Competitive Immunosensor Research Articles

A competitive photoelectrochemical immunosensor for the detection of diethylstilbestrol based on an Au/UiO-66(NH2)/CdS matrix and a direct Z-scheme Melem/CdTe heterojunction as labels

A novel photoelectrochemical (PEC) bioassay protocol for diethylstilbestrol (DES) was systematically proposed. The Au/UiO-66(NH2) hybrid with countless active sites was fabricated using an in situ reduction strategy and was subsequently decorated with CdS nanoparticles (NPs) by a sequential chemical bath deposition process. The resulting Au/UiO-66(NH2)/CdS composites were exploited as a sensing matrix for the first time. The excellent photocurrent response of the as-prepared hybrid under visible light is attributed to its improved photo-electric conversion efficiency due to the local surface plasma resonance (LSPR) effect of Au and the matching energy-level structure between UiO-66(NH2) and CdS. In addition, the competitive strategy for the detection of DES was devised by employing the direct Z-scheme Melem/CdTe heterojunction covalently conjugated with bovine serum albumin (BSA)-DES as signal tags. As a consequence, a cascade-like band-edge level was obtained, which could effectively promote the transfer of photogenerated charges and markedly improve the photocurrent. On the basis of the above strategy, the concentrations of DES could be detected through the competitive binding of DES antibodies (Anti-DES) with either Melem/CdTe/BSA-DES or free DES. Under optimal conditions, the linear range for the detection of DES was 0.1 pg mL−1 to 20 ng mL−1. The detection limit was as low as 0.06 pg mL−1 (S/N = 3). Furthermore, the designed PEC DES-sensing approach has acceptable selectivity, reproducibility, and stability and offers great promise for the detection of small molecules in biomedical, food and environmental samples.

A competitive-type photoelectrochemical immunosensor for aflatoxin B1 detection based on flower-like WO3 as matrix and Ag2S-enhanced BiVO4 for signal amplification

A competitive photoelectrochemical (PEC) immunosensor was successfully utilized for quantitative detection of aflatoxin B1 (AFB1). The flower-like tungsten oxide (WO3) hierarchical architectures with large specific surface areas and porous nanostructure was prepared as a matrix material for immobilizing of BSA-AFB1. The Ag ions-modified bismuth vanadate nano-polyhedron (BiVO4@Ag+) was utilized as labels for immobilization anti-AFB1 through affinity specific binding. The Ag2S was prepared in-situ growth by immediately deposition the S2− onto the BiVO4@Ag+. The high photocurrent intensity was generated by the obtained BiVO4@Ag2S under visible-light irradiation. Moreover, excellent sensitivity was achieved by using the WO3 as the matrix and BiVO4@Ag2S as labels to enhance photocurrent response and improve the photocurrent conversion efficiency. Under optimal conditions, the competitive PEC immunosensor showed wide linear range from 1.0 pg mL−1 to 120 ng mL−1 with limit detection of 0.28 pg mL−1 for AFB1. The biosensor was performed with good reproducibility, acceptable stability, and high specificity, which indicated potential application in the detection of other toxic small molecules.

Ultrasensitive competitive method-based electrochemiluminescence immunosensor for diethylstilbestrol detection based on Ru(bpy)32+ as luminophor encapsulated in metal–organic frameworks UiO-67

In this work, Ru(bpy)32+ encapsulated in metal–organic frameworks (MOFs) UiO-67 (Ru(bpy)32+/UiO-67) as luminophor was easily prepared and firstly applied in constructing an electrochemiluminescence (ECL) immunosensor to efficiently estimate diethylstilbestrol (DES). The competitive method-based ECL immunosensor platform was fabricated by amino-silicon dioxide which possesses large surface area. The poriness of UiO-67 was splendid so that Ru(bpy)32+ could be easily encapsulated. Ru(bpy)32+/UiO-67 with excellent ECL luminescence signal existed large specific surface area for easily labeled with antibodies. DES competed with bovine serum albumin-diethylstilbestrol (BSA-DES) for binding to antibody-specific sites in the constructed immunosensor. However DES was micromolecule, which was easier to bond to antibodies than BSA-DES. The ECL signal was gradually decreases with the increase of the concentration of DES. Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 0.01 pg mL−1 to 50 ng mL−1 with a low detetion limit of 3.27 fg mL−1 (S/N = 3). The novel fabricated immunosensor with interference immunity and high stability may cause an attractive approach for the other targets determination.

Enzyme immunoassay (ELISA/immunosensor) for a sensitive detection of pork adulteration in meat

ELISA/immunosensor were developed for a sensitive detection of pork adulteration in meat. Two formats of ELISA were performed. First, an extracted IgG was directly immobilized in the microplate. This assay allowed an identification of 0.01% as level of pork adulteration in 14 h15 min. In order to decrease the time of the assay, a competitive ELISA was developed by immobilizing IgG standard, which compete with the extracted IgG. This assay allowed a detection of 0.1% of pork adulteration in 45 min.Furthermore, two formats of electrochemical immunosensors were elaborated using the electro-entrapment of IgG in polymer modified graphite paste electrode. First, a direct immunosensor was capable of identifying 0.1 and 1% in raw and cooked meat respectively in 2 h. The second format was based on a competitive immunosensor, which was able to detect 0.01% of pork adulteration within 20 min.Both competitive immunoassays revealed high sensitivity, good specify and reduced analysis time.

An ultrasensitive competitive immunosensor using silica nanoparticles as an enzyme carrier for simultaneous impedimetric detection of tetrabromobisphenol A bis(2-hydroxyethyl) ether and tetrabromobisphenol A mono(hydroxyethyl) ether

Based on our produced polyclonal antibody capable of recognizing tetrabromobisphenol A bis(2-hydroxyethyl) ether (TBBPA-DHEE) and tetrabromobisphenol A mono(hydroxyethyl) ether (TBBPA-MHEE) (cross-reactivity, 100% for TBBPA DHEE; 98.7% for TBBPA MHEE), an important derivative and byproduct of tetrabromobisphenol A (TBBPA), respectively, a novel ultrasensitive competitive immunosensor was established using an electrochemical impedimetric strategy for the simultaneous detection of both chemicals. A significantly amplified electrochemical impedance spectroscopy (EIS) for quantitative target analysis was obtained through (i) the biocatalytic precipitation of 4-chloro-1-naphthol (CN) on the electrode surface triggered by horseradish peroxidase (HRP) and (ii) increased amounts of the enzyme with HRP-loaded silica nanoparticles carrying poly-brushes (SiO2@PAA) as labels, achieving a remarkable improvement in catalytic performance. Under the optimized conditions, the immunosensor showed satisfactory accuracy (recovery, 84.6–118%) and a good linear range (0.21- 111.31ng/mL) with a limit of detection (LOD) of 0.08ng/mL (S/N = 3) for TBBPA DHEE and TBBPA MHEE. In addition, the proposed approach was used to analyse real environmental water samples, and our results indicated that this immunosensor had great potential for the determination of the trace pollutants in aquatic environments.

Electrochemiluminescent competitive immunosensor based on polyethyleneimine capped SiO2 nanomaterials as labels to release Ru(bpy)32+ fixed in 3D Cu/Ni oxalate for the detection of aflatoxin B1

In this work, a highly-efficient competitive method-based electrochemiluminescence (ECL) immunosensor was proposed based on {[Ru(bpy)3][Cu2xNi2(1−x)(ox)3]}n (Cu/Ni/Ru) as luminophor to efficiently detect aflatoxins B1 (AFB1). Cu/Ni/Ru exhibited excellent ECL behavior. While polyethyleneimine capped silicon dioxide (PEI@SiO2) could decrease the ECL performance of Cu/Ni/Ru due that PEI could destroy the structure of Cu/Ni/Ru via producing the complex between PEI and metal ions (Cu(II)/Ni(II)), inducing the release of Ru(bpy)32+. Since Au nanoparticles can directly combine with antibody and antigen, the Cu/Ni/Ru and PEI@SiO2 was functionalized by Au nanoparticles. The quantitative detection of AFB1 was based on the competitive binding between AFB1-bovine serum albumin labeled Au-PEI@SiO2 (Au-PEI@SiO2-AFB1-BSA) and free AFB1 with antibody-AFB1 which immobilized on Au-Cu/Ni/Ru. The ECL signal increased with augmenting the concentrations of the free AFB1 due to less Au-PEI@SiO2-AFB1-BSA combining with antibodies. Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 0.01ngmL−1 to 100ngmL−1 with a detection limit of 0.0039ngmL−1 (S/N = 3). The proposed immunosensor also provides a promising approach for ultrasensitive detection of other mycotoxins.

Competitive amperometric immunosensor for determination of p53 protein in urine with carbon nanotubes/gold nanoparticles screen-printed electrodes: A potential rapid and noninvasive screening tool for early diagnosis of urinary tract carcinoma

Since p53 protein has become recognized biomarker for both diagnostic and therapeutic purposes in oncological diseases with particular relevance for bladder cancer, it is highly desirable to search for a novel sensing tool for detecting the patient's p53 level at the early stage. Here we report the first study on the development and validation of a novel disposable competitive amperometric immunosensor for determination of p53 protein at subnanomolar levels, based on p53 immobilization on gold nanoparticles/carbon nanotubes modified screen-printed carbon electrodes. The assay protocol requires the use of single anti-p53 mouse monoclonal antibody (DO-7 clone), able to recognize both wild-type and mutant p53. The developed immunosensor as well as the protocol of the electrochemical immunoassay were optimized by means of an experimental design procedure to assess the suitability of the device to be validated and applied for the determination of p53 in untreated and undiluted urine samples. It was found that the developed competitive immunodevice was able to achieve wide linear range detection of wild-type p53 from 20 pM to 10 nM with a low detection limit of 14 pM in synthetic urine samples, suggesting the sensor's capability of working in a complex sample matrix. The excellent performance results also in terms of selectivity, trueness and precision, coupled with the advantages of an easy preparation and low-cost assay in contrast to other methods which require very complex, time-consuming and costly nanostructured architectures, makes the developed competitive immunosensor an analytically robust diagnostic tool, valuable for implementation of screening and follow-up programs in patients with urologic malignancies.

Enzymatic DMFC device used for direct analysis of chloramphenicol and a comparison with the competitive immunosensor method

This paper aims at investigating the possibility to use a catalytic “direct methanol fuel cell” (DMFC), previously optimized, for the analysis of chloramphenicol. The alcohol dehydrogenase enzyme was immobilized within it, for a direct analysis of the antibiotic. The analytical results confirmed the validity of this idea and highlighted the advantages, especially those associated with a remarkable decrease in the response time. A comparison with the use of the same device, but without the alcohol dehydrogenase enzyme (ADH), was made. The LOD was 8×10−7M, while a 1.0–5.0mM linear range was obtained in the presence of chloramphenicol. This also shows a high specificity for chloramphenicol. Finally, the main analytical data were compared and discussed with those obtained using a conventional amperometric immunosensor, assembled by us to this purpose, which uses a typical competitive format.

Electrochemical indirect competitive immunoassay for ultrasensitive detection of zearalenone based on a glassy carbon electrode modified with carboxylated multi-walled carbon nanotubes and chitosan

The authors describe an electrochemical immunoassay for ultrasensitive detection of the mycotoxin zearalenone (ZEA). A nanocomposite was prepared from carboxy-functionalized multi-walled carbon nanotubes and chitosan (cMWCNTs/Chit). The morphology and electrochemical performance of the materials was characterized by field-emission scanning electron microscopy, atomic force microscopy, differential pulse voltammetry, cyclic voltammetry, and electrochemical impedance spectroscopy. In this assay, ZEA–BSA conjugated covalently to activated cMWCNTs/Chit film, then the indirect competition between ZEA–BSA and free ZEA when immobilization of excess anti-ZEA. The secondary antibody is labeled with the enzyme alkaline phosphatase which can hydrolyze the substrate 1-naphthylphosphate to produce 1-naphthol which gives a stable and strong anodic electrochemical signal at a low working voltage of 0.3 V (vs. Ag/AgCl). The use of the modified GCE results in a strongly enhanced electrochemical current response. Compared with conventional methods, the established immunosensor exhibited a high level of sensitivity. Under optimal conditions, this immunoassay can quantify ZEA in the 10 pg·mL−1 to 1000 ng·mL−1 concentration range with a detection limit of 4.7 pg·mL−1 and the sensitivity is 0.51 μA·μM−1·cm−2. The method was applied to the determination of ZEA in cereal and feedstuff samples. Results showed satisfactory recovery and good consistency with high-performance liquid chromatography. Therefore, the indirect competitive electrochemical immunosensor provide a viable tool based on bioanalysis.

Indirect competitive electrochemical immunosensor for hepatitis A virus antigen detection

Indirect competitive electrochemical immunosensor for hepatitis A virus antigen (HAV) detection was developed by immobilizing HAV on the surface of carbon nanopowder paste electrode. The monitoring of the target HAV antigen was performed using a secondary antibody labeled with peroxidase. The analytical signal corresponds to an amperometric current resulting from the addition of hydrogen peroxide and hydroquinone as a redox mediator. A calibration curve was obtained by plotting the intensity of the amperometric signal versus the concentrations of HAV antigens. A low detection limit for HAV antigen was attained and estimated to be 26·10−5IU·mL−1. The sensitivity was also investigated and compared with indirect competitive ELISA. Moreover, the immunosensor provides exact results over a broad linear range from 2·10−4 to 5·10−3IU·mL−1, (R2=0.998). The average relative standard deviation (RSD) of this method was less than 3% for the assays performed on the same day and 7% for the measurements made on different days. The immunosensor exhibits a good stability and high biological selectivity for HAV antigen detection and was successfully applied for the determination of HAV in water samples.

Competitive voltammetric morphine immunosensor using a gold nanoparticle decorated graphene electrode

The authors describe a voltammetric immunosensor for morphine (MOR) that is based on a graphene screen printed electrode (GSPE) modified with gold nanoparticles (AuNP) which were electrodeposited on its surface. The gold nanoparticles were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy and cyclic voltammetry. Cysteamine was then self-assembled on the gold-modified electrodes via thiol interaction in order to introduce terminal amino groups to the electrode surface. The electrodes were then used to fabricate the immunosensor by covalent immobilization of antibodies against MOR. The sensor works on the basis of a competition between MOR and the morphine-bovine serum albumin (MOR-BSA) conjugate for the immobilized antibodies on the sensor surface and the resulting change in the square wave voltammetry reduction current using the hexacyanoferrate system as an electrochemical probe. The competitive immunoassay enables sensitive and selective detection of MOR in the 0.1 to 100 ng·mL−1 MOR concentration range, with a 90 pg·mL−1 detection limit. The method was applied to the determination of MOR in spiked saliva samples and showed high recoveries. We believe that this immunosensor offers a simple, rapid, sensitive and accurate MOR test.

Innovative gold-free carbon nanotube/chitosan-based competitive immunosensor for determination of HIV-related p24 capsid protein in serum

The first competitive immunosensor for the determination of HIV-related p24 capsid protein, realized and validated in undiluted human serum.

Silicon phthalocyanine-decorated TiO2 mesocrystal coupled with multifunctional all-carbon structure for multistep cascade signal amplifier in photoelectrochemical immunoassay

Herein, a new photoelectrochemical sensor for aflatoxin B1 (AFB1) detection was established based on the all-carbon nanohybrids as transducer scaffold accompanied with quasi-octahedral TiO2 mesocrystal (QOTM) absorbed dentritic Si phthalocyanines (SiPcs) as the efficient bioprobe. Specifically, the all-carbon nanocomposites exhibited fine photoelectric properties due to the photo-generated electrons of carbon quantum dots could be rapidly accepted by conductive carbon nanohorns, which evidently expedited the electron transport, and the nanohybrids were first employed as affinity support to anchor AFB1 antibody. The dentritic SiPcs sensitized QOTM presented better photocurrent response and stability in comparison with QOTM alone due to impressive light-harvesting capability of the dentritic SiPcs. Under light irradiation, the photo-induced electrons of SiPcs migrated to the QOTM, and successively transferred to carbon quantum dots as electron relay mediator then via carbon nanohorns to the electrode surface based on suitable band alignment. This hierarchical charge transport cascade paradigm efficiently impeded the charge recombination, resulting in noticeably increased photocurrent. In this competitive immunosensor, the AFB1 standards compete with the labeled AFB1, leading to photocurrent decreased with the increasing target concentration in a wide linear range of 10−6–102ng/mL. The newly developed methodology provided a versatile approach for ultrasensitive detection of small molecules.

Investigate electrochemical immunosensor of cortisol based on gold nanoparticles/magnetic functionalized reduced graphene oxide

A sensitively competitive electrochemical immunosensor for the detection of cortisol was successfully developed based on gold nanoparticles and magnetic functionalized reduced graphene oxide (AuNPs/MrGO). In order to construct the base of the immunosensor, the MrGO was initially fabricated by chemical cross-linking and used to modify the nafion pretreated glassy carbon electrode. Subsequently, the surface of electrode was modified by AuNPs via electrochemical deposition. A variety of cortisol (Cor) can be firmly loaded in the AuNPs/MrGO with large specific surface area and good bioactivity to construct the basic electrode (Cor/AuNPs/MrGO/Nafion@GCE), which was characterized by the cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), respectively. Due to the cortisol on the surface of basic electrode and samples can competitively combine with the cortisol antibody labelled by horseradish peroxidase (HRP-Strept-Biotin-Ab). Finally, the detection signal of electrochemical immunosensor (HRP-Strept-Biotin-Ab-Cor/AuNPs/MrGO/Nafion@GCE) in the test liquid had negative correlations with the concentration of cortisol in samples. The AuNPs/MrGO with excellent electrical conductivity being applied, the electrochemical response of the immunosensor was immensely amplified. The immunosensor displayed excellent analytical performance for the detection of cortisol range from 0.1 to 1000ng/mL with a detection limit of 0.05ng/mL at 3σ. Moreover, compared the developed immunoassay with commercially available enzyme linked immunosorbent assay, the proposed method showed good precision, acceptable stability and reproducibility, indicating the immunosensor could be used for the sensitive, efficient and real-time detection of cortisol in real samples. Therefore, the present strategy provides a novel and convenient method for clinical determination of cortisol.

A competitive luminol chemiluminescence immunosensor based on a microfluidic chip for the determination of ractopamine.

Herein, a competitive luminol chemiluminescence immunosensor based on a microfluidic chip was developed to detect ractopamine (RCT) both in phosphate buffer and swine urine samples. The immunosensor can provide a liner range of 0.5-40 ng/mL and a high sensitivity with a limit of detection of 0.97 ng/mL for RCT detection in swine urine. Good rates of recovery in negative swine urine samples were achieved over the RCT concentration ranging from 0.5 to 40 ng/mL. The proposed method offered a promising analytical scheme for the on-site determination of RCT.

Optical waveguide lightmode spectroscopy technique-based immunosensor development for aflatoxin B1 determination in spice paprika samples

Optical waveguide lightmode spectroscopy (OWLS) technique has been applied to label-free detection of aflatoxin B1 in a competitive immunoassay format, with the aim to compare the analytical goodness of the developed OWLS immunosenor with HPLC and enzyme-linked immunosorbent assay (ELISA) methods for the detection of aflatoxin in spice paprika matrix. We have also assessed applicability of the QuEChERS method prior to ELISA measurements, and the results were compared to those obtained by traditional solvent extraction followed by immunoaffinity clean-up. The AFB1 content of sixty commercial spice paprika samples from different countries were measured with the developed and optimized OWLS immunosensor. Comparing the results from the indirect immunosensor to that obtained by HPLC or ELISA provided excellent correlation (with regression coefficients above 0.94) indicating that the competitive OWLS immunosensor has a potential for quick determination of aflatoxin B1 in paprika samples.

A competitive love wave immunosensor for detection of okadaic acid based on immunogold staining method

This work describes an immunosensor for the determination of okadaic acid (OA) using a Love-wave sensor with the immunogold staining method. In this assay, OA-BSA was captured by the anti-BSA antibody (BSA-MAb) bond with staphylococcal protein A (SPA) on the sensor surface via Fc regions. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were employed to monitor the fabrication steps of immunosensor using the redox probe of K3Fe(CN)6. An indirect competitive immunoassay format was employed to detect OA after the immunosensor was prepared and gold nanoparticles (AuNPs) were introduced to amplify the phase signal. The results demonstrated that developed immunosensor was able to specifically respond to OA ranging from 10 to 150ng/mL, providing limit of detection of 5.5ng/mL. When applied to determine OA concentration in real sample, the results agreed well with those obtained by the commercial ELISA assay. The developed immunosensor is therefore suitable and has great application potential in detection of OA in shellfish.

An ultrasensitive competitive immunosensor for impedimetric detection of microcystin-LR via antibody-conjugated enzymatic biocatalytic precipitation

Herein we design a sensitive impedimetric immunosensor for quantitative detection of microcystin-LR (MC-LR) by combining antibody-conjugated enzymatic biocatalytic precipitation reaction and a competitive format. MC-LR-bovine serum albumin (MC-LR-BSA), which conjugated onto the nanogold-modified glassy carbon electrode through cysteamine and glutaraldehyde, was introduced as an analyte competitor. Competitive assays were performed by incubating the target MC-LR and the analyte competitor with horseradish peroxidase-labeled monoclonal anti-MC-LR antibodies (HRP-mAb). After the competitive reaction, HRP were introduced as the signal labels onto the immunosensor, accelerating the biocatalytic precipitation reaction of substrate 4-chloro-1-naphthol with the aid of H2O2. The enzymatically generated insoluble precipitate formed an insulting layer on the electrode surface and efficiently restricted the electron transfer of redox probe Fe(CN)64−/3−, resulting in an enhanced amplification of impedimetric signal. The impedimetric signal decreased with the increase of concentration of MC-LR, and exhibited a dynamic linear range from 0.01 to 100μgL−1 with a detection limit of 0.004μgL−1. The proposed immunosensing method can be applied for rapid detection of MC-LR in real water samples with accuracy comparable to the high-performance liquid chromatography. The platform shows good precision, high sensitivity and specificity, which opening a promising horizon for monitoring of cyanobacterial pollution in environmental matrices.

Competitive immunosensor based on gliadin immobilization on disposable carbon-nanogold screen-printed electrodes for rapid determination of celiotoxic prolamins

The first competitive disposable amperometric immunosensor based on gliadin-functionalized carbon/nanogold screen-printed electrodes was developed for rapid determination of celiotoxic prolamins. To date, no competitive spectrophotometric or electrochemical immunoassays have yet been successfully applied to gluten detection in processed food samples, which require the use of complex prolamin extraction solutions containing additives with denaturing, reducing and disaggregating functions. Thus, in this work, great effort was put into the optimization and performance evaluation of the immunosensor in terms of suitability as a screening tool for analysis of cereal-based food samples. For this purpose, aqueous ethanol or complex extraction mixtures, as the patented Cocktail Solution®, were proved effective in the extraction of gliadin. Good sensitivity was achieved after optimization of the immunocompetitive assay, giving limit of detection and limit of quantitation of 8 and 22ng/ml of gliadin, respectively, for ethanol extracts. The immunosensor was proved to be suitable also for samples extracted with Cocktail Solution® after a proper dilution. Analysis of real samples of different flours proved the suitability of the immunosensing device as a powerful tool for safety assessment of raw materials used for the formulation of dietary products for celiac disease patients. This immunosensor combines good analytical performance using a very simplified set-up protocol with suitability for rapid screening analysis performed using inexpensive and portable instrumentation. Graphical abstract Depiction of the development and working principle of the competitive immunosensor.

Dual-responsive competitive immunosensor for sensitive detection of tumor marker on g-CN/rGO conjugation

A new dual mode competitive immunosensing platform for sensitive determination of α-fetoprotein (AFP) combined photoelectrochemical and electrochemical methods was designed on graphite carbon nitride and reduced graphene oxide (g-CN/rGO) conjugate support. To construct such versatile support, polyamidoamine dendrimer (PAAD) decorated graphene oxide (GO) as substrate was reduced via potentiostatic technology, and the prepared rGO-PAAD nanocomposite was utilized as matrix to immobilize g-CN and capture antigen, and as electron transporter to mediate electron transfer for enhancement of photoelectrochemical and electrochemical signals. And then, horseradish peroxidase labeled antibody was competitively captured onto sensing interface with free antigen in incubated solution. Photoelectrochemical signal originated from g-CN, an excellent photoactive material with large photo-induced electric response, and was amplified by rGO-PAAD nanocomposite. The resultant concentration positively related linear calibration range was from 1pg/mL to 40ng/mL with ultralow detection limit of 1pg/mL. On the other hand, the enzyme-induced electrocatalytical signal amplification was produced by using hydroquinone as reactive substrate and horseradish peroxidase as reactive enzyme in existence of H2O2, which displayed a wide dynamic linear range from 0.1ng/mL to 160ng/mL with low detection limit of 0.1ng/mL. Additionally, the satisfying selectivity, good reproducibility and high consistency of dual-signal outputs of this designed dual-responsive immunosensor demonstrated the promising application in developing sensitive and accurate immunosensor for clinical test and disease diagnosis.