Label Of Secondary Antibody Research Articles

Target-induced photoelectrochemistry and colorimetric dual-mode platform for HIgG based on Ag2S/SnO2 composites and CoOOH nanoflakes

Human immunoglobulin G (HIgG) plays a significant role in diagnosing infections and monitoring autoimmune diseases. However, the development of sensitive and reliable sensing platforms remains a challenge. Based on the Ag2S/SnO2 composites, a dual-mode sensing platform was developed using multifunctional CoOOH nanoflakes for the sensitive detection of HIgG through photoelectrochemistry (PEC) and colorimetry. In the PEC mode, Ag2S QDs boost the PEC activity of SnO2 NFs and facilitate the subsequent antibody immobilization. Ascorbic acid (AA) captures holes in Ag2S/SnO2 to enhance the photocurrent. CoOOH, acting as a secondary antibody label, forms a sandwich structure with the electrode. The loading of HIgG and CoOOH-Ab2 due to specific recognition of antigen-antibody, leads to the quenching of PEC signal. Various amounts of HIgG will bind with different amounts of CoOOH-Ab2, displaying a linear response within the range of 100 pg/mL∼10 μg/mL, with the detection limit of 2.2 pg/mL. In the colorimetric mode, CoOOH efficiently catalyzes the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB), and various amount of the sandwich biocomplexes formed with different HIgG levels lead to distinct color changes for visual detection. The linear response range is 500 pg/mL∼9.375 μg/mL with a lower detection limit of 0.29 ng/mL. The two independent modes with different mechanisms, based on the same PEC electrode, can validate each other and provide more scientific and reliable results in response to the targets. Thus, this dual-mode sensing platform for targets-induced signaling changes presents a novel strategy for bioanalysis and detection.

Ultrasensitive electrochemiluminescence immunosensor based on Co-doped MoOx as co-reactant generator for the detection of amyloid β-protein

Herein, a novel electrochemiluminescence (ECL) immunosensor was constructed to detect amyloid β-protein (Aβ) by using Co2+ doped MoOx (Co@MoOx) as both sensing substrate and coreactant generator, Au nanoclusters and isoluminol (Au NCs-ABEI) as secondary antibody label. Co@MoOx showed good electrolysis performance of aquatic oxygen, and the dissolved oxygen just acted as a coreactant of the ABEI emitter in this system. Au NCs was synthesized from reduced glutathione, which had ultrasmall size and good biocompatibility. Bioconjugate Au NCs-ABEI was constructed on Au NCs surface by cross-linking ABEI with amino group on ABEI surface. Through modifying Co@MoOx and Au NCs-ABEI on the electrode successively, the ECL signal intensity of immunosensor increased, which showed high sensitivity for the detection of Aβ, with the detection range from 1.00 fg·mL−1 to 500 pg·mL−1 and the detection limit as low as 0.330 fg·mL−1. This strategy will provide some reference for the early diagnosis and prevention of Alzheimer's disease (AD) and detection of other disease markers.

Preparation of a pH-responsive controlled-release electrochemical immunosensor based on polydopamine encapsulation for ultrasensitive detection of alpha-fetoprotein.

To accomplish ultra-sensitive detection of alpha-fetoprotein(AFP), a novel electrochemical immunosensor using polydopamine-coated Fe3O4 nanoparticles (PDA@Fe3O4 NPs) as a smart label and polyaniline (PANI) and Au NPs as substrate materials has been created. The sensor has the following advantages over typical immunoassay technology: (1) The pH reaction causes PDA@Fe3O4 NPs to release Prussian blue (PB) prosoma while also destroying the secondary antibody label and immunological platform and lowering electrode impedance; (2) PB has a highly efficient catalytic effect on H2O2, allowing for the obvious amplification of electrical impulses; (3) PANI was electrodeposited on the electrode surface to avoid PB loss and signal leakage, which effectively absorbed and fixed PB while considerably increasing electron transmission efficiency. The sensor's detection limit was 0.254pg·mL-1 (S/N = 3), with a detection range of 1pg·mL-1 to 100ng·mL-1. The sensor has a high level of selectivity, repeatability, and stability, and it is predicted to be utilized to detect AFP in real-world samples.

Perylene diimide and g-C3N4 nanosheet as potential-resolved cathode luminophores for ultrasensitive ratiometric electrochemiluminescence immunosensor

In electrochemiluminescence(ECL) field, the shortage of potential-resolved luminophores severiously limited the development of biosensors for accurate assay such as ratiometric biosensors. In this work, N,N′-dimethyl-3,4,9,10-perylenedicarboximide(PDI-CH3) and graphite-like carbon nitride(g-C3N4) nanosheet was firstly reported to produce potential-resolved ECL with coreactant K2S2O8 between 0 and − 1.2 V. And there was good overlap between the ECL spectrum of g-C3N4 and the absorption spectra of PDI-CH3. Moreover, PDI-CH3 and g-C3N4 were used to construct ECL ratiometric system for sandwiched immunosensors to detect tumor marker carcinoembryonic antigen (CEA), in which g-C3N4 was used as ECL platform and PDI-CH3 was used as ECL label of secondary antibody(Ab2). Increasing the CEA concentration, the loading of PDI-CH3 on the electrode stepwisely increased, which leaded to gradual increase of ECLPDI-CH3 at − 0.5 V and significant decrease of ECLg-C3N4 at − 1.2 V due to the high quenching effects of PDI-CH3 to g-C3N4 through dual-quenching mechanism: competitive consumption of coreactant and ECL resonance energy transfer. And the ECLPDI-CH3/ECLg-C3N4 showed ultrasensitive linear response for CEA between 0.1 fg mL−1 and 1 ng mL−1 with detection limit 0.052 fg mL−1 according to IUPAC criterion. In addition, the proposed ratiometric biosensor exhibited good recovery in human serum samples.

Design and Evaluation of a Competitive Phosphorescent Immunosensor for Aflatoxin M1 Quantification in Milk Samples Using Mn:ZnS Quantum Dots as Antibody Tags

Aflatoxin M1 (AFM1) is one of the most widespread aflatoxins that can be present in the milk of lactating mammals. It can cause carcinogenicity, mutagenesis, teratogenesis, genotoxicity and immunosuppression. The WHO recommends reducing the AFM1 concentration in food products, so the European Commission has set a maximum allowable limit of 0.05 µg L−1 in milk and its products. Thus, there is a need to develop new methodologies to satisfy the demand for reliable, cost-effective, robust and sensitive AFM1 routine controls. In the present work, a competitive phosphorescent immunosensor for AFM1 quantification in milk, based on antibody–antigen recognition and Mn:ZnS quantum dots (d-QDs) as photoluminescent labels, has been developed. Two different assay strategies based on the use of d-QDs as labels of secondary antibodies (direct assay), or of a derivative species of the antigen AFM1-Bovine Serum Albumin (indirect assay) were compared in terms of analytical performance for AFM1 quantification. The best analytical results were obtained with the immunoassay format that uses d-QDs as tags of secondary antibodies (direct assay), and said design was finally selected. The selected immunosensor provided a detection limit for AFM1 quantification of only 0.002 µg L−1, which greatly satisfied the maximum tolerable limit of AFM1 in milk of 0.05 µg L−1. The accuracy, calculated as recovery of AFM1 in fortified skimmed milk samples, ranged from 81 to 90%, with relative standard deviations from 3% to 14%. These results bring to light the good performance of such phosphorescent biosensors as simple and fast alternatives to conventional chromatographic analytical methods.

Sandwich-type photoelectrochemical immunosensor for procalcitonin detection based on Mn2+ doped CdS sensitized Bi2WO6 and signal amplification of NaYF4:Yb, Tm upconversion nanomaterial

In this paper, we constructed a sandwich-type photoelectrochemical (PEC) immunosensor for the quantitative detection of procalcitonin (PCT) based on the sensitization of Mn2+ doped CdS (CdS:Mn) nanocomposites to Bi2WO6 and the signal amplification effect of an upconversion material NaYF4:Yb,Tm. Bi2WO6 was synthesized with a three-dimensional flowered structure. CdS:Mn reduced the recombination of photogenerated carriers, and significantly improved photocurrent response. Lanthanide-doped upconversion nanomaterials were used as the label of secondary antibody. NaYF4:Yb,Tm have two functions, not only connected with the secondary antibody, but also can further amplify the photocurrent response. The proposed immunosensor for detecting PCT provided a desired linear range of 0.5 pg mL−1-100 ng mL−1 and a detection limit of 0.13 pg mL−1 under optimal experimental conditions. Besides, the PEC immunosensor demonstrated good reproducibility, specificity and stability. The results of determination of PCT in real human serum samples were satisfactory. Thus, the immunosensor may be applied in the clinical diagnosis of PCT and other biomarkers.

A Sandwich-Type Electrochemical Immunosensor based on Pd Nanocubes Functionalized MoO2 Nanospheres for Highly Sensitive Detection of CEA

In this study, a sandwich-type electrochemical immunosensor was developed for detecting carcinoembryonic antigen (CEA) effectively. The proposed electrochemical immunosensor was based on gold nanocrystals (Au NDs) as the substrate material for capturing primary antibodies and using palladium nanoscale cubics (Pd NCs) loaded on amino-functionalized MoO2 nanospheres (Pd NCs/NH2-MoO2 NSs) as a secondary antibody label. Au NDs had dendritic structures that were more conducive to capturing Ab1 and can accelerate electron transfer. MoO2 had the good catalytic capacity for reduction of H2O2 and favourable electrical conductivity. Similarly, Pd NCs had excellent catalytic performance for hydrogen peroxide reduction. Hence, the obtained Pd NCs/NH2-MoO2 NSs were more effective than Pd NCs and NH2-MoO2 NSs in catalytic reduction of hydrogen attribute to a synergistic effect, showing excellent catalysis. The proposed electrochemical immunosensor had a lower detection limit of 3.3 fg ml−1 jand a wider detection range from 10 fg ml−1 to 100 ng ml−1 (S/N = 3) for the detection of CEA under optimal experimental conditions. The proposed electrochemical immunosensor showed good sensitivity, stability, selectivity, reproducibility and its good detection performance of the immunosensor indicated that it had a broad application prospect in clinical detection.

Electrochemiluminescence immunosensor based on the quenching effect of CuO@GO on m-CNNS for cTnI detection

A sandwich-type electrochemiluminescence (ECL) immunosensor based on the resonance energy transfer (RET) was proposed for ultrasensitive detection of cardiac troponin I (cTnI). The RET behavior could be generated between graphite carbon nitride nanosheets (m-CNNS) as donor and copper oxide@graphene oxide (CuO@GO) as acceptor, achieving the quenching effect of CuO@GO on m-CNNS for cTnI detection. The m-CNNS synthesized by mechanical grinding of the graphite carbon nitride (CN) not only has better dispersion and higher specific surface area, but also has high luminous efficiency and stable chemical properties. Therefore, m-CNNS was used as the matrix material and luminophore. As the acceptor, CuO@GO prepared by in-situ chemical synthesis of CuO NPs onto GO sheets also has a high specific surface area, which could be used as a label of secondary antibody (Ab2). Under optimal conditions, cTnI could be determined within the linear range of 0.1 pg mL−1 to 100 ng mL−1 and had a low detection limit (0.028 pg mL−1, S/N = 3). Meanwhile, the prepared ECL immunosensor possessed great stability, specificity and reproducibility, providing a new method for detecting cTnI and other biomarkers.

Ultrasensitive ratiometric electrochemical immunoassay of N-terminal pro-B-type natriuretic peptide based on three-dimensional PtCoNi hollow multi-branches/ferrocene-grafted-ionic liquid and Co[sbnd]N[sbnd]C nanosheets

Developing a simple and sensitive method for detecting tumor biomarkers is practically important for early diagnosis and clinical treatment of diseases. However, it is still the major challenge to improve the sensitivity and accuracy. Here, “a dual-signal read out mode” ratiometric electrochemical immunosensor was developed based on well-dispersed hierarchical PtCoNi hollow multi-branches (HMBs)/ferrocene-grafted-ionic liquid (Fc-g-IL, synthesized via Schiff-base reaction) and CoNC nanosheets (NSs, obtained by a simple pyrolysis method) as signal amplification. Specifically, the PtCoNi HMBs were synthesized by solvothermal treatment and then etched with acetic acid. The PtCoNi HMBs/Fc-g-IL showed improved catalytic property and superior biocompatibility. Meanwhile, Au nanostars/thionine decorated CoNC NSs (Au-Thi-Co-N-C NSs) performed as the label of secondary antibody (Ab2) to greatly enhance the signals. This immunoassay for N-terminal pro-B-type natriuretic peptide (NT-proBNP) was realized by boosting the Thi signals while inversely decaying the Fc responses, in turn showing the linear relationship towards the analyte in the range of 0.001–10.0 ng mL–1 with the low detection limit down to 0.35 pg mL–1 (S/N = 3). This study displays a promising platform for determination of other tumor biomarkers in real medical applications.

Immunosensing of NT‐proBNP via Cu2+‐based MOFs Biolabeling and in situ Microliter‐droplet Anodic Stripping Voltammetry

AbstractImmunoassay of amino‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP) was conducted using Cu2+‐1,3,5‐benzenetricarboxylic acid metal‐organic frameworks (HKUST‐1 MOFs) as the secondary antibody label, and in situ microliter‐droplet anodic stripping voltammetry detection of Cu2+ in 0.1 M HNO3+1 M NaCl directly on the glassy carbon immunoelectrode. Electrochemical methods, quartz crystal microbalance, scanning electron microscopy and energy dispersive spectroscopy were employed for material and electrode characterizations. Under optimized conditions, the limit of detection (S/N=3) was 0.33 fg mL−1, and the analysis of NT‐proBNP in clinical serum samples returned good results.

Electrochemical Immunosensors for Sensitive Detection of Neuron-Specific Enolase Based on Small-Size Trimetallic Au@Pd^Pt Nanocubes Functionalized on Ultrathin MnO2 Nanosheets as Signal Labels.

Medically, neuron-specific enolase (NSE) as a specific tumor marker has become an important indicator to diagnose small-cell lung carcinoma. In this study, a sandwich-type electrochemical immunosensor was designed to determine NSE sensitively. Au nanoparticle (Au NP)-embedded zinc-based metal-organic frameworks (Au@MOFs) were prepared as the substrate materials to modify the electrode and immobilize the primary antibody (Ab1). The Au@MOFs with the free amino groups on the MOF surface could effectively increase the immobilization amount of Ab1 through covalent linkage. Simultaneously, the embedding of Au NPs improved the conductivity of MOFs and accelerated interface electron transfer. Sub-30 nm trimetallic Au@Pd^Pt nanocubes (Au@Pd^Pt NCs) loaded onto ultrathin MnO2 nanosheets (MnO2 UNs/Au@Pd^Pt NCs) acted as the labels of secondary antibodies. The small-size Au@Pd^Pt NCs enhanced atomic utilization efficiency and offered more catalytic active sites. The MnO2 UNs with high external surface areas could improve the dispersion of Au@Pd^Pt NCs. The MnO2 UNs/Au@Pd^Pt NCs could catalyze the H2O2 reduction and promote the oxidation of hydroquinone to quinone effectively because of their synergistic effect; thus, the generated quinone achieved amplification of the highly reductive peak current. Furthermore, under the optimal conditions, the immunosensor exhibited a low detection limit (4.17 fg/mL) and broad linear range (10 fg/mL to 100 ng/mL). The results were satisfactory for NSE detection in human serum samples, implying that the presented method had great application potential in clinical bioanalysis.

Mo2C combined with carbon material nanosphere as an electrochemiluminescence super-enhancer and antibody label for ultrasensitive detection of cardiac troponin I

To sensitively detect cardiac troponin I (cTnI), a novel electrochemiluminescence (ECL) immunosensor was constructed on the basis of the core-shell Mo2C combined with carbon material (Mo2C@C) nanosphere as an ECL super-enhancer and a label of secondary antibody (Ab2). Mo2C@C nanosphere with excellent conductivity and high chemical stability could increase the ECL intensity of the tris-(2,2′-bipyridine)ruthenium (Ru(bpy)32+). Mo2C@C/polyethylenimine (Mo2C@C/PEI) firmly anchored Ab2 by covalent bonding. Otherwise, amino-modified multiwall carbon nanotubes@nafion (MWCNTs-NH2@N) composite film, as substrate material, could connect with the Ru(bpy)32+ by ion exchange and immobilize primary antibodies (Ab1) by amide bond. In addition, the boron nitride quantum dots (BNQDs) were utilized as the co-reactant, which were synthesized by using low-cost material melamine as a nitrogen source. Under optimal experimental conditions, the proposed ECL immunosensor possessed a low detection limit of 0.03 pg/mL (S/N = 3) and a wide linear range from 0.1 pg/mL to 50 ng/mL. The ultrasensitive ECL immunosensor had excellent stability, outstanding specificity and great reproducibility. It was also used to detect cTnI in human serum with satisfactory results.

Sandwich-type electrochemical immunosensor based on Au@Pt DNRs/NH2-MoSe2 NSs nanocomposite as signal amplifiers for the sensitive detection of alpha-fetoprotein

A sandwich-type electrochemical immunosensor was fabricated for the quantitation of alpha fetoprotein (AFP). To this end, the Au@Pt dendritic nanorods loaded with amino functionalized molybdenum selenide nanosheets (Au@Pt 41 DNRs/NH2-MoSe2 NSs) with enhanced peroxidase-like properties were selected as the secondary antibody label (Ab2) to achieve signal amplification. The as-obtained Au@Pt DNRs/NH2-MoSe2 NSs exhibited better catalytic activity toward hydrogen peroxide reduction and offered rich anchors for bioconjugation. Meanwhile, gold nanoparticles anchored on an amino functionalized graphene (Au NPs/NH2-GS) composite with admirable conductivity and biocompatibility was used as the matrix material to improve sensitivity. Under optimal conditions, amperometric current responses had a good linear relationship with the logarithm values of AFP concentration in the range 10 fg mL–1 to 200 ng mL–1 with a detection limit of 3.3 fg mL–1 (S/N = 3). Additionally, the immunosensor had excellent reproducibility, selectivity, and stability, which indicated superior performance for AFP detection compared with previous reports. The satisfactory results of human serum samples analysis showed that the designed immunosensor has potential applicability for the sensitive detection of other tumor markers.

Electrochemical immunosensor for HBe antigen detection based on a signal amplification strategy: The co-catalysis of horseradish peroxidase and nanoporous gold

The amplification of the electrochemical signal is crucial to improving the sensing performance of an immunosensor. In this study, a signal amplification strategy based on the co-catalysis of horseradish peroxidase (HRP) and nanoporous gold (NPG) was used to fabricate an electrochemical immunosensor for the determination of Hepatitis B e antigen (HBeAg). For the resulting immunosensor, HRP catalyzed catechol and thus served as a signal label of secondary antibody. NPG with unique properties was selected as a carrier for capture antibody immobilization. Further, NPG had an efficient electrocatalytic activity towards catechol (the substrate of HRP), which thus significantly enhanced the electrochemical signal produced by the HRP-labeled secondary antibody. Based on the co-catalysis of HRP and NPG, the immunosensor exhibited a good linear relationship between peak current density and HBeAg concentration in a wide HBeAg detection range from 1 pg mL−1 to 1 ng mL−1 with a high sensitivity of 23.51 μA mL ng−1 cm−2 as well as a low detection limit of 0.064 pg mL−1 (S/N = 3). Additionally, the immunosensor presented a strong anti-interference capability against human serum, human serum/dopamine, human serum/tryptophan, human serum/NO2−, human serum/D-glucose, and human serum/IgG. For the detection of HBeAg in human serum, the concentrations detected by the immunosensor were in good agreement with the standard concentrations that added in the human serum samples. These unique properties potentially make the proposed immunosensor a good choice for HBeAg detection.

Determining the Spatial Relationship of Membrane-Bound Aquaporin-4 Autoantibodies by STED Nanoscopy

Determining the spatial relationship of individual proteins in dense assemblies remains a challenge for superresolution nanoscopy. The organization of aquaporin-4 (AQP4) into large plasma membrane assemblies provides an opportunity to image membrane-bound AQP4 antibodies (AQP4-IgG) and evaluate changes in their spatial distribution due to alterations in AQP4 isoform expression and AQP4-IgG epitope specificity. Using stimulated emission depletion nanoscopy, we imaged secondary antibody labeling of monoclonal AQP4-IgGs with differing epitope specificity bound to isolated tetramers (M1-AQP4) and large orthogonal arrays of AQP4 (M23-AQP4). Imaging secondary antibodies bound to M1-AQP4 allowed us to infer the size of individual AQP4-IgG binding events. This information was used to model the assembly of larger AQP4-IgG complexes on M23-AQP4 arrays. A scoring algorithm was generated from these models to characterize the spatial arrangement of bound AQP4-IgG antibodies, yielding multiple epitope-specific patterns of bound antibodies on M23-AQP4 arrays. Our results delineate an approach to infer spatial relationships within protein arrays using stimulated emission depletion nanoscopy, offering insight into how information on single antibody fluorescence events can be used to extract information from dense protein assemblies under a biologic context.

A novel sandwich-type immunosensor for detection of carcino-embryonic antigen using silver hybrid multiwalled carbon nanotubes/manganese dioxide

A novel sensitive sandwich-type nonenzymatical electrochemical immunosensor was fabricated for quantitative determination of the carcino-embryonic antigen (CEA). β-cyclodextrin/multiwalled carbon nanotubes (β-CD/MWCNTs) that were used as electrochemical sensing platform improved the surface of electrode to immobilize first antibodies (Ab1) and enhanced the electrical conductivity. The synthetic silver nanoparticles-carbon nanotube/manganese dioxide (Ag NPs-MWCNTs/MnO2) was used as labels of secondary antibodies (Ab2) can improve the surface of electrode and enhance the electrochemical signal. MnO2 and Ag NPs achieve dual signal amplification to improve the sensitivity effectively. Under the optimal conditions, the electrochemical immunosensor exhibited a wide linear range of 0.0001–0.5ng·mL−1 and 0.5–10ng·mL−1 respectively with a low detection limit of 0.03pg·mL−1 for CEA. The immunosensor was also successfully applied to detect the actual sample. This novel method may find huge potential application for the detection of CEA in the clinical diagnosis.

Primary cilia are present on human blood and bone marrow cells and mediate Hedgehog signaling.

Primary cilia are nonmotile, microtubule-based organelles that are present on the cellular membrane of all eukaryotic cells. Functional cilia are required for the response to developmental signaling pathways such as Hedgehog (Hh) and Wnt/β-catenin. Although the Hh pathway has been shown to be active in leukemia and other blood cancers, there have been no reports describing the presence of primary cilia in human blood or leukemia cells. In the present study, we show that nearly all human blood and bone marrow cells have primary cilia (97-99%). In contrast, primary cilia on AML cell lines (KG1, KG1a, and K562) were less frequent (10-36% of cells) and were often shorter and dysmorphic, with less well-defined basal bodies. Finally, we show that treatment of blood cells with the Hh pathway ligand Sonic Hedgehog (SHh) causes translocation of Smoothened (SMO) to the primary cilia and activation of Hh target genes, demonstrating that primary cilia in blood cells are functional and participate in Hh signaling. Loss of primary cilia on leukemia cells may have important implications for aberrant pathway activation and response to SMO inhibitors currently in clinical development.

Ultrasensitive sandwich-type prostate specific antigen immunosensor based on Ag overgrowth in Pd nano-octahedrons heterodimers decorated on amino functionalized multiwalled carbon nanotubes

A novel and ultrasensitive sandwich-type electrochemical immunosensor was designed for quantitative detection of prostate specific antigen (PSA). The Ag overgrowth in Pd nano-octahedrons, developed Pd-Ag heterodimers (Pd-Ag HDs), loaded on amino functionalized multiwalled carbon nanotubes (MWCNTs-NH2) were employed as labels of secondary antibodies (Ab2). The synergistic effect of Pd-Ag HDs exhibited high electrocatalytic activity towards the reduction of H2O2. MWCNTs-NH2 with good dispersion, plentiful amino groups and large specific surface area, could load a large number of Pd-Ag HDs to enhance the sensitivity and amplify the signal of the immunosensor. Moreover, the electroplated gold nanoparticles (Au NPs) were employed for the immobilization of primary antibodies (Ab1). Au NPs with excellent conductivity could enhance the electron transport between substrate material and electrode surface, and improve the sensitivity of immunosensor. Under optimal conditions, the electrochemical immunosensor exhibited a wide linear range of 0.1pg/mL–30ng/mL towards PSA, and a low detection limit of 0.03pg/mL (S/N=3). The proposed immunosensor exhibited good sensitivity, reproducibility and stability for the quantitative detection of PSA, which hold a great potential in clinical and diagnostic applications.

A novel electrochemiluminescent immunosensor based on the quenching effect of aminated graphene on nitrogen-doped carbon quantum dots

Nitrogen-doped carbon quantum dots (N-CQDs) with an average diameter of 2 nm were synthesized by carbonization of diethylene triamine pentacetate acid (DTPA). The simple prepared N-CQDs showed excellent electrochemiluminescence (ECL) property and were used as luminophors to fabricate a sandwich-type ECL immunosensor. Aminated graphene (NH2-G) was also synthesized and used as a label of secondary antibody. The labeled NH2-G could effectively quench the ECL of N-CQDs modified on electrodes due to ECL resonance energy transfer (ERET). Immunological recognition which induced ECL quenching enabled the quantitative determination of biomarkers. Alpha fetoprotein (AFP) was selected as a model analyte to investigate the analytical performance of the proposed immunosensor. Under optimal conditions, a good linear relationship between ECL intensity and the logarithm of AFP concentration was obtained in the range of 0.01–100 ng mL−1 with the detection limit of 3.3 pg mL−1. The proposed ECL immunosensor showed good stability, acceptable selectivity and reproducibility.

Ultrasensitive enzyme-free electrochemical immunoassay for free thyroxine based on three dimensionally ordered macroporous chitosan–Au nanoparticles hybrid film

The measurement of free thyroxine concentration in serum is considered to be an essential indicator of thyroid function. Here, a novel enzyme-free sandwich electrochemical immunosensor for the detection of FT4 antigen based on the immobilization of primary antibody (Ab1) on three dimensional ordered macroporous chitosan–Au nanoparticles hybrid (3DOM CS–AuNPs) film electrode, and magnetic multiwall carbon nanotubes (MMWCNTs) were used as label of secondary antibody (Ab2). The 3DOM CS–AuNPs film electrode was constructed by one-step electrodeposition of CS–AuNPs composite onto Au electrode with silica opal template. MMWCNTs were prepared by chemical co-precipitation of Fe2+ and Fe3+ salts on carboxylated MWCNTs. Ru(bpy)32+ labeled anti-FT4 (Ru(bpy)32+–Ab2) was covalently attached to MMWCNTs through the formation of amide bond between the carboxylic groups of MWCNTs and the amine groups of antibody. Under the optimal conditions, FT4 was detected in a concentration range from 0.71fgmL−1 to 1.15pgmL−1 with a correlation coefficient of 0.998 and a detection limit of 0.20fgmL−1. Moreover, the immunosensor showed excellent selectivity, good stability, satisfactory reproducibility and regeneration. Importantly, the developed method was used to assay clinical serum specimens, achieving a good relation with those obtained from the commercialized electrochemiluminescent method.