CE-based simultaneous liquid-phase noncompetitive enzyme immunoassay for three tumor markers in human serum using electrochemical detection

A method for indirectly detecting horseradish peroxidase (HRP) was described by CE with electrochemical detection. Details of selection for optimum conditions were presented. The detection limit of free HRP was 1.09 x 10(-12) M or 0.94 zmol (S/N = 3). A novel CE-based liquid-phase binding noncompetitive enzyme immunoassay (CE-EIA) was developed. In this method, after the noncompetitive immunoreaction in liquid phase, the free enzyme (HRP)-labeled antibody (Ab*) and the bound enzyme-labeled complex (Ag-Ab*) were separated and then the system of HRP catalyzing H(2)O(2)/o-aminophenol (OAP) reaction was adopted. Prostate specific antigen (PSA), carcinoembryonic antigen (CEA), and human chorionic gonadotropin (HCG) in human serum samples were detected without any sample preparation, with the detection limits (S/N = 3) of 0.22, 0.17 and 0.30 ng/mL, respectively. This technique has been successfully applied to detect simultaneously PSA, CEA, and HCG in 12 min, upon adding these three antigens into human serum to simulate patient serum. It proves that the CE-EIA technique proposed could be developed into a sensitive and new method for simultaneous clinical assay of multianalytes.     

Cation-exchange antibody labeling for simultaneous electrochemical detection of tumor markers CA15-3 and CA19-9

We report on a new kind of non-covalent multi-label electrochemical immunoassay that was applied to simultaneously quantify the tumor markers CA15-3 and CA19-9. The method employs a nanohybrid composed of an ionomer and conductive titanium dioxide nanoparticles that act as a matrix support for the antibodies. The two antibodies (anti-CA153 and anti-CA199) were labeled (a) with a cobaltous dipyridine complex, and (b) with methylene blue. Labeling is based on cation-exchange interaction rather than on covalent conjugation. The redox potentials of the two labels are separated by an interval of 0.3 V. The resulting sandwich-type immunosensor was read out by differential pulse voltammetry. The potential sites and currents of the two redox probes reflect the concentration of the two analytes. The two analytes were determined with a detection limit of 1.6 U?mL^?1 for CA19-9, and of 0.3 U?mL^?1 for CA15-3.

Multiplex detection of tumor markers with photonic suspension array

A novel photonic suspension array was developed for multiplex immunoassay. The carries of this array were silica colloidal crystal beads (SCCBs). The codes of these carriers are the characteristic reflection peak originated from their structural periodicity, and therefore they do not suffer from fading, bleaching, quenching, and chemical instability. In addition, because no dyes or materials related with fluorescence are included, the fluorescence background of SCCBs is very low. With a sandwich format, the proposed suspension array was used for simultaneous multiplex detection of tumor markers in one test tube. The results showed that the four tumor markers, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), carcinoma antigen 125 (CA 125) and carcinoma antigen 19-9 (CA 19-9) could be assayed in the ranges of 1.0-500 ng mL⁻¹, 1.0-500 ng mL⁻¹, 1.0-500 U mL⁻¹ and 3.0-500 U mL⁻¹ with limits of detection of 0.68 ng mL⁻¹, 0.95 ng mL⁻¹, 0.99 U mL⁻¹ and 2.30 U mL⁻¹ at 3σ, respectively. The proposed array showed acceptable accuracy, detection reproducibility, storage stability and the results obtained were in acceptable agreement with those from parallel single-analyte test of practical clinical sera. This technique provides a new strategy for low cost, automated, and simultaneous multiplex immunoassay.     

Sensitive and simultaneous detection of different disease markers using multiplexed gold nanorods

A multiplexed bioanalytical assay is produced by incorporating two types of gold nanorods (GNRs). Besides retaining the desirable features of common GNRs LSPR sensors, this sensor is easy to fabricate and requires only a visible–NIR spectrometer for detection. This assay can simultaneously detect different acceptor–ligand pairs by choosing the proper GNRs possessing various LPWs in a wide detection wavelength range and can be developed into a high-throughput detection method. This bioanalytical assay allows easy detection of human serum specimens infected by S. japonicum and tuberculosis (TB) from human serum specimens (human serum/Tris–HCl buffer ratio = 1:10⁴) without the need for sample pretreatment. The technique is very sensitive compared to other standard methods such as indirect hemagglutination assays (IHA) that require a serum concentration ratio of larger than 1:20 and enzyme-linked immunosorbent assays (ELISA) requiring a ratio larger than 1:100. This methodology can be readily extended to other immunoassays to realize wider diagnostic applications.     

Sensitive electrochemical detection of ozone

An amperometric sensor capable of detecting ozone in the low ppb range was developed. The most suitable electrochemical cell was based on a gold-Nafion electrode with 0.5 M H₂SO₄ as internal electrolyte solution. It is demonstrated, that by careful selection of the experimental conditions such as electrode materials, electrolyte solution and applied potential the determination of low concentrations of ozone in air is possible with detection limits of 0.6 ppb. Cross-sensitivities to the major inorganic gaseous species found in the atmosphere, are also presented, and the use of a chemical filter to circumvent the interference by nitrogen dioxide is described.     

Aptamer/quantum dot-based simultaneous electrochemical detection of multiple small molecules

A novel strategy for “signal on” and sensitive one-spot simultaneous detection of multiple small molecular analytes based on electrochemically encoded barcode quantum dot (QD) tags is described. The target analytes, adenosine triphosphate (ATP) and cocaine, respectively, are sandwiched between the corresponding set of surface-immobilized primary binding aptamers and the secondary binding aptamer/QD bioconjugates. The captured QDs yield distinct electrochemical signatures after acid dissolution, whose position and size reflect the identity and level, respectively, of the corresponding target analytes. Due to the inherent amplification feature of the QD labels and the “signal on” detection scheme, as well as the sensitive monitoring of the metal ions released upon acid dissolution of the QD labels, low detection limits of 30 nM and 50 nM were obtained for ATP and cocaine, respectively, in our assays. Our multi-analyte sensing system also shows high specificity to target analytes and promising applicability to complex sample matrix, which makes the proposed assay protocol an attractive route for screening of small molecules in clinical diagnosis.     

An enzyme immunosensor for the electrochemical determination of the tumor antigen α-fetoprotein

A specific sensor for a tumor antigen, α-fetoprotein (AFP) can be prepared from a membrane with immobilized antibody and an oxygen probe with a permeable teflon membrane. Anti-AFP antibody is covalently immobilized on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane and glutaraldehyde. The sensor is applied to enzyme immunoassay based on competitive antigen-antibody reaction with catalase-labelled antigen. After competitive binding of free and catalase-labelled AFP, the sensor is examined for catalase activity by amperometric measurement after addition of hydrogen peroxide. AFP can be determined in the range 10^-11–10^-8 g ml^-1.     

Mucin 4 detection with a label-free electrochemical immunosensor

Mucin 4 (MUC4) is a useful biomarker for endometriosis and cancers of the pancreas, esophagus and breast. The very first electrochemical immunosensor for the detection of MUC4 is reported, using carbon-based screen-printed electrodes modified by reaction with the diazonium salt of p-aminophenylacetic acid. Electrochemical impedance spectroscopy and cyclic voltammetry were used to characterize and optimize the electrografting process. The in situ surface modification through diazotation with phenylacetic groups enables the chemical binding of the specific antibody, followed by its affinity reaction with MUC4. The immunosensor was optimized with respect to several parameters and is very promising for clinical applications, having a limit of detection of 0.33 μg mL^− 1 and a linear domain between 1 and 15 μg mL^− 1 obtained by electrochemical impedance spectroscopy measurements.     

Electrochemical genosensor array for the simultaneous detection of multiple high-risk human papillomavirus sequences in clinical samples

An electrochemical genosensor array for the simultaneous detection of three high-risk human papillomavirus (HPV) DNA sequences, HPV16, 18 and 45, exhibiting high sensitivity and selectivity is presented. The electrodes of a 4 × 4 array were modified via co-immobilization of a 1:100 (mol/mol) mixture of a thiolated probe and an oligoethyleneglycol-terminated bipodal thiol. Detection of synthetic and PCR products was carried out in a sandwich type format, with the target hybridized between a surface immobilized probe and a horseradish peroxidase-labelled secondary reporter probe. The detection limits obtained in the detection of each individual target were in the pM range, allowing the application of this sensor for the detection of samples obtained from PCR amplification of cervical scrape samples. The results obtained exhibited an excellent correlation with the HPV genotyping carried out within a hospital laboratory. Multiplexing and cross-reactivity studies demonstrated high selectivity over potential interfering sequences, facilitating application of the developed platform for the high-throughput screening of multiple high-risk DNA sequences.     

Simultaneous electrochemical detection of multiple tumor markers based on dual catalysis amplification of multi-functionalized onion-like mesoporous graphene sheets

In this work, a sandwich-type electrochemical immunosensor for simultaneous sensitive detection of prostate specific antigen (PSA) and free prostate specific antigen (fPSA) is fabricated. Gold nanoparticles (AuNPs) modified Prussian blue and nickel hexacyanoferrates nanoparticles were firstly prepared, respectively, and then decorated onion-like mesoporous graphene sheets (denoted as Au@PBNPs/O-GS and Au@NiNPs/O-GS) as distinguishable signal tags to label different detection antibodies. Subsequently, streptavidin and biotinylated alkaline phosphatase (bio-AP) were employed to block the possible remaining active sites. With the employment of the as prepared nanohybrids, the dual catalysis amplification can be achieved by catalysis of the ascorbic acid 2-phosphate to in situ produce AA in the presence of bio-AP, and then AA was further catalyzed by Au@PBNPs/O-GS and Au@NiNPs/O-GS nanohybrids, respectively, to obtain the higher signal responses. The experiment results show that the linear range of the proposed immunosensor for simultaneous determination of fPSA is from 0.02 to 10 ng mL⁻¹ with a detection limit of 6.7 pg mL⁻¹ and PSA is from 0.01 to 50 ng mL⁻¹ with a detection limit of 3.4 pg mL⁻¹ (S/N = 3). Importantly, the proposed method offers promise for rapid, simple and cost-effective analysis of biological samples.     

A label-free electrochemical immunosensor based on gold nanoparticles for detection of paraoxon

An electrochemical immunosensor for the direct determination of paraoxon has been developed based on the biocomposites of gold nanoparticles loaded with paraoxon antibodies. The biocomposites are immobilized on the glassy carbon electrode (GCE) using Nafion membrane. On the immunosensor prepared paraoxon shows well-shaped CV with reduction and oxidation peaks located −0.08 and −0.03 mV versus SCE, respectively. The detection of paraoxon performed at −0.03 mV is beneficial for guaranteeing sufficient selectivity. The amount of the biocomposite consisting gold nanoparticles loaded with antibodies and the volume of Nafion solution used for fabricating the immunosensor have been studied to ensure sensitivity and conductivity of the immunosensor. The immunosensor has been employed for monitoring the concentrations of paraoxon in aqueous samples up to 1920 μg l⁻¹ with a detection limit of 12 μg l⁻¹.     

Development of an electrochemical immunosensor for the detection of HbA1c in serum

An electrochemical immuno-biosensor for detecting glycosylated haemoglobin (HbA1c) is reported based on glassy carbon (GC) electrodes with a mixed layer of an oligo(phenylethynylene) molecular wire (MW) and an oligo(ethylene glycol) (OEG). The mixed layer is formed from in situ-generated aryl diazonium cations. To the distal end of the MW, a redox probe 1,1'-di(aminomethyl)ferrocene (FDMA) was attached followed by the covalent attachment of an epitope N-glycosylated pentapeptide (GPP), an analogon to HbA1c, to which an anti-HbA1c monocolonal antibody IgG can selectively bind. HbA1c was detected by a competitive inhibition assay based on the competition for binding to anti-HbA1c IgG antibodies between the analyte in solution, HbA1c, and the surface bound epitope GPP. Exposure of the GPP modified sensing interface to the mixture of anti-HbA1c IgG antibody and HbA1c results in the attenuation of ferrocene electrochemistry due to free antibody binding to the interface. Higher concentrations of analyte led to higher Faradaic currents as less anti-HbA1c IgG is available to bind to the electrode surface. It was observed that there is a good linear relationship between the relative Faradaic current of FDMA and the concentration of HbA1c from 4.5% to 15.1% of total haemoglobin in serum without the need for washing or rinsing steps.     

Automated microsystem for electrochemical detection of cancer markers

The development of a fully automated microsystem housing an amperometric immunosensor is presented. The microfluidic cell integrates reagent storage and electrochemical immunodetection and was applied for the detection of breast cancer markers. The main advantage of this system is that no external fluidic storage is required and the instrumental setup is thus greatly simplified. The fluidics of the microsystem is computer controlled and requires minimal end-user intervention. The analytical performance of the device was compared with a manually driven system and applied for the amperometric detection of the carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA15-3). This automation methodology greatly improves the analytical performance of the immunosensor in terms of accuracy and reproducibility as evidenced by a reduction of LOD observed for CEA and CA15-3 with respect to a manually driven system. Finally, the automated microsystem was applied for the analysis of real patient serum samples, demonstrating excellent correlation with a commercial ELISA.     

Portable smartphone-based microfluidic electrochemical immunosensor for ultrasensitive detection of alpha-fetoprotein in human serum

Rapid and accurate tracing of biomarkers is essential for early detecting and diagnosing of cancer. Therefore, a valid and convenient strategy needs to be developed for efficient monitoring of cancer biomarkers. Herein, we constructed a portable microfluidic electrochemical immunosensor based on three-dimensional reduced graphene oxide (3D rGO) doped with gold nanoparticles (Au NPs) for ultrasensitive determination of alpha-fetoprotein (AFP). The designed microfluidic chip, with the advantages of small injection volume, detachable structure and high integration, was fabricated by 3D printing, which only needed 9 μL of reagent to realize the high sensitivity detection. In addition, the 3D Au NPs-rGO composites with high specific surface area and electrons transfer capacity can effectively increase electroactive sites and enhance electrochemical signals. Benefiting from these features, the 3D Au NPs-rGO microfluidic electrochemical immunochip showed a wide detection range between 0.1 pg/mL–200 ng/mL and a best detection limit of 0.045 pg/mL with the high sensitivity of 175.008 μA (ng/mL)⁻¹ cm⁻². Meanwhile, the proposed immunosensor exhibited reliable AFP detection in human serum samples, which demonstrated that this portable smartphone-based microfluidic electrochemical immunosensor hold great promises in clinical detection and huge potential in personalized healthcare.     

Magnetic beads-based electrochemical immunosensor for detection of pseudorabies virus antibody in swine serum

A novel magnetic electrochemical immunosensor has been developed for the detection of pseudorabies virus antibody in swine serum. The magnetic glass carbon electrode was fabricated to manipulate magnetic beads for the direct sensing applications. Magnetic beads were employed as the platforms for the immobilization and immunoreaction process, and gold nanoparticles were chosen as electroactive labels for the electrochemical detection. The parameters concerning the assay strategy were carefully investigated. Under the optimal conditions, the linear response range of pseudorabies virus antibody dilution ratio (standard positive serum) was 1:250 to 1:1000 with a detection limit of 1:1000. Finally, this developed immunoassay method was successfully applied in the detection of pseudorabies virus antibody in swine serum, and had a good diagnostic accordance in comparison with ELISA.     

A monoclonal antibody-based immunosensor for detection of Sudan I using electrochemical impedance spectroscopy

Sudan I monoclonal antibodies (Mabs) were prepared by hybridoma technique and firstly used to develop a Sudan I immunosensor by immobilizing the Mabs on a gold electrode. o-Mercaptobenzoic acid (MBA) was covalently conjugated on the gold electrode to form a self-assembled monolayer (SAM). The immobilization of Sudan I Mabs to the SAM was carried out through a stable acyl amino ester intermediate generated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide (NHS), which could condense antibodies reproducibly and densely on the SAM. The changes of the electrode behavior after each assembly step were investigated by cyclic voltammetric (CV) technique. The Sudan I concentration was measured through the increase of impedance values in the corresponding specific binding of Sudan I and Sudan I antibody. A linear relationship between the increased electron-transfer resistance (Ret) and the logarithmic value of Sudan I concentrations was found in the range of 0.05-50 ng mL⁻¹ with the detection limit of 0.03 ng mL⁻¹. Using hot chili as a model sample, acceptable recovery of 96.5-107.3% was obtained. The results were validated by conventional HPLC method with good correlation. The proposed method was proven to be a feasible quantitative method for Sudan I analysis with the properties of stability, highly sensitivity and selectivity.     

Disposable electrochemical immunosensor for environmental applications

An Immunosensor for the detection of polychlorinated biphenyls (PCB) has been developed, using carbon-based screen-printed electrodes as solid-phase and signal transducers. The immunosensor realised is based on a direct competitive immunoassay scheme, in which the antibody against PCB was directly immobilised onto the carbon surface of the screen-printed electrode. Competition between the PCBs present in the sample and a fixed concentration of an enzyme-labelled congener was realised and evaluated by electrochemical detection. The immunosensor developed was tested on Arochlor mixtures (1242 and 1248), and was applied in environmental and food analysis by testing some real samples (from animal tissues and marine sediments). Results obtained demonstrate the ability of this device to detect PCBs in complex matrices.     

Fluorescence immunosensor based on p-acid-encapsulated silica nanoparticles for tumor marker detection

A novel, simple and highly sensitive amplified fluorescence label-free immunosensor by using p-acid-encapsulated silica nanomaterials has been developed for the first time. 4,4'-(2,5-Dimethoxy-1,4-phenylene)bis(ethyne-2,1-diyl)dibenzoic acid (p-acid) and p-acid-encapsulated silica were prepared, and characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance, ultraviolet visible spectroscopy (UV-vis) and fluorescence spectroscopy. In layer-by-layer self-assembling processes using (3-aminopropyl)triethoxysilane, p-acid@SiO(2) was assembled on the glass substrate. Antibody was immobilized on the surface of p-acid@SiO(2) with N,N'-carbonyldiimidazole. The functional nanomaterials present good analytical properties with a calibration range of 0.1-100 ng mL(-1), and allow the detection of carcinoembryonic antigen (CEA) at a concentration as low as 0.04 ng mL(-1). What is important is that the as-synthesized p-acid@SiO(2) nanomaterials could be further extended for the detection of other biomarkers or biocompounds.     

A label-free electrochemical immunosensor based on multi-functionalized graphene oxide for ultrasensitive detection of microcystin-LR

In this paper, a label-free electrochemical immunosensor for ultrasensitive detection of microcystin-leucine-arginine (MC-LR) based on multi-functionalized graphene oxide was constructed. The graphene oxide has a large surface area for the immobilization of the antibody. Meanwhile, the introduction of the AuNPs and 1-butyl-3-methylimidazolium hexafluorophosphate could enhance the response of the current by improving the electrical conductivity. Thus the electrochemical immunosensor could be prepared through a one-step process and differential pulse voltammetry was employed to detect sensitively MC-LR. Under optimal conditions, the current response of the immunosensor decreased proportionally to the logarithmic concentrations of MC-LR in the range of 0.1–1000 ng/mL with a detection limit of 0.1 ng/mL (S/N = 3). This one-step label-free electrochemical immunosensor showed good performance in specificity, stability, reproducibility, and application.