激光拉曼光谱技术在生物分子DNA研究中的应用和进展

激光技术的兴起使拉曼光谱成为激光分析中最活跃的研究领域之一,已被广泛地用于物质成分的分析和分子结构的鉴定。本文综述了拉曼技术在DNA研究中近年来的最新进展,包括：DNA的常规拉曼光谱分析;DNA的激光共振拉曼光谱分析;DNA在金属表面或电极上吸附行为的表面增强拉曼光谱研究;DNA的傅立叶变换拉曼光谱研究等。并对拉曼光谱技术在DNA等生物大分子领域中的研究前景做了进一步的展望。     

唇腺炎性病变组织的共聚焦显微拉曼光谱特征研究

目的:研究唇腺炎性病变组织的拉曼光谱指纹特征,为拉曼光谱技术临床鉴别诊断唇腺炎性病变提供理论基础。方法:收集舍格伦综合征病变唇腺30例、唇腺急性炎症组织18例及正常唇腺组织30例,应用激光共聚焦显微拉曼光谱仪对唇腺组织进行拉曼光谱检测。应用主成分分析法(Principal component analysis,PCA)及判别函数(Discrimination function analysis,DFA)对光谱数据进行分析,研究唇腺组织光谱指纹诊断价值。结果:唇腺炎症组织与正常组织光谱间存在光谱指纹差异,这些差异代表了某些蛋白、核酸及脂类物质等生物大分子发生改变。PCA-DFA分析发现这些差异性拉曼光谱具有鉴别诊断价值,可以区分不同唇腺组织,总体诊断准确率达91.8%,经交互验证后准确率为89.4%。结论:不同唇腺炎症组织及正常组织间拉曼光谱存在差异,不仅揭示生物大分子改变,还具有临床鉴别诊断价值。拉曼光谱技术在唇腺炎性病变组织鉴别诊断具有巨大应用潜力。     

单细胞拉曼光谱在微生物研究中的应用

拉曼显微光谱是一种能够提供0.5–1.0μm空间分辨率的单个微生物细胞内化学结构信息的研究技术。近几年来,拉曼显微光谱被越来越多地应用于微生物单细胞的研究中,它可以快速无损地检测微生物细胞内的特征化学组分。典型的单个微生物细胞的拉曼光谱包含核酸、蛋白质、碳水化合物、脂质和色素(例如类胡萝卜素)等信息,这些信息能够表征微生物细胞的基因型、表型和生理状态。所以单细胞拉曼显微光谱是一种可用于区分微生物样品的"全生物指纹"技术,它可用于研究单个微生物细胞生命阶段的转变、鉴定微生物单细胞中的色素及其他化合物的含量变化等。本文综述了目前拉曼显微光谱在微生物单细胞研究上的应用,主要包括与稳定同位素标记(stable isotope probing,SIP)、拉曼成像、光谱分类和细胞分选技术结合来探究微生物单细胞对物质吸收后特征峰的变化、推导物质循环过程、进行微生物分类鉴定和探索基因型与表型的关系。拉曼显微光谱作为微生物单细胞研究的手段之一,在代谢过程的研究、活细胞分选和细胞对物质的利用上具有广泛的应用前景。     

非标记表面增强拉曼光谱在DNA检测中的应用

表面增强拉曼光谱(SERS)是一种超灵敏的生化分析技术,已经被广泛运用于细胞、核酸、蛋白质等生物分子的检测,在生物医学领域表现出了巨大的应用潜力。近年来,将表面增强拉曼光谱技术应用于遗传物质DNA的精准检测,引起了人们广泛的关注。本文简要叙述了表面增强拉曼光谱技术的基本原理及其在DNA检测中的优势,主要介绍了非标记的DNA-SERS检测应用进展,其中包括本项目组的相关工作。研究表明,非标记DNA-SERS技术有望成为一种快速、准确的临床诊断方式。     

家兔晶状体拉曼光谱空间分布研究

晶状体透明性的维持与其蛋白的结构成分极为密切,因此研究晶状体内的蛋白空间分布变化有重要意义.由实验中得到的450 cm~(-1)～2 000 cm~(-1)范围内健康家兔晶状体拉曼光谱,计算了家兔晶状体蛋白中三条氨基酸侧链和两条蛋白质主链拉曼谱峰沿赤道部和视轴部的光谱强度,根据实验结果讨论了这五条拉曼光谱在家兔晶状体中的空间分布特性.     

SERS标记方法在生化分析中的应用

表面增强拉曼散射(SERS)标记方法结合现代生物标记方法与SERS光谱技术,使吸附到金或银等贵金属表面的标记分子的拉曼信号显著增强,并将其作为标记示踪信号,具有生物兼容性好、灵敏度高、分子特征性强和快速简便等优点,已成为新颖的标记示踪技术的研究热点之一。本文综述近年来SERS标记技术应用于基因分析、蛋白质检测、微生物检测、肿瘤靶向和小分子物质的最新进展,着重介绍蛋白质和小分子物质的检测,并展望了今后的发展方向。     

皮肤组织显微共聚焦拉曼光谱成像研究

显微共聚焦拉曼光谱成像技术(Confocal Raman Microspectroscopy Imaging,CRMI)能够对样品微区进行精确无损的拉曼光谱分析和光谱图像扫描,提供生物样品的无损高分辨光学信息。本项研究工作,利用CRMI技术实验获取了正常人体离体皮肤组织的拉曼光谱特征,并结合典型特征峰的扫描图像,探讨了脂类、蛋白质等成分在皮肤真皮层的分布特点。实验发现皮肤组织真皮层内胶原蛋白的拉曼特征峰1 248 cm-1强度及其空间分布尤为突出,这一实验结果与组织学中胶原纤维占真皮结缔组织95%的事实相符。实验结果显示,CRMI技术能够全面诠释生物组织内部生化组成与分布信息,在实验描述皮肤组织病理变化的分子生物学机制方面具有广阔的应用前景。     

拉曼光谱技术在微生物学中的应用

拉曼光谱具有快速、灵敏、无损、实时监测等显著特点,在微生物学领域得到广泛应用。分别介绍共焦显微拉曼光谱、共振拉曼光谱、表面增强拉曼光谱、拉曼成像、相干反斯托克斯拉曼光谱、激光镊子拉曼光谱和Raman-FISH的原理和特点,并重点总结和分析不同拉曼光谱技术在微生物的结构、化学组成,以及代谢过程等相关研究中的应用优势。合理利用这些技术在基础微生物、发酵微生物和微生物诊断等方面具有潜在的应用价值。     

基于拉曼光谱的鼻咽癌细胞株总蛋白放射敏感性研究

本文以鼻咽癌细胞株CNE2为放射敏感性的研究对象,经不同剂量X射线照射及不同时间培养后分别提取总蛋白,用共聚焦显微拉曼光谱仪检测其拉曼光谱。统计分析表明:被测样品的拉曼光谱中观察到一些可以归属于蛋白质物质的较为明显的基团频率振动峰;不同剂量的X射线照射后,总蛋白质的平均拉曼光谱与对照组谱形基本一致,但与对照组间的光谱存在着对应峰信号强度的不同。实验提示:照射后谱峰强度的增大或减小,提示着相关物质含量有所改变。分析照射后癌细胞总蛋白拉曼光谱的变化情况,结合数学统计方法,以探寻放射敏感性的特征拉曼标志,可以作为研究肿瘤放射敏感性的手段之一。     

拉曼光谱检测微生物的研究方法和进展北大核心

快速准确地识别和鉴定微生物对于环境科、食品质量以及医学诊断等领域研究至关重要。拉曼光谱(Raman spectroscopy)已经被证明是一种能够实现微生物快速诊断的新技术,在提供微生物指纹图谱信息的同时,能够快速、非标记、无创、敏感地在固体和液体环境中实现微生物单细胞水平的检测。本文简单介绍了拉曼光谱的基本概念和原理,重点综述了拉曼光谱微生物检测应用中的样品处理方法及光谱数据处理方法。除此之外,本文概括了拉曼光谱在细菌、病毒和真菌中的应用,其中单独概括了拉曼在细菌快速鉴定和抗生素药敏检测中的应用。最后,本文阐述了拉曼光谱在微生物检测中的挑战和展望。     

Monitoring of microbial proteome dynamics using Raman stable isotope probing

Abnormal protein kinetics could be a cause of several diseases associated with essential life processes. An accurate understanding of protein dynamics and turnover is essential for developing diagnostic or therapeutic tools to monitor these changes. Raman spectroscopy in combination with stable isotope probes (SIP) such as carbon-13, and deuterium has been a breakthrough in the qualitative and quantitative study of various metabolites. In this work, we are reporting the utility of Raman-SIP for monitoring dynamic changes in the proteome at the community level. We have used ¹³C-labeled glucose as the only carbon source in the medium and verified its incorporation in the microbial biomass in a time-dependent manner. A visible redshift in the Raman spectral vibrations of major biomolecules such as nucleic acids, phenylalanine, tyrosine, amide I, and amide III were observed. Temporal changes in the intensity of these bands demonstrating the feasibility of protein turnover monitoring were also verified. Kanamycin, a protein synthesis inhibitor was used to assess the feasibility of identifying effects on protein turnover in the cells. Successful application of this work can provide an alternate/adjunct tool for monitoring proteome-level changes in an objective and nondestructive manner.     

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.     

拉曼光谱在皮肤领域的研究进展

拉曼光谱是一种新型的光学检测技术,常用于材料鉴定。近年来,随着无创检测需求的增加,拉曼光谱逐渐应用于疾病诊断、物质鉴别等生物领域。综述了拉曼光谱在皮肤领域的研究进展,及其对皮肤组织成分鉴别和皮肤疾病诊断的价值,以期推动拉曼光谱广泛应用于皮肤病学的机理研究和临床诊断。     

激光喇曼光谱技术在食品科学中的应用

激光喇曼光谱技术是一种非侵入、非弹性的光散射技术,它能够无损地提供丰富的分子结构和物质成分的信息。近来它在食品工业领域表现出很大的应用潜力。本文综述了激光喇曼光谱技术在食品科学中的应用及其新进展。主要包括果蔬农药残留的检测、肉类产品质量检测、伪劣食品鉴定、食物蛋白的研究以及食品加工监控等方面的应用。并对喇曼光谱技术在这些方面的应用前景作了进一步的展望。     

Comparative Evaluation Of Raman Spectroscopy At Different Wavelengths For Extremophile Exemplars

Raman spectra have been obtained for extremophiles from several geological environments; selected examples have been taken from hot and cold deserts comprising psychrophiles, thermophiles and halophiles. The purpose of this study is the assessment of the effect of the wavelength of the laser excitation on the ability to determine unique information from the Raman spectra about the specificity of detection of biomolecules produced as a result of the survival strategies adopted by organisms in extreme terrestrial environments. It was concluded that whereas FT-Raman spectroscopy at 1064 nm gave good quality results the time required to record the data was relatively large compared with other wavelengths of excitation but that better access to the CH stretching region for organic molecules was given. Shorter wavelength excitation of biomolecules in the blue-green regions of the visible spectrum using a conventional dispersive spectrometer was more rapid but very dependent upon the type of chemical compound being studied; most relevant biomolecules fluoresced at these wavelengths but carotenoids exhibited a resonance effect which resulted in an improved detection capability. Minerals and geological materials, in contrast, were best studied at these visible wavelengths. In general, the best compromise system for the excitation of the Raman spectra of both geological and biological materials was provided using a 785 nm laser coupled with a dispersive spectrometer, especially for accessing the 1800–200 cm⁻¹ wavenumber shift region where much of the definitive analytical information resides. This work will have conclusions relevant to the use of miniaturised Raman spectrometers for the detection of biomolecules in extraterrestrial planetary exploration.     

拉曼光谱分析技术在细胞生物学研究中的应用进展

细胞是生物体结构和功能的基本单位,自被发现以来新的研究方法不断涌现。单细胞拉曼光谱能提供细胞内核酸、蛋白质、脂质含量等大量信息,可在不损伤细胞的条件下实时动态地监测细胞分子结构变化,亦可获得细胞的“分子指纹”,具有敏感性高、实时检测、活样品不需固定或染色、不损伤细胞等众多特点。近年来国内外研究者将拉曼光谱应用于细胞药物处理、细胞水平疾病诊断、单细胞生命活动监测、亚细胞结构等研究,取得了不同程度的进展。随着研究的深入,拉曼光谱分析技术必将在干细胞,癌症研究、细胞分选、药物筛选等领域大有作为。     

In‐line monitoring of amino acids in mammalian cell cultures using raman spectroscopy and multivariate chemometrics models

The application of PAT for in‐line monitoring of biopharmaceutical manufacturing operations has a central role in developing more robust and consistent processes. Various spectroscopic techniques have been applied for collecting real‐time data from cell culture processes. Among these, Raman spectroscopy has been shown to have advantages over other spectroscopic techniques, especially in aqueous culture solutions. Measurements of several process parameters such as glucose, lactate, glutamine, glutamate, ammonium, osmolality and VCD using Raman‐based chemometrics models have been reported in literature. The application of Raman spectroscopy, coupled with calibration models for amino acid measurement in cell cultures, has been assessed. The developed models cover four amino acids important for cell growth and production: tyrosine, tryptophan, phenylalanine and methionine. The chemometrics models based on Raman spectroscopy data demonstrate the significant potential for the quantification of tyrosine, tryptophan and phenylalanine. The model for methionine would have to be further refined to improve quantification.     

Surface‐enhanced Raman spectroscopy for on‐site analysis: A review of recent developments

On‐site identification and quantification of chemicals is critical for promoting food safety, human health, homeland security risk assessment, and disease diagnosis. Surface‐enhanced Raman spectroscopy (SERS) has been widely considered as a promising method for on‐site analysis due to the advantages of nondestructive, abundant molecular information, and outstanding sensitivity. However, SERS for on‐site application has been restricted not only by the cost, performance, and portability of portable Raman instruments, but also by the sampling ability and signal enhancing performance of the SERS substrates. In recent years, the performance of SERS for on‐site analysis has been improved through portable Raman instruments, SERS substrates, and other combined technologies. In this review, popular commercial portable Raman spectrometers and the related technologies for on‐site analysis are compared. In addition, different types of SERS substrates for on‐site application are summarized. SERS combined with other technologies, such as electrochemical and microfluidics are also presented. The future perspective of SERS for on‐site analysis is also discussed.