共聚焦显微拉曼光谱鼻咽癌检测中的研究进展

共聚焦显微拉曼光谱技术是将拉曼光谱技术与共聚焦显微分析技术结合起来的一种检测方法,可以提供细胞、组织或体液的化学指纹信息,具有灵敏度高、非侵入性、免标记、水干扰性小等优势。共聚焦显微拉曼光谱在肿瘤细胞间细微差异的检测及肿瘤生物标志物探索方面具有重要的应用价值。本文综述了当前共聚焦显微拉曼光谱在鼻咽癌检测中的应用现状及研究进展,探讨其成为鼻咽癌临床诊断方法的价值及前景。     

拉曼光谱用于细菌快速药敏检测的研究进展

致病菌对人类健康构成重大威胁,抗菌药物的滥用导致了细菌耐药性的发展和传播。目前,临床微生物室常用的药敏试验,如纸片扩散法、浓度梯度纸条扩散法、肉汤稀释法和全自动药敏分析等都是基于细菌生长的方法,且操作流程繁琐,需要8～16 h才能出结果。该文从快速药敏检测(RAST)的研究出发,重点叙述了拉曼光谱在RAST领域的研究进展。利用拉曼光谱对细菌进行基于代谢表型的药敏检测,检测时间明显缩短,优于常规基于细菌生长的药敏方法。但该方法缺少大规模的临床分离株验证,而且难以实现临床标本中细菌的免分离检测。该文对RAST技术的临床验证、可重复性评估、临床适用性评估、准确性评估、拉曼光谱与电阻抗及微流控技术的创新结合及其在复杂临床标本中直接药敏检测等方面进行展望。     

基于显微共焦拉曼技术对胃癌血清样本诊断的应用研究

目的探讨显微共焦拉曼光谱技术在胃癌(GC)诊断中的应用价值。方法本研究一共入组111份血清样本,其中38例经病理学确诊的GC患者,33例胃良性病患者,40例体检健康者的血清;利用XploRA PLUS型全自动显微共焦拉曼系统分析不同血清拉曼光谱的特征,采用主成分分析(PCA)、正交偏最小二乘法判别分析(OPLS-DA)、分层聚类分析(HCA)等方法进行统计学处理。结果受试样本可以检测到重复性较好的8个峰,分别于1 003、1 155、1 446、1 517、1 655、2 873、2 932、3 059/cm位移处,且平均拉曼光谱峰值的强弱存在差异。利用OPLS-DA统计方法得到该模型的R2X(cum)=0.988,R2Y(cum)=0.749,可预测性Q(cum)=0.642。基于OPLS-DA的AUC分别为0.996、0.999、0.997,HCA结果显示聚类GC、胃部良性病、健康者的正确率分别为97.37%、87.88%、100%。结论基于显微共焦拉曼技术联合OPLS-DA统计方法,对GC、胃良性病及健康人的血清样本可做鉴别区分,该技术有望成为GC辅助诊断的新方法。     

万古霉素修饰玻片用于金黄色葡萄球菌的表面增强拉曼光谱检测

目的建立基于表面增强拉曼光谱(SERS)技术的金黄色葡萄球菌检测方法。方法在碳二亚胺催化下,氨基化玻片与万古霉素形成共价结合,用紫外分光光度计测量玻片偶联万古霉素含量。利用万古霉素修饰玻片捕获并固定金黄色葡萄球菌,在银纳米颗粒增强下,用激光显微拉曼光谱仪检测其SERS光谱。结果傅里叶红外光谱仪检测修饰玻片在1 538 cm~(-1)处具有红外特征峰,紫外分光光度法测定修饰玻片万古霉素含量达0.02 mg/cm~2。在激光显微拉曼光谱仪633 nm激光下,检测出金黄色葡萄球菌在724 cm~(-1)、780 cm~(-1)、1 001 cm~(-1)、1 050 cm~(-1)、1 100 cm~(-1)、1 240 cm~(-1)具有SERS特征峰。结论万古霉素修饰玻片成功捕获金黄色葡萄球菌并检测出其SERS光谱,为直接从人体体液中捕获、鉴定金黄色葡萄球菌提供基础依据。     

宫颈癌及宫颈重度上皮内瘤变拉曼光谱的特征分析

目的:利用拉曼光谱在分子水平上研究宫颈重度上皮内瘤变组织与宫颈癌组织病变细胞与正常细胞的差别.方法:取手术切除或活检的宫颈重度上皮内瘤变及宫颈鳞癌各20例,并取其相应病变旁正常组织作为对照组.利用显微共焦拉曼光谱仪测定病变组织,以及其(和)相应病变旁相对正常组织的光谱.结果:病变组织和病变旁组织的显微共焦拉曼光谱差异有显著性,癌组织具有特征峰.结论:宫颈癌病变组织与癌旁相对正常组织的拉曼光谱均有明显差异,拉曼光谱仪可以用来判断宫颈癌病变与正常宫颈组织,有望成为宫颈癌早期诊断的新方法.     

周围神经显微结构的拉曼光谱和超光谱成像特征

目的:研究周围神经内部显微结构的显微拉曼光谱和超光谱成像特征。方法:取新西兰大白兔10只,解剖并获取其骶1脊髓前根和后根标本,随机选取各10例标本作厚度为30μm冷冻切片。用波长633 nm的激光扫描神经纤维断面;随机各取3例前根和后根标本作厚度为10μm冷冻切片,用超光谱成像系统采集标本的超光谱数据。每个断面对轴突和髓鞘各取15个感兴趣区域(regions of interest,ROI)进行分析。结果:周围神经纤维的拉曼光谱在550 cm-1、1080 cm-1、1280 cm-1、1440cm-1、1660 cm-1处有明显的拉曼散射,以1440 cm-1处最为显著;运动和感觉神经纤维的拉曼光谱相似;周围神经髓鞘与轴突的超光谱曲线有区别。结论:周围神经感觉与运动神经纤维的显微拉曼光谱和超光谱曲线相似,难以独立进行区分。超光谱成像技术可直接对周围神经髓鞘进行识别。     

拉曼光谱技术在检验医学中的研究进展及应用

拉曼光谱技术以其"指纹图谱"性、无损检测、灵敏度高、简便快速等独特的技术优势, 在生物大分子、病原微生物检测、肿瘤精准分子诊断等领域展现出良好的应用潜力。该文立足于临床检验应用,简要阐述了拉曼光谱检测的技术原理,从生物大分子、细胞、组织、病原微生物、体液检测等多层次详细介绍了拉曼光谱技术在生物医学应用方面的技术发展及最新研究成果,探讨了临床转化亟需克服的问题及发展前景。     

表面增强拉曼光谱在肿瘤实验室诊断中的应用

拉曼光谱技术是一种通过光与物质的相互作用产生的非弹性散射光来进行检测的光学技术,可以提供细胞、组织或生物流体的化学指纹信息;表面增强拉曼光谱(SERS)技术是在拉曼光谱技术基础上发展起来的具有更高灵敏度、高分析效率的新技术,其基本原理是探寻与疾病相关的SERS特征谱线,从分子水平研究新陈代谢的小分子含量及种类的变化。本文简述SERS在肿瘤实验室诊断中的应用。     

支原体肺炎儿童与健康儿童血清拉曼光谱检测的差异性研究

目的 探讨血清拉曼光谱检测技术在支原体肺炎儿童与健康儿童的差异性。方法 收集商洛市中心医院2017年4～12月收治的支原体肺炎患儿血清100例作为实验组,选取同期健康儿童的血清100例作为对照组,使用全自动显微拉曼系统检测两组血清的拉曼光谱信号,对差异性进行分析。结果 与对照组相比,实验组血清在位移1 653.34 cm^-1处拉曼光谱峰发生了减弱,差异有统计学意义(t=2.137,P<0.05),在位移1 152.93和1 515.33 cm^-1处拉曼光谱峰得到了增强,差异有统计学意义(t=2.027和2.028,均P<0.05)。结论 初步研究表明支原体肺炎儿童与健康儿童血清拉曼光谱信号存在差异。     

表面增强拉曼散射技术在生物医学检测中的研究及应用

拉曼光谱在检验医学领域的应用正在不断进步和发展, 基于拉曼光谱技术的生物传感平台为疾病精准分子诊断提供了一种新的手段, 特别是表面增强拉曼散射(SERS)技术作为一种快速无损的检测方法, 具有样品制备简单、受水的干扰小、实时检测等优点, 在医学检验领域显示出巨大的应用潜力。同时, 随着SERS与其他技术包括电化学、纳米新材料、微流控、生物芯片、DNA纳米机器、人工智能和机器学习等技术的集成结合, 其在医学检验领域将发挥越来越重要的作用。相信随着对SERS研究的不断深入以及多个学科领域之间的交叉融合, 其在生物医学检测领域将得到广泛应用, 并有望成为下一代精准诊断的重要技术平台。     

MALDI-TOF mass spectrometry tools for bacterial identification in clinical microbiology laboratory

Since the early 1980s, mass spectrometry has emerged as a particularly powerful tool for analysis and characterization of proteins in research. Recently, bacteriologists have focused their attention on the use of mass spectrometry (MS) for bacterial identification, especially Matrix Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF). Moreover, recent publications have evaluated MALDI-TOF in microbiology laboratory for routine use. MALDI-TOF-MS is a rapid, precise, and cost-effective method for identification of intact bacteria, compared to conventional phenotypic techniques or molecular biology. Furthermore, it allows identification of bacteria directly from clinical samples (blood cultures for example).The goal of this review was to update recent data concerning routine identification of microorganisms by MALDI-TOF in the clinical microbiology laboratory.     

Weighted spectral reconstruction method for discrimination of bacterial species with low signal-to-noise ratio Raman measurements

Raman spectroscopy is a label-free and non-destructive spectroscopic technique that has been explored for bacterial identification. However, noise often interferes with the interesting Raman peaks because the Raman signal is inherently weak, especially for bacterial samples. Although this problem can be solved by increasing the exposure time or the power of the excitation laser, a longer acquisition time is required or the risk of sample damage is increased. In contrast, short exposure time and low laser power often lead to inadequate acquisition of Raman scattering, in which the Raman spectra with low signal-to-noise ratio (SNR) is difficult to be further analyzed. In order to quickly and accurately characterize biological samples by using low SNR Raman measurements, a weighted spectral reconstruction based method was developed and tested on Raman spectra with low SNR from 20 bacterial samples of two species. Principal component analysis followed by support vector machine was applied on the reference Raman spectra and the spectra recovered from the low SNR Raman measurements by the proposed method, the traditional spectral reconstruction method, and four other commonly used de-noising methods for the discrimination of bacterial species. The results showed that a classification accuracy of 90% was achieved based on our method, which was comparable to that of the reference Raman spectra and showed significant advantages over other spectral recovery methods. Therefore, the weighted spectral reconstruction method can preserve the most biochemical information for the bacterial species'' identification while removing the noise from the low SNR Raman spectra, in which the advantages of lesser sample damage and shorter acquisition time would promote wider biomedical applications of Raman spectroscopy.

Raman spectra recovered from low SNR Raman measurements by weighted spectral reconstruction method show excellent preservation of information about bacterial identification.     

克罗诺杆菌分种方法研究进展

克罗诺杆菌，原名阪崎肠杆菌，是污染婴幼儿配方奶粉的主要致病菌，能感染新生儿，并导致严重的坏死性小肠结肠炎、菌血症和脑膜炎等疾病。随着对该菌认识的深入，阪崎肠杆菌被命名为肠杆菌科独立的新属——克罗诺杆菌属。最新分类表明克罗诺杆菌属共包含7个种，每个种之间基因组构成和致病性存在差异，因此分种是克罗诺杆菌研究的基础，对进一步认识和了解克罗诺杆菌具有重要意义。近年来，除了传统生化方法分种，一系列分子生物学方法也应用于克罗诺杆菌的分种。本文综述了克罗诺杆菌的生物学特性和目前用于克罗诺杆菌分种的各种方法，以期增加对克罗诺杆菌分种方法的认识，为今后的研究工作提供依据。     

Confocal Raman micro-spectroscopy for rapid and label-free detection of maleic acid-induced variations in human sperm

Confocal Raman microspectroscopy is a valuable analytical tool in biological and medical research, allowing the detection of sample variations without external labels or extensive preparation. To determine whether this method can assess the effect of maleic acid on sperm, we prepared human sperm samples incubated in different concentrations of maleic acid, after which Raman spectra from the various regions of sperm cells were recorded. Following the maleic acid treatment, Raman spectra indicated significant changes. Combined with other means, we found that the structures and chemical compositions of sperm membranes were damaged, and even the sperm DNA was damaged by the incorporation of maleic acid. Thus, this technique can be used for detection and identification of maleic acid-induced changes in human sperm at a molecular level. Although this particular application of Raman microspectroscopy still requires further validation, it has potentially promise as a diagnostic tool for reproductive medicine.OCIS codes: (300.6450) Spectroscopy, Raman; (170.0170) Medical optics and biotechnology; (170.5660) Raman spectroscopy; (170.1530) Cell analysis     

A look into the use of Raman spectroscopy for brain and breast cancer diagnostics: linear and non-linear optics in cancer research as a gateway to tumor cell identity

ABSTRACT

Introduction: Currently, intensely developing of linear and non-linear optical methods for cancer detection provides a valuable tool to improve sensitivity and specificity. One of the main reasons for insufficient progress in cancer diagnostics is related to the fact that most cancer types are not only heterogeneous in their genetic composition but also reside in varying microenvironments and interact with different cell types. Until now, no technology has been fully proven for effective detecting of invasive cancer, which infiltrating the extracellular matrix.

Areas covered: This review investigates the current status of Raman spectroscopy and Raman imaging for brain and breast cancer diagnostics. Moreover, the review provides a comprehensive overview of the applicability of atomic force microscopy (AFM), linear and non-linear optics in cancer research as a gateway to tumor cell identity.

Expert commentary: A combination of linear and non-linear optics, particularly Raman-driven methods, has many additional advantages to identify alterations in cancer cells that are crucial for their proliferation and that distinguish them from normal cells.     

Bacteria detection with thin wetting film lensless imaging

Lensless on-chip imaging is a promising technique to count and monitor cells and micro-objects in liquid sample. In this paper we apply this technique to the observation of μL sample containing bacteria evaporated onto a microscope slide. Compared with previously reported techniques, a large improvement in signal to noise ratio is obtained due to the presence of a few μm thick wetting film creating a micro-lens on top of each bacteria. In these conditions, standard CMOS sensor are able to detect micro-objects as small as few μm, e.g. E.coli and Bacillus subtilis bacteria and 1 μm polymer beads with a large signal to noise ratio of 45 ± 10. An overall detection efficiency of 85 ± 7% and a co-localization error of σ(1D) = 1.1μm compared with reference fluorescence microscopy images are achieved. This novel technique will be used as a pre-positioning tool prior to other optical identification methods, e.g. Raman spectroscopy.     

An update on the use of Raman spectroscopy in molecular cancer diagnostics: current challenges and further prospects

Introduction: Cancer is responsible for an extraordinary burden of disease, affecting 90.5 million people worldwide in 2015. Outcomes for these patients are improved when the disease is diagnosed at an early, or even precancerous, stage. Raman spectroscopy is demonstrating results that show its ability to detect the molecular changes that are diagnostic of precancerous and cancerous tissue. This review highlights the new advances occurring in this domain.

Areas covered: PubMed searches were undertaken to identify new research in the utilisation of Raman spectroscopy in cancer diagnostics. The areas in which Raman spectroscopy is showing promise are covered, including improving the accuracy of identifying precancerous changes, using the technology in real time, in vivo modalities, the search for a biomarker to aid potential screening and predicting the response of the cancer to the treatment regimen.

Expert commentary: Many of the examples in this review are focused on Barrett’s oesophagus and oesophageal adenocarcinoma as this is my area of expertise and perfectly exemplifies where Raman spectroscopy could be utilised in clinical practise. The authors discuss the areas where they believe current knowledge is lacking and how Raman spectroscopy could answer the dilemmas that are still faced in the management of cancer.     

Sensitive and specific discrimination of pathogenic and nonpathogenic Escherichia coli using Raman spectroscopy—a comparison of two multivariate analysis techniques

The determination of bacterial identity at the strain level is still a complex and time-consuming endeavor. In this study, visible wavelength spontaneous Raman spectroscopy has been used for the discrimination of four closely related Escherichia coli strains: pathogenic enterohemorrhagic E. coli O157:H7 and non-pathogenic E. coli C, E. coli Hfr K-12, and E. coli HF4714. Raman spectra from 600 to 2000 cm⁻¹ were analyzed with two multivariate chemometric techniques, principal component-discriminant function analysis and partial least squares-discriminant analysis, to determine optimal parameters for the discrimination of pathogenic E. coli from the non-pathogenic strains. Spectral preprocessing techniques such as smoothing with windows of various sizes and differentiation were investigated. The sensitivity and specificity of both techniques was in excess of 95%, determined by external testing of the chemometric models. This study suggests that spontaneous Raman spectroscopy with visible wavelength excitation is potentially useful for the rapid identification and classification of clinically-relevant bacteria at the strain level.OCIS codes: (300.6450) Spectroscopy, Raman; (170.0170) Medical optics and biotechnology; (170.1420) Biology; (170.1580) Chemometrics; (170.5660) Raman spectroscopy; (170.1530) Cell analysis     

关注分子技术在临床微生物检验中的应用

本期"微生物分子诊断专题"精心组织了6篇文章,包括1篇综述和5篇专著。内容主要围绕临床常见的细菌、真菌和分枝杆菌感染等的诊断、治疗(耐药)、预防和控制问题,重点介绍了分子生物学技术在临床微生物标本的直接检测、基因分型、耐药基因研究、抗原制备及血清学诊断等方面的应用研究。血流感染是一种严重的全身感染性疾病,但血培养存在检验周期长、阳性率低等问题,应用分子生物学技术能够对血流感染中的常见或少见病原菌、分枝杆菌、苛养菌等进行快速鉴定及耐药分析,可以缩短检验周期。艰难梭菌是一种重要的医院感染病原菌,引起抗菌药物相关性腹泻,GeneXpert实时荧光定量聚合酶链反应(PCR)能够直接检测粪便标本中的艰难梭菌,具有快速、操作简便等优点。光滑念珠菌可引起血流感染等多种感染,且耐药性强,是医院感染中常见的侵袭性真菌,微卫星多态性分析法简便快速,分辨力高于多位点序列分析(MLST),可作为临床实验室光滑念珠菌基因分型方法。近年来白念珠菌对唑类抗真菌药的耐药性有增高趋势。"白念珠菌对唑类耐药的机制和生物膜相关基因的表达"一文提示ERG11基因的过度表达为白念珠菌对唑类耐药的重要机制。菌丝细胞壁蛋白HWP1基因的高表达与白念珠菌生物膜形成相关。肺炎克雷伯菌对碳青霉烯类耐药的主要机制是产生质粒介导的2型肺炎克雷伯菌碳青霉烯酶(KPC-2),"同源重组敲除临床肺炎克雷伯菌质粒blaKPC-2基因"一文建立的λred同源重组法能可靠敲除其质粒上的blaKPC-2,为进一步研究blaKPC-2在肺炎克雷伯菌对碳青霉烯类药物耐药中所起的作用奠定基础。"结核分枝杆菌rdESA7-6抗原在耻垢分枝杆菌中的制备及其血清学诊断研究"一文研究制备的结核分枝杆菌融合蛋白-6用于结核病血清学检测具有较好的敏感性和特异性,可用于结核病患者的血清学诊断。