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，以区分癌症的不同疾病状态。筛选方法是基于高分辨率的微芯片电泳与激光诱导荧光检测。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，而不是单个生物标志物，以提供对照个体、患有不同阶段癌症的患者和患有癌前疾病的患者之间的差异。具有荧光检测的微芯片电泳提供了聚糖结构及其异构体的优异分辨率，并且具有巨大的检测灵敏度。初步结果表明，长度为<$20 cm且分离场强为<$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品的N-聚糖，例如，来自乳腺癌、卵巢癌和食管腺癌患者。在基于毛细管或芯片的仪器上的有效分离和灵敏检测允许低丰度和高丰度N-聚糖有助于疾病状态区分的统计分析。 在该应用中，微流体装置将用作筛选与癌症相关的N-聚糖的方便且可靠的平台。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-聚糖将被鉴定。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-聚糖的微芯片电泳是用于各种癌症的快速、可靠的筛选方法，（2）开发一套N-聚糖标准品以用作微芯片电泳中标准品添加的尺寸梯度，（3）通过质谱分析确定N-聚糖结构，和（4）将整体相整合到微流体装置中用于透析、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.