Dual-Mechanism-Driven Ratiometric Electrochemiluminescent Biosensor for Methicillin-Resistant Staphylococcus aureus.

Design of a ratiometric method is a promising pathway to improve the sensitivity and reliability of electrochemiluminescent (ECL) assay, for which the signals produced at two distinct potentials change reversely as it is applied to the target analyte. Herein, a biosensor for ECL assay of methicillin-resistant Staphylococcus aureus (MRSA) was constructed by immobilizing porcine IgG for capturing MRSA onto an electrode that was precoated with ß-cyclodextrin-conjugated luminol nanoparticles (ß-CD-Lu NPs) as an anodic luminophore. MOF PCN 224 loaded with an atomically distributed Zn element (PCN 224/Zn) was conjugated with phage recombinant cellular-binding domain (CBD) to act as a cathodic luminophore for tracing MRSA. After the formation of the sandwich complex of ß-CD-Lu NPs-porcine IgG/MRSA/PCN 224/Zn-CBD on the biosensor, two ECL reactions were triggered with cyclic voltammetry. The anodic process of the ß-CD-Lu NPs-H2O2 system and the cathodic process of the PCN 224/Zn-S2O82- system competed to react with reactive oxygen species (ROS) for producing ECL emission, which led to a reverse change of the two signals. Meanwhile, the overlap of the ß-CD-Lu NPs emission spectrum and PCN 224/Zn absorption spectrum effectively triggered ECL resonance energy transfer between the donor (ß-CD-Lu NPs) and the acceptor (PCN 224/Zn). Thus, a ratiometric ECL method was proposed for assaying MRSA with a dual-mechanism-driven mode. The detection limit for assaying MRSA is as low as 12 CFU/mL. The biosensor was applied to assay MRSA in various biological samples with recoveries ranging from 84.9 to 111.3%.

Preparation of Co dual atomic site catalysts loaded on defect-engineered MOFs material with superb chemiluminescent enhancement effect for sensitive detection of bacteria.

BACKGROUND: Dual atomic site catalysts (DASCs) have aroused extensive interest in analytical chemistry on account of the superb catalytic activity caused by the highly-exposed active centers and synergistic effect of adjacent active centers. The reported protocols for preparing DASCs usually involve harsh conditions such as acid/base etching and high-temperature calcination, leading to unfavorable water dispersity and restricted application. It is crucial to develop DASCs with satisfactory water dispersity, improved stability, and mild preparation procedures to facilitate their application as signal probes in analytical chemistry. RESULTS: Formic acid was adopted as a modulator for preparing MOF-808 with abundant defective sites, which was used as the carrier for implanting Co atoms. Co DASCs with a special coordination structure of Co2-O10 and a high loading efficiency of 11.1 wt% were prepared with a mild solvothermal protocol. The resultant Co DASCs can significantly accelerate decay of H2O2 for forming numerous reactive oxygen radicals and boost chemiluminescent (CL) signal. Co DASCs at 1.0 µg mL-1 can enhance the CL signal of luminol-H2O2 system by about 5800 times. Thanks to their satisfactory water dispersity and excellent CL enhancement performance, they were used as ultra-sensitive CL signal probes for monitoring methicillin-resistant Staphylococcus aureus. The method shows a detection range of 102-107 CFU mL-1 and a detection limit of 47 CFU mL-1. Antibiotic susceptibility test was performed with the established CL method to prove its practicality. SIGNIFICANCE: The water dispersible Co DASCs prepared with facile and mild solvothermal protocol exhibit prominent peroxidase-like activity and can promote the production of reactive oxygen radicals for boosting CL signal. Therefore, this study paves an avenue for implanting DASCs in defect-engineered carrier to prepare signal probes suitable for development of ultra-sensitive CL analytical methods.

Paper-based analytical device integrated with bacteriophage tail fiber protein for bacteria detection and antimicrobial susceptibility test.

Severe antimicrobial resistance calls for developing rapid, sensitive and affordable methodological platform for clinical diagnosis of bacterial infection. Herein, a paper-based analytical device (PAD) for fluorescent (FL) detection and antimicrobial susceptibility test (AST) of Pseudomonas aeruginosa (P. aeruginosa) was fabricated by defining 96 hydrophilic microzones on a filter paper using a handheld stamp dipped with liquid wax. The size of microzones was designed to be consistent with traditional 96-well microplate, thus the FL signals can be collected by a commercialized microplate reader. Streptavidin was immobilized into the microzones by chitosan-glutaraldehyde crosslinking reaction, and then biotinylated bacteriophage tail fiber protein (TFP) was conjugated through biotin-streptavidin affinity system. TFP and fluorescein isothiocyanate (FITC) labeled antimicrobial peptide were used as capture agent and signal probe, respectively, for FL detection of P. aeruginosa on the PAD. The linear range for quantifying P. aeruginosa is 1.0 × 103 CFU/mL to 1.0 × 107 CFU/mL, with a detection limit of 137 CFU/mL. The PAD was also applied to conduct AST of P. aeruginosa for imipenem, meropenem, cefepime, amikacin, and gentamicin, and the results are consistent with the traditional broth dilution method. The PAD provides an affordable diagnosis platform for bacterial infection, especially in resource-limited institutes and countries.

PEGylated Ni Single-Atom Catalysts as Ultrasensitive Electrochemiluminescent Probes with Favorable Aqueous Dispersibility for Assaying Drug-Resistant Pathogens.

Ni single-atom catalysts (SACs) were synthesized by high-temperature calcination of nickel ions and 1,10-phenanthroline on carbon black as a carrier. Benefiting from the ultrahigh atom utilization efficiency, Ni SACs can significantly accelerate decay of dissolved oxygen to generate abundant reactive oxygen species through an oxygen reduction reaction occurring on cathodes. The generated reactive oxygen species can vastly enhance the electrochemiluminescent (ECL) signal of luminol without participation of exogenous co-reactants. To overcome the inherent unfavorable aqueous dispersibility of Ni SACs prepared by the calcination protocol, they were functionalized with highly hydrophilic PEG 2000. Thanks to the abundant carboxyl groups on PEG 2000, the PEGylated Ni SACs (Ni@PEG) can be used as ECL probes to tag biorecognition molecules. In this proof-of-principle work, an ECL biosensor for assaying methicillin-resistant Staphylococcus aureus was developed by using porcine IgG as capture molecule and phage cell-binding domain tagged with Ni@PEG as signal tracer. It shows a broad linear range of 73-7.3 × 106 CFU/mL and a low detection limit of 25 CFU/mL. The recovery values for assaying spiked samples are between 80.8 and 119.2%. It was also utilized to assess MRSA susceptibility to four antibiotics, with results consistent with those obtained by the standard broth microdilution technique. To the best of our knowledge, it is the first time to utilize aqueous dispersible SACs as highly sensitive ECL probes for developing biosensors.

Small ubiquitin-related modifier-fused bacteriophage tail fiber protein with favorable aqueous solubility for lateral flow assay of Pseudomonas aeruginosa.

Rapid and sensitive assay of pathogenic bacteria is critical for minimizing the risk of infectious diseases. Inspired by the interaction between bacteriophages and host bacteria, we obtained a gene sequence of tail fiber protein (TFP) from Pseudomonas aeruginosa (P. aeruginosa) bacteriophage. Then the gene sequence was used to express a recombinant TFP, which can act as a potential capture molecule for P. aeruginosa. Small ubiquitin-related modifier (SUMO) tag was fused onto the TFP fragment to overcome its unfavorable aqueous solubility. The obtained SUMO tag-fused TFP (STFP) can be uniformly distributed onto a nitrocellulose membrane to form a test line due to the improved aqueous solubility, which facilities fabrication of a lateral flow assay strip. Thus we developed a lateral flow assay method by using STFP as a capture molecule and AuCo nanoparticles-labeled aptamer as a signal tracer for point-of-care testing of P. aeruginosa. By using the test strip, P. aeruginosa can be semi quantified with color band and quantified with chemiluminescent (CL) signal catalyzed by AuCo nanoparticles. The concentration range for quantification is 3.3 × 102 CFU/mL to 3.3 × 107 CFU/mL. The test strip was applied to assay P. aeruginosa in different sample matrixes including cerebrospinal fluid, physiological salt solution, drinking water and pear juice. The results demonstrate the application potential of the STFP-based lateral flow assay for medical diagnosis, food and drug safety monitoring.

Risk Factors for Antimicrobial Resistance of Staphylococcus Species Isolated from Dogs with Superficial Pyoderma and Their Owners.

The microbial communities on the skin of dogs include several species of bacteria, which contribute to skin health and disease. Staphylococcus pseudintermedius, cultured at high frequency from the skin of dogs, is an opportunistic pathogen causing superficial pyoderma. Effective treatment against S. pseudintermedius infections is an important issue in veterinary medicine. However, multiple antibiotic-resistant mechanisms gradually developed by bacteria make treatment more challenging nowadays. Drug-resistant genes may have the chance to be transferred from infected dogs to other staphylococci in humans. The objective of this survey is to investigate the bacterial species that cause canine superficial pyoderma and characterize the antibiotic-resistant profiles and drug-resistant genes of isolated S. pseudintermedius. In addition, the possible risk factors causing S. pseudintermedius colonizing owners were also evaluated by a questionnaire survey. Sixty-five bacteria were isolated from dogs with superficial pyoderma, which included 47 S. pseudintermedius (72.3%), 12 other staphylococci (18.5%), 4 other Gram-positive bacteria (6.2%) and 2 Gram-negative bacteria (3.1%). Strains containing mecA and blaZ genes showed multiple-drug resistance characteristics. Dogs that received antimicrobial treatment within a recent month were at significantly higher risk of MRSP infections. Only five S. pseudintermedius strains (8.33%) were isolated from 60 samples of owners. Risk factor analysis indicated there was no significant association between S. pseudintermedius isolated from dogs and owners, but the "Keeping three or more dogs" and "Dogs can lick the owner's face" have high odds ratios of 3.503 and 5.712, respectively. MRSP isolates belonged to three different dru types, including dt11y (29.41%), dt11a (47.06%) and dt10cp (23.53%). In conclusion, the major pathogen of canine superficial pyoderma is found to be S. pseudintermedius in Taiwan, and isolates which are mecA- or blaZ-positive are generally more resistant to commonly used antibiotics. Although S. pseudintermedius isolated from the owners might be transferred from their dogs, definite risk factors should be examined in the future study.

An electrochemical immunosensor coupling a bamboo-like carbon nanostructure substrate with toluidine blue-functionalized Cu(ii)-MOFs as signal probes for a C-reactive protein assay.

In this paper, a novel sandwich immunosensor based on a toluidine blue (Tb) loaded metal organic framework (Cu(ii)-HKUST-1/Tb) as the signal element and a nitrogen-doped 3D carbon nanostructure as the electrode substrate was constructed for the detection of C-reactive protein (CRP). Tb as an electrochemically active agent usually forms a polymer by aggregation in the solvent, causing a poor electrochemical response. Therefore, in order to overcome this obstacle, Cu(ii)-HKUST-1 with a porous nanostructure and large specific surface area as a carrier could adsorb a large number of Tb molecules on its surface to improve its electrochemical performance. In addition, the high electron transfer efficiency of the N-doped bamboo-like carbon nanotubes (CoFe/N-GCT) improves the sensitivity of the biosensor. Differential pulse voltammetry (DPV) was used to detect the current signal of Tb at -0.2 V. The current response increased with the increase in concentration of CRP, ranging from 0.5 to 200 ng mL-1. The detection limit is 166.7 pg mL-1 (S/N = 3). Moreover, the proposed biosensor can be applied in real serum sample detection. It has potential applications in the field of biomedicine assays.

Electrochemical detection of C-reactive protein using functionalized iridium nanoparticles/graphene oxide as a tag.

C-reactive protein (CRP) has become a recognized indicator of inflammation. CRP concentration in serum is an important indicator for monitoring early heart damage, and it is also a newly discovered coronary heart disease-associated inflammatory factor. A conductive nano-hybrid material composed of Au NPs and ionic liquid functionalized molybdenum disulfide (Au NPs/IL-MoS2) was prepared and utilized to immobilize primary CRP antibodies. Subsequently, 1,5-diaminonaphthalene (DN) was adsorbed onto graphene oxide (GO) through π-π stacking, which was used to load iridium nanoparticles (Ir NPs) as a tag to label secondary CRP antibodies. The large surface area of Au NPs/IL-MoS2 and the excellent electrocatalytic properties of Ir NPs/GO-DN toward the reduction of H2O2 resulted in a highly sensitive assay for CRP antigens. This immunosensor exhibited wide linear ranges from 0.01 to 100 ng mL-1 and a lower detection of limit of 3.3 pg mL-1 (S/N = 3). This CRP immunosensor can be applied in real serum sample analysis with satisfactory results, indicating that the immunosensor has potential applications in biomedical detection.

An Enzyme-Linked Immunosorbent Assay for C-Reactive Protein Based on Gold Nanoparticles@Metal Porphyrin Porous Compound.

In this paper, an enzyme-linked immunosorbent assay (ELISA) using gold nanoparticles/metal porphyrin porous compound (Au NPs@PAF-40-Fe) as a marker was developed. Quantitative detection of C-reactive protein (CRP) was achieved by UV-visible spectrophotometry under optimal conditions. The sensor has a good linear relationship in the range of 0.05-2000 ng/mL. The lower limit of detection was 0.017 ng/mL (S/N = 3), the linear correlation coefficient was 0.9921, and the average recovery was 100.74%. The ELISA has good selectivity and provides a sensitive way for detection of CRP.

2D-porphrinic covalent organic framework-based aptasensor with enhanced photoelectrochemical response for the detection of C-reactive protein.

In this study, a novel photoelectrochemical (PEC) aptasensor based on two-dimensional (2D) porphyrinic covalent organic frameworks (p-COFs) for the label-free detection of C-reactive protein (CRP) is presented. The obtained p-COFs possess high conductivity and an improved stability due to strong and rigid covalent linkages. The introduction of p-COFs hinder the recombination of electrons and holes, decreasing their band gap (Eg), thereby which improved the photocurrent conversion efficiency. Compared with pure porphyrin, p-COFs exhibited enhanced photocurrent intensity. An amplified photocurrent conversion efficiency and enhanced photocurrent results from H2O2, which act as active molecules and electron donors. As an unprecedented application of COFs in PEC bioanalysis, the detection of CRP with a PEC aptasensor is presented. The assembly of a CRP aptamer on the surface of Ag nanoparticles hinders the electron transfer, resulting in the decrease of the photocurrent response. This PEC aptasensor exhibits good analytical performances such as a rapid response, high stability, wide linear range and excellent selectivity, making COFs promising candidates for PEC bioanalysis.

The Preparation of C-Reactive Protein Immunosensor Based on Nano-Mimetic Enzyme Co3O4.

In this work, a nano-mimetic enzyme Co3O4 nanoparticles with catalytic effect on the reduction of H2O2, was synthesized and used to label C-reactive protein antibody. Au nanoparticles (Au NPs) were loaded on COF-TpPa-1 to form Au NPs@COF-TpPa-1 with good biocompatibility and adsorption ability. A sandwich C-reactive protein immunosensor was prepared using Au NPs @COF-TpPa-1 as the immobilization matrix. The catalytic current of Co3O4 nanoparticles to the reduction of H2O2 was measured by the chronoamperometry which is in proportion to the concentration of CRP. The linear range of this immunosensor was 0.05-80 ng/mL with the linear correlation coefficient of 0.9955. The method demonstrated a good sensitivity with a detection limit of 0.017 ng/mL (S/N = 3) for CRP. When the C-reactive protein immunosensor was applied to real serum samples analysis, the satisfied results were obtained, indicating that the immunosensor is of prospect of application.