Microfluidic Devices for Cancer Screening by N-Glycan Analysis

描述（由申请人提供）：将开发一种基于微流体的筛查方法来区分不同的癌症疾病状态。筛选方法是基于高分辨率微芯片电泳结合激光诱导荧光检测。生成所有样品的n -聚糖谱，并通过统计分析进行比较。该方法依赖于对整个n -聚糖谱的分析，而不是单一的生物标志物，以在对照个体、患有不同阶段癌症的患者和患有恶性前疾病的患者之间提供充分的区分。微芯片电泳荧光检测对多糖结构及其异构体有很好的分辨能力，具有很高的检测灵敏度。初步结果表明，长度为20 cm、分离场强为750 V/cm的分离通道能够快速有效地分离来自临床相关血清样本的n -聚糖，例如乳腺癌、卵巢癌和食管腺癌患者的血清样本。基于毛细管或芯片的仪器的高效分离和灵敏检测允许低丰度和高丰度的n -聚糖有助于疾病状态分化的统计分析。在这个应用中，微流控装置将作为一个方便可靠的平台来筛选与癌症相关的n -聚糖。随着基于微流体的方法的优化，那些对疾病状态分化贡献最大的n -聚糖将被识别出来。这些分子结构的确定将通过改进电泳分析和检测灵敏度来优化和验证筛选方法。此应用程序的具体目的是：(1)证明N-聚糖的微芯片电泳是一种快速、可靠的筛选各种癌症的方法；(2)开发一套N-聚糖标准，用作微芯片电泳标准添加的分级阶梯；(3)通过质谱分析确定N-聚糖结构；(4)将单片相集成到微流控装置中，用于透析、固相萃取、电色谱和酶促测序。

DESCRIPTION (provided by applicant): A microfluidic-based screening method will be developed to differentiate different disease states of cancer. The screening method is based on high-resolution microchip electrophoresis coupled with laser-induced fluorescence detection. N-Glycan profiles are generated for all samples and compared by statistical analysis. The method relies on the analysis of the entire N-glycan profile, not a single biomarker, to provide sufficien differentiation among control individuals, patients suffering from various stages of cancer, and patients with pre-malignant diseases. Microchip electrophoresis with fluorescence detection provides excellent resolution of glycan structures and their isomers and has tremendous detection sensitivity. Preliminary results suggest that separation channels ¿20 cm in length and separation field strengths ¿750 V/cm are able to rapidly and efficiently separate N-glycans derived from clinically relevant serum samples, e.g., from patients with breast cancer, ovarian cancer, and esophageal adenocarcinoma. Efficient separation and sensitive detection on capillary- or chip-based instruments permit both low and high abundance N-glycans to contribute to the statistical analysis for disease-state differentiation. In this application, microfluidic devices will be used as a convenient and reliable platform to screen N-glycans associated with cancer. As microfluidic-based methods are optimized, those N-glycans that contribute most to disease-state differentiation will be identified. Determination of these molecular structures will enable optimization and validation of the screening method through improvements to electrophoretic analysis and detection sensitivity. The specific aims of this application are to: (1) demonstrate that microchip electrophoresis of N- glycans is a rapid, reliable screening method for various cancers, (2) develop a suite of N-glycan standards to be used as sizing ladders for standard addition in microchip electrophoresis, (3) determine N-glycan structures by mass-spectrometric analysis, and (4) integrate monolithic phases into the microfluidic devices for dialysis, solid-phase extraction, electrochromatography, and enzymatic sequencing.