A gold nanoparticle-based lateral flow immunoassay for atrazine point-of-care detection using a handhold scanning device as reader.

A method is described to achieve accurate quantitative detection of atrazine (ATZ) in maize by using lateral flow strips based on gold nanoparticles (GNPs) and a handheld scanning reader. GNPs of 15 nm in diameter were applied as label, and a lateral flow immune assay strip was prepared. The linear range was 5.01-95.86 ng mL-1 with a detection limit of 4.92 ng mL-1 in phosphate buffer, 4 times better than the readout by the naked eye. ATZ-spiked corn samples were also analysed. The accuracy of results of spiked samples was confirmed by ELISA and liquid chromatography-tandem mass spectrometry (HPLC), which proved the reliability of the proposed method. A handhold device with an optical scanning system was designed for on-site quantitative detection. Combined with the pretreatment, the assay could be completed in less than 20 min.

Universal and Ultrasensitive Immunochromatographic Assay by Using an Antigen as a Bifunctional Element and Antialbumin Antibody on a Test Line.

A universal and ultrasensitive immunochromatographic assay (ICA) was established using antigen as a bifunctional element and antialbumin antibody in a test line. Preincubation was introduced for competitive recognition. After optimization, the linear detection of aflatoxin M1 (AFM1) with quantum dot bead (QB)-based ICA (QB-ICA) sensor ranged from 10 to 52 pg mL-1, with a 50% inhibitory concentration (IC50) of 23 pg mL-1, which was nearly 49.6-fold lower than those of ICA on a traditional structure with traditional pretreatment (IC50 = 1.10 ng mL-1) and 10-fold lower than those of ICA on a traditional structure with acid aid pretreatment (IC50 = 0.25 ng mL-1). The limit of detection (LOD) for AFM1 was 16 pg mL-1 in milk, which was approximately 16.3-fold times higher than those of ICA on a traditional structure with traditional pretreatment and 6.3-fold higher than those of ICA on a traditional structure with acid aid pretreatment. The LOD improved by 20-fold by using the proposed structure compared to that of conventional enzyme-linked immunosorbent assay (ELISA) for AFM1-spiked milk samples (IC10 = 0.12 ng mL-1). The performance and practicability of the established QB-ICA sensor were validated with a commercial ELISA kit. To evaluate universality, we successfully detected chloramphenicol, with IC50 of 0.42 ng mL-1. Given its high sensitivity and universality, the proposed QB-ICA can be used as an alternative for rapid, sensitive, and universal quantitative detection of all small-molecule analytes.

An anti-BSA antibody-based immunochromatographic assay for chloramphenicol and aflatoxin M1 by using carboxy-modified CdSe/ZnS core-shell nanoparticles as label.

A lateral-flow immunochromatographic assay with excellent sensitivity and wide application potential is described. The bovine serum albumin (BSA) antibody was immobilized in the test line for universality, and preincubation was introduced for high method sensitivity. Carboxy-modified CdSe/ZnS core-shell nanoparticles were used as label, and the fluorescence peaking at 605 nm was detected. The fluorescence in the test line was negative against the relevant analyte content. The chloramphenicol (CAP) and the aflatoxin M1 (AFM1) in milk were detected using the same strip to validate the universality. After optimization, the detection limit for CAP is 10 pg·mL-1, which is three times less that of a conventional assay (30 pg·mL-1). The detection limit for AFM1 was 6 pg·mL-1, which was 13 times less than that of a conventional assay (8 pg·mL-1). The method was applied in the analysis of spiked milk samples. The performance was compared with that of the commercial ELISA kit, and good agreement was observed. Graphical abstractSchematic representation of the universal and sensitive combined immunochromatographic assay (USICA) and conventional immunochromatographic assay (TICA) of chloramphenicol (CAP) and aflatoxin M1.

Ovalbumin antibody-based fluorometric immunochromatographic lateral flow assay using CdSe/ZnS quantum dot beads as label for determination of T-2 toxin.

This work describes an anti-ovalbumin antibody-based lateral flow immunoassay (LFI) for T-2 toxin. The antibody uses a coating antigen as a bifunctional element for universality and introduces preincubation to improve the detection limits of the method. T-2 toxin and ovalbumin-modified T-2 toxin competitively binds on the anti-T-2 toxin monoclonal antibody modified on CdSe/ZnS quantum dot beads during preincubation. The modified T-2 toxin acts as a bifunctional element that forms immuno complexes during preincubation and combines with anti-ovalbumin antibody coated in the test line through the ovalbumin terminal. Fluorescence is detected at 610 nm on the test zone following photoexcitation at 365 nm. It has a reverse dose-effect relationship with the amount of T-2 toxin. The calibration plot is linear in the 20-110 fg mL-1 T-2 toxin concentration range, and the limit of detection (LOD) is 10 fg mL-1, which is lower by 8-fold than that of the traditional LFI system (LOD 80 fg mL-1) and one order of magnitude than those of LFIs with labels of colloidal gold nanoparticles (LOD 150 fg mL-1) or fluorophores (LOD 190 ng mL-1). Universality was verified through aflatoxin B1 detection using the established ovalbumin antibody-based LFI system (LOD 10 fg mL-1). The performance of the method was compared with that of established systems and a commercial ELISA kit (LOD 360 fg mL-1). Graphical abstractSchematic representation of ovalbumin antibody-based immunochromatographic lateral flow assay for T-2 toxin. Preincubation is introduced for high sensitivity. T-2- anti-ovalbumin acts as a bi-functional element for universality. CdSe/ZnS quantum dot beads act as label. Fluorometric signal is detected at 610 nm.

[Rapid detection of diethylstilbestrol using a quartz crystal microbalance with gold nanoparticals amplification].

OBJECTIVE: To develop a quartz crystal microbalance (QCM) immunosensor with high sensitivity and selectivity for the rapid detection of diethylstilbestrol. METHODS: Dextran was used as reducing agent for preparing gold nanoparticles (AuNPs) with the size of 40 nm. The AuNPs were coupled with anti-DES antibody after amination. A monolayer was generated after immersing the quartz crystal into the solution of 5 mmol/L 11-mercaptoundecanoic acid(MUA) for 16 hours. After the monolayer was activated by 1-ethyl-3-(3-dimethylaminopropry) carbodiimide hydrochloride (EDC·HCl) and N-hydrosuccinimide (NHS), 20 µl of 2.2 mg/ml DES-HS-BSA was dropped onto the surface of crystal to prepare a sensitive membrane which can recognize DES specifically. Then, 50 µl of 1 mol/L ethanolamine (pH 8.5) was used to seal the carboxylic groups to make the sensitive membrane which could identify DES specifically. QCM immunosensor was used as detection platform to optimize the reaction conditions. Under the optimized conditions, 10 µl of 28 µg/ml AuNPs-antibody was mixed with 10 µl of 0.03-2.5 µg/ml DES, and the mixture was added on the sensitive membrane. QCM immunosensor was used to detect the signals and the standard curve was obtained at the same time. The detection limit was calculated based on the standard curve. The specificity was evaluated by testing DES and its analogues with the same concentration. RESULTS: The optimized concentration for the immobilization of DES-HS-BSA on the surface of QCM was 2.2 mg/ml. The optimized concentration for coupling anti-DES antibody with AuNPs was 7 µg/ml and 15 nmol/L, respectively. The optimized concentration of AuNPs-antibody was 14 µg/ml. The logarithm of DES concentration was proportional to the frequency shift in the range of 0.16-500 ng/ml, Δf=-24.170 lgCDES+69.71, R(2)=0.998. The detection limit of this method was 0.13 ng/ml. DES analogues could not influence the detection of DES obviously, so the sensor had good specificity. CONCLUSION: The quartz crystal microbalance immunosensor with gold nanoparticals amplification could detect DES sensitively and rapidly.

A core-shell-structured molecularly imprinted polymer on upconverting nanoparticles for selective and sensitive fluorescence sensing of sulfamethazine.

A core-shell structured molecularly imprinted polymer on upconverting nanoparticles (UCNPs@MIP) was synthesized for the fluorescence (FL) sensing of sulfamethazine (SMZ). Hexagonal UCNPs were synthesized by the solvothermal method, then coated with a thin silica shell and modified with vinyl groups. Finally, surface polymerization was initiated among the vinyl groups, the functional monomers and cross-linking agents by the initiator. The MIP synthesized by this procedure was anchored on the surface of UCNPs, possessed better site accessibility and lower transfer resistance for the target molecule compared to bulk imprinted materials. The obtained UCNPs@MIP showed good binding capacity, fast response, high selectivity and specificity to the SMZ. The FL intensity of the UCNPs@MIP decreased sensitively with the increasing concentration of SMZ in the range of 50-700 ng mL(-1), the detection limit was 34 ng mL(-1) (S/N = 3). The UCNPs@MIP was successfully applied to the detection of SMZ in chicken samples. Thanks to the unique near-infrared (NIR) excitation nature of UCNPs, the chicken meat only needed some simple extraction procedures before FL detection, no complex purifications were required. The average recoveries ranged from 96.01% to 98.90%, with relative standard deviations (RSDs) below 4.5%. This work offers a novel sensing system that combined the advantages of upconverting nanotechnology and molecularly imprinted technology.

Sensitive detection of atrazine in tap water using TELISA.

A highly sensitive flow injection analysis (FIA)-based thermal enzyme-linked immunoassay, TELISA, was developed for the rapid detection of atrazine (ATZ) in tap water. ATZ and ß-lactamase-labeled ATZ were employed in a competitive immunoassay using a monoclonal antibody (mAb). After the off-column liquid-phase competition, the mAb was captured on the Protein G Sepharose™ 4 Fast Flow (PGSFF) column support material. Injected ß-lactamase substrate ampicillin was degraded by the column-bound ATZ-ß-lactamase, generating a detectable heat signal. Several assay parameters were optimized, including substrate concentration, flow rates and regeneration conditions, as well as the mAb and ATZ-ß dilution ratios and concentrations. The assay linear range was 0.73-4.83 ng mL(-1) with a detection limit of 0.66 ng mL(-1). An entire heat signal requires 10 min for generation, and the cycle time is less than 40 min. The results were reproducible and stable. ATZ-spiked tap water samples exhibited a recovery rate of 103%-116%, which correlated with the UHPLC-MS/MS measurements. We attributed this significant increase in sensitivity over our previously published work to the following factors: (i) the capture of already-formed immune complexes on the column via immobilized Protein G, which eliminated chemical immobilization of the antibody; (ii) off-column preincubation allows the formation of immune complexes under nearly ideal conditions; and (iii) multiple buffers can be used to, in one case, enhance immune-complex formation and in the other to maximize enzymatic activity. Furthermore, the scheme creates a universal assay platform in which sensing is performed in the off-column incubation and detection after capture in the enzyme thermistor (ET) detector, which opens up the possibility of detecting any antigen for which antibodies were available.

Fast detection of atrazine in corn using thermometric biosensors.

Fast detection is important in screening large-scale samples. This study establishes a direct competitive ELISA method (dcTELISA) based on an enzyme thermistor for fast atrazine (ATZ) detection. ATZ competes with ß-lactamase-labeled ATZ (ATZ-E) for the binding sites on anti-ATZ monoclonal antibody (mAb). The mAb are covalently bound to Controlled Pore Glass (CPG) in an immunoreactor to form immunocomplexes with ATZ and ATZ-E. Several parameters of biosensor performance were optimized, such as the ATZ-E concentration, concentration and nature of the substrate, flow rate, and effect of temperature on the sensor response. After optimization, the assay time for a single sample was 12 min. The work process and result were compared with those of high-performance liquid chromatography (HPLC). The detection results exhibited a recovery rate of 88% to 107% in ATZ-spiked fresh cut corn stalks and silage samples. The results obtained via dcTELISA had good correlation with that of HPLC, and the biosensor response was reproducible and stable even when used continuously for over 4 months. All these properties suggested that the fast detection method, dcTELISA, may be used to detect pesticide residue in large-scale samples.

Factors Associated with Benzodiazepines Prolonged-Term Use in Post-Stroke Subjective Sleep Disturbance: A Single-Centre Retrospective Study from China.

PURPOSE: To investigate the prevalence of short- and long-term benzodiazepine and z-drugs (BZD) for the treatment of post-stroke subjective sleep disturbance (SSD) and to evaluate the risk factors associated with prolonged BZD treatment in this patient body. PATIENTS AND METHODS: Between 1st January 2018 and 1st December 2018, we identified 542 inpatients suffering from acute stroke in Heyuan People's Hospital. Of these, 290 inpatients were included in our final analysis. These patients were divided into three groups according to the treatment they received: non/occasional BZD (non-BZD), short-term BZD (short-term) and prolonged-term BZD (prolonged-term) treatment. We investigated the prevalence of each BZD treatment term and identified differences between the groups. Univariate logistic regression analysis was used to identify potential predictors for the prolonged use of BZD. Multinomial logistic regression analysis was used to assess the correlation between the prolonged use of BZD and potential predictors. RESULTS: The prevalence of cases receiving short and prolonged BZD treatments were 40.35% and 31.72%, respectively; none of the patients received polysomnography (PSG) screening from obstructive sleep apnoea (OSP). Treatment strategies were limited to BZD and traditional Chinese medicine; none of the patients received cognitive-behavioral treatment (CBT) or other forms of treatment. Logistic regression analysis showed that the short-term use was associated with z-drugs (odds ratio [OR]: 2.189, 95% confidence interval [CI]: 1.419-3.378), non-communication barriers (OR =0.535, 95% CI: 0.325-0.880) and posterior circulation infarct (POCI) (OR =2.199, 95% CI: 1.112-4.349). The prolonged-term use was associated with z-drugs (OR =3.012, 95% CI: 1.637-5.542), non-communication barriers (OR =0.530, 95% CI: 0.307-0.916), partial anterior circulation infarct (PACI) (OR =0.455, 95% CI: 0.250-0.827), and non pain after stroke (OR =0.315, 95% CI: 0.207-0.480). CONCLUSION: The status of BZD abuse for post-stroke SSD is worrying. Additional research attention and treatment options are needed for the treatment of post-stroke SSD. In particular, the potential combination of stroke and OSP appears to be underestimated and neglected. Post-stroke SSD patients should receive more comprehensive assessment and rigid follow-up to avoid the prolonged use of BZD. Additional and effective therapeutic strategies (such as positive pressure ventilation treatment or CBT) are urgently needed for cause-specific intervention.

Author Correction: Pretreatment-Integration for Milk Protein Removal and Device-Facilitated Immunochromatographic Assay for 17 Items.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Pretreatment-Integration for Milk Protein Removal and Device-Facilitated Immunochromatographic Assay for 17 Items.

Accurate and comprehensive immunochromatographic assay (ICA) data are urgently required in the daily supervision of plants, schools, testing institutions, and law-enforcing departments. Through pretreatment-integration and device-facilitated operation, a quantitative ICA with high sensitivity and throughput was realized on the basis of a commercialized semi-quantitative ICA strip. Three pretreatment methods, namely, acid base, heavy metal salt, and organic solvent methods, have less than three steps. The pretreatment was established for protein removal. A total of 17 pretreated ICA items in milk were considered for the identification of the most suitable pretreatment method. The items are composed of six items pretreated by the acid-base method, six by the heavy salt method, and five by the organic solvent method. Then, the ICA results with pretreatment were compared with those without pretreatment. After pretreatment, the signal intensity increased by 39%, the detection limit decreased to 12%, the half maximal inhibitory concentration decreased to 18%, and the detection range increased fourfold. A device with mixing and centrifugation functions was designed for the pretreatment-related operations. A pre-incubation sampling device was used to facilitate incubation in batch and high-throughput detection. An ICA reader was used. The detection throughput reached 8 samples per batch or 32 samples per hour. The designed devices were printed through 3D printing and rapid prototyping.

Immunochromatographic assay for T-2 toxin based on luminescent quantum dot beads.

A quantum dot bead based immunochromatographic assay (QB-ICA) system was established for T-2 toxin (T-2), which widely occurs in agriculture and could be used as a potential biological warfare agent. After optimization, the dynamic linear detection range of T-2 calculated from a calibration curve was from 0.12 to 0.67 ng mL-1 and the limit of detection (LOD) was 0.08 ng mL-1, which is lower than those of the ICA based on colloidal gold nanoparticles or a fluorescent material or an antibody-based biochip in other reports. The performance and practicability of the established ICA system were validated with a commercial ELISA kit and the two methods were comparable. The proposed QB-ICA for T-2 could be an alternative for rapid, sensitive, and quantitative on-site detection of this toxin in biosafety monitoring in agriculture and for susceptibility testing of the potential release of this biological warfare agent.

Dual dye-loaded Au@Ag coupled to a lateral flow immunoassay for the accurate and sensitive detection of Mycoplasma pneumoniae infection.

We present an attractive model of surface-enhanced Raman scattering-based lateral flow immunoassay (SERS-LFIA) for the sensitive and accurate detection of Mycoplasma pneumoniae (MP) infection in human serum. The SERS-LFIA strip uses Au@Ag nanoparticles (Au@Ag NPs) loaded with two layers of Raman dye 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) as SERS tags. The advantages of the dual dye-loaded SERS tags (Au/DTNB@Ag/DTNB) are the high sensitivity and the bioconjugation flexibility of the detection antibody. As determined from our SERS-LFIA strip, human IgM was quantified by monitoring the SERS signal on the test line. The limit of detection for human IgM was 0.1 ng mL-1, which was 100 times more sensitive than that by using the colorimetric method. Our assay results for 20 MP-specific IgM positive serum specimens showed 100% accuracy and detection rate, whereas the parallel enzyme-linked immunosorbent assay only showed 85% detection rate. The SERS-LFIA strip also exhibited high specificity and potential clinical applications. Therefore, our SERS-based LFIA strip has strong potential for practical applications in the sensitive and rapid detection of MP.

Mulberry extracts alleviate aß 25-35-induced injury and change the gene expression profile in PC12 cells.

Mulberry, which contained high amounts of anthocyanins, has been used in traditional Chinese medicine. Mulberry fruit extracts (ME) have demonstrated the antioxidant activity and neuroprotection. The study was to investigate the neuroprotective efficacy of ME against ß-amyloid 25-35- (Aß 25-35-) induced PC12 cells injury. Cells preincubated with or without ME (200 µg/mL) for 24 h were treated with Aß 25-35 (20 µmol/L) for another 24 h. Cell viability was assessed by MTT, gene expression profiles were examined by cDNA microarrays, and RT-PCR were used to confirm the results of microarray assays. ME pretreatment was found to neutralize the cytotoxicity and prevent Aß 25-35-induced cells injury. Analyses of gene expression profile revealed that genes involving cell adhesion, peptidase activity, cytokine activity, ion binding activity, and angiogenesis regulation were significantly modulated by ME pretreatment. Among those genes, Apaf1, Bace2, and Plcb4 were enriched in the "Alzheimer's disease-reference pathway" and downregulated after ME intervention. RT-PCR results showed that ME preincubation could significantly inhibit Aß 25-35 increased mRNA levels of these three genes. Overall, ME pretreatment could substantially alleviate PC12 cells injury and downregulate expression of AD-related genes, such as Apaf1, Bace2, and Plcb4. This study has a great nutrigenomics interest and brings new and important light in the field of AD intervention.

Development of gold nanoparticle-based rapid detection kit for melamine in milk products.

A reliable and sensitive kit for the rapid detection of melamine (Mel) was developed. The kit is based on gold nanoparticle (Au NP) probe and includes a standard colorimetric card. The Au NPs were prepared by sodium borohydride reduction and characterized by transmission electron microscopy, which revealed particle sizes of approximately 5 nm. The performance of the kit in terms of aggregation kinetics, cross-reactivity, anti-interference, and sample pretreatment was investigated. The standard colorimetric card was then fabricated by chromatic aberration of a series of standard Mel-spiked milk reacts with the 5 nm Au NPs. The working range of the kit is 1-120 mg/L, and its performance is visibly more rapid and reliable by comparison with the standard colorimetric card. As low as 1 mg/L of Mel levels in milk can be determined, with the assay taking only about 10 min, including sample pretreatment. The kit can be stored for a year at room temperature. Samples were also detected by the kit, yielding results close to those obtained by high-performance liquid chromatography/mass spectrometry. Thus, the kit is applicable to qualitative and semiquantitative field detection, as well as naked-eye screening without the aid of any instrumentation.