Development of a disposable amperometric immunosensor for the detection of ecstasy and its analogues using screen-printed electrodes

An amperometric immunosensor for the specific and simple detection of 3,4-methylenedioxyamphetamine (MDA) and its analogues, 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) in saliva and urine was developed. A direct competitive assay in which free analyte and horseradish peroxidase labelled species were simultaneously added to an immobilised polyclonal antibody was employed. Both MDA and MDMA could be labelled with the enzyme and the use of an MDMA-HRP tracer greatly enhanced the sensitivity of the assay. Amperometric detection was performed at +100 mV versus Ag/AgCl, using tetramethylbenzidine (TMB)/H₂O₂ as substrate. The antibody, raised specifically against the methylenedioxy moiety of an MDA-BSA immunogen allowed highly specific detection of these analogues with negligible cross-reactivity towards any other amphetamine related compounds. Total assay time was 45 min and the standard curve using MDA could be evaluated within the range 0.61-400 ng ml⁻¹ with corresponding limit of detection (LOD) of 0.36 and 0.042 ng ml⁻¹ for saliva and urine, respectively. The cross-reactivity pattern of the analytes was determined and showed the order of sensitivity increased with increased alkyl chain length (MDA < MDMA < MDEA). The overall performance of the sensor, working range, precision and sensitivity demonstrate its usefulness for rapid and direct measurement of methylenedioxy analogues of ecstasy in saliva and urine. The sensor has better specificity than any previous method for ecstasy, with greater sensitivity than ELISA methods, is less expensive/assay with an “easier to use” format than previous methods. The detection works in saliva or urine, eliminating requirement of blood sampling, with improved precision.     

Amperometric immunosensor for the detection of Vibrio cholerae O1 using disposable screen-printed electrodes.

A disposable amperometric immunosensor was studied for the rapid detection of Vibrio cholerae (V. cholerae), the causative agent of cholera, employing an indirect sandwich enzyme linked immunosorbent assay (ELISA) principle. Screen-printed electrodes (SPEs) were fabricated (by using commercial and homemade carbon inks), electrochemically characterized and the assay conditions were optimized for capturing antibodies and antigen. Whole cell lysate (WCL) of V. cholerae was used to raise antibodies in rabbits and mice. The antibodies raised against WCL of V. cholerae were found to be specific, and no cross reactivity was observed with other enteric bacteria. 1-Naphthyl phosphate was used as a substrate with the amperometric detection of its enzymatic hydrolysis product 1-naphthol at a potential of +400 mV vs. Ag/AgCl reference electrode. A comparison between the amperometric detection technique and the standard ELISA was made in terms of the total assay time, the amount of biological materials used and the sensitivity of detection. The minimum detection limit of the amperometric immunosensor for V. cholerae was found to be 10(5) cells/ml in 55 min, while ELISA detected 10(6) cells/ml in 4 h.     

Biomolecule-free,selective detection of clenbuterol based on disposable screen-printed carbon electrode

We report here the development of a selective clenbuterol sensor made of disposable screen-printed carbon electrode (SPCE) without the need of adding any biorecognition element. Good analytical performance was achieved through the proper function of both the oxygen functionalities and edge plane sites on the “preanodized” SPCE (SPCE*). It is the amino group of clenbuterol to effectively form hydrogen bond with the SPCE* to induce the adsorption of clenbuterol. The edge plane sites enhance the electron transfer process and further help the dimer formation of clenbuterol to generate electroactivity for analysis. Square wave voltammetry was applied to increase the detection sensitivity with a linear response in the range of 7–1000 ppb and a detection limit of 0.51 ppb (S/N = 3). In the real sample analysis, results observed were satisfactory with meat, human blood, and human urine. High reproducibility in sensor fabrication further favors the disposable purpose of applications.     

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 disposable, screen-printed electrode for the amperometric determination of azide based on the immobilization with catalase or tyrosinase.

A disposable, screen-printed electrode based on the immobilization of catalase or tyrosinase was developed to construct biosensors for the amperometric determination of azide. The determination principles for azide by these two methods are based on inhibiting the enzymatic consumption of an electrode-detectable substance (hydrogen peroxide or catechol) on an enzyme-immobilized electrode. Both of these methods show a sensitive detection range and a short measuring time.     

Rolling circle amplification based amperometric aptamer/immuno hybrid biosensor for ultrasensitive detection of Vibrio parahaemolyticus

Microchimica Acta - The authors describe an antibody-aptamer based hetero-sandwich amperometric biosensor for the foodborne pathogen Vibrio parahaemolyticus. Antibody on the surface of a gold...     

Direct detection of myoglobin in whole blood using a disposable amperometric immunosensor

An amperometric immunosensor for the rapid detection of myoglobin in whole blood was developed. Due to its rapid kinetics, myoglobin is a useful biochemical marker for the early assessment of acute myocardial infarction (AMI). A one-step indirect sandwich assay was employed using a polyclonal goat anti-human cardiac myoglobin antibody with monoclonal mouse anti-myoglobin and goat anti-mouse IgG conjugated to alkaline phosphatase (AP), as the detecting antibodies. The final sensor required 30 min for incubation. The standard curve was linear between 85 and 925 ng/ml. The intra- and inter-assay coefficients of variation were below 8%. No cross-reactivity of the antibodies was found with other cardiac proteins. The overall performance of the sensor, rapid analysis time, wide working range, good precision and specificity demonstrate its potential usefulness for early assessment of AMI.     

A disposable immunosensor based on gold colloid modified chitosan nanoparticles-entrapped carbon paste electrode

A new strategy is described to construct disposable electrochemical immunosensors for the assay of human immunoglobulin. It is based on a carbon paste electrode constructed from chitosan nanoparticles modified with colloidal gold. The stepwise assembly process of the immunosensor was characterized by means of cyclic voltammetry and electrochemical impedance spectroscopy. Assay conditions that were optimized included the amount of chitosan nanoparticles in the preparation of carbon paste electrode, antibody concentration, and the incubation time of the antibody immobilization. Using hexacyanoferrate as a mediator, the current change increased with the concentration of human immunoglobulin G. A linear relationship in the concentration range 0.3 to 120 ng mL^?1 was achieved, with a detection limit of 0.1 ng mL^?1 (S/N?=?3). The method combines the specificity of the immunological reaction with the sensitivity of the gold colloid amplified electrochemical detection, and it has potential application in clinical immunoassay.     

A sensitive amperometric immunosensor for alpha-fetoprotein based on carbon nanotube/DNA/Thi/nano-Au modified glassy carbon electrode

A novel amperometric immunosensor for the determination of alpha-fetoprotein (AFP) was constructed using films of multi-wall carbon nanotubes/DNA/thionine/gold nanoparticles (nano-Au). Firstly, multi-wall carbon nanotubes (MWCNT) dispersed in poly(diallydimethlammonium chloride) (PDDA) were immobilized on the nano-Au film which was electrochemically deposited on the surface of glassy carbon electrode. Then a negatively charged DNA film was absorbed on the positively charged PDDA. Subsequently, thionine was attached to the electrode via the electrostatic interaction between thionine and the DNA. Finally, the nano-Au was retained on the thionine film for immobilization of AFP antibody (anti-AFP). The modification process was characterized by cyclic voltammetry (CV) and scanning electron microscope (SEM). The factors possibly influenced the performance of the proposed immunosensors were studied in detail. Under optimal conditions, the proposed immunosensor exhibited good electrochemical behavior to AFP in a two concentration ranges: 0.01–10.0 and 10.0–200.0 ng/mL with a relatively low detection limit of 0.04 ng/mL at three times the background noise. Moreover, the selectivity, repeatability and stability of the proposed immunosensor were acceptable.     

An electrochemical immunosensor based on agarose hydrogel films for rapid determination of ractopamine

A novel label-free electrochemical immunosensor for rapid determination of ractopamine was constructed by incorporating ractopamine–bovine thyroglobulin antigen in agarose hydrogel films modified on a glassy carbon electrode. Cyclic voltammetry and differential pulse voltammetry were employed to investigate the electrochemical character of the immunosensor during different modified stages using a redox probe system of K₃Fe(CN)₆/K₄Fe(CN)₆. In the presence of polyclonal antibody against ractopamine, K₃[Fe(CN)₆] electron transfer is inhibited, presumably due to that antibody in solution could adsorb on the electrode surface modified antigen. A competitive immunoreaction system was applied to determinate the free ractopamine in phosphate buffer solution. The selected polyclonal antibody showed very high sensitivity and specificity for ractopamine, and was used for the detection and quantitative determination of trace amounts of ractopamine in spiked animal feeds.     

A label-free amperometric immunosensor for detection of zearalenone based on trimetallic Au-core/AgPt-shell nanorattles and mesoporous carbon

A novel label-free amperometric immunosensor is proposed for the ultrasensitive detection of zearalenone (ZEN) based on mesoporous carbon (MC) and trimetallic nanorattles (core/shell particles with movable cores encapsulated in the shells). The nanorattles are composed of special Au-core and imperfect AgPt-shell structure (Au@AgPt). The Au@AgPt nanorattles are loaded onto the MC by physical adsorption. The structure of the Au@AgPt nanorattles was characterized by using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Energy dispersive X-ray spectroscopy (EDS) confirmed the composition of the synthesized nanorattles. Compared with monometallic and bimetallic nanoparticles (NPs), Au@AgPt nanorattles show a higher electron transfer rate due to the synergistic effect of the Au, Ag and Pt NPs. MC further improves the sensitivity of the immunosensor because of its extraordinarily large specific surface area, suitable pore arrangement and outstanding conductivity. The large specific surface area of MC and MC@Au@AgPt were characterized by the BET method. ZEN antibodies are immobilized onto the nanorattles via Ag–NH₂ bonds and Pt–NH₂ bonds. Cyclic voltammetry and square wave voltammetry were used to characterize the recognizability of ZEN. Under optimum experimental conditions, the proposed immunosensor exhibited a low detection limit (1.7 pg mL⁻¹), a wide linear range (from 0.005 to 15 ng mL⁻¹) as well as good stability, reproducibility and selectivity. The sensor can be used in clinical analysis.     

A sandwich HIV p24 amperometric immunosensor based on a direct gold electroplating-modified electrode

Acquired immune deficiency syndrome (AIDS) is a severe communicable immune deficiency disease caused by the human immune deficiency virus (HIV). The analysis laboratory diagnosis of HIV infection is a crucial aspect of controlling AIDS. The p24 antigen, the HIV-1 capsid protein, is of considerable diagnostic interest because it is detectable several days earlier than host-generated HIV antibodies following HIV exposure. We present herein a new sandwich HIV p24 immunosensor based on directly electroplating an electrode surface with gold nanoparticles using chronoamperometry, which greatly increased the conductivity and reversibility of the electrode. Under optimum conditions, the electrochemical signal showed a linear relationship with the concentration of p24, ranging from 0.01 ng/mL to 100 ng/mL (R > 0.99), and the detection limit was 0.008 ng/mL. Compared with ELISA, this method increased the sensitivity by more than two orders of magnitude (the sensitivity of ELISA for p24 is about 1 ng/mL). This immunosensor may be broadly applied to clinical samples, being distinguished by its ease of use, mild reaction conditions, guaranteed reproducibility, and good anti-interference ability.     

An efficient label-free immunosensor based on ce-MoS2/AgNR composites and screen-printed electrodes for PSA detection

Journal of Solid State Electrochemistry - Label-free electrochemical immunosensors (EIs) are simple and sensitive in cancer diagnosis. Electrode materials which can offer a large specific area and...     

Rapid hydrolysis and electrochemical detection of trace carbofuran at a disposable heated screen-printed carbon electrode

A disposable heated screen-printed carbon electrode (HSPCE) is successfully fabricated. It demonstrates rapid responses to electrical heating and is easily elevated above the water boiling point by a high frequency alternating current. The temperature rise at the HSPCE was found to be strongly dependent on the square of the heating current and the electrode width. Carbofuran (CAF) could be rapidly hydrolyzed to carbofuran phenol at the HSPCE with raised temperature, and then determined at the same electrode at room temperature by differential potential voltammetry (DPV). The factors influencing the detection were examined, including pH, hydrolytic temperature and heating time. Under the optimum conditions, the detection linear range of CAF was from 4.0 x 10(-7) to 4.0 x 10(-4) mol L(-1) and the detection limit was 5.0 x 10(-8) mol L(-1) (S/N = 3). This method was successfully applied to the analysis of CAF residues in real samples (spiked water, soil and vegetables), and satisfactory recoveries were obtained.     

A disposable immunosensor for detection of 17beta-estradiol in non-extracted bovine serum

This paper reports the assembly of a disposable immunosensor based on the direct competitive enzyme-linked immunosorbent assay (ELISA), for simple and fast measurement of 17β-estradiol (17β-E₂) in bovine serum, using screen-printed electrodes (SPEs) and a Palm-Sens portable electrochemical detector. The immunosensor strip was assembled immobilising, by passive adsorption, anti-rabbit IgG onto the surface of the working SPE electrode. After the interaction between anti-rabbit IgG and rabbit anti-17β-E₂ polyclonal antibodies (PAb), the competition was performed using 17β-estradiol-alkaline phosphatase conjugate (17β-E₂-AP) synthesised in our laboratory. The enzymatic substrate used for signal generation was 1-naphthylphosphate and its conversion to an electroactive product (1-naphthol) was measured using differential pulse voltammetry (DPV). To develop a prototype for field measurements, the entire competitive protocol has been optimised directly in a blank non-extracted bovine serum.According to the new EU criteria established by the Commission Decision 2002/657/EC for qualitative and quantitative screening methods, the detection capability (CCβ), was determined. The CCβ value resulted below the action limit (40 pg mL⁻¹) fixed for 17β-E_2.Spiked and real samples were analysed using the electrochemical immunostrips obtaining precision values (relative standard deviation, R.S.D.%) ranging from 8.6 to 17.0% and a recovery (R%) from 88.2 to 120.0%.Results obtained on real samples were confirmed by liquid chromatography coupled on-line with tandem mass spectrometry (LC-MS/MS) using an atmospheric pressure chemical ionisation (APCI) source and a heated nebulizer (HN) interface; this is the method currently used to confirm illegal hormone administration for regulatory purposes. The disposable immunosensor appears suitable as a screening tool for field analysis of bovine serum estradiol.     

Copper-modified gold electrode specific for monosaccharide detection Use in amperometric determination of phenylmercury based on invertase enzyme inhibition

The electrochemical oxidation of mono- and disaccharides at various copper-modified electrodes is reported: glassy carbon modified at open circuit or by electrochemical deposition of copper, gold modified by electrochemical deposition, and at bulk copper electrodes. A comparative study of these four electrodes was made by linear sweep voltammetry and amperometry. The maximum oxidation peak separation between disaccharides and monosaccharides is about 200 mV. After optimization, amperometric determination of monosaccharides was done at +0.30 versus Ag/AgCl in 0.15 M NaOH at the copper-modified gold electrode.

Using the developed method, the enzymatic activities of invertase and β-galactosidase were determined through their reaction with sucrose and lactose, respectively. Validation was carried out by a spectrophotometric method based on 3,5-dinitrosalicylic acid, and it was shown that the proposed electrochemical method is more sensitive.

The analytical utility of the copper-modified gold electrode was tested for the determination of organic mercury. Addition of phenylmercury standards to the invertase solution caused a decrease in the enzyme activity, and allowed the determination of phenylmercury in pharmaceutical samples. The concentration has been determined in the 10–55 ng ml⁻¹ range.     

A disposable amperometric biosensor for rapid screening of anticholinesterase activity in soil extracts

A disposable amperometric biosensor for the determination of anticholinesterase activity in soil extracts is described. The sensitive membrane was obtained by co-crosslinking acetylcholinesterase and choline oxidase with bovine serum albumin using glutaraldehyde. The anticholinesterase activity of the soil extract was measured using chronoamperometry at 650 mV vs. Ag/AgCl to monitor the biocatalytically produced H2O2 before and after the inhibition step. An inhibition percentage of 38 +/- 4% was recorded for soil extracts spiked with 10 ppb of ethyl parathion. The device has the potential to be used as a gross sensor for the assessment of anticholinesterase activity in soil extracts.     

Indirect determination of alkaline phosphatase based on the amperometric detection of indigo carmine at a screen-printed electrode in a flow system.

Amperometric analysis of indigo carmine at a bare screen-printed electrode placed in an FIA system is reported. This compound is easily detected at a potential of -0.3 V (vs. Ag pseudo-reference electrode) without observing any fouling of the electrode surface, thus allowing the repetitive use of the same electrode in a reproducible manner (coefficients of variation down to 7% for more than 20 consecutive determinations). A linear range of three orders of magnitude and a limit of detection in the sub-micromolar range were attained for this molecule. Based on these studies, indirect amperometric measurements of alkaline phosphatase (ALP) activity in solution were easily carried out using 3-indoxyl phosphate substrate. Its hydrolysis catalyzed by ALP gave rise to indigo product. This product is insoluble in aqueous solutions but it was easily converted into its soluble parent compound, indigo carmine, by addition of fuming sulfuric acid to the reaction media. Using this approach, we achieved a linear range of more than one order of magnitude and a limit of detection of 1 U/l ALP, for an enzymatic reaction time of 60 min.