Multiplexed Spectral Signature Detection for Microfluidic Color-Coded Bioparticle Flow

Abstract

Here, we report a high-speed photospectral detection technique capable of discriminating subtle variations of spectral signature among fluorescently labeled cells and microspheres flowing in a microfluidic channel. The key component used in our study is a strain-tunable nanoimprinted grating microdevice coupled with a photomultiplier tube (PMT). The microdevice permits acquisition of the continuous spectral profiles of multiple fluorescent emission sources at 1 kHz. Optically connected to a microfluidic flow chamber via a multimode optical fiber, our multiwavelength detection platform allows for cytometric measurement of cell groups emitting nearly identical fluorescence signals with a maximum emission wavelength difference as small as 5 nm. The same platform also allows us to demonstrate microfluidic flow cytometry of four different microsphere types in a wavelength bandwidth as narrow as 40 nm at a high (>85%) confidence level. Our study shows that detection of fluorescent spectral signatures at high speed and high resolution can expand specificity of multicolor flow cytometry. The enhanced capability enables multiplexed analysis of color-coded bioparticles based on single-laser excitation and single-detector spectroscopy in a microfluidic setting. The fluorescence signal discrimination power achieved by the optofluidic technology holds great promise to enable quantification of cellular parameters with higher accuracy as well as enumeration of a larger number of cell types than conventional flow cytometric methods.Fluorescently color-coded cells, biomolecules, and microspheres enable multiparameter detection in disease screening, 1 molecular imaging, 2 DNA sequencing, 3 microarray applications, 4,5 cellular function studies, 6 and multiplexed bead-based assays. 7 The ability to identify multiple fluorescence emissions with high specificity is needed to gain sufficient multiplicity in biological analytical methods. Color decoding achieved by analyzing the detailed spectral characteristics of fluorophores can meet this demand, allowing fluorescence emissions with significant spectral overlap to be distinguished. 6, 8 -10 However, detection of multiple spectral signatures generally reduces measurement speed and throughput due to a larger volume of required information and suffers from the added complexity and cost of © 2010 American Chemical Society * To whom correspondence should be addressed. katsuo@umich.edu. SUPPORTING INFORMATION AVAILABLEAdditional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Here, selective identification of multiple fluorescent molecular markers is also essential to discriminate diverse analyte species in the original sample mixture. However, a large hurdle against achieving high specificity and accuracy in multicolor flow cytometry arises when there are similarities and overlaps in the emission spectra of different dyes.14 For example, flow cytometers using as many as 11 fluorescent probe colors have been demonst...

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