Deoxyribonucleic acid was covalently immobilized onto oxidized glassy carbon electrode surfaces that had been activated using 1-[3-(dimethylamino)-propyl]-3-ethylcarbodimide hydrochloride and N-hydroxysulfosuccinimide. This reaction is selective for immobilization through deoxyguanosine (dG) residues. Immobilized DNA was detected voltammetrically, using tris (2,2'-bipyridyl)cobalt(III) perchlorate and tris (1,10-phenanthroline)cobalt(III) perchlorate (Co(bpy)3(3+) and Co(phen)3(3+). These complexes are reversibly electroactive (1e-) and preconcentrate at the electrode surface through association with double-stranded DNA. Voltammetric peak currents obtained with a poly(dG)poly(dC)-modified electrode depend on [Co(bpy)3(3+)] and [Co(phen)3(3+)] in a nonlinear fashion and indicate saturation binding with immobilized DNA. Voltammetric peak currents for Co(phen)3(3+) reduction were used to estimate the (constant) local DNA concentration at the modified electrode surface; a binding site size of 5 base pairs and an association constant of 1.74 x 10(3) M(-1) yield 8.6 +/- 0.2 mM base pairs. Cyclic voltammetric peak separations indicate that heterogeneous electron transfer is slower at DNA-modified electrodes than at unmodified glassy carbon electrodes. A prototype sequence-selective DNA sensor was constructed by immobilizing a 20-mer oligo (deoxythymidylic acid) (oligo(dT)20), following its enzymatic elongation with dG residues, which yielded the species oligo(dT)20(dG)98. Cyclic voltammograms of 0.12 mM Co(bpy)3(3+) obtained before and after hybridization with poly-(dA) and oligo(dA)20 show increased cathodic peaks after hybridization. The single-stranded form is regenerated on the electrode surface by rinsing with hot deionized water. These results demonstrate the use of electroactive hybridization indicators in a reusable sequence-selective biosensor for DNA.

Carbon paste electrodes modified by the inclusion of either octadecylamine or stearic acid were used as solid phases to which DNA was covalently bound. Immobilized DNA was detected by voltammetry of solutions containing submillimolar quantities of Co(bpy)3(ClO4)3, Co(phen)3(ClO4)3, and Os(bpy)3-Cl2 (bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline), all of which associate reversibly with immobilized DNA and yield increased peak currents at DNA-modified electrodes. Immobilization onto octadecylamine-modified electrodes was performed using a water-soluble carbodiimide, and at high DNA concentrations in the reaction mixture, it resulted in visible polymerization of DNA on the surface. Optimization of the deoxyguanosine- (dG-) selective immobilization reaction for stearic acid-modified electrodes, using water-soluble carbodiimide and N-hydroxysulfosuccinimide reagents to activate carboxylate groups on the surface, yielded conditions of 4.5% (w/w) stearic acid and 10 micrograms/mL DNA. Polythymidylic acid of 4000-base average length (poly(dT)4000) was immobilized at stearic acid-modified electrodes following enzymatic elongation with dG residues at the 3'-end. These DNA-modified electrodes were used to study hybridization with analyte poly(dA)4000 by in situ voltammetry of 60 microM Co(bpy)3(ClO4)3 at low ionic strength (20 mM NaCl), and by voltammetry of the same complex, following exposure of the electrode to poly(dA)4000 in a separate hybridization step conducted at high ionic strength (0.5 M NaCl). Results indicate slow (> or = 1 h) hybridization at low ionic strength and fast (< or = 10 min) hybridization at high ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Background The pandemic due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has tremendous consequences for our societies. Knowledge of the seroprevalence of SARS-CoV-2 is needed to accurately monitor the spread of the epidemic and to calculate the infection fatality rate (IFR). These measures may help the authorities to make informed decisions and adjust the current societal interventions. The objective was to perform nationwide real-time seroprevalence surveying among blood donors as a tool to estimate previous SARS-CoV-2 infections and the population based IFR. Methods Danish blood donors aged 17–69 years giving blood April 6 to May 3 were tested for SARS-CoV-2 immunoglobulin M and G antibodies using a commercial lateral flow test. Antibody status was compared between geographical areas and an estimate of the IFR was calculated. The seroprevalence was adjusted for assay sensitivity and specificity taking the uncertainties of the test validation into account when reporting the 95% confidence intervals (CI). Results The first 20,640 blood donors were tested and a combined adjusted seroprevalence of 1.9% (CI: 0.8-2.3) was calculated. The seroprevalence differed across areas. Using available data on fatalities and population numbers a combined IFR in patients younger than 70 is estimated at 89 per 100,000 (CI: 72-211) infections. Conclusions The IFR was estimated to be slightly lower than previously reported from other countries not using seroprevalence data. The IFR is likely several fold lower than the current estimate. We have initiated real-time nationwide anti-SARS-CoV-2 seroprevalence surveying of blood donations as a tool in monitoring the epidemic.

A review of reported biosensors for the sequence-selective detection of analyte DNA sequences shows that signal transduction based on electrogenerated chemiluminescence, potentiometry and voltammetry are all capable of detecting analyte DNA at attomole levels. In contrast, optical and piezoelectric transducers detect DNA at picomole and femtomole levels, respectively. The principles and applications of these promising electrochemical DNA biosensors are described and discussed.

Serological SARS-CoV-2 assays are needed to support clinical diagnosis and epidemiological investigations. Recently, assays for large-scale detection of total antibodies (total-Ab) and immunoglobulin (Ig) G and M against SARS-CoV-2 antigens have been developed, but there are limited data on the diagnostic accuracy of these assays. This study was a Danish national collaboration and evaluated fifteen commercial and one in-house anti-SARS-CoV-2 assays in sixteen laboratories. Sensitivity was evaluated using 150 samples from individuals with asymptomatic, mild or moderate COVID-19; nonhospitalized or hospitalized, confirmed by nucleic acid amplification tests (NAAT), collected 13-73 days either from symptom onset or from positive NAAT (patients without symptoms). Specificity and cross reactivity were evaluated in samples collected prior to the SARS-CoV-2 epidemic from >586 blood donors and patients with autoimmune diseases, cytomegalovirus or Epstein-Barr virus infections and acute viral infections. A specificity of ≥99% was achieved by all total-Ab and IgG assays except one, Diasorin/LiaisonXL-IgG (97.2%). Sensitivities in descending order were: Wantai/ELISA total-Ab (96.7%), CUH-NOVO/in-house ELISA total-Ab (96.0%), Ortho/Vitros total-Ab (95.3%), YHLO/iFlash-IgG (94.0%), Ortho/Vitros-IgG (93.3%), Siemens/Atellica total-Ab (93.2%), Roche/Elecsys total-Ab (92.7%), Abbott/Architect-IgG (90.0%), Abbott/Alinity-IgG (median 88.0%), Diasorin/LiaisonXL-IgG (median 84.6%), Siemens/Vista total-Ab (81.0%), Euroimmun/ELISA-IgG (78.0%), and Snibe/Maglumi-IgG (median 78.0%). However, confidence intervals overlapped for several assays. The IgM results were variable, with the Wantai/ELISA-IgM showing the highest sensitivity (82.7%) and specificity (99%). The rate of seropositivity increased with time from symptom onset and symptom severity.

Detailed knowledge of how diversity in the sequence of the human genome affects phenotypic diversity depends on a comprehensive and reliable characterization of both sequences and phenotypic variation. Over the past decade, insights into this relationship have been obtained from whole-exome sequencing or whole-genome sequencing of large cohorts with rich phenotypic data1,2. Here we describe the analysis of whole-genome sequencing of 150,119 individuals from the UK Biobank3. This constitutes a set of high-quality variants, including 585,040,410 single-nucleotide polymorphisms, representing 7.0% of all possible human single-nucleotide polymorphisms, and 58,707,036 indels. This large set of variants allows us to characterize selection based on sequence variation within a population through a depletion rank score of windows along the genome. Depletion rank analysis shows that coding exons represent a small fraction of regions in the genome subject to strong sequence conservation. We define three cohorts within the UK Biobank: a large British Irish cohort, a smaller African cohort and a South Asian cohort. A haplotype reference panel is provided that allows reliable imputation of most variants carried by three or more sequenced individuals. We identified 895,055 structural variants and 2,536,688 microsatellites, groups of variants typically excluded from large-scale whole-genome sequencing studies. Using this formidable new resource, we provide several examples of trait associations for rare variants with large effects not found previously through studies based on whole-exome sequencing and/or imputation.

Glucose oxidase (GOx) was covalently modified at pH 7.2 with ferrocenecarboxylic acid (FCA), ferrocenedicarboxylic acid (FDA), and ferroceneacetic acid (FAA) using 1-ethyl-3-( 3-(dimethy1amino)propyl)-carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide to promote selective coupling to surface lysines.Reagent ratios were varied to obtain derivatives with 5-12 ferrocene groups per GOx dimer and 260% activity. For comparison, GOx was derivatized with FCA in the presence of 3 M urea using only EDC as a promoter. Varying reagent ratios yielded derivatives with 4-39 FCA groups per GOx and 530% activity; linear sweep voltammetry results showed a slow but readily detectable release of FCA upon storage of these derivatives. Tryptophan fluorescence quenching in two media, 0.1 M phosphate buffer (pH 7.0) and 8 M urea, confirmed that GOx was covalently modified and not merely associated with ferrocene. In all cases, the GOx derivatives exhibited significantly greater quenching than controls containing native GOx with free ferrocenes. The results of voltammetric dilution experiments (performed in oxygen-free solutions in the presence of excess glucose) were consistent with rate-limiting intramolecular electron transfer from the reduced flavin centers to bound ferricinium. Using an expression derived here, values between 0.16 and 0.90 s-1 were obtained for intramolecular electron transfer in the FCA derivatives, suggesting that the location (rather than the number) of bound ferrocene groups is rate-determining. Approximately lo3-fold slower intramolecular electron transfer was measured in an FDA derivative, consistent with fluorescence quenching data which indicated that bound FDA is more solvent-exposed than bound FCA. The results of lysine-targeted modification of GOx are interpreted in light of the recently published 3-D structure of GOx; since the critical flavin-lysine separations are all >23 A, an alternative approach is necessary to obtain GOx derivatives for use in a practical, reagentless glucose sensor.

A novel neutral ferrocene-containing polyacrylamidebased redox gel was prepared from the copolymerization of vinylferrocene (VF) with acrylamide and A^iV'-methylenebisacrylamide in a one-step procedure. Hydroxypropyl-/?-cyclodextrin (HPCD) was used to convert waterinsoluble VF into the water-soluble VF-HPCD inclusion complex, allowing the copolymerization to be performed in aqueous solution. A new ternary catalyst, consisting of flavin mononucleotide, H2O2, and -tetra-