Evaluation of PhenoMATRIX and PhenoMATRIX PLUS for the screening of MRSA from nasal and inguinal/perineal swabs using chromogenic media.

The objective of this study was to assess the clinical performances of PhenoMATRIX and PhenoMATRIX PLUS for the screening of methicillin-resistant Staphylococcus aureus (MRSA) from nasal and inguinal/perineal ESwabs using chromogenic media. The automated performances were compared to the manual reading. Additionally, we evaluated PhenoMATRIX PLUS for the automatic release of the negative results to the Laboratory Information System (LIS) and the automatic discharge of the negative plates from the incubators. A total of 6,771 non-duplicate specimens were used by PhenoMATRIX as a machine learning model. The validation of these settings was performed on 17,223 non-duplicate specimens. The MRSA positivity rate was 5% (866/17,223). Validated settings were then used by PhenoMATRIX PLUS on another 1,409 non-duplicate specimens. The sensitivities of PhenoMATRIX and PhenoMATRIX PLUS were 99.8% [95% confidence interval (CI), 99.2%-99.9%] and 100% (95% CI, 92.1%-100%), respectively. The specificities of PhenoMATRIX and PhenoMATRIX PLUS were 99.1% (95% CI, 99.0%-99.2%) and 95.2% (95% CI, 93.8%-96.1%), respectively. All the 1,297 MRSA-negative specimens analyzed by PhenoMATRIX PLUS were automatically released and sent to the LIS immediately after availability of the culture image on the WASPLab (100% accuracy). All negative media plates were automatically discarded. PhenoMATRIX PLUS decreases the time spent by technologists on negative plates and ensures optimal usage of the incubators' capacity.

A global comparative field study to evaluate the factor VIII activity of efanesoctocog alfa by one-stage clotting and chromogenic substrate assays at clinical haemostasis laboratories.

INTRODUCTION: Structural and chemical modifications of factor VIII (FVIII) products may influence their behaviour in FVIII activity assays. Hence, it is important to assess the performance of FVIII products in these assays. Efanesoctocog alfa is a new class of FVIII replacement therapy designed to provide both high sustained factor activity levels and prolonged plasma half-life. AIM: Evaluate the accuracy of measuring efanesoctocog alfa FVIII activity in one-stage clotting assays (OSAs) and chromogenic substrate assays (CSAs). METHODS: Human plasma with no detectable FVIII activity was spiked with efanesoctocog alfa or a full-length recombinant FVIII product comparator, octocog alfa, at nominal concentrations of 0.80 IU/mL, 0.20 IU/mL, or 0.05 IU/mL, based on labelled potency. Clinical haemostasis laboratories (N = 35) tested blinded samples using in-house assays. Data from 51 OSAs (14 activated partial thromboplastin time [aPTT] reagents) and 42 CSAs (eight kits) were analyzed. RESULTS: Efanesoctocog alfa activity was reliably (±25% of nominal activity) measured across all concentrations using OSAs with Actin FSL and multiple other aPTT reagents. Under- and overestimation of FVIII activity occurred with some reagents. No specific trend was observed for any class of aPTT activators. A two- to three-fold overestimation was consistently observed using CSAs and the OSA with Actin FS as the aPTT reagent across evaluated concentrations. CONCLUSION: Under- or overestimation occurred with some specific OSAs and most CSAs, which has been previously observed with other modified FVIII replacement products. Efanesoctocog alfa FVIII activity was measured with acceptable accuracy and reliability using several OSA methods and commercial plasma standards.

Decoupling of the Confused Complex in Oxidation of 3,3',5,5'-Tetramethylbenzidine for the Reliable Chromogenic Bioassay.

Regulation of the reaction pathways is a perennial theme in the field of chemistry. As a typical chromogenic substrate, 3,3',5,5'-tetramethylbenzidine (TMB) generally undertakes one-electron oxidation, but the product (TMBox1) is essentially a confused complex and is unstable, which significantly hampers the clinic chromogenic bioassays for more than 50 years. Herein, we report that sodium dodecyl sulfate (SDS)-based micelles could drive the direct two-electron oxidation of TMB to the final stable TMBox2. Rather than activation of H2O2 oxidant in the one-electron TMB oxidation by common natural peroxidase, activation of the TMB substrate by SDS micelles decoupled the thermodynamically favorable complex between TMBox2 with unreacted TMB, leading to an unusual direct two-electron oxidation pathway. Mechanism studies demonstrated that the complementary spatial and electrostatic isolation effects, caused by the confined hydrophobic cavities and negatively charged outer surfaces of SDS micelles, were crucial. Further cascading with glucose oxidase, as a proof-of-concept application, allowed glucose to be more reliably measured, even in a broader range of concentrations without any conventional strong acid termination.

Nanozyme's catalytic activity at neutral pH: reaction substrates and application in sensing.

Nanozymes exhibit their great potential as alternatives to natural enzymes. In addition to catalytic activity, nanozymes also need to have biologically relevant catalytic reactions at physiological pH to fit in the definition of an enzyme and to achieve efficient analytical applications. Previous reviews in the nanozyme field mainly focused on the catalytic mechanisms, activity regulation, and types of catalytic reactions. In this paper, we discuss efforts made on the substrate-dependent catalytic activity of nanozymes at neutral pH. First, the discrepant catalytic activities for different substrates are compared, where the key differences are the characteristics of substrates and the adsorption of substrates by nanozymes at different pH. We then reviewed efforts to enhance reaction activity for model chromogenic substrates and strategies to engineer nanomaterials to accelerate reaction rates for other substrates at physiological pH. Finally, we also discussed methods to achieve efficient sensing applications at neutral pH using nanozymes. We believe that the nanozyme is catching up with enzymes rapidly in terms of reaction rates and reaction conditions. Designing nanozymes with specific catalysis for efficient sensing remains a challenge.

Enzyme-free colorimetric detection of biothiols based on the photoinduced oxidation of 3,3',5,5'-tetramethylbenzidine.

Realizing the rapid and on-site detection of biothiols in complex biological and food samples using simple assays and devices remains a major challenge. In this study, biothiols containing sulfhydryl groups were found to be able to inhibit the photo-triggered oxidation of 3,3',5,5'-tetramethylbenzidine (TMB). Based on the discovery, using the commercially available and low-cost TMB as the chromogenic substrate, an enzyme-free colorimetric approach was developed for the rapid determination of biothiols. The method does not involve the introduction of any natural enzymes, nanoenzymes, and external oxidants. The mechanisms of the photoinduced oxidation of TMB and the detection of biothiols were proposed. Furthermore, a smartphone-based portable device integrated with test strips was constructed by the 3D printing technique. This device can simultaneously meet the requirements of the photocatalytic oxidation reaction of TMB and the detection of biothiols. The entire process only takes less than 5 min. The successful detection of cysteine in urine and milk samples demonstrates the great potential of the device in the on-site assays.

A modified, optimized kinetic photometric thrombin generation assay components to measure the potency of thrombin in plasma.

The purpose of the present study was to improve, optimize and modification, a kinetic photometric assay of Factor IIa activity with regard to blood sample types, calibration, analytic corrections, and activation reagents, in order to establish a rapid routine, less time consuming, and suitable method for the evaluation of large numbers of clinical samples. Our modified method was based on the continuous monitoring of the Factor IIa released from the reacting substance, in the presence of a specific chromogenic peptide substrate (S2238). The optimization of pH and of the concentrations of substrate, activator (CaCl2), EDTA, and tissue factor was carried out as well as blood sample types (Platelet-rich and platelet-poor plasma samples) so as to ensure the best compromise between reliability, specificity, and sensitivity, However, the Factor IIa activity was compared in the plasma of different donors. In conclusion, our modified and optimized FIIa generation assay is a rapid and simple method for the diagnosis and monitoring of different clinical cases, at the same time it can be easily adapted to clinical chemistry analyzers.

Photo-isomerization of the isolated photoactive yellow protein chromophore: what comes before the primary step?

Photoactive proteins typically rely on structural changes in a small chromophore to initiate a biological response. While these changes often involve isomerization as the "primary step", preceding this is an ultrafast relaxation of the molecular framework caused by the sudden change in electronic structure upon photoexcitation. Here, we capture this motion for an isolated model chromophore of the photoactive yellow protein using time-resolved photoelectron imaging. It occurs in <150 fs and is apparent from a spectral shift of ∼70 meV and a change in photoelectron anisotropy. Electronic structure calculations enable the quantitative assignment of the geometric and electronic structure changes to a planar intermediate from which the primary step can then proceed.

Self-coordinated nanozyme on Cu3BiS3 nanorods for high-performance aptasensing.

A novel strategy is reported to access high-performance nanozymes via the self-coordination of ferrocyanides ([Fe(CN)6]4-) onto the surface of the Cu3BiS3 (CBS) nanorods. Notably, the in situ formed nanozymes had high catalytic activity, good stability, low cost, and easy mass production. The formed nanozyme catalyzed the oxidation of the typical chromogenic substrate of 3,3',5,5'-tetramethylbenzidine (TMB) with a distinctive absorption peak at 652 nm, accompanied by a blue color development. Moreover, the attachment of deoxyribonucleoside 5'-monophosphates (dNMP) beforehand onto the surface of CBS prevented coordination of ferrocyanides and resulted in the tunable formation of the nanozyme, thereby enabling the construction of an exquisite biosensing platform. Taking the aptasensing of chloramphenicol (CAP) as an example, the engineered nanozyme allowed the construction of a homogenous, label-free, and high-performance bioassay in terms of its convenience and high sensitivity. Under the optimal conditions, changes in the absorption intensity at 652 nm for the oxidized TMB provides a good linear correlation with the logarithm of CAP concentrations in the range 0.1 pM to 100 nM, and the limit of detection was 0.033 pM (calculated from 3σ/s). Considering a vast number of bioreactions can be connected to dNMP production, we expect the engineerable nanozyme as a universal signal transduction scaffold for versatile applications in bioassays. Through the attachment of deoxyribonucleoside 5'-monophosphate (dNMP) on the surface of CBS to regulate the generation of self-coordinated nanozyme CBS/BiHCF, a homogeneous, label-free, and high-performance universal aptasensing platform was constructed.

Sensitive colorimetric assay of hydrogen peroxide and glucose in humoral samples based on the enhanced peroxidase-mimetic activity of NH2-MIL-88-derived FeS2@CN nanocomposites compared to its precursors.

By employing NH2-MIL-88 as a template, we synthesized the intermediate Fe@CN under high-temperature calcination and further fabricated the FeS2@CN nanocomposites in the presence of sulfur powder. Under varying temperatures (300-600 °C) and Fe@CN-to-S ratios (1:3-6), FeS2@CN500-5 nanocomposites had the highest peroxidase-mimetic activity. Under optimized conditions (incubation temperature 40 °C; solution pH 4.0 and nanocomposite concentration 10 µg/mL; 652-nm absorption), the Michaelis-Menten constant (Km) of FeS2@CN was much lower than that of horseradish peroxidase (HRP), therefore demonstrating that it had a higher affinity for both chromogenic substrates than conventional HRP. The limits of detection for H2O2 and glucose were 0.15 and 0.30 µmol/L, respectively, and the recoveries for glucose were 91.8-103% with RSDs <5.2%. The novelty of this study lies in (1) the FeS2@CN was confirmed to possess stronger enzyme-mimetic activity than its precursors (NH2-MIL-88 and Fe@CN); (2) the enhanced activity resulted from the unsaturated sites of N and S doping and the plentiful defects on the porous carbon surface; and (3) free radical trapping experiments evidenced that •OH played a major role in the catalytic reaction, while h+ and •O2- simultaneously participated in the catalytic process. These convincing performance metrics lead us to postulate that the FeS2@CN-based colorimetric biosensor provides a promising approach for several real-world applications, such as point-of-care diagnosis and workplace health evaluations.

Performance of Two Novel Chromogenic Media for the Identification of Multidrug-Resistant Candida auris Compared with Other Commercially Available Formulations.

Non-albicans Candida species are emerging in the nosocomial environment, with the multidrug-resistant (MDR) species Candida auris being the most notorious example. Consequently, rapid and accurate species identification has become essential. The objective of this study was to evaluate five commercially available chromogenic media for the presumptive identification of C. auris Two novel chromogenic formulations, CHROMagar Candida Plus (CHROMagar) and HiCrome C. auris MDR selective agar (HiMedia), and three reference media, CandiSelect (Bio-Rad), CHROMagar Candida (CHROMagar), and Chromatic Candida (Liofilchem), were inoculated with a collection of 9 genetically diverse C. auris strains and 35 strains from closely related comparator species. After 48 h of incubation, the media were evaluated for their ability to detect and identify C. auris All media had the same limitations in the differentiation of the more common species Candida dubliniensis and Candida glabrata Only on CHROMagar Candida Plus did C. auris colonies develop a species-specific coloration. Nevertheless, the closely related pathogenic species Candida pseudohaemulonii and Candida vulturna developed a similar appearance as C. auris on this medium. CHROMagar Candida Plus was shown to be superior in the detection and identification of C. auris, with 100% inclusivity for C. auris compared to 0% and 33% for the reference media and HiCrome C. auris MDR selective agar, respectively. Although C. vulturna and C. pseudohaemulonii can cause false positives, CHROMagar Candida Plus was shown to be a valuable addition to the plethora of mostly molecular methods for C. auris detection and identification.

Single molecule chromogenic in situ hybridization assay for RNA visualization in fixed cells and tissues.

Visualization of gene expression at single RNA molecular level represents a great challenge to both imaging technologies and molecular engineering. Here we show a single molecule chromogenic in situ hybridization (smCISH) assay that enables counting and localizing individual RNA molecules in fixed cells and tissue under bright-field microscopy. Our method is based on in situ padlock probe assays directly using RNA as a ligation template and rolling circle amplification combined with enzyme catalyzed chromogenic reaction for amplification product visualization. We show potential applications of our method by detecting gene expression variations in single cells, subcellular localization information of expressed genes, and gene expression heterogeneity in formalin-fixed, paraffin-embedded tissue sections. This facile and straightforward method can in principle be applied to any type of RNA molecules in different samples.

Colorimetric detection of homocysteine by a pyridylazo dye-based Cu2+ complex via indicator displacement mechanism.

A BrPAPS based Cu2+ complex has been developed as a colorimetric probe for the selective recognition of homocysteine (Hcy) over cysteine (Cys) and glutathione (GSH) in an aqueous solution via the indicator displacement assay. BrPAPS formed a complex with Cu2+ in a 1:1 ratio (BrPAPS-Cu2+) accompanied by the color change from yellow to red. Detecting Hcy is based on high affinity of Hcy for Cu2+. The addition of Hcy to BrPAPS-Cu2+ caused the complex formation of Hcy with Cu2+ in a 2:1 stoichiometry, resulting a hypsochromic shift with change back of color from red to yellow by the release of BrPAPS from BrPAPS-Cu2+. The absorption response is linear with the Hcy concentration in the range of 0-20 µM with a detection limit of 1.46 µM. Moreover, the detection of Hcy was not significantly affected by other amino acids from the competition experiments. Thus, BrPAPS-Cu2+ can be used as a simple probe for Hcy in aqueous solution.

A functional chromogen gene C from wild rice is involved in a different anthocyanin biosynthesis pathway in indica and japonica.

KEY MESSAGE: we identified a functional chromogen gene C from wild rice, providing a new insight of anthocyanin biosynthesis pathway in indica and japonica. Accumulation of anthocyanin is a desirable trait to be selected in rice domestication, but the molecular mechanism of anthocyanin biosynthesis in rice remains largely unknown. In this study, a novel allele of chromogen gene C, OrC1, from Oryza rufipongon was cloned and identified as a determinant regulator of anthocyanin biosynthesis. Although OrC1 functions in purple apiculus, leaf sheath and stigma in indica background, it only promotes purple apiculus in japonica. Transcriptome analysis revealed that OrC1 regulates flavonoid biosynthesis pathway and activates a few bHLH and WD40 genes of ternary MYB-bHLH-WD40 complex in indica. Differentially expressed genes and metabolites were found in the indica and japonica backgrounds, indicating that OrC1 activated the anthocyanin biosynthetic genes OsCHI, OsF3H and OsANS and produced six metabolites independently. Artificial selection and domestication of C1 gene in rice occurred on the coding region in the two subspecies independently. Our results reveal the regulatory system and domestication of C1, provide new insights into MYB transcript factor involved in anthocyanin biosynthesis, and show the potential of engineering anthocyanin biosynthesis in rice.

Evaluation of CHROMagar™-Serratia agar, a new chromogenic medium for the detection and isolation of Serratia marcescens.

A comparative analysis of the performance of the new selective chromogenic CHROMagar™-Serratia culture medium for detection and isolation of Serratia marcescens was undertaken. A total of 134 clinical isolates (95 S. marcescens with and without carbapenemase production and 39 non-S. marcescens isolates) and 96 epidemiological samples (46 rectal swabs and 50 from environmental surfaces) were studied. Diagnostic values when compared with CHROMagar™-Orientation medium were 96.8% sensitivity, 100% specificity, 100% positive predictive value and 88.5% negative predictive value. In conclusion, CHROMagar™-Serratia shows an excellent ability for differentiation of S. marcescens among clinical isolates and in environmental samples.

CRISPR-Cas experiments for schools and the public.

The "gene scissors" CRISPR-Cas currently revolutionize the field of molecular biology with an enormous impact on society due to the broad application potentials in biomedicine, biotechnology and agriculture. We have developed simple CRISPR-Cas experiments that can serve to introduce pupils, students and non-scientists alike to the fascinating power of targeted gene editing. The experimental course is divided into two parts. In part 1, we target plasmid borne lacZ to convert blue E. coli to white E. coli. In part 2, we analyse the CRISPR-Cas9 mediated double strand breaks in the lacZ gene by a) colony PCR, b) colony cracking gel or c) restriction digest of the plasmids. Experimental work is embedded in short theoretical lecture parts that provide background of CRISPR-Cas and a step-by-step tutorial for the practical work. Though the experiment is robust, inexpensive and simple it should be noted that guidance by an expert instructor is required. Based on our experience, a full day lab course has a positive influence on the participants' attitude towards research in general. This is true for high school students as well as non-scientists (age groups 16-70 years).

Salt-template preparation of Mo5N6 nanosheets with peroxidase- and catalase-like activities and application for colorimetric determination of 4-aminophenol.

Mo5N6 nanosheets were synthesized by a nickel-induced growth method and were found to possess peroxidase-like activity in acidic condition and catalase-like activity in weak basic condition. In acidic condition, Mo5N6 nanosheets can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 to form a blue color product (TMBOX). At the co-existence of 4-aminophenol (4-AP), 4-AP can react with H2O2 and TMBOX, resulting in the decrease of TMBOX and the fading of blue color. Therefore, a facile, sensitive colorimetric method for the quantitative detection of 4-AP was developed. The linear range for 4-AP was 1.0 to 80.0 µmol⋅L‒1 (R2 = 0.999), and the detection limit was 0.56 µmol⋅L‒1 based on 3σ/k. Resorcinol, aniline, humic acid, and common ions and anions in surface water did not interfere the determination of 4-AP. This colorimetric method was applied to measure the 4-AP in real water sample from Wulong River in Fujian Province of China. The relative standard deviation for the determination of 4-AP was ranged from 0.03 to 1.88%, and the recoveries from spiked samples were ranged between 99.2 and 107.6%. The determination results were consistent with those obtained by HPLC.

Peroxidase-mimetic activity of FeOCl nanosheets for the colorimetric determination of glutathione and cysteine.

For the first time the enzyme mimic activity of iron oxychloride (FeOCl) nanosheets has been studied. The intrinsic peroxidase-mimetic activity of the nanosheets in the presence of H2O2 was approved by the efficient oxidation of tetramethylbenzidine (TMB). The Michaelis-Menten constant of the nanosheets toward TMB was about six times lower than that of natural horseradish peroxidase. The superiority of the nanosheets' catalytic property ascribes to their H2O2 activation ability. Based on the inhibition of the nanozymes' catalytic reaction, an assay was developed for the quantitative measurement of glutathione (GSH) and cysteine (Cys). The linear range for both biomolecules was over the range of 3-33 µM. The LOD values (3σ/slope) for GSH and Cys were 2.23 µM and 2.76 µM, respectively. Importantly, we succeeded in colorimetric discrimination of GSH and Cys kinetically. We achieved high selectivity toward GSH and Cys. This work extends the feasibility of using FeOCl as nanozymes to construct biosensors, colorimetric probes for medical diagnosis, and nanozyme-based cancer therapy.

Nanozyme based on CoFe2O4 modified with MoS2 for colorimetric determination of cysteine and glutathione.

A nanozyme based on CoFe2O4 modified with MoS2 was constructed for colorimetric determination of cysteine (Cys) and glutathione (GSH). Firstly, ferrite CoFe2O4 is synthesized, and it is then modified by MoS2 to form a flower-like polymer (MoS2@CoFe2O4). In the presence of H2O2, a redox interaction takes place, and the resulting hydroxyl promoted a colorimetric conversion from colorless to blue in the presence of 3,3',5,5'-tetramethylbenzidine (TMB). However, once Cys or GSH is added, they are capable to compete with the interaction of the hydroxyl with TMB, resulting in an inhibition of the colorimetric conversion. The colorimetric distinction is sensitive to the amount of target. The results obtained proved that the catalytic efficiency of MoS2@CoFe2O4 is 4.4-fold and 1.8-fold to that of MoS2 and CoFe2O4. Meanwhile, the Km values to TMB and H2O2 are 0.067 and 0.048 mM, respectively, which are 6.5-fold and 77-fold, respectively smaller than those of natural peroxidase such as HPR. This indicates that the MoS2@CoFe2O4 possesses a favorable interaction affinity. Additionally, the colorimetric distinction caused by the competition between TMB and cysteine or glutathione is obvious. The signal responses to cysteine and glutathione are linear in the range 0.5~15 µM and 0.5~35 µM, and the LODs are 0.10 and 0.21 µM, respectively. In practical assay of Cys in serum, the RSD of the sample tests is 4.6%, and the recoveries for the spiked assays are 95.3% and 96.0% with the RSD of 2.1% and 4.2%, respectively.

Fluorescence-active and peroxidase-like expanded mesoporous silica-encapsulated ultrasmall Pt nanoclusters: a novel colorimetric/fluorescent dual-mode nanosensor for sensitive detection of mercury in medicinal and edible Pueraria lobata.

A novel colorimetric/fluorescent dual-mode nanosensor for Hg2+ detection was constructed using expanded mesoporous silica (EMSN)-encapsulated ultrasmall platinum nanoclusters (EMSN@Pt NCs) with improved peroxidase-like and stable fluorescent activities. The sensing technique was based on the mechanism that the peroxidase mimetic activity and fluorescence intensity of EMSN@Pt NCs can be inhibited in the presence of Hg2+. In this sensing platform, a linear range of 5-50 nM with a detection limit of 1.78±0.38 nM and quantification limit of 5.93 nM was obtained via fluorescent analysis. A linear calibration curve from 0.25 to 200 nM with a detection limit of 8.25±0.51 nM and quantification limit of 27.47 nM was achieved via colorimetric analysis. The proposed dual-mode probe possesses excellent selectivity and reliability for Hg2+ detection, which can function as an efficient nanosensor for the quantitative determination of Hg2+ in Pueraria lobata.

Determination of Cr(VI) based on the peroxidase mimetic catalytic activity of citrate-capped gold nanoparticles.

Highly negatively charged gold nanoparticles (AuNPs) are shown to have strong simulated oxidase activity and effectively boosted the oxidation of enzyme substrate 3,3',5,5'-tetramethylbenzidine (TMB) by hexavalent chromium ion Cr(VI), resulting in the formation of oxidation product with blue color. Based on this, a facile colorimetric assay was developed to detect Cr(VI) at a range 0.008~0.156 mg/L with r = 0.996. The detection limit was estimated to be 0.52 µg/L. In addition, the colorimetric assay showed high selectivity against 28 other interfering ions. It was performed at room temperature and required about half an hour including the preparation of AuNPs. The assay was successfully applied to the determination of Cr(VI) in spiked water samples, and recoveries in the range 95.00-105.40% were obtained. This work paves a way for design of high performance sensor based on highly active nanozymes and also provides an extremely practical analytical tool for the monitoring of Cr(VI) in the environment.