Hoffmann Katrin, Behnke Thomas, Grabolle Markus, Resch-Genger Ute
BAM Federal Institute for Materials Research and Testing, Richard-Willstaetter-Str. 11, 12489, Berlin, Germany.
Anal Bioanal Chem. 2014 May;406(14):3315-22. doi: 10.1007/s00216-013-7597-3. Epub 2014 Jan 16.
Bioanalytical, clinical, and security applications increasingly require simple, efficient, and versatile strategies to measure an ever increasing number of analytes or events in parallel in a broad variety of detection formats as well as in conjunction with chromatographic separation techniques or flow cytometry. An attractive alternative to common optical multiplexing and encoding methods utilizing spectral multiplexing/color encoding and intensity encoding is lifetime multiplexing, which relies on the discrimination between different fluorescent reporters based on their fluorescence decay kinetics. Here, we propose a platform of surface-functionalizable polymeric nanoparticles stained with fluorophores differing in their fluorescence lifetimes as a new multiplexing and encoding approach. Proof-of-concept measurements with different sets of lifetime-encoded polystyrene nanoparticles are presented, obtained via staining of preformed particles with visible (vis)- and near-infrared (NIR)-emissive organic dyes, which display very similar absorption and emission spectra to enable excitation and detection at the same wavelengths, yet sufficiently different fluorescence decay kinetics in suspension, thereby minimizing instrumentation costs. Data analysis was performed with a linear combination approach in the lifetime domain. Our results and first cell experiments with these reporter sets underline the suitability of our multiplexing strategy for the discrimination between and the quantification of different labels. This simple and versatile concept can be extended to all types of fluorophores, thereby expanding the accessible time scale, and can be used, e.g., for the design of labels and targeted probes for fluorescence assays and molecular imaging, cellular imaging studies, and barcoding applications, also in conjunction with spectral and intensity encoding.
生物分析、临床和安全应用越来越需要简单、高效且通用的策略，以便在广泛的检测形式中，以及与色谱分离技术或流式细胞术结合使用时，并行测量越来越多的分析物或事件。与利用光谱复用/颜色编码和强度编码的常见光学复用和编码方法相比，寿命复用是一种有吸引力的替代方法，它基于不同荧光报告分子的荧光衰减动力学进行区分。在这里我们提出了一个表面功能化聚合物纳米颗粒的平台，用荧光寿命不同的荧光团染色，作为一种新的复用和编码方法。展示了使用不同组寿命编码聚苯乙烯纳米颗粒的概念验证测量结果，这些结果是通过用可见光（vis）和近红外（NIR）发射有机染料对预制颗粒进行染色获得的，这些染料显示出非常相似的吸收和发射光谱，以便在相同波长下进行激发和检测，但在悬浮液中的荧光衰减动力学足够不同，从而将仪器成本降至最低。在寿命域中采用线性组合方法进行数据分析。我们的结果以及使用这些报告组进行的首次细胞实验强调了我们的复用策略适用于区分和量化不同的标记。这个简单通用的概念可以扩展到所有类型的荧光团，从而扩大可及的时间尺度，并且可以用于例如设计用于荧光测定和分子成像、细胞成像研究以及条形码应用的标记和靶向探针，也可与光谱和强度编码结合使用。
