Detection Of Α-fetoprotein Research Articles

Novel electrochemical immune sensor based on Hep-PGA-PPy nanoparticles for detection of α-Fetoprotein in whole blood

A simple and accurate immune sensor for quantitative detection of α-Fetoprotein (AFP) was developed based on the immobilization of antigen on the surface of Hep-PGA-PPy nanoparticles modified glassy carbon electrodes (GCE). The obtained Hep-PGA-PPy nanoparticles were characterized by fourier transform infrared (FT-IR) spectra and transmission electron microscopy (TEM). And the blood compatibility of Hep-PGA-PPy nanoparticles was investigated by invitro coagulation tests, hemolysis assay and whole blood adhesion tests. Combining the conductive property of polypyrrole (PPy) and the biocompatibility of heparin (Hep), the Hep-PGA-PPy nanoparticles could improve not only the anti-biofouling effect the electrode, but also improved the electrochemical properties of the immune sensor. Under optimal conditions, the proposed immune sensor could detect AFP in a linear range from 0.1 to 100ngmL-1 with a detection limit of 0.099ngmL-1 at the signal-to-noise ratio of 3, and it also possessed good reproducibility and storage stability. Furthermore, the detection of AFP in five human blood samples also showed satisfactory accuracy with low relative errors. Thus, the developed immune sensor which showed acceptable reproducibility, selectivity, stability and accuracy could be potentially used for the detection of whole blood samples directly.

A Novel Current-suppression-type Immunoassay of Tumor Markers Based on Gold Nanorods and Silver Nanoflowers

A new current-suppression-type electrochemical immunosensor with enhanced sensitivity for the detection of α-fetoprotein was developed based on the ease of preparing gold nanorods to immobilize α-fetoprotein antibody coupled with silver nanoflower deposition, to act as a redox mediator, on a Au electrode. The interaction of antigen and antibody on the electrode interface makes a barrier for electrons and inhibits electro-transfer, resulting in decreased DPV signals of nano-Ag.

Label-free immunosensor based on hyperbranched polyester for specific detection of α-fetoprotein

A novel label-free immunosensor based on hyperbranched polyester nanoparticles with nitrite groups (HBPE-NO2), which were synthesized through a simple one-step chemical reaction, was first developed for specific detection of α-fetoprotein (AFP), the tumor marker for liver cancer. The obtained HBPE-NO2 nanoparticles (NPs) were characterized by the proton nuclear magnetic resonance spectroscopy (1H NMR), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). And the fabricated process of immunosensor was investigated by attenuated total reflection Fourier-transform infrared spectra (ATR-FTIR), static water contact angles, scanning electron microscope (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The electrochemical performances of the AFP immunosensor were studied. Results indicated the prepared HBPE-NO2-modified immunosensor showed excellent electrochemical properties and satisfactory accuracy for the detection of AFP of the real clinical samples that attributed to the properties of the HBPE-NO2 NPs, which had nanosized structure to increase the specific surface area and unique chemical reactivity for loading capacity of protein molecules. Construction of biosensors using the structure and properties of hyperbranched molecules will offer ideal electrode substrates, which provided more possibilities for the design of biosensor.

A photoelectrochemical immunosensor for detection of α-fetoprotein based on Au-ZnO flower-rod heterostructures

In this work, a novel label free photoelectrochemical (PEC) immunosensor has been developed for the detection of α-fetoprotein (AFP). The immunosensor was based on Au-ZnO flower-rods (FRs) heterostructure, where Au nanoparticles (NPs) were firstly electrodeposited by cyclic voltammetry methods. Scanning electron microscopy (SEM), X-ray diffraction (XRD), Mott-Schottky plot (MS), UV–vis diffuse reflectance spectrum and fluorescence emission spectrum were used for the characterizations of Au-ZnO FRs. The results demonstrated that Au NPs not only obviously enhanced the visible light absorption of ZnO FRs due to surface plasmon resonance (SPR) but also improved the separation of photo-generated electron-hole pairs. Therefore, the photocurrent of Au-ZnO FRs was increased under simulated sunlight. The photocurrent was reduced after the specific antibody-antigen immune reaction. And the photocurrent decrement was linear with the logarithm of AFP antigen concentration in the range from 0.005 ng mL−1 to 50 ng mL−1 with a low detection limit of 0.56 pg mL−1 (S/N = 3). The PEC immunosensor also exhibited high anti-interference property and acceptable stability. This work would provide a promising photoelectrochemical strategy for the detection of other proteins in clinical diagnosis.

The Value of GPC3 and GP73 in Clinical Diagnosis of Hepatocellular Carcinoma.

The incidence of hepatocellular carcinoma (HCC) has increased over the past decades in China. Current screening methods of HCC such as detection of α-fetoprotein (AFP) combined with liver ultrasonography remain unsatisfactory. Many HCC patients have already missed the optimal treatment period when diagnosed. Our study aimed to evaluate the value of Glypican 3 (GPC3) and Golgi protein 73 (GP73) in the detection of HCC. Thirty-nine patients with HCC and 31 patients with liver cirrhosis were enrolled. The level of serum GPC3 and GP73 were determined by ELISA. The expression of GPC3 mRNA and GP73 mRNA in peripheral blood mononuclear cell (PBMC) and liver tissues were also measured with qRT-PCR. Then, receiving operating characteristic (ROC) curves were plotted to detect the sensitivity and specificity of serum GPC3 and GP73 in the diagnosis of HCC. The levels of serum GPC3 and GP73 in the HCC group were significantly higher than in the cirrhosis group (p < 0.0001). Patients with GPC3 > 9.3 μg/L and GP73 > 77.68 ng/mL had a risk of HCC of 92.31%. The HCC diagnosis ROC curve analysis indicated that when setting the GPC3 cutoff value > 9.3 μg/L, AUC = 0.956. The sensitivity and specificity of GPC3 were 89.74% and 96.77%, respectively, with a positive predictive value of 97.2%, negative predictive value of 88.2%, + LR of 27.82 and - LR of 0.11. When setting GP73 cutoff value > 77.68 ng/mL, AUC = 0.937. The sensitivity and specificity of GP73 were 92.31% and 83.87%, respectively, with positive predictive value of 87.8%, negative predictive value of 89.7%, + LR of 5.72 and - LR of 0.092. No significant difference (p > 0.05) was found between GPC3 and GP73 AUC in ROC curves, indicating that these two biomarkers were equivalent in the prediction of HCC. The expression of serum GPC3 and GP73 was significantly higher in the HCC patients compared with the cirrhosis patients. GPC3 and GP73 might be effective non-invasive diagnostic indicators of HCC.

Preparation of gold nanoparticle coated NaYF4:Yb,Er,Gd nanoparticles and their application for AFP detection in the red region

A novel surface plasmon resonance (SPR) enhanced upconversion (UC) luminescence system was designed for the detection of α-fetoprotein (AFP) by using gold nanoparticle (NP) coated NaYF4:Yb,Er,Gd upconversion nanoparticles (UCNPs) as probes.

Ultrasensitive Detection of α-Fetoprotein by Total Internal Reflection Scattering-Based Super-Resolution Microscopy for Superlocalization of Nano-Immunoplasmonics.

Superlocalization of immunoplasmonic nanotags on antibody-bound gold-nanoislands (GNIs) along the x and y coordinates was determined using total internal reflection scattering-based super-resolution microscopy (TIRS-SRM) at subdiffraction limit resolution. Individual immunoplasmonic nanotags (20 nm silver nanoparticles) and 100 nm GNIs were selectively acquired in the evanescent field layer by wavelength-dependent plasmonic scattering using two illumination lasers (405 and 635 nm, respectively). α-Fetoprotein (AFP), a liver cancer-related model protein, was immobilized as a target molecule on the GNI arrays. The centroid position of a localized immunoplasmonic nanotag on the GNI was resolved at less than 10 nm of spatial resolution by applying 2D Gaussian fitting to its point spread function. This method showed enhanced sensitive quantification with a limit of detection (LOD) of 7.04 zM (1-2 molecules of AFP/GNI), which was 100-5000000000 times lower than detection limits obtained with previous AFP detection methods. Furthermore, the method was also successfully applied to quantify AFP molecules at the single-molecule level in human serum samples. The wavelength-dependent TIRS-SRM method was demonstrated to be an effective tool for superlocalizing individual protein molecules and interactions in nanoscale regions and was a reliable method for the ultrasensitive quantitative detection of disease-related protein molecules as a nanosensor and for diagnosis at the single-molecule level.

Nonenzymatic Electrochemical Immunosensor Using Ferroferric Oxide–Manganese Dioxide–Reduced Graphene Oxide Nanocomposite as Label for α-Fetoprotein Detection

A novel nonenzymatic electrochemical immunosensor was fabricated for quantitative detection of [Formula: see text]-fetoprotein (AFP). The immunosensor was constructed by modifying gold electrode with electrochemical reduction of graphene oxide-carboxyl multi-walled carbon nanotube composites (ERGO–CMWCNTs) and electrodeposition of gold nanoparticles (AuNPs) for effective immobilization of primary antibody (Ab[Formula: see text]. Ferroferric oxide–manganese dioxide–reduced graphene oxide nanocomposites (Fe3O4@MnO2–rGO) were designed as labels for signal amplification. On one hand, the excellent electroconductivity and outstanding electron transfer capability of ERGO–CMWCNTs/AuNPs improved the sensitivity of the immunosensor. On the other hand, introduction of rGO could not only increase the specific surface area for immobilization of secondary antibody (Ab[Formula: see text] but also build a synergetic effect to reinforce the electrocatalytic properties of catalysts. Fe3O4@MnO2–rGO nanocomposites were characterized by scanning electron microscope, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Using AFP as a model analyte, the proposed sandwich-type electrochemical immunosensor exhibited a wide linear range of 0.01–50[Formula: see text][Formula: see text] with a low detection limit of 5.8[Formula: see text][Formula: see text]. Moreover, the Fe3O4@MnO2–rGO-based peroxidase mimetic system displayed an excellent analytical performance with low cost, satisfactory reproducibility and high selectivity, which could be further extended for detecting other disease-related biomarkers.

A SPR biosensor based on signal amplification using antibody-QD conjugates for quantitative determination of multiple tumor markers.

The detection of tumor markers is very important in early cancer diagnosis; however, tumor markers are usually present at very low concentrations, especially in the early stages of tumor development. Surface plasmon resonance (SPR) is widely used to detect biomolecular interactions; it has inherent advantages of being high-throughput, real-time, and label-free technique. However, its sensitivity needs essential improvement for practical applications. In this study, we developed a signal amplification strategy using antibody-quantum dot (QD) conjugates for the sensitive and quantitative detection of α-fetoprotein (AFP), carcinoembryonic antigen (CEA) and cytokeratin fragment 21-1 (CYFRA 21-1) in clinical samples. The use of a dual signal amplification strategy using AuNP-antibody and antibody-QD conjugates increased the signal amplification by 50-folds. The constructed SPR biosensor showed a detection limit as low as 0.1 ng/mL for AFP, CEA, and CYFRA 21-1. Moreover, the results obtained using this SPR biosensor were consistent with those obtained using the electrochemiluminescence method. Thus, the constructed SPR biosensor provides a highly sensitive and specific approach for the detection of tumor markers. This SPR biosensor can be expected to be readily applied for the detection of other tumor markers and can offer a potentially powerful solution for tumor screening.

Signal-on electrochemiluminescent immunosensor based on poly(amidoamine) dendrimer functionalized carbon nanodots amplification for ultrasensitive detection of α-fetoprotein

Abstract An ultrasensitive sandwich-type electrochemiluminescent biosensor based on carbonaceous based nanomaterials was fabricated for alpha-fetoprotein detection. Herein, the composite of fullerene, graphene oxide and chitosan with specific surface area, good water dispersibility, and excellent biocompatibility was first modified onto the electrode surface and then it was in situ electrochemical reduced to improve the conductivity. After the capturing antibody was immobilized onto the modified electrode surface, the detection antibody together with poly(amidoamine) dendrimers functionalized carbon nanodots as the recognizable signal tags was self-assembled onto the electrode by typical sandwich immune reaction. By this design, the abundant functional groups, good water dispersibility and good electroconductivity of poly(amidoamine) dendrimers functionalized carbon nanodots make the composite not only an immobilizing platform to carrier numerous detection antibodies, but also an promising promoter for the electrochemiluminescent signal of luminol. With the aforementioned amplify factor, the established electrochemiluminescent biosensor under the optimal conditions demonstrated a wider dynamic response from 1 fg mL−1 to 80 ng mL−1 with a low detection limit of 0.33 fg mL−1. Additionally, the experimental results exhibited the immunosensor possessed excellent stability, reproducibility and selectively. Accordingly, this immunosensing strategy for alpha-fetoprotein with the assistance of poly(amidoamine) dendrimers functionalized carbon nanodots provided a promising approach in clinical disease scanning.

Immobilization of antibodies on aldehyde-functionalized polymer/graphene films for the fabrication of a label-free electrochemical immunosensor

Abstract Using poly-(epichlorohydrin) and 4-pyridinecarboxaldehyde, we synthesized a new aldehyde-functionalized polymer, which was applied to simplify antibody immobilization on the electrode surface, in which no additional chemical cross-linking is required. Combined with the advantages of graphene, a label-free electrochemical immunosensor based on graphene/aldehyde-containing polymer film for α-fetoprotein detection was fabricated. α-Fetoprotein was detected within the range from 0.05 to 35 ng mL − 1 with a detection limit of 0.02 ng mL − 1 obtained by 3 S/N. The proposed immunosensor is easy, label-free, specific and thus provides a good diagnostic tool for detection of α-fetoprotein and other cancer markers.

Branched zinc oxide nanorods arrays modified paper electrode for electrochemical immunosensing by combining biocatalytic precipitation reaction and competitive immunoassay mode.

Branched zinc oxide nanorods (BZR) arrays, an array with high charge carries collection efficiency and specific surface area, are grown on the reduced graphene oxide-paper working electrode for the first time to construct a paper-based electrochemical (EC) immunosensor. Typically, the BZR are fabricated via a simple hydrothermal process, which can provide abundant sites for antibodies loading. By combining the large surface area of porous zinc oxide (PZS) and good biocompatibility of gold nanoparticles (AuNPs), PZS-AuNPs (PZS@Au) nanocomposites are designed to label horseradish peroxide (HRP) and antigens. After a competitive reaction between antigens and PZS@Au nanocomposites labeled antigens, the signal labels are introduced into the immunosensor, in which, HRP participate in biocatalytic precipitation process. The produced precipitate reduces the electrode surface area and hinders the electron transfer. With the increase of concentration of antigens, the signal labels introduced into the sensor decrease, thus, a signal-on immunoassay for α-fetoprotein detection is constructed. The proposed paper-based EC immunosensor combines enzymatic biocatalytic precipitation reaction and competitive immunoassay mode for the first time, and possesses a wide linear range from 0.2 pg mL(-1) to 500 ng mL(-1) with a detection limit of 0.08 pg mL(-1). In addition, the proposed method is simple, sensitive and specific and can be a promising platform for other protein detection.

Detection of α-fetoprotein with an ultrasensitive electrochemiluminescence paper device based on green-luminescent nitrogen-doped graphene quantum dots

Abstract A facile one-pot solvothermal method based on exfoliating and disintegrating treatments for graphite oxide was developed to prepare water-soluble graphene quantum dots. Dimethylformamide served as a solvent and source of nitrogen, the as-prepared nitrogen-doped graphene quantum dots (N-GQDs) exhibited bright green emission under ultraviolet irradiation (∼365 nm). More importantly, an ultrasensitive paper-based electrochemiluminescence (ECL) immunoassay was developed with green-luminescent N-GQDs as ECL labels and α-fetoprotein antigen (AFP) as model protein. The N-GQDs were promising ECL labels because of their low anodic ECL potential of +0.84 V and high biocompatibity, which can facilitate highly sensitive ECL bioassays. A novel paper working electrode was fabricated through a seed-mediated growth approach and served as a promising platform for antibodies attachment. Upon the immunorecognition of the immobilized AFP to its antibody labeled with N-GQDs, the proposed immunoassay displayed increasing ECL intensity, leading to a wide calibration range of 0.005–100 ng mL −1 with a detection limit of 1.2 pg mL −1 [signal-to-noise ratio (S/N) = 3]. The ECL immunoassay for real samples displayed very similar results with the commercial ECL immunoassay, which provided a powerful avenue for the design of the ultrasensitive detection method for clinical application.

Sensitive detection of a tumor marker, α-fetoprotein, with a sandwich assay on a plasmonic chip.

Two types of plasmonic silver- and gold-coated grating biosensor chips (plasmonic chip) were applied in the detection of α-fetoprotein (AFP) with a sandwich imunoassay and surface plasmon field-enhanced fluorescence. On the plasmonic chip, unlabeled marker in the sandwich immunoassay was first quantitatively detected over a wide range between 10(-12) and 10(-8) g/mL. The affinity constants between AFP and anti-AFP antibody, which were obtained by fitting the experimental data to the Langmuir isotherm adsorption curve, were 1 × 10(8) g(-1) mL regardless of the kind of metal in the plasmonic chips. Although the fluorescence intensity on the silver plasmonic chip was 5 times larger than that on the gold plasmonic chip, the limit of detection (LOD) was on the order of 10(-11) g/mL and not improved with a silver plasmonic chip. Herein, we used a new setup that generated less dispersions of both the fluorescence intensity for nonspecific adsorption and the background (optical blank) signal and improved the LOD of AFP to 4 pg/mL (55 fM) with the silver plasmonic chip. With the highly sensitive detection in the sandwich immunoassay, the development of a plasmonic chip for clinical diagnosis by a blood test is promising.

A sandwich-type electrochemical immunosensor based on multiple signal amplification for α-fetoprotein labeled by platinum hybrid multiwalled carbon nanotubes adhered copper oxide

Abstract In this work, we designed a novel and ultrasensitive sandwich-type electrochemical immunosensor for quantitative detection of α-fetoprotein (AFP), using β-cyclodextrin functionalized graphene (CD-GS) as the platform and platinum hybrid multiwalled carbon nanotubes adhered copper oxide (Pt@CuO-MWCNTs) as labels. CD-GS has high specific surface area, ideal dispersibility and good biocompatibility, which was used to efficiently capture more primary antibodies (Ab 1 ). Pt, CuO and multiwalled carbon nanotubes (MWCNTs) can be used as catalysts for the decomposition of hydrogen peroxide. The Pt@CuO-MWCNTs realized multiple amplified signals due to their synergy. Therefore, Pt@CuO-MWCNTs was used as the marker, combined with secondary antibodies (Ab 2 ) and this has the effect of multiple amplified signals to catalyze hydrogen peroxide. Under optimal conditions the designed immunosensor exhibited a wide linear range from 1 pg mL −1 to 20 ng mL −1 with a low detection limit of 0.33 pg mL −1 (S/N = 3) for AFP. The designed immunosensor showed good selectivity, reproducibility and stability, which could provide potential applications in clinical monitoring of AFP.

Sensitive colorimetric detection of protein by gold nanoparticles and rolling circle amplification.

An ultrasensitive method for the detection of protein is critically important in fundamental research and practical applications due to the low abundance of disease markers in body fluids or tissues. To detect the trace levels of disease markers with high sensitivity and specificity, a sensitive colorimetric biosensor for protein assay was developed using gold nanoparticles (AuNPs) and rolling circle amplification (RCA). After binding the biotinylated primer/circular template to the streptavidin-conjugated sandwich ELISA immunocomplex, the biotinylated primer was isothermally extended to generate single-stranded DNA (ssDNA). Sequentially, the padlock DNA was added and hybridized with the RCA products. The aggregation of the additional AuNPs in the supernatant containing the surplus padlock DNA and a certain concentration of salt could then be observed. The established sensor allowed for the specific detection of α-fetoprotein (AFP) with a detection limit of 33.45 pg mL(-1). It was also demonstrated that this method could distinguish 500 pg mL(-1) AFP with the naked eye. In addition, this biosensor could be applied to complex sample analysis and could be further used as a universal method for any protein or virus determination by changing the corresponding antibodies.

A novel photoelectrochemical sensor for the detection of α-fetoprotein based on a mesoporous TiO2–CdS QD composite film

A label-free photoelectrochemical (PEC) immunosensor based on a CdS-modified mesoporous TiO2 film is reported to detect α-fetoprotein (AFP). The assay platform exhibits excellent selectivity and sensitivity. The detection limit of 0.02 ng mL−1 for AFP is obtained.

Immunosensor based on carbon nanotube/manganese dioxide electrochemical tags

This article reports on carbon nanotube/manganese dioxide (CNT-MnO2) composites as electrochemical tags for non-enzymatic signal amplification in immunosensing. The synthesized CNT-MnO2 composites showed good electrochemical activity, electrical conductivity and stability. The electrochemical signal of CNT-MnO2 composites coated glassy carbon electrode (GCE) increased by nearly two orders of magnitude compared to bare GCE in hydrogen peroxide (H2O2) environment. CNT-MnO2 composite was subsequently validated as electrochemical tags for sensitive detection of α-fetoprotein (AFP), a tumor marker for diagnosing hepatocellular carcinoma. The electrochemical immunosensor demonstrated a linear response on a log-scale for AFP concentrations ranging from 0.2 to 100 ng mL(-1). The limit of detection (LOD) was estimated to be 40 pg mL(-1) (S/N=3) in PBS buffer. Further measurements using AFP spiked plasma samples revealed the applicability of fabricated CNT-MnO2 composites for clinical and diagnostic applications.

Electrochemical immunosensor for α-fetoprotein detection using ferroferric oxide and horseradish peroxidase as signal amplification labels

An electrochemical immunosensor for quantitative detection of α-fetoprotein (AFP) in human serum was developed using graphene sheets (GS) and thionine (TH) as electrode materials and mesoporous silica nanoparticles (MSNs) loaded with ferroferric oxide (Fe3O4) nanoparticles and horseradish peroxidase (HRP) as labels for signal amplification. In this study, the compound of GS and TH (GS-TH) was used as a substrate for promoting electron transfer and immobilization of primary antibody of AFP (Ab1). MSNs were used as a carrier for immobilization of secondary antibody of AFP (Ab2), Fe3O4, and HRP. The synergistic effect occurred between Fe3O4 and HRP and greatly improved the sensitivity of the immunosensor. This method could detect AFP over a wide concentration range from 0.01 to 25 ng ml(-1) with a detection limit of 4 pg ml(-1). This strategy may find wide potential application in clinical analysis or detection of other tumor markers.

A novel solid-state Ru(bpy)32+ electrochemiluminescence immunosensor based on poly(ethylenimine) and polyamidoamine dendrimers as co-reactants

In this study, a novel solid-state Ru(bpy)3(2+) electrochemiluminescence (ECL) sandwiched immunosensor for sensitive detection of α-fetoprotein (AFP) was constructed based on poly(ethylenimine) (PEI) functionalized reduced graphene oxide (PEI-rGO) and Au nanoparticles (AuNPs) decorated polyamidoamine (PAMAM) dendrimers. Both PEI and PAMAM are polymers with a lot of amino groups, which are able to serve as good co-reactant to remarkably enhance the ECL signal of Ru(bpy)3(2+). For improving the poor conductivity of PAMAM, the AuNPs were decorated on the amino groups of PAMAM. Through Au-N bonds, the formed AuNPs-PAMAM was decorated on the PEI-rGO. The obtained AuNPs-PAMAM/PEI-rGO was introduced to immobilize the detection antibody (Ab2). Then, the Ab2 labeled AuNPs-PAMAM/PEI-rGO was modified onto the glass carbon electrode surface via sandwiched immunoreactions. The ECL substrate was prepared by mixing nafion and the complex (Ru-PtNPs) of Pt nanoparticles (PtNPs) and Ru(bpy)3(2+), which could reduce the consumption of Ru complex, simplify the operation and enhance the ECL efficiency. The experimental results demonstrated that the proposed immunosensor had good response to AFP. The linear range was from 0.01 pg mL(-1) to 10 ng mL(-1) with a low detection limit of 3.3 fg mL(-1). Meanwhile, with satisfying stability, selectivity and reproducibility, the proposed sandwiched immunosensor was presented to possess good potential in clinical detection.