A ratiometric fluorescent dark box and smartphone integrated portable sensing platform based on hydrogen bonding induction for on-site determination of enrofloxacin.

Enrofloxacin (ENR) residues in animal-derived food and water threaten human health. Simple, low-cost and on-site detection methods are urgently needed. Blue emitting carbon quantum dots (CQDs) and orange rhodamine B (RhB) were used as recognition and reference signals, respectively, to construct a ratiometric fluorescence sensor. After the addition of ENR, the color of the sensor changed from orange to blue because hydrogen bonding induced a considerable increase in CQDs fluorescence. Based on this mechanism, a simple and low cost on-site portable sensing platform was constructed, which integrated a stable UV light strip and a smartphone with voice-controlled phototaking function and an RGB app. The t-test results of spiked ENR recoveries for diluted milk, honey and drinking water revealed no significant differences between the ratiometric fluorescent sensor and portable sensing platform. Thus, this portable sensing platform provides a novel strategy for on-site quantification of quinolone antibiotics in foodstuffs and environmental water.

Evolution of Antidrug Antibody Assays During the Development of Anti-Tissue Factor Pathway Inhibitor Monoclonal Antibody Marstacimab.

Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of the extrinsic coagulation pathway. In patients with hemophilia A or B, inhibition of TFPI is an alternative therapeutic approach that augments the extrinsic coagulation pathway. Marstacimab is an investigational fully human monoclonal antibody that binds and neutralizes TFPI and is being evaluated as a prophylactic treatment to prevent or reduce the frequency of bleeding episodes in patients with severe hemophilia A or B, with or without inhibitors (antibodies against coagulation factors). However, the efficacy, safety, and pharmacokinetics of marstacimab may be affected by the induction of antidrug antibody (ADA) responses. Here, we describe the evolution and validation of three quasi-quantitative electrochemiluminescence-based methods to detect marstacimab ADAs, starting from their use in a first-in-human phase 1 study to their use in phase 2 and 3 clinical studies of patients with severe hemophilia. For all three methods, validation criteria evaluated the performance of the assays in screening and confirmatory cut points, precision, selectivity, drug tolerance, target interference, and stability. Additional criteria for validation were dilution linearity (Methods 1 and 2) and low positive control concentration, prozone effect, plate homogeneity, and robustness (Method 3). The three methods met validation criteria and are a potentially valuable tool in detecting the induction of marstacimab ADAs during treatment in patients with hemophilia.

Three-dimensional fluorinated covalent organic frameworks coated capillary for the separation of fluoroquinolones by capillary electrochromatography.

In this work, a three-dimensional fluorinated covalent organic frameworks (3D FCOFs) JUC-515 was synthesized from tetra(4-aminophenyl)methane (TAM) and 2,3,5,6-tetrafluoroterephthalol (TFA) by an ionic liquid method. JUC-515 was introduced into the capillary column and bonded to the inner wall of the capillary column by chemical bonding. Through a variety of characterization results, JUC-515 was successfully synthesized and introduced into the capillary column. The effects of buffer solution concentration, organic additive content and pH of the buffer solution on the separation of fluoroquinolones (FQs) were investigated in detail. The JUC-515-coated capillary column showed good resolution (>1.5) and reproducibility. The relative standard deviations (RSDs) of the retention time for intraday, interday, column-to-column and interbatch precision were less than 0.88%, 2.45%, 2.74% and 3.32%, respectively. The RSDs of the peak area for intraday, interday, column-to-column and interbatch precision were less than 3.79%, 4.31%, 3.33% and 5.62%, respectively. The JUC-515-coated capillary column could be used no less than 150 times. The results showed that the JUC-515-coated capillary column had good separation performance. In addition, by separating fluorinated ß-phenylalanine analogs, ß-phenylalanine and trifluoromethyl ß-phenylalanine analogs, the separation mechanism based on fluorine interactions was discussed. In conclusion, JUC-515 had good potential as a stationary phase for capillary electrochromatography.

Systematic study of tissue section thickness for MALDI MS profiling and imaging.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has become a powerful method for studying the spatial distribution of molecules. Preparation of tissue sections is a critical step for obtaining high-quality imaging data. The thickness of the slice of tissue affects the feature quality of MALDI MSI. However, few studies involved in-depth and systematic examination of slice thickness. Herein, we investigate the effect of tissue slice thickness on MALDI MSI detection. We found that the thicker the slice, the worse the results obtained by MALDI MS, which we attributed to the charging effect. The optimal slice thickness of brain tissue obtained in this work is 2-6 µm. Comparisons of the effects of slice thickness on atmospheric pressure and vacuum MALDI assays indicated that the ion signals and imaging quality of vacuum MALDI were more seriously affected by the thickness, with atmospheric pressure (AP) MALDI having a greater tolerance for slice thickness than vacuum MALDI. The MALDI MSI of peptides after enzymatic digestion of tissue sections of different thicknesses was also studied, revealing that the most suitable tissue thickness for enzyme digestion is about 10 µm. Finally, we optimized the slice thicknesses of six tissues in mice to provide a reference for MALDI MSI studies. It is worth mentioning that in our study the values of slice thickness range from the nanometer level (400 nm) at the minimum to 150 µm at the maximum, values which were unprecedented. Detailed in-depth and systematic studies of slice thickness will promote the development of sample preparation technology of AP and vacuum MALDI MSI, which will provide important references for the selection of tissue section thickness.

Fluorinated covalent organic frameworks as a stationary phase for separation of fluoroquinolones by capillary electrochromatography.

A fluorinated covalent organic framework composed of 2,3,5,6-tetrafluoroterephthaldehyde (TFA) and 1,3,5-tri(4-aminophenyl)benzene (TAPB) is proposed for electrochromatographic separation. TFA-TAPB is for the first time regarded as the stationary phase of capillary electrochromatography. The TFA-TAPB-coated capillary columns exhibited satisfactory separation (resolution values > 1.5) and good reproducibility towards fluoroquinolones. The intraday relative standard deviations (RSDs) of retention time and peak areas were 0.54-0.68% and 1.69-2.82%, respectively. The interday RSDs of retention time and peak areas were less than 1.79% and 2.30%, respectively. The column-to-column RSDs of retention time and peak areas were 0.22-0.73% and 0.74-1.86%, respectively. And, the inter-batch RSDs of retention time and peak areas were less than 0.39% and 1.67%, respectively. Moreover, the possible separation mechanism was discussed, and it was found that the π-π stacking effect, hydrophobic interaction, hydrogen bonding, and fluorous interactions were the main factors. Overall, these results demonstrated that TFA-TAPB has high prospect for CEC separation.

Adsorption behavior and mechanism of sulfonamides on controllably synthesized covalent organic frameworks.

In this work, four kinds of covalent organic framework (COF) materials (TpPa-1, TpBD, TpDT, and TFBBD) with different pore sizes or functional groups were synthesized by an ultrasonic method for the adsorption of five sulfonamides. Optimization experiments regarding the adsorption time, vortex speed, and pH were carried out to improve adsorption efficiency. In addition, kinetic and thermodynamic experiments were conducted to explore the adsorption mechanism of the sulfonamides on the different COFs. The adsorption processes of the five sulfonamides on the four COFs fit the pseudo-second-order kinetic model and Langmuir adsorption isotherm model. Additionally, pore filling, hydrogen bond interactions, and electrostatic attraction were found to be the main adsorption mechanisms.

Capillary coated with three-dimensional covalent organic frameworks for separation of fluoroquinolones by open-tubular capillary electrochromatography.

The Schiff-base reaction of 1,3,5-triformylphloroglucinol (Tp) and tetra(4-aminophenyl)methane (TAM) was performed for the synthesis of a three-dimensional covalent organic framework named 3D TpTAM, which was obtained by an ultrasound-assisted method for the first time. The morphology and structure of the synthesized TpTAM were characterized through various methods. Then, TpTAM-coated capillary columns were subsequently prepared by a covalent bonding method within a short time and applied for the separation of fluoroquinolones by capillary electrochromatography (CEC) with good resolution and reproducibility. The intraday relative standard deviations (RSDs) of the retention time and peak areas were 0.88%-0.95% and 2.27%-3.81%, respectively. The interday RSDs of retention time and peak areas were 0.71%-0.89% and 0.88%-3.60%, respectively. The column-to-column RSDs of retention time and peak areas were less than 1.90% and 13.56%, respectively. The interbatch RSDs of retention time and peak areas were less than 3.48% and 3.89%, respectively. The TpTAM-coated capillary columns could be used for no less than 100 runs with no observable changes in the separation efficiency. The separation mechanism was also studied, which indicated that π-π stacking effects, hydrophobic interactions and hydrogen bonding were the main factors. The results revealed that 3D TpTAM should have superior potential as the stationary phase in CEC for chromatographic separation.

Molybdenum disulfide-graphene oxide composites as dispersive solid-phase extraction adsorbents for the enrichment of four paraben preservatives in cosmetics.

Molybdenum disulfide-graphene oxide composite (MoS2/GO) was synthesized and used as the adsorbent in dispersive solid-phase extraction. Four paraben preservatives, namely, methylparaben, ethylparaben, propylparaben, and butylparaben, were enriched with MoS2/GO and determined by ultra-high-performance liquid chromatography. Molybdenum disulfide was intercalated into graphene oxide layers to reduce self-aggregation by using the solvothermal method. The experimental results indicated that the as-prepared MoS2/GO composite exhibited great enrichment capability toward those four paraben preservatives, and the adsorption time was 10 min and the elution time was as short as 1 min. The mechanism of MoS2/GO composite and parabens is attributed to hydrogen bonding and electrostatic attraction. The relative standard deviation (RSD, n = 9) of this method was below 7.6%. Limits of detection and limits of quantification were in the range 0.4-2.3 ng/mL and 1.4-7.6 ng/mL, respectively. The recoveries obtained from the parabens of cosmetic sample were in the range 91.3-124% with RSDs below 10%. The developed method has great potential for the determination of emerging contaminants with low cost and high sensitivity.

A graphene oxide-molybdenum disulfide composite used as stationary phase for determination of sulfonamides in open-tubular capillary electrochromatography.

A graphene oxide-molybdenum disulfide (GO-MoS2) composite was synthesized and utilized as the highly efficient stationary phase of open-tubular capillary electrochromatography (OT-CEC). The characterization results indicated that GO-MoS2 composite was successfully synthesized. The GO-MoS2-coated capillary column was prepared by covalent immobilization method for the determination of seven sulfonamides. The baseline separation of seven sulfonamides was achieved by GO-MoS2-coated capillary column. The linear range was 0.05-100 µg/mL for sulfisomidine, sulfathiazole, sulfamerazine, phthalylsulfathiazole and sulfacetamide, 0.1-100 µg/mL for sulfamonomethoxine and sulfachloropyridazine with a satisfactory correlation coefficients (R2) > 0.9994. This developed OT-CEC method was successfully applied to determinate of seven sulfonamides in environmental water and milk samples with good recoveries of 85.77% - 109.10% and 80.03% - 109.97%, respectively. These results indicated that GO-MoS2-coated capillary column possessed good stability and repeatability.

Influences of Daily Life Habits on Risk Factors of Stroke Based on Decision Tree and Correlation Matrix.

PURPOSE: To explore the influences of smoking, alcohol consumption, drinking tea, diet, sleep, and exercise on the risk of stroke and relationships among the factors, present corresponding knowledge-based rules, and provide a scientific basis for assessment and intervention of risk factors of stroke. METHODS: The decision tree C4.5 algorithm was optimized and utilized to establish a model for stroke risk assessment; then, the main risk factors of stroke (including hypertension, dyslipidemia, diabetes, atrial fibrillation, body mass index (BMI), history of stroke, family history of stroke, and transient ischemic attack (TIA)) and daily habits (e.g., smoking, alcohol consumption, drinking tea, diet, sleep, and exercise) were analyzed; corresponding knowledge-based rules were finally presented. Establish a correlation matrix of stroke risk factors and analyze the relationship between stroke risk factors. RESULTS: The accuracy of the established model for stroke risk assessment was 87.53%, and the kappa coefficient was 0.8344, which was superior to that of the random forest and Logistic algorithm. Additionally, 37 knowledge-based rules that can be used for prevention of risk factors of stroke were derived and verified. According to in-depth analysis of risk factors of stroke, the values of smoking, exercise, sleep, drinking tea, alcohol consumption, and diet were 6.00, 7.00, 8.67, 9.33, 10.00, 10.60, and 10.75, respectively, indicating that their influence on risk factors of stroke was reduced in turn; on the one hand, smoking and exercise were strongly associated with other risk factors of stroke; on the other hand, sleep, drinking tea, alcohol consumption, and diet were not firmly associated with other risk factors of stroke, and they were relatively tightly associated with smoking and exercise. CONCLUSIONS: Establishment of a model for stroke risk assessment, analysis of factors influencing risk factors of stroke, analysis of relationships among those factors, and derivation of knowledge-based rules are helpful for prevention and treatment of stroke.

Analysis of behavioral disability and construction of a disability scale for the elderly in Shanghai.

BACKGROUND: With the gradual aging of China's population development, the number of disabled elderly has increased significantly. OBJECTIVE: In order to better solve the problem of life care for these elderly people, it is necessary to conduct in-depth and detailed research on the specific conditions of disabled elderly people, in order to differentiate different conditions for care and set appropriate insurance provisions. METHODS: Based on the detailed analysis of the basic behavioral ability of the elderly, and referring to the International Disability standards, this paper refines the three basic living ability indicators of physiological behavior, cognitive behavior and interpersonal behavior, and integrates the cultural elements of assimilation, continuity, fusion and cohesion with Chinese characteristics. A more systematic and perfect five-level disability scale which conforms to the national conditions of China is designed. RESULTS: The disability of the elderly in Shanghai was investigated with the newly constructed scale, and a detailed analysis and five-level division were made. CONCLUSION: Experiments show that the results of this study can more effectively establish the disability level of the elderly in China.

Ambient temperature fabrication of a covalent organic framework from 1,3,5-triformylphloroglucinol and 1,4-phenylenediamine as a coating for use in open-tubular capillary electrochromatography of drugs and amino acids.

A covalent organic framework (COF) named TpPa-1 was designed and synthesized at ambient temperature by an ultrasound-assisted method from 1,3,5-triformylphloroglucinol (Tp) and 1,4-phenylenediamine (Pa-1). It was utilized as a stationary phase in open-tubular capillary electrochromatography (OT-CEC). The column was coated with TpPa-1 using a covalent bonding strategy. The coated capillary was characterized by morphology, crystallography, and mesoporous analysis to confirm the successful fabrication. The OT-CEC method was utilized for the analysis of tetracyclines, sulfonamides, cephalosporins and amino acids with high-resolution (Rs > 1.81) and good precision (RSD < 4.9%). It takes about 12 h from COF preparation to OT-CEC separation. Graphical abstract A covalent organic framework (COF) named TpPa-1 was synthesized at ambient temperature by an ultrasound-assisted method from 1,3,5-triformylphloroglucinol (Tp) and 1,4-phenylenediamine (Pa-1). COF-TpPa-1 modified capillary column was utilized for the analysis of tetracyclines, sulfonamides, cephalosporins and amino acids with high-resolution and good precision.

Label free aptasensor for ultrasensitive detection of tobramycin residue in pasteurized cow's milk based on resonance scattering spectra and nanogold catalytic amplification.

TOB aptamer can be adsorbed on the AuNPs surface to form AuNPs-aptamer complexation to prevent AuNPs aggregation in high salt solution. When TOB was added to the AuNPs solution, the aptamer would bind with TOB and depart from the AuNPs surface. The amount of the AuNPs-aptamer complexation depends on the TOB concentration. Different concentration of AuNPs-aptamer can catalyze the reduction reaction of CuSO4 to produce different size Cu2O particle. The resonance scattering peak intensities are correlated with the Cu2O size. Large size Cu2O particle as a resonance scattering spectroscopy probe can remarkable improve the TOB detection sensitivity. We have succeeded to detect the trace TOB in aqueous solutions. The linear range and limit of detection were 0.50-17 nM and 0.19 nM, respectively. This simple and inexpensive method exhibited high sensitivity and selectivity, which was successfully used to detect TOB in milk. The results indicated the accuracy and precision were satisfied.

Instrument-free enrichment and detection of phosphopeptides using paper-based Phos-PAD.

The facile detection of phosphopeptides is important for clinical screening and phosphoproteomic research. This work develops an instrument-free, cost-effective, convenient paper-based method for quantitative analysis of phosphorylated peptides. With a novel portable device, Phos-PAD, this method can achieve selective enrichment and colorimetric detection of phosphopeptides within 15 min TiO2 nanoparticle-based chemisorption and tetrabromophenol blue-based colorimetric assay were integrated into the single paper-based analytical device. The color change can indicate the presence of phosphopeptides and the mean pixel intensity of the red channel can be used for phosphopeptide quantification. With capability of quantifying phosphopeptides in serum samples, this Phos-PAD assisted phosphopeptide assay may attract significant attention to clinical analysis of endogenous serum phosphopeptides.

Neutralizing Antibody Assay Development with High Drug and Target Tolerance to Support Clinical Development of an Anti-TFPI Therapeutic Monoclonal Antibody.

Immunogenicity is a major challenge for protein therapeutics which can potentially reduce drug efficacy and safety and is often being monitored by anti-drug antibody (ADA) and neutralizing antibody (NAb) assays. Circulating targets and residual drugs in matrices can have significant impacts on accuracy of results from ADA and NAb assays, and sufficient drug and target tolerance for these assays are necessary. Here, we report the development of a competitive ligand binding (CLB) NAb assay for an anti-TFPI (tissue factor pathway inhibitor) monoclonal antibody (PF-06741086) with high drug and target tolerance to support ongoing clinical studies. A double acid affinity capture elution approach was used to mitigate drug interference, and a robust target removal strategy was employed to enhance target tolerance. The validated NAb assay has sensitivity of 313 ng/mL, drug tolerance of 50 µg/mL, and target tolerance of 1200 ng/mL. A step-by-step tutorial of assay development is described in this manuscript along with the rationale for using a high drug/target tolerant NAb assay. The NAb assay cut point factor obtained was 0.78. Other assay performance characteristics, e.g., precision and selectivity, are also discussed. This validated method demonstrated a superior drug and target tolerance to warrant specific and precise characterization of the NAb responses in support of ongoing clinical studies.

Synthesis, Anti-Varicella-Zoster Virus and Anti-Cytomegalovirus Activity of 4,5-Disubstituted 1,2,3-(1H)-Triazoles.

BACKGROUND: Clinical drugs for herpesvirus exhibit high toxicity and suffer from significant drug resistance. The development of new, effective, and safe anti-herpesvirus agents with different mechanisms of action is greatly required. OBJECTIVE: Novel inhibitors against herpesvirus with different mechanisms of action from that of clinical drugs. METHODS: A series of novel 5-(benzylamino)-1H-1,2,3-triazole-4-carboxamides were efficiently synthesized and EC50 values against Human Cytomegalovirus (HCMV), Varicella-Zoster Virus (VZV) and Herpes Simplex Virus (HSV) were evaluated in vitro. RESULTS: Some compounds present antiviral activity. Compounds 5s and 5t are potent against both HCMV and VZV. Compounds 5m, 5n, 5s, and 5t show similar EC50 values against both TK+ and TK- VZV strains. CONCLUSION: 5-(Benzylamino)-1H-1, 2,3-triazole-4-carboxamides are active against herpesviruses and their activity is remarkably affected by the nature and the position of substituents in the benzene ring. The results indicate that these derivatives are independent of the viral thymidine kinase (TK) for activation, which is indispensable for current drugs. Their mechanisms of action may differ from those of the clinic anti-herpesvirus drugs.

Approaches to Resolve False Reporting in Neutralizing Antibody Assays Caused by Reagent Leaching from Affinity Capture Elution Solid Phase.

Insufficient drug tolerance presents a major challenge in the development of neutralizing antibody (NAb) assays for biotherapeutics. Sample pre-treatment using solid-phase extraction with acid dissociation (SPEAD) is widely reported to improve drug tolerance. In this paper, a case study is presented in which SPEAD was used in conjunction with a competitive ligand binding NAb assay format. A significant degree of biotin-drug conjugate leaching was observed resulting in the reporting of both false positive and false negative results in NAb assay. Mitigation steps have been evaluated to address drug/biotin-drug conjugate leaching. These steps included assessment of the streptavidin-coated plate in conjunction with biotin-drug conjugates at various biotin molar challenge ratios (MCR). In addition, an alternative method based on covalent capture of the drug on an aldehyde-activated plate was assessed. Both approaches were compared for the degree of drug/biotin-drug conjugate leaching during the second elution step of the SPEAD procedure. Moreover, the impact of various conditions on the assay performance was assessed, including elution pH, sample incubation time, and biotin MCR. For the covalent drug capture method, capture conditions were evaluated. Optimized conditions in both streptavidin capture and covalent capture methods enabled a significant reduction of drug/biotin-drug conjugate leaching. A streptavidin high binding capacity approach using biotin-drug conjugate with a MCR of 50:1 was chosen as the optimal method yielding a NAb assay with a fit for purpose sensitivity (153 ng/mL) and a drug tolerance of up to 50 µg/mL with 500 ng/mL PC.

Identification of aminoglycoside antibiotics in milk matrix with a colorimetric sensor array and pattern recognition methods.

Aminoglycoside antibiotics (AAs) abused in animal husbandry can cause antibiotic residues in animal-derived foods, which do harm to human beings' health. Therefore the detection of AAs residues in the animal-origin foods, such as milk, eggs and meat is necessary. We used two single-stranded DNA (ssDNA) oligonucleotides as nonspecific receptors to develop a simple colorimetric sensor array based on gold nanoparticles (AuNPs) for identification and quantification the AAs. Different AA addition triggered the DNA detaching from AuNPs then resulted in different degree salt induced aggregation of AuNPs. The aggregation induced spectral changes of AuNPs with five AA addition were analyzed based on pattern recognition techniques, fisher linear discriminant analysis (FLD) and hierarchical cluster analysis (HCA). The results indicated that colorimetric sensor array has successfully identified five AAs at a concentration range of 120-280 nM. Five AAs in aqueous solution and complex milk matrix can be identified with an accuracy of 100%. More importantly, our developed sensor array is sufficiently sensitive for the discrimination of pure streptomycin (STR), binary mixtures of STR and gentamicin (GEN) at a total concentration of 120 nM.

A Simple Approach to Determine a Curve Fitting Model with a Correct Weighting Function for Calibration Curves in Quantitative Ligand Binding Assays.

In ligand binding assays (LBA), the concentration to response data is a nonlinear relationship driven by the law of mass action. Four parameter logistic (4PL) and five parameter logistic (5PL) curve fitting models are two widely accepted and validated models for LBA calibration curve data. Selection of the appropriate regression model and weighting function are key components of LBA development. Assessment of selected model and weighting function should be performed during assay development and confirmed later during validation. There has been limited published work on practical approaches to determining an appropriate weighting function and selection of a regression model for ligand binding assays. Herein, a structured scheme is presented to determine both. By applying commonly available software, assay performance data were analyzed to determine weighting functions and associated choice of a curve fitting model in three presented case studies. As a result, assay ranges of quantification were improved by reducing lower limit of quantification (from 1.00 to 0.317 ng/mL in one case study and from 2.06 to 1.37 ng/mL in another) or extending both low and upper limits of quantification(e.g., 1.04 to 48.3 ng/mL improved to 0.602 to 145 ng/mL). In addition, assay calibration curve performance demonstrated improved assay accuracy (%RE) and precision (%CV). Recommendations on decision flow when determining appropriate weighting function and curve fit model are presented.

Colorimetric aptasensors for determination of tobramycin in milk and chicken eggs based on DNA and gold nanoparticles.

Colorimetric aptasensors were designed for detection of tobramycin (TOB) based on unmodified gold nanoparticles (AuNPs) and single-strand DNA (ssDNA). In the absence of TOB, the DNA aptamer was coated on the surface of AuNPs to keep it against salt-induced aggregation. In the presence of TOB, aptamer will bind with TOB and detach from the surface of AuNPs because of higher affinities between aptamer and TOB. Then less protection of DNA may result in the aggregation of AuNPs by salt and an apparent color change from red to purple-blue. The developed aptasensors showed a high selectivity and sensitivity for TOB detection. The linearity range and the detection limit were 40-200 nM and 23.3 nM respectively. The validity of the procedure and applicability of aptasensors were successfully used to detect TOB in milk and chicken eggs, and the results were excellent in accord with the values obtained by spectrofluorimetric detection.