新型悬浮式液态生物芯片检测系统的构建及其在癌症早期诊断上的应用

Early diagnosis of cancer is of great significance to the cancer therapy, and the joint detection of multiple tumor markers is proved to be an effective way for early diagnosis of malignant tumors. Because conventional tumor markers determination methods usually use single indicator, it is difficult for them to meet the clinical requirements for early cancer diagnosis, differential efficacy and prognostic evaluation. Due to the features of high-throughput, fast and multiplex detection, the suspension array liquid biochip is a good candidate for the joint detection of multiple tumor markers. However, patent protection, high price and the technical defects constraint its development. This project attempts to combine red to near infrared emitting quantum dots encoded fluorescent microspheres with controllable optical properties and the ordinary flow cytometry to build a novel suspension array liquid biochip platform. Hot injection method will be utilized to prepare the highly-fluorescent red to near infrared quantum dots in liquid paraffin, and SPG membrane emulsification with solvent evaporation method will be used to achieve the controllable preparation of quantum dots encoded fluorescent microspheres. After successfully achieved the ways of encoding and decoding, the novel suspension liquid biochip platform will be used to realize high-throughput, fast joint detection of the multi-tumor markers, which will lay the foundation for further development of suspension array liquid biochip with independent intellectual property and provide an efficient new way for early diagnosis of cancer.

癌症的早期诊断对癌症的治疗具有重要意义，而多肿瘤标志物联合检测是早期诊断恶性肿瘤的有效途径。常规肿瘤标志物的测定多采用单一指标检测方法，难以满足临床对癌症早期诊断、鉴别疗效及预后评估的要求，而悬浮式液态生物芯片由于其高通量、快速、多指标的检测特点可满足多肿瘤标志物联合检测的要求。但是目前商用悬浮式液态生物芯片由于专利保护、成本昂贵及自身的技术缺陷制约了它的发展。本项目尝试将光学性能优异可控的红光-近红外量子点荧光微球结合普通流式细胞仪构建新型悬浮式液态生物芯片检测平台，利用液体石蜡体系研究红光-近红外量子点的高效制备技术，采用SPG膜乳化结合溶剂挥发法实现红光-近红外量子点荧光微球的可控制备。研究在该检测平台成功实现编码和解码的方法和途径，并实现多肿瘤标志物的高通量、快速联合检测，为进一步研发具有自主知识产权的新型悬浮式液态生物芯片打下基础，为癌症的早期诊断提供高效新途径。

本项目的工作主要围绕着基于功能纳米颗粒/聚合物编码微球的液相芯片构建及其应 用进行相关研究,得到了如下创新研究结果: 1) 首次使用无Cd近红外量子点来进行(磁 性/非磁性)荧光编码微球的制备及液相芯片平台的建立,所用量子点具有低毒性、缓解因磁性颗粒吸收导致荧光性能骤降现象的优势; 2) 提出“单波长”编码方式,并建立数学模型进行近红外量子点的编码指导,有效地扩展了编码的方式,提高了宽发射峰染料的编码能力及其在液相芯片中的适用能力，获得了目前文献报导的编码数量最多（48种）的磁性荧光微球，且微球及编码信号性能优越，适用于发展液相悬浮式生物芯片; 3) 利用自制CuInS2/ZnS量子点编码磁性荧光微球和非磁性荧光微球成功建立针对五种肿瘤标志物AFP、CEA、CA199、CA125、CA242的多元检测的液相悬浮式生物芯片体系。结果表明，基于磁性荧光微球的多因子免疫检测比传统的ELISA免疫检测灵敏度大幅提高，最高可达108倍。临床血清样本检测结果进一步证明我们的液相芯片系统针对五种标志物的灵敏度和特异度均较高，其中灵敏度达到70%~87.5%，而特异度达到91.1%~ 100%; 4) 开发出了基于PEG接枝双亲性聚合物的量子点编码微球, 并成功验证了其在抑制非特异性吸附及提高检测灵敏度方面的效果。以上研究成果为进一步研发具有自主知识产权的新型悬浮式液态生物芯片及其临床转化打下基础,为癌症的早期诊断提供高效新途径。