Sandwich-type Electrochemical Immunoassay Research Articles

Electrochemical immunoassay for one-pot detection of thyroxin (T4) and thyroid-stimulating hormone (TSH) using magnetic and Janus nanoparticles.

Concurrent measurement ofthyroid-stimulating hormone (TSH) and thyroxine (T4) hormones profoundly help clinicians diagnose hyper- and hypothyroidism. This work demonstrates the development of a sandwich-type electrochemical immunoassay using Janus and magnetic nanoparticles for one-pot detection of thyroxine (T4) and thyroid-stimulating hormone (TSH). The signaling probe was developed by preparing Janus cadmium (CdO) and zinc oxide (ZnO) NPs decorated by T4/TSH-specific molecularly imprinted polymers (MIPT4-CdO and MIPTSH-ZnO). The capture probe was obtained by coating magnetic Fe3O4 NPs with 1,3-Bis(3-carboxy propyl) tetramethyl disiloxane and activating using N-hydroxy succinimide (NHS) and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), and finally conjugating with T4/TSH-specific antibodies. To analyze T4 and TSH in actual samples, MIPT4-CdO and MIPTSH-ZnO were added to the sample solutions, and after incubation, capture probes (Fe3O4-AbTSH and Fe3O4-AbT4) were added. An external magnetic field was used to separate the sandwiched nanosystem, followed by adding a dilute solution of nitric acid (HNO3) to dissolve CdO and ZnO NPs and free Cd(II) and Zn(II) cations. The concentration of these cations was determined using constant-current potentiometric stripping analysis (cc-PSA) on screen-printed electrodes (SPE) modified with multi-walled carbon nanotubes (MWCNT). The obtained signals for Cd(II) and Zn(II) were proportional to T4 and TSH concentrations. Limits of detection (LOD) for T4 and TSH analyses were respectively 0.02ng.dL-1 and 0.0002 µU.mL-1 with a linear range of 0.05-50ng.dL-1 and 0.001-100 µU.mL-1. The proposed nanosystem's main advantage is the simultaneous detection of T4 and TSH in clinical samples with high sensitivity, selectivity, and stability.

Highly sensitive detection of thyroglobulin based on sandwich-type electrochemical immunoassay.

As a dimeric protein, thyroglobulin (Tg) is an important biomarker for different thyroid cancer (DTC), so designing effective method to detect Tg is of great significance. In this work, by preparing β-cyclodextrin (CD) functionalized carbon nanotubes (CNTs) nanohybrid (CD-CNTs) as carrier to immobilize primary antibody (Ab1) of Tg, assembling sulfydryl ferrocene (Fc) and secondary antibody (Ab2) on the surface of nanogold (Au) as signaling amplifier (Ab2-Au-Fc), a simple and sensitive sandwich-type electrochemical immunoassay (STEM) of Tg was designed herein for the first time. In brief, CNTs show large surface area and conductivity, while CD offers superior host-guest recognition capability that can bound with Ab1; meanwhile, Fc probe can offer stable electrochemical signal that is proportionable to the concentration of Tg. Under the optimum conditions, the proposed STEM platform shows excellent sensing results for Tg detection: a considerable low analytical detection (0.5ngmL-1) and wide linearity (2 to 200ngmL-1), suggesting the designed STEM platform offers potential real applications for detect Tg.

Electrochemical immunoassay based on choline oxidase-peroxidase enzymatic cascade

Horseradish peroxidase (HRP)-based electrochemical immunoassays are considered promising techniques for point-of-care clinical diagnostics, but the necessary addition of unstable H2O2 in the enzymatic system may hinder their practical application. Although glucose oxidase (GOx) has been widely explored for in situ generation of H2O2 in HRP-based immunoassay, the GOx-catalyzed reduction of oxidized peroxidase substrate may limit the immunosensing performance. Here, we report a sensitive electrochemical immunosensor based on a choline oxidase (ChOx)-HRP cascade reaction. In this design, ChOx catalyzes the oxidation of choline, during which H2O2 is generated in situ and thus oxidizes acetaminophen (APAP) in the presence of HRP. The electrochemical behavior of APAP in the ChOx-HRP cascade was compared with that of the commonly used GOx-HRP cascade, which confirmed that ChOx could be a superior preceding enzyme for sensitive immunoassay based on the bienzymatic cascade. The developed ChOx-HRP cascade was also further explored for a sandwich-type electrochemical immunoassay of parathyroid hormone in artificial and clinical serum. The calculated detection limit was ~3 pg/mL, indicating that the ChOx-HRP cascade is especially promising for highly sensitive electrochemical immunoassays when APAP is used as the peroxidase substrate.

Dual labeled mesoporous silica nanospheres based electrochemical immunosensor for ultrasensitive detection of carcinoembryonic antigen

Carcinoembryonic antigen (CEA) is a well-known cancer biomarker for the detection of several malignancies. The development of ultrasensitive CEA diagnostic tools is crucial for early detection and progression observation of tumors. Herein, a dual signal amplified sandwich-type electrochemical immunoassay was developed based on dual-labeled mesoporous silica nanospheres as a signal amplifier, combined with NiO@Au decorated graphene as a conductive layer for ultrasensitive and rapid determination of CEA. The dual-labeled mesoporous silica (DLMS) nanosphere, which was synthesized by entrapping Au nanorod (Au NR) and horseradish peroxidase (HRP) in the channels of amine-functionalized SBA-15 followed by subordinate antibody (Ab2) conjugation which was denoted as Au NR@SBA-15/Ab2-HRP. The dual signal amplification from Au NR@SBA-15 and HRP enhanced the sensitivity of the proposed immunoassay. Consequently, the developed DLMS based immunosensor displayed ultra-low limits of detection of 5.25 fg/mL and a wide range of linearity (0.1–5 pg/mL), which was extended for CEA determination in real-time samples with improved recoveries of >98%. Therefore, this dual amplification prototype would cater to the clinical requirements for the ultrasensitive detection of CEA biomarkers.

Amperometric immunoassay for the carcinoembryonic antigen by using a peroxidase mimic consisting of palladium nanospheres functionalized with glutathione-capped gold nanoparticles on graphene oxide.

A composite nanoenzyme was used in a sandwich-type electrochemical immunoassay for the carcinoembryonic antigen (CEA). Hierarchically porous palladium nanospheres (Pd NPs) were functionalized with glutathione-capped gold nanoparticles (G-Au NPs) and then loaded onto graphene oxide (GO) to obtain a peroxidase mimicking nanoenzyme of type GO-supported G-Au/Pd. The composite can catalyze the oxidation of the substrate tetramethylbenzidine (TMB) by H2O2 to give blue-colored oxidized TMB within only 20s. This strong peroxidase activity, good conductivity and high specific surface area of the material make it a useful label for secondary antibodies (Ab2) for the detection of CEA. The cotton-like electrodeposited gold nanoparticles with good electrical conductivity were used to immobilize primary antibody (Ab1). The amperometric immunoassay has a detection range that extends from 10fg·mL-1 to 100ng·mL-1 at a working potential of -0.4V with addition of 5mmol·L-1 H2O2 as electrochemically active substrate, and the detection limit is as low as 3.2fg·mL-1 (S/N= 3). Graphical abstract Schematic of sandwich electrochemical immunosensor for the carcinoembryonic antigen. Electrodeposited gold used as substrate material, and Graphene oxide supported G-Au NPs functionalized porous Pd nanospheres (GO supported G-Au/Pd) as signal amplification platform, which catalyze the oxidation of tetramethylbenzidine (TMB).

Dually functional polyethylenimine-coated gold nanoparticles: a versatile material for electrode modification and highly sensitive simultaneous determination of four tumor markers.

A novel sandwich-type electrochemical multiplex immunoassay is described for simultaneous detection of the tumor biomarkers alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA) and interleukin-8 (IL-8). Polyethylenimine-coated gold nanoparticles (PEI-AuNPs) were used for both modification of a screen-printed carbon electrode (SPCE) and as labeling tags. The coated AuNPs can be easily adsorbed on the electrodes which also areloaded with the electroactive metal ions cadmium(II), lead(II) copper(II) and silver(I) and related secondary antibodies (Ab2). These give distinct voltammetric signals at -0.80, -0.55, -0.20 and + 0.05V, respectively (vs Ag/AgCl). Four corresponding capture antibodies (Ab1) were then conjugated to one of the electrodes. After a sandwich-type structure was formed by binding of the analytes and the labeling AuNPs, the electrochemical signal responses were recorded. Under the optimized testing conditions, there is a linear relationship in range from 0.25-10ngmL-1 for AFP, CEA and PSA, and from 0.50-100pgmL-1 for IL-8. The corresponding detection limits are 1.7, 1.6, 0.9 and 1.0fgmL-1, respectively. Cross reactivity, interferences and stability of the modified electrodes and of the signal nanotags are satisfying in that they can be stored for >4weeks without significant signal reduction. The method was successfully applied to the determination of the biomarkers in spiked human serum. Graphical abstract Poly(ethylenimine)-coated gold nanoparticles were used in a sandwich-type multiplex electrochemical immunosensor. Thecoated gold nanoparticles were used for both electrode modification and aselectrochemical nanotags. The resultingvmmunosensor exhibits excellent sensitivity for the four analytes studied,and also displaysselectivity and long-term stability.

Amperometric sandwich immunoassay for determination of myeloperoxidase by using gold nanoparticles encapsulated in graphitized mesoporous carbon.

An ultrasensitive sandwich-type electrochemical immunosensor was developed for the amperometric determination of serum myeloperoxidase (MPO). The method is making use of (a) gold nanoparticles encapsulated in graphitized mesoporous carbons (AuNP@GMC); and (b) horseradish peroxidase (HRP) labeled secondary antibody (HRP@Ab2) immobilized on AuNP@GMC. MPO capture antibody (Ab1) was immobilized on the electrode modified with an AuNP-graphene oxide nanocomposite. The sandwich immunoreaction leads to the formation of the complex composed of Ab1, MPO, and HRP@Ab2. An amplified electrochemical signal is produced by electrocatalytic reduction of H2O2 (at a typical voltage of -0.18V vs. Ag/AgCl) in the presence of enzymatically oxidized thionine. The peak current of thionine was measured using differential pulse voltammetry. Under optimized steady-state conditions, the reduction peak increases in the 1 to 300pg.mL-1 MPO concentration range, and the detection limit is 0.1pg.mL-1 (at S/N= 3). Graphical abstract Schematic presentation of AuNP-GO based sandwich-type electrochemical immunoassay for the determination of myeloperoxidase by using gold nanoparticles encapsulated in graphitized mesoporous carbons (AuNP@GMC) as a carrier for horseradish peroxidase (HRP) labeled secondary antibody (HRP@Ab2).

A non-enzymatic electrochemical immunoassay for quantitative detection of Escherichia coli O157:H7 using Au@Pt and graphene

Herein, a non-enzymatic sandwich-type electrochemical immunoassay was fabricated for quantitative monitoring of Escherichia coli O157:H7 (E. coli O157:H7). Silica coated Fe3O4 magnetic nanoparticles (Fe3O4@SiO2) were modified with mouse anti-E. coli O157:H7 monoclonal antibody (Ab1) to act as capture probes to reduce detection time and increase the sensitivity of the immunoassay. The Au@Pt nanoparticles were loaded on neutral red (NR) functionalized graphene to form composite complex rGO-NR-Au@Pt. rGO-NR-Au@Pt has high specific surface area and good biocompatibility. rGO-NR-Au@Pt was used as the carriers of detection antibodies (Ab2). Au@Pt catalyzed the reduction of hydrogen peroxide (H2O2) to detection of E. coli O157:H7 with the thionine (TH) as electron mediator to effectually amply the current signal. Under the optimized conditions, a linear relationship between the reduction peak current change (ΔIpc) and the logarithm of the E. coli O157:H7 concentration is obtained in the range from 4.0 × 103 to 4.0 × 108 CFU mL−1 and the limit of detection (LOD) is 4.5 × 102 CFU mL-1 at a signal-to-noise ratio of 3. The immunoassay exhibits acceptable specificity, reproducibility and stability on the detection of E. coli O157:H7. Furthermore, the immunoassay showed good performance in pork and milk samples. The results suggest that this immunoassay will be promising in the food safety area.

Sandwich-type electrochemical immunoassay based on Co3O4@MnO2-thionine and pseudo-ELISA method toward sensitive detection of alpha fetoprotein

In this study, a sensitive sandwich-type electrochemical immunosensor was fabricated for the detection of alpha fetoprotein (AFP) based on Co3O4@MnO2-thionine (Co3O4@MnO2-Th) and the screen printing technique. Meanwhile, the pseudo enzyme-linked immunosorbent assay (pseudo-ELISA) method was applied in fabricating the immunosensor. Screen-printed carbon electrode (SPCE) was applied for achieving the detection of AFP. Simultaneously, the amino functionalized Co3O4@MnO2-Th was employed as secondary label and could greatly improve the electrochemical response signal, which was beneficial for detecting AFP. Differential pulse voltammetry (DPV) was applied for the detection of AFP and electrochemical impedance spectroscopy (EIS) confirmed the successful fabrication of the immunosensor. Under optimal conditions, the immunosensor exhibited a linear response toward AFP in the range of 0.001–100 ng/mL, with a low detection limit of 0.33 pg/mL. Simultaneously, the proposed immunosensor displayed acceptable selectivity, excellent stability and well reproducibility. Furthermore, this proposed strategy may open up new ideas and find many potential applications in the detection of other tumor markers.

Ultrasensitive immunoassay of proteins based on in-situ enzymatic formation of quantum dots and microliter-droplet anodic stripping voltammetry

We report an ultrasensitive sandwich-type electrochemical immunoassay of proteins, on the basis of in situ enzymatic formation of CdS quantum dots (QDs), simultaneous chemical dissolution of CdS and cathodic preconcentration of Cd, and microliter-droplet anodic stripping voltammetry (ASV) detection directly on the immunoelectrode. The antibody 2 labeled with alkaline phosphatase (ALP) and Au nanoframes (Ab2-ALP-AuNFs) can catalyze hydrolysis of ascorbic acid 2-phosphate to form ascorbic acid and inorganic phosphate, and the latter can stabilize the formation of CdS QDs on the immunoelectrode through Cd2+-S2− reaction. A beforehand “potential control” and then an injection of 7 μL 0.1 M aqueous HNO3 lead to dissolution of the CdS QDs for simultaneous electrodeposition of metallic Cd, and ASV directly on the immunoelectrode is then conducted to quantify the antigen analyte. The use of AuNFs can increase the enzyme load and activity for improved signaling. Under optimized conditions, this method is used for ultrasensitive analysis of human immunoglobulin G (IgG) and human cardiopathy biomarker cardiac troponin I (cTnI), giving limits of detection (LODs, S/N = 3) of 1.2 fg mL−1 for IgG and of 1.1 fg mL−1 for cTnI (equivalent to 173 molecules in the 6 μL sample employed for cTnI analysis). The LOD of IgG is ca. 3 orders of magnitude lower than that of the fluorescent method by experimentally recording the emission spectrum of the CdS QDs at λex = 290 nm and λem = 500 nm.

Magnetoimmunosensor for simultaneous electrochemical detection of carcinoembryonic antigen and α-fetoprotein using multifunctionalized Au nanotags

We report a sandwich-type electrochemical immunoassay for simultaneous determination of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP), using multifunctionalized gold nanoparticles as signal tags. Two nanotags including thionine-Au nanoparticles and ferrocene-Au nanoparticles were synthesized and used as distinguishable indicators. The sensor was fabricated by means of co-immobilization of primary anti-CEA (AbCEA) and anti-AFP (AbAFP) antibodies on the surface of Fe3O4 nanoparticle, the strategy is based on observation distinct voltammetric peak derived from distinguish probe (AbCEA-Au-thionine and AbAFP-Au-ferrocene nanotags). Experimental results revealed that this immunosensor enabled simultaneous determination of CEA and AFP in the linear ranges of (0.05–120ngmL−1) and (0.05–100ngmL−1), respectively. The detection limits (DL) for AFP and CEA were 0.012ngmL−1 and 0.018ngmL−1, respectively (S/N=3). Furthermore, the methodology was also evaluated for the analysis of AFP and CEA in the clinical human serum specimens, the obtained results was in desirable agreement with clinical data.

A sandwich-type electrochemical immunoassay for ultrasensitive detection of non-small cell lung cancer biomarker CYFRA21-1

Many studies confirm that the aberrant expression of Cytokeratin 19 fragment 21-1 (CYFRA21-1) is highly correlated with non-small cell lung cancer (NSCLC), especially for squamous cell carcinoma. Herein, we report a sandwich-type electrochemical immunosensor based on signal amplification strategy of multiple nanocomposites to test CYFRA21-1 selectively and sensitively. The proposed immunosensor fabricated by three-dimensional graphene (3D–G), chitosan (CS) and glutaraldehyde (GA) composite on the glass carbon electrode (GCE) with a large surface area is prepared to immobilize primary antibodies (Ab1) and provide excellent conductivity. To further amplify the electrochemical signal, the trace tag on the foundation of gold nanoparticles (AuNPs) is coated with amino-functionalized carbon nanotube (MWCNT-NH2) nanocomposite through thionine linking, which provides more amino groups to capture more horseradish peroxidase-labeled antibodies (HPR-Ab2) and enhances the conductivity. Under optimal conditions, the developed immunosensor exhibits excellent analytical performance for the determination of CYFRA21-1 with a wide linear range from 0.1 to 150ng·mL−1 and a low detection limit (LOD) of 43pg·mL−1. Furthermore, satisfactory results are obtained for the determination of CYFRA21-1 in real clinical serum samples, indicating the potential of the immunoassay to be applied in clinical analysis.

Amperometric sandwich immunoassay for the carcinoembryonic antigen using a glassy carbon electrode modified with iridium nanoparticles, polydopamine and reduced graphene oxide

The authors describe a sandwich-type electrochemical immunoassay for sensitive determination of the carcinoembryonic antigen (CEA). It is based on the use of iridium nanoparticles (Ir NPs) acting as electrochemical signal amplifier on the surface of a glassy carbon electrode. At first, polydopamine-reduced graphene oxide (PDA-rGO) was employed to immobilize primary antibody (Ab1) against CEA. Secondly, Ir-NPs were used as a support for the immobilization of secondary antibody (Ab2) to afford signal labels. The large surface area of PDA-rGO and the excellent electro-oxidative H2O2-sensing properties of Ir NPs result in a sensitive assay for CEA. Operated best at a working voltage of −0.6 V (vs. SCE), the assay has a linear range that extends from 0.5 pg⋅mL−1 to 5 ng·mL−1, and the lower detection limit is 0.23 pg⋅mL−1. The immunosensor displays satisfactory reproducibility and stability, thus demonstrating a reliable immunoassay strategy for tumor biomarkers. It was applied to the determination of CEA in spiked serum samples.

Functional Magnetic Nanoparticles for Clinical Application: Electrochemical Immunoassay of Hepatitis B Surface Antigen and α-Fetoprotein

This work reports an efficient method to quantify the Hepatitis B surface antigen and α-fetoprotein in human serum using a functional magnetic nanoparticle-assisted sandwich-type electrochemical immunoassay. The Fe 3 O 4 magnetic nanoparticles were first modified with carboxyl functional groups to permit stable bioconjugation to the amine groups of most biological targets. The primary antibodies were then covalently stained on the surface of the functional magnetic nanoparticles, followed by the analyte and secondary antibodies, resulting in a sandwich-type (antibody-antigen-antibody/enzyme) immune complex. The secondary antibodies were labeled with horseradish peroxidase for the catalytic oxidation of 2-aminophenol to yield electrochemically reducible molecules. The separation using an external magnetic field guaranteed fast and reliable purification and enrichment of analytes. Quantitative analysis was performed upon representative clinical targets: Hepatitis B surface antigen and α-fetoprotein in human serum. The detection limits were 0.06 ng/mL for the former and 0.5 ng/mL for the latter, which were about 10 times lower than values obtained by conventional enzyme-linked immunosorbent assays. The reported method may be adopted as a general strategy for the sensitive and selective determination of additional proteins and biological molecules.

Poly(o-phenylenediamine)-carried nanogold particles as signal tags for sensitive electrochemical immunoassay of prolactin

A novel class of redox-active molecular tags, poly(o-phenylenediamine)-carried nanogold particles (GPPDs), was first synthesized and functionalized with horseradish peroxidase-anti-prolactin conjugates (HRP-anti-PRL). Thereafter, a specific sandwich-type electrochemical immunoassay was designed for determination of prolactin (PRL) by using GPPD-labeled HRP-anti-PRL conjugates as molecular tags on anti-PRL antibody-modified glassy carbon electrode. Compared with pure gold nanoparticles and poly(o-phenylenediamine) microspheres, the as-prepared GPPDs increased the surface coverage of the nanostructures, and enhanced the immobilization amount of biomolecules. Several labeling protocols compromising GPPD-labeled HRP-anti-PRL, nanogold particles-labeled HRP-anti-PRL and poly(o-phenylenediamine) microspheres-labeled HRP-anti-PRL, were investigated for detection of PRL, and improved analytical features were obtained with the GPPD-based strategy. With the GPPD labeling method, dependence of the electrochemical signals on the incubation time and pH of the assay solution were also studied. The strong attachment of HRP-anti-PRL to the GPPDs resulted in a good repeatability and intermediate reproducibility down to 9.8%. The dynamic concentration range spanned from 0.5 to 180ngmL−1 PRL with a detection limit of 0.1ngmL−1 at the 3Sblank level. No significant differences at the 95% confidence level were encountered in the analysis of 10 spiked blank cattle serum samples between the developed immunoassay and enzyme-linked immunosorbent assay method for determination of PRL.

Carbon nanospheres-promoted electrochemical immunoassay coupled with hollow platinum nanolabels for sensitivity enhancement

Two nanostructures including carbon nanospheres-graphene hybrid nanosheets (CNS-GNS) and hollow platinum nanospheres (HPtNS) were first synthesized by using direct electrolytic reduction and wet chemistry methods, respectively. Thereafter, a specific sandwich-type electrochemical immunoassay was designed for determination of carcinoembryonic antigen (CEA) by using HPtNS-labeled horseradish peroxidase-anti-CEA conjugates (HRP-anti-CEA) as molecular tags and anti-CEA-assembled CNS-GPS as sensing probes. Compared with pure graphene nanosheets, the presence of carbon nanospheres on the graphene increased the surface coverage of the substrate, and enhanced the immobilized amount of primary antibodies. Several labeling protocols, such as HRP-anti-CEA, solid platinum nanoparticle-labeled HRP-anti-CEA, and hollow platinum nanospheres-labeled HRP-anti-CEA, were investigated for determination of CEA and improved analytical features were obtained with hollow platinum nanosphere labeling. With the HPtNS labeling method, the effects of incubation time and pH on the current responses of the immunosensors were also studied. The strong attachment of biomolecules to the CNS-GPS and HPtNS resulted in a good repeatability and intermediate precision down to 10.2%. The dynamic concentration range spanned from 0.001ngmL−1 to 100ngmL−1 CEA with a detection limit of 1.0pgmL−1 at the 3Sblank level. No significant differences at the 0.05 significance level were encountered in the analysis of 10 clinical serum samples between the developed immunoassay and the commercially available electrochemiluminescent method for determination of CEA.

Ultrasensitive electrochemical immunoassay for BRCA1 using BMIM·BF4-coated SBA-15 as labels and functionalized graphene as enhancer

BRCAl is an anti-oncogene in women, who are genetically predisposed to breast and ovary cancer. The detection of BRCA1 can offer an opportunity to characterize the function of genetic features in breast and ovarian cancer and to screen breast or ovarian cancer patients. In this study, we designed a new label and fabricated a novel sandwich-type electrochemical immunoassay for the ultrasensitive detection of BRCAl. Horseradish peroxidase (HRP) was entrapped in the pores of amino-group functionalized SBA-15 and the secondary antibody (Ab2) combined with SBA-15 by covalent bond. Ionic liquid (IL) was added into the mixed solution of SBA-15/HRP/Ab2 and application of IL increased the electrochemical activity of HRP and promoted electron transport. The synergistic effect between IL, SBA-15, Ab2 and HRP could retain the bioactivity of HRP and Ab2. The sensitivity of the sandwich-type immunosensor using SBA-15/HRP/Ab2/BMIM·BF4 as labels for BRCA1 detection was much higher than that using either SBA-15/HRP/Ab2 or SBA-15/Ab2 as labels. Under optimal conditions, the electrochemical immunoassay exhibited a wide working range from 0.01 to 15 ng/mL with a detection limit of 4.86 pg/mL BRCA1. The precision, reproducibility, and stability of the immunoassay were acceptable.

Ultrasensitive Electrochemical Immunoassay of Staphylococcal Enterotoxin B in Food Using Enzyme-Nanosilica-Doped Carbon Nanotubes for Signal Amplification

A new sandwich-type electrochemical immunoassay for ultrasensitive detection of staphylococcal enterotoxin B (SEB) in food was developed using horseradish peroxidase-nanosilica-doped multiwalled carbon nanotubes (HRPSiCNTs) for signal amplification. Rabbit polyclonal anti-SEB antibodies immobilized on the screen-printed carbon electrode (SPCE) and covalently bound to the HRPSiCNTs were used as capture antibodies and detection antibodies, respectively. In the presence of SEB analyte, the sandwich-type immunocomplex could be formed between the immobilized anti-SEB on the SPCE and anti-SEB-labeled HRPSiCNTs, and the carried HRP could catalyze the electrochemical reduction of H2O2 with the help of thionine. The high content of HRP in the HRPSiCNTs could greatly amplify the electrochemical signal. Under optimal conditions, the reduction current increased with the increase of SEB in the sample, and exhibited a dynamic range of 0.05-15 ng/mL with a low detection limit (LOD) of 10 pg/mL SEB (at 3σ). Intra- and interassay coefficients of variation were below 10%. In addition, the assay was evaluated with SEB spiked samples including watermelon juice, soymilk, apple juice, and pork food, receiving excellent correlation with results from commercially available enzyme-linked immunosorbent assay (ELISA).

Highly sensitive electrochemical immunoassay for human IgG using double-encoded magnetic redox-active nanoparticles

A new sandwich-type electrochemical immunoassay was developed for the detection of human IgG using doubly-encoded and magnetic redox-active nanoparticles as recognition elements on the surface of a glassy carbon electrode modified with anti-IgG on nanogold particles. The recognition elements were synthesized by coating magnetic Fe3O4 nanoparticles with Prussian blue nanoparticles and then covered with peroxidase-labeled anti-IgG antibodies (POx-anti-IgG) on Prussian blue nanoparticles. The immunoelectrode displays very good electrochemical properties towards detection of IgG via using double-encoded magnetic redox-active nanoparticles as trace and hydrogen peroxide as enzyme substrate. Its limit of detection (10 pmol·L−1) is 10-fold better than that of using plain POx-anti-IgG secondary antibodies. The method was applied to the detection of IgG in serum samples, and an excellent correspondence with the reference values was found.

Chemical/Biochemical Preparation of New Polymeric Bionanocomposites with Enzyme Labels Immobilized at High Load and Activity for High-Performance Electrochemical Immunoassay

We report on the chemical/biochemical preparation of novel polymeric bionanocomposites (PBNCs) material consisting of polydopamine (PDA), Pt nanoparticles (PtNPs), glucose oxidase (GOx), Au nanoparticles (AuNPs), and antibody for high-performance sandwich-type electrochemical immunoassay. The preparation includes chemical synthesis of PDA−PtNPs−GOx bionanocomposites with abundant GOx and PtNPs immobilized in/on the PDA matrix and biochemical synthesis of AuNPs/PDA−PtNPs−GOx bionanocomposites with highly dispersed AuNPs on the PDA−PtNPs−GOx for successive efficient adsorption of antibody and GOx, finally yielding GOxads/antibody/AuNPs/PDA−PtNPs−GOx bionanocomposites (PBNCs 1). The thus-prepared PBNCs 1 exhibit high load and activity of the enzyme label immobilized both in the interior and on the surface for ultrasensitive signal readout, as well as high load of antibody on the surface for significant immuno-recognition efficiency. Transmission electron microscopy, atomic force microscopy, UV−vis spectrosco...