Highly efficient dual-channel cytometric-detection of micron-sized particles in microfluidic device
Introduction
Rapid and reliable detections of concentration and size distribution of biological particles in suspensions are essential for many biochemical applications, such as in food safety inspection and quality control [1], [2], drug discovery [3], environmental monitoring [4], and biomedicines [5]. Among all currently available methods, optical flow cytometry is one of the most commonly used and has been widely employed to analyze many important characteristics of biological particles by providing rapid, quantitative, and multi-parameter measurements [6]. Conventional flow cytometers are designed to handle micron-sized particles in suspension at throughputs of tens of thousands particles per second with 6–8-parameter analyses [7], [8]; however due to large consumptions of expensive labeling agents in each testing run, high operating costs and limited levels of portability and integrability, these full-sized bench-top instruments are generally dedicated to some very limited applications [9].
As a miniaturized version of conventional flow cytometers, the microchip-based flow cytometers have shown some clear advantages in many areas [10]. By replacing glass tubes with microfabricated channel as the flow channel, this miniaturized flow cytometer can not only process small volumes of biological samples but also integrate many functions, and its portability has also been improved [11], [12]. Over the last few years, microchip-based flow cytometers has rapidly become one of the intensively studied subjects in the field of micro total analysis systems (μTAS) [13], [14], [15], and significant progress have been made in the manipulation, detection, and analysis of biological cells or particles in related microfluidic devices [16], [17]. However, the measurable particles throughput and detection efficiency of these systems are still well below that of modern flow cytometers, particularly for some applications [18], in which fluorescent and light scattering signals need to be detected simultaneously to indicate different characteristics of cells at high throughput with good detection efficiency. To evaluate these capabilities in microfluidic devices, Ramsey and his colleagues [19], [20] firstly developed microchip-based flow cytometers using electrokinetic focusing to detect latex beads and bacterium Escherichia coli (E. coli) at rates of 85 Hz using both fluorescence emission and light scattering and reported a detection efficiency of 94%. Similarly Holmes et al. [21] reported a microchip-based flow cytometers in combination with dielectrophoretic focusing to detect latex beads based on their fluorescence emission at a throughput rate of 250 particles/s. Recently, Simonnet and Groisman [22] constructed a microfluidic device with a complicated hydrodynamic focusing in both in-plane and out-of-plane directions to further improve detection efficiency of fluorescent latex beads in suspension to achieve a detection efficiency of 97% with a sample linear flow rate of up to 8 m/s. In addition to testing micro-scale beads in suspensions as model particles, Sakamoto et al. [23] detected both cultured bacterial cells and bacteria in natural environment with good results to verify the detection accuracy of micro flow cytometer for the real microbiological application. While some others attempts, such as parallelizing [24] and multiplexing microchannels [25], were utilized in electrophoretic analyses and micro coulter counter to improve the throughput of microfluidic device, efficient detection and quantification based on these technologies had not been realized yet.
In this work, we present the development of highly efficient count and identification for artificial latex beads and bacterial cells in a microfluidic device using dual-channel cytometric detection incorporating one dimensional hydrodynamic focusing to spatially confine particles. Using mixtures of fluorescently labeled and unlabeled beads, quantitative count and identification were demonstrated in the microfabricated channel at different particle throughputs up to 416 particles/s along with the high detection efficiencies and good correlation between fluorescence emission and light scattering signals. In addition, efficiently identifying the “targeted” fluorescently labeled particles in suspension mixed with various “interfering” unlabeled particles with dual-channel detection was reported here as well. Finally, the system was applied to test FITC-labeled nonpathogenic E. coli DH5α to verify its detection efficiency and accuracy.
Section snippets
Chemicals and sample preparation
All reagents used were of analytical grade and were purchased from Sigma–Aldrich (St. Louis, MO) unless otherwise indicated. Fluorescently labeled YG polystyrene latex beads (referred to as labeled beads from herein) with average diameter of 1.013 μm and 1.900 μm, and non-fluorescently labeled polystyrene beads (referred to as unlabeled beads from herein) with average diameters of 0.989 μm and 2.077 μm, were all purchased from Polysciences Inc. (Warrington, PA). All beads were separately suspended
Results and discussion
In order to demonstrate the potentials to detect and identify biological samples, such as mammalian cells, yeast, and bacteria, using cytometric detection in the current microfabricated channel, fluorescently labeled beads with an average size of 1.013 μm in diameter, similar to those of smaller cells [30], were interrogated firstly through detecting the fluorescence emission and light scattering signals to demonstrate the detection efficiency and correlation percentage. Subsequently, the
Conclusions
Efficient count and identification of the micro-scaled particles in microfabricated channel using dual-channel cytometric detection has the potential to be a cost-effective and portable alternative to conventional flow cytometer. A robust microfluidic device equipped with a free-space optics setup was utilized in this study to handle the measurement of fluorescence emission and light scattering signals generated from artificial latex beads and bacterial cells. The results showed the detection
Acknowledgments
This research is supported by a Strategic Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC). The authors thank Dr. Yan Du from University of Ottawa for her help on sample preparation, Ms. Ping Zhao and Mr. Eric Estwick from the National Research Council Canada (NRC) for their help on device fabrication and packaging.
Canjun Mu received his Microelectronics and Solid-electronics Ph.D. in 2008 from Institute of Microsystem and Information Technology, Chinese Academy of Science, China. He is currently a postdoctoral research fellowship of University of Ottawa with a research focus on photonics, microfluidics and microelectromechanical systems for pathogen detection and environmental monitoring.
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Canjun Mu received his Microelectronics and Solid-electronics Ph.D. in 2008 from Institute of Microsystem and Information Technology, Chinese Academy of Science, China. He is currently a postdoctoral research fellowship of University of Ottawa with a research focus on photonics, microfluidics and microelectromechanical systems for pathogen detection and environmental monitoring.
Feiling Zhang received her Master degree in Electronic Science and Technology in 2007 from Xidian university (China). From 2007 to 2009, she worked for Spansion (China) Ltd., Qimonda Technologies (Xi’an) Co., Ltd. (China), and National Research Council (Ottawa, ON) respectively. Since 2010, she has been a test engineer in Adeptron Technologies Corporation (Ottawa, ON). Her research interests include the high-speed instrumental measurement and control, real-time signal process.
Zhiyi Zhang obtained his polymer Ph.D. in 1990 from Zhongshan University (China). From 1990 to 2001, he worked with Zhongshan University, Loughborough University (UK), McMaster University (Canada), National Research Council Canada (NRC), Woodbridge Group Co. (Canada), and Zenastra Photonics Inc. (Canada). Since 2002, he has been a research officer at NRC. His current research at NRC is focused on microfluidics, photonics, and chemical/biomedical sensors.
Min Lin received the Ph.D. degree in Biochemistry in 1991 from Queen's University (Kingston, ON, Canada). He is currently a senior research scientist at the Canadian Food Inspection Agency (Ottawa, ON) and an adjunct professor with the Department of Biochemistry, Microbiology and Immunology, University of Ottawa (Ottawa, ON). His research interests include the role of Listeria monocytogenes immunogenic surface proteins in pathogenesis and immunity, biosensor for foodborne pathogen detection, and antibody detection for infectious disease diagnosis.
Xudong Cao received his Ph.D. degree in Chemical Engineering in 2001 from University of Toronto (Toronto, ON). Subsequently, he received his postdoctoral training at Harvard University (Cambridge, MA) and Brown University (Providence, RI) before he joined the University of Ottawa (Ottawa, ON) as a faculty member in 2005. His current research interests are polymeric material development for tissue engineering, microfabrications and pathogen detections.