基于二维阵列多孔硅芯片的低分子量生物标志物分析技术及其在疾病诊断中的应用

Human plasma or serum contains thousands of low molecular weight protein or peptides （LMWP）, which are shed from tissues and cells or derive from proteolytic degradation of larger serum proteins. It has been proposed that the LMWP represents a rich untapped source of disease-specific diagnostic information. However, two major hurdles limit the mining of information from the pool of the LMWP: (1) the extremely low concentration of disease-relevant biomarkers that enter circulation early in the course of a disease may be masked by other proteins, including high-abundance species such as albumin and immunoglobulins. Conventional methods, such as two-dimensional (2-D) gel electrophoresis, do not have the sensitivity and resolution to detect and quantify these low-abundance, low molecular weight proteins. (2) Degradation of protein biomarkers can occur immediately following the collection of blood or other bodily fluids as a result of ex-vivo proteolytic processes. Therefore, It is highly important to develope a practical method to capture,enrich,protect,and detect the low abundant LMWP in the disease information mining and clinical diagnosis.In this poject, we propose to construct a two dimentional Porous Silicon chip (2D-PSi),which have a capability to fractionate, capture, and protect the LMWP from serum, urine and cell lysate samples in one step. The 2D-PSi will combine the microarray and microfluidics technology. One dimension of the chip will be patterned with etched porous silicon array with pore size ladder, while another dimension is a chemical pattern with a variety of affinity group and surface modification. PDMS chip with microfluidics channel are designed to transport biofluids samples to each spot of 2D-PSi chip. The capability of LMWP capture and fraction, as well as the behavior of laser desorption and ionization on the 2D-PSi chip will be investigated. Determination of seized peptides and classification of LMWP pattern on this chip will be achieved by MALDI-TOF/TOF imaging and stastical analysis.The goal of this project is to estabilsh a new method for disease diagnosis and management based on the recognition of peptides profiles and discovery of peptides biomarkers in the untapped LMWP fraction of human biofluids.

人血清及体液中肽类物质或低分子量蛋白（LMWP）蕴藏着大量与疾病有关的信息，构成了一类潜在的新型疾病标志物。然而低含量的LMWP极易被血清及体液中的高丰度蛋白质掩盖。因此，如何去捕获、富集 、保护及检测血清或体液中的LMWP是摆在我们面前的一个重要问题。本项目拟构建不同孔径与不同表面电荷特征的二维多孔硅（2D-PSi）阵列芯片。研究该阵列芯片的刻蚀与表面修饰方法，在芯片的一向维度上排列孔道直径呈阶梯分布的多孔硅阵列，在另一个维度上赋予多孔硅阵列不同的表面电荷特征及亲疏水性。此外，拟引入微流控技术将血液或体液样品分别通过2D-PSi的各个点阵，研究该2D-PSi 芯片对血液与体液试样中LMWP的捕获与筛分能力，及作为质谱离子化基质的性能。通过MALDI-TOF/TOF质谱检测与成像技术并结合数学统计模型，建立LMWP谱识别及肽生物标志物发现的新方法，为疾病诊断与疾病信息的跟踪提供重要的手段。

通过四年的研究，课题组建立了用于肽指纹图谱识别的多种技术手段，成功地将所建立的技术手段应用于多种肿瘤患者血清样本的判别。 主要取得的成果如下: (1) 提出了采用多孔硅（PSi）富集及保存血清肽的新技术，极大地降低了血清样本在存储和运输过程中肽谱的不稳定性，有助于延长血清采样后的保存时间，减少生物信息的丢失。有望作为未来生物样本库中保存血样的一种新材料。（2）建立了基于多孔硅颗粒的原位质谱分析技术。通过电化学刻蚀制备了孔径可调控的PSi颗粒，建立了PSi孔径与截留蛋白质分子量的关系。同时采用表面化学修饰的方法提高了PSi颗粒的稳定性及选择性。将PSi用于血清样本的肽谱分析表现出多方面的优势，兼具筛分、捕获、富集、保护等多多重功能。同时利用了多孔硅的半导体特性进行原位质谱分析。被捕获的肽段无需洗脱，直接在颗粒孔道通内通过MALDI技术离子化。该技术极大地加速了复杂样本的分析速度，同时提高了分析的灵敏度，减少了肽谱信息的丢失。（3）建立了多维富集颗粒原位质谱分析（On-particle mass spectrometric detection）及多孔硅芯片质谱分析技术。在上述工作基础之上，制备了多种表面化学修饰的的PSi颗粒，由于不同表面化学的多孔硅对不同肽段的选择性差异，对同一血清样本进行富集和颗粒原位检测后可获得更高的肽谱覆盖率。同时提出了贵金属修饰PSi芯片增强质谱分析信号的原理。探讨了LSPR效应及半导体-金属复合材料的电荷转移对MALDI信号的影响。（4）采用所建立的PSi颗粒原位质谱分析技术及PSi芯片质谱分析获取了超过100例人血清样本的肽谱数据，用student-t test 挑选出了与疾病关联的多个特征肽段，并以上述特征肽段对不同样本进行聚类分析。结果表明所有样本的判别准确率都超过90%。该技术不仅可判别肿瘤患者与健康人群的血清，同时可高准确率地判别不同肿瘤患者，肿瘤转移与原发病患者等，为疾病跟踪及治疗提供了有效的分析手段。（5）发现了肽谱识别技术不仅可有效区分不同肿瘤患者的血清样本，还可准确地判别不同种类的细胞甚至不同亚型的细胞。同时建立了基于特征肽谱的肿瘤组织成像技术。该研究结果将为精准医疗提供了新的技术手段，有望用于肿瘤患者的亚型区分及靶向药物的筛选。