Magneto-controlled graphene immunosensing platform for simultaneous multiplexed electrochemical immunoassay using distinguishable signal tags.

A novel flow-through multiplexed immunoassay protocol for simultaneous electrochemical determination of carcinoembryonic (CEA) and alpha-fetoprotein (AFP) in biological fluids was designed using biofunctionalized magnetic graphene nanosheets (MGO) as immunosensing probes and multifunctional nanogold hollow microspheres (GHS) as distinguishable signal tags. The probes were fabricated by means of co-immobilization of primary anti-CEA (Ab(1)) and anti-AFP (Ab(2)) antibodies on the Fe(3)O(4) nanoparticle-coated graphene nanosheets (MGO-Ab(1,2)). The reverse-micelle method was used for the synthesis of distinguishable signal tags by encapsulation of horseradish peroxide (HRP)-thionine and HRP-ferrocene into nanogold hollow microspheres, respectively, which were utilized as labels of the corresponding GHS-Ab(1) and GHS-Ab(2). A sandwich-type immunoassay format was employed for the online detection of CEA and AFP by coupling a flow-through detection cell with an external magnet. The assay was based on the catalytic reduction of H(2)O(2) at the various peak potentials in the presence of the corresponding mediators. Experimental results revealed that the multiplexed electrochemical immunoassay enabled the simultaneous monitoring of AFP and CEA in a single run with wide working ranges of 0.01-200 ng mL(-1) for AFP and 0.01-80 ng mL(-1) for CEA. The detection limits (LODs) for both analytes at 1.0 pg mL(-1) (at 3s(B)) were very low. No obvious nonspecific adsorption and cross-talk were observed during a series of analyses to detect target analytes. Intraassay and interassay coefficients of variation were <10%. Importantly, the methodology was evaluated for the analysis of clinical serum specimens, receiving a good correlation between the flow-through multiplexed electrochemical immunoassay and an electrochemiluminescence method as a reference.

[1]  H. Ju,et al.  Flow-through multianalyte chemiluminescent immunosensing system with designed substrate zone-resolved technique for sequential detection of tumor markers. , 2006, Analytical chemistry.

[2]  D. Juncker,et al.  Hydrogel droplet microarrays with trapped antibody-functionalized beads for multiplexed protein analysis. , 2011, Lab on a chip.

[3]  R. Behm,et al.  X-ray photoelectron spectrum in surface interfacing of gold nanoparticles with polymer molecules in a hybrid nanocomposite structure , 2009, Nanotechnology.

[4]  Zhimin Zhang,et al.  Nanogold-enwrapped graphene nanocomposites as trace labels for sensitivity enhancement of electrochemical immunosensors in clinical immunoassays: Carcinoembryonic antigen as a model. , 2010, Biosensors & bioelectronics.

[5]  Y. Chai,et al.  Reverse-micelle synthesis of electrochemically encoded quantum dot barcodes: application to electronic coding of a cancer marker. , 2010, Analytical chemistry.

[6]  S. Stankovich,et al.  Chemical analysis of graphene oxide films after heat and chemical treatments by X-ray photoelectron and Micro-Raman spectroscopy , 2009 .

[7]  R. Levy,et al.  Analytical chemistry: The matrix neutralized , 2009, Nature.

[8]  Alexander P Hsiao,et al.  Multiplexed protein detection using antibody-conjugated microbead arrays in a microfabricated electrophoretic device. , 2010, Lab on a chip.

[9]  I. Willner,et al.  Dual biosensing by magneto-controlled bioelectrocatalysis. , 2002, Angewandte Chemie.

[10]  R. Niessner,et al.  Magnetic bead-based fluorescence immunoassay for aflatoxin B1 in food using biofunctionalized rhodamine B-doped silica nanoparticles. , 2010, The Analyst.

[11]  Jin Chang,et al.  Structural design and preparation of high-performance QD-encoded polymer beads for suspension arrays , 2011 .

[12]  J. Choo,et al.  Highly sensitive immunoassay of lung cancer marker carcinoembryonic antigen using surface-enhanced Raman scattering of hollow gold nanospheres. , 2009, Analytical chemistry.

[13]  D. Russell,et al.  Multiplexed detection of metabolites of narcotic drugs from a single latent fingermark. , 2010, Analytical chemistry.

[14]  Steven C Kazmierczak,et al.  Nanodiagnostics: a new frontier for clinical laboratory medicine. , 2006, Clinical chemistry.

[15]  Michael S. Wilson,et al.  Multiplex measurement of seven tumor markers using an electrochemical protein chip. , 2006, Analytical chemistry.

[16]  Jie Wu,et al.  Disposable immunosensor array for ultrasensitive detection of tumor markers using glucose oxidase-functionalized silica nanosphere tags. , 2011, Biosensors & bioelectronics.

[17]  Andrew N Hoofnagle,et al.  Simultaneous quantification of apolipoprotein A-I and apolipoprotein B by liquid-chromatography-multiple- reaction-monitoring mass spectrometry. , 2010, Clinical chemistry.

[18]  Ruo Yuan,et al.  Ultrasensitive electrochemical immunosensor for clinical immunoassay using thionine-doped magnetic gold nanospheres as labels and horseradish peroxidase as enhancer. , 2008, Analytical chemistry.

[19]  Dan Wu,et al.  A novel label-free electrochemical immunosensor based on graphene and thionine nanocomposite , 2010 .

[20]  Huang-Hao Yang,et al.  A graphene platform for sensing biomolecules. , 2009, Angewandte Chemie.

[21]  Feng Yan,et al.  Electric field-driven strategy for multiplexed detection of protein biomarkers using a disposable reagentless electrochemical immunosensor array. , 2008, Analytical chemistry.

[22]  F. Geiger,et al.  Specific and nonspecific metal ion-nucleotide interactions at aqueous/solid interfaces functionalized with adenine, thymine, guanine, and cytosine oligomers. , 2011, Journal of the American Chemical Society.

[23]  Itamar Willner,et al.  Magnetic control of electrocatalytic and bioelectrocatalytic processes. , 2003, Angewandte Chemie.

[24]  Marco Giannetto,et al.  A voltammetric immunosensor based on nanobiocomposite materials for the determination of alpha-fetoprotein in serum. , 2011, Biosensors & bioelectronics.

[25]  Jun Liu,et al.  Sensitive immunosensor for cancer biomarker based on dual signal amplification strategy of graphene sheets and multienzyme functionalized carbon nanospheres. , 2010, Analytical chemistry.

[26]  Feng Yan,et al.  Automated support-resolution strategy for a one-way chemiluminescent multiplex immunoassay. , 2009, Analytical chemistry.

[27]  Feng Yan,et al.  Dual signal amplification of glucose oxidase-functionalized nanocomposites as a trace label for ultrasensitive simultaneous multiplexed electrochemical detection of tumor markers. , 2009, Analytical chemistry.

[28]  Seung-Woo Lee,et al.  Highly sensitive biosensing using arrays of plasmonic Au nanodisks realized by nanoimprint lithography. , 2011, ACS nano.

[29]  Jason Y. Park,et al.  Magnetism and magnetoresistance: attractive prospects for point-of-care testing? , 2009, Clinical chemistry.

[30]  S. Freedland.  Screening, risk assessment, and the approach to therapy in patients with prostate cancer , 2011, Cancer.

[31]  Chad A Mirkin,et al.  The bio-barcode assay for the detection of protein and nucleic acid targets using DTT-induced ligand exchange , 2006, Nature Protocols.

[32]  Fengping Wang,et al.  Fabrication and characterization of Fe3O4 thin films deposited by reactive magnetron sputtering , 2005 .

[33]  Wolfgang Knoll,et al.  In situ antibody detection and charge discrimination using aqueous stable pentacene transistor biosensors. , 2011, Journal of the American Chemical Society.

[34]  Chad A. Mirkin,et al.  Drivers of biodiagnostic development , 2009, Nature.

[35]  R. Larsson,et al.  Aminopeptidase N (CD13) as a target for cancer chemotherapy , 2011, Cancer science.

[36]  K. I. Vasu,et al.  Electrochemical and oxygen reduction behaviour of solid silver-bismuth/antimony electrodes in KOH solutions , 1993 .

[37]  Jing-Juan Xu,et al.  Label-free photoelectrochemical immunoassay for alpha-fetoprotein detection based on TiO(2)/CdS hybrid. , 2009, Biosensors & bioelectronics.

[38]  F. L. Deepak,et al.  Synthesis, Morphology, and Optical Characterization of Nanocrystalline Er3+:Y2O3 , 2010 .

[39]  Xiaohong Fang,et al.  Single-molecule detection of proteins using aptamer-functionalized molecular electronic devices. , 2011, Angewandte Chemie.

[40]  Feng Yan,et al.  Biomedical and clinical applications of immunoassays and immunosensors for tumor markers , 2007 .

[41]  Luis A Tortajada-Genaro,et al.  Multiplexed microimmunoassays on a digital versatile disk. , 2009, Analytical chemistry.

[42]  C. Mirkin,et al.  Nanoparticle-Based Bio-Bar Codes for the Ultrasensitive Detection of Proteins , 2003, Science.

[43]  M. Linder,et al.  Hydrophilic modification of polystyrene with hydrophobin for time-resolved immunofluorometric assay. , 2010, Biosensors & bioelectronics.

[44]  Thermally addressed immunosorbent assay for multiplexed protein detections using phase change nanoparticles. , 2010, Analytical chemistry.

[45]  Xiaohong Wang,et al.  Assembly of folate-polyoxometalate hybrid spheres for colorimetric immunoassay like oxidase. , 2011, Chemical communications.

[46]  Juan Tang,et al.  Nanoparticle-based sandwich electrochemical immunoassay for carbohydrate antigen 125 with signal enhancement using enzyme-coated nanometer-sized enzyme-doped silica beads. , 2010, Analytical chemistry.

[47]  Pi-Tai Chou,et al.  Carbon nanoparticle-enhanced immunoelectrochemical detection for protein tumor marker with cadmium sulfide biotracers. , 2009, Analytical chemistry.

[48]  A. Bergel,et al.  Electrochemical reduction of oxygen on glassy carbon: catalysis by catalase , 2000 .

[49]  A. Baldi,et al.  Electrical readout of protein microarrays on regular glass slides. , 2011, Analytical chemistry.

[50]  Y. Chai,et al.  Amperometric immunosensor based on multiwalled carbon nanotubes/Prussian blue/nanogold-modified electrode for determination of α-fetoprotein. , 2010, Analytical biochemistry.

[51]  S. Tarucha,et al.  Tuneable electronic properties in graphene , 2011 .

[52]  Chao Gao,et al.  Supraparamagnetic, conductive, and processable multifunctional graphene nanosheets coated with high-density Fe3O4 nanoparticles. , 2010, ACS applied materials & interfaces.

[53]  Juan Tang,et al.  Carbon nanotube-based symbiotic coaxial nanocables with nanosilica and nanogold particles as labels for electrochemical immunoassay of carcinoembryonic antigen in biological fluids. , 2011, Talanta.

[54]  Arben Merkoçi,et al.  Double-codified gold nanolabels for enhanced immunoanalysis. , 2007, Analytical chemistry.

[55]  Dianping Tang,et al.  In situ amplified electrochemical immunoassay for carcinoembryonic antigen using horseradish peroxidase-encapsulated nanogold hollow microspheres as labels. , 2008, Analytical chemistry.

[56]  Feng Yan,et al.  Disposable reagentless electrochemical immunosensor array based on a biopolymer/sol-gel membrane for simultaneous measurement of several tumor markers. , 2008, Clinical chemistry.

[57]  X. Jia,et al.  Graphene edges: a review of their fabrication and characterization. , 2011, Nanoscale.

[58]  Juan Tang,et al.  Ultrasensitive electrochemical immunoassay of staphylococcal enterotoxin B in food using enzyme-nanosilica-doped carbon nanotubes for signal amplification. , 2010, Journal of agricultural and food chemistry.

[59]  Ángel Maquieira,et al.  Direct hapten-linked multiplexed immunoassays on polycarbonate surface. , 2011, Biosensors & bioelectronics.

[60]  Jianzhong Lu,et al.  Simultaneous detection of two lung cancer biomarkers using dual-color fluorescence quantum dots. , 2011, The Analyst.