Study Of New Methods To Assay Protein By Using Graphene

Protein,as the key component of life organism,is an important executor of life functions such as cellular metabolism and regulation and is the most important acting sites of external factors such as pathogenic factors and drugs.In recent years,with the deepening of the research on mechanism of various diseases,the detection of disease-related proteins has become an important means and basis for disease diagnosis,prognostic and efficacy evaluation and received great concern and attention.The development of nanomaterial and technique has provided powerful tool for the construction of kinds of biosensors that continues to emerge for the detection of disease-related proteins,but there is still many drawbacks of these developed biosensors.With the above-mentioned challenges in mind,this paper focuses on the development of a series of novel functional nanoprobes and detecting strategies that are based on graphene nanomaterial as scaffold and biomolecule as functional elements for detecting disease-related nucleic acid and proteins.Through the analysis of real samples,we have preliminarily demonstrated the feasibility and dominance compared with traditional test methods.The detailed content was described as follows:(1)A colorimetric array-based strategy has been developed to discriminate different kinds of protein molecules and two subtypes and three stages of non-small cell lung cancer(NSCLC)based on the interaction between graphene oxide(GO)and single strand DNA modified gold.nanoparticles(ssDNA-GNPs).Here,amounts of ssDNA-GNPs can adsorb onto the surface of GO due to the strong ?-stacking interaction between the ring structures in the nucleobases and the hexagonal cells of the graphene.Because of the spherical nature of GNPs,different GO layers can be cross-linked by ssDNA-GNPs and bulky aggregate will finally form,leading to the precipitation of both ssDNA-GNPs and GO.However,when the DNA strands on the surface of GNPs become double strands or other rigid structures,the GNPs will not interact with GO because of the embedding of free bases and the precipitate will not form,leaving a wine-red color in the solution.Furthermore,different kinds of proteins can differently influence the interaction between ssDNA-GNPs and GO.Some proteins enhance the interaction of the two nanomaterials,increasing the formation of precipitation.In contrast,some proteins inhibit the interaction between the two nanomaterials,retarding the formation of the precipitation.While some other proteins do not affect the interaction.Taking advantage of this phenomenon,the developed array-based sensor can distinguish different proteins as low as 0.5 nM using different ssDNA-GNPs as sensor elements and can be used to discriminate the subtypes and stages of NSCLC patients,demonstrating its ability of analyzing complex biological samples.Besides,also on the basis of interaction of ssDNA-GNPs and GO,our proposed method can be expanded to detect nucleic acid and metal ion and obtains satisfactory results.(2)An immunoassay strategy based on the pH-responsive molecule modified carboxyl graphene oxide(cGO)has been developed to detect human immunoglobulin G(IgG)with high sensitivity and three tumor biomarkers of lung cancer with multicolor readout.Here,kinds of pH-responsive nanoprobes have been prepared by conjugation of antibody to the edge of cGO through a carbodiimide chemistry and adsorption of allochroic molecules such as malachite green carbinol base,methyl red,phenolphthalein and thymolphthalein onto the surface of cGO.Making use of these nanoprobes and sandwich-type structure,IgG has been firstly detected to demonstrate the analytical ability of the proposed method.The results indicate that the naked-eye detection of limit(LOD)is down to 100 pg/mL which is 400-fold higher than the traditional enzyme-linked immunosorbent assay(ELISA).Moreover,the final detection is stop buffer needless and can be completed within 3 minutes,simplifying the experimental procedure.Then,these nanoprobes have been used to simultaneously detect three tumor biomarkers of lung cancers,including arcino-embryonic antigen(CEA),neuron-specific enolase(NSE),and cytokeratin-19 fragment(Cyfra21-1),demonstrating that the positive rate of lung cancer can be significantly enhanced by simultaneously analyzing three biomarkers in patients' sera.(3)An electrochemical strategy based on a composite material containing graphene oxide and metal nanoparticle the has been developed.Firstly,we describe a simple and effective method to fabricate uniform and well dispersed silver n anoparticles(AgNPs)on GO,based on a facial-induced co-reduction strategy.The synthesized nanohybrid may show ultrafine dispersed AgNPs(2.9±1.4 nm),individually separated GO sheets,as well as highly covered surface(5250 nanocrystals per square micrometer),indicating the formation of a high-quality GO-based nanohybrid.Then,GO-AgNP modified electrode shows excellent catalytic activity for oxygen reduction reaction(ORR)and exhibits enhanced signal readout for biomolecules(drugs,proteins,enzymes etc.),demonstrating the potential application of this newly synthesized inorganic hybrid with strong synergistic coupling effects on advanced functional systems and graphene-based sensors.Finally,making use of this two dimensional composite nanomaterial,we propose a novel electrochemical assay for protein detection.Specifically,electrode co-modified with phospholipid bilayer and DNA aptamer not only can efficiently capture target protein to the surface of gold electrode,but also achieve amplified signal due to the fact that graphene with the high surface area can enrich large amounts of AgNPs.Taking thrombin as model target,the detection of limit can be down to 1 pM with a linear range from 1 pM to 10 nM.Also of note,this method has considerable selectivity and antijamming capability,making it suitable for real sample analysis.Moreover,this method can be adapted into other targets such as protein,nucleic acid and small molecules through replace thrombin aptamer with other recognition elements.