Division Biophotonics (BAM-1.2), Federal Institute for Materials Research and Testing (BAM), Richard-Willstätter-Str. 11, 12489, Berlin, Germany.
Department of Physics, Humboldt-Universität zu Berlin, Newtonstr. 15, 12489, Berlin, Germany.
Sci Rep. 2020 Nov 10;10(1):19477. doi: 10.1038/s41598-020-76150-x.
To demonstrate the potential of time-resolved flow cytometry (FCM) for bioanalysis, clinical diagnostics, and optically encoded bead-based assays, we performed a proof-of-principle study to detect biomolecular interactions utilizing fluorescence lifetime (LT)-encoded micron-sized polymer beads bearing target-specific bioligands and a recently developed prototype lifetime flow cytometer (LT-FCM setup). This instrument is equipped with a single excitation light source and different fluorescence detectors, one operated in the photon-counting mode for time-resolved measurements of fluorescence decays and three detectors for conventional intensity measurements in different spectral windows. First, discrimination of bead-bound biomolecules was demonstrated in the time domain exemplarily for two targets, Streptavidin (SAv) and the tumor marker human chorionic gonadotropin (HCG). In a second step, the determination of biomolecule concentration levels was addressed representatively for the inflammation-related biomarker tumor necrosis factor (TNF-α) utilizing fluorescence intensity measurements in a second channel of the LT-FCM instrument. Our results underline the applicability of LT-FCM in the time domain for measurements of biomolecular interactions in suspension assays. In the future, the combination of spectral and LT encoding and multiplexing and the expansion of the time scale from the lower nanosecond range to the longer nanosecond and the microsecond region is expected to provide many distinguishable codes. This enables an increasing degree of multiplexing which could be attractive for high throughput screening applications.
为了展示时间分辨流式细胞术（FCM）在生物分析、临床诊断和光编码珠基分析中的潜力，我们进行了一项原理验证研究，利用带有靶特异性生物配体的荧光寿命（LT）编码微尺寸聚合物珠和最近开发的原型寿命流式细胞仪（LT-FCM 装置）来检测生物分子相互作用。该仪器配备了单个激发光源和不同的荧光探测器，一个探测器在光子计数模式下用于荧光衰减的时间分辨测量，另外三个探测器用于不同光谱窗口中的常规强度测量。首先，在时间域中对两种靶标，链霉亲和素（SAv）和肿瘤标志物人绒毛膜促性腺激素（HCG），进行了珠结合生物分子的区分的示例。在第二步中，利用 LT-FCM 仪器的第二个通道中的荧光强度测量，代表性地解决了炎症相关生物标志物肿瘤坏死因子（TNF-α）的生物分子浓度水平的确定问题。我们的结果强调了 LT-FCM 在悬浮液分析中测量生物分子相互作用的时间域中的适用性。在未来，预计光谱和 LT 编码的组合、多路复用以及时间尺度从较低的纳秒范围扩展到较长的纳秒和微秒区域，将提供许多可区分的代码。这使得多路复用的程度增加，这可能对高通量筛选应用具有吸引力。
