One-step immobilization of antibodies for α-1-fetoprotein immunosensor based on dialdehyde cellulose/ionic liquid composite

A novel immunosensor for α-1-fetoprotein based on dialdehyde cellulose/ionic liquid composite film as a matrix has been developed. Microcrystalline cellulose was activated by sodium metaperiodate to produce dialdehyde cellulose. Antibodies can be immobilized on the electrode by a one-step method through covalent bonding of the aldehyde groups of dialdehyde cellulose with the amino groups of antibodies, in which no additional chemical cross-linking step is required. Moreover, ionic liquid added can improve the conductivity of the sensing interface and, therefore, can enhance the electrochemical signal. In this work, α-1-fetoprotein was detected within the range from 0.1 to 60 ng ml⁻¹ with a detection limit of 0.07 ng ml⁻¹ (signal/noise = 3). The proposed immunosensor had good specificity and reproducibility. It was used to determine real samples with satisfactory results.     

The development of an electrochemical immunosensor using a thiol aromatic aldehyde and PAMAM-functionalized Fe3O4@Au nanoparticles

In this work, a novel thiol aromatic aldehyde was synthesized. It can be used as a substrate to directly immobilize antibodies on a gold electrode, for which no additional chemical cross-linker is required. It was also applied as a linker to prepare Fe₃O₄@Au/PAMAM/Ab₂–horseradish peroxidase bioconjugates, which introduced multiple enzymes onto a sensing interface owing to the high surface-to-volume ratio of Fe₃O₄@Au nanoparticles and many functional groups of the poly(amidoamine) dendrimer (PAMAM). The introduced multiple enzymes greatly improved the detection signal. Under optimal conditions, the proposed electrochemical immunosensor exhibited desirable performance for detection of IgG in the range 0.005–50 ng ml⁻¹ with a detection limit of 3 pg ml⁻¹ based on a signal-to-noise ratio of 3. It has great potential application in the area of clinical analysis.     

Sensitive amperometric immunosensor for α-fetoprotein based on carbon nanotube/gold nanoparticle doped chitosan film

A novel strategy for the fabrication of sensitive immunosensor to detect α-fetoprotein (AFP) in human serum has been proposed. The immunosensor was prepared by immobilizing AFP antigen onto the glassy carbon electrode (GC) modified by gold nanoparticles and carbon nanotubes doped chitosan (GNP/CNT/Ch) film. GNP/CNT hybrids were produced by one-step synthesis based on the direct redox reaction. The electrochemical properties of GNP/CNT/Ch films were characterized by impedance spectroscopy and cyclic voltammetry. It was indicated that GNP/CNT nanohybrid acted as an electron promoter and accelerated the electron transfer. Sample AFP, immobilized AFP, and alkaline phosphatase (ALP)-labeled antibody were incubated together for the determination based on a competitive immunoassay format. After the immunoassay reaction, the bound ALP label on the modified GC led to an amperometric response of 1-naphthyl phosphate (1-NP), which was changed with the different antigen concentrations in solution. Under the optimized experimental conditions, the resulting immunosensor could detect AFP in a linear range from 1 to 55 ng ml⁻¹ with a detection limit of 0.6 ng ml⁻¹. The proposed immunosensor, by using GNP/CNT/Ch as the immobilization matrix of AFP, offers an excellent amperometric response of ALP-anti-AFP to 1-NP. The immunosensor provided a new alternative to the application of other antigens or other bioactive molecules.     

The fabrication of a label-free electrochemical immunosensor using Nafion/carbon nanotubes/charged pyridinecarboxaldehyde composite film

A label-free electrochemical immunosensor based on Nafion/carbon nanotubes (CNTs)/charged pyridinecarboxaldehyde composite film was developed for the detection of hepatitis B surface antigen. Nafion/CNTs/charged pyridinecarboxaldehyde nanocomposites were prepared by dispersing charged pyridinecarboxaldehyde and CNTs in Nafion solution. The nanocomposites were cast on the electrode surface to form aldehyde-terminated composite film that can covalently bind antibody on the film without using other reagent. The immunosensor response was linearly changed with hepatitis B surface antigen concentration in the range from 0.1 to 25 ng ml⁻¹ with a detection limit (signal/noise ratio = 3) of 0.04 ng ml⁻¹. Some important advantages such as simple preparation, good stability, reproducibility, and selectivity of the immunosensor were achieved.     

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.     

An electrochemical enzyme bioaffinity electrode based on biotin-streptavidin conjunction and bienzyme substrate recycling for amplification

A signal amplificatory electrochemical immunoassay with biotin-streptavidin conjunction and multienzymatic-based substrate recycling was developed in this work. Biotinylated secondary antibody (bio-IgG) was preliminarily assembled onto the immunosensor interface based on the sandwich format. Streptavidin was then loaded based on biotin-streptavidin conjunction. Owing to four identical binding sites of streptavidin to biotin, amounts of biotinylated alkaline phosphatase (bio-AP) were attached, and this improved the catalytic performance of the proposed immunosensor. Under the enzyme catalysis of AP, the electroinactive p-aminophenylphosphate (PAPP) substrate was rapidly hydrolyzed into the electroactive p-aminophenol (PAP) product, which next oxidized at the electrode surface into p-quinoneimine (PQI). In the presence of diaphorase (DI), PQI was reduced back to PAP, leading to a reversible cycle of PAP. Then the oxidized state of DI was regenerated into its reduced native state by its natural substrate, nicotinamide adenine dinucleotide (NADH). With the several amplification factors mentioned above, a wider linear ranged from 10⁻¹⁴ to 10⁻⁵ g ml⁻¹ was acquired with a relatively low detection limit of 3.5 × 10⁻⁵ g ml⁻¹ for human IgG. In addition, the nonspecific adsorption of proposed immunosensor was also investigated here.     

Enhanced electrochemiluminescence from luminol at carboxyl graphene for detection of α-fetoprotein

In this study, a novel sensitive electrochemiluminescence (ECL) immunosensor was constructed by carboxyl graphene (GR) for enhancing luminol–O₂ system emission. Here, carboxyl GR was used to enhance the ECL intensity of luminol that had excellent electron transfer ability and good solubility. The sensing platform was constructed by depositing carboxyl GR on electrodes and immobilizing antibodies on the surface of carboxyl GR through amidation. The specific immunoreaction between α-fetoprotein (AFP) and antibodies resulted in a decrease of ECL intensity, and the intensity decreased linearly with AFP concentrations in the range of 5 pg ml⁻¹ to 14 ng ml⁻¹ with a detection limit of 2.0 pg ml⁻¹. The proposed immunosensor exhibits high specificity, good reproducibility, and longtime stability. It may become a promising technique for protein detection.     

Voltammetric determination of cefixime in pharmaceuticals and biological fluids

Electroreduction and adsorption of cefixime was studied in phosphate buffer by cyclic voltammetry (CV), differential pulse cathodic adsorptive stripping voltammetry (DPCAdSV), and square-wave cathodic adsorptive stripping voltammetry (SWCAdSV) at hanging mercury drop electrode (HMDE). These fully validated sensitive and reproducible cathodic adsorptive stripping voltammetric procedures were applied for the trace determination of the bulk drug in pharmaceutical formulations and in human urine. The optimal experimental parameters were as follows: accumulation potential = −0.1 V (vs. Ag/AgCl, 3 M KCl), accumulation time = 50 s, frequency = 140 Hz, pulse amplitude = 0.07 V, and scan increment = 10 mV in phosphate buffer (pH 2.6). The first peak current showed a linear dependence with the drug concentration over the range of 50 ng ml⁻¹ to 25.6 μg ml⁻¹. The achieved limit of detection and limit of quantitation were 3.99 and 13.3 ng ml⁻¹ by SWCAdSV and 7.98 and 26.6 ng ml⁻¹ by DPCAdSV, respectively. The procedure was applied to assay the drug in tablets. Applicability was also tested in urine samples. Peak current was linear with the drug concentration in the range of 1 to 60 μg ml⁻¹ of the urine, and minimum detectability was found to be 12.6 ng ml⁻¹ by SWCAdSV and 58.4 ng ml⁻¹ by DPCAdSV.     

Graphene oxide supported rhombic dodecahedral Cu2O nanocrystals for the detection of carcinoembryonic antigen

In this work, a simple electrochemical immunosensor was developed for the detection of carcinoembryonic antigen (CEA) based on rhombic dodecahedral Cu₂O nanocrystals–graphene oxide–gold nanoparticles (rCu₂O–GO–AuNPs). GO as the template and surfactant resulting in rCu₂O exhibit improved rhombic dodecahedral structure uniformity and excellent electrochemical performance. Moreover, GO was found to be able to effectively improve the long stability of rCu₂O on the electrode response. Under optimal conditions, the immunosensor showed a low limit of detection (0.004 ng ml⁻¹) and a large linear range (0.01–120 ng ml⁻¹). This work presents a potential alternative for the diagnostic applications of GO-supported special morphology materials in biomedicine and biosensors.     

Electrochemical immunosensor for α-fetoprotein detection using ferroferric oxide and horseradish peroxidase as signal amplification labels

An electrochemical immunosensor for quantitative detection of α-fetoprotein (AFP) in human serum was developed using graphene sheets (GS) and thionine (TH) as electrode materials and mesoporous silica nanoparticles (MSNs) loaded with ferroferric oxide (Fe₃O₄) nanoparticles and horseradish peroxidase (HRP) as labels for signal amplification. In this study, the compound of GS and TH (GS–TH) was used as a substrate for promoting electron transfer and immobilization of primary antibody of AFP (Ab₁). MSNs were used as a carrier for immobilization of secondary antibody of AFP (Ab₂), Fe₃O₄, and HRP. The synergistic effect occurred between Fe₃O₄ and HRP and greatly improved the sensitivity of the immunosensor. This method could detect AFP over a wide concentration range from 0.01 to 25 ng ml⁻¹ with a detection limit of 4 pg ml⁻¹. This strategy may find wide potential application in clinical analysis or detection of other tumor markers.     

Screening of boldenone and methylboldenone in bovine urine using disposable electrochemical immunosensors

Electrochemical based immunosensors for the detection of boldenone and methylboldenone in bovine urine were described in this paper. The immunosensors were fabricated by immobilizing boldenone-bovine serum albumin conjugate on the surface of screen-printed electrodes (SPEs), and followed by the competition between the free analyte and coating conjugate with corresponding antibodies. The use of anti-species IgG-horseradish peroxidase conjugate determined the degree of competition. The electrochemical technique chosen was chronoamperometry, performed at a potential of +100 mV whereby the product of the catalysis of 3,3′,5,5′-tetramethylbenzidine undergoes reduction produced by the enzyme label. The limits of detection of assay were 30.9 ± 4.3 pg ml⁻¹ for boldenone and 120.2 ± 8.2 pg ml⁻¹ for methylboldenone, respectively. Results of repeated analysis of each androgen carried out using three different batches of electrodes indicate suitable repeatability (EC₅₀ = 1.0 ± 0.3 ng ml⁻¹ (n = 3, N = 3), R² = 0.969, R.S.D. = 9.6% for boldenone and 1.5 ± 0.3 ng ml⁻¹, 0.971, 10.5% for methylboldenone, respectively). Urine samples were determined directly after a single dilution step, omitting extraction and hydrolysis. This method offers the advantage to pick up both boldenone and its major metabolites in an efficient manner due to the high cross-reactivity pattern of α-boldenone with this antibody. The concentration of methylboldenone in urine detected by developed methods does indicate methylboldenone administration to heifers. Gas chromatography coupled to mass spectrometry analysis was performed to quantitate the individual metabolites present in urine samples, and results were validated with both ELISA and immunosensor data.     

Electrochemical immunosensor based on Pd–Au nanoparticles supported on functionalized PDDA-MWCNT nanocomposites for aflatoxin B1 detection

This paper reports a label-free electrochemical immunosensor for the determination of aflatoxin B1 (AFB1), which is based on a gold electrode modified by a biocompatible film of carbon nanotubes/poly(diallyldimethylammoniumchloride)/Pd–Au nanoparticles (CNTs/PDDA/Pd–Au). The nanocomposite was characterized by transmission electron microscopy and the electrochemical behavior of modified electrodes was investigated by cyclic voltammetry. The CNTs/PDDA/Pd–Au nanocomposites film showed good electron transfer ability, which ensured high sensitivity to detect AFB1 in a range from 0.05 to 25 ng mL⁻¹ with a detection limit of 0.03 ng mL⁻¹ obtained at 3σ (where σ is the standard deviation of the blank solution, n = 10). The proposed immunosensor provides a simple tool for AFB1 detection. This strategy can be extended to any other antigen detection by using the corresponding antibodies.     

Determination of 17β-oestradiol by fluorescence immunoassay with streptavidin-conjugated quantum dots as label

A sensitive and selective method for the determination of 17β-oestradiol by fluorescence immunoassay (FIA) was established on the basis of quantum dots (QDs) as label. The complex of biotin-labelled anti-rabbit IgG and strepavidin conjugated by quantum dots (QD-SA) was regarded as a probe in this system and the strepavidin-biotin system as signal amplification system. After optimising the conditions of the immunoreaction, such as the concentration of the reagent and the pH of the buffer solution, the linear range and the limit of detection of 17β-oestradiol were 0.01-10,000 ng ml⁻¹ and 0.00542 ng ml⁻¹, respectively. This method was applied to determine oestradiol in water samples, with the percent recoveries in the range of 86-113%.     

Detection of Bacillus thuringiensis Cry1Ab protein based on surface plasmon resonance immunosensor

Two novel surface plasmon resonance immunosensors were fabricated for detection of the Bacillus thuringiensis Cry1Ab protein and to demonstrate their performance in analyzing Cry1Ab protein in crop samples. Sensor 2 was modified by 1,6-hexanedithiol, Au/Ag alloy nanoparticles, 3-mercaptopropionic acid, and protein A (or not [sensor 1]), with Cry1Ab monoclonal antibody. As a result, both of the immunosensors exhibited satisfactory linear responses in the Cry1Ab protein concentration ranges of 10 to 500 ng ml⁻¹ and 8 to 1000 ng ml⁻¹, and the detection limits were 5.0 and 4.8 ng ml⁻¹, respectively. The immunosensors possessed good specificity and acceptable reproducibility. In addition, crop samples could be analyzed after a simple treatment. The transgenic crops could be easily identified from the conventional ones by the two immunosensors.     

Human serum albumin interference on plasmon-based immunokinetic assay for antibody screening in model blood sera

The effect of human serum albumin (HSA) on an immunokinetic assay for an antibody to bovine serum albumin has been determined in model serum solutions with HSA concentrations in the range 0 to 450 μM (0-30 mg ml⁻¹). The assay is performed on two plasmon-based detection platforms: a continuous gold surface and a nanoparticle-based array reader. The assay has a minimum detection concentration of 760 ± 160 pM (120 ± 25 ng ml⁻¹) in phosphate-buffered saline, falling to 2.5 ± 0.7 nM (380 ± 100 ng ml⁻¹) in physiological HSA concentration. The concentration of HSA correlates with the refractive index of the solution, and this may be used to calibrate assay response. The addition of the charged chaotrope SCN⁻ in 150 mM concentration improves the reproducibility and consistency of the assay, with a minimum detection concentration of 2.9 ± 0.5 nM (440 ± 80 ng ml⁻¹). The effect of high concentrations of HSA on the immunokinetic assay can be corrected with a measurement of bulk refractive index in a reference channel.     

Digoxin derivatives substituted by alkylidene at the butenolide part

A series of digoxin derivatives containing the γ-alkylidene butenolide moiety were synthesised by way of stereoselective vinylogous aldol reaction of the unactivated butenolide in simple conditions. The structures of compounds synthesised were characterised by infrared (IR), nuclear magnetic resonance (NMR) and HR-MS. Preliminary bioassay shows that some of them have cardiac functions, especially compound 2g that induced a marked increase in myocardial contractility at 10 ng ml⁻¹ and 20 ng ml⁻¹ concentrations without digitalis toxicity.     

One-step kinetics-based immunoassay for the highly sensitive detection of C-reactive protein in less than 30 min

This article reveals a rapid sandwich enzyme-linked immunosorbent assay (ELISA) for the highly sensitive detection of human C-reactive protein (CRP) in less than 30 min. It employs a one-step kinetics-based highly simplified and cost-effective sandwich ELISA procedure with minimal process steps. The procedure involves the formation of a sandwich immune complex on capture anti-human CRP antibody-bound Dynabeads in 15 min, followed by two magnet-assisted washings and one enzymatic reaction. The developed sandwich ELISA detects CRP in the dynamic range of 0.3 to 81 ng ml⁻¹ with a limit of detection of 0.4 ng ml⁻¹ and an analytical sensitivity of 0.7 ng ml⁻¹. It detects CRP spiked in diluted human whole blood and serum with high analytical precision, as confirmed by conventional sandwich ELISA. Moreover, the results of the developed ELISA for the determination of CRP in the ethylenediaminetetraacetic acid plasma samples of patients are in good agreement with those obtained by the conventional ELISA. The developed immunoassay has immense potential for the development of rapid and cost-effective in vitro diagnostic kits.     

Amperometric immunosensor for carcinoembryonic antigen detection with carbon nanotube-based film decorated with gold nanoclusters

A new amperometric immunosensor for the determination of carcinoembryonic antigen (CEA) was constructed. First, the uniform nanomultilayer film was fabricated via layer-by-layer (LBL) assembly of positively charged carbon nanotubes wrapped by poly(diallyldimethylammonium chloride) and negatively charged poly(sodium-p-styrene-sulfonate), which could provide a high accessible surface area and a biocompatible microenvironment. Subsequently, gold nanoclusters were electrodeposited on the electrode to immobilize anti-CEA. The fabricated process and electrochemical behaviors of the immunosensor were characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM). Under optimal conditions, the proposed immunosensor could detect CEA in two linear ranges from 0.1 to 2.0 ng mL⁻¹ and from 2.0 to 160.0 ng mL⁻¹, with a detection limit of 0.06 ng mL⁻¹.     

Label-free electrochemical immunosensor based on Nile blue A-reduced graphene oxide nanocomposites for carcinoembryonic antigen detection

In this article, a novel, label-free, and inherent electroactive redox immunosensor for carcinoembryonic antigen (CEA) based on gold nanoparticles (AuNPs) and Nile blue A (NB) hybridized electrochemically reduced graphene oxide (NB–ERGO) is proposed. The composite of NB–graphene oxide (NB–GO) was prepared by π–π stacking interaction. Then, chronoamperometry was adopted to simultaneously reduce HAuCl₄ and nanocomposites of NB–GO for synthesizing AuNPs/NB–ERGO. The immunosensor was fabricated by capturing CEA antibody (anti-CEA) at this nanocomposite modified electrode. The immunosensor determination was based on the fact that, due to the formation of antigen–antibody immunocomplex, the decreased response currents of NB were directly proportional to the concentrations of CEA. Under optimal conditions, the linear range of the proposed immunosensor was estimated to be from 0.001 to 40 ng ml⁻¹ and the detection limit was estimated to be 0.00045 ng ml⁻¹. The proposed immunosensor was used to determine CEA in clinical serum samples with satisfactory results. The proposed method may provide promising potential application in clinical immunoassays with the properties of facile procedure, stability, high sensitivity, and selectivity.     

Horseradish peroxidase functionalized gold nanorods as a label for sensitive electrochemical detection of alpha-fetoprotein antigen

In this study, a novel tracer, horseradish peroxidase (HRP) functionalized gold nanorods (Au NRs) nanocomposites (HRP–Au NRs), was designed to label the signal antibodies for sensitive electrochemical measurement of alpha-fetoprotein (AFP). The preparation of HRP–Au NRs nanocomposites and the labeling of secondary antibody (Ab₂) were performed by one-pot assembly of HRP and Ab₂ on the surface of Au NRs. The immunosensor was fabricated by assembling carbon nanotubes (CNTs), Au NRs, and capture antibodies (Ab₁) on the glassy carbon electrode. In the presence of AFP antigen, the labels were captured on the surface of the Au NRs/CNTs via specific recognition of antigen–antibody, resulting in the signal intensity being clearly increased. Differential pulse voltammetry (DPV) was employed to record the response signal of the immunosensor in phosphate-buffered saline (PBS) containing hydrogen peroxide (H₂O₂) and 3,3′,5,5′-tetramethylbenzidine (TMB). Under optimal conditions, the signal intensity was linearly related to the concentration of AFP in the range of 0.1–100 ng ml⁻¹, and the limit of detection was 30 pg ml⁻¹ (at signal/noise [S/N] = 3). Furthermore, the immunoassay method was evaluated using human serum samples, and the recovery obtained was within 99.0 and 102.7%, indicating that the immunosensor has potential clinical applications.