A SELF-CONTAINED MICROFLUIDIC CELL SORTING SYSTEM FOR HIV DIAGNOSIS

Y. Sun1, A. Q. Liu1†, P. H. Yap2and T. C. Ayi2

1School of Electrical & Electronic Engineering

Nanyang Technological University, Nanyang Avenue, Singapore 639798 2Defence Medical Research Institute, DSO National Laboratories

20 Science Park Drive, Singapore 118230 (†Corresponding author: Tel (65) 6790-4336, Fax (65) 6793-3318, Email eaqliu@ntu.edu.sg )

ABSTRACT

This paper described a self-contained microfluidic system that is capable of monitoring the progression of the HIV infection by counting and sorting CD4 cells. The system can handle raw blood sample without off-chip sample preparation step and by using laser diode as the light source, the need for big and expensive laser is eliminated. Blood analysis is currently being performed in central labs on expensive, high-maintenance equipment and can be time consuming, whereas our system is portable, low-cost and could be used in each doctor’s office, as a point-of-care instrument at home or in places with high risk of exposure to biowarfare agents. Keywords: Microfluidics, Cell Sorting, HIV Diagnosis, Lab-on-a-Chip

INTRODUCTION

Counting the number of a specific type of T cells, known as CD4 cells, is the current standard used to monitor the progression of the HIV infection to AIDS. During the course of an infection, the number of CD4 cells present in the blood fall precipitously. 150 CD4 cells/µl is a sign of disease progression. Number of CD4 cells in patients already being treated with pharmacological agents for the virus can be an important indicator of whether their system has developed drug-resistant viral strains.

Currently, the samples need to be sent to a central laboratory for analysis, and the results become available after several hours, sometimes days, which may hamper timely diagnosis. Therefore there is a need for portable, fast and easy-to-operate cytometer system for small clinics or ever household use.

At present some research groups are developing miniaturized cell interrogation and sorting tools for appliance in portable diagnostic instruments. Kruger et al. demonstrated their latest results on the development of a miniaturized flow cytometer which can perform the key

functions of detection, enumeration and sorting of fluorescent species [1]. Fu et al. described their efforts in developing a microfabricated elastometric cell sorter. Using electrokinetic flow, they demonstrated sample dispensing, interrogation, automation, sorting and recovery [2]. Dittrich et al. reported an integrated micro total analysis system based on a microfluidic on-chip device for reaction, high-sensitivity detection and sorting of fluorescent cells and particles [3]. And previously, we described a high-accuracy cell sorter using electroosmotically-driven microfluidic system [4]. But for all these miniaturized flow cytometers, blood samples need to be pre-processed before they can be put into the chip. And another problem is that external laser has to be used as the light source for optical detection, which results in the difficulty of integration.

This paper presents a novel self-contained microfluidic system fabricated in poly(dimethyl siloxane) (PDMS) to monitor the progression of the HIV infection. Reagents are preloaded in the respect reservoirs such that the system can handle raw blood sample without off-chip sample preparation step. Laser diode is integrated in the chip as the light source, which eliminates the need for big and expensive laser and makes the optical system more compact.

DESIGN OF THE CELL SORTER

The heart of the system is a disposable micro-fabricated fluidic device. Fig. 1 shows the schematic diagram of the cell sorting system and Fig. 2 shows a photograph of the fabricated device. The chip is fabricated in poly(dimethylsiloxane) (PDMS) by means of rapid prototyping technology, and is then sealed with a glass coverslip by plasma bonding.

The hybrid silicone elastomer/glass chip contains microfluidic circuitry for whole-blood sample acquisition, fluorescent-labeling of CD4 cells, continuous lysing of red blood cells, electrokinetic focusing of leukocytes into a cell-sized narrow stream, and counting and sorting of

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CD4 cells. A drop of blood is drawn into the middle input reservoir by the user. The reservoir is preloaded with ethylene diamine tetra acetic acid (EDTA) anticogulants, CD4+ antibody tagged with fluorescent dye and phosphate buffered saline (PBS) solution. Each side channel is filled with red blood cells (RBC) lysis buffer. At the input reservoir, CD4 cells bind to fluorescent labeled CD4+ antibody. Sample solution is transported with electoosmotic flow, which is controlled by platinum electrodes at the input and output wells. At the intersection between the main and side channels, continuous lysising of RBC and electrokinetic focus of the cell suspension are initiated. All the RBCs are lysised and CD4 cells are labeled with green fluorescent dye. The focusing effect enables a single cell suspension along the center line of the micro-channel and through the detection region, which permits more sensitive measurements to be made.

Fig. 1. Schematic diagram of the cell sorting system. The pre-focused sample moves down to the detection

region where fluorescence is excited by the focused laser light then measured by an on-chip photodetector. The micro optics system comprises of laser diode, micro lens array and photodetector. CD4 cells are identified by their distinct fluorescent responses. Peaks of fluorescent signal are counted using DAQ card, which is corresponding to the number of CD4 cells. By replacing laser to a laser diode, the cost and space for peripheral equipment are greatly reduced, hence the highly integrated and self-contained cell sorter is realized.

The sorting process is realized by biasing the direction of the electroosmotic flow through electrically switching the voltages at output reservoirs. Sample solution is defaulted to flow to waste reservoir. When a CD4 cell is detected, the flow is switched to the other direction for 5 ms then switched back. Since the electroosmotic flow responds to the direction of the applied field instantaneously, electrical manipulation provides much shorter switching times.

Fig. 2. Photograph of the microchip device.

MICROFLUIDICS FOR CELL MANIPULATION

Electroosmosis was chosen as the pumping mechanism to transport sample solution through the channel systems. Electroosmosis provides great advantages for use in microfluidic systems. Through selective applications of electric fields, fluid pumping, handling and control can be achieved, eliminating moving mechanical components such as valves and flow switches. In microscales, electroosmosis can replace pressure-driven flows and alleviate the difficulties associated with exceedingly large pressure gradients occurring with mechanical pumping for microscale channels. Furthermore, since the response of electroosmotic flow to the direction of the applied field is instantaneous, electrical manipulation provides much shorter switching times and thus, yields much higher sorting speeds.

To achieve optimized microchannel structure, both simulation and experimental study of different configurations with varying channel widths and angles were carried out. A microscale particle image velocimetry (µ-PIV) system was developed to measure electroosmotic flow in microchannel. The use of the µ-PIV enables spatially resolved measurements of instantaneous velocity fields [5]. Focusing effect

As mentioned above, incorporation of transverse spatial confinement of the sample permits more sensitive measurements to be made. Here electroosmotic flow was exploited to focus leukocytes into a cell-sized narrow stream. Different configurations with varying channel widths and angles were simulated. The result showed that the ratio of flow rates of sheath and sample fluids, which equals to the ratio of electric fields, directly determines the lateral expansion of the focused fluid, whereas the

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angle of side channels to the intersection did not show any significant effect. Fig. 3 shows the relationship between the focusing ratio and normalized electric potential at focusing channel (voltage at input reservoir is taken as 1).

Fig. 3. Variation of focusing ratio with normalized electric potential at focusing channel.

The µ-PIV utilizes flow tracing particles to map the flow in the micro-geometries. We mixed 500 nm fluorescent micro-spheres with PBS buffer to visualize and measure confinement of sample. Fig. 4 shows simulated and experimental data of electrokinetic focusing. Initially cells were very disorganized, but after being focused at the channel intersection, their velocity increased and yielded in flow constriction along the centre of the outgoing channel. Electrical field strengths in the sample focusing and the outgoing channels are 20, 40 and 100 V/cm, respectively. The sample to outgoing field strength ratio was 0.2 and yielded a sample stream width of ~16 µm. Fig. 5 shows simulated and measured velocity vector. (a) (b) Fig. 4. Electrokinetic focusing effect. (a) Simulation and (b) CCD image.

(a) (b) Fig. 5. Velocity vector. (a) Simulated results and (b) Measured results using µ-PIV system. Switching effect

The sorting process is realized by biasing the direction of the electroosmotic flow through electrically switching the voltages at output reservoirs. The sorting routine was run in an automated mode. The electrodes connected to the computer could be activated via a feedback loop from the detection module. Sample solution is defaulted to flow to waste reservoir. When a CD4 cell is detected, the flow is switched to the other direction for 5 ms then switched back. Both simulated and measured velocity vectors for upward flow at Y-junction are shown in Fig. 6. The sorted cells show relatively constant speed in electric fields up to 300 V/cm, corresponding to velocity of 5 mm/sec. For clinical diagnosis, generally few micro liter blood sample is needed for each test. This microfluidic system has a throughput up to 70 cells per second and each test can be finished within ten minutes. Throughout can be further improved in future systems by increasing the electric field or paralleling the operation of a array of cell sorters. (a)

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Fig. 6. Velocity vectors at Y-junction for upward flow. (a) Simulated and (b) Measured results using µ-PIV system.

OPTICAL DETECTION

Besides the manipulation, another important task for

micro cell sorter is the precise detection of manipulated fluid samples. The key problem hindering the miniaturization of flow cytometry is making the optical detection system more compact. This problem is tackled here by integrating laser diode in the chip as the light source. Fig. 7 is the SEM of the micro optics system which comprises of laser diode, micro lens array and photodetector. Light from laser diode was focused to a cell-size spot by microlens arrays. The pre-focused sample moves down to the detection region where fluorescence is excited by the focused laser light and then measured by an on-chip photodetector. Fig. 8 shows the emission signal collected by the photo-detector. CD4 cells are identified by their distinct fluorescent responses. Peaks of fluorescent signal are counted using DAQ card, which is corresponding to the number of CD4 cells. By replacing laser to a laser diode, the cost and space for peripheral equipment are greatly reduced, hence the highly integrated and self-contained cell sorter is realized.

Fig. 7. SEM micrograph of micro-optic system.

Fig. 8. Optical signals of cells detection using photodetector.

CONCLUSIONS

This paper presents a self-contained microfluidic cell

sorting system which is capable of monitoring the progression of the HIV infection by counting and sorting CD4 cells. Electroosmosis was used as the driving mechanism to transport, focus and switch cells. Laser diode was integrated in the chip as the light source, which eliminated the need for bulk laser. Blood samples provided by hospital are used to test the fidelity of the system. It is found that the system can provide very high accuracy in judging the HIV progression period of patients.

REFERENCES [1] J. Kruger, K. Singh, C. Jackson and A. Morrison,

“Development of a microfluidic device for fluorescence activated cell sorter,” Micromech. Microeng. 12, 486-494 (2002).

[2] A. Y. Fu, C. Spence, A. Scherer and S. R. Quake, “A microfabricated fluorescence-activated cell sorter,” Nat. Biotechnol, 17, 1109-1111 (1999).

[3] P. S. Dittrich and P. Schwille, “An Integrated Microfluidic System for Reaction, High-Sensitivity Detection, and Sorting of Fluorescent Cells and Particles,” Anal. Chem, 75, 5767-5774 (2003).

[4] Y. Sun, L. C. Ng, S. K. Chua, X. M. Zhang, P. Droge, T. C. Ayi, P. H. Yap and A. Q. Liu, “A high-accuracy cell sorter using electroosmotically-driven microfluidic system,” Proceedings of Asia-Pacific Conference of Transducers and Micro-Nano Technology, pp. 267-271, Sapporo, Japan, Jul. 4-8, 2004.

[5] M. J. Kim, A. Beskok and K. D. Kihm, “Electroosmosis-driven Micro-channel Flows: a Comparative Study of µ-PIV Measurements and Numerical Simulations,” Exp. Fluids, 33, 170-180 (2002).

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