Signal Tags Research Articles

Ultrasensitive electrochemical immunoassay for CEA through host–guest interaction of β-cyclodextrin functionalized graphene and Cu@Ag core–shell nanoparticles with adamantine-modified antibody

A novel non-enzymatic immunoassay was designed for ultrasensitive electrochemical detection of carcino-embryonic antigen (CEA) using β-cyclodextrin functionalized Cu@Ag (Cu@Ag-CD) core–shell nanoparticles as labels and β-cyclodextrin functionalized graphene nanosheet (CD-GN) as sensor platform. CD-GN has excellent conductivity which promoted the electric transmission between base solution and electrode surface and enhanced sensitivity of immunosensor. In addition, owing to supramolecular recognition of CD-GN for the guest molecule, quite a few synthesized adamantine-modified primary antibodies (ADA-Ab1) were immobilized on the CD-GN by supramolecular host–guest interaction between CD and ADA. Cu@Ag-CD as a signal tag could be captured by ADA-modified secondary antibody (ADA-Ab2) through a host–guest interaction, leading to a large loading of Cu@Ag nanoparticles with high electrical conductivity and catalytic activity. The fabricated immunosensor exhibits excellent analytical performance for the measurement of CEA with wide range linear (0.0001–20ng/mL), low detection limit (20fg/mL), good sensitivity, reproducibility and stability, which provide an enormous application prospect in clinical diagnostics.

Multiplexed electrochemical immunoassay using streptavidin/nanogold/carbon nanohorn as a signal tag to induce silver deposition

An ultrasensitive multiplexed immunoassay method was developed by using streptavidin/nanogold/carbon nanohorn (SA/Au/CNH) as a novel signal tag to induce silver enhancement for signal amplification. The Au/CNH was prepared by in situ growth of nanogold on carboxylated CNH and functionalized with streptavidin. The SA/Au/CNH showed well dispersibility in physiological buffer and could sever as a common tracing tag to recognize biotinylated signal antibody. The immunosensor array was prepared on disposable screen-printed electrodes. Through sandwich-type immunoreaction and biotin-streptavidin affinity reaction, the SA/Au/CNH tag was captured on the immunoconjugates to induce silver deposition and amplify the electrochemical stripping signals. Using α-fetoprotein and carcinoembryonic antigen as model analytes, the proposed method showed wide linear ranges with the detection limits down to 0.024pgmL−1 and 0.032pgmL−1, respectively, and eliminated completely signal cross-talk between adjacent immunosensors. It provided a convenient, high-efficient and ultrasensitive electrochemical detection route for biological analytes, showing great potential in clinical application.

An Ultrasensitive Simultaneous Multianalyte Immunoassay Based on Arsenic and Mercury Ions Labeled SiO2@Au Nanoparticle Probes

Abstract An ultrasensitive immunoassay was developed on the basis of As3+ and Hg2+ labeled SiO2@Au nanoparticles (NPs) probe and hydride generation-atomic fluorescence spectrometry (HG-AFS) for the detection of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9). Firstly, amino SiO2@Au NPs were synthesized for selective absorption of As3+ and Hg2 to form As(Hg)-SiO2@Au NPs signal tags, respectively. Subsequently, the prepared probes were labeled on the secondary antibody (Ab2) of CEA and CA 19-9, respectively. Based on the sandwich immunoassay scheme, corresponding Ab1, antigen and Ab2 modified with probes, were conjugated to form the immunocomplex on the bottom of 96-well plate at room temperature. After the immunocomplex were dissolved in an alkali solution, As3+ and Hg2+ were released into the solution and detected by HG-AFS, the atomic fluorescence intensity of As3+ and Hg2+ were proportional with logarithms of CEA and CA 19-9 concentration, then indirect quantitative analysis of CEA and CA 19-9 was realized. This assay was based on the determination of the concentration of As3+ and Hg2+ by HG-AFS for quantization of the corresponding CEA and CA 19-9 antigen. The assay showed a wide linear range of 0.001–100.0 µg L−1 for CEA and 0.01–80 U mL−1 for CA 19-9, with the detection limit of 0.5 ng L−1 and 0.005 U mL−1, respectively. This method was used in the determination of real serum samples, and the results were consistent with that from ELISA. This assay showed three orders of magnitude of sensitivity higher than that of ELISA, which provided promising simultaneous multianalyte immunoassays (SMIAs) in the early diagnosis of pancreatic cancer.

Simultaneous electrochemical detection of multiple tumor markers using functionalized graphene nanocomposites as non-enzymatic labels

In this work, we designed a novel sandwich-type electrochemical immunosensor for simultaneous detection of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) using non-enzymatic multiple-label method. Firstly, gold nanoparticles (AuNPs) decorated reduced graphene oxide (rGO)-carried prussian blue (PB) and AuNPs decorated rGO-carried thionine (Thi) (denoted as AuNPs-PB@rGO and AuNPs-Thi@rGO, respectively) were employed as distinguishable signal tags, which were utilized to load anti-CEA (Ab2,1) and anti-AFP (Ab2,2), respectively. Subsequently, hierarchically aloe-like gold microstructures (HAG) with large effective area were used to immobilize the capture antibodies anti-CEA (Ab1,1) and anti-AFP (Ab1,2), which effectively enhanced the performance of the immunosensor. Experimental results revealed that the sandwich-type immunosensor can permit the simultaneous detection of CEA and AFP with linear range of 0.6–80ngmL−1 for two analytes, the detection limit was 0.12ngmL−1 for CEA and 0.08ngmL−1 for AFP (at a signal to noise ratio of 3), respectively. The immunosensor was applied to real sample analysis, and the recovery was within 95.9%–105.6% for CEA and 95.6%–105.4% for AFP. In addition, the immunosensor exhibited good reproducibility and stability, suggesting the immunosensor offered potential in analysis of biological samples.

Procalcitonin sensitive detection based on graphene–gold nanocomposite film sensor platform and single-walled carbon nanohorns/hollow Pt chains complex as signal tags

Septicemia is a serious disease that requires early diagnosis, and procalcitonin (PCT) serves as a diagnostic biomarker for this disease. Traditional clinical detection (via immune-gold chips) remains difficult and expensive. An electrochemical immunosensor based on new nanomaterials may provide a solving approach. Herein, an ultrasensitive sandwich electrochemical strategy for PCT detection was developed. Firstly, reduced graphene oxide (rGO)–gold (Au) nano-composite film was used as the immunosensor platform to increase the amount of PCT antibody 1(Ab1) immobilized. Next, single-walled carbon nanohorns (SWCNHs)/hollow Pt chains (HPtCs) complex was firstly utilized to label PCT Ab2 as signal tags. For SWCNHs with few side effects, high surface area and HPtCs with higher specific surface, better catalytic activity, complex synthesized from both may provide more advantages. Moreover, to amplify signal, HPtC catalytic activity with H2O2 was enhanced by horseradish peroxidase (HRP) for dual synergy amplification. The whole results demonstrated that the proposed immunosensor exhibited fast operation, high sensitivity, good reproducibility, acceptable stability and ideal selectivity compared with traditional method. The linear calibration of the immunosensor ranged from 1.00pg/mL to 2.00×101ng/mL with a detection limit of 0.43pg/mL. Analytical application results revealed that the immunosensor matched with the real concentrations of serum samples. Overall this immunosensor may provide a new alternative strategy for PCT detection.

Signal amplification for multianalyte electrochemical immunoassay with bidirectional stripping voltammetry using metal-enriched polymer nanolabels

A novel bidirectional (anodic and cathodic) stripping voltammetric immunoassay (SVI) was designed for the simultaneous determination of multiple model cancer biomarkers (AFP, CEA and CA19-9) in a single run, based on the integration of Envision complex loaded metal nanoparticles as signal tags and immunomagnetic beads as capture probes. The Envision complex, which is a long branching polymer consisting of numerous secondary antibodies and horseradish peroxidase (HRP), was employed for signal amplification by labeling corresponding detection antibodies and further loading metal nanoparticles (CdS, PbS and gold) to prepare distinguishable metal signal tags. Herein, the generation of immunosensing probes involved the co-immobilization of three types of primary anti-AFP, anti-CEA, and anti-CA19-9 antibodies onto a single magnetic Dynabead. After a two-binding step sandwich-type immunoassay, the Envision/CdS, Envision/PbS and Envision/Au signal tags were introduced onto the surface of the Dynabeads. The subsequent bidirectional voltammetric analysis of stripping metal components from immunocomplexes for quantification of tumor biomarkers was performed in a microcell with minimum capacity of 50μL. Experimental results showed the immunoassay enabled the simultaneous determination of multiple biomarkers over a broad range of concentrations (AFP, 1pgmL−1–50ngmL−1; CEA, 1pgmL−1–50ngmL−1; CA19-9, 5pgmL−1–100ngmL−1) with detection limits reaching 0.02pgmL−1 for AFP, 0.05pgmL−1 for CEA, and 0.3pgmL−1 for CA19-9. The results indicated that the proposed bidirectional multiplexed immunoassay can increase the number of analytes by SVI and has high sensitivity, excellent stability, and great promise for applications in clinical cancer diagnosis.

Simultaneous electrochemical detection of multiple tumor markers using metal ions tagged immunocolloidal gold

In this work, a sandwich-format electrochemical immunosensor has been fabricated in the aim of simultaneous detection of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) using metal ions tagged immunocolloidal gold nanocomposites as signal tags. The capture anti-CEA and anti-AFP were immobilized onto the chitosan–Au nanoparticles (CHIT–AuNPs) membrane modified glassy carbon electrode through glutaraldehyde (GA). The metal ion labels could be detected directly through differential pulse voltammetry (DPV) without metal pre-concentration, and the distinct voltammetric peaks had a close relationship with each sandwich-type immunoreaction. Under the optimized conditions, the multiplexed immunoassay exhibited good sensitivity and selectivity for the simultaneous determination of CEA and AFP with linear ranges of 0.01–50ngmL−1. The detection limits for CEA and AFP are 4.6pgmL−1 and 3.1pgmL−1, respectively. The method was successfully applied for the determination of AFP and CEA levels in clinical serum samples, and the results were in good agreement with standard enzyme linked immunosorbent assay (ELISA). This approach gives a promising simple and sensitive immunoassay strategy for clinical applications.

A novel strategy of procalcitonin detection based on multi-nanomaterials of single-walled carbon nanohorns–hollow Pt nanospheres/PAMAM as signal tags

Procalcitonin (PCT) is a common clinical biomarker of septicemia, and precise detection of PCT is critical to the treatment of septicemia. Conventional detection methods suffer various shortcomings that hinder precise PCT detection. An electrochemical immunosensor based on the antigen–antibody immune-reaction may provide a solution. We developed a novel electrochemical immunosensor for PCT using single-walled carbon nanohorns (SWCNHs)–hollow Pt nanospheres (HPtNPs) as signal tags. Given the high specific surface area of SWCNHs and the interesting catalytic properties of HPtNPs, the successful synthesis of SWCNHs–HPtNPs provided not only a high specific surface area for loading substantial antigen but also good catalytic properties to enhance the electrochemical signal with high sensitivity. In addition, to increase the quantity of the electrochemically active compound thionine immobilized on the surface, PAMAM, which is a class of hyper-branched polymers with a diamine core and an amido amine branching structure, was assembled on the SWCNH–HPtNP surface. A series of results demonstrated the higher sensitivity, improved stability and ideal selectivity of the novel immunosensor compared with a sensor prepared using traditional methods. Furthermore, a broad linear response from 10 pg mL−1 to 20 ng mL−1 with a detection limit of 1.74 pg mL−1 was observed under the optimized conditions of the assay. This immunosensor may provide a new option for clinical septicemia diagnosis through PCT detection.

Multiplexed electrochemical immunoassay of biomarkers using chitosan nanocomposites

In this work, a novel and sensitive multiplexed immunoassay protocol for simultaneous electrochemical determination of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) was designed using functionalized chitosan composites. The immunosensing platform was prepared via immobilizing capture anti-AFP and anti-CEA on chitosan-Au nanoparticles (AuNPs) through EDC/NHS linking. The signal tags were fabricated by immobilizing electroactive redox probes – Prussian blue (PB) and ferrocenecarboxylic acid (Fc) on chitosan (CHIT), following by absorbing AuNPs to immobilize labeled anti-AFP and anti-CEA, respectively. A sandwich-type immunoassay format was employed for the simutaneous detection of AFP and CEA. The assay was based on the electrochemical oxidation/reduction of the redox species in signal tags, which has a relationship with the concentration of analytes. Experimental results revealed that the multiplexed electrochemical immunoassay enabled the simutaneous monitoring of AFP and CEA with a wide range of 0.05–100ngmL−1 for both AFP and CEA. The detection limits (LOD) was 0.03ngmL−1 for AFP and 0.02ngmL−1 for CEA (S/N=3). The assay results of serum samples with the proposed method were in a good agreement with the reference values from standard ELISA method. And the negligible cross-reactivity between the two analytes makes it possesses potential promise in clinical diagnosis.

Highly sensitive voltammetric detection of DNA hybridization in sandwich format using thionine-capped gold nanoparticle/reporter DNA conjugates as signal tags

A highly sensitive sandwich DNA detection method based on voltammetric detection of thionine-capped gold nanoparticle (AuNP)/reporter DNA conjugate tags on gold particle-modified screen-printed carbon electrode (SPCE) was developed. The SPCE was modified with gold particle by electrodeposition of gold on SPCE surface. The DNA sensor was prepared by self-assembly of a thiolated DNA probe on gold particle-modified SPCEs. The sandwich-type system was formed by specific recognition of biosensor surface-confined probe DNA to target DNA, followed by attachment of thionine-capped AuNPs/reporter DNA conjugates. The biosensor is very sensitive because of the large number of electroactive thionine molecules in the thionine-capped AuNPs/reporter DNA conjugates. Under optimal conditions, the dynamic detection range of target DNA was from 1.0 × 10−16 to 1.0 × 10−14 mol L−1, and the detection limit was 0.5 × 10−16 mol L−1. The DNA sensor exhibited selectivity against single-base mismatched DNA.

Platinum porous nanoparticles hybrid with metal ions as probes for simultaneous detection of multiplex cancer biomarkers

In this work, platinum porous nanoparticles (PtPNPs) absorbed metal ions as electrochemical signals were fabricated. Clean-surface PtPNPs were prepared by a surfactant-free method and decorated with amino groups via 2-aminoethanethiol. Amino capped PtPNPs complexation with Cd2+ and Cu2+ to form PtPNPs-Cd2+ and PtPNPs-Cu2+ hybrids, respectively. Anti-CEA and Anti-AFP separately labeled with PtPNPs-Cd2+ and PtPNPs-Cu2+ were used as distinguishable signal tags for capturing antigens. The metal ions were detected in a single run through differential pulse voltammetry (DPV) without acid dissolution, electric potentials and peak heights of which reflected the identity and concentrations of the corresponding antigen. Ionic liquid reduced graphene oxide (IL-rGO) modified glassy carbon electrode (GCE) was used as a substrate, which was rich in amino groups to immobilize antibodies by glutaraldehyde through cross-link between aldehyde groups and amino groups. Using the proposed probes and platform, a novel sandwich-type electrochemical immunosensor for simultaneous detecting carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) was successfully developed. This immunoassay possessed good linearity from 0.05ngmL−1 to 200ngmL−1 for both CEA and AFP. The detection limit of CEA was 0.002ngmL−1 and that of AFP was 0.05ngmL−1 (S/N=3). Furthermore, analysis of clinical serum samples using this immunosensor was well consistent with the data determined by the enzyme-linked immunosorbent assay (ELISA). It suggested that the proposed electrochemical immunoassay provided a potential application of clinical screening for early-stage cancers.

Highly Efficient Visual Detection of Trace Copper(II) and Protein by the Quantum Photoelectric Effect

This work presented a photocurrent response mechanism of quantum dots (QDs) under illumination with the concept of a quantum photoelectric effect. Upon irradiation, the photoelectron could directly escape from QDs. By using nitro blue tetrazolium (NBT) to capture the photoelectron, a new visual system was proposed due to the formation of an insoluble reduction product, purple formazan, which could be used to visualize the quantum photoelectric effect. The interaction of copper(II) with QDs could form trapping sites to interfere with the quantum confinement and thus blocked the escape of photoelectron, leading to a "signal off" visual method for sensitive copper(II) detection. Meanwhile, by using QDs as a signal tag to label antibody, a "signal on" visual method was also proposed for immunoassay of corresponding protein. With meso-2,3-dimercaptosuccinic-capped CdTe QDs and carcino-embryonic antigen as models, the proposed visual detection methods showed high sensitivity, low detection limit, and wide detectable concentration ranges. The visualization of quantum photoelectric effect could be simply extended for the detection of other targets. This work opens a new visual detection way and provides a highly efficient tool for bioanalysis.

Simultaneous detection of four biomarkers with one sensing surface based on redox probe tagging strategy

In this paper, a simple and sensitive amperometric immunosensor for simultaneous detection of four biomarkers by using distinguishable redox-probes as signal tags was proposed for the first time. In sandwich immunoassay format, four kinds of capture antibodies (C-Ab) were immobilized by gold nanoparticles (AuNPs) electro-deposited on the surface of glass carbon electrode (GCE); four kinds of detection antibodies (D-Ab) labeled with different redox probes (including anthraquinone 2-carboxylic acid (Aq), thionine (Thi), ferrocenecarboxylic acid (Fc) and tris(2,2’-bipyridine-4,4’-dicarboxylic acid) cobalt(III) (Co(bpy)33+)), were combined with 3,4,9,10-perylenetetracarboxylic acid (PTCA), poly(diallyldimethylammonium chloride) (PDDA) and AuNPs functionalized carbon nanotubes, and served as signal tracer. When four target antigens were present, differential pulse voltammetry (DPV) scan exhibited four well-resolved peaks, each peak indicated one antigen, and its intensity was quantitative correlational to the concentration of corresponding analyte. To verify the strategy, four biomarkers for diagnosis of colorectal carcinoma, including carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9 CA125, and CA242, were used as model analytes, the immunosensor exhibited high selectivity and sensitivity, and peak current displayed good linear relationship to logarithm concentration in the ranges from 0.016 to 15ngmL−1 for CEA; 0.008 to 10ngmL−1 for CA19-9; 0.012 to 12ngmL−1 for CA125; 0.010 to 10ngmL−1 for CA242, and low detection limits of 4.2, 2.8, 3.3 and 3.8pgmL−1, respectively.

Au–ionic liquid functionalized reduced graphene oxide immunosensing platform for simultaneous electrochemical detection of multiple analytes

In this work, an Au–ionic liquid functionalized reduced graphene oxide nanocomposite (IL-rGO–Au) was fabricated via the self-assembly of ionic liquid functionalized reduced graphene oxide (IL-rGO) and gold nanoparticles (AuNPs) by electrostatic interaction. The IL-rGO can be synthesized and stabilized by introducing the cations of the amine-terminated ionic liquids (IL-NH2) into the graphene oxide (GO). With the assistance of IL-NH2, AuNPs were uniformly and densely absorbed on the surfaces of the IL-rGO. The proposed IL-rGO–Au nanocomposite can be used as an immunosensing platform because it can not only facilitate the electrons transfer of the electrode surface but also provide a large accessible surface area for the immobilization of abundant antibody. To assess the performance of the IL-rGO–Au nanocomposite, a sandwich-type electrochemical immunosensor was designed for simultaneous multianalyte detection (carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) as model analytes). The chitosan (CS) coated prussian blue nanoparticles (PBNPs) or cadmium hexacyanoferrate nanoparticles (CdNPs) and loaded with AuNPs were used as distinguishable signal tags. The resulting immunosensor exhibited high selectivity and sensitivity in simultaneous determination of CEA and AFP in a single run. The linear ranges were from 0.01 to 100ngmL−1 for both CEA and AFP. The detection limits reached 0.01ngmL−1 for CEA and 0.006ngmL−1 for AFP, respectively. No obvious nonspecific adsorption and cross-talk was observed during a series of analyses to detect target analytes. In addition, for the detection of clinical serum samples, it is well consistent with the data determined by the ELISA, indicating that the immunosensor provides a possible application for the simultaneous multianalyte determination of CEA and AFP in clinical diagnostics.

Implementation of an RFID-Based Virtual Signal Mechanism for Indoor Location Sensing System

The variety in wireless technology and mobile computing devices has caused a growing application of location sensing systems and correspondence services. The indoor location sensing system which applies radio frequency identification technology (RFID) is the most popular research topic in the area of location sensing systems, and LANDMARC is the most representative one. However, LANDMARC suffers the drawback of inaccuracy in unstable signal intensity. A Virtual Signal Location System (VSLS) is designed and developed to overcome some drawbacks of LANDMARC. The concept of VSLS is based on the additional setting of virtual signal tags as well as the normal distribution of signal strength, analysis of sampling rate and equalization to decrease signal intensity error. Comparisons were made with LANDMARC; results showed that VSLS effectively increased the sampling quality of the signal as well as the precision of positioning.

Electrochemical immunoassay for thyroxine detection using cascade catalysis as signal amplified enhancer and multi-functionalized magnetic graphene sphere as signal tag

This paper constructed a reusable electrochemical immunosensor for the detection of thyroxine at an ultralow concentration using cascade catalysis of cytochrome c (Cyt c) and glucose oxidase (GOx) as signal amplified enhancer. It is worth pointing out that numerous Cyt c and GOx were firstly carried onto the double-stranded DNA polymers based on hybridization chain reaction (HCR), and then the amplified responses could be achieved by cascade catalysis of Cyt c and GOx recycling with the help of glucose. Moreover, multi-functionalized magnetic graphene sphere was synthesized and used as signal tag, which not only exhibited good mechanical properties, large surface area and an excellent electron transfer rate of graphene, but also possessed excellent redox activity and desirable magnetic property. With a sandwich-type immunoreaction, the proposed cascade catalysis amplification strategy could greatly enhance the sensitivity for the detection of thyroxine. Under the optimal conditions, the immunosensor showed a wide linear ranged from 0.05pgmL−1 to 5ngmL−1 and a low detection limit down to 15fgmL−1. Importantly, the proposed method offers promise for reproducible and cost-effective analysis of biological samples.

Mass spectrometry signal amplification for ultrasensitive glycoprotein detection using gold nanoparticle as mass tag combined with boronic acid based isolation strategy

We describe a novel method for rapid and ultrasensitive detection of intact glycoproteins without enzymatic pretreatment which was commonly used in proteomic research. This method is based on using gold nanoparticle (AuNP) as signal tag in laser desorption/ionization mass spectrometry (LDI-MS) analysis combined with boronic acid assisted isolation strategy. Briefly speaking, target glycoproteins were firstly isolated from sample solution with boronic acid functionalized magnetic microparticles, and then the surface modified gold nanoparticles were added to covalently bind to the glycoproteins. After that, these AuNP tagged glycoproteins were eluted from magnetic microparticles and applied to LDI-MS analysis. The mass signal of AuNP rather than that of glycoprotein was detected and recorded in this strategy. Through data processing of different standard glycoproteins, we have demonstrated that the signal of AuNP could be used to quantitatively represent glycoprotein. This method allows femtomolar detection of intact glycoproteins. We believe that the successful validation of this method on three different kinds of glycoproteins suggests the potential use for tracking trace amount of target glycoproteins in real biological samples in the near future.

Host–Guest Interaction of Adamantine with a β-Cyclodextrin-Functionalized AuPd Bimetallic Nanoprobe for Ultrasensitive Electrochemical Immunoassay of Small Molecules

A modular labeling strategy was presented for electrochemical immunoassay via supramolecular host-guest interaction between β-cyclodextrin (β-CD) and adamantine (ADA). An ADA-labeled antibody (ADA-Ab) was synthesized via amidation, and the number of ADA moieties loaded on a single antibody was calculated to be ~7. The β-CD-functionalized gold-palladium bimetallic nanoparticles (AuPd-CD) were synthesized in aqueous solution via metal-S chemistry and characterized with transmission electron microscopy and X-ray photoelectron spectra. After the ADA-Ab was bound to the antigen-modified electrode surface with a competitive immunoreaction, AuPd-CD as a signal tag was immobilized onto the immunosensor by a host-guest interaction, leading to a large loading of AuPd nanoparticles. The highly efficient electrocatalysis by AuPd nanoparticles for NaBH4 oxidation produced an ultrasensitive response to chloramphenicol as a model of a small molecule antigen. The immunoassay method showed a wide linear range from 50 pg/mL to 50 μg/mL and a detection limit of 4.6 pg/mL. The specific recognition of antigen by antibody resulted in good selectivity for the proposed method. The host-guest interaction strategy provided a universal labeling approach for the ultrasensitive detection of small molecule targets.

Amperometric Immunosensor for Determination of Clenbuterol Based on Enzyme-Antibody Coimmobilized ZrO2 Nano Probes as Signal Tag

Abstract A novel amperometic immunosensor for Clenbuterol (CLB) detection based on enzyme-antibody co-immobilized zirconium dioxide (ZrO2) nanoprobes for signal amplification was developed. First, ZrO2 nanoparticles (ZNPs) were used to adsorb glucose oxidase (GOD) enzyme and CLB antibody (anti-CLB) to prepare the signal tag (ZNPs/GOD-anti CLB). While poly(diallyldimethylammonium chloride) (PDDA), Hemin and Au nanoparticles (AuNPs) were assembled layer by layer on the surface of multi-walled carbon nanotubes (MWCNTs) which was modified by screen-printed electrodes (SPCE). By this means, the sensor (SPCE|WCNTs/PDDA/Hemin/AuNPs), which can catalyze H2O2 into generate reduction current, was obtained. When CLB was detected, the samples and ZNPG were embedded to the bottom of the 96-well plate in advance. On the basis of the reaction mechanism of competitive enzyme-linked immunoassay, CLB in samples would compete with slab CLB for the combination with the nanoprobe ZNPG. After reaction, glucose (Glu) was added to the 96-well plate, and then the GOD catalyze Glu to generate H2O2, which could be detected by the immunosensor for quantification. Current decline in value (δI) was inversely proportional to a certain concentration range of CLB. Under the optimal conditions (pH 7.0, incubation time 30 min at 37 °C), the immunosensor provided an excellent response for CLB ranging from 0.003 to 100 μg L−1 with a detection limit of 1 ng L−1 (3σ). The sensitivity of this method was 2 orders of magnitude higher than that of the ELISA method due to the high density of GOD on the signal tag, which could greatly amplify the detection signal. The average recovery of the samples was 93.6%, the coefficient of intragroup variation was less than 2.5%, which confirmed that the amperometic immunosensor prepared had a high precision. The immunosensor was reusable, sensitive and rapid for the analysis of ultra trace amounts of CLB in food samples in real time.

Redox-active and Catalytic-efficient PAMAM Dendrimer Nanostructures for Sensing Low-abundance Protein with Signal Amplification

Abstract Functionalized poly(amido amine) (PAMAM) dendrimer nanostructures with redox activity and catalytic ability were for the first time utilized as detectable signal tags for highly efficiency electrochemical immunosensing of low-abundance protein (prostate-specific antigen, PSA, used as a model) on a disposable immunosensor, based on an in situ amplification strategy of bioelectrocatalytic reaction.