Ultrasensitive Raman spectroscopy-based virus detection using glycan-coated plasmonic substrates

Hepatitis viral infections are the most common cause of hepatitis liver disease, which eventually leads to cancer and fibrosis if undetected early. Therefore, early detection would allow for preventive and therapeutic actions. Here, a surface-enhanced Raman spectroscopy (SERS)-based biosensor was developed using plasmonic molybdenum trioxide quantum dots (MoO3-QDs) as theSERS substrates. The nanostructured substrate of MoO3-QDs was functionalized with a proteoglycan (Syndecan-1) as a novel bioreceptor for target hepatitis E virus (HEV). The innovative bio-detection system achieved a detection limit of 1.05 fg/mL for tested HEV target (ORF2), indicating superb clinically relevant sensitivity and performance. The designed biosensing system incorporating a glycan motif as a bioreceptor instead of the conventional antibodies or aptamers,presents new insights for the ultrasensitive detection of HEV and other infectious viruses.<br/

Plasmonic Oleylamine-Capped Gold and Silver Nanoparticle-Assisted Synthesis of Luminescent Alloyed CdZnSeS Quantum Dots

We report on a novel strategy to tune the structural and optical properties of luminescent alloyed quantum dot (QD) nanocrystals using plasmonic gold (Au) and silver (Ag) nanoparticles (NPs). Alloyed CdZnSeS QDs were synthesized via the organometallic synthetic route with different fabrication strategies that involve alternative utilization of blends of organic surfactants, ligands, capping agents, and plasmonic oleylamine (OLA)-functionalized AuNPs and AgNPs. Ligand exchange with thiol l-cysteine (l-cyst) was used to prepare the hydrophilic nanocrystals. Analysis of the structural properties using powder X-ray diffraction revealed that under the same experimental condition, the plasmonic NPs altered the diffractive crystal structure of the alloyed QDs. Depending on the fabrication strategy, the crystal nature of OLA-AuNP-assisted CdZnSeS QDs was a pure hexagonal wurtzite domain and a cubic zinc-blende domain, whereas the diffraction pattern of OLA-AgNP-assisted CdZnSeS QDs was dominantly a cubic zinc-blende domain. Insights into the growth morphology of the QDs revealed a steady transformation from a heterogeneous growth pattern to a homogenous growth pattern that was strongly influenced by the plasmonic NPs. Tuning the optical properties of the alloyed QDs via plasmonic optical engineering showed that the photoluminescence (PL) quantum yield (QY) of the AuNP-assisted l-cyst-CdZnSeS QDs was tuned from 10 to 31%, whereas the PL QY of the AgNP-assisted l-cyst-CdZnSeS QDs was tuned from 15 to 90%. The low PL QY was associated with the surface defect state, while the remarkably high PL QY exhibited by the AgNP-assisted l-cyst-CdZnSeS QDs lends strong affirmation that the fabrication strategy employed in this work provides a unique opportunity to create single ensemble, multifunctional, highly fluorescent alloyed QDs for tailored biological applications

Purification of functional baculovirus particles from silkworm larval hemolymph and their use as nanoparticles for the detection of human prorenin receptor (PRR) binding

<p>Abstract</p> <p>Background</p> <p>Baculovirus, which has a width of 40 nm and a length of 250-300 nm, can display functional peptides, receptors and antigens on its surface by their fusion with a baculovirus envelop protein, GP64. In addition, some transmembrane proteins can be displayed without GP64 fusion, using the native transmembrane domains of the baculovirus. We used this functionality to display human prorenin receptor fused with GFP_uv(GFP_uv-hPRR) on the surface of silkworm <it>Bombyx mori </it>nucleopolyhedrovirus (BmNPV) and then tested whether these baculovirus particles could be used to detect protein-protein interactions.</p> <p>Results</p> <p>BmNPV displaying GFP_uv-hPRR (BmNPV-GFP_uv-hPRR) was purified from hemolymph by using Sephacryl S-1000 column chromatography in the presence of 0.01% Triton X-100. Its recovery was 86% and the final baculovirus particles number was 4.98 × 10⁸pfu. Based on the results of enzyme-linked immunosorbent assay (ELISA), 3.1% of the total proteins in BmNPV-GFP_uv-hPRR were GFP_uv-hPRR. This value was similar to that calculated from the result of western blot by a densitometry (2.7%). To determine whether BmNPV-GFP_uv-hPRR particles were bound to human prorenin, ELISA results were compared with those from ELISAs using protease negative BmNPV displaying β1,3-<it>N</it>-acetylglucosaminyltransferase 2 fused with the gene encoding GFP_uv(GGT2) (BmNPV-<it>CP</it>^--GGT2) particles, which do not display hPRR on their surfaces.</p> <p>Conclusion</p> <p>The display of on the surface of the BmNPV particles will be useful for the detection of protein-protein interactions and the screening of inhibitors and drugs in their roles as nanobioparticles.</p

A localized surface plasmon resonance-amplified immunofluorescence biosensor for ultrasensitive and rapid detection of nonstructural protein 1 of Zika virus

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High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV) displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization

<p>Abstract</p> <p>Background</p> <p>Budded baculoviruses are utilized for vaccine, the production of antibody and functional analysis of transmembrane proteins. In this study, we tried to produce and purify the recombinant <it>Bombyx mori </it>nucleopolyhedrovirus (rBmNPV-hPRR) that displayed human (pro)renin receptor (hPRR) connected with FLAG peptide sequence on its own surface. These particles were used for further binding analysis of hPRR to human prorenin. The rBmNPV-hPRR was produced in silkworm larvae and purified from its hemolymph using size exclusion chromatography (SEC).</p> <p>Results</p> <p>A rapid method of BmNPV titer determination in hemolymph was performed using quantitative real-time PCR (Q-PCR). A correlation coefficient of BmNPV determination between end-point dilution and Q-PCR methods was found to be 0.99. rBmNPV-hPRR bacmid-injected silkworm larvae produced recombinant baculovirus of 1.31 × 10⁸plaque forming unit (pfu) in hemolymph, which was 2.8 × 10⁴times higher than transfection solution in Bm5 cells. Its purification yield by Sephacryl S-1000 SF column chromatography was 264 fold from larval hemolymph at 4 days post-injection (p.i.), but 35 or 39 fold at 4.5 or 5 days p.i., respectively. Protein patterns of rBmNPV-hPRR purified at 4 and 5 days were the same and ratio of envelope proteins (76, 45 and 35 kDa) to VP39, one of nucleocapsid proteins, increased at 5 days p.i. hPRR was detected in only purified rBmNPV-hPRR at 5 days p.i..</p> <p>Conclusion</p> <p>The successful purification of rBmNPV-hPRR indicates that baculovirus production using silkworm larvae and its purification from hemolymph by Sephacryl S-1000 SF column chromatography can provide an economical approach in obtaining the purified BmNPV stocks with high titer for large-scale production of hPRR. Also, it can be utilized for further binding analysis and screening of inhibitors of hPRR.</p

Bioconversion of paper sludge to biofuel by simultaneous saccharification and fermentation using a cellulase of paper sludge origin and thermotolerant Saccharomyces cerevisiae TJ14

<p>Abstract</p> <p>Background</p> <p>Ethanol production from paper sludge (PS) by simultaneous saccharification and fermentation (SSF) is considered to be the most appropriate way to process PS, as it contains negligible lignin. In this study, SSF was conducted using a cellulase produced from PS by the hypercellulase producer, <it>Acremonium cellulolyticus </it>C-1 for PS saccharification, and a thermotolerant ethanol producer <it>Saccharomyces cerevisiae </it>TJ14 for ethanol production. Using cellulase of PS origin minimizes biofuel production costs, because the culture broth containing cellulase can be used directly.</p> <p>Results</p> <p>When 50 g PS organic material (PSOM)/l was used in SSF, the ethanol yield based on PSOM was 23% (g ethanol/g PSOM) and was two times higher than that obtained by a separate hydrolysis and fermentation process. Cellulase activity throughout SSF remained at around 60% of the initial activity. When 50 to 150 g PSOM/l was used in SSF, the ethanol yield was 21% to 23% (g ethanol/g PSOM) at the 500 ml Erlenmeyer flask scale. Ethanol production and theoretical ethanol yield based on initial hexose was 40 g/l and 66.3% (g ethanol/g hexose) at 80 h, respectively, when 161 g/l of PSOM, 15 filter paper units (FPU)/g PSOM, and 20% inoculum were used for SSF, which was confirmed in the 2 l scale experiment. This indicates that PS is a good raw material for bioethanol production.</p> <p>Conclusions</p> <p>Ethanol concentration increased with increasing PSOM concentration. The ethanol yield was stable at PSOM concentrations of up to 150 g/l, but decreased at concentrations higher than 150 g/l because of mass transfer limitations. Based on a 2 l scale experiment, when 1,000 kg PS was used, 3,182 kFPU cellulase was produced from 134.7 kg PS. Produced cellulase was used for SSF with 865.3 kg PS and ethanol production was estimated to be 51.1 kg. Increasing the yeast inoculum or cellulase concentration did not significantly improve the ethanol yield or concentration.</p