A fast, ultrasensitive SERS immunoassay to detect SARS-CoV-2 in saliva.

The COVID-19 pandemic has emphasized the need for accurate, rapid, point-of-care diagnostics to control disease transmission. We have developed a simple, ultrasensitive single-particle surface-enhanced Raman spectroscopy (SERS) immunoassay to detect the SARS-CoV-2 spike protein in saliva. This assay relies on the use of single chain Fv (scFv) recombinant antibody expressed in E. coli to bind the SARS-CoV-2 spike protein. Recombinant scFv labeled with a SERS-active dye in solution is mixed with unlabeled scFv conjugated to gold-coated magnetic nanoparticles and a sample to be tested. In the presence of the SARS-CoV-2 spike protein, immunocomplexes form and concentrate the labeled scFv close to the gold surface of the nanoparticles, causing an increased SERS signal. The assay detects inactivated SARS-CoV-2 virus and spike protein in saliva at concentrations of 1.94 × 103 genomes mL-1 and 4.7 fg mL-1, respectively, making this direct detection antigen test only 2-3 times less sensitive than some qRT-PCR tests. All tested SARS-CoV-2 spike proteins, including those from alpha, beta, gamma, delta, and omicron variants, were detected without recognition of the closely related SARS and MERS spike proteins. This 30 min, no-wash assay requires only mixing, a magnetic separation step, and signal measurements using a hand-held, battery-powered Raman spectrometer, making this assay ideal for ultrasensitive detection of the SARS-CoV-2 virus at the point-of-care.

Rapid, Point-of-Care scFv-SERS Assay for Femtogram Level Detection of SARS-CoV-2.

Rapid, sensitive, on-site identification of SARS-CoV-2 infections is an important tool in the control and management of COVID-19. We have developed a surface-enhanced Raman scattering (SERS) immunoassay for highly sensitive detection of SARS-CoV-2. Single-chain Fv (scFv) recombinant antibody fragments that bind the SARS-CoV-2 spike protein were isolated by biopanning a human scFv library. ScFvs were conjugated to magnetic nanoparticles and SERS nanotags, followed by immunocomplex formation and detection of the SARS-CoV-2 spike protein with a limit of detection of 257 fg/mL in 30 min in viral transport medium. The assay also detected B.1.1.7 ("alpha"), B.1.351 ("beta"), and B.1.617.2 ("delta") spike proteins, while no cross-reactivity was observed with the common human coronavirus HKU1 spike protein. Inactivated whole SARS-CoV-2 virus was detected at 4.1 × 104 genomes/mL, which was 10-100-fold lower than virus loads typical of infectious individuals. The assay exhibited higher sensitivity for SARS-CoV-2 than commercial lateral flow assays, was compatible with viral transport media and saliva, enabled rapid pivoting to detect new virus variants, and facilitated highly sensitive, point-of-care diagnosis of COVID-19 in clinical and public health settings.

Inactivation of the uptake hydrogenase in the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS enables a biological water-gas shift platform for H2 production.

Biological H2 production has potential to address energy security and environmental concerns if produced from renewable or waste sources. The purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS produces H2 while oxidizing CO, a component of synthesis gas (Syngas). CO-linked H2 production is facilitated by an energy-converting hydrogenase (Ech), while a subsequent H2 oxidation reaction is catalyzed by a membrane-bound hydrogenase (MBH). Both hydrogenases contain [NiFe] active sites requiring 6 maturation factors (HypA-F) for assembly, but it is unclear which of the two annotated sets of hyp genes are required for each in R. gelatinosus CBS. Herein, we report correlated expression of hyp1 genes with Ech genes and hyp2 expression with MBH genes. Moreover, we find that while Ech H2 evolving activity is only delayed when hyp1 is deleted, hyp2 deletion completely disrupts MBH H2 uptake, providing a platform for a biologically driven water-gas shift reaction to produce H2 from CO.

Genome annotation provides insight into carbon monoxide and hydrogen metabolism in Rubrivivax gelatinosus.

We report here the sequencing and analysis of the genome of the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS. This microbe is a model for studies of its carboxydotrophic life style under anaerobic condition, based on its ability to utilize carbon monoxide (CO) as the sole carbon substrate and water as the electron acceptor, yielding CO2 and H2 as the end products. The CO-oxidation reaction is known to be catalyzed by two enzyme complexes, the CO dehydrogenase and hydrogenase. As expected, analysis of the genome of Rx. gelatinosus CBS reveals the presence of genes encoding both enzyme complexes. The CO-oxidation reaction is CO-inducible, which is consistent with the presence of two putative CO-sensing transcription factors in its genome. Genome analysis also reveals the presence of two additional hydrogenases, an uptake hydrogenase that liberates the electrons in H2 in support of cell growth, and a regulatory hydrogenase that senses H2 and relays the signal to a two-component system that ultimately controls synthesis of the uptake hydrogenase. The genome also contains two sets of hydrogenase maturation genes which are known to assemble the catalytic metallocluster of the hydrogenase NiFe active site. Collectively, the genome sequence and analysis information reveals the blueprint of an intricate network of signal transduction pathways and its underlying regulation that enables Rx. gelatinosus CBS to thrive on CO or H2 in support of cell growth.

Draft genome sequence of Rubrivivax gelatinosus CBS.

Rubrivivax gelatinosus CBS, a purple nonsulfur photosynthetic bacterium, can grow photosynthetically using CO and N(2) as the sole carbon and nitrogen nutrients, respectively. R. gelatinosus CBS is of particular interest due to its ability to metabolize CO and yield H(2). We present the 5-Mb draft genome sequence of R. gelatinosus CBS with the goal of providing genetic insight into the metabolic properties of this bacterium.

The role of the bidirectional hydrogenase in cyanobacteria.

Cyanobacteria have tremendous potential to produce clean, renewable fuel in the form of hydrogen gas derived from solar energy and water. Of the two cyanobacterial enzymes capable of evolving hydrogen gas (nitrogenase and the bidirectional hydrogenase), the hox-encoded bidirectional Ni-Fe hydrogenase has a high theoretical potential. The physiological role of this hydrogenase is a highly debated topic and is poorly understood relative to that of the nitrogenase. Here the structure, assembly, and expression of this enzyme, as well as its probable roles in metabolism, are discussed and analyzed to gain perspective on its physiological role. It is concluded that the bidirectional hydrogenase in cyanobacteria primarily functions as a redox regulator for maintaining a proper oxidation/reduction state in the cell. Recommendations for future research to test this hypothesis are discussed.

Xenopus DNA2 is a helicase/nuclease that is found in complexes with replication proteins And-1/Ctf4 and Mcm10 and DSB response proteins Nbs1 and ATM.

We have used the Xenopus laevis egg extract system to study the roles of vertebrate Dna2 in DNA replication and double-strand-break (DSB) repair.  We first establish that Xenopus Dna2 is a helicase, as well as a nuclease.  We further show that Dna2 is a nuclear protein that is actively recruited to DNA only after replication origin licensing.  Dna2 co-localizes in foci with RPA and is found in a complex with replication fork components And-1 and Mcm10.  Dna2 interacts with the DSB repair and checkpoint proteins Nbs1 and ATM.  We also determine the order of arrival of ATM, MRN, Dna2, TopBP1, and RPA to duplex DNA ends and show that it is the same both in S phase and M phase extracts.  Interestingly, Dna2 can bind to DNA ends independently of MRN, but efficient nucleolytic resection, as measured by RPA recruitment, requires both MRN and Dna2.  The nuclease activity of Mre11 is required, since its inhibition delays both full Dna2 recruitment and resection. Dna2 depletion inhibits but does not block resection, and Chk1 and Chk2 induction occurs in the absence of Dna2.