Quantum dot-based lateral flow immunoassay for detection of chloramphenicol in milk

A novel rapid (20 min) fluorescent lateral flow test for chloramphenicol (CAP) detection in milk was developed. The chosen format is a binding-inhibition assay. Water-soluble quantum dots with an emission peak at 625 nm were applied as a label. Milk samples were diluted by 20 % with phosphate buffer to eliminate the matrix effect. The result of the assay could be seen by eye under UV light excitation or registered by a portable power-dependent photometer. The limit of CAP detection by the second approach is 0.2 ng/mL, and the limit of quantitation is 0.3 ng/mL.

Integration of lateral flow and microarray technologies for multiplex immunoassay: application to the determination of drugs of abuse

We report on a lateral flow microarray that combines multi-spot immunochip technology and immunochromatography. It can serve as a tool for the simultaneous detection of multiple analytes. The test zone of the nitrocellulose support comprises a microarray spotted with up to 32 antigens that can capture labeled gold-antibodies after lateral flow. The detection limits and detectable concentration ranges of the assay were characterized. The method was applied to the determination of drugs of abuse (and their metabolites) in urine, specifically of morphine, amphetamine, methamphetamine, and benzoylecgonine. The assay format is rapid (10 min), and has both a low relative standard deviation (< 9 %) and high recoveries (95–114 %). The detection limits (2–20 ng mL^–1 for drugs of abuse) are comparable to those of conventional single-analyte strip methods.

Multiplex bead-array competitive immunoassay for simultaneous detection of three pesticides in vegetables

We report on a multiplex bead-based competitive immunoassay using suspension array technology for the simultaneous detection of the pesticides triazophos, carbofuran and chlorpyrifos. Three hapten-protein conjugates were covalently bound to carboxylated fluorescent microspheres to serve as probes. The amount of conjugates and antibodies were optimized. The new multi-analyte assay has dynamic ranges of 0.02–50 ng?mL^?1, 0.5–500 ng?mL^?1 and 1.0–1000 ng?mL^?1 for triazophos, carbofuran and chlorpyrifos, respectively, and the detection limits are 0.024, 0.93 and 1.68 ng?mL^?1. This new multiplex assay is superior to the traditional ELISA in possessing a wider detection range, better reproducibility and the feature of multi-target detection. Cross-reactivity studies indicated that the bead-array method is highly selective for the three target pesticides, and that individual analyses have no significant influence between each other, also without cross-reactions from other structurally related pesticides. The method was applied to analyze vegetables spiked with the three pesticides, and the recoveries were in ranges of 78.5–112.1 %, 72.2–120.2 % and 70.2–112.8 %, respectively, with mean coefficients of variation of <15 %.

A highly sensitive caffeine immunoassay based on a monoclonal antibody

A new immunoassay has been developed based on a commercially available anti-caffeine monoclonal antibody and a de novo synthesized tracer, using horseradish peroxidase and UV–visible detection. Caffeine, which is frequently found in surface waters, can be quantified with a relative error lower than 20% for concentrations above 0.025 μg L^?1 (limit of quantitation, direct analysis). The limit of detection is 0.001 μg L^?1 and can be reduced by solid-phase extraction (SPE). Moreover, with minor adaptations, the assay can be used to quantify caffeine in several beverages, shampoo, and caffeine tablets. The results obtained by ELISA correlate well with those from liquid chromatography–tandem mass spectrometry (LC–MS–MS) for the tested matrices. Several surface waters from Berlin were analysed and all tested positive for caffeine, with concentrations higher than 0.030 μg L^?1. In one run 66 samples can be analysed within 2 h.

Development of a lateral flow fluorescent microsphere immunoassay for the determination of sulfamethazine in milk

The fluorescent microsphere has been increasingly used as detecting label in immunoassay because of its stable configuration, high fluorescence intensity, and photostability. In this paper, we developed a novel lateral flow fluorescent microsphere immunoassay (FMIA) for the determination of sulfamethazine (SMZ) in milk in a quantitative manner with high sensitivity, selectivity, and rapidity. A monoclonal antibody to SMZ was covalently conjugated with the carboxylate-modified fluorescent microsphere, which is polystyrene with a diameter of 200 nm. Quantitative detection of SMZ in milk was accomplished by recording the fluorescence intensity of microspheres captured on the test line after the milk samples were diluted five times. Under optimal conditions, the FMIA displays a rapid response for SMZ with a limit of detection of as low as 0.025 ng mL^?1 in buffer and 0.11 μg L^?1 in milk samples. The FMIA was then successfully applied on spiked milk samples and the recoveries ranged from 101.1 to 113.6 % in the inter-batch assay with coefficient of variations of 6.0 to 14.3 %. We demonstrate here that the fluorescent microsphere-based lateral flow immunoassay (LFIA) is capable of rapid, sensitive, and quantitative detection of SMZ in milk.

Mixed immunoassay design for multiple chemical residues detection

In this research, a mixed immunoassay design for multiple chemical residues detection based on combined reverse competitive enzyme-linked immunosorbent assay (ELISA) procedure was developed. This method integrated two reverse ELISA reactions in one assay by labeling horseradish peroxidase to deoxynivalenol (DON) and orbifloxacin. Within this method, IC50 of the two mAbs for each analyte we produced ranged from 23?～?68 ng?mL^?1 for DONs and 4.1?～?49 ng?mL^?1 for quinolones (QNs). The limit of detection measured by IC10 was achieved at 0.45–1.3 ng?mL^?1 for DONs and 0.59–6.9 ng?mL^?1 for QNs, which was lower than the maximum residue levels. Recoveries in negative samples spiked at concentrations of 100, 200, and 500 ng?mL^?1 ranged from 91.3 to 102.2 % for DONs and 88.7–98.05 % for QNs with relative standard deviation less than 9.88 and 12.67 %. The results demonstrated that this developed immunoassay was suitable for screening of low molecular weight contaminants.

A highly sensitive europium nanoparticle-based lateral flow immunoassay for detection of chloramphenicol residue

A europium nanoparticle-based lateral flow immunoassay for highly sensitive detection of chloramphenicol residue was developed. The detection result could be either qualitatively resolved with naked eye or quantitatively analyzed with the assistance of a digital camera. In the qualitative mode, the limit of detection (LOD) was found to be 0.25 ng/mL. In the quantitative mode, the half-maximal inhibition concentration (IC50) was determined to be 0.45 ng/mL and the LOD can reach an ultralow level of 0.03 ng/mL, which is ~100 times lower than that of the conventional colloidal gold-based lateral flow immunoassay. Potential application of the established method was demonstrated by analyzing representative cow milk samples.

Competitive amperometric immunosensor based on covalent linking of a protein conjugate to dendrimer-functionalised nanogold substrate for the determination of 2,4,6-trinitrotoluene

A new amperometric immunosensor for 2,4,6-trinitrotoluene based on the working principle of competitive enzyme-linked immunosorbent assay was developed and characterised. An electrodeposited nanogold substrate was functionalised by deposition of self-assembled monolayers of 2-aminoethanethiol as linkers for the subsequent immobilisation of polyamidoaminic dendrimers. Our approach makes use of those dendrimers to anchor a trinitrobenzene-ovalbumin conjugate on the electrode surface. The immunosensor was tested and validated for the determination of 2,4,6-trinitrotoluene showing high selectivity with respect to other nitroaromatic compounds, a limit of detection of 4.8 ng/mL and a limit of quantitation of 6 ng/mL. The immunosensor was tested for the quantification of the analyte in spiked soils and in a real sample of post-blast soil, evidencing a good recovery rate (113 %).

ATPase inhibitor based luciferase assay for prolonged and enhanced ATP pool measurement as an efficient fish freshness indicator

The nucleotide degradation pathway in somatic cells leads to the accumulation of products such as hypoxanthine and inosine, which are commonly used as fish and meat freshness indicators. Assays based on these molecules cannot differentiate the postmortem time over a short period of time (5–10 h). Further, quantification of these degradation products is cumbersome, costly and time-consuming. For the proposed assay, optimal concentrations of 30 and 2 mM, respectively, for the ATPase inhibitors sodium orthovanadate and EDTA were found. Further, it was observed that a firefly luciferase based assay could enhance the sensitivity levels up to 165-fold at 30 °C. In addition, it was observed that the sensitivity for ATP assay was enhanced up to 60-fold even after 12 h. The limit of detection for the ATP assay was 1 pM, unlike other conventional methods, which are sensitive only up to micromolar levels. Moreover, as little as 0.044 g fish fillet was required for the assay, and no time-consuming sample preparation was necessary. Luminescence of prolonged duration was observed in harvested fish kept at -20 °C in comparison with fish kept at 4 and 30 °C, which reflects the shelf life of fish preserved at lower temperatures.

Simultaneous serum desalting and total protein determination by macroporous reversed-phase chromatography

Macroporous reversed-phase (mRP) chromatography was successfully used to develop an accurate and precise method for total protein in serum. The limits of detection (0.83 μg, LOD) and quantification (2.51 μg, LOQ) for the mRP method are comparable with those of the widely used micro BCA protein assay. The mRP method can be used to determine the total protein concentration across a wide dynamic range by detecting chromatographic peaks at 215 nm and 280 nm. The method has the added advantage of desalting and denaturing proteins, leading to more complete digestion by trypsin and to better LC–MS–MS identification in shotgun proteomics experiments.

Fluorescence-based immunoassay for human chorionic gonadotropin based on polyfluorene-coated silica nanoparticles and polyaniline-coated Fe3O4 nanoparticles

We report on an ultrasensitive fluorescence immunoassay for human chorionic gonadotrophin antigen (hCG). It is based on the use of silica nanoparticles coated with a copolymer (prepared from a fluorene, a phenylenediamine, and divinylbenzene; PF@SiO₂) that acts as a fluorescent label for the secondary monoclonal antibody to β-hCG antigen. In parallel, Fe₃O₄ nanoparticles were coated with polyaniline, and these magnetic particles (Fe₃O₄@PANI) served as a solid support for the primary monoclonal antibody to β-hCG antigen. The PF@SiO₂ exhibited strong fluorescence and good dispersibility in water. A fluorescence sandwich immunoassay was developed that enables hCG concentrations to be determined in the 0.01–100 ng·mL^?1 concentration range, with a detection limit of 3 pg·mL^?1.

Highly sensitive electrochemical immunoassay for zearalenone in grain and grain-based food

We have developed a highly sensitive electrochemical immunoassay for the quantitation of zearalenone (ZEN), a mycotoxin produced by Fusarium species. In this enzyme linked immunosorbent assay, the enzymatic conversion of the substrate p-nitrophenylphosphate is detected by a microplate reader and the signal subsequently converted into an electrical signal. The concentrations of coating antigen (ZEN-ovalbumin), of monoclonal antibody, and of goat anti-mouse antibody labeled with alkaline phosphatase were optimized. In terms of electrochemical detection, the types and pH values of the buffers, the conditions for agitating, and scanning frequency were optimized. The effective detection range of this immunoassay is quite wide (0.004 to 9.5 ng?mL^?1), and the limit of detection is 2 pg?mL^?1. ZEN-free corn, wheat, and grain-based food samples were spiked with ZEN and analyzed by this method, and recoveries were found to range from 91.6 % to 113.0 %. Unlike previously described electrochemical methods, this method is both highly sensitive and has a wide working range. The method is fast and thus provides a platform for high-throughput analysis that meets the current need to monitor trace levels of analytes in grain and grain-based food.

Validation of a novel method to identify in utero ethanol exposure: simultaneous meconium extraction of fatty acid ethyl esters,ethyl glucuronide,and ethyl sulfate followed by LC-MS/MS quantification

Presence of fatty acid ethyl esters (FAEE), ethyl glucuronide (EtG), and ethyl sulfate (EtS) in meconium, the first neonatal feces, identifies maternal alcohol consumption during pregnancy. Current meconium alcohol marker assays require separate analyses for FAEE and EtG/EtS. We describe development and validation of the first quantitative liquid chromatography tandem mass spectrometry assay for 9 FAEEs, EtG, and EtS in 100 mg meconium. For the first time, these alcohol markers are analyzed in the same meconium aliquot, enabling comparison of the efficiency of gestational ethanol exposure detection. 100 mg meconium was homogenized in methanol and centrifuged. The supernatant was divided, and applied to two different solid phase extraction columns for optimized analyte recovery. Limits of quantification for ethyl laurate, myristate, linolenate, palmitoleate, arachidonate, linoleate, palmitate, oleate, and stearate ranged from 25–50 ng/g, with calibration curves to 2,500–5,000 ng/g. EtG and EtS linear dynamic ranges were 5–1,000 and 2.5–500 ng/g, respectively. Mean bias and between-day imprecision were <15 %. Extraction efficiencies were 51.2–96.5 %. Matrix effects ranged from ?84.7 to 16.0 %, but were compensated for by matched deuterated internal standards when available. All analytes were stable (within ±20 % change from baseline) in 3 authentic positive specimens, analyzed in triplicate, after 3 freeze/thaw cycles (?20 °C). Authentic EtG and EtS also were stable after 12 h at room temperature and 72 h at 4 °C; some FAEE showed instability under these conditions, although there was large inter-subject variability. This novel method accurately detects multiple alcohol meconium markers and enables comparison of markers for maternal alcohol consumption.

Prussian blue-doped nanogold microspheres for enzyme-free electrocatalytic immunoassay of p53 protein

We report on a new enzyme-free electrochemical immunoassay for the sensitive detection of the p53 protein (p53; a model analyte) by using a screen-printed carbon electrode modified with monoclonal mouse anti-human p53 antibody tagged with gold nanoparticles. First, nanogold microspheres doped with Prussian Blue were synthesized by a reverse micelle method. The resulting microspheres were used to label polyclonal anti-p53 antibody which then was applied in a sandwich immunoassay in pH 6.5 buffer solution using the Prussian Blue in the particles as the redox-active reporter. The electrochemical signal of the immunosensor is shown to increase with the concentration of the analyte (p53 protein) in the range from 0.5 to 80 U mL^?1, with a detection limit of 0.1 U mL^?1. No non-specific adsorption was observed. Coefficients of variation for intra-assay and inter-assay were below 8.5 % and 11.5 %, respectively. In addition, the method was applied to the analysis of 15 human serum samples, and a good relationship was found between the new immunoassay and the referenced electro-chemiluminescence method.

Quantitative turbidity assay for lipolytic enzymes in microtiter plates

A clearing assay for lipolytic enzymes has been realized in 96-well microtiter plates. A thin layer containing emulsified tributyrin as turbidity-generating substrate was placed on a thicker supporting aqueous layer. Both layers were stabilized by a gel-forming agent. Enzyme addition leads to clearing of the emulsion detected with a standard microtiter plate reader as a decrease of extinction. Dependencies of the signal kinetics on the substrate and enzyme concentrations were studied. For 0.5–1 % tributyrin content the reaction rate is not substrate-limited. An initial slope of the signal kinetics is proportional to the lipase activity. A detailed characterization of the assay was performed. Lipolysis of tributyrin was confirmed by glycerol detection. Various gel-forming agents were compared and diffusion conditions in these gels were analyzed. Agar and agarose were found to be the most suitable gel-forming agents, which do not affect enzyme diffusion whereas polyacrylamide gels block lipase diffusion and therefore are not suitable for the assay. The optimized assay prepared from 1 % tributyrin emulsion in 2 % agar gel was tested with six microbial lipases and porcine pancreatic lipase. The detection limit is 20–60 ng/well which is equivalent to 30 μU/well for T. lanuginosus lipase.

Pressurised hot water extraction in continuous flow mode for thermolabile compounds: extraction of polyphenols in red onions

Extraction and analysis of labile compounds in complex sample matrices, such as plants, is often a big analytical challenge. In this work, the use of a “green and clean” pressurised hot water extraction (PHWE) approach performed in continuous flow mode is explored. Experimental data for extraction and degradation kinetics of selected compounds were utilised to develop a continuous flow extraction (CFE) method targeting thermolabile polyphenols in red onions, with detection by high-performance liquid chromatography (HPLC)–diode array detection (DAD)–mass spectrometry (MS). Water containing ethanol and formic acid was used as extraction solvent. Method performance was focused on extraction yield with minimal analyte degradation. By adjusting the flow rate of the extraction solvent, degradation effects were minimised, and complete extraction could be achieved within 60 min. The CFE extraction yields of the polyphenols investigated were 80–90 % of the theoretically calculated quantitative yields and were significantly higher than the yields obtained by conventional methanol extraction and static batch extraction (70–79 and 58–67 % of the theoretical yields, respectively). The precision of the developed method was lower than 8 % expressed as relative standard deviation.

Biotin-avidin-conjugated metal sulfide nanoclusters for simultaneous electrochemical immunoassay of tetracycline and chloramphenicol

We report on a protocol for a simultaneous competitive immunoassay for tetracycline (TC) and chloramphenicol (CAP) on the same sensing interface. Conjugates of TC and of CAP with bovine serum albumin were first co-immobilized on a glassy carbon electrode modified with gold nanoparticles. In parallel, monoclonal anti-TC and anti-CAP antibodies were conjugated onto CdS and PbS nanoclusters, respectively. In a typical assay, the immobilized haptens and the added target analytes competed for binding to the corresponding antibodies on the nanoclusters. Subsequently, Cd(II) and Pb(II) ions are released from the surface of the corresponding nanoclusters by treatment with acid and then were detected by square wave anodic stripping voltammetry. The currents at the peak potentials for Cd(II) and Pb(II) were used as the sensor signal for TC and CAP, respectively. This multiplex immunoassay enables the simultaneous determination of TC and CAP in a single run with dynamic ranges from 0.01 to 50 ng mL^?1 for both analytes. The detection limits for TC and for CAP are 7.5 pg mL^?1 and 5.4 pg mL^?1, respectively. No obvious nonspecific adsorption and cross-reactivity was observed in a series of analyses. Intra-assay and inter-assay coefficients of variation were less than 10 %. The method was evaluated by analyzing TC and CAP in spiked samples of milk and honey. The recoveries range from 88 % to 107 % for TC, and from 91 % to 119 % for CAP.

Determination of five quinolone antibiotic residues in foods by micellar electrokinetic capillary chromatography with quantum dot indirect laser-induced fluorescence

A new assay was developed for the determination of five quinolone antibiotic residues in foods, loxacin, enrofloxacin, ciprofloxacin, lomefloxacin, and norfloxacin, by micellar electrokinetic capillary chromatography with indirect laser-induced fluorescence, in which cadmium telluride quantum dots were used as a fluorescent background substance. Some factors that affected the peak height and the resolution were examined. The optimized running buffer was composed of 20 mM SDS, 7.2 mg/L quantum dots, and 10 mM borate at pH 8.8. The separation voltage was 20 kV. Under these conditions, five quinolone antibiotic residues were separated successfully within 8 min. The detection limits ranged from 0.003 to 0.008 mg/kg; the linear dynamic ranges were all 0.01?～?10 mg/kg; and the average recoveries of the spiked samples were 81.4?～?94.6 %. The assay can meet the requirement of maximum residue limits to these five quinolone antibiotics in the regulations of the European Union and Japan and has been applied for determining their residues in animal-derived food.

Simple optical determination of silver ion in aqueous solutions using benzo crown-ether modified gold nanoparticles

We describe a method for the modification of gold nanoparticles (Au-NPs) with benzo-15-crown-5 that led to the development of a colorimetric assay for Ag(I) ion. The brown color of a solution of the modified Au-NPs turns to purple on addition of Ag(I) ion. The ratio of the UV–vis absorption at 600 nm and 525 nm is proportional to the concentration of Ag(I) ions in the range from 20 to 950 nM, and the detection limit is 12.5 nM. Other metal ions do not interfere if present in up to millimolar concentrations. The method enables a rapid determination of Ag(I) in lake and drinking water and is amenable to bare-eye readout.

Fluorescent assay for oxytetracycline based on a long-chain aptamer assembled onto reduced graphene oxide

We report on a fluorescent assay for oxytetracycline (OTC) using a fluorescein-labeled long-chain aptamer assembled onto reduced graphene oxide (rGO). The π-π stacking interaction between aptamer and rGO causes the fluorescence of the label to be almost completely quenched via energy transfer so that the system has very low background fluorescence. The addition of OTC leads to the formation of G-quadruplex OTC complexes and prevents the adsorption of labeled aptamer on the surface of rGO. As a result, fluorescence is restored, and this effect allows for a quantitative assay of OTC over the 0.1–2 μM concentration range and with a detection limit of 10 nM. This method is simple, rapid, selective and sensitive. It may be applied to other small molecule analytes by applying appropriate aptamers.