Search

The Virus BioResistor (VBR) is a biosensor capable of the rapid and sensitive detection of small protein disease markers using a simple dip-and-read modality. For example, the bladder cancer-associated protein DJ-1 (22 kDa) can be detected in human urine within 1.0 min. with a limit-of-detection (LOD) of 10 pM. The VBR uses engineered virus particles as receptors to recognize and selectively bind the protein of interest. These virus particles are entrained in a conductive poly(3,4 ethylenedioxythiophene) or PEDOT channel. The electrical impedance of the channel increases when the target protein is bound by the virus particles. But VBRs exhibit a sensitivity that is inversely related to the molecular weight of the protein target. Thus, large proteins, such as IgG antibodies (150 kDa), can be undetectable even at high concentrations. We demonstrate that the electrochemical over-oxidation of the VBR’s PEDOT channel increases its electrical impedance, conferring enhanced sensitivity for both small and large proteins. Over-oxidation makes possible the detection of two an antibody, undetectable at a normal VBR, with a limit-of-detection of 40 ng/mL (250 pM), and a dynamic range for quantitation extending to 600 ng/mL.

The 2018 Nobel Prize in Chemistry recognized in vitro evolution, including the development by George Smith and Gregory Winter of phage display, a technology for engineering the functional capabilities of antibodies into viruses. Such bacteriophage solve inherent problems with antibodies including their high cost, thermal lability, and their propensity to aggregate. While phage display accelerated the discovery of peptide and protein motifs for recognition and binding to proteins in a variety of applications, the development of biosensors using intact phage particles was largely unexplored in the early 2000’s. Virus particles, 16.5 MDa in size and assembled from thousands of proteins, could not simply be substituted for antibodies in any existing biosensor architectures. Incorporating viruses into biosensors required us to answer several questions: What process will allow the incorporation of viruses into a functional bioaffinity layer? How can the binding of a protein disease marker to a virus particle be electrically transduced to produce a signal? Will the variable salt concentration of a bodily fluid interfere with electrical transduction? A completely new biosensor architecture, and a new scheme for electrically transducing the binding of molecules to viruses, was required. This Account describes the highlights of a research program, launched in 2006, that answered these questions. These efforts culminated in 2018, in the invention of a biosensor specifically designed to interface with virus particles – the Virus BioResistor (VBR). The VBR is a resistor, consisting of a conductive polymer matrix into which M13 virus particles are entrained. The electrical impedance of this resistor, measured across four orders of magnitude in frequency, simultaneously measures the concentration of a target protein and the ionic conductivity of the medium in which the resistor is immersed. Large signal amplitudes coupled with the inherent simplicity of the VBR sensor design results in high signal-to-noise (S/N >100) and excellent sensor-to-sensor reproducibility. Using this new device, we have measured the urinary bladder cancer biomarker, nucleic acid deglycase (DJ-1) in urine samples. This optimized VBR is characterized by extremely low sensor-to-sensor coefficients-of-variation in the range of 3-7% across the DJ-1 binding curve down to a 30 pM limit-of-quantitation (LOQ), encompassing four orders of magnitude in concentration.

Unlocking the potential of personalized medicine in point-of-care settings requires a new generation of biomarker and proteomic assays. Ideally, assays could inexpensively perform hundreds of quantitative protein measurements in parallel at the bedsides of patients. This goal greatly exceeds current capabilities. Furthermore, biomarker assays are often challenging to translate from benchtop to clinic due to difficulties achieving and assessing the necessary selectivity, sensitivity, and reproducibility. To address these challenges, we developed an efficient (70 %) immunoassay method. Specifically, the immunoblot membrane is dotted with the sample and then developed in a vortex fluidic device (VFD) reactor. All assay steps-blocking, binding, and washing-leverage the unique thin-film microfluidics of the VFD. The approach can accelerate direct, indirect, and sandwich immunoblot assays. The applications demonstrated include assays relevant to both the laboratory and the clinic.

The prevalence rate of Cigar, Little Cigar, and Cigarillo (CLCC) use among youth rose above the rate of cigarettes for the first time in 2019, and Black and Hispanic youth remain disproportionately more susceptible and likely to use these products compared to White youth. Addressing this disparity through education requires a clearer understanding of the ways youth refer to, and group, CLCCs. Twenty-eight virtual focus groups with youth ages 13–17 (n = 105) were conducted across the U.S. Groups were split by race/ethnicity, with 14 Black and 14 Hispanic groups, and further divided by CLCC experimenters and susceptible users. Youth participants discussed CLCC use behaviors, harm and risk perceptions, and knowledge, attitudes, and beliefs about CLCC products. When shown photos of CLCC products during focus groups, youth across groups identified and labeled these products into four subcategories. Large cigars were universally labeled “cigars”. Little cigars were unfamiliar and guessed to be cigarettes. Tipped cigarillos were synonymous with the brand Black and Mild and considered tobacco. Untipped cigarillos were most referred to as “blunts” and used with marijuana. Understanding the nuances of language and use patterns of CLCCs is critical to ensuring the accuracy of measurement and impact of public health communications.

A previous report demonstrated the strong association between the presence of antibodies binding to an epitope region from SARS-CoV-2 nucleocapsid, termed Ep9, and COVID-19 disease severity. Patients with anti-Ep9 antibodies (Abs) had hallmarks of antigenic interference (AIN), including early IgG upregulation and cytokine-associated injury. Thus, the immunological memory of a prior infection was hypothesized to drive formation of suboptimal anti-Ep9 Abs in severe COVID-19 infections. This study identifies a putative primary antigen capable of stimulating production of cross-reactive, anti-Ep9 Abs. Binding assays with patient blood samples directly show cross-reactivity between Abs binding to Ep9 and only one bioinformatics-derived, homologous putative antigen, a sequence derived from the neuraminidase protein of H3N2 influenza A virus. This cross-reactive binding is highly influenza strain specific and sensitive to even single amino acid changes in epitope sequence. The neuraminidase protein is not present in the influenza vaccine, and the anti-Ep9 Abs likely resulted from the widespread influenza infection in 2014. Therefore, AIN from a previous infection could underlie some cases of COVID-19 disease severity.

IntroductionStudies show that tobacco use among sexual and gender minority (SGM) populations is disproportionately higher than heterosexual or cisgender populations. However, few studies have examined tobacco use among SGM subgroups by race/ethnicity or associations between SGM-specific discrimination and connection to SGM identity and tobacco use.MethodsThis study analysed survey data from 11 313 SGM (gay, lesbian, bisexual, other sexual minority or gender minority) young adults in the USA and reported current cigarette, e-cigarette, other tobacco (cigar, smokeless tobacco, hookah) and polytobacco use. We used multinomial logistic regression to estimate associations between (a) SGM subgroup, race/ethnicity, SGM-specific discrimination and SGM identity connection and (b) each tobacco use outcome (vs never use of tobacco). We conducted postestimation testing to assess predicted probabilities of tobacco use against the sample average.ResultsLesbian females (particularly black lesbian females) had higher-than-average probability of polytobacco use. White bisexual and lesbian participants had higher-than-average probability of cigarette and e-cigarette use, respectively. Higher levels of discrimination were associated with polytobacco use. Higher levels of identity connectedness were protective against certain tobacco use behaviours among gender minority participants and participants with high levels of discrimination experience.ConclusionsWe found variations in tobacco use by SGM subgroups overall and by race/ethnicity. Discrimination may be a risk factor for certain tobacco use behaviours. However, SGM identity connectedness may be protective against tobacco use among gender minority individuals and individuals experiencing SGM-specific discrimination. These findings can inform targeted approaches to reach SGM subgroups at greater risk of tobacco use.

Limited data exists for longitudinal growth outcomes in neonates with a history of necrotizing enterocolitis (NEC). We aimed to study 20-year growth outcomes in NEC survivors.A retrospective matched cohort study included neonates diagnosed with NEC and control subjects matched for birth year, birth weight, and gestational age who had at least one post-discharge follow-up. The primary outcome was growth, measured by length and weight until 20 years. Logistic regression was used to test the change in growth from birth until most recent encounter.Five hundred twenty-seven neonates were included: 294 with NEC, and 233 controls. Sixty-eight percent of NEC cases were Bell's stage I, 25% were stage II, and 7% were stage III. Median gestational age was 29 weeks, and median birth weight was 1237 g. Infants with NEC had a longer NICU stay (p 0.0001) and increased number of comorbidities (p 0.0001). Compared to overall and sex-matched controls, infants with NEC had a significantly slower growth rate in terms of weight (p 0.0068) but not length (p = 0.09). Neither group exhibited failure to thrive.These results suggest that non-surgical NEC may have a more profound impact on long-term growth than previously considered.Retrospective Cohort-Matched Study.Level III.

A previous report demonstrated the strong association between the presence of antibodies binding to an epitope region from SARS-CoV-2 nucleocapsid, termed Ep9, and COVID-19 disease severity. Patients with anti-Ep9 antibodies (Abs) had hallmarks of antigenic imprinting (AIM), including early IgG upregulation and cytokine-associated injury. Thus, the immunological memory of a previous infection was hypothesized to drive formation of suboptimal anti-Ep9 Abs in severe COVID-19 infections. This study identifies a putative primary antigen capable of stimulating production of cross-reactive, anti-Ep9 Abs. Binding assays with patient blood samples directly show cross-reactivity between Abs binding to Ep9 and only one bioinformatics-derived, homologous potential antigen, a sequence derived from the neuraminidase protein of H3N2 Influenza A virus. This cross-reactive binding is highly influenza strain specific and sensitive to even single amino acid changes in epitope sequence. The neuraminidase protein is not present in the influenza vaccine, and the anti-Ep9 Abs likely resulted from the widespread influenza infection in 2014. Therefore, AIM from a previous infection could underlie some cases of COVID-19 disease severity.ImportanceInfections with SARS-COV-2 result in diverse disease outcomes, ranging from asymptomatic to fatal. The mechanisms underlying different disease outcomes remain largely unexplained. Previously, our laboratory identified a strong association between the presence of an antibody and increased disease severity in a subset of COVID-19 patients. Here, we report that this severity-associated antibody cross-reacts with viral proteins from an influenza A viral strain from 2014. Therefore, we speculate that antibodies generated against previous infections, like the 2014 influenza A, play a significant role in directing some peoples’ immune responses against SARS-COV-2. Such understanding of the sources and drivers of COVID-19 disease severity can help early identification and pre-emptive treatment.

The COVID-19 pandemic has resulted in over two million deaths worldwide. Despite efforts to fight the virus, the disease continues to overwhelm hospitals with severely ill patients., Effective methods for predicting COVID-19 disease trajectories are urgently needed. Here, enzyme-linked immunosorbent assay (ELISA) and coronavirus antigen microarray (COVAM) analysis mapped antibody epitopes in the plasma of COVID-19 patients (n = 86) experiencing a wide range of disease states. The experiments identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including admission to the intensive care unit (ICU), requirement for ventilators, or death. Importantly, anti-Ep9 antibodies can be detected within 6 days post-symptom onset and sometimes within 1 day. Furthermore, anti-Ep9 antibodies correlate with various comorbidities and hallmarks of immune hyperactivity. We introduce a simple-to-calculate, disease risk factor score to quantitate each patient’s comorbidities and age. For patients with anti-Ep9 antibodies, scores above 3.0 predict more severe disease outcomes with a 13.42 likelihood ratio (96.7% specificity). The results lay the groundwork for a new type of COVID-19 prognostic to allow early identification and triage of high-risk patients. Such information could guide more effective therapeutic intervention. IMPORTANCE The COVID-19 pandemic has resulted in over two million deaths worldwide. Despite efforts to fight the virus, the disease continues to overwhelm hospitals with severely ill patients. Diagnosis of COVID-19 is readily accomplished through a multitude of reliable testing platforms; however, prognostic prediction remains elusive. To this end, we identified a short epitope from the SARS-CoV-2 nucleocapsid protein and also a disease risk factor score based upon comorbidities and age. The presence of antibodies specifically binding to this epitope plus a score cutoff can predict severe COVID-19 outcomes with 96.7% specificity.

A polymer-based electrode capable of specific detection of human serum albumin, and its glycated derivatives, is described. The sensor is constructed from a glass microscope slide coated with a synthesized, polythiophene film bearing a protected, iminodiacetic acid motif. The electrode surface is then further elaborated to a functional biosensor through deprotection of the iminodiacetic acid, followed by metal-affinity immobilization of a specific and high-affinity, albumin ligand. Albumin was then quantified in buffer and synthetic urine via electrochemical impedance spectroscopy. Glycated albumin was next bound to a boronic acid-modified, single-cysteine dihydrofolate reductase variant to quantify glycation ratios by square-wave voltammetry. The platform offers high sensitivity, specificity, and reproducibility in an inexpensive arrangement. The detection limits exceed the requirements for intermediate-term glycemic control monitoring in diabetes patients at 5 and 1 nM for albumin and its glycated forms, respectively.

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. But 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 one minute. The VBR consists of a pair of millimeter-scale gold electrodes that measure the electrical impedance of an ultra-thin (≈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(styrene sulfonate)). 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 results 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.

Mechano-activated chemistry is a powerful tool for remodeling of synthetic polymeric materials, however, few reactions are currently available. Here we show that using piezochemical reduction of a CuII -based pre-catalyst, a step-growth polymerization occurs via the copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction to form a linear polytriazole. Furthermore, we show that a linear polymer can be crosslinked mechanochemically using the same chemistry to form a solid organogel. We envision that this chemistry can be used to harness mechanical energy for constructive purposes in polymeric materials.

This study assesses the water quality of the Upper Santa Cruz Watershed in southern Arizona in terms of fecal coliform and Escherichia coli (E. coli) bacteria concentrations discharged as treated effluent and from nonpoint sources into the Santa Cruz River and surrounding tributaries. The objectives were to (1) assess the water quality in the Upper Santa Cruz Watershed in terms of fecal coliform and E. coli by comparing the available data to the water quality criteria established by Arizona, (2) to provide insights into fecal indicator bacteria (FIB) response to the hydrology of the watershed and (3) to identify if point sources or nonpoint sources are the major contributors of FIB in the stream. Assessment of the available wastewater treatment plant treated effluent data and in-stream sampling data indicate that water quality criteria for E. coli and fecal coliform in recreational waters are exceeded at all locations of the Santa Cruz River. For the wastewater discharge, 13%–15% of sample concentrations exceeded the 800 colony forming units (cfu) per 100 mL sample maximum for fecal coliform and 29% of samples exceeded the full body contact standard of 235 cfu/100 mL established for E. coli; while for the in-stream grab samples, 16%–34% of sample concentrations exceeded the 800 cfu/100 mL sample maximum for fecal coliforms and 34%–75% of samples exceeded the full body contact standard of 235 cfu/100 mL established for E. coli. Elevated fecal coliform and E. coli concentrations were positively correlated with periods of increased streamflow from rainfall. FIB concentrations observed in-stream are significantly greater (p-value < 0.0002) than wastewater treatment plants effluent concentrations; therefore, water quality managers should focus on nonpoint sources to reduce overall fecal indicator loads. Findings indicate that fecal coliform and E. coli concentrations are highly variable, especially along urban streams and generally increase with streamflow and precipitation events. Occurrences of peaks in FIB concentrations during baseflow conditions indicate that further assessment of ecological factors such as interaction with sediment, regrowth, and source tracking are important to watershed management.

[1] Liquid latex was used as a method to seal visible surface-connected preferential flow pathways (PFPs) in the field in an effort to block large surface-connected preferential flow and force water to move through the soil matrix. The proposed approach allows for the quantification of the contribution of large surface-connected cracks and biological pores to infiltration at various soil moisture states. Experiments were conducted in a silty clay loam soil in a field under a no-till corn-soybean rotation planted to corn. Surface intake rates under ponding were measured using a simplified falling head technique under two scenarios: (1) natural soil conditions with unaltered PFPs and (2) similar soil conditions with latex-sealed large macropores at the surface. Results indicated that the contribution of flow from large surface-connected macropores to overall surface intake rates varied from approximately 34% to 99% depending on the initial moisture content and macroporosity present. However, evidence of preferential flow continued to appear in latex-sealed plots, suggesting significant contributions to preferential flow from smaller structural macropores, particularly in two out of four tests where no significant differences were observed between control and latex-sealed plots. Citation: Sanders, E. C., M. R. Abou Najm, R. H. Mohtar, E. Kladivko, and D. Schulze (2012), Field method for separating the contribution of surface-connected preferential flow pathways from flow through the soil matrix, Water Resour. Res., 48, W04534