HSA binding is as high as 200 Ω, contributing to low sensor-to-sensor coefficients-of-variation (<15%) across the entire calibration curve for HSA from 7.5 nM to 900 nM. The response time for the VBR is 3-30 s.

Hollow Pd-Ag Composite Nanowires for Fast Responding and Transparent Hydrogen Sensors.

Pd based alloy materials with hollow nanostructures are ideal hydrogen (H2) sensor building blocks because of their double-H2 sensing active sites (interior and exterior side of hollow Pd alloy) and fast response. In this work, for the first time, we report a simple fabrication process for preparing hollow Pd-Ag alloy nanowires (Pd@Ag HNWs) by using the electrodeposition of lithographically patterned silver nanowires (NWs), followed by galvanic replacement reaction (GRR) to form palladium. By controlling the GRR time of aligned Ag NWs within an aqueous Pd2+-containing solution, the compositional transition and morphological evolution from Ag NWs to Pd@Ag HNWs simultaneously occurred, and the relative atomic ratio between Pd and Ag was controlled. Interestingly, a GRR duration of 17 h transformed Ag NWs into Pd@Ag HNWs that showed enhanced H2 response and faster sensing response time, reduced 2.5-fold, as compared with Ag NWs subjected to a shorter GRR period of 10 h. Furthermore, Pd@Ag HNWs patterned on the colorless and flexible polyimide (cPI) substrate showed highly reversible H2 sensing characteristics. To further demonstrate the potential use of Pd@Ag HNWs as sensing layers for all-transparent, wearable H2 sensing devices, we patterned the Au NWs perpendicular to Pd@Ag HNWs to form a heterogeneous grid-type metallic NW electrode which showed reversible H2 sensing properties in both bent and flat states.

Accelerating Palladium Nanowire H2 Sensors Using Engineered Nanofiltration.

The oxygen, O2, in air interferes with the detection of H2 by palladium (Pd)-based H2 sensors, including Pd nanowires (NWs), depressing the sensitivity and retarding the response/recovery speed in air-relative to N2 or Ar. Here, we describe the preparation of H2 sensors in which a nanofiltration layer consisting of a Zn metal-organic framework (MOF) is assembled onto Pd NWs. Polyhedron particles of Zn-based zeolite imidazole framework (ZIF-8) were synthesized on lithographically patterned Pd NWs, leading to the creation of ZIF-8/Pd NW bilayered H2 sensors. The ZIF-8 filter has many micropores (0.34 nm for gas diffusion) which allows for the predominant penetration of hydrogen molecules with a kinetic diameter of 0.289 nm, whereas relatively larger gas molecules including oxygen (0.345 nm) and nitrogen (0.364 nm) in air are effectively screened, resulting in superior hydrogen sensing properties. Very importantly, the Pd NWs filtered by ZIF-8 membrane (Pd NWs@ZIF-8) reduced the H2 response amplitude slightly (ΔR/R0 = 3.5% to 1% of H2 versus 5.9% for Pd NWs) and showed 20-fold faster recovery (7 s to 1% of H2) and response (10 s to 1% of H2) speed compared to that of pristine Pd NWs (164 s for response and 229 s for recovery to 1% of H2). These outstanding results, which are mainly attributed to the molecular sieving and acceleration effect of ZIF-8 covered on Pd NWs, rank highest in H2 sensing speed among room-temperature Pd-based H2 sensors.