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Flow PDMS Glass Objectives accomplished toward point-of-care diagnostic instrument: • Developed conjugation protocol for anti-CD4 antibodies to europium nanoparticles • Recirculation of blood using a hand-cranked peristaltic pump • Separation of WBCs from whole blood finger stick sample • On-chip CD4 labeling with FITC fluorescent particles • TRF imaging of europium nanoparticles in trap devices Cost estimates show a total expense of $1.68 per test, well within the target price range. The only technical skill required is the initial finger prick and cartridge loading. The imaging system is portable and affordable, with low energy requirements. In conclusion, significant progress has been made toward achieving the design criteria established for an effective point-of-care CD4 counter for application in low-resource settings. Figure 2. Hand-crankable peristaltic pump for point-of-care device. A novel microfluidic pump was designed which allows manual pumping of inputs through the device. The revolution of the pump forces fluid through the device, and the hand-crank mechanism allows for the pump to be operated with no electrical power. The device is composed of inlet ports, mixer elements, and the circular array of channels that comprise the PDMS portion of the pump. Point-of-care diagnostic device for monitoring CD4 levels of HIV patients in resource-poor settings Lina Aboulmouna 1 , Peter DelNero 1 , Parker Gould 2 , Rosalynne Korman 3 , Christopher Madison 2,3 , Stephen Schumacher 1 Advisers: Dr. Kevin Seale 3 , Dr. John Wikswo 3 Departments of 1 Chemical and Biomolecular Engineering, 2 Electrical Engineering, and 3 Biomedical Engineering Acknowledgements Results Methodology Problem Statement Future Work • Refine blood filtering system • Improve protocol for conjugating anti-CD4 antibodies to europium nanoparticles • Streamline recirculation protocol for robust sample analysis • Develop automated image analysis to quantify CD4 count Conclusions According to the World Health Organization, more than 35 million people were living with human immunodeficiency virus (HIV) in developing countries in 2005. Global health initiatives emphasize the urgent need for affordable and effective technologies to obtain CD4 counts in resource-poor areas in order to identify patients, monitor disease progression, determine treatment regimen, and track patient response. In developed countries, CD4 counts are obtained by flow cytometry, which requires large capital investment, skilled technicians, and reliable infrastructure. Limited clinical access in developing countries inhibits treatment efficiency, burdens the patient, and prevents effective distribution of resources. A successful device would measure low CD4 cell counts accurately, cheaply, quickly and require low technical expertise to operate. Inspired by global HIV treatment initiatives, alternative technologies are emerging for accurate, low-cost diagnostic assays. Among these, microfluidic point-of-care instruments propose a viable alternative to clinical equipment like flow cytometry. This project aims to prototype a novel diagnostic platform to acquire CD4 counts while meeting the design limitations of low-resource settings. Our special thanks go to our mentors Prof. Kevin Seale and Prof. John Wikswo, of the Vanderbilt Institute for Integrative Biosystems Research and Education, for their efforts to guide our work. In addition, we extend our sincere gratitude to Dan Morrow and Dr. Bob Buck of Gauge Scientific for providing their technical expertise in time-resolved fluorescent imaging. We also thank Loi Hoang for his help with pump operation and troubleshooting. Finally, we thank Prof. Paul King for his feedback and guidance in completing our project. Figure 3. Microfluidic trap device. An array of U-shaped structures captures cells as they flow through the device. Inset: Magnified diagram is of a cell trap containing two cells (green). Inset by K. Seale. Cell Trap Filter Array Peristaltic Pump & Mixer References Figure 4. Time-resolved fluorescence (TRF). TRF imaging generates a fluorescent excitation but does not capture an image until after a short time delay. This time delay allows cellular auto-fluorescence and background fluorescence (blue line) to decay to minimal levels, while the fluorescence from europium nanoparticles (red line) remains strong. 1. O’Brien, WA, Hartigan, PM, Daar, ES, Simberkoff, MS, and Hamilton, JD. Changes in plasma HIV RNA levels and CD4þ lymphocyte counts predict both response to antiretroviral therapy and therapeutic failure, VA Cooperative Study Group on AIDS. Ann Intern Med 126: 939–945 (1997). 2. http://www.who.int/hiv/pub/guidelines/artadultguidelines.pdf 3. Rodriguez, WR, et al. A Microchip CD4 Counting Method for HIV Monitoring in Resource-Poor Settings, PLoS Medicine, July 2005, Volume 2, Issue 7. Table 1. CD4 lymphocyte counts are used to stage the progression of HIV disease according to standards set by the Center for Disease Control (CDC). Device Components Vanderbilt Reader: Point-of-care TRF Identification of CD4+ cells in whole blood on chip Our specific goals for the project include: • Conversion of DC electric pump to hand-cranked mechanical pump • Integration of microfluidic platform with camera and pumps • Concentration of white blood cells in whole blood sample • Conjugation of anti-CD4 antibodies to fluorescent europium nanoparticles • Fluorescent antibody labeling of CD4 cells • Digital image acquisition and analysis • Quantification of CD4 cell counts per microliter of a blood sample Design Objectives Figure 7. CD4 cells can be labeled on chip. FITC-CD4 antibodies were flowed into a device containing Jurkat T cells. The excess antibodies were rinsed from the device, revealing the presence of newly-labeled CD4 Jurkat cells. A series of fluorescent pictures (A) is given, as well as the overlay of the fluorescent and DIC images (B). This is proof of concept that CD4 cells can be labeled while trapped in the device. Figure 8. TRF images of cell traps. Europium nanoparticles were pumped into a trap array at various concentrations, and TRF images were taken. This figure demonstrates that europium phosphorescence can be detected at dilutions as low as 1:25. On-chip fluorescent labeling of CD4 in Jurkat T cells 1 10 100 1000 1 1:10 1:25 1:50 1:100 Pixel intensity Eu dilution TRF/Eu Calibration Europium nanoparticles and TRF for CD4 detection C 1) Obtain and clean a silicon wafer. 2) Pour a dollop of SU-8 on the wafer center. 3) Spin-coat the SU-8 to desired thickness. 4) Remove the edge bead, if necessary. 5) Expose through mask to UV light. 6) Inspect and post expose bake. 7) Develop away un- cross-linked SU-8. 8) Cast PDMS onto SU-8 master. 9) Cut cured device away from master. 10) Punch necessary inlet and outlet ports. 11) Clean device and plasma bond to glass. CD4+ count Stage Patient’s status >500 cells/mL Stage 1 HIV-infected 200-500cells/mL Stage 2 HIV-infected <200cells/mL Stage 3 AIDS Figure 1. Step-by-step procedures for photolithography and replica molding. 1. Stepper Motor 2. Alignment Screws 3. Set Screw 4. Shaft Coupler 5. Fluid Tubing 6. PDMS Washer 7. Thrust Bearing 8. PDMS Device 9. Polycarbonate Base 2. 4. 6. 9. 8. 7. 5. 1. Antibodies Buffer Whole blood Pump track Mixer 3. Figure 5. FITC-CD4 antibodies identify CD4 cells in whole blood. Although the CD4 cells are initially indistinguishable in a sea of red blood cells (left), they become discrete when illuminated with fluorescence (right). Figure 6. CD4 cells are captured from the whole blood sample. As whole blood is flowed through the device, red blood cells pass through while some white blood cells are trapped. Differential Interference Contrast (DIC) (A) and FITC (B) images were taken at 0 sec, 6 sec, and 6 min. FITC images were thresholded (C) and overlaid on top of the corresponding DIC images (D). The number of trapped CD4 cells increases over time from 1 to 2 to 4, suggesting that the number of trapped CD4 cells will continue to increase as the whole blood is recirculated through the device. A B C D t = 0 sec t = 6 sec t = 6 min A B Image analysis and detection threshold requirements The cells captured during a test on a patient’s blood must be concentrated by the trap device such that their fluorescent signal can be read by the TRF reader. Our experiments suggest that recirculation of the whole blood sample through the device is necessary in order to achieve this concentration. The CD4 count per volume of sample under recirculation is given by where A and a c are the cross sectional areas of the trap chamber and a single CD4 cell, respectively, V s is the sample volume, f is the trapping efficiency of the device, n is the number of times the sample volume is recirculated through the device, and l m is the illumination measured by the TRF reader. Figure 9. Simulation of healthy and diseased fluorescent response. The trap device captured Jurkat T cells (immortalized T lymphocytes), as shown in these images. After counting the cells in the field of view, 12% were marked with red spots (left), which corresponded to a healthy person’s CD4 cell count. Then, the number of marked cells was reduced to simulate the cell count of a person with AIDS (right, green spots). 10 cm 1 1:10 1:25 1:50 1:100 20 μm 20 μm 20 μm 20 μm 20 μm 20 μm 20 μm 20 μm 40 μm 40 μm
