Horseradish peroxidase functionalized gold nanorods as a label for sensitive electrochemical detection of alpha-fetoprotein antigen

In this study, a novel tracer, horseradish peroxidase (HRP) functionalized gold nanorods (Au NRs) nanocomposites (HRP–Au NRs), was designed to label the signal antibodies for sensitive electrochemical measurement of alpha-fetoprotein (AFP). The preparation of HRP–Au NRs nanocomposites and the labeling of secondary antibody (Ab₂) were performed by one-pot assembly of HRP and Ab₂ on the surface of Au NRs. The immunosensor was fabricated by assembling carbon nanotubes (CNTs), Au NRs, and capture antibodies (Ab₁) on the glassy carbon electrode. In the presence of AFP antigen, the labels were captured on the surface of the Au NRs/CNTs via specific recognition of antigen–antibody, resulting in the signal intensity being clearly increased. Differential pulse voltammetry (DPV) was employed to record the response signal of the immunosensor in phosphate-buffered saline (PBS) containing hydrogen peroxide (H₂O₂) and 3,3′,5,5′-tetramethylbenzidine (TMB). Under optimal conditions, the signal intensity was linearly related to the concentration of AFP in the range of 0.1–100 ng ml⁻¹, and the limit of detection was 30 pg ml⁻¹ (at signal/noise [S/N] = 3). Furthermore, the immunoassay method was evaluated using human serum samples, and the recovery obtained was within 99.0 and 102.7%, indicating that the immunosensor has potential clinical applications.     

Development of a highly sensitive,high-throughput assay for glycosyltransferases using enzyme-coupled fluorescence detection

Glycosyltransferases catalyze transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. Identification of selective modulators of glycosyltransferases is important both to provide new tools for investigating pathophysiological roles of glycosylation reactions in cells and tissues, and as new leads in drug discovery. Here we describe a universal enzyme-coupled fluorescence assay for glycosyltransferases, based on quantification of nucleotides produced in the glycosyl transfer reaction. GDP, UDP, and CMP are phosphorylated with nucleotide kinase in the presence of excess ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase, glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for conversion of resazurin to resorufin, which is determined by fluorescence measurement. The method was validated by comparison with an HPLC method, and employed to screen the LOPAC1280 library for inhibitors in a 384-well plate format. The assay performed well, with a Z′-factor of 0.80. We identified 12 hits for human galactosyltransferase B4GALT1 after elimination of false positives that inhibited the enzyme-coupled assay system. The assay components are all commercially available and the reagent cost is only 2 to 10 US cents per well. This method is suitable for low-cost, high-throughput assay of various glycosyltransferases and screening of glycosyltransferase modulators.     

Development of a sequential injection analysis system for monitoring of trehalose concentrations

A sequential injection analysis (SIA) system was developed to on-line monitor the concentrations of trehalose in biological processes. The system is based on the enzymatic reaction of trehalose to glucose in an immobilized trehalase reactor and the subsequent reaction of glucose in the presence of glucose oxidase. The peroxide produced is detected using horseradish peroxidase with a redox indicator (ABTS, 2,2′-azino-di(3-ethylbezthiazoli-sulfonic acid-6)) and a spectrophotometer set at 435 nm. The SIA was fully automated using software written in the LabVIEW^TM development environment. A number of system variablese.g. flow rate of the carrier buffer solution and volume ratio of sample to reagents, were evaluated to increase the sensitivity and performance of the SIA system. The performance of the trehalose-SIA system was linear under partially optimized operating conditions in the concentration range of 0.3 to 2.0 g/L (R²=0.999) with a sample frequency of 6 1/h. The SIA system was successfully employed to on-line monitor the concentrations of trehalose in a continuously stirred tank reactor. The on-line monitored data were in good agreement with the off-line data measured by a HPLC with a refractive index detector (n=14, R²=0.9865).     

Nitrogen-doped carbon dots as a fluorescent probe for the highly sensitive detection of bilirubin and cell imaging

Nitrogen-doped carbon dots (NCDs) with bright blue fluorescence were constructed by a hydrothermal method using sucrose and l- proline as raw materials. The NCDs were characterized by transmitted electron microscopy, X-ray diffraction, Fourier-transform infrared spectrometry, X-ray photoelectron spectroscopy, and ultraviolet-visible absorption and fluorescence spectroscopy to investigate the morphology, elemental composition, and optical properties. The NCDs had good water solubility, high dispersibility with an average diameter of only 1.7 nm, and satisfactory optical properties with a fluorescence quantum yield of 23.4%. The NCDs were employed for the detection of bilirubin. A good linear response of the NCDs in the range 0.35–9.78 μM was obtained for bilirubin with a detection limit of 33 nM. The NCDs were also applied to the analysis of real samples, serum and urine, with a recovery of 95.34% to 104.66%. The low cytotoxicity and good biocompatibility of the NCDs were indicated by an MTT assay and cell imaging of HeLa cells. Compared with other detection systems, using NCDs for bilirubin detection was a facile and efficient method with good selectivity and sensitivity.     

Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein

The current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N) localizes to the cytoplasm and the nucleolus and is required for viral RNA synthesis. N is the most abundant coronavirus protein, so it is of utmost importance to develop specific antibodies for its detection. In this study, we developed a sandwich immunoassay to recognize the SARS-CoV-2 N protein. We immunized one alpaca with recombinant SARS-CoV-2 N and constructed a large single variable domain on heavy chain (VHH) antibody library. After phage display selection, seven VHHs recognizing the full N protein were identified by ELISA. These VHHs did not recognize the nucleoproteins of the four common human coronaviruses. Hydrogen Deuterium eXchange–Mass Spectrometry (HDX-MS) analysis also showed that these VHHs mainly targeted conformational epitopes in either the C-terminal or the N-terminal domains. All VHHs were able to recognize SARS-CoV-2 in infected cells or on infected hamster tissues. Moreover, the VHHs could detect the SARS variants B.1.17/alpha, B.1.351/beta, and P1/gamma. We propose that this sandwich immunoassay could be applied to specifically detect the SARS-CoV-2 N in human nasal swabs.     

Development of a sensitive flow‐injection–chemiluminescence detection method for the determination of laevodopa

A simple chemiluminometric method using flow injection has been developed for the determination of laevodopa, based on its sensitizing effect on the weak chemiluminescence (CL) reaction between Na₂SO₃ and acidic KMnO₄. Under optimum experimental conditions, the CL intensity was linearly related to the concentration of laevodopa from 3.4 × 10^–8 to 2.4 × 10^–5 mol/L and the detection limit was 1.1 × 10^–8 mol/L (s:n = 3). The relative standard deviation (RSD) of the proposed method calculated from 20 replicate injection of 3 × 10^–7 mol/L laevodopa was 3.3%. The correlation coefficient was 0.997. The method was successfully applied to the determination of laevodopa in commercial pharmaceutical formulations and spiked urine samples. Copyright © 2008 John Wiley & Sons, Ltd.     

Development of a highly sensitive nested-PCR method using a single closed tube for detection of Fusarium culmorum in cereal samples

AIMS: The aim of the study was to develop a sensitive detection method of Fusarium culmorum contamination in cereal samples. METHODS AND RESULTS: A nested-PCR method using a single closed tube was developed for the detection of F. culmorum in infected cereal samples. The concentrations of the first primer pair was diluted 10,000 times compared to the concentration used for the second primer pair. Differing annealing temperatures allowed both first and second polymerase chain reaction (PCR) reactions to be performed subsequently in the same closed tube. The detection limit was 5-50 fg of purified target DNA and allowed the detection of 1% infected seeds of wheat in a mixture with uninfected grains. CONCLUSIONS: F. culmorum can be specifically detected in cereal samples by the highly sensitive method of nested-PCR in a single closed tube. SIGNIFICANCE AND IMPACT OF THE STUDY: This work describes the detection of F. culmorum in cereal samples that is approximately 100 times more sensitive than previous PCR methods, involves low risk of cross contaminations between samples, low costs and reduced hands-on time as compared to standard nested-PCR protocols.     

A highly sensitive and rapid method for the detection of DNA fragments using the photoprotein obelin as a reporter

The recombinant Ca²⁺-activated photoprotein obelin was used as a reporter protein in a solid-phase bioluminescent hybridization DNA assay. Oligonucleotide probes were immobilized on the surface of polymer methacrylate beads or microbiological plates of different types. A 30-mer oligonucleotide or its derivative with the biotin residue on the 3′-terminus, as well as a denatured double-stranded PCR fragment of the hepatitis C virus with the sequence of the 30-mer oligonucleotide was used as a DNA template. The probe in the hybridization complex was labeled by the elongation of the chain using a Taq DNA polymerase in the presence of biotinylated deoxyuridine triphosphate. The results of the bioluminescent assay were compared with the results of colorimetric analysis obtained with alkaline phosphatase as a reporter protein. It was shown that the use of the bioluminescent obelin label substantially accelerates the DNA detection procedure, provides a high sensitivity of the assay (no less than 10^?15 mol of DNA template), and ensures a quantitative determination of the amount of DNA template in the tested sample.     

Nitrogen‐doped carbon dots as a fluorescent probe for the highly sensitive detection of Ag+ and cell imaging

An easy hydrothermal synthesis strategy was applied to synthesize green‐yellow emitting nitrogen‐doped carbon dots (N‐CDs) using 1,2‐diaminobenzene as the carbon source, and dicyandiamide as the dopant. The nitrogen‐doped CDs resulted in improvement in the electronic characteristics and surface chemical activities. N‐CDs exhibited bright fluorescence emission and could response to Ag⁺ selectively and sensitively. Other ions produced nearly no interference. A N‐CDs based fluorescent probe was then applied to sensitively determine Ag⁺ with a detection limit of 5 × 10^?8 mol/L. The method was applied to the determination of Ag⁺ dissolved in water. Finally, negligibly cytotoxic, excellently biocompatibile, and highly fluorescent carbon dots were applied for HepG2 cell imaging and the quenched fluorescence by adding Ag⁺, which indicated its potential applications.     

Development of a flow‐injection analysis system with fluorescence detection for gatifloxacin determination in organized medium

This work reports the development and optimization of a flow injection analysis system with fluorescence detection (FIA–FLUO) for gatifloxacin (GFX) determination in organized medium. The analytical system was based on the enhanced fluorescence of gatifloxacin in micellar medium containing sodium dodecyl sulfate (SDS) at pH 6.0. The influence of physical (carrier flow rate, sample volume and volume of reaction coil) and chemical (pH, concentration of buffer and concentration of SDS) parameters that could affect the performance of the FIA system was evaluated in order to reach optimum conditions in terms of sensitivity and analytical throughput. Under optimized conditions, the FIA–FLUO system allowed the injection of 40 samples per hour with a limit of quantification of 72 µg/L and a RSD of 3.5% at 0.20 mg/L. Real samples of commercial pharmaceutical formulations containing GFX were analyzed, and no statistical difference was observed between the results obtained using the developed system and those obtained using the reference method based on high‐performance liquid chromatography with UV detection. Copyright © 2014 John Wiley & Sons, Ltd.     

Immobilization of bovine serum albumin as a sensitive biosensor for the detection of trace lead ion in solution by piezoelectric quartz crystal impedance

A biosensor based on bovine serum albumin (BSA) for the detection of lead (Pb(2+)) ion was developed and characterized. BSA was immobilized onto a colloidal Au-modified piezoelectric quartz crystal (PQC) as a biosensor for the detection of Pb(2+) ion by piezoelectric quartz crystal impedance (PQCI). Calibration curves for the quantification of Pb(2+) ion showed excellent linearity throughout the concentration range from 1.0 x 10(-7) to 3.0 x 10(-9)mol/L. The interaction between the Pb(2+) ions and the sensor chip is influenced significantly by the pH of the reaction buffer, and the optimal pH for the experiment was 5.4. Under the optimal conditions, the detection limit of 1.0 x 10(-9)mol/L for Pb(2+) was obtained. Kinetic parameters of the Pb(2+)-BSA interactions were also determined by using this chip. The sensor chip could be regenerated for use by dipping in the ethylenediaminetetraacetic acid (EDTA) solution for approximately 2h, and the chip was used to detect Pb(2+) ion for eight times without obvious signal attenuation.     

Development of a highly efficient gene targeting system for Fusarium graminearum using the disruption of a polyketide synthase gene as a visible marker

We cloned a polyketide synthase gene (pks12) from Fusarium graminearum, a devastating fungal pathogen of cereals. Transformation-mediated gene disruption led to an easily detectable albino phenotype of the disruptants. We used the disruption of the pks12 gene as a visible marker for transformation-mediated homologous recombination and optimized the transformation procedure to achieve a high rate of homologous recombination. In combination with the published genomic sequence data and the generation of expressed sequence tags (ESTs) for F. graminearum, this is a useful tool to investigate this important plant pathogen on a molecular level. Optimized transformation of F. graminearum resulted in at least 93% homologous recombination events when the homologous genomic DNA fragment in the vector had a size of approximately 800bp and was linearized in the middle. Using a genomic sequence of approximately 500bp in the transformation vector, 70% of the transformants still exhibited homologous recombination. On the contrary, no more than 10% homologous recombination events were observed when less than 400bp DNA fragments were used. We co-transformed F. graminearum with two different vectors. One vector harboured a DNA insert homologous to the pks12 gene, while the other vector consisted of the same vector backbone carrying the selection marker specific for F. graminearum. About 70% of the transformants had a disrupted pks12 gene, and all of these showed an integration of the second vector into the pks disruption vector. Therefore, the time-consuming construction of a single transformation vector can be avoided; furthermore, it is now easily feasible to express a gene construct at a defined and mutated genomic site.