集成微流体驱动泵的微流控微珠阵列芯片用于基因突变检测的研究

A Microfluidic Microbeads Array Chip Integrated with Micro-fluid Driven Micro-pump for Discrimination of Gene Mutation

建立了一种基于微泵集成微流控微珠阵列芯片及三磷酸腺苷双磷酸酶(Apyrase)介导的等位基因特异性延伸的基因突变检测方法。将微流控芯片、引物修饰微珠阵列及基于毛细和蒸发作用的微流体驱动泵集成构建检测芯片,待测目标序列流过装配的微球阵列并与微球表面延伸引物杂交,在Apyrase和去除外切酶活性的Klenow DNA聚合酶协同作用下,引物3'末端碱基与目标序列包含的基因突变检测位点匹配则能够发生延伸,并将生物素化的dCTP掺入到引物的延伸序列中并固定在微球表面,链霉亲和素修饰量子点能与微球表面引物延伸序列中的生物素结合并提供荧光信号,而引物3'末端与目标序列存在单碱基不匹配则不能发生延伸。结果表明:采用这种单碱基识别技术,微泵驱动的芯片内可以检测0.2 pmol/L目标序列(信背比>3),液压驱动的芯片内能识别0.5 pmol/L目标序列,而芯片外检测只能识别0.1 nmol/L目标序列,微泵集成芯片在检测基因突变时其灵敏度较芯片外基因突变分析提高了500倍,并在0.5～30 pmol/L目标序列浓度范围内待测序列浓度与检测信号呈良好的线性关系。测定了一个人基因组样本中多药耐药蛋白基因1(MDR1)的两个多态性位点C3435T及G2677T,结果显示该样本具有3435CT及2677TT的基因型组合,此结果与DNA测序结果一致。本方法用于基因突变分析,具有良好的特异性、灵敏性及稳定性。

A new approach was developed for the single-nucleotide detection based on the micropump-integrated microfluidic microbeads array chip and an apyrase-mediated primer extension process.The microfluidic chip,primers-modified microbeads array and the micro-pump driven by evaporation and capillary effect were integrated to construct the detection chip.The target DNA flowed across fabricated microfluidic beads array and hybridized with immobilized primer sequences.When the 3′ terminus of primer was matched with the target DNA in the single-base mutation site of interest,under synergistic effect of apyrase and exonuclease-deficient Klenow DNA polymerase,the matched primer could be extended along the template DNA sequence and the biotin-dCTP could be incorporated into extended primers immobilized on the surface of microbeads.However,single-base mismatched duplexes in the 3′ terminus of primer could not be extended.Streptavidin-labeled quantum dots were then allowed to bind to the deposited biotin moieties and displayed the signal.The results indicated the on-chip single-nucleotide discrimination driven by micro-pump and liquid pressure could detect 0.2 pmol/L target DNA（S/N3） and 0.5 pmol/L target DNA respectively,but off-chip assay only discriminated 0.1 nmol/L of target DNA.The chip-based signal enhancement for single-nucleotide discrimination using micro-pump integrated microfluidic chip resulted in 500 times higher sensitivity than that of an off-chip test.The fluorescence signals were correlated to the concentration of the target DNA in the range of 0.5-30 pmol/L.Meanwhile,two multi-drug resistance gene 1（MDR1）-associated SNP sites（C3435T and G2677T） from a human genomic sample were determined using the proposed method.The fluorescence signals indicated the subject used here possessed the MDR1 3435CT and MDR1 2677TT genotypes,which were consistent with the results by DNA sequencing.This approach displays good specificity,sensitivity and stability for discrimination of gene mutation.