Simultaneous electrochemical immunoassay using graphene-Au grafted recombinant apoferritin-encoded metallic labels as signal tags and dual-template magnetic molecular imprinted polymer as capture probes.

A novel electrochemical multiplexed immunoassay was designed for simultaneous determination of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) using recombinant apoferritin-encoded metallic nanoparticles (rApo-M) as labels and dual-template magnetic molecularly imprinted polymers (MMIPs) as capture probes. The labels were prepared by loading recombinant apoferritin (r-Apo) and separately immobilize primary antibodies (anti-AFP and anti-CEA) via Au nanoparticles of in site growth on graphene (G). The capture probes were synthesized by self-polymerization of dopamine (DA) on the Fe3O4 nanoparticles (Fe3O4 NPs) and using AFP and CEA as the template proteins, which were used to enrich the targets simultaneously. After a sandwich-type immunoreaction, the labels were captured to the surface of MMIPs. The subsequent electrochemical stripping analysis of the metal components from the immunocomplex provide a means for quantification of targets based on the peak currents of Cd and Pb. Experimental results showed the immunoassay enabled the simultaneous determination of AFP and CEA in a single run with wide dynamic ranges of 0.001-5ngmL(-1). And the detection limits of AFP and CEA were 0.3 and 0.35pgmL(-1) (S/N=3), respectively. These results suggested that the proposed multiplexed immunoassay would be applied for clinical screening of other biomarkers.

[1]  A. Salimi,et al.  Au nanoparticles/PAMAM dendrimer functionalized wired ethyleneamine-viologen as highly efficient interface for ultra-sensitive α-fetoprotein electrochemical immunosensor. , 2014, Biosensors & bioelectronics.

[2]  Huang-Hao Yang,et al.  Mussel-inspired molecularly imprinted polymer coating superparamagnetic nanoparticles for protein recognition , 2010 .

[3]  Feng Yan,et al.  Streptavidin‐Functionalized Silver‐Nanoparticle‐Enriched Carbon Nanotube Tag for Ultrasensitive Multiplexed Detection of Tumor Markers , 2011 .

[4]  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.

[5]  Yuehe Lin,et al.  Electroactive silica nanoparticles for biological labeling. , 2006, Small.

[6]  Chenghua Li,et al.  A Ferritin from Dendrorhynchus zhejiangensis with Heavy Metals Detoxification Activity , 2012, PloS one.

[7]  Huangxian Ju,et al.  Surface molecularly imprinted nanowire for protein specific recognition. , 2008, Chemical communications.

[8]  Hongyuan Chen,et al.  Simultaneous electrochemical immunoassay using CdS/DNA and PbS/DNA nanochains as labels. , 2013, Biosensors & bioelectronics.

[9]  Shouzhuo Yao,et al.  Apoferritin protein nanoparticles dually labeled with aptamer and horseradish peroxidase as a sensing probe for thrombin detection. , 2013, Analytica chimica acta.

[10]  Yuehe Lin,et al.  Apoferritin-templated synthesis of encoded metallic phosphate nanoparticle tags. , 2007, Analytical chemistry.

[11]  Songqin Liu,et al.  Simultaneous detection of dual proteins using quantum dots coated silica nanoparticles as labels. , 2011, Biosensors & bioelectronics.

[12]  Tae Gwan Park,et al.  Substrate‐Independent Layer‐by‐Layer Assembly by Using Mussel‐Adhesive‐Inspired Polymers , 2008, Advanced materials.

[13]  Xiwen He,et al.  A self-assembled polydopamine film on the surface of magnetic nanoparticles for specific capture of protein. , 2012, Nanoscale.

[14]  Jun‐Jie Zhu,et al.  Graphene-assisted dual amplification strategy for the fabrication of sensitive amperometric immunosensor. , 2011, Biosensors & bioelectronics.

[15]  Xindong Wang,et al.  A novel pH-controlled immunosensor using hollow mesoporous silica and apoferritin combined system for target virus assay. , 2014, Biosensors & bioelectronics.

[16]  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.

[17]  Peng Xue,et al.  Highly specific and ultrasensitive graphene-enhanced electrochemical detection of low-abundance tumor cells using silica nanoparticles coated with antibody-conjugated quantum dots. , 2013, Analytical chemistry.

[18]  Yuehe Lin,et al.  Versatile apoferritin nanoparticle labels for assay of protein. , 2006, Analytical chemistry.

[19]  Zhanfang Ma,et al.  Au-ionic liquid functionalized reduced graphene oxide immunosensing platform for simultaneous electrochemical detection of multiple analytes. , 2014, Biosensors & bioelectronics.

[20]  Yan Zhang,et al.  Triple catalysis amplification strategy for simultaneous multiplexed electrochemical immunoassays based on cactus-like MnO2 functionalized nanoporous gold , 2013 .

[21]  Haeshin Lee,et al.  Mussel-Inspired Surface Chemistry for Multifunctional Coatings , 2007, Science.

[22]  Lehui Lu,et al.  Polydopamine and its derivative materials: synthesis and promising applications in energy, environmental, and biomedical fields. , 2014, Chemical reviews.

[23]  Dan Du,et al.  Nanoparticle-based immunosensor with apoferritin templated metallic phosphate label for quantification of phosphorylated acetylcholinesterase. , 2011, Biosensors & bioelectronics.

[24]  Dan Ran,et al.  Polydopamine-based molecular imprinting on silica-modified magnetic nanoparticles for recognition and separation of bovine hemoglobin. , 2013, The Analyst.

[25]  Xing Li,et al.  A cost-effective sandwich electrochemiluminescence immunosensor for ultrasensitive detection of HIV-1 antibody using magnetic molecularly imprinted polymers as capture probes. , 2014, Biosensors & bioelectronics.

[26]  M. Baraibar,et al.  Iron loading-induced aggregation and reduction of iron incorporation in heteropolymeric ferritin containing a mutant light chain that causes neurodegeneration. , 2011, Biochimica et biophysica acta.

[27]  Electrochemical detection of individual single nucleotide polymorphisms using monobase-modified apoferritin-encapsulated nanoparticles. , 2012, Biosensors & bioelectronics.

[28]  Dan Du,et al.  Magnetic particle-based immunoassay of phosphorylated p53 using protein cage templated lead phosphate and carbon nanospheres for signal amplification , 2012 .