Sung-Yi Yang

A cell counting/sorting system incorporated with a microfabricated flow cytometer chip

Flow cytometry is a popular technique for counting and sorting individual cells. This study presents and demonstrates a new cell counting/sorting system integrated with several essential components including a micromachined flow cytometer chip device, an optical detection system and a data analysis and control system to achieve the functions of cell sample injection, optical signal detection and cell collection. By using MEMS technology, we have integrated several microfluidic components such as micro pneumatic pumps/valves onto a polymer-based chip device. Three pneumatic micropumps are used to provide the hydrodynamic driving force for both sample and sheath flows such that hydrodynamic flow focusing can be achieved, and a micro flow switch device comprising three pneumatic microvalves located downstream of the micro sample flow channel is used for cell collection. Cell samples of human lung cancer cells labelled with commercially available fluorescent dyes have been detected and collected successfully utilizing the developed device. The real-time image of dye-labelled cell samples being excited and detected can be monitored and observed through the LCD panel by a custom designed CCD/APD holder and moving stage. Finally, micro flow switch devices were used to successfully sort the cells into the desired outlet channel, and the counting results of the specific cell samples were monitored through the counting panel. The current study focuses on the setup of the overall system. The proposed flow cytometer system has several advantages such as portability, low cost and easy operation process. The size of the system is 37 cm × 16 cm × 18 cm and the weight is 3.5 kg. The error rate of counting and sorting was 1.5% and 2%, respectively. The sorting frequency of the microvalve device is calculated to be 120 cells min−1. The developed microfluidic chip device could be a promising tool for cell-based application fields such as profiling, counting and sorting.

Micro flow cytometry utilizing a magnetic bead-based immunoassay for rapid virus detection☆

The current study presents a new miniature microfluidic flow cytometer integrated with several functional micro-devices capable of viral sample purification and detection by utilizing a magnetic bead-based immunoassay. The magnetic beads were conjugated with specific antibodies, which can recognize and capture target viruses. Another dye-labeled anti-virus antibody was then used to mark the bead-bound virus for the subsequent optical detection. Several essential components were integrated onto a single chip including a sample incubation module, a micro flow cytometry module and an optical detection module. The sample incubation module consisting of pneumatic micropumps and a membrane-type, active micromixer was used for purifying and enriching the target virus-bound magnetic beads with the aid of a permanent magnet. The micro flow cytometry module and the optical detection module were used to perform the functions of virus counting and collection. Experimental results showed that virus samples with a concentration of 10(3)PFU/ml can be automatically detected successfully by the developed system. In addition, the entire diagnosis procedure including sample incubation and virus detection took only about 40min. Consequently, the proposed micro flow cytometry may provide a powerful platform for rapid diagnosis and future biological applications.

A vortex-type micromixer utilizing pneumatically driven membranes

Micromixers are commonly employed for chemical or biological analysis in micro-total-analysis-system applications. Mixing performance is important since it allows for rapid and efficient chemical or biological reactions. This study, therefore, reports a new vortex-type micromixer which utilizes pneumatically driven membranes to generate a swirling flow in a mixing chamber. The micromixer chip is fabricated by using micro-electro-mechanical-systems technology as well as a computer-numerically controlled machine for rapid prototyping. Two different membrane layouts and driving frequencies are evaluated to determine if there is a significant improvement in the mixing performance. Experimental results indicate that the mixing efficiency increases with increasing driving frequencies and the mixing time is reduced by approximately tenfold as the driving frequency increases from 1 to 6 Hz. A mixing efficiency as high as 95% can be achieved, in time periods as short as 0.6 and 0.7 s for the two- and four-membrane layouts, respectively. Furthermore, numerical simulations are also employed to characterize the swirling flow field, the concentration distribution and the mixing mechanism as well. Combined experimental data and numerical results illustrate the fluid dynamic phenomena that allow for rapid mixing in this vortex-type micromixer.

An SU-8 microlens array fabricated by soft replica molding for cell counting applications

This paper reports a new polymeric, aspheric SU-8 microlens array using a soft replica molding method and its application to cell counting. A bio-detection system comprising the SU-8 microlens array with its three-dimensional convex geometry, a micro flow cytometer chip and an optical detection module is demonstrated. A polymethyl methacrylate (PMMA) template is first fabricated and then the SU-8 microlens array is replicated using a new fabrication process. The developed array has four sizes of microlens, with diameters of 50, 100, 150 and 200 ?m. Experimental results show that the surface roughness of the microlens was only 10.2 nm for the SU-8 polymer material. The microlens had good surface uniformity and excellent optical properties with calculated focal lengths ranging from tens to hundreds of micrometers, depending on their dimensions. A microlens with a high numerical aperture ranging from 0.3 to 0.75 was achieved. The microlens array can be used to increase the efficiency of the optical detection, allowing high-resolution detection and providing a high signal-to-noise ratio. The microlens array has the potential to be widely used for optical or biophotonic applications and for the integration of microfluidic devices. In order to demonstrate its capability, the developed microlens array was integrated with a micro flow cytometer for cell counting applications. Successful counting of fluorescent-labeled human lung cancer cells is demonstrated using the developed method.

Synthesis of gold nanoparticles using a vortex-type micro-mixing system

A new microfluidic reaction chip capable of mixing, transporting and reacting is developed for synthesis of gold nanoparticles with tunable sizes. It allows for a rapid and a cost-effective approach to accelerate the synthesis of gold nanoparticles. The microfluidic reaction chip was made of CNC machining and PDMS casting process to integrate a vortex-type micro-mixer and a micro-pump on a single chip. The micro-mixer is capable of generating a vortex-type flow field to achieve a mixing efficiency as high as 94% within 1 second. Successful synthesis of dispersed gold nanoparticles has been demonstrated within a shorter period of time (5 minutes), as compared to traditional methods. The dispersed gold nanoparticles had an average diameter of 17 nm, 23 nm and 48 nm, respectively. The size of the nanoparticles can be fine-tuned by using reagents with different volumes. The development of the microfluidic reaction system is promising for synthesis of functional nanoparticles for further biomedical applications.

Vancomycin-resistant gene identification from live bacteria on an integrated microfluidic system by using low temperature lysis and loop-mediated isothermal amplification

Vancomycin-resistant Enterococcus (VRE) is a kind of enterococci, which shows resistance toward antibiotics. It may last for a long period of time and meanwhile transmit the vancomycin-resistant gene (vanA) to other bacteria. In the United States alone, the resistant rate of Enterococcus to vancomycin increased from a mere 0.3% to a whopping 40% in the past two decades. Therefore, timely diagnosis and control of VRE is of great need so that clinicians can prevent patients from becoming infected. Nowadays, VRE is diagnosed by antibiotic susceptibility test or molecular diagnosis assays such as matrix-assisted laser desorption ionization/time-of-flight mass spectrometry and polymerase chain reaction. However, the existing diagnostic methods have some drawbacks, for example, time-consumption, no genetic information, or high false-positive rate. This study reports an integrated microfluidic system, which can automatically identify the vancomycin resistant gene (vanA) from live bacteria in clinical samples. A new approach using ethidium monoazide, nucleic acid specific probes, low temperature chemical lysis, and loop-mediated isothermal amplification (LAMP) has been presented. The experimental results showed that the developed system can detect the vanA gene from live Enterococcus in joint fluid samples with detection limit as low as 10 colony formation units/reaction within 1 h. This is the first time that an integrated microfluidic system has been demonstrated to detect vanA gene from live bacteria by using the LAMP approach. With its high sensitivity and accuracy, the proposed system may be useful to monitor antibiotic resistance genes from live bacteria in clinical samples in the near future.

An integrated microfluidic system for rapid isolation and detection of live bacteria in periprosthetic joint infections

Periprosthetic joint infection (PJI) is difficult to treat and the incidence is between 1% and 2% in primary arthroplasties. Implant-associated infections usually arise via either primary infections from bacterial invasion at the time of implant surgery or secondary infections from hematogenous sources. The two-stage re-implantation protocol that consists of extensive debridement at the first stage followed by delayed re-implantation is currently the standard process for chronic PJI with a success rate between 82% to 95%. Furthermore, re-implantation arthroplasty should be only performed after ensuring the complete eradication of bacterial infection to avoid devastating complications. However, it is still a challenge in clinical practice to accurately determine the eradication of infections before or during implantation. Conventional diagnostic methods such as measurements of serum C-reactive protein or interleukin-6 levels, culture of joint aspirates, and microscopic examination of tissue biopsy are either non-specific or relatively time-consuming. For critical decision-making before or during the re-implantation surgery, a quick method with high sensitivity and specificity is therefore of great need. Previous studies reported bacterial ribosomal ribonucleic acids (rRNAs) as a target for the diagnosis of infections since rRNAs are highly conserved among bacterial species and abundant in amount. By using universal primers, the presence of bacterial rRNA could be amplified by using reverse-transcription polymerase chain reaction (RT-PCR). Currently the RT-PCR method for detection of bacterial rRNA is highly sensitive with a limit of detection (LOD) as low as a pictogram level. However, RT-PCR signals could only indirectly distinguish live from dead bacteria based on the degradation of rRNA in the tissue. Furthermore, the whole detection procedure of 16s rRNA RT-PCR is labor-intensive. Therefore, an integrated microfluidic system was presented in this work, which could distinguish the existence of live bacteria within 1 hour with a LOD of 104 colony formation unit (CFU). In this study, the fabrication of the microfluidic chip was improved so that the consistency of the transported liquid volume was increased. Moreover, by using an ethidium monoazide (EMA) assay, the cumbersome pre-treatment process of rRNA in live bacteria can be alleviated. This is the first time that a microfluidic platform was reported to detect live bacteria successfully in PJI samples.

An integrated microfluidic system for antibiotic resistance gene identification capable differentiating live and dead of vancomycin-resistant enterococcus

This study presents an integrated microfluidic system which can identify the vancomycin resistant gene (vanA) from live hetero-bacteria automatically. In this study, a new approach to diagnose the vanA gene from live hetero-bacteria by using ethidium monoazide (EMA) and loop-mediated isothermal amplification (LAMP) was proposed and its feasibility was tested and verified. In addition, an integrated microfluidic system including a microfluidic chip and a home-made control system was also demonstrated. The experimental results showed that the developed system can detect the vanA gene from “live” Enterococcus but not “dead” Enterococcus successfully with a detection limit of 10 colony formation units (CFU). The entire process including the sample pre-treatment process could be automated within 1 hour. This is the first time that an integrated microfluidic system was demonstrated to diagnose the vanA gene from live bacteria by using a molecular diagnosis approach. With its high sensitivity and accuracy, the proposed system may be promising to verify antibiotic resistance genes from live hetero-bacteria which cannot be achieved by using the existing diagnostic methods.

A suction-type, pneumatic microfluidic device for rapid DNA extraction

This current study presents a new miniature, integrated system capable of rapid extraction of genomic DNA (gDNA) from saliva samples. Three major components of the DNA extraction chip, including six symmetrical, normally-closed microvalves, a sample transport/mixing unit and a waste unit are integrated in this device. The microfluidic DNA extraction chip was made of CNC machining and PDMS casting processes to integrate a liquid channel layer, an air chamber layer and a glass layer on a single chip. Liquid samples can be transported and mixed by the suction-type sample transport/mixing unit by using micropumps, micromixers and normally-closed microvalves. Experimental results showed the pumping rate can be as high as 400 µL/min at an applied pressure of −70 KPa for an air chamber with a depth of 1.5 mm. A mixing efficiency as high as 96.5% can be achieved within 3 sec. Experimental data also showed that gDNA with an average concentration of 45 ± 3 ng/µL and an optical intensity (OD) value (260/280) of 1.5 ± 0.2 from ten measurements can be successfully achieved within 25 minutes. Consequently, the proposed miniature system can provide a powerful platform for automatic, rapid DNA extraction.