Small volume bead assay for ovalbumin with electrochemical detection

A bead based sandwich enzyme immunoassay coupled to electrochemical detection for ovalbumin has been developed. The enzyme label alkaline phosphatase was used to convert the substrate 4-aminophenyl phosphate to electroactive product 4-aminophenol. The detection was done in a microdrop by continuously monitoring the enzyme turnover with a rotating disk electrode. This reduces dilution of the enzyme product, a key to achieving low detection limits. The assay developed has a detection limit of 0.1 ng ml-1. Assay sensitivity in complex matrices such as food and serum was compared.     

A rapid assay method for catechol-O-methyltransferase activity by flow injection analysis

A rapid assay employing flow injection analysis (FIA) to determine the activity of purified catechol-O-methyltransferase (COMT) from porcine liver is described. The method was based on the determination of normetanephrine, the 3-O-methyl metabolite of the substrate norepinephrine. Excess norepinephrine was removed from the incubation mixture by alumina extraction twice to allow normetanephrine to be subjected to flow injection analysis, coulometrical oxidation, fluorogenic reaction with ethylenediamine and fluorescence detection. K(m) and V(max) values for COMT obtained with the system were 503 microM and 4.51 nmol/min/mg protein, respectively. The method is suitable for screening of COMT inhibitors or activators, as a large number of samples, up to 200, can be processed in one working day.     

Monoclonal-based enzyme-linked immunosorbent assay and immunochromatographic rapid assay for monensin.

Monensin, a member of the ionophoric polyether antibiotics, is used primarily as a coccidiostat. A protein conjugate of monensin was prepared and utilized to produce monoclonal antibodies in the BALB/c-P3X63Ag8U.1 fusion system. Only one hybridoma that produces monoclonal antibody against monensin was isolated from one in 329 wells. The monoclonal antibody was used to develop quantitative assays for monensin by means of an enzyme-linked immunosorbent assay (ELISA). The detection limit was 1 ng ml-1 and the relative standard deviations were 2.1-6.3% intra-assay and 5.9-12.9% inter-assay. All ELISA results for assay of chicken plasma and cattle milk were confirmed using a bioassay to be used as the official method. The ELISA and bioassay results showed close correlations for plasma (r2 = 0.98, n = 25) and milk (r2 = 0.95, n = 25). Using the anti-monensin monoclonal antibodies produced, a rapid test kit based on the immunochromatographic method was developed. Detection limits of monensin for cattle milk, cattle plasma and chicken plasma were about 40, 40 and 160 ppb. respectively.     

The application of magnetic bead hybridization for the recovery and STR amplification of degraded and inhibited forensic DNA

A common problem in the analysis of forensic DNA evidence is the presence of environmentally degraded and inhibited DNA. Such samples produce a variety of interpretational problems such as allele imbalance, allele dropout and sequence specific inhibition. In an attempt to develop methods to enhance the recovery of this type of evidence, magnetic bead hybridization has been applied to extract and preconcentrate DNA sequences containing short tandem repeat (STR) alleles of interest. In this work, genomic DNA was fragmented by heating, and sequences associated with STR alleles were selectively hybridized to allele-specific biotinylated probes. Each particular biotinylated probe-DNA complex was bound to streptavidin-coated magnetic beads using enabling enrichment of target DNA sequences. Experiments conducted using degraded DNA samples, as well as samples containing a large concentration of inhibitory substances, showed good specificity and recovery of missing alleles. Based on the favorable results obtained with these specific probes, this method should prove useful as a tool to improve the recovery of alleles from degraded and inhibited DNA samples.     

A novel screen-printed electrode array for rapid high-throughput detection

A novel multi-channel electrode array sensing device was fabricated by screen-printing techniques using 96-well plate as the template. To confirm its practical value, we developed a one-step preparation of multi-walled carbon nanotubes (MWCNTs) doped electrode array by an ink containing MWCNTs, which was applied to the simultaneous detection of a variety of biological samples and environmental pollutants. Results demonstrated that the designed sensing device could carry out the multiple measurements of different analytes at the same time, while MWCNTs enhanced the electrocatalytic activity of electrodes toward electroactive molecules. The required amount of each sample was only ～200 μL. Moreover, the excellent differential pulse voltammetric (DPV) response toward dopamine, hydroquinone and catechol was obtained and the detection limits was determined to be 0.337, 0.289 and 0.369 μM, respectively. Comparing it with the traditional screen-printed electrode (SPE), this sensing device possesses the advantages of high-throughput, fast electron transfer rate for electrodes, short-time analysis and low sample consumption.     

A rapid nuclear staining test using cationic dyes contributes to efficient STR analysis of telogen hair roots

Telogen hairs presented in the crime scene are commonly encountered as trace evidence. However, short tandem repeat (STR) profiling of the hairs currently have low and limited use due to poor success rate. To increase the success rate of STR profiling of telogen hairs, we developed a rapid and cost‐effective method to estimate the number of nuclei in the hair roots. Five cationic dyes, Methyl green (MG), Harris hematoxylin (HH), Methylene blue (MB), Toluidine blue (TB), and Safranin O (SO) were evaluated in this study. We conducted a screening test based on microscopy and the percentage of loss with nuclear DNA, in order to select the best dye. MG was selected based on its specific nuclei staining and low adverse effect on the hair‐associated nuclear DNA. We examined 330 scalp and 100 pubic telogen hairs with MG. Stained hairs were classified into five groups and analyzed by STR. The fast staining method revealed 70% (head hair) and 33.4% (pubic hair) of full (30 alleles) and high partial (18–29 alleles) STR profiling proportion from the lowest nuclei count group (one to ten nuclei). The results of this study demonstrated a rapid, specific, nondestructive, and high yield DNA profiling method applicable for screening telogen hairs.     

Design and validation of an STR hexaplex assay for DNA profiling of grapevine cultivars

Although the analysis of length polymorphism at STR loci has become a method of choice for grape cultivar identification, the standardization of methods for this purpose lags behind that of methods for DNA profiling in human and animal forensic genetics. The aim of this study was thus to design and validate a grapevine STR protocol with a practically useful level of multiplexing. Using free bioinformatics tools, published primer sequences, and nucleotide databases, we constructed and optimized a primer set for the simultaneous analysis of six STR loci (VVIi51, scu08vv, scu05vv, VVMD17, VrZAG47, and VrZAG83) by multiplex PCR and CE with laser‐induced fluorescence, and tested it on 90 grape cultivars. The new protocol requires subnanogram quantities of the DNA template and enables automated, high‐throughput genetic analysis with reasonable discriminatory power. As such, it represents a step toward further standardization of grape DNA profiling.     

A rapid spectrophotometric method for iodimetric α-amylase assay

A kinetic, two-point method for the assay of α-arnylase in serum, involving spectrohotometric measurement of a starch-iodine complex, is described. This approach avoids interferences by serum proteins and other substances that react with iodine. The method requires less than 4 min per assay, only 10 μl of sample is used, and precision and accuracy are comparable to those of established procedures.     

A multiplexed bead assay for profiling glycosylation patterns on serum protein biomarkers of pancreatic cancer

A multiplexed bead-based immunoassay was developed to simultaneously profile glycosylation patterns of serum proteins to investigate their usefulness as biomarkers for pancreatic cancer. The multiplex assay utilized protein-specific capture antibodies chemically coupled individually to beads labeled with specific amounts of fluorescent dye. Captured proteins were detected based on the extent and specific type of glycosylation as determined by successive binding of fluorescent lectin probes. Advantages to this technique include the fact that antibodies coupled to the beads had minimal nonspecific binding to the lectins ConA/SNA, avoiding the step of chemically blocking the antibody glycans and the bead assays were performed in a 96-well filter plate enabling high-throughput screening applications with improved reproducibility. The assay was tested with ConA and SNA lectins to examine the glycosylation patterns of α-1-β glycoprotein (A1BG) and serum amyloid p (SAP) component for use as potential biomarkers for the detection of pancreatic cancer based on the results from prior biomarker studies. The results showed that the SNA response on the captured A1BG protein could distinguish chronic pancreatitis samples from pancreatic cancer with a p-value of 0.035 and for the SAP protein with SNA, a p-value of 0.026 was found between the signal of normal controls and the pancreatic cancer samples. For the ConA response, a decline in the signal for both proteins in the serum samples was found to distinguish pancreatic cancer from normal controls and renal cell carnoma samples (A1BG, p<0.05; and SAP, p<0.0001).     

Ceramic-based multisite microelectrode array for rapid choline measures in brain tissue

A ceramic-based multisite microelectrode array to measure choline in vivo in brain tissues is described. The microelectrodes were linear to 200 μM choline (R²=0.999±0.001) with a detection limit of approximately 0.4 μM (S/N of 3) in both single microelectrode and self-referencing amperometric recording modes. The 90% rise time of the sensor was 1.4 s, allowing for rapid measures of choline. Good selectivity (>300:1) was observed over interferents such as ascorbic acid, uric acid, and DOPAC in the single microelectrode mode. However, a self-referencing recording mode was needed to remove potassium-evoked dopamine signals in rat striatum. In vivo measurements of choline in the rat brain are presented.     

A displacement assay for the sensing of protein interactions using sugar-tetraphenylethene conjugates

A displacement sensor array based on sugar-substituted tetraphenylethenes with the aggregation-induced emission feature for proteins to perform screening of protein-protein interactions is demonstrated.     

A novel fluorescent indicator displacement assay for sensing the anticancer drug gefitinib

Fluorescent indicator displacement assays have become popular for converting synthetic receptors into optical sensors. We have now shown that a 1:2 host–guest complex between cucurbit[8]uril (Q[8]) and proflavine (PF) can be used as a fluorescent indicator for sensing the anticancer drug gefitinib. The 2PF@Q[8] complex can be used to detect gefitinib with high selectivity using fluorescence spectrometry with a detection limit of 6.63 × 10^?8 mol^?L. The proposed sensing mechanism was investigated using ¹H nuclear magnetic resonance spectroscopy (¹H NMR), isothermal titration calorimetry and electrospray ionisation mass spectrometry. The 2PF@Q[8] complex was shown to be suitable for imaging gefitinib in prostate cancer (PC3) cells, which may help to elucidate relevant biological processes at the molecular level. We have developed a novel F-IDA to detect the anticancer drug GEF with high selectivity. The new indicator has excellent selectivity and a low detection limit for GEF. We have also demonstrated that the F-IDA can be used for the practical determination of drugs in living cells.     

A selective ATP chromogenic sensor for use in an indicator displacement assay

We synthesized a dinuclear Zn²⁺ complex that is useful as a sensor for ATP in a DMSO/H₂O (1:9, v/v) solvent system via a simple indicator displacement assay (IDA). This chromogenic sensor method can be used to analyze 0.1-2.0 μM of ATP with no interference from ADP or AMP.     

DNA aptamer selection and aptamer-based fluorometric displacement assay for the hepatotoxin microcystin-RR

Microcystin-RR (MC-RR) is a highly acute hepatotoxin produced by cyanobacteria. It is harmful to both humans and the environment. A novel aptamer was identified by the systemic evolution of ligands by exponential enrichment (SELEX) method as a recognition element for determination of MC-RR in aquatic products. The graphene oxide (GO) SELEX strategy was adopted to generate aptamers with high affinity and specificity. Of the 50 aptamer candidates tested, sequence RR-33 was found to display high affinity and selectivity, with a dissociation constant of 45.7 ± 6.8 nM. Aptamer RR-33 therefore was used as the recognition element in a fluorometric assay that proceeds as follows: (1) Biotinylated aptamer RR-33 is immobilized on the streptavidinylated wells of a microtiterplate, and carboxyfluorescein (FAM) labelled complementary DNA is then allowed to hybridize. (2) After removal of excess (unbound) cDNA, sample containing MC-RR is added and incubated at 37 °C for 2 h. (3) Displaced free cDNA is washed away and fluorescence intensity measured at excitation/emission wavelengths of 490/515 nm. The calibration plot is linear in the 0.20 to 2.5 ng·mL^?1 concentration range, and the limit of detection is 80 pg·mL^?1. The results indicate that the GO-SELEX technology is appropriate for the screening of aptamers against small-molecule toxins. The detection scheme was applied to the determination of MC-RR in (spiked) water, mussel and fish and gave recoveries between 91 and 98 %. The method compares favorably to a known ELISA. Conceivably, this kind of assay is applicable to other toxins for which appropriate aptamers are available.

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Graphical abstract The aptamer RR-33 specific for microcystin (MC-RR) was identified by using the graphene oxide (GO) SELEX process. This aptamer was used for determination of MC-RR by a fluorometric displacement assay.

     

A strand displacement signal amplification-assisted fluorescence assay for sensitive detection of microRNA

MicroRNA is a vital biomarker because of its abnormal expression in the emergence and development of diseases, especially in cancers. Herein, a label-free fluorescent sensing platform is proposed for detecting microRNA-21, coupled with the cascade toehold-mediated strand displacement reaction and magnetic beads. Target microRNA-21 acts as an initiator to trigger the cascade toehold-mediated strand displacement reaction and it outputs double-stranded DNA. After magnetic separation, the double-stranded DNA is intercalated by SYBR Green I, resulting in an amplified fluorescent signal. Under the optimal conditions, a wide linear range (0.5–60 nmol/L) and low limits of detection (0.19 nmol/L) are exhibited. What's more, the biosensor shows great specificity and reliability between microRNA-21 and other microRNAs involved in cancer (microRNA-34a, microRNA-155, microRNA-10b, and let-7a). Owing to the properties of fabulous sensitivity, high selectivity, and simplicity of operator, the proposed method paves a promising way for microRNA-21 detection in cancer diagnosis and biological research.     

A rapid modified gas chromatographic assay for esterase activity.

A rapid modified procedure for the gas chromatogrpahic determination of esterase activity was studied. Aliphatic esters such as ethyl n-butyrate, n-propul n-butyrate, n-butyl n-butyrate and n-amyl n-butyrate were used as substrates and acetone was chosen as the most suitable solvent for dissolving the substrates in order to avoid alcoholysis. The enzyme reaction was started in a mixture of 0.03 M phosphate buffer, pH 7.90, containing an adequals, an aliquot of the reaction solutionwas injected directly on to a gas chromatograph and the alcohols produced were separated.     

An ELIME assay for the rapid diagnosis of coeliac disease

Coeliac disease (CD) is a gluten-induced autoimmune enteropathy found in genetically susceptible subjects. Because of the high number of undetected cases, rapid and cheaper screening methods are needed. Currently, the CD diagnosis involves the detection of anti-transglutaminase IgA antibodies (anti-tTG IgA) in blood serum through the use of ELISA systems with confirmation by histology of the intestinal mucosa. A new, rapid magneto-electrochemical immunosensor for CD diagnosis has been developed and applied to serum sample analysis. The system uses magnetic beads coated with tTG antigen to detect anti-tTG antibodies in positive serum samples and an alkaline phosphatase-conjugated anti-human IgA as label. An electrochemical readout, using magnetized screen-printed electrodes coupled with a portable instrument, is made after the addition of α-naphtyl phosphate, which is enzymatically converted into the electrochemically active α-naphthol product. The work involved the following considerations: (1) optimization of analytical parameters; (2) recovery evaluation, adding known concentrations of anti-tTG IgA to “blank” sera; (3) analysis of 107 blood serum samples; (4) calculation of the ROC curve, resulting in a cut-off of 1.0 U/ml, 100% of clinical sensitivity and 98.36% of clinical specificity; evaluation of the agreement between electrochemical and ELISA kit values (r ² of 0.943). The system developed could be an useful tool for a correct and rapid CD diagnosis. This method is simple, cheap, rapid, and suitable for screening analyses performed outside of the classical diagnostic laboratory.     

Colloidal gold probe based rapid immunochromatographic strip assay for cortisol

A rapid and semi-quantitative immunochromatographic strip (ICS) test for cortisol analysis in serum was developed. The test strip was based on a competitive assay format. Colloidal gold nanoparticles were synthesized and coupled with cortisol-3-carboxymethyloxime-adipic acid dihydrazide-bovine serum albumin (F-3-CMO-ADH-BSA) antigen to directly compete with cortisol in human serum samples. F-3-CMO-ADH-BSA-gold label and uncoupled colloidal gold nanoparticles were appropriately characterized using UV-vis spectroscopy, transmission electron microscopy and atomic force microscopy. Anticortisol antibody raised against F-3-CMO-BSA immunogen in New Zealand white rabbits was coated on the NC membrane as test line. Anti-BSA antibody was used as control line. The lower detection limit of the ICS test was 30 ngmL(-1) with visual detection and was completed in 10 min. About 30 human serum samples were also analyzed by the developed strip test and their range of cortisol concentration was established. The developed ICS test is rapid, economic and user friendly.     

Simple and rapid high-performance liquid chromatographic assay for esmolol.

A procedure for determining esmolol concentrations in blood is described. Dichloromethane was used to extract esmolol from the blood and to inhibit the activity of blood esterases. Blood esmolol concentrations were determined by high-performance liquid chromatography using 3-methoxy-O-demethylencainide as the internal standard. The limit of detection of this assay was 5 ng/ml. The relationship between the peak-height ratio of esmolol and the internal standard was linear in the concentration ranges 10-30,000 ng/ml. The mean absolute and relative recoveries of esmolol from blood were 84 and 89%, with coefficients of variation less than 3%. This method has been used in our laboratory for pharmacokinetic and pharmacodynamic studies.     

A fluorescence displacement assay for antidepressant drug discovery based on ligand-conjugated quantum dots

The serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) protein plays a central role in terminating 5-HT neurotransmission and is the most important therapeutic target for the treatment of major depression and anxiety disorders. We report an innovative, versatile, and target-selective quantum dot (QD) labeling approach for SERT in single Xenopus oocytes that can be adopted as a drug-screening platform. Our labeling approach employs a custom-made, QD-tagged indoleamine derivative ligand, IDT318, that is structurally similar to 5-HT and accesses the primary binding site with enhanced human SERT selectivity. Incubating QD-labeled oocytes with paroxetine (Paxil), a high-affinity SERT-specific inhibitor, showed a concentration- and time-dependent decrease in QD fluorescence, demonstrating the utility of our approach for the identification of SERT modulators. Furthermore, with the development of ligands aimed at other pharmacologically relevant targets, our approach may potentially form the basis for a multitarget drug discovery platform.