2D-IR spectroscopy for serum diagnostics

生物流体（例如血清）的生化分析是医疗保健环境中的重要信息来源。生物流体可以快速获得，患者的不适感最小，而蛋白质，脂质，糖和其他代谢物的水平会随着身体化学反应而波动，在特定症状变得明显之前提供健康恶化的早期预警信号。血清中的蛋白质含量是整体分析的理想底物。人血清含有约70 mg/mL蛋白质，由白蛋白（约35-50 mg/mL）和球蛋白（约25-35 mg/mL）组成。在诊断上，白蛋白与球蛋白比率（AGR）的测量是有用的，因为AGR的变化与炎症反应有关。球蛋白可能提供更多信息，因为它们包含大量蛋白质。大部分是所谓的γ-球蛋白，但特定球蛋白的水平，如免疫球蛋白-G（IgG，约80%的γ-球蛋白），伊加（约13%）和IgM（约6%）与特定的健康相关问题。使用红外（IR）光谱法对血清进行光谱分析具有潜在的变革性。这些测量提供了样品的广泛化学指纹，有助于有效地对样品进行分类，确定是否需要进行深入诊断，然后指导后续的深入诊断。一个单一的光谱测量也将比一组基于抗体的测定更快和更经济，每一个都针对一个单一的生物流体成分。然而，IR方法还没有发展到临床应用，因为生物流体是含水的，水掩盖了来自蛋白质的信号，这些蛋白质组成了大部分血清样品，因此是一个主要的诊断标志物。在这里，我们将开发一种先进的光谱技术，超快2D-IR光谱作为血清定量、无标记分析的工具。我们的初步数据（Chem Sci doi：10.1039/C9 SC 01590 F）显示，来自一系列超短（100 fs持续时间）激光脉冲的2D-IR信号完全抑制了相对于蛋白质响应的水背景，允许在未处理的湿血清样品上进行透射测量。使用蛋白质的特征性2D光谱特征，该特征性2D光谱特征产生于其二级结构中固有的振动耦合，我们已经从血清中的白蛋白和球蛋白质组分光谱解析信号，并测量了具有生物医学重要性的白蛋白与球蛋白的比率，其在临床相关范围内的准确度为+/- 4%。我们还证明了2D-IR光谱可以区分来自结构相似的球蛋白IgG、伊加和IgM的信号，从而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获得的血清蛋白水平。我们将超越这一概念验证，并奠定科学基础，推动未来将2D-IR技术转化为医疗保健-相关的血清诊断应用。我们将开发使用2D-IR分析所需的样品处理，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浓度所需的内部校准方法，精确度为+/-1%。我们将利用2D-IR光谱的丰富分子信息内容来测量血清的低分子量组分，例如糖、磷脂和核酸，从而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筛选患者血清样本，以区分健康和患病样本。

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.