Fabrication Of GoldMag Nanoparticles-based Genotyping System And Its Application In Medication Guide Of Clopidogrel

Background:The effectiveness of drug metabolism and its toxicity affects the disease’s treatment effect and the drug’s safety. Pharmacogenetics, pharmacogenomics and other related disciplines shows gene polymorphism of drug metabolizing enzymes are the reason for individual differences of drug metabolism. Therefore, a new detection technology that can ascertain the drug metabolism enzyme gene polymorphism and provided instant information on individual differences is of great significance to improve the efficacy and safety of drug and guide individual treatment.Point-of-Care Testing (POCT), a technology developed from the medical laboratory automation and simplification, and owning to its convenience, rapidity, no need for expensive instruments and operations staff, is widely applied to medical health, customs and quarantine, farming and animal husbandry, forestry, environmental and food detection with the need of high-efficient and fast-paced society. However, the POCT are mostly used in rapid chromatographic detection based on the immunology and is seldom used for genotyping.2010report on cardiovascular disease in China shows that3million people die of cardiovascular disease in every year and which is the top1leading causes of death for patients, therefore, much attention has been paid to its medication safety and effectiveness. Clopidogrel sulfate is a clinical first-line drug for antithrombotic therapy. However, about30%of patients after taking the drug have poor curative effect, even causing serious adverse reactions. Studies have shown that clopidogrel resistance is associated with the cytochrome P450enzyme CYP2C19gene polymorphisms, in which CYP2C19*2(c.681G>A) and CYP2C19*3(c.636G>A) is the main influence factors to increasing myocardial infarction, stroke, and stent thrombosis. FDA announced the clopidogrel early security public information which require to add black box warning in specification that the clopidogrel has a potential risk of increasing the cardiovascular events owing to being decreased drug efficacy. No functional CYP2C19*2(c.681G> A) and CYP2C19*3(c.636G> A) allele have the important effects on drug metabolism and the drug are unable to give full play to the role in the patients with weak metabolism.Aim:To fabrication a gold magnetic composite nanoparticle (GoldMag NP) suitable for POCT system of CYP2C19genotyping for clopidogrel individual medication of cardiovascular and cerebrovascular disease. Furthermore, taken the335blood samples of cardiovascular patients as the research object, the CYP2C19*2and CYP2C19*3alleles of P450enzyme involved in the metabolism of clopidogrel were genotyped with the new POCT system and evaluated by gene sequencing technology which is an existing gold standard method. From the analysis of genotype by statistical method, the results revealed allele frequency of CYP2C19*2and CYP2C19*3. At last, the results of statistical correlation analysis between gene mutation and the type of clopidogrel metabolism provide a theoretical and technical support for clopidogrel individual medication.Methods and Results:1. The Fe3O4NPs were first prepared using the hydrothermal method, there are oleic molecules on the surface and the mean diameter of FeaO4NPs is8nm, with saturation magnetization55.1emu/g. To render the NPs water-soluble, we oxidized the oleic molecules on the surface of Fe3O4NPs and added CTAB surfactant to make the hydrophobic groups to hydrophilic groups on the surface of the nanoparticles. as-synthesized Fe3O4NPs as the core, prepared the new structure of Fe3O4/Au/Fe3O4petal shaped GoldMag NPs. As revealed by XRD、FT-IR、UV-Vis、VSM、EDS、ICP and HTEM, the mean diameter of the composite particles is38nm, characteristic absorption peak of the Fe3O4/Au composite nanoparticles is at540nm, with saturation magnetization40.7emu/g and coupling capacity of goat anti-mouse IgG307μg permilligram GoldMag NPs. TEM images illustrate GoldMag NPs were in30nm-sized sphere morphology, with large amount of8nm-sized Fe3O4nanoparticles dissolved. These as-synthesized nanoflowers dispersed in water possess good dispersion and stabilization and exhibit a zeta-potential above+30mV.2. A SNP fast and sensitive chromatography strip for clopidogrel individual medication was designed and prepared. This novel fast chromatographic model was constructed through specific binding the gold magnetic composite nanoparticle marked digoxin monoclonal antibodies and the gene mutation primer of CYP2C19*2(681G>A) and CYP2C19*3(636G>A) alleles marked digoxin molecule. Condition optimization, performance evaluation and parameter confirmation were done through the constructed plasmid DNA and ultraviolet spectrophotometer. The results showed that this novel SNP fast chromatographic test strip has high specificity and the sensitivity can reach0.1%.3. The335blood samples of cardiovascular and cerebrovascular patients in Shaanxi province were taken as the research object. The gene mutations of CYP2C19*2and CYP2C19*3allele of the samples were determined by the developed SNP fast chromatography technology and the sequencing method which is an existing gold standard method. The statistical results showed that the gene mutations frequency distributions of the two alleles are both comply with the laws of genetic equilibrium, and the accuracy of the novel technology are98%comparing the sequencing method and two methods do not exist significant differences in accuracy. However,the detectable rate of the novel method is much higher than the sequencing method and two methods exist significant differences. The statistical genotypes of these blood samples showed that the population percentage of clopidogrel metabolism type is40.8%for the fast metabolism,43.2%for the metabolism and16%for the slow metabolism, respectively.Herein, simple, convenience and fast technology with high specificity and sensitivity genotyping method was developed based on the GoldMag NPs. This technology has no complex operation and doesn’t need expensive instrument, so it can be easily popularized in all levels of medical institutions in a specifications PCR lab, and promote the individual medication detection to the standardization and industrialization, as well as lay a foundation for the research of clinical drug and disease treatment.