Microfluidic carbon-blackened polydimethylsiloxane device with reduced ultra violet background fluorescence for simultaneous two-color ultra violet/visible-laser induced fluorescence detection in single cell analysis

Abstract

In single cell analysis (SCA), individual cell-specific properties and inhomogeneous cellular responses are being investigated that is not subjected to ensembleaveraging or heterogeneous cell population effects. For proteomic single cell analysis, ultra-sensitive and reproducible separation and detection techniques are essential. Microfluidic devices combined with UV laser induced fluorescence (UV-LIF) detection have been proposed to fulfill these requirements. Here, we report on a novel microfluidic chip fabrication procedure that combines straightforward production of polydimethylsiloxane (PDMS) chips with a reduced UV fluorescence background (83%-reduction) by using PDMS droplets with carbon black pigments (CBP) as additives. The CBP-droplet is placed at the point of detection, whereas the rest of the chip remains transparent, ensuring full optical control of the chip. We systematically studied the relation of the UV background fluorescence at CBP to PDMS ratios (varying from 1:10 to 1:1000) for different UV laser powers. Using a CBP/PDMS ratio of 1:20, detection of a 100 nM tryptophan solution (S/N ¼ 3.5) was possible, providing a theoretical limit of detection of 86 nM (with S/N ¼ 3). Via simultaneous two color UV/VIS-LIF detection, we were able to demonstrate the electrophoretic separation of an analyte mixture of 500 nM tryptophan (UV) and 5 nM fluorescein (VIS) within 30 s. As an application, two color LIF detection was also used for the electrophoretic separation of the protein content from a GFPlabeled single Spodoptera frugiperda (Sf9) insect cell. Thereby just one single peak could be measured in the visible spectral range that could be correlated with one single peak among others in the ultraviolet spectra. This indicates an identification of the labeled protein c-PKC and envisions a further feasible identification of more than one single protein in the future.

The Poisson distribution and beyond: methods for microfluidic droplet production and single cell encapsulation

“…9a). 54,142,[167][168][169] The addition of a fluorescent or chemical reporter can increase sensitivity to the detection of cells and their function, [170][171][172][173] however label-free detection is also feasible, permitting the high-speed analysis of cells without the requirement for added reporters. Ondemand sorting methods such as those using localized fluorescence-activated dielectrophoretic (DEP) forces can further expand the versatility of cell sorting to include a measure of cell function.…”

“…140 Other excellent examples of microfluidic fluorescenceactivated cell sorting have been reported. 54,142,[167][168][169] The addition of a fluorescent or chemical reporter can increase sensitivity to the detection of cells and their function, [170][171][172][173] however label-free detection is also feasible, permitting the high-speed analysis of cells without the requirement for added reporters. These on-chip detection methods, including optical and electrical ones, are covered in a recent review article.…”

Frontier microfluidic techniques for short and long-term single cell analysis

“…Galla et al embedded carbon black in PDMS to increase the SNR of UV signals in the analysis area of a PDMS device. 26 Analysis of fluorescent signals from cells in bulk is difficult and typically limited in throughput due to sample preparation yielding random cell locations, thereby forming clumps or overlaps, which can be solved by immobilizing cells in an array. Although droplet based microfluidics has many advantages for cell arraying, many assays can be done without droplets in non-compartmentalized cell array platforms, which enable easy, high throughput image processing.…”

Two‐photon excitation in chip electrophoresis enabling label‐free fluorescence detection in non‐UV transparent full‐body polymer chips

“…utilized a PDMS/quartz hybrid chip that allowed label‐free detection of tryptophan and two proteins with detection limits in the micromolar range . In following work, they added carbon black to the PDMS in the fabrication process and were thereby able to further reduce the fluorescence background of the chip material .…”