Reduced Graphene Oxide-tetraethylene Pentamine Research Articles

Electrochemical DNA aptamer platform based on CuAlO2/rGO-TEPA@AuPt nanocomposites for dopamine detection

Dopamine (DA) is a neurotransmitter that plays an important role in the central nervous system and has a significant impact on an individual's attention, mood, and cognition. Therefore, real-time monitoring of DA levels could play a key role in the management and treatment of many psychiatric diseases. Herein, we report the development of a sensitive and selective electrochemical aptamer immunosensor for detection of DA. DNA aptamers are used as model protein biomarkers to avoid the effects of interfering substances and to analyze the performance of immunosensors. Copper aluminate (CuAlO2)/reduced graphene oxide-tetraethy lenepentamine (rGO-TEPA) and gold-platinum bimetallic nanoparticles (AuPtNPs) were utilized as modifiers for the sensor fabrication. Notably, rGO-TEPA in the composite was able to induce excess oxygen to CuAlO2, resulting in a strong promotion between CuAlO2 and rGO-TEPA, significantly improving the electron transfer on the immunosensor surface. The bimetallic AuPtNPs would provid more attachment sites for the aptamers, and further improved the sensitivity of the biosensor. Under optimized experimental conditions, the constructed immunosensor was capable of real-time monitoring capabilities within a concentration range of 0.05 nM–10 μM, with a detection limit of 0.017 nM at S/N = 3, highlighting its potential for health management and clinical diagnosis applications.

Electrospun Nanofibrous Membranes Based on Citric Acid-Functionalized Chitosan Containing rGO-TEPA with Potential Application in Wound Dressings.

The present research work is focused on the design and investigation of electrospun composite membranes based on citric acid-functionalized chitosan (CsA) containing reduced graphene oxide-tetraethylene pentamine (CsA/rGO-TEPA) as materials with opportune bio-properties for applications in wound dressings. The covalent functionalization of chitosan (CS) with citric acid (CA) was achieved through the EDC/NHS coupling system and was checked by 1H-NMR spectroscopy and FTIR spectrometry. The mixtures to be electrospun were formulated by adding three concentrations of rGO-TEPA into the 1/1 (w/w) CsA/poly (ethylene oxide) (PEO) solution. The effect of rGO-TEPA concentration on the morphology, wettability, thermal stability, cytocompatibility, cytotoxicity, and anti-biofilm activity of the nanofibrous membranes was extensively investigated. FTIR and Raman results confirmed the covalent and non-covalent interactions that appeared between the system’s compounds, and the exfoliation of rGO-TEPA sheets within the CsA in the presence of PEO (CsA/P) polymer matrix, respectively. SEM analysis emphasized the nanofibrous architecture of membranes and the presence of rGO-TEPA sheets entrapped into the CsA nanofiber structure. The MTT cellular viability assay showed a good cytocompatibility with the highest level of cell development and proliferation registered for the CsA/P composite nanofibrous membrane with 0.250 wt.% rGO-TEPA. The designed nanofibrous membranes could have potential applications in wound dressings, given that they showed a good anti-biofilm activity against Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus bacterial strains.

A sandwich-type electrochemical immunosensor using rGO-TEPA-Thi-Au as sensitive platform and CMK-3@AuPtNPs as signal probe for AFP detection

Alpha-fetoprotein (AFP) plays an important role as a marker in the clinical diagnosis of cancer. However, the sensitive detection of AFP in clinical samples is still challenging. Therefore, we developed a highly sensitive electrochemical immunosensor for the effective detection of AFP. In this study, thionine (Thi) and gold nanoparticles (AuNPs) co-modified reduced graphene oxide-tetraethylene pentamine (rGO-TEPA-Thi-Au), which was used as the immunosensor platform to fix the primary antibody (Ab1), leading to a large improvement in the sensitivity of the immunosensor. Furthermore, ordered mesoporous carbon (CMK-3) modified with AuPt binary nanoparticles (CMK-3@AuPtNPs) was used as the signal probe for further signal amplification because CMK-3 can provide a large specific surface area for in-situ reduction of AuPtNPs with good conductivity. Therefore, a dual signal amplification was observed in the amperometric i-t curve, and the detection limit of the proposed immunosensor was effectively enhanced. The constructed immunosensor showed a wide linear range of 0.005 to 100 ng mL−1 and a low detection limit of 0.0022 ng mL−1 at S/N = 3. Furthermore, the proposed immunosensor exhibited excellent stability and recovery, demonstrating its potential for clinical applications.

2D/3D material amplification strategy for disposable label-free electrochemical immunosensor based on rGO-TEPA@Cu-MOFs@SiO2@AgNPs composites for NMP22 detection

Nuclear matrix protein 22 (NMP22) is a crucial bladder cancer marker, the accurate detection of which is conductive to improving the effectiveness of tumor prevention and treatment. However, due to low sensitivity, it is impossible for conventional immunoassay to detect the trace of NMP22 at the early stages of tumor progression. It is crucial to develop a simple, sensitive and accurate detection method of NMP22. As a variety of two-dimensional (2D) material, reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) demonstrates excellent conductivity and contains abundant amino groups. As a sort of 3D material, silica-modified Cu-based metalorganic frameworks deposited silver nanoparticles (Cu-MOFs@SiO2@AgNPs) composites are characterized by large surface area, excellent biocompatibility and water stability, which allows the immobilization of anti-NMP22 in large amounts via Ag-N bonds. Through a combination of the advantages exhibited by 2D material, 3D material, AgNPs and disposable screen-printed electrode, a disposable and sensitive label-free electrochemical immunosensor has been successfully assembled for NMP22 detection. In addition to exhibiting a wide linear range (0.1 pg∙mL−1-1000 ng∙mL−1) and a low detection limit (33.33 fg∙mL−1, S/N = 3), the proposed immunosensor also demonstrated satisfactory selectivity, strong reproducibility and high stability. Moreover, the excellent performance in detecting human urine samples makes it applicable in practice.

Ultrasensitive label-free electrochemical immunosensor based on core-shell Au@PtNPs functionalized rGO-TEPA/PB nanocomposite for HBsAg detection

In this study, a sensitive and quantitative label-free electrochemical immunosensor based on bimetallic [email protected] nanoparticles, Prussian blue (PB), and reduced graphene oxide-tetraethylene pentamine nanocomposite (rGO-TEPA) was constructed to achieve sensitive detection of HBsAg. A one-pot green method was used to synthesize interlocked rGO-TEPA/PB hybrid composites as high-performance materials with good conductivity. PB was the electron transfer mediator used to provide electrochemical signals. By utilizing rGO-TEPA as supporting material, the stability and intensity of PB were greatly improved. The incorporation of highly conductive [email protected] nanoparticles could further amplify the electrochemical signal, and the high adsorption capacity could provide an efficient substrate for hepatitis B surface antibody (HBsAb) immobilization. Under optimal conditions, the immunoassay had a wide linear range from 0.25 pg mL−1 to 400 ng mL−1 and a low detection limit of 0.08 pg mL−1 (S/N = 3). This method was also applied to the analysis of human serum samples, and the obtained results were on par with those of electrochemiluminescence immunoassay (ECLIA), suggesting significant potential for this new method in clinical research.

Label-Free Electrochemical Immunosensor Based on Gold and Iron-Oxide Nanoparticle Co-modified rGO-TEPA Hybrid for Sensitive Detection of Carcinoembryonic Antigen

Herein we report a label-free amperometric immunosensor based on reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) hybrid material co-modified by gold and iron-oxide magnetic nanoparticles (Au/MNPs-rGO-TEPA) for detection of carcinoembryonic antigen (CEA). The Au/MNPs-rGO-TEPA nanomaterials were prepared and coated on the working electrode as a sensing platform for antibody (anti-CEA) immobilization and signal amplification. This nanomaterial exhibited some excellent functionalities, such as magnetic and enhanced electrical properties due to its large specific surface area and favorable conductivity. Thionine (Thi) molecule was attached onto rGO-TEPA via π-π stacking and was used as an electrochemically active substance. The immunosensor was developed based on the increase in quantity of CEA captured, proportional to the decrease in peak currents of Thi. Under optimal conditions, the Au/MNPs-rGO-TEPA/Thi-based immunosensor showed impressive linear range from 0.1 pg/mL to 150 ng/mL. In addition, the proposed method had excellent linearity, high sensitivity, and relatively good stability, thereby offering a good platform for early clinical analysis.

A novel 3D paper-based microfluidic electrochemical glucose biosensor based on rGO-TEPA/PB sensitive film

A novel 3D paper-based microfluidic screen-printed electrode (SPE) composed of two layers was constructed by photolithography and screen-printing technology. Aldehyde functionalized hydrophilic zone of the counter and reference electrodes layer was prepared for glucose oxidase immobilization. Highly conductive prussian blue deposited reduced graphene oxide-tetraethylene pentamine (rGO-TEPA/PB) modified paper working electrode layer can be used as an electrochemical sensitive membrane for quantitative detection of hydrogen peroxide (H2O2), which was the enzyme-catalyzed reaction product. Therefore, this 3D paper-based microfluidic electrochemical biosensor can be used for quantitative detection of glucose. Under optimum conditions, the proposed biosensor can be used for quantitative determination of glucose over a wide linear range of 0.1 mM–25 mM with detection limit of 25 μM. Finally, the 3D paper-based microfluidic electrochemical biosensor was applied to determine glucose in human sweat and blood, and the obtained results were in good consistency with values measured by Roche’s blood glucose meter. In addition, the proposed 3D paper-based electrochemical device showed good repeatability, stability, and anti-interference, which would be of great potential to monitor glucose in complex biological fluids.

Establishment of electrical immunosensor for the detection of nuclear matrix protein-22

Objective A label-free electrochemical immunosensor was developed for the detection of nuclear matrix protein-22 (NMP22) as a biomarker of bladder cancer. Methods The study was based on the establishment and validation of the methodology. Urine samples were collected from 20 patients with bladder cancer and 20 controls in the affiliated Hongqi hospital of Mudanjiang medical university from September in 2017 to July in 2019 to validate the developed method. A screen-printed electrode (SPE) was modified with a film of a composite made from the reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) immobilized Zn-based-Metal-organic frameworks deposited with Au nanoparticles (rGO-TEPA@Au-ZIF8). Primary antibody against NMP22 was immobilized on the Au nanoparticles on the surface of the modified SPE, which then was blocked with bovine serum albumin to elimiate nonspecific binding sites. The process of the construction of the proposed sensorwas characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Differential pulse voltammetry was used to evaluate the linear range, recovery, precision, selectivity and stability. The data were analyzed by Mann-Whitney U test. Results Under optimal conditions, the immunosensor exhibited a linear range of 0.01-1000 ng/mlwith a detection limit of 3.33 pg/ml (S/N=3) and a standard recovery of 97.65%-107.05%. The levels of NMP22 in urine samples from patients with bladder cancer [66.03 (4.34, 91.74)]ng/ml determined by the proposed sensor were significantly higher than those of controls 0.54(0.06, 8.84) ng/ml(P=0.001). Conclusion The immunosensor can achieve sensitive, rapid and acucurate detection of NMP22, and has potential application prospects in monitoring tumor markers. Key words: Urinary bladder neoplasms; Nuclear proteins; Biosensing techniques; Electrochemical techniques; Immunoassay; Graphite; Polyamines; Biomarkers, tumor

A highly sensitive label-free electrochemical immunosensor based on AuNPs-PtNPs-MOFs for nuclear matrix protein 22 analysis in urine sample

A reliable and non-invasive electrochemical immunosensor for nuclear matrix protein 22 (NMP22) was presented based on reduced graphene oxide-tetraethylenepentamine (rGO-TEPA) and gold nanoparticles‑platinum nanoparticles-metal organic frameworks (AuNPs-PtNPs-MOFs) nanomaterial. rGO-TEPA is a promising template to enhance the loading ability of electrode for its large specific surface area and abundance in amino groups. AuNPs-PtNPs-MOFs nanomaterial was synthesized by NaBH4 reduction method and applied to immobilize biomolecules due to their good biocompatibility, well conductivity, and excellent H2O2 electrocatalytic ability. The electrochemical response signal was primarily originated from the obstruction of immobilized NMP22 antigen towards electrocatalytic reduction of H2O2 and the signal was recorded by differential pulse voltammetry. Under optimum conditions, the immunosensor showed a sensitive response towards NMP22 at a broad concentration range from 0.005ng·mL−1 to 20ng·mL−1. Two strong linear ranges could be observed at 0.005ng·mL−1 to 0.5ng·mL−1 (R2=0.9833), and 0.5ng·mL−1 to 20ng·mL−1 (R2=0.9909), with a low LOD of 1.7pg·mL−1 (S/N=3). Satisfactory reproducibility, stability and selectivity were obtained for NMP22 detection in real urine samples compared with standard ELISA assay, demonstrating the good analytical performance and potential non-invasive clinical application of this label-free electrochemical immunosensor.

A disposable paper-based microfluidic immunosensor based on reduced graphene oxide-tetraethylene pentamine/Au nanocomposite decorated carbon screen-printed electrodes

A novel, disposable and sensitive microfluidic paper-based electrochemical immunosensor (μ-PEI) was developed by using gold nanoparticles (Au NPs) decorated reduced graphene oxide-tetraethylene pentamine (rGO-TEPA/Au nanocomposite) as electrode materials. The rGO-TEPA greatly amplified the current response due to its excellent conductivity and large surface area. Au NPs was decorated on the surface of rGO-TEPA to increase the biocompatibility and retained good stability for rGO-TEPA/Au nanocomposite, which was used as an effective sensor platform for anchoring the capturing antibodies (Ab1) and accelerating the electron transfer to the screen-printed electrodes (SPEs). The simple and disposable paper-based microfluidic channel patterned on the rGO-TEPA/Au nanocomposite modified SPEs (SPEs/rGO-TEPA/Au) was designed for combination of immunochromatography and immunofiltration simultaneously. With the combination of portable SPEs/rGO-TEPA/Au and simple paper-based microfluidic devices, horseradish peroxidase (HRP) and labeled signal antibodies (Ab2) co-immobilized gold nanorods (HRP-GNRs-Ab2) was explored as the tracers for square wave voltammetry (SWV) detection. Using Alpha-Fetoprotein (AFP) as a model analyte, the proposed μ-PEI exhibited a satisfactory performance like simple fabrication, high stability, selectivity, wide linear range (0.01ngmL−1–100.0ngmL−1) with a low detection limit (0.005ngmL−1). Furthermore, human serum were applied to the obtained μ-PEI and determined with satisfactory results.

A novel non-invasive detection method for the FGFR3 gene mutation in maternal plasma for a fetal achondroplasia diagnosis based on signal amplification by hemin-MOFs/PtNPs

The small amount of cell-free fetal DNA (cffDNA) can be a useful biomarker for early non-invasive prenatal diagnosis (NIPD) of achondroplasia. In this study, a novel non-invasive electrochemical DNA sensor for ultrasensitive detecting FGFR3 mutation gene, a pathogenic gene of achondroplasia, based on biocatalytic signal materials and the biotin-streptavidin system are presented. Notably encapsulation of hemin in metal-organic frameworks-based materials (hemin-MOFs) and platinum nanoparticles (PtNPs) were used to prepare hemin-MOFs/PtNPs composites via a one-beaker-one-step reduction. We utilized hemin-MOFs/PtNPs for signal amplification because the promising hemin-MOFs/PtNPs nanomaterial has remarkable ability of catalyze H2O2 as well as excellent conductivity. To further amplify the electrochemical signal, reduced graphene oxide-tetraethylene pentamine (rGO-TEPA), gold nanoparticles and streptavidin were selected for modification of the electrode to enhance the conductivity and immobilize more biotin-modified capture probe (Bio-CP) through the high specificity and superior affinity between streptavidin and biotin. The electrochemical signal was primarily derived from the synergistic catalysis of H2O2 by hemin and PtNPs and recorded by Chronoamperometry. Under the optimal conditions, this newly designed biosensor exhibited sensitive detection of FGFR3 from 0.1fM to 1nM with a low detection limit of 0.033fM (S/N=3). We proposed that this ultrasensitive biosensor is useful for the early non-invasive prenatal diagnosis of achondroplasia.

Ultrasensitive electrochemical immunosensor for quantitative detection of tumor specific growth factor by using Ag@CeO2 nanocomposite as labels

In this paper, an ultrasensitive electrochemical immunosensor was developed for the detection of tumor specific growth factor (TSGF). Reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) was used to modify the surface of glassy carbon electrode (GCE). Meanwhile, Ag@CeO2 nanocomposite was synthesized and applied as secondary-antibody (Ab2) labels for the fabrication of the immunosensor. The amperometric response of the immunosensor for the reduction of H2O2 was recorded. Simultaneously, electrochemical impedance spectroscopy (EIS) and Cyclic voltammetry (CV) were used to characterize the fabrication process of the immunosensor. The anti-TSGF primary antibody (Ab1) was immobilized onto the rGO-TEPA modified GCE via cross-linking with glutaraldehyde (GA). And then the TSGF antigen and Ab2-Ag@CeO2 were modified onto the electrode surface in sequence. Under the optimal conditions, the immunosensor exhibited a wide linear range (0.500–100pg/mL), a low detection limit (0.2pg/mL), good reproducibility, acceptable selectivity and excellent stability. The proposed sensing strategy may provide a potential application in the detection of other cancer biomarkers.

Electrosynthesis of acetate from CO2by a highly structured biofilm assembled with reduced graphene oxide–tetraethylene pentamine

A highly structured biofilm assembled with reduced graphene oxide–tetraethylene pentamine and the bacteriumSporomusa ovataperformed high-rate microbial electrosynthesis.

A novel electrochemical immunosensor based on the rGO-TEPA-PTC-NH2 and AuPt modified C60 bimetallic nanoclusters for the detection of Vangl1, a potential biomarker for dysontogenesis

The aberrant expression of Vangl1 is highly correlated with dysontogenesis, especially for neural tube defects. Therefore, the ultrasensitive detection of Vangl1 would provide a new approach for the specific early diagnostics in dysembryoplasia. However, no quantitative detection method is currently available. Herein, we describe the development of a new approach to fill this assay gap. We utilized C60-templated AuPt bimetallic nanoclusters for signal amplification because the promising C60 nanomaterial provides a large surface area for the in site reduction of bimetallic nanocomposites as well as excellent conductivity. To further amplify the electrochemical signal, reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) and a derivative of 3,4,9,10-perylenetetracarboxylicdianhydride (PTC-NH2) were selected for modification of the electrode to provide more amino groups for the immobilization of antibodies and to enhance the conductivity. The electrochemical signal was primarily derived from the catalysis of H2O2 by C60–AuPt. Chronoamperometry was applied to record the electrochemical signals. Under optimal conditions, the prepared immunosensor exhibited a wide linear range from 0.1pgmL−1 to 450pgmL−1 and a low detection limit of 0.03pgmL−1. Moreover, the proposed method exhibited good stability and recovery, suggesting its potential for use in clinical research.

Ultrasensitive electrochemical biosensor based on reduced graphene oxide-tetraethylene pentamine-BMIMPF6 hybrids for the detection of α2,6-sialylated glycans in human serum.

In this paper, a simple, ultrasensitive and label-free electrochemical α2,6-sialylated glycans biosensor based on reduced graphene oxide-tetraethylene pentamine-1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF6) hybrids was developed. Due to the abundance of amino groups from reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) and the electrostatic interaction of BMIMPF6, bimetallic gold platinum alloy nanoparticles (AuPtNPs) were densely adsorbed onto the surface of the nanocomposite, providing a large surface area available for the immobilization of Sambucus nigra agglutinin (SNA). AuPtNPs have excellent conductivity and catalytic activity, which can promote electron transfer between the electrolyte solution and the surface of electrode and can enhance the sensitivity of biosensor. SNA, which specifically binds α2,6-sialylated glycans, was covalently immobilized on AuPtNPs for specific detection of α2,6-sialylated glycans in human serum. Under optimal experimental conditions, amperometric response changes were used to detect α2,6-sialylated glycans with a broad linear range of 10 fg mL(-1) -1 μg mL(-1) and a low detection limit of 3 fg mL(-1) (S/N=3). When applied to spiked serum samples, the recovery of the developed biosensor ranged from 100.8% to 101.4%, suggesting that the electrochemical biosensor would be suitable for the practical detection of α2,6-sialylated glycans.

Electrochemical immunosensor for detecting typical bladder cancer biomarker based on reduced graphene oxide–tetraethylene pentamine and trimetallic AuPdPt nanoparticles

A highly sensitive electrochemical immunosensor for detection of typical bladder cancer biomarker-nuclear matrix protein 22 (NMP22) was developed by using reduced graphene oxide–tetraethylene pentamine (rGO–TEPA) and trimetallic AuPdPt nanoparticles (NPs). rGO–TEPA was used as the ideal material for signal amplification and AuPdPt NPs immobilization due to its excellent conductivity and large surface area. An effective platform was constructed for antibodies anchoring by using AuPdPt NPs, which kept the antibodies’ high stability and bioactivity. Moreover, AuPdPt NPs could accelerate the electron transfer and enhance the signal response, which assisted by the synergistic effect of the three different metals (Au, Pd and Pt). The proposed immunosensor showed satisfied performance such as simple fabrication, low detection limits (0.01U/mL), wide linear range (from 0.040 to 20U/mL), short analysis time (2min), high stability and selectivity in the detection of NMP22. Furthermore, the proposed immunosensor was employed to test real urine samples with satisfactory results.

Ultrasensitive electrochemical detection of secretoneurin based on Pb(2+)-decorated reduced graphene oxide-tetraethylene pentamine as a label.

In this work, a novel electrochemical immunosensor for the detection of secretoneurin (SN), which uses metal ion functionalised reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) as a label, is reported for the first time. rGO-TEPA contains a large number of amino groups, which makes it an ideal templet for the loading of metal ions. rGO-TEPA-Pb(2+) was employed to immobilise secondary secretoneurin (SN) antibody (Ab2), and the resulting nanocomposite (Ab2-rGO-TEPA-Pb(2+)) was used as a trace tag for signal amplification. A modified electrode consisting of functionalised graphene nanosheets (Au@GS) was used as a substrate to immobilise the antibodies. Under the optimal conditions, the immunoassay exhibited high sensitivity, acceptable stability and reproducibility with a wide linear range from 0.001 to 100ngmL(-1) (R=0.996), and an ultra-low detection limit of 0.33pgmL(-1) (S/N=3). Furthermore, the immunosensor could be employed to detect SN in clinical serum samples. The proposed sensing strategy enriches the electrochemical immunoassay and exhibits potential for the point-of-care diagnostic application of the clinical screening of biomarkers.

Rapidly accomplished femtomole soluble CD40 ligand detection in human serum: a “green” homobifunctional agent coupled with reduced graphene oxide-tetraethylene pentamine as platform

A reliable and sensitive electrochemical immunosensor based on a “green” conjunction agent coupled with an amino-group qualified material was used for the analysis of soluble CD40 ligand.

Sandwich-type electrochemical immunosensor using dumbbell-like nanoparticles for the determination of gastric cancer biomarker CA72-4

A novel and sensitive nonenzymatic sandwich-type electrochemical immunosensor for the detection of gastric cancer biomarker CA72-4 was fabricated using dumbbell-like PtPd-Fe3O4 nanoparticles (NPs) as a novel kind of label. The signal amplification strategy, using the synergetic effect present in PtPd-Fe3O4 to increase the reduction ability of the NPs toward H2O2, improved the sensitivity of the immunosensor. The immunosensor was constructed by modifying glassy carbon electrode with reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) for effective immobilization of primary anti-CA72-4 antibody (Ab1). Secondary anti-CA72-4 antibody (Ab2) was adsorbed onto the PtPd-Fe3O4 NPs. The proposed immunosensor displayed a wide linear range (0.001–10U/mL) with the low detection limit (0.0003U/mL). The immunosensor was evaluated for serum samples, receiving satisfactory results. Therefore, the immunosensor possesses excellent clinical value in cancer screening as well as convenient point-of-care diagnostics.

An electrochemical immunosensor for ultrasensitive detection of carbohydrate antigen 199 based on Au@CuxOS yolk–shell nanostructures with porous shells as labels

A novel and sensitive electrochemical immunosensor for ultrasensitive detection of pancreatic cancer biomarker carbohydrate antigen 199 (CA199) was proposed by using Au@CuxOS yolk–shell nanostructures with porous shells as labels for signal amplification. Au@CuxOS yolk–shell nanostructures exhibit high electrocatalytic activity toward the reduction of hydrogen peroxide (H2O2) as analytical signal. Moreover, secondary antibody (Ab2) can adsorb on the surface of Au@CuxOS with porous shells which has large surface area and could greatly increase the probability of Ab2–antigen interactions thereby leading to higher sensitivity. Reduced graphene oxide–tetraethylene pentamine (rGO–TEPA), containing abundant amine groups, was supported Au nanoparticles as a support platform to immobilize the primary antibody (Ab1). The resulting sensing interface of rGO–TEPA/AuNPs could provide a large electroconductive surface area, allowing high loadings of the biological recognition elements as well as the occurrence of electrocatalytic and electron-transfer processes. Under optimal conditions, the immunosensor exhibited a wide linear response to CA199 ranging from 0.001 to 12U/mL with a low detection limit of 0.0005U/mL. The designed immunosensor displayed good precision, high sensitivity, acceptable stability and reproducibility, and has been applied to the analysis of serum with satisfactory results. The proposed method provides a new promising platform of clinical immunoassay for other biomolecules.