Microfluidic Devices for Cancer Screening by N-Glycan Analysis

描述(申请人提供)：将开发一种基于微流控的筛查方法，以区分癌症的不同疾病状态。该筛选方法基于高分辨率芯片电泳法和激光诱导荧光检测法。为所有样品生成N-葡聚糖图谱，并通过统计分析进行比较。该方法依赖于对整个N-糖链图谱的分析，而不是单一的生物标记物，以在对照组、癌症不同阶段的患者和癌前疾病患者之间提供充分的区分。微芯片电泳法结合荧光检测技术能够很好地分辨糖链结构及其异构体，并具有极高的检测灵敏度。初步结果表明，长度为20 cm的分离通道和分离电场强度为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-糖链，例如乳腺癌、卵巢癌和食管腺癌患者的血清样本。毛细管或芯片仪器的有效分离和灵敏检测使低丰度和高丰度N-葡聚糖都有助于疾病状态区分的统计分析。在这项应用中，微流控设备将被用作一个方便和可靠的平台来筛选与癌症相关的N-糖链。随着基于微流控的方法得到优化，那些对疾病状态区分贡献最大的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。本申请的具体目的是：(1)证明N-葡聚糖的微芯片电泳法是一种快速、可靠的癌症筛查方法；(2)开发一套N-葡聚糖标准品，用作微芯片电泳法中标准添加的尺寸梯；(3)通过质谱分析确定N-葡聚糖的结构；以及(4)将整体相集成到微流控设备中，用于透析、固相萃取、电色谱和酶测序。

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.