A Simple and Rapid Methicillin-Resistant Staphylococcus aureus (MRSA) Screening Test Using a Mannose-Binding Lectin (MBL)-Conjugated Gold Nanoparticle Probe.

Rapid diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) is essential for guiding clinical treatment and preventing the spread of MRSA infections. Herein, we present a simple and rapid MRSA screening test based on the aggregation effect of mannose-binding lectin (MBL)-conjugated gold nanoparticles (AuNP), called the MRSA probe. Recombinant MBL protein is a member of the lectin family and part of the innate immune system. It can recognize wall teichoic acid (WTA) on the membrane of MRSA more specifically than that of methicillin-sensitive Staphylococcus aureus (MSSA) under optimized salt conditions. Thus, the MRSA probe can selectively bind to MRSA, and the aggregation of the probes on the surface of the target bacteria can be detected and analyzed by the naked eye within 5 min. To demonstrate the suitability of the method for real-world application, we tested 40 clinical S. aureus isolates (including 20 MRSA specimens) and recorded a sensitivity of 100%. In conclusion, the MRSA probe-based screening test with its excellent sensitivity has the potential for successful application in the microbiology laboratory.

A rapid quantitative on-site coronavirus disease 19 serological test.

On-site severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) serological assays allow for timely in-field decisions to be made regarding patient status, also enabling population-wide screening to assist in controlling the coronavirus disease 2019 (COVID-19) pandemic. Here we propose a rapid microfluidic serological assay with two unique functions of nanointerstice filling and digitized flow control, which enable the fast/robust filling of the sample fluid as well as precise regulation of duration and volume of immune reaction. Developed microfluidic assay showed enhanced limit of detection, and 91.67% sensitivity and 100% specificity (n = 152) for clinical samples of SARS CoV-2 patients. The assay enables daily monitoring of IgM/IgG titers and patterns, which could be crucial parameters for convalescence from COVID-19 and provide important insight into how the immune system responds to SARS CoV-2. The developed on-site microfluidic assay presented the mean time for IgM and IgG seroconversions, indicating that these titers plateaued days after seroconversion. The mean duration from day 0 to PCR negativity was 19.4 days (median 20 d, IQR 16-21 d), with higher IgM/IgG titres being observed when PCR positive turns into negative. Simple monitoring of these titres promotes rapid on-site detection and comprehensive understanding of the immune response of COVID-19 patients.

Rapid and highly sensitive pathogen detection by real-time DNA monitoring using a nanogap impedimetric sensor with recombinase polymerase amplification.

Fast detection of pathogens is important for protecting our health and society. Herein, we present a high-performance nanogap impedimetric sensor for monitoring nucleic acid amplification in real time using isothermal recombinase polymerase amplification (RPA) for rapid pathogen detection. The nanogap electrode chip has two pairs of opposing gold electrodes with a 100 nm gap and was fixed to a PCB. Then, the nanogap impedimetric sensor was immersed in RPA reaction solution for the detection of E. coli O157:H7, and target DNA amplification was evaluated through bulk solution impedance changes using impedance spectroscopy every minute during RPA. In addition, target gene amplification in the sample solution during RPA was confirmed with a 2% DNA agarose gel. Our nanogap impedimetric sensor can detect down to a single copy of the eae A gene in gDNA extracted from E. coli O157:H7 as well as a single cell of pathogenic E. coli O157:H7 strain within 5 min during direct RPA, which was performed with the pathogen itself and without the extraction and purification of target gDNA. The miniaturized nanogap impedimetric sensor has potential as a cost-effective point-of-care device for fast and accurate portable pathogen detection via real-time nucleic acid analysis.

Simple, rapid, and accurate malaria diagnostic platform using microfluidic-based immunoassay of Plasmodium falciparum lactate dehydrogenase.

This work reports on a rapid diagnostic platform for the detection of Plasmodium falciparum lactate dehydrogenase (PfLDH), a representative malaria biomarker, using a microfluidic microplate-based immunoassay. In this study, the microfluidic microplate made it possible to diagnose PfLDH with a small volume of sample (only 5 µL) and short time (< 90 min) compared to conventional immunoassays such as enzyme-linked immunosorbent assay (ELISA). Moreover, the diagnostic performance of PfLDH showed high sensitivity, specificity, and selectivity (i.e., 0.025 pg/µL in phosphate-buffered saline and 1 pg/µL in human serum). The microfluidic-based microplate sensing platform has the potential to adapt simple, rapid, and accurate diagnoses to the practical detection of malaria.

Staphylococcus aureus Specific FRET Probe-Based Antibacterial Susceptibility Testing (SF-AST) by Detection of Micrococcal Nuclease Activity.

In this study, we describe a simple and rapid antibacterial susceptibility testing (AST) method for Staphylococcus aureus called S. aureus specific fluorescence resonance energy transfer (FRET) probe-based AST (SF-AST), which is based on an S. aureus specific FRET probe (SF probe) that detects micrococcal nuclease (MNase) activity secreted from S. aureus. The SF-AST was tested with an S. aureus quality control (QC) strain against six relevant antibiotics, and the minimum inhibitory concentration (MIC) values obtained with the broth microdilution (BMD) method were compared, as a gold standard AST. Results were obtained with high accuracy in 4-6 h. The MIC for the methicillin resistance using 20 clinical S. aureus isolates of SF-AST showed 100% sensitivity, specificity, positive predictive value, and negative predictive value, as compared to BMD. Thus, the SF-AST method is a simple, rapid, and useful antibiotic resistance test for S. aureus, and it provides a basis for clinical treatment in a short time.

FRET probe-based antibacterial susceptibility testing (F-AST) by detection of bacterial nucleases released by antibiotic-induced lysis.

This study demonstrates a novel and rapid antibacterial susceptibility testing (AST) method, called fluorescence resonance energy transfer (FRET) probe-based AST (F-AST), which relies on a nuclease-activated FRET probe that detects bacterial nucleases released by antibiotic-induced bacterial lysis. Three quality control (QC) strains and two additional clinically important strains were tested, and the minimum inhibitory concentration (MIC) values from both gold standard AST method (broth microdilution (BMD)) and the new F-AST method were compared. The resulting fluorescence signals from the F-AST method were obtained within 3-6 h and were consistent with MIC values obtained from the BMD method, which took more than 16 h. Thus, the F-AST method is a simple and rapid tool to detect antibacterial susceptibility, including MIC values, and provides a basis for rapid clinical treatments.

Multivalent Antibody-Nanoparticle Conjugates To Enhance the Sensitivity of Surface-Enhanced Raman Scattering-Based Immunoassays.

Multivalent immunoprobes can improve the sensitivity of biosensors because increased valency can strengthen the binding affinity between the receptor and target biomolecules. Here, we report surface-enhanced Raman scattering (SERS)-based immunoassays using multivalent antibody-conjugated nanoparticles (NPs) for the first time. Multivalent antibodies were generated through the ligation of Fab fragments fused with Fc-binding peptides to immunoglobulin G. This fabrication method is easy and fast because of the elimination of heterologous protein expression, high degrees of antibody modifications, and covalent chemical ligation steps. We constructed multivalent antibody-NP conjugates (MANCs) and employed them as SERS immunoprobes. MANCs improved the sensitivity of SERS-based immunoassays by 100 times compared to standard antibody-NP conjugates. MANCs will increase the feasibility of practical SERS-based immunoassays.

A scanometric antibody probe for facile and sensitive immunoassays.

We have developed a novel scanometric antibody probe for rapid, sensitive, and naked-eye-visible immunoassays. Using this probe, we clearly demonstrated the successful scanometric detection and identification of influenza A viruses on a microarray. In addition, the sensitivity of the scanometric immunoassay was comparable to that of the fluorescence-based method.

Gold patterned biochips for on-chip immuno-MALDI-TOF MS: SPR imaging coupled multi-protein MS analysis.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of immuno-captured target protein efficiently complements conventional immunoassays by offering rich molecular information such as protein isoforms or modifications. Direct immobilization of antibodies on MALDI solid support enables both target enrichment and MS analysis on the same plate, allowing simplified and potentially multiplexing protein MS analysis. Reliable on-chip immuno-MALDI-TOF MS for multiple biomarkers requires successful adaptation of antibody array biochips, which also must accommodate consistent reaction conditions on antibody arrays during immuno-capture and MS analysis. Here we developed a facile fabrication process of versatile antibody array biochips for reliable on-chip MALDI-TOF-MS analysis of multiple immuno-captured proteins. Hydrophilic gold arrays surrounded by super-hydrophobic surfaces were formed on a gold patterned biochip via spontaneous chemical or protein layer deposition. From antibody immobilization to MALDI matrix treatment, this hydrophilic/phobic pattern allowed highly consistent surface reactions on each gold spot. Various antibodies were immobilized on these gold spots both by covalent coupling or protein G binding. Four different protein markers were successfully analyzed on the present immuno-MALDI biochip from complex protein mixtures including serum samples. Tryptic digests of captured PSA protein were also effectively detected by on-chip MALDI-TOF-MS. Moreover, the present MALDI biochip can be directly applied to the SPR imaging system, by which antibody and subsequent antigen immobilization were successfully monitored.

Detection of mutant p53 using field-effect transistor biosensor.

We assessed the abilities of wild p53 and mutant p53 proteins to interact with the consensus DNA-binding sequence using a MOSFET biosensor. This is the first report in which mutant p53 has been detected on the basis of DNA-protein interaction using a FET-type biosensor. In an effort to evaluate the performance of this protocol, we constructed the core domain of wild p53 and mutant p53 (R248W), which is DNA-binding-defective. After the immobilization of the cognate DNA to the sensing layer, wild p53 and mutant p53 were applied to the DNA-coated gate surface, and subsequently analyzed using a semiconductor analyzer. As a consequence, a significant up-shift in drain current was noted in response to wild p53, but not mutant p53, thereby indicating that sequence-specific DNA-protein interactions could be successfully monitored using a field-effect-based biosensor. These data also corresponded to the results obtained using surface plasmon resonance (SPR) measurements. Taken together, our results show that a FET-type biosensor might be promising for the monitoring of mutant p53 on the basis of its DNA-binding activity, providing us with very valuable insights into the monitoring for diseases, particularly those associated with DNA-protein binding events.

Monitoring of cleavage preference for caspase-3 using recombinant protein substrates.

The apoptotic caspases have been classified in accordance with their substrate specificities, as the optimal tetrapeptide recognition motifs for a variety of caspases have been determined via positional scanning substrate combinatorial library technology. Here, we focused on two proteolytic recognition motifs, DEVD and IETD, owing to their extensive use in cell death assay. Although DEVE and IETD have been generally considered to be selective for caspase-3 and -8, respectively, the proteolytic cleavage of these substrates does not display absolute specificity for a particular caspase. Thus, we attempted to monitor the cleavage preference for caspase-3, particularly using the recombinant protein substrates. For this aim, the chimeric GST:DEVD:EGFP and GST:IETD:EGFP proteins were genetically constructed by linking GST and EGFP with the linkers harboring DEVD and IETD. To our best knowledge, this work constitutes the first application for the monitoring of cleavage preference employing the recombinant protein substrates that simultaneously allow for mass and fluorescence analyses. Consequently, GST: IETD:EGFP was cleaved partially in response to caspase-3, whereas GST:DEVD:EGFP was completely proteolyzed, indicating that GST:DEVD:EGFP is a better substrate than GST:IETD:EGFP for caspase-3. Collectively, using these chimeric protein substrates, we have successfully evaluated the feasibility of the recombinant protein substrate for applicability to the monitoring of cleavage preference for caspase-3.

Detection of conformationally changed MBP using intramolecular FRET.

The principal objective of this study was to explore protein conformational changes using fluorescence resonance energy transfer (FRET) technology. Maltose binding protein (MBP) was adopted as a target model, due to its well-characterized structure and ligand specificity. To the best of our knowledge, this is the first report to provide information regarding the biological distance between the two lobes of MBP upon maltose binding. For the FRET pair, ECFP and EYFP were used as the donor and the acceptor, and were linked genetically to the C-terminal and N-terminal regions of MBP (ECFP:MBP:EYFP), respectively. After the FRET reaction, maltose-treated MBP was shown to exhibit a considerable energy transfer (FRET efficiency (E)= approximately 0.11, Distance (D)= approximately 6.93 nm) at the ensemble level, which was regarded as reflective of the increase in donor quenching and the upshift in acceptor emission intensity, thereby suggesting that the donor and the acceptor had been brought close together as the result of structural alterations in MBP. However, upon glucose treatment, no FRET phenomenon was detected, thereby implying the specificity of interaction between MBP and maltose. The in vitro FRET results were also confirmed via the acceptor photobleaching method. Therefore, our data showed that maltose-stimulated conformational changes of MBP could be measured by FRET, thereby providing biological information, including the FRET efficiency and the intramolecular distance.

Novel application of surface plasmon resonance biosensor chips for measurement of advanced glycation end products in serum of Zucker diabetic fatty rats.

Advanced glycation end products (AGEs) have been implicated in diabetic complications. To measure AGEs, especially N(epsilon)-(carboxymethyl)lysine (CML), in sera from Zucker diabetic fatty rats (ZDF) and Zucker lean rats (ZL), we used a novel method of protein chip and surface plasmon resonance imaging (SPRI). Serum samples were obtained from male ZDF and ZL rats at 20 weeks of age. Antibodies to AGEs or CML were immobilized on a gold surface, which was modified by cysteine-tagged, protein-G constructs. The gold chip upon which the serum was spotted was optically coupled with a prism coupler. The reflected images from the gold chip were obtained using a charge-coupled device (CCD) camera. The direct analysis of the glycated proteins and products using SPRI showed that AGEs and CML levels were elevated in ZDF serum, compared with ZL serum. The lowest detection limit of AGEs was 10 ng/ml, with a working range covering the physiological range. These results indicate that the protein chip and SPRI system is very suitable for the measurement of glycated proteins and end products in serum samples. This system offers high sensitivity without any fluorescent or other labeling of the components and saves a substantial amount of time, resources, and labor. Our results suggest that SPRI systems can be used as a tool to diagnose diabetic complications.

A potent reporter applicable to the monitoring of caspase-3-dependent proteolytic cleavage.

In this study, we developed a chimeric caspase-3 substrate (GST:DEVD:EGFP) comprised of glutathione-S transferase (GST) and enhanced green fluorescent protein (EGFP) with a specialized linker peptide harboring the caspase-3 cleavage sequence, DEVD. Using this reporter, we assessed the proteolytic cleavage of the artificial caspase-3 substrate for caspase-3. The common feature of this approach is that the presence of the DEVD sequence between GST and EGFP allows for caspase-3-dependent cleavage after the Asp (D) residue, resulting in the elimination of EGFP from the GST:DEVD:EGFP reporter. To the best of our knowledge, this study reports the first application employing a chimeric protein substrate, with the similar accuracy level compared to the conventional methods such as fluorometric assays. As a result, using this GST:DEVD:EGFP reporter, caspase-3 activation based on proteolytic properties could be monitored via a variety of bioanalytical techniques such as immunoblot analysis, glutathione-agarose bead assay, and on-chip visualization, providing both technical and economical advantages over the extensively utilized fluorogenic peptide assay. Our results convincingly showed that this versatile reporter (GST:DEVD:EGFP) constitutes a useful system for the monitoring of caspase-3 activation, potentially enabling the monitoring of the proteolytic activities of different intra-cellular proteases via the substitution of the cleavage sequence within the same schematic construct.

Directed immobilization of DNA-binding proteins on a cognate DNA-modified chip surface.

Here we describe a useful method for the site-directed immobilization of proteins with a DNA-binding domain (DNA-BD) on the cognate DNA-coated gold surface for surface plasmon resonance (SPR) imaging analyses. In order to assess the performance of this procedure, we utilized two DNA-BDs, yeast GAL4 DNA-BD, and bacterial LexA DNA-BD. After the immobilization of the cognate double-stranded DNAs (dsDNAs) to a gold chip surface with a monolayer of poly(l-lysine) for sequence-specific DNA-protein interaction, purified recombinant GAL4 DNA-BD:EGFP and LexA DNA-BD:RFP fusion proteins were applied to a dsDNA-spotted gold chip, and were subsequently analyzed using an SPR imaging system. Consequently, the recombinant DNA-binding proteins, GAL4 DNA-BD:EGFP and LexA DNA-BD:RFP, were shown to bind selectively to their cognate DNA sequences on the gold chip. Collectively, our results revealed that sequence-specific dsDNA microarray approach could prove useful in performing the site-directed immobilization of DNA-binding proteins onto a gold thin film in a parallel format, and thereby potentially allowing for the analysis of transcription factor binding profiling as well as for the monitoring of protein-protein interactions between target proteins with DNA-binding domain as a fusion tag and their binding partners.

SPR imaging-based monitoring of caspase-3 activation.

The activation of caspase-3 plays an important role in the apoptotic process. In this study, we describe a novel method by which caspase-3-dependent proteolytic cleavage can be monitored, using a surface plasmon resonance (SPR) imaging protein chip system. To the best of our knowledge, this is the first report regarding the SPR imaging-based monitoring of caspase-3 activation. In order to evaluate the performance of this protocol, we constructed a chimeric caspase-3 substrate (GST:DEVD:EGFP) comprised of glutathione S transferase (GST) and enhanced green fluorescent protein (EGFP) with a specialized linker peptide harboring the caspase-3 cleavage sequence, DEVD. Using this reporter, we assessed the cleavage of the artificial caspase-3 substrate in response to caspase-3 using an SPR imaging sensor. The purified GST:DEVD:EGFP protein was initially immobilized onto a glutathionylated gold chip surface, and subsequently analyzed using an SPR imaging system. As a result, caspase-3 activation predicated on the proteolytic properties inherent to substrate specificity could be monitored via an SPR imaging system with a detection performance similar to that achievable by the conventional method, including fluorometric assays. Collectively, our data showed that SPR imaging protein chip system can be effectively utilized to monitor the proteolytic cleavage in caspase-3, thereby potentially enabling the detection of other intracellular protease activation via the alteration of the protease recognition site in the linker peptides.

Stress-governed expression and purification of human type II hexokinase in Escherichia coli.

The full encoding sequence for human type II hexokinase (HXK II) was cloned into the E. coli expression vector pET 21b and expressed as a C-terminally hexahistidine-tagged protein in the BL21 (DE3) strain. The IPTG-induced HXK II approximately accounted for 17% of the total E. coli proteins, and 81% of HXK II(6xHis) existed in inclusion bodies. To improve the production of soluble recombinant HXK II protein, in the functionally active form, we used low temperature, and the osmotic stress expression method. When expressed at 18 degrees C, about 83% of HXK II(6xHis) existed in the soluble fraction, which amounted to a 4.1-fold yield over that expressed at 37 degrees C. The soluble form of HXK II(6xHis) was also highly produced in the presence of 1 M sorbitol under the standard condition (37 degrees C), which indicated that temperature downshift and low water potentials were required to improve the yield of active recombinant HXK II protein. The expressed protein was purified by metal chelate affinity chromatography performed in an IDA Excellose column charged with Ni2+ ions, resulting in about 40 mg recombinant HXK II protein obtained with purity over 89% from 5 l of E. coli culture. The identity of HXK II(6xHis) was confirmed by Western blotting analysis. Taken together, using the stress-governed expression described in this study, human active HXK II can be purified in sufficient amounts for biochemical and biomedical studies.

A split enhanced green fluorescent protein-based reporter in yeast two-hybrid system.

We have developed a novel reporter system involving a yeast two-hybrid assay, which utilizes the reconstitution of the split EGFP reporter in order to characterize the relevant protein-protein interactions. To our knowledge, this study represents the first application of the split EGFP system as a read-out in a yeast two-hybrid assay. In comparison with the existing two-hybrid system, the bait and prey vectors were improved with regard to the reporter and the replication control element. As a result, the reconstituted EGFP has been observed to evidence a restored fluorescence upon protein-protein interactions in yeast, thereby allowing for the characterization of its interactor. The use of a split EGFP reporter has some salient advantages. Firstly, no substrates are required for the production of fluorescence. Secondly, low copy number plasmids may help to solve the protein toxicity problem, via the reduction of expression. Thirdly, this technique may prove useful in overcoming the autoactivation problem, due to the fact that the read-out of the yeast two-hybrid system is transcription-independent. Collectively, our results showed that the split EGFP reporter system might potentially be applied in yeast two-hybrid assays for the high-throughput screening of protein-protein interactions, with a simple and direct fluorescent read-out.