Development of a quantum dot-based fluorescent immunoassay for progesterone determination in bovine milk

The use of semiconductor quantum dots (QDs) as fluorescent labels to develop a competitive immunoassay for sensitive detection and quantification of progesterone in cow's milk is described. Colloidal water-soluble CdSe/ZnS QDs are conjugated to an antigen derivative (progesterone-BSA conjugate) and a simple methodology is optimised to determine the antigen concentration in the final bioconjugate. The obtained QD-linked antigens were then employed together with unlabelled anti-progesterone monoclonal antibodies, as the biological recognition elements, in the development of the quantitative QDs-based fluorescent immunoassay for progesterone in bovine milk. After optimization, the developed immunoassay proved to cover a progesterone concentration range from 0.3 to 14.5 ng/mL in cow milk. Milk samples were just diluted 10-fold with deionised water and directly analysed with the proposed immunoassay, without additional sample pre-treatment or analyte extraction. The minimum detectable level (IC(10)) of the developed immunoassay turned out to be 0.1 ng/mL of progesterone in bovine milk. The sensitivity (IC(50)) achieved was 2.2 ng/mL with a reproducibility of 3.5% RSD as obtained from the results of the analysis of the triplicate of same samples but in three different days. Applicability of the proposed methodology was evaluated by analyzing cow's milk samples enriched with known concentrations of progesterone and recoveries better than 90% were achieved.     

Simultaneous detection of multiple chemical residues in milk using broad-specificity antibodies in a hybrid immunosorbent assay

In this study, a novel immunoassay using 2 types of sensors (QDs and an enzyme) were simultaneously used for detecting multiple structurally different molecules in milk. The method integrates the fluorescence-linked immunosorbent assay (FLISA) using QD605 and QD655 as probes and an enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase (HRP) labeled secondary antibody. The FLISA was produced by anti-sulfonamide and anti-quinolone broad-specificity monoclonal antibodies (MAbs) for simultaneously detecting 6 sulfonamides and 11 quinolones. Combined with the FLISA, an ELISA was utilized for detecting melamine from the same milk samples. The cross-reactivity of the MAbs was retained while binding the QDs by using avidin and a secondary antibody as bridges. Milk samples were detected using this hybrid immunoassay, with limits of detection (LOD) of the quinolones (0.18 ng mL(-1)), sulfonamides (0.17 ng mL(-1)) and melamine (7.5 ng mL(-1)), respectively. The results demonstrated that the detection limits of the integrated methods were better than required and simplified the sample pretreatment process. The developed immunoassay is suitable for high-throughput screening of low-molecular weight contaminants.     

Development of a TIRF-based biosensor for sensitive detection of progesterone in bovine milk

A total internal reflectance fluorescence (TIRF)-based biosensor for progesterone in bovine milk was developed and tested by measuring the progesterone level in daily milk samples for 25 days, covering a whole estrus cycle. The detection is based on total internal reflectance fluorescence. The assay has been designed as a binding-inhibition test with a progesterone derivative covalently immobilized on the sensor surface and a monoclonal anti-progesterone antibody as biological recognition element. First an existing progesterone assay was optimized by reducing the assay time per measurement, resulting in an assay time of about 5 min and reaching a limit of detection (LOD) of 0.04 ng mL(-1) and a quantification limit (LOQ) of 0.34 ng mL(-1). After calibration the assay was tested by measuring the progesterone level in daily milk samples over several weeks. An estrus cycle of a cow could be measured. As results become available within minutes without any preparation or pre-concentration of the milk samples the fully automated TIRF-based biosensor for progesterone can be used in-line in the milking parlor and thus could be an important tool for reproductive management of dairy cattle detecting heat and predicting pregnancy, which are critical parameters in milk production.     

Enzyme immunoassay of ampicillin in milk

An indirect immunoassay for quantitative determination of ampicillin (range, 10-1000 ng/ml) in buffer or milk has been developed. Polyclonal antibodies were obtained against ampicillin conjugated with bovine serum albumin; the conjugate was synthesized by direct condensation using carbodiimide. The antibodies were specific for ampicillin and exhibited low cross-reactivity to other penicillins (azlocillin, 17%; penicillin G, 10%; piperacillin, 5%; and carbenicillin, 4%). Matrix effects were minimized by combining the use of a casein-supplemented buffer (content of casein, 1%) with sample dilution. The threshold of ampicillin detection in milk (diluted tenfold) was equal to 5.0 ng/ml (which corresponded to 50 ng/ml of the original sample).     

Enzyme-Linked Immunosorbent Assay of Ampicillin in Milk

An indirect immunoassay for quantitative determination of ampicillin (range, 10–1000 ng/ml) in buffer or milk has been developed. Polyclonal antibodies were obtained against ampicillin conjugated with bovine serum albumin; the conjugate was synthesized by direct condensation using carbodiimide. The antibodies were specific for ampicillin and exhibited low cross-reactivity to other penicillins (azlocillin, 17%; penicillin G, 10%; piperacillin, 5%; and carbenicillin, 4%). Matrix effects were minimized by combining the use of a casein-supplemented buffer (content of casein, 1%) with sample dilution. Limit of detection for ampicillin in milk (diluted tenfold) was equal to 5.0 ng/ml (which corresponded to 50 ng/ml of the original sample).     

Monoclonal antibody technology for mycotoxins

Specific monoclonal antibodies (MABs) against aflatoxins, ochratoxin A, zearalenone, diacetoxyscirpenol and T-2 toxin have been prepared in various laboratories by the application of hybridoma technology to mycotoxins. These antibodies can be selected for sensitivity, reduced cross-reactivity, reliability and ease of production. When a suitable antibody is chosen it can then be used in a rapid immunological method such as an enzyme-linked or radio-immunoassay or immunoaffinity chromatography system. These assays have a lower limit of mycotoxin detection in the ng/ml range and have been applied to the determination of mycotoxins in samples such as maize, peanuts, peanut butter, milk and porcine kidneys. Using these immunoassay techniques, sample preparation has generally been simplified to a matter of solvent extraction of mycotoxins from the sample followed by dilution; under these conditions, levels of 1-5ug of mycotoxins/kg of sample can be found. The application and advantages of MABs to mycotoxins and the use of these antibodies in various assay techniques is discussed.     

Quantification of strontium in human serum by ICP-MS using alternate analyte-free matrix and its application to a pilot bioequivalence study of two strontium ranelate oral formulations in healthy Chinese subjects

A rapid, sensitive and accurate ICP-MS method using alternate analyte-free matrix for calibration standards preparation and a rapid direct dilution procedure for sample preparation was developed and validated for the quantification of exogenous strontium (Sr) from the drug in human serum. Serum was prepared by direct dilution (1:29, v/v) in an acidic solution consisting of nitric acid (0.1%) and germanium (Ge) added as internal standard (IS), to obtain simple and high-throughput preparation procedure with minimized matrix effect, and good repeatability. ICP-MS analysis was performed using collision cell technology (CCT) mode. Alternate matrix method by using distilled water as an alternate analyte-free matrix for the preparation of calibration standards (CS) was used to avoid the influence of endogenous Sr in serum on the quantification. The method was validated in terms of selectivity, carry-over, matrix effects, lower limit of quantification (LLOQ), linearity, precision and accuracy, and stability. Instrumental linearity was verified in the range of 1.00–500 ng/mL, corresponding to a concentration range of 0.0300–15.0 μg/mL in 50 μL sample of serum matrix and alternate matrix. Intra- and inter-day precision as relative standard deviation (RSD) were less than 8.0% and accuracy as relative error (RE) was within ±3.0%. The method allowed a high sample throughput, and was sensitive and accurate enough for a pilot bioequivalence study in healthy male Chinese subjects following single oral administration of two strontium ranelate formulations containing 2 g strontium ranelate.     

A very simple high‐performance liquid chromatographic method with fluorescence detection for the determination of gemifloxacin in human breast milk

A high‐performance liquid chromatographic method with fluorescence detection was developed and validated for the determination of gemifloxacin in human breast milk. The proposed method allows the determination of gemifloxacin in breast milk samples without complex sample preparation. The samples were mixed with a mobile phase and filtered with a 0.45 µm polytetrafluoroethylene filter before analysis. Chromatographic separation was carried out on a C18 column (150 × 4.6 mm, 5 µm I.D.) using methanol:50 mM ortho‐phosphoric acid solution (40:60) as the mobile phase with a 1.0 mL/min flow rate. Quantitation was performed using fluorescence detection with an excitation wavelength at 272 nm and an emission wavelength at 395 nm. The linear range was found to be 0.1–2.5 µg/mL. The method was applied successfully for the determination of gemifloxacin in breast milk obtained from a breastfeeding mother after oral administration of a single tablet that included 320 mg gemifloxacin per gemifloxacin tablet. Copyright © 2015 John Wiley & Sons, Ltd.     

Fluorescence quenching investigation on the interaction of glutathione‐CdTe/CdS quantum dots with sanguinarine and its analytical application

Water‐soluble glutathione (GSH)‐capped core/shell CdTe/CdS quantum dots (QDs) were synthesized. In pH 5.4 sodium phosphate buffer medium, the interaction between GSH‐CdTe/CdS QDs and sanguinarine (SA) was investigated by spectroscopic methods, including fluorescence spectroscopy and ultraviolet‐visible absorption spectroscopy. Addition of SA to GSH‐CdTe/CdS QDs results in fluorescence quenching of GSH‐CdTe/CdS QDs. Quenching intensity was in proportion to the concentration of SA in a certain range. Investigation of the quenching mechanism, proved that the fluorescence quenching of GSH‐CdTe/CdS QDs by SA is a result of electron transfer. Based on the quenching of the fluorescence of GSH‐CdTe/CdS QDs by SA, a novel, simple, rapid and specific method for SA determination was proposed. The detection limit for SA was 3.4 ng/mL and the quantitative determination range was 0.2–40.0 µg/mL with a correlation coefficient of 0.9988. The method has been applied to the determination of SA in synthetic samples and fresh urine samples of healthy human with satisfactory results. Copyright © 2013 John Wiley & Sons, Ltd.     

Investigation of dual-layer membrane cloaking method by surface plasmon resonance for direct chronoamperometric immunoassay of serum sample

A "dual-layer membrane cloaking" (DLMC) method was developed to construct disposable electrochemical immunosensor for direct determination of serum sample. Mouse IgG (MIgG) molecules were firstly immobilized on a substrate. After the formation of a didodecyldimethylammonium bromide (DDAB) membrane on the MIgG modified substrate, an additional bovine serum albumin (BSA) thin layer was formed to build a BSA/DDAB dual-layer membrane (DLM). When alkaline phosphatase conjugated anti-mouse IgG antibodies (anti-MIgG-ALP) in human serum were incubated on the substrate, anti-MIgG-ALP was recognized specifically by the immobilized MIgG while all nonspecifically adsorbed proteins were selectively removed together with BSA/DDAB DLM by 5% Triton X-100 (v/v) before final measurements. The BSA/DDAB DLM was characterized and optimized by surface plasmon resonance (SPR) technique, and further employed in a disposable immunoassay based on an ITO chip. Under optimal conditions, MIgG in human serum was directly detected in the range of 2.0-18.0 ng mL(-1) without dilution or separation. A limit of detection as low as 0.922 ng mL(-1) (6.15 pM) was obtained. The proposed DLMC method can efficiently prevent the penetration of matrix proteins through single cloaking membrane and completely eliminate nonspecific adsorption. It has great potential in providing a versatile way for direct determination of serum sample with ultra-sensitivity.     

Direct determination of selenium and other trace elements in serum samples by ICP-MS.

Selenium belongs to a group of trace elements of special interest in biological samples for clinical diagnosis. Selenium has antioxidizing functions and is essential for providing the organism with triiodothyronine produced from thyroxine. Among several analytical techniques used to determine the Se concentration in serum, Inductively Coupled Plasma Mass Spectrometry (ICP-MS) has been used in the past because of its high sensitivity. Interference problems originating from different ions on the major Se isotopes have been described to be a limiting factor for the direct determination of Se in these matrices. Standard addition calibration or isotope dilution is often required to overcome carbon-enhanced ionisation effects in biological sample matrices. In most cases, the typical serum sample volume which is available for the analysis is limited to 0.5 ml or less, making multiple sample preparation for standard addition calibration impractical. Isotope dilution requires enriched isotopes and substantial sample preparation. Furthermore, the approximate Se concentration in every sample has to be known to adjust the appropriate amount of spike to each sample. Matrix matching with methanol has been described to overcome ionisation effects but we found limiting factors of this application when other trace elements are also determined within one sample run. This paper describes an effective sample preparation method which allows the direct determination of Se in serum without limiting the analytical capabilities for the additional determination of Al, Cu, Ni, Co, Cd, Mn and Zn in a single sample run by ICP-MS. Optimization procedures are presented and results of the analysis of reference samples are discussed, with a comparison of more than 150 serum data with those obtained by the GF-AAS method.     

Rapid method for detection of Salmonella in milk by surface plasmon resonance (SPR)

The Plasmonic surface plasmon resonance (SPR) device was used to develop a rapid, simple and specific immunoassay for detection of Salmonella in milk. Rapid detection of Salmonella contamination is a major challenge for the food industry. Salmonella contamination is well known in all foods including pasteurised milk. The SPR assay was developed as a sandwich model using a polyclonal antibody against Salmonella as capture and detection antibody. Milk spiked with Salmonella typhimurium cells, killed by thimerosal (1%, w/w) treatment was used. Using the Plasmonic SPR assay it was possible to detect S. typhimurium down to a concentration of 1.25 x 10(5) cells ml(-1) in both milk and buffer system. The results obtained are comparable with existing, approved rapid Salmonella detection techniques. No negative effects on the sensitivity of the assay are encountered due to the milk matrix. Hence, no sample preparation or clean-up steps are required. The sample volume requirement for the assay is only 10 microl. Using the assay S. typhimurium was detected in milk within 1h, whereas the cultural techniques require 3-4 days for presumptive positive isolates and further time for confirmation. The rapid tests require at least 24h for the results. The Plasmonic SPR device operates on the Kretschmann configuration and is a cuvette-based system with the advantage of having eight channels on one single SPR chip.     

Analysis of amphetamines and metabolites in urine with ultra performance liquid chromatography tandem mass spectrometry

A simple, rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry method was developed and fully validated for the quantitative determination of seven amphetamines and metabolites in urine. The method was validated for selectivity, linearity, LOQ, LOD, imprecision, bias, analyte and processed sample stability, matrix effect, recovery, carryover and dilution integrity. A classic liquid–liquid extraction with ethyl acetate was used as sample preparation procedure. The compounds were separated on an Acquity UPLC HSS C₁₈ column in 6.8 min. The linear dynamic range was established from 25 to 500 ng/mL. The limit of quantification was fixed to the lowest calibrator level and the limit of detection ranged from 0.125 to 2.5 ng/mL. The method presented an excellent intra- and inter-assay imprecision and bias (<10.7%) at each measured concentration of two external quality controls (QC) and three “in house” QC. No matrix effects were observed and good recoveries (>70%) were obtained for all the compounds. No carryover was observed after the analysis of high concentrated samples (8000 ng/mL). The method was subsequently applied to authentic samples.     

Quantitative analysis of the response of an electrochemical biosensor for progesterone in milk

An electrochemical biosensor for progesterone in cow's milk was developed and used in a competitive immunoassay by Hart et al. (1977, Studies towards a disposable screenprinted amperometric biosensor for progesterone, Biosens. Bioelectron. 12, 1113-1121). The sensor was fabricated by depositing anti-progesterone monoclonal antibody (mAb) onto screen-printed carbon electrodes (SPCEs) which were coated with rabbit anti-sheep IgG (rIgG). This sensor was operated following the steps of competitive binding between sample and conjugate (alkaline-phosphatase-labelled progesterone) for the immobilised mAb sites and measurements of an amperometric signal in the presence of p-nitrophenylphosphate using either colorimetric assays or cyclic voltammetry. The hook effect of the progesterone biosensor was found in the concentration range of milk progesterone between 0 and 5 ng/ml when the sensor was fabricated using a loading of 25 ng rIgG per electrode of a diameter of 3 mm and a 1/50 dilution of mAb. A computer model has been developed in this study to simulate the operation of this progesterone biosensor with consideration of the fabrication processes. This paper presents the results of validating the computer model and the model has predicted the hook effect as observed in tests. The model thus reveals that the hook effect is determined by the total number of binding sites available and the rates of labelled and unlabelled progesterone diffusing towards the sensor surface and the binding rates.     

Optimization of the trace element determination by ICP-MS in human blood serum

The ICP-MS for simultaneous trace element determination in human blood has prevailed as the most suitable methodology for clinical aims because of its rapidity, detection limits and minimal sample quantity needed for analysis.As the proteic matrix is high, it is necessary to fine-tune the ICP-MS Agilent 7500i with autosampler CETAC ASX-500 and ISIS System connected, and further we have to the sample pre-treatment in order to obtain good results.The study of the results shows that the best pre-treatment for blood serum samples consists of a basic treatment by 1/10 dilution with a solution of EDTA and NH₄OH, with a detection limit of the order of μg/L and a reduction of the necessary patient sample volume to 2 mL.     

Characterization of the caprine estrous cycle using enzyme immunoassay for the determination of whole blood progesterone concentration

Eighteen mature female dairy goats were used to determine the feasibility of enzyme immunoassay for the measurement of progesterone in this species. Both quantitative and qualitative enzyme immunoassay kits were used to measure progesterone concentration in unextracted whole blood. Progesterone profiles were similar to those previously reported using either protein-binding or radioimmunoassay as the test. A Pearson's correlation coefficient comparison of our enzyme immunoassay values with radioimmunoassay values gave a correlation coefficient of 0.95. Using the qualitative test, 100% of the samples with high progesterone concentrations had quantitative values greater than 4.00 ng/ml progesterone with a mean of 12.13 ng/ml. Estrus samples had a mean progesterone concentration of 0.70 ng/ml.     

Spectrofluorimetric determination of amisulpride and bumidazone in raw materials and tablets

A highly sensitive, simple and rapid spectrofluorimetric method was developed for the determination of amisulpride (AMS) and bumidazone (BUM) in tablet form. The proposed method is based on measuring the native fluorescence of the studied drugs in methanol at 360 and 344 nm after excitation at 276 and 232 nm for AMS and BUM, respectively. The fluorescence–concentration plots were rectilinear over the ranges of 5.0–60.0 ng/mL for AMS and 0.5–5.0 µg/mL for BUM. The lower detection limits were 0.70 ng/mL and 0.06 µg/mL, and the lower quantification limits were 2.0 ng/mL and 0.18 µg/mL for AMS and BUM, respectively. The method was successfully applied for the analysis of AMS and BUM in commercial tablets. Statistical evaluation and comparison of the data obtained using the proposed and comparison methods revealed good accuracy and precision for the proposed method. Copyright © 2014 John Wiley & Sons, Ltd.     

Preparation and Biological Effect of Nucleotide-Capped CdSe/ZnS Quantum Dots on Tetrahymena thermophila

In this paper, we described the preparation and characterization of different types of modified CdSe/ZnS quantum dots (QDs) and explored the biological effects of QDs with different surface modifications on the whole growth of unicellular protozoan Tetrahymena thermophila BF(5) using a thermal activity monitor air isothermal microcalorimeter. Our results demonstrated that adenosine 5'-monophosphate (AMP) showed stronger interaction with QDs than other types of nucleotide. AMP-QDs could stimulate the growth of T. thermophila while mercaptoacetic acid-capped CdSe/ZnS quantum dots inhibited it. In addition, the population density determination and fluorescence imaging of T. thermophila BF(5) also confirmed the results obtained from microcalorimetry. It is believed that this approach will provide a more convenient methodology for the kinetics and thermodynamics of microorganism when coexisting with QDs in real time, and all of which are very significant to understanding the effect of QDs to organism.     

Paper supported immunosensor for detection of antibiotics

Paper supports were used to develop a simple, inexpensive, fast and sensitive electrochemical immunosensor for the analysis of antibiotic residues in milk samples, where single-walled carbon nanotubes (SWNTs) and a simple dip-dry coating method were employed to prepare the highly sensitive biosensor. Well-dispersed SWNTs were impregnated with an antibody against neomycin to obtain a composite coating solution, followed by dipping the filtration paper in the solution to fabricate the sensitive biosensor which had high electrical conductivity. Based on the impedance change in the entire paper supported biosensor with increased concentrations of neomycin, the limit detection of the optimized method was 0.04 ng mL(-1) and a linear detection range from 0.2 to 125 ng mL(-1), well below the European Union regulations for neomycin in this matrix. This paper supported biosensor was applied to determine neomycin in milk samples after a simple sample treatment, with spiked recoveries which ranged from 93.25 to 110.47%. A variety of antibiotic residues in milk samples could be determined following similar sensor preparation.     

Flow injection fluorescence immunoassay for gentamicin using sol-gel-derived mesoporous biomaterial

Sol-gel-derived mesoporous biomaterials were used for the first time in the flow-injection fluorescence immunoassay system. Anti-gentamicin antibody was immobilized in a mesoporous sol-gel material using tetramethoxysilane as a precursor and poly(ethylene glycol) as a template. The sol-gel glass was used to develop an immunoaffinity column for the flow-injection immunoassay of gentamicin. Little unspecific adsorption of gentamicin on the sol-gel and no antibody leaching under harsh elution conditions were found. The immunoassay is based on the competition between gentamicin and fluorescein isothiocyanate-labeled gentamicin for a limited number of encapsulated antibody binding sites. NaOH solution of 5 x 10(-3)mol/L is used for the regeneration of encapsulated antibody binding sites after each measurement, which allows the immunoreactor to be used for up to 20 times without any loss of reactivity. Sample preconcentration is not needed and a single assay can be performed within 10 min. The calibration for gentamicin has a working range of 250-5000 ng/mL with a detection limit of 200 ng/mL, which is close to that of the fluorescence immunoassay and fluorescence polarization immunoassay using the same reactants. Comparison of the results from this method with that obtained from HPLC showed an excellent correlation.