2D-IR spectroscopy for serum diagnostics

血清等生物体液的生化分析是医疗保健环境中重要的信息来源。获得生物液的速度很快，患者的不适感最小，而蛋白质、脂类、糖和其他代谢物的水平会随着身体化学的变化而波动，在特定症状显现之前提供健康恶化的早期预警信号。仅血清的蛋白质含量就是进行整体分析的理想底物。人血清含有约70 mg/毫升的蛋白质，由白蛋白(~35-50毫克/毫升)和球蛋白(~25-35毫克/毫升)组成。在诊断方面，测量白蛋白/球蛋白比率(AGR)是有用的，因为AGR的变化与炎症反应有关。球蛋白甚至可能提供更多的信息，因为它们包含大量的蛋白质。大部分是所谓的丙种球蛋白，但特定球蛋白的水平，如免疫球蛋白-G(免疫球蛋白，约占丙种球蛋白的80%)、免疫球蛋白A(~13%)和免疫球蛋白M(~6%)与特定的健康相关问题有关。使用红外光谱对血清进行光谱分析具有潜在的变革性。这些测量提供了样本的广泛化学指纹，有助于对样本进行有效的分类，确定是否需要，然后指导后续的深入诊断。单一的光谱测量也将比一组基于抗体的分析更快、更经济。然而，IR方法尚未进入临床应用，因为生物流体是水，而水掩盖了构成血清样本主体的蛋白质的信号，因此蛋白质是主要的诊断标记物。在这里，我们将开发一种先进的光谱技术，超快2D-IR光谱，作为一种对血清进行定量、无标记分析的工具。我们的初步数据(Chem Sci DOI：10.1039/C9SC01590F)显示，由一系列超短(100ps-持续时间)激光脉冲产生的2D-IR信号完全抑制了相对于蛋白质响应的水背景，从而允许在传输过程中对未经处理的潮湿血清样本进行测量。利用蛋白质二级结构固有的振动耦合产生的特征2D光谱特征，我们获得了来自血清中白蛋白和球蛋白部分的光谱分辨信号，并在临床相关范围内以+/-4%的精度测量了具有生物医学重要性的白蛋白/球蛋白比率。我们还展示了2D-IR光谱可以将信号与结构相似的球蛋白Ig G、Ig A和Ig M区分开来，开辟了一种直接的光谱方法来测量目前只能通过生物医学实验室测试才能获得的血清蛋白质水平。在这项提议中，我们将超越这一概念验证，并为推动2D-IR技术未来转化为与医疗保健相关的血清诊断应用奠定科学基础。我们将制定分析使用2D-IR所需的样品处理、数据收集、处理和分析协议。我们将开发测量六种关键血清蛋白质浓度所需的内部校准方法，准确度为+/-1%。我们将利用2D-IR光谱丰富的分子信息含量来测量血清中的低分子组分，如糖、磷脂和核酸，提供血清样本的广泛生物医学指纹。最终，我们将使用这些方法来筛选患者血清样本，以区分健康和疾病样本。

Biochemical analysis of biofluids, such as blood serum, is an important source of information in the healthcare environment. Biofluids are obtained quickly and with minimal patient discomfort, while levels of proteins, lipids, sugars and other metabolites fluctuate in response to body chemistry, providing early warning signals of deterioration in health before specific symptoms become apparent. The protein content of blood serum alone is an ideal substrate for holistic analysis. Human serum contains ~70 mg/mL of proteins, composed of albumin (~35-50 mg/mL) and the globulins (~25-35 mg/mL). Diagnostically, measurement of the albumin to globulin ratio (AGR) is useful because changes in the AGR are linked to an inflammatory response. The globulins are potentially even more informative because they encompass a huge number of proteins. The bulk are so-called gamma-globulins, but levels of specific globulin proteins such as immunoglobulin-G (IgG, ~ 80% of the gamma-globulins), IgA (~13%) and IgM (~6%) are associated with specific health-related issues. Spectroscopic analysis of serum using infrared (IR) spectroscopy is potentially transformative. The measurements provide the broad chemical fingerprint of the sample that makes for effective triage of samples, determining the need for and then guiding subsequent in-depth diagnosis. A single spectroscopic measurement would also be faster and more economical than a panel of antibody-based assays, each targeting a single biofluid component.IR methods have yet to progress to clinical applications however, because biofluids are aqueous and water obscures signals from the proteins that comprise the bulk of serum samples and which are therefore a major diagnostic marker.Here, we will develop an advanced spectroscopy technique, ultrafast 2D-IR spectroscopy as a tool for quantitative, label-free analysis of blood serum. Our preliminary data (Chem Sci doi: 10.1039/C9SC01590F) shows that the 2D-IR signal, which derives from a series of ultrashort (100 fs-duration) laser pulses, completely suppress the water background relative to the protein response, allowing measurements to be made in transmission on unprocessed, wet, serum samples. Using the characteristic 2D spectral signature of a protein that arises from the vibrational couplings inherent in its secondary structure, we have spectrally resolved signals from albumin and globulin protein fractions in serum and measured the biomedically-important albumin to globulin ratio with an accuracy of +/- 4% across a clinically-relevant range. We have also demonstrated that 2D-IR spectroscopy can differentiate signals from the structurally similar globulin proteins IgG, IgA and IgM, opening up a straightforward spectroscopic approach to measuring levels of serum proteins that are currently only accessible via biomedical laboratory testing.In this proposal, we will go beyond this proof of concept and lay the scientific groundwork to drive future translation of 2D-IR technology into healthcare-related serum diagnostics applications. We will develop the sample handling, data collection, processing and analysis protocols needed to use 2D-IR analytically. We will develop internal calibration approaches needed to measure the concentration of six key serum proteins to an accuracy of +/- 1%. We will use the rich molecular information content of 2D-IR spectroscopy to measure low molecular weight fractions of serum such as sugars phospholipids and nucleic acids, delivering a broad biomedical fingerprint of a serum sample. Ultimately, we will use these methods to screen patient serum samples in order to differentiate between healthy and diseased samples.