Nanoelectrode ensembles as recognition platform for electrochemical immunosensors

In this study we demonstrate the possibility to prepare highly sensitive nanostructured electrochemical immunosensors by immobilizing biorecognition elements on nanoelectrode ensembles (NEEs) prepared in track-etch polycarbonate membranes. The gold nanodisk electrodes act as electrochemical transducers while the surrounding polycarbonate binds the antibody-based biorecognition layer. The interaction between target protein and antibody is detected by suitable secondary antibodies labelled with a redox enzyme. A redox mediator, added to the sample solution, shuttles electrons from the nanoelectrodes to the biorecognition layer, so generating an electrocatalytic signal. This allows one to fully exploit the highly improved signal-to-background current ratio, typical of NEEs. In particular, the receptor protein HER2 was studied as the target analyte. HER2 detection allows the identification of breast cancer that can be treated with the monoclonal antibody trastuzumab. NEEs were functionalized with trastuzumab which interacts specifically with HER2. The biorecognition process was completed by adding a primary antibody and a secondary antibody labelled with horseradish peroxidase. Hydrogen peroxide was added to modulate the label electroactivity; methylene blue was the redox mediator generating voltammetric signals. NEEs functionalized with trastuzumab were tested to detect small amounts of HER2 in diluted cell lysates and tumour lysates.     

Ultrasensitive detection of adrenocorticotropin hormone (ACTH) using disposable phenylboronic-modified electrochemical immunosensors

This work reports for the first time an electrochemical immunosensor for the determination of adrenocorticotropin hormone (ACTH). The immunoelectrode design involves the use of amino phenylboronic acid for the oriented immobilization of anti-ACTH antibodies onto screen-printed carbon modified electrode surfaces. A competitive immunoassay between the antigen and the biotinylated hormone for the binding sites of the immobilized antibody was performed. The electroanalytical response was generated by using alkaline phosphatase-labelled streptavidin and 1-naphtyl phosphate as the enzyme substrate. The electrochemical oxidation of the enzyme reaction product, 1-naphtol, measured by differential pulse voltammetry was employed to monitor the affinity reaction. Under the optimized working conditions, an extremely low detection limit of 18 pg/L was obtained. Cross-reactivity was evaluated against other hormones (cortisol, estradiol, testosterone, progesterone, hGH and prolactin) and the obtained results demonstrated an excellent selectivity. The developed immunosensor was applied to a human serum sample containing a certified amount of ACTH with good results.     

Criteria to distinguish between natural situations and illegal use of boldenone, boldenone esters and boldione in cattle: 2. Direct measurement of 17β-boldenone sulpho-conjugate in calf urine by liquid chromatography–high resolution and tandem mass spectrometry

Boldenone is banned in the European Union (Directive 96/22/EC) as growth promoter for meat producing animals. Boldione (ADD), boldenone and boldenone esters (mainly the undecylenate form) are commercially available as anabolic preparations, either to the destination of human, horse or cattle. Since the late 90s, the natural occurrence of boldenone metabolites has been reported in cattle. According to EU regulation, the unambiguous demonstration of boldenone administration in bovine urine should be provided on the basis of boldenone identification in the corresponding conjugate fraction. An analytical method has been developed and validated according to current standards with main concern to the measurement of intact 17β-boldenone-sulphate. The analytical procedure included direct extraction–purification of target analyte on octadecylsilyl cartridges and direct detection of phase II metabolite by liquid chromatography (negative electrospray), tandem mass spectrometry (QqQ) or high resolution mass spectrometry (Orbitrap™). Decision limit (CCα) and detection capability (CCβ) were respectively 0.2 μg L⁻¹ and 0.4 μg L⁻¹ on triple quadrupole and 0.1 μg L⁻¹ and 0.2 μg L⁻¹ on hybrid system. The method was successfully applied to the analysis of incurred samples collected in different experiments. 17β-Boldenone-sulphate was measurable up to 36 h after oral administration of boldione, and 30 days after 17β-boldenone undecylenate intra-muscular injection. This conjugate form was never detected in non-treated animals, confirming its status of definitive candidate marker for boldenone administration in calf.     

Highly sensitive near-simultaneous assay of multiple "lean meat agent" residues in swine urine using a disposable electrochemiluminescent immunosensors array

Nowadays, β(2)-agonists are abused illegally as "lean meat agents" for food-producing animals, and cause increasing food-safety accidents in some countries. Due to their hazard to the human health, "lean meat agents" are banned in most countries and required to be routinely monitored. We herein report a disposable electrochemiluminescent immunosensors array for near-simultaneous assay of multiple β(2)-agonist residues in swine urine, by using ractopamine and salbutamol as the models. In this investigation, a screen-printed carbon electrodes array was assembled and acted as the substrate of the immunosensors array. Then the immunosensors array was constructed by site-selectively immobilizing the antigens of ractopamine and salbutamol on the working electrodes of array. After the competitive immuno-binding, with the aid of a homemade single-pore-four-throw switch, the electrochemiluminescent signals of the two β(2)-agonists were sequentially detected using a non-array detector. The limits of detection for ractopamine and salbutamol were 8.5 and 17pg/mL, respectively, which were much lower than those of the most previous reports. Compared with other routine methods based on chromatography and ELISA, this method is more suitable for screening of multiple β(2)-agonists in quantities of samples, owing to its merits of low cost, user-friendliness and high throughput, and shows great promise in food safety and agonist surveillance.     

Criteria to distinguish between natural situations and illegal use of boldenone, boldenone esters and boldione in cattle 1. Metabolite profiles of boldenone, boldenone esters and boldione in cattle urine

Boldenone is an androgenic steroid that improves the growth and food conversion in food producing animals. In most countries worldwide, this anabolic steroid is forbidden for meat production. Until recently, the control of its illegal use was based either on 17beta-boldenone or 17alpha-boldenone (its main metabolite in cattle) identification in edible tissues, hair, faeces or urine. Recent observations and data tend to demonstrate the natural occurrence (but not ubiquitous) in cattle of these steroids, making the analytical strategy of the control more complicated. We investigated the metabolism of boldenone in cattle after intramuscular and oral treatment of boldenone, boldenone esters and boldione. The central objective was to elucidate the structures of the main metabolites (phase I and phase II) in urine, with main objective to be further in position to compare boldenone urinary profiles of treated and non-treated animals. Nine metabolites have been identified, only four were present whatever the treatment and the administered boldenone source. Nevertheless, all of them have been detected at least once in non-treated animals which did not permit us to use them as biomarkers of an illegal treatment. At last, but not at least, all metabolites were found mainly glucuro-conjugated, and rarely sulfo-conjugated, with the only exception of 17beta-boldenone. Current investigations are showing the absence of 17beta-boldenone sulfoconjugate in non-treated animals; that would permit to distinguish non-treated from treated animals with boldione, boldenone and boldenone esters.     

Analysis of environmental biomarkers in urine using an electrochemical detector

Phenols are present in the environment and are prevalent in human populations, as environmental contaminants, dietary components, or their metabolites. Many are potential endocrine-altering agents. Currently available methods analyze single components or single families of chemicals as biomarkers of exposure. In order to assess multiple biologically relevant exposures to such substances, we evaluated the feasibility of determining several phenols simultaneously in urine, using an electrochemical detector (ECD) in combination with high performance liquid chromatography (LC). Based on reported analyses in the literature and the ECD response, we selected four xenobiotic residues, including three phytoestrogens (enterolactone, daidzein, and genistein) and bisphenolA [BPA]. These compounds had detection limits below 1 microg/L in urine using the cleanup procedure (glucuronidase hydrolysis and C18 column) and the urine volume (2 mL) we employed. As a pilot study to demonstrate the method's utility, we determined urinary enterolactone, daidzein, genistein and BPA in samples from nine children and 24 adults.     

A recombinant Fab fragment-based electrochemical immunosensor for the determination of testosterone in bovine urine

This work describes the development of an electrochemical, recombinant Fab fragment-based immunosensor for the detection of testosterone in bovine urine. The sensor comprised of a testosterone conjugate on the surface of screen-printed electrodes, and recognition followed by an anti-testosterone Fab fragment. The use of an IgG-horseradish peroxidase conjugate determined the degree of competition. Chronoamperometry at a potential of +100 mV, was chosen to reductively measure the product of the catalysis of 3,3',5,5'-tetramethylbenzidine catalysis. ELISA was primarily used to investigate the assay system, prior to transferring to SPEs. The final Fab-based sensor exhibited the linear range of 300-40,000 pg/ml with limit of detection of 90+/-13 pg/ml. Furthermore, the developed Fab sensor allowed for the determination of testosterone in bovine urine directly after dilution, omitting the necessity of extraction and hydrolysis. Comparison of administrated bovine urine samples between the developed Fab sensor and GC-MS data showed quantitative or semi-quantitative results and enabled identification of suspicious samples for further extensive analysis by established analytical techniques. With simple sample preparation, low limit of detection, and good repeatability, the proposed method can offer alternative advantages as a primary screening tool for meat quality control.     

Multi residue screening of intact testosterone esters and boldenone undecylenate in bovine hair using liquid chromatography electrospray tandem mass spectrometry

The abuse of esters of natural androgenic steroids in cattle fattening and sports is hard to control via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. In veterinary control strange findings of 17beta-testosterone and 17alpha-testosterone in urine are often ignored because of the lack of statistically sound reference data of naturally occurring levels. An interesting alternative for inconclusive urine analyses in veterinary control can be provided by the analysis of the administered steroids themselves, i.e. the analysis of intact steroid esters in hair. Unfortunately, the analysis of intact steroid esters is complicated not only by the vulnerability of the esters which precludes alkaline hydrolysis of the hair, but also by the wide polarity range of short and long-chain esters yielding very poor recoveries for either the one or the other. In this study, a multi-steroid esters LC/MS/MS screening method is presented for trace analysis of the synthetic intact esters of 17beta-testosterone and the undecylenate ester of 17beta-boldenone in bovine hair. The method, requiring only 200 mg of pulverised hair, features a mild digestion procedure using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and the use of four deuterium-labelled steroid esters as internal standards covering the wide polarity range of the analytes. In spiked hair samples for most of the analytes the limit of detection and the accuracy using isotope dilution were 2-5 ng/g and 97-105%, respectively. The applicability was demonstrated using hair samples from a controlled experiment in which six bovines were injected intramuscularly with two different doses of two commercial mixtures of testosterone esters, and with two different doses of boldenone undecylenate. Depending on the dose all administered testosterone- and boldenone esters were found to be incorporated in bovine hair following a single intramuscular injection, except testosterone propionate which dose might have been too low.     

Development of immunosensors for direct detection of three wound infection biomarkers at point of care using electrochemical impedance spectroscopy

A method for label-free, electrochemical impedance immunosensing for the detection and quantification of three infection biomarkers in both buffer and directly in the defined model matrix of mock wound fluid is demonstrated. Triggering Receptor-1 Expressed on Myeloid cells (TREM-1) and Matrix MetalloPeptidase 9 (MMP-9) are detected via direct assay and N-3-oxo-dodecanoyl-l-HomoSerineLactone (HSL), relevant in bacterial quorum sensing, is detected using a competition assay. Detection is performed with gold screen-printed electrodes modified with a specific thiolated antibody. Detection is achieved in less than 1h straight from mock wound fluid without any extensive sample preparation steps. The limits of detection of 3.3 pM for TREM-1, 1.1 nM for MMP-9 and 1.4 nM for HSL are either near or below the threshold required to indicate infection. A relatively large dynamic range for sensor response is also found, consistent with interaction between neighbouring antibody-antigen complexes in the close-packed surface layer. Together, these three novel electrochemical immunosensors demonstrate viable multi-parameter sensing with the required sensitivity for rapid wound infection detection directly from a clinically relevant specimen.     

Label electrochemical immunosensor for prostate-specific antigen based on graphene and silver hybridized mesoporous silica

Prostate-specific antigen (PSA), as the specificity of prostate cancer markers, has been widely used in prostate cancer diagnosis and screening. In this study, we fabricated an electrochemical immunosensor for PSA detection using the amino-functionalized graphene sheet–ferrocenecarboxaldehyde composite materials (NH₂-GS@FCA) and silver hybridized mesoporous silica nanoparticles (Ag@NH₂-MCM48). Under optimal conditions, the fabricated immunosensor showed a wide linear range with PSA concentration (0.01–10.0 ng·ml⁻¹). Low detection limit (2 pg·ml⁻¹) proved the high sensitivity. In addition, the immunosensor possessed good stability and reproducibility. Moreover, the application to PSA analysis in serum samples yielded satisfactory results.     

Direct detection of boldenone sulfate and glucuronide conjugates in horse urine by ion trap liquid chromatography-mass spectrometry

A study of the equine phase II metabolism of the anabolic agent boldenone is reported. Boldenone sulfate, boldenone glucuronide and their C17-epimers were synthesised as reference standards in our lab and a method was developed for their detection in a horse urine matrix. Solid phase extraction was used to purify the analytes, which were then detected by ion trap LC/MS. Negative and positive ionisation mode MS(2) were used for the detection of sulfate and glucuronide conjugates, respectively. Boldenone sulfate and 17-epiboldenone glucuronide were detected as the major and minor phase II metabolites, respectively, in horse urine samples collected following the administration of boldenone undecylenate by intramuscular injection.     

Detection of 2,4,6-trinitrotoluene in seawater using a reversed-displacement immunosensor

Reported in this study are the experimental design and results of an immunosensor for the detection of the explosive, 2,4,6-trinitrotoluene (TNT) in seawater using a reversed-displacement format. This reversed-displacement immunosensor methodology has successfully measured TNT in seawater by direct injection, eliminating the need for preconcentration or pretreatment of samples. A microcolumn containing an Affi-Gel resin derivatized with a 2,4,6-trinitrobenzene (TNB) moiety and a fluorophore-labeled anti-TNT antibody composed the immunoassay reactive chamber. Fluorophore-labeled anti-TNT antibody was incubated with the modified Affi-Gel resin until binding equilibrium was reached. Under a constant flow, samples containing TNT were introduced into the flow stream displacing the fluorophore-labeled TNT antibody. Limits of detection were 2.5ng/mL or part-per-billion (ppb) for TNT in saline buffer and 25ppb in seawater with an analysis time of 10 min. Two anti-TNT antibodies with differing binding affinities were compared in the reversed-displacement assay format, and a correlation between affinity and detection limits was observed. Furthermore, we have demonstrated that the reversed-displacement format can be used to screen seawater samples containing TNT, remains effective after dozens of cycles, and provides significant fluorescence response before regeneration is required.     

Development of a screen-printed carbon electrochemical immunosensor for picomolar concentrations of estradiol in human serum extracts

Investigations into the development of a prototype electrochemical immunosensor for estradiol (E(2)) are described. After optimising reagent loadings in a 96-well enzyme-linked immunosorbent assay (ELISA), antibodies (rabbit anti-mouse IgG and monoclonal mouse anti-E(2)) were immobilised by passive adsorption onto the surface of screen-printed carbon electrodes (SPCEs). A competitive immunoassay was then performed using an alkaline-phosphatase (ALP)-labelled E(2) conjugate. Calibration plots for E(2) buffer standards, performed colorimetrically on the SPCEs using a para-nitrophenyl phosphate substrate solution, were in good agreement with ELISA calibration plots. Electrochemical measurements were then performed using differential pulse voltammetry (DPV) following the production of 1-naphthol from 1-naphthyl phosphate. The calibration plot of DPV peak current versus E(2) concentration showed a measurable range of 25-500 pg/ml with a detection limit of 50 pg/ml. A coefficient of variation of between 13.0 and 15.6% was obtained for repeat measurements. The immunosensor was applied to the determination of E(2) in spiked serum, following an extraction step with diethyl ether. A mean recovery for the method of 102.5% was obtained with a CV of 19.1%. The options available for further development of the sensor regarding precision, limit of detection and direct sample analysis are discussed.     

Determination of isoflavones in soybean food and human urine using liquid chromatography with electrochemical detection

A highly sensitive high-performance liquid chromatographic method with electrochemical detection (HPLC-ED) was developed for the determination of isoflavones. Electrochemical behaviour of daidzein and genistein was studied on carbon paste electrode (CPE) by adsorptive transfer stripping square wave voltammetry. The obtained electrochemical results were used for the development of HPLC-ED method. Furthermore, isoflavones were separated on an Atlantis dC18 column using a mobile phase consisting of acetonitrile (solvent A) and 0.15M acetate buffer of pH 5.5 (solvent B) at a flow rate 0.4 mL/min. A linear gradient profile (solvent B) was at 0-2 min 87%; 22 min 60%; 27 min 50%; 31 min 45%; 47 min 87%. Full scan of multi-channel coulometric detection was tested and optimal potential at 450 mV was chosen for our purposes. Calibration curves were linear (daidzein R(2) = 0.9993 and genistein R(2) = 0.9987). The detection limit for daidzein/genistein was 480/394 pg/mL (1.8/1.5 nM) and per column 2.4/1.9 pg. Isoflavones extracted from soybean products (farina, meat, milk) by the accelerated solvent extraction (ASE) procedure and isoflavones present in human urine were determined by the HPLC-ED method.     

Comparative study of thiolated protein G scaffolds and signal antibody conjugates in the development of electrochemical immunosensors

To achieve a high efficiency of analyte capture by a capture antibody attached to an electrochemical immunosensor, we have immobilised an analyte-specific antibody on a self-assembled layer of recombinant Protein G that was thiolated with succinimidyl-6-[3'-(2-pyridyldithio)-propionamido] hexanoate (LC-SPDP). Then two techniques were employed for conjugating a second antigen-specific antibody to alkaline phosphatase (mAb2-AP) using either LC-SPDP or the biotin-streptavidin interaction as the mode of cross-linking the antibody and enzyme. After characterising the two mAb2-AP preparations (mAb2-(LC-SPDP)-AP and mAb2-(Biotin-SA)-AP), they were each used as the signal antibody for immunosensors formatted for two-site immunoassays where the capture antibody was attached to a Protein G-(LC-SPDP) scaffold on gold electrodes. The antibodies and assays were specific for the clinically important hormone, human chorionic gonadotrophin (hCG). Protein G-(LC-SPDP) provided a stable scaffold, while mAb2-(LC-SPDP)-AP and mAb2-(Biotin-SA)-AP performed well as the signal antibodies. Immunosensors with mAb2-(Biotin-SA)-AP were characterised by a limit of detection of 216 I UL(-1) for hCG and a linear response up to approximately 2000 I UL(-1). Conversely, immunosensors with mAb2-(LC-SPDP)-AP exhibited a limit of detection of 240 I UL(-1) and a linear response up to 4000 I UL(-1).     

Chronopotentiometry and Faradaic impedance spectroscopy as signal transduction methods for the biocatalytic precipitation of an insoluble product on electrode supports: routes for enzyme sensors, immunosensors and DNA sensors

The biocatalyzed precipitation of an insoluble product produced on electrode supports is used as an amplification path for biosensing. Enzyme-based electrodes, immunosensors and DNA sensors are developed using this biocatalytic precipitation route. Faradaic impedance spectroscopy and chronopotentiometry are used as transduction methods to follow the precipitation processes. While Faradaic impedance spectroscopy leads to the characterization of the electron-transfer resistance at the electrode, chronopotentiometry provides the total resistance at the interfaces of the modified electrodes. A horseradish peroxidase, HRP, monolayer-functionalized electrode is used to sense H₂O₂ by the biocatalyzed oxidation of 4-chloro-1-naphthol (1), to the insoluble product benzo-4-chlorohexadienone (2). An antigen monolayer electrode is used to sense the dinitrophenyl antibody, DNP-Ab, applying an anti-antibody–HRP conjugate as a biocatalyst for the oxidative precipitation of 1 by H₂O₂ to yield the insoluble product 2. An oligonucleotide (3) functionalized monolayer electrode is used to sense the DNA-analyte (4), that is one of the Tay–Sachs genetic disorder mutants. Association of a biotin-labeled oligonucleotide to the sensing interface, followed by the association of the avidin–HRP conjugate and the biocatalyzed precipitation of 2 leads to the amplified sensing of 4. The amount of the precipitate accumulated on the conductive support is controlled by the concentration of the respective analytes and the time intervals employed for the biocatalytic precipitation of 2. The electron-transfer resistances of the electrodes covered by the insoluble product (2) are derived from Faradaic impedance measurements, whereas the total electrode resistances are extracted from chronopotentiometric experiments. A good correlation between the total electrode resistances and the electron-transfer resistances at the conducting supports are found. Chronopotentiometry is suggested as a rapid transduction means (a few seconds). The precautions needed to apply chronopotentiometry in biosensors are discussed.     

Screening for oestrogenic activity of plant and food extracts using in vitro trout hepatocyte cultures

The use of in vitro trout hepatocyte cultures is shown to provide a simple and effective way to screen plant and food products for oestrogenic activity. The relative oestrogenic activities of 0.1 g each of extracts of phytosterol, soy isoflavone, red clover, kudzu and soybean extracts were determined using this assay and found to be equivalent to 212, 1, 3.2, 132 and 1025 nM of 17beta-estradiol, respectively. Controls were performed on soybean and kudzu extracts using specific ELISAs for isoflavones and these confirmed the validity of the cell culture assay. The method described offers an advantage over current methods in that it can detect increased oestrogenic activity that may occur as a result of metabolic activation of pre- or pro-oestrogens liver cells.     

幽门螺旋杆菌单链抗体的筛选及鉴定

为了获得幽门螺旋杆菌特异性单链抗体scFv,通过噬菌体展示技术,首次直接用幽门螺旋杆菌细胞Hp对噬菌体单链抗体文库Tomlinson进行单链抗体的筛选,经5轮筛选后,通过ELISA方法检测,从随机挑选的96个克隆中获得了8株阳性克隆。再分别将阳性克隆与10种常见菌进行ELISA的交叉反应,最终得到1株特异性表达抗Hp的scFv的噬菌体JH1。随后又进一步对JH1所表达的scFv基因进行PCR扩增,分别得到scFv的VH片段、VL片段,全长基因分别为527bp、368bp和935bp,这些包含着部分载体序列的DNA片段与理论值相符。通过对scFv全长基因进行测序,在NCBI中进行基因序列比对,与已报道的一种植物RNA病毒的复制酶单链抗体基因序列有96％同源性。