An Ultra-sensitive Electrochemiluminescent Detection of Carcinoembryonic Antigen Using a Hollowed-out Electrode

Ultrasensitive detection of cancer biomarkers has attracted considerable attention recently in academic research and clinic diagnostics. Here, we use a hollowed-out carbon nanotube sponge (CNTSP) electrode to fabricate an immunosensor to realize the sensitive detection of carcinoembryonic antigen (CEA). Nitrogen-doped carbon quantum dots (N-CDs) are combined with antibody that can specially recognize CEA and used as the electrochemiluminescent (ECL) probes in this work. The hollowed-out and permeable CNTSP facilitates chemical species exchange on the surface of electrode, offering an enhanced ECL signal. The resulting ECL immunosensor enables the determination of CEA concentration to be in a wide linear range from 0.005 to 50 pg mL⁻¹ with a detection limit of 1.4 fg mL⁻¹. Furthermore, with good stability, acceptable precision and reproducibility, the proposed ECL assay strategy offers a wide application potential in clinical analysis.     

Electrochemical Biosensor Based on Nanocomposites Film of Thiol Graphene‐Thiol Chitosan/Nano Gold for the Detection of Carcinoembryonic Antigen

In this paper, a thiol graphene‐thiol chitosan‐gold nanoparticles (thGP‐thCTS‐AuNPs) nanocomposites film with porous structure was fabricated by electrochemically depositing on glassy carbon electrode (GCE), which exhibited good biocompatibility and improved conductivity, to construct immunosensor free label for detection of carcinoembryonic antigen (CEA). The electrochemical behavior of this immunosensor was investigated by cyclic voltammetry. Under the optimum conditions, the immunosensor revealed a good amperometric response to CEA in two linear ranges (0.3–8.0 ng mL^?1 and 8.0–100 ng mL^?1) with a detection limit of 0.03 ng mL^?1. The results indicated that the immunosensor has the advantages of good selectivity, high sensitivity, and good stability for the determination of CEA.     

Electrochemiluminescence biosensor for carcinoembryonic antigen detection based on Au-Ag/g-C3N4 nanocomposites

Herein, an electrochemiluminescence (ECL) aptasensor for carcinoembryonic antigen (CEA) detection was developed based on Au-Ag/g-C₃N₄ nanocomposites (NCs), which were synthesized by decorating graphitic carbon nitride (g-C₃N₄) nanosheets with alloy-structured Au-Ag bimetallic nanoparticles (NPs) via one-step in situ chemical reduction. As ECL sensing platform, Au-Ag/g-C₃N₄ NCs could significantly improve the ECL intensity of luminol due to the good conductivity of Au-Ag NPs, electrocatalytic activity for oxygen evolution reaction (OER) and the ability to adsorb luminol via π stacking interaction. In addition, it could load the thiol terminated aptamers of CEA via Au-S or Ag-S bonds. In the presence of CEA, the ECL response of the proposed biosensor decreased significantly due to the fact that the assembled protein layers hindered the electron transfer and the diffusion of ECL reactants toward the electrode surface. The proposed ECL sensor exhibited a good linear relationship with CEA in the range of 1.0–1.0 × 10^?6 ng/mL with a detection limit of 8.9 × 10^?7 ng/mL. The satisfactory results were obtained in the detection of CEA in human serum samples.     

Ultrasensitive Eletrogenerated Chemiluminescence Immunoassay by Magnetic Nanobead Amplification

An ultrasensitive electrogenerated chemiluminescence (ECL) immunoassay was proposed by using magnetic nanobeads (MNBs) as the carrier of ECL labels for ECL emission amplification. Carcinoembryonic antigen (CEA) and MNBs were initially immobilized on a platform in 1 : 1 molar ratio via sandwich immunoreaction. Subsequently, the MNBs were released from the platform and labeled with Ru(bpy)₃²⁺ species. After the MNBs with Ru(bpy)₃²⁺ were immobilized on an Au electrode, ECL of the Ru(bpy)₃²⁺ was measured for CEA determination. A linear relation between the ECL intensity and CEA concentration was obtained in a range of 1×10^?14 to 3×10^?13 mol/L (2.0 to 60 pg/mL) with a limit of detection of 8.0×10^?15 mol/L (1.6 pg/mL).     

An ultrasensitive luminol cathodic electrochemiluminescence immunosensor based on glucose oxidase and nanocomposites: Graphene–carbon nanotubes and gold-platinum alloy

In the present study, a novel and ultrasensitive electrochemiluminescence (ECL) immunosensor based on luminol cathodic ECL was fabricated by using Au nanoparticles and Pt nanoparticles (nano-AuPt) electrodeposited on graphene–carbon nanotubes nanocomposite as platform for the detection of carcinoembryonic antigen (CEA). For this introduced immunosensor, graphene (GR) and single wall carbon nanotubes (CNTs) dispersed in chitosan (Chi-GR-CNTs) were firstly decorated on the bare gold electrode (GE) surface. Then nano-AuPt were electrodeposited (DpAu-Pt) on the Chi-GR-CNTs modified electrode. Subsequently, glucose oxidase (GOD) was employed to block the non-specific sites of electrode surface. When glucose was present in the working buffer solution, GOD immediately catalyzed the oxidation of glucose to in situ generate hydrogen peroxide (H₂O₂), which could subsequently promote the oxidation of luminol with an amplified cathodic ECL signal. The proposed immunosensor was performed at low potential (−0.1 to 0.4 V) and low concentration of luminol. The CEA was determined in the range of 0.1 pg mL⁻¹ to 40 ng mL⁻¹ with a limit of detection down to 0.03 pg mL⁻¹ (S N⁻¹ = 3). Moreover, with excellent sensitivity, selectivity, stability and simplicity, the as-proposed luminol-based ECL immunosensor provided great potential in clinical applications.     

Luminescence‐Functionalized Metal–Organic Frameworks Based on a Ruthenium(II) Complex: A Signal Amplification Strategy for Electrogenerated Chemiluminescence Immunosensors

Novel luminescence‐functionalized metal–organic frameworks (MOFs) with superior electrogenerated chemiluminescence (ECL) properties were synthesized based on zinc ions as the central ions and tris(4,4′‐dicarboxylicacid‐2,2′‐bipyridyl)ruthenium(II) dichloride ([Ru(dcbpy)₃]²⁺) as the ligands. For potential applications, the synthesized MOFs were used to fabricate a “signal‐on” ECL immunosensor for the detection of N‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP). As expected, enhanced ECL signals were obtained through a simple fabrication strategy because luminescence‐functionalized MOFs not only effectively increased the loading of [Ru(dcbpy)₃]²⁺, but also served as a loading platform in the ECL immunosensor. Furthermore, the proposed ECL immunosensor had a wide linear range from 5 pg mL^?1 to 25 ng mL^?1 and a relatively low detection limit of 1.67 pg mL^?1 (signal/noise=3). The results indicated that luminescence‐functionalized MOFs provided a novel amplification strategy in the construction of ECL immunosensors and might have great prospects for application in bioanalysis.     

Highly sensitive luminol electrochemiluminescence immunosensor based on ZnO nanoparticles and glucose oxidase decorated graphene for cancer biomarker detection

In this work, we reported a sandwiched luminol electrochemiluminescence (ECL) immunosensor using ZnO nanoparticles (ZnONPs) and glucose oxidase (GOD) decorated graphene as labels and in situ generated hydrogen peroxide as coreactant. In order to construct the base of the immunosensor, a hybrid architecture of Au nanoparticles and graphene by reduction of HAuCl₄ and graphene oxide (GO) with ascorbic acid was prepared. The resulted hybrid architecture modified electrode provided an excellent platform for immobilization of antibody with good bioactivity and stability. Then, ZnONPs and GOD functionalized graphene labeled secondary antibody was designed for fabricating a novel sandwiched ECL immunosensor. Enhanced sensitivity was obtained by in situ generating hydrogen peroxide with glucose oxidase and the catalysis of ZnONPs to the ECL reaction of luminol–H₂O₂ system. The as-prepared ECL immunosensor exhibited excellent analytical property for the detection of carcinoembryonic antigen (CEA) in the range from 10 pg mL⁻¹ to 80 ng mL⁻¹ and with a detection limit of 3.3 pg mL⁻¹ (S N⁻¹ = 3). The amplification strategy performed good promise for clinical application of screening of cancer biomarkers.     

Electrochemiluminescence Immunosensor Based on Functionalized Graphene/Fe3O4‐Au Magnetic Capture Probes for Ultrasensitive Detection of Tetrodotoxin

An ultrasensitive electrochemiluminescence (ECL) immunosensor for the detection of tetrodotoxin (TTX) is proposed, which are composed of the branched poly‐(ethylenimine) (BPEI) functionalized graphene (BGNs)/Fe₃O₄‐Au magnetic capture probes and luminol‐capped gold nanocomposites (luminol‐AuNPs) as the signal tag. Herein, a typical sandwich immunecomplex was constructed on the glassy carbon electrode. The BGNs/Fe₃O₄‐Au hybrids could efficiently conjugate primary antibody via the Au−S chemical bonds or Au−N chemical bonds and rapidly separate under external magnetic field. The introduction of BPEI to GO could enhance the luminol‐ECL intensity. Meanwhile, the multifunctional nanocomposites have been proved with good water‐solubility, excellent electron transfer, outstanding stability, etc. The luminescent luminol‐AuNPs, a high efficient electrochemiluminescence marker, can be assembled on the second antibody, which can produce the ECL signal to achieve the determination of TTX. This proposed ECL immunosensor with a linear range from 0.01–100 ng/mL can be applied in the detection of TTX in real samples with satisfactory results.     

A novel lable-free electrochemical immunosensor for carcinoembryonic antigen based on gold nanoparticles-thionine-reduced graphene oxide nanocomposite film modified glassy carbon electrode

In this paper, gold nanoparticle-thionine-reduced graphene oxide (GNP-THi-GR) nanocomposites were prepared to design a label-free immunosensor for the sensitive detection of carcinoembryonic antigen (CEA). The nanocomposites with good biocompatibility, excellent redox electrochemical activity and large surface area were coated onto the glassy carbon electrode (GCE) surface and then CEA antibody (anti-CEA) was immobilized on the electrode to construct the immunosensor. The morphologies and electrochemistry of the formed nanocomposites were investigated by using scanning electron microscopy (SEM), ultraviolet-visible (UV-vis) spectrometry, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). CV and differential pulse voltammetry (DPV) studies demonstrated that the formation of antibody-antigen complexes decreased the peak current of THi in the GNP-THi-GR nanocomposites. The decreased currents were proportional to the CEA concentration in the range of 10-500 pg/mL with a detection limit of 4 pg/mL. The proposed method was simple, fast and inexpensive for the determination of CEA at very low levels.     

Highly enhanced electrochemiluminescent strategy for tumor biomarkers detection with in situ generation of l-homocysteine for signal amplification

In this work, an ultrasensitive peroxydisulfate electrochemiluminescence (ECL) immunosensor using in situ generation of l-homocysteine (l-Hcys) for signal amplification was successfully constructed for detection of carcinoembryonic antigen (CEA). In the reaction of biological methylation, S-adenosyl-l-homocysteine hydrolase (SAHH) catalyzed the reversible hydrolysis of S-adenosyl-l-homocysteine (SAH) to produce l-Hcys, which was inducted into ECL system to construct the immunosensor for signal amplification in this work. Simultaneously, Gold and palladium nanoparticles functionalized multi-walled carbon nanotubes (Au-PdNPs@MWCNTs) were prepared, which were introduced to immobilize the secondary antibody (Ab₂) and SAHH with high loading amount and good biological activity due to their improved surface area and excellent biocompatibility. Then the proposed ECL immunosensor was developed by a sandwich-type format using Au-PdNPs@MWCNTs-SAHH-Ab₂ as tracer and graphene together with AuNPs as substrate. Besides the enhancement of Au-PdNPs, the enzymatic catalysis reaction also amplified the ECL signal dramatically, which was achieved by efficient catalysis of the SAHH towards the hydrolysis of SAH to generate improved amount of l-Hcys in situ. Furthermore, due to the special interaction between Au-PdNPs and -SH or -NH₂ in l-Hcys, l-Hcys would gradually accumulate on the surface of the immunosensor, which greatly enhanced the concentration of l-Hcys on the immunosensor surface and further improved the ECL intensity. With the amplification factors above, a wide linear ranged from 0.1 pg mL⁻¹ to 80 ng mL⁻¹ was acquired with a relatively low detection limit of 33 fg mL⁻¹ for CEA.     

A designer ormosil gel for preparation of sensitive immunosensor for carcinoembryonic antigen based on simple direct electron transfer

The excellent direct electron transfer (DET) of enzyme labeled to antibody immobilized in designer organically modified silicate (ormosil) sol–gel was achieved at an electrode, which was used to construct a novel reagentless immunosensor for antigen determination. The synthesized ormosil architecture provided a hydrophilic interface for retaining the activity of immobilized enzyme labeled immunocomponent. The proposed immunosensor for carcinoembryonic antigen (CEA) prepared by immobilizing horseradish peroxidase-labeled CEA antibody (HRP-anti-CEA) in the architecture showed a surface-controlled electrode process attributed to the DET between electrode and HRP with a rate constant of 5.94 ± 0.40 s⁻¹. The formation of immunocomplex upon incubation in CEA or sample solution led to block of DET and linearly decrease in voltammetric response over CEA concentration ranging from 0.5 to 3.0 and 3.0 to 120 ng ml⁻¹. The limit of detection for CEA was 0.4 ng ml⁻¹. The immunosensor showed good accuracy and acceptable storage stability, precision and reproducibility. The proposed method was simple, low-cost and potentially attractive for clinical immunoassays.     

A Solid‐State Electrochemiluminescence Immunosensor Based on MWCNTs‐Nafion and Ru(bpy)32+/Nano‐Pt Nanocomposites for Detection of α‐Fetoprotein

A approach was successfully employed for constructing a solid‐state electrochemiluminescence (ECL) immunosensor by layer‐by‐layer self‐assembly of multiwall carbon nanotubes (MWCNTs)‐Nafion composite film, Ru(bpy)₃²⁺/nano‐Pt aggregates (Ru‐PtNPs) and Pt nanoparticles (PtNPs). The influence of Pt nanoparticles on the ECL intensity was quantitatively evaluated by calculating the electroactive surface area of different electrodes with or without PtNPs to immobilize Ru(bpy)₃²⁺. The principle of ECL detection for target α‐fetoprotein antigen (AFP) was based on the increment of resistance after immunoreaction, which led to a decrease in ECL intensity. The linear response range was 0.01–10 ng mL^?1 with the detection limit of 3.3 pg mL^?1. The immunosensor exhibited advantages of simple preparation and operation, high sensitivity and good selectivity.     

Study on an immunosensor based on gold nanoparticles and a nano-calcium carbonate/Prussian blue modified glassy carbon electrode

An amperometric carcinoembryonic antigen (CEA) immunosensor was fabricated based on Prussian blue (PB), nano-calcium carbonate (nano-CaCO₃) and nano-gold modified glassy carbon electrode. First, PB as a mediator was deposited on glassy carbon electrode to obtain a negatively charged surface. Then, positive nano-CaCO₃ was adsorbed on the PB modified electrode through electrostatic interaction. Subsequently, gold nanoparticles were deposited on the nano-CaCO₃/PB modified electrode. The use of two kinds of nanomaterials (nano-CaCO₃ and nano-gold) with good biocompatibility as immobilization matrixes not only provides a biocompatible surface for protein loading but also avoids the leaking of PB. The size of nano-CaCO₃ was characterized by transmission electron microscopy (TEM). The factors influencing the performance of the immunosensor presented were studied in detail. Under the optimized conditions, cyclic voltammograms (CV) determination of CEA showed a specific response in two concentration ranges from 0.3 to 20 ng mL^?1 and from 20 to 100 ng mL^?1 with a detection limit of 0.1 ng mL^?1 at a signal-to-noise ratio of 3. The immunosensor presented exhibited high selectivity, sensitivity and good stability.     

CdSe Quantum Dots Based Electrochemiluminescence Immunosensor with Simple Structure for Ultrasensitive Detection of Salbutamol

A rapid and ultrasensitive electrochemiluminescence (ECL) competitive immunoassay based on CdSe quantum dots (QDs) and the shorter chain as possible (cysteamine and glutaraldehyde) has been designed for the detection of salbutamol (SAL). Cysteamine and glutaraldehyde made coating antigen immobilize well on the gold electrode surface through the reaction between functional groups, which brought about the simplicity of the immunosensor to some extent. Transmission electron microscopy image, dynamic light scattering, photoluminescence, ultraviolet‐visible absorption and electrochemical impedance spectra were used to characterize the prepared CdSe QDs and the cysteamine/glutaraldehyde/Ovalbumin‐SAL/anti‐SAL‐QDs immunosensor. In the air‐saturated PBS buffer containing 0.1 M K₂S₂O₈ and 0.1 M KCl (pH 9.0), a strong ECL emission of QDs can be observed which depended linearly on the logarithm of the salbutamol concentration with a wide range from 0.05 ng mL^?1 to 100 ng mL^?1, and a detection limit of 0.0056 ng mL^?1. The sensitivity, repeatability, and specificity of the ECL immunosensor have been evaluated. The sensor has been applied to real samples with satisfactory results. This work will open new ways of detecting food additive residue based on QDs ECL in immunoassays.     

Ultrasensitive electrochemiluminescence immunoassay for tumor marker detection using functionalized Ru-silica@nanoporous gold composite as labels

In this work, we reported a simple and sensitive sandwich-type electrochemiluminescence (ECL) immunosensor for carcinoembryonic antigen (CEA) on a gold nanoparticles (AuNPs) modified glassy carbon electrode (GCE). The Ru-silica (Ru(bpy)(3)(2+)-doped silica) capped nanoporous gold (NPG) (Ru-silica@NPG) composite was used as an excellent label with amplification techniques. The NPG was prepared with a simple dealloying strategy, by which silver was dissolved from silver/gold alloys in nitric acid. The primary antibody was immobilized on the AuNPs modified electrode through l-cysteine and glutaraldehyde, and then the antigen and the functionalized Ru-silica@NPG composite labeled secondary antibody were conjugated successively to form a sandwich-type immunocomplex through the specific interaction. The concentrations of CEA were obtained in the range from 1 pg mL(-1) to 10 ng mL(-1) with a detection limit of 0.8 pg mL(-1). The as-proposed ECL immunosensor has the advantages of high sensitivity, specificity and stability and could become a promising technique for tumor marker detection.     

Ultrasensitive electrochemiluminescent immunosensor based on dual signal amplification strategy of gold nanoparticles-dotted graphene composites and CdTe quantum dots coated silica nanoparticles

A facile and ultrasensitive electrochemiluminescent (ECL) immunosensor for detection of prostate-specific antigen (PSA) was designed by using CdTe quantum dots coated silica nanoparticles (SiO₂@QDs) as bionanolabels. To construct such an electrochemiluminescence immunosensor, gold nanoparticles-dotted graphene composites were immobilized on the working electrode, which can increase the surface area to capture a large amount of primary antibodies as well as improve the electronic transmission rate. The as-prepared SiO₂@QDs used as bionanolabels, showed good ECL performance and good ability of immobilization for secondary antibodies. The approach provided a good linear response ranging from 0.005 to 10 ng?mL^?1 with a low detection limit of 0.0032 ng?mL^?1. Such immunosensor showed good precision, acceptable stability, and reproducibility. Satisfactory results were obtained for determination of PSA in human serum samples. Therefore, the proposed method provides a new promising platform of clinical immunoassay for other biomolecules.     

A Highly Sensitive Electrochemiluminescence Choline Biosensor Based on Poly(aniline‐luminol‐hemin) Nanocomposites

Poly(aniline‐luminol‐hemin) nanocomposites are prepared on an electrode surface through electropolymerization, and a highly sensitive electrochemiluminescence (ECL) biosensor for choline is developed based on the poly(aniline‐luminol‐hemin) nanocomposites and an enzyme catalyzed reaction of choline oxidase (CHOD). The obtained nanocomposites are characterized by scanning electron microscopy (SEM), atomic absorption spectrometry (AAS) and ECL. The results indicate that hemin can be incorporated into the poly(aniline‐luminol) nanocomposites using the facile electropolymerization method, and the poly(aniline‐luminol‐hemin) nanocomposites are rod shaped porous nanostructure. Moreover, the poly(aniline‐luminol‐hemin) nanocomposites exhibit higher ECL intensity than poly(aniline‐luminol) nanocomposites in alkaline media due to the catalytic effect of hemin on the ECL of the polymerized luminol and the electron transfer ability of hemin in the nanocomposites. CHOD is immobilized on the surface of the poly(aniline‐luminol‐hemin) nanocomposites modified electrode with glutaraldehyde, and the ECL biosensor based on poly(aniline‐luminol‐hemin)/CHOD exhibits a wider linear range for the choline detection. The enhanced ECL signals are linear with the logarithm of concentration of choline over the range of 1.0×10^?11～1.0×10^?7 mol L^?1 with a low detection limit of 1.2×10^?12 mol L^?1. Moreover, the proposed biosensor is successfully applied to the detection of choline in milk.     

Electrochemiluminescence immunoassay for the carcinoembryonic antigen using CdSe:Eu nanocrystals

CdSe:Eu nanocrystals were successfully synthesized and characterized by transmission electron microscopy, X-ray powder diffraction, and X-ray photoelectric spectroscopy. The CdSe:Eu nanocrystals showed enhanced green electrochemiluminescence (ECL) intensity when compared to pure CdSe nanocrystals. Further, the nanocrystals were used to design an ECL immunosensor for the detection of carcinoembryonic antigen (CEA) that has a linear response over the 1.0 fg·mL^?1 to 100 ng·mL^?1 CEA concentration range with a 0.4 fg·mL^?1 detection limit. The assay was applied to the determination of CEA in human serum samples.

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Graphical abstract Schematic of the assay: GCE-glassy-carbon electrode, Ab- Antibody, BSA- Bovine serum albumin, Ag- Antigen. CdSe:Eu nanocrystals were used to design an ECL immunosensor for the detection of carcinoembryonic antigen.

     

Preparation of Fe3O4@C@CNC multifunctional magnetic core/shell nanoparticles and their application in a signal-type flow-injection photoluminescence immunosensor

We describe here the preparation of carbon-coated Fe₃O₄ magnetic nanoparticles that were further fabricated into multifunctional core/shell nanoparticles (Fe₃O₄@C@CNCs) through a layer-by-layer self-assembly process of carbon nanocrystals (CNCs). The nanoparticles were applied in a photoluminescence (PL) immunosensor to detect the carcinoembryonic antigen (CEA), and CEA primary antibody was immobilized onto the surface of the nanoparticles. In addition, CEA secondary antibody and glucose oxidase were covalently bonded to silica nanoparticles. After stepwise immunoreactions, the immunoreagent was injected into the PL cell using a flow-injection PL system. When glucose was injected, hydrogen peroxide was obtained because of glucose oxidase catalysis and quenched the PL of the Fe₃O₄@C@CNC nanoparticles. The here proposed PL immunosensor allowed us to determine CEA concentrations in the 0.005–50 ng?·?mL^-1 concentration range, with a detection limit of 1.8 pg?·?mL^-1.

Modification of Gold Electrode via Electrografting of the in situ Generated 3‐Carboxy‐1,2,4‐triazoldiazonium Salt for Label‐free Determination of Carcinoembryonic Antigen

In present study electrografting of the in situ generated 3‐carboxy‐1,2,4‐triazoldiazonium chloride on the Au disk electrode have been studied. The electrode film thickness differences between electrodeposited due to the aryl radical structure 3‐carboxy‐1,2,4‐triazoldiazonium chloride and 4‐carboxyphenyldiazonium salt was shown. The mechanism of 3‐carboxy‐1,2,4‐triazoldiazonium chloride electrografting has been proposed. 4‐nitroanilin was used to investigate the carbodiimide crosslinking capacity. It was established that electrodeposited films are suitable for carbodiimide crosslinking but the reaction proceed only on the “external” electrografted layer. Under the chosen optimal parameters, the label‐free electrochemical immunosensor have been developed. 3‐carboxy‐1,2,4‐triazoldiazonium chloride electrografting provided improvement of analytical characteristics in respect to electrodeposited 4‐carboxyphenyldiazonium salt. The linear range for carcinoembryonic antigen (CEA) detection is 10–10⁴ ng ? ml^?1, the limit of detection estimated as 0.2 ng ? ml^?1. The developed immunosensor is stable during 30 day′s storage and selective against excess of bovine serum albumin as an interfering reagent.