Spectral analysis of multiplex Raman probe signatures.
Spectral analysis of multiplex Raman probe signatures.
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DOI：
10.1021/nn800243g
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发表时间：
2008-11-25
期刊：
影响因子：
17.1
通讯作者：
Knudsen, Beatrice S.
中科院分区：
文献类型：
--
作者：
Lutz, Barry R.;Dentinger, Claire E.;Nguyen, Lienchi N.;Sun, Lei;Zhang, Jingwu;Allen, April N.;Chan, Selena;Knudsen, Beatrice S.
关键词：
拉曼纳米粒子探针是一种新兴的新型光学标记，用于生物测定、细胞和组织中的生理和病理过程的询问。虽然它们独特的发射特征对于多路复用是理想的，但是这些探针的全部潜力尚未实现，因为常规分析方法是不充分的。我们报告了一种新的光谱拟合方法，利用整个光谱特征定量提取单个探针信号的多重光谱。我们使用未结合和抗体结合的复合有机-无机纳米颗粒（COIN）在一系列多重测定中评价该方法。结果显示小信号（<总信号的2%）的灵敏的多重检测，以及在相应的4重和单重板结合测定中的相似的检测限。在福尔马林固定的人前列腺组织的三重测定中，两个抗体缀合的COIN和常规荧光团用于成像前列腺特异性抗原、细胞角蛋白-18和DNA的表达。光谱分析方法有效地去除了组织的自发荧光和其他未知背景，允许在亚细胞空间分辨率下准确和可重复的成像（ROC曲线下的面积为0.89 +/-0.03）。在所有测定系统中，光谱分析引起的误差≤总信号的2%。总之，光谱拟合方法提供了（1）来自具有重叠峰的多重光谱的信号的定量，（2）未知背景的稳健逐点去除，（3）定量评估分析误差的机会，（4）消除操作员偏差，以及（5）适用于高通量分析的简单自动化。该方法的简单实施和普遍适用性显著扩展了拉曼探针用于定量体内和体外多重分析的潜力。
Raman nanoparticle probes are an emerging new class of optical labels for interrogation of physiological and pathological processes in bioassays, cells, and tissues. Although their unique emission signatures are ideal for multiplexing, the full potential of these probes has not been realized because conventional analysis methods are inadequate. We report a novel spectral fitting method that exploits the entire spectral signature to quantitatively extract individual probe signals from multiplex spectra. We evaluate the method in a series of multiplex assays using unconjugated and antibody-conjugated Composite Organic-Inorganic Nanoparticles (COINs). Results show sensitive multiplex detection of small signals (<2% of total signal), and similar detection limits in corresponding 4-plex and singlet plate binding assays. In a triplex assay on formalin-fixed human prostate tissue, two antibody-conjugated COINs and a conventional fluorophore are used to image expression of prostate-specific antigen, cytokeratin-18, and DNA. The spectral analysis method effectively removes tissue autofluorescence and other unknown background, allowing accurate and reproducible imaging (area under ROC curve 0.89+/−0.03) at subcellular spatial resolution. In all assay systems, the error attributable to spectral analysis constitutes ≤2% of total signal. In summary, the spectral fitting method provides (1) quantification of signals from multiplex spectra with overlapping peaks, (2) robust spot-by-spot removal of unknown background, (3) the opportunity to quantitatively assess the analysis error, (4) elimination of operator bias, and (5) simple automation appropriate for high-throughput analysis. The simple implementation and universal applicability of this approach significantly expands the potential of Raman probes for quantitative in-vivo and ex-vivo multiplex analysis.
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