The Preparation And Application Of Visible Protein Chip Based On GoldMag Particles Detection

Protein chip, the technology of the highly parallel analysis of protein, allowsdetecting thousands of proein moleculars in a single experiment. It is characterized byits high throughput, micromation and automatization. In recent years, protein chip hasbecome an important tool in proteomics, diagnostics and drug discovery programs.In recent years, the fluorescent and nano-gold detection are widely used inprotein chip. Although fluorescent detection is highly sensitive, expensiveinstrumentations as well as complex labeling procedures have limited its applicationsin clinical diagnosis. The Gold-conjugated probe was highly sensitive, especially thesignal is greatly amplified by the reduction of silver promoted by the gold particles.However, the procedure of protein labeled with colloidal gold was time-consuming,laborious and the labeled protein was difficult to be quantified. Therefore, there is anurgent need to develop detection techniques that do not rely on organic dyes orcolloidal gold.In this paper, we used the GoldMag nano-particles as labels to develop a newtype of colorimetric detection for analyzing protein chips coupled with silverenhancement. GoldMag nano-particles are a new type of super-paramagneticFe₃O₄/Au composite nano-particles with core/shell structure, in which the shell is alayer of gold and the core is a magnetic particle. The core/shell-structure particlescombine property of colloidal gold particles for convenient binding and detection ofbiomolecules with separation power of superparamagnetic core. GoldMagnano-particles labeling method is characterized by its considerably simple labelingprocess and higher labeling efficiency, especially the labeled protein can be easilyquantifiedIn this experiment, the human IgG were immobilized on the epoxy-coated slides.After blocking and incubating with gold-conjugated goat anti-human IgG, the slides were submerged in silver enhancer solution to amplify the detection signal, and thenproduced black inmage on the slides, which could be seen by naked eyes. Theoptimum conditions were as follows:(1) The epoxy-coated slides could produce better signal.(2) The optimum spotting concentration was 0.1-0.2mg/mL.(3) The condition of immobilization was 37℃for 1-2h.(4) The incubation time of antigen-antibody was 30 min.(5) Using 1%BSA as the blocking reagent.(6) The silver enhancement time was 10-15 min.According to optimum conditions, the HCV antigen protein chips had beenfabricated. The detection limit and specificity were analyzed. In addition, the HCVantigen protein chip was used to test 10 healthy sera and 6 sera from HCV patients,which had been diagnosed by ELISA. The result revealed that this method was simple,quik, and highly specific, and the concordance between protein chip and ELISA was100%.Through comparing the labeling protein technologies between the GoldMagparticles and colloidal gold, the result showed that colloidal gold had higher sensitivity.However, colloidal gold suffered from false positive because of nonspecific absorption;Due to GoldMag's larger diameter and lower density of gold particles, the silverenhancement was lower than the one of colloidal gold. But, GoldMag not only almosthad no false positive, resulting from low nonspecific absorption, but also possessed theproperty of simple labeling process, quantification of labelled protein and lowerbackground. With the development of preparation of GoldMag particles, thecolorimentric detection of protein chip based on GoldMag probe coupled with silverenhancement will apply in more fields, especially promote the application in clinicaldiagnosis.