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Group 14: Lina Aboulmouna Peter DelNero Parker Gould Rosie Korman Chris Madison

A point of care diagnostic device for monitoring CD4 levels in HIV patients in a resource poor setting. Group 14: Lina Aboulmouna Peter DelNero Parker Gould Rosie Korman Chris Madison Stephen Schumacher Advisor: Dr. Kevin Seale, BME Vanderbilt University VIIBRE/ SyBBURE Nashville, TN.

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Group 14: Lina Aboulmouna Peter DelNero Parker Gould Rosie Korman Chris Madison

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  1. A point of care diagnostic device for monitoring CD4 levels in HIV patients in a resource poor setting Group 14: LinaAboulmouna Peter DelNero Parker Gould Rosie Korman Chris Madison Stephen Schumacher Advisor: Dr. Kevin Seale, BME Vanderbilt University VIIBRE/SyBBURE Nashville, TN

  2. Problem Statement • The Gates Foundation has identified low-cost HIV testing as a primary global health goal (“Grand Challenges in Global Health”). • Current HIV testing methods are slow and expensive • Flow cytometers are not suitable for the point-of-care needs for developing countries • ~$30,000-$150,000 per flow cytometer. • Limitations include energy scarcity, untrained technicians, high capital-cost equipment, patient proximity, low throughput and long feedback period

  3. What is a CD4+ Lymphocyte? CD4 Antigen CD4 White blood cell CD4 Other surface markers Note: Image not to scale

  4. CD4+ counts and Stage of HIV Infection • Patients with HIV who have CD4+ counts above 500cells/uL are in stage 1, CD4+ counts between 500cells/uL and 200cells/uL are in stage 2, and CD4+ counts of 200cells/uL and below are in stage 3 and are classified as having AIDS.

  5. Performance Criteria • Prototype accurately determines CD4 count • Meets $2/test Gates Foundation challenge • Small sample volume (single finger-stick) • Generate results in minutes • Disposable and portable • Minimal energy requirements • Low technical expertise

  6. Primary objective • Create a working prototype that accomplishes the specified goals and meets the performance criteria

  7. Diagnostic Device Design Input PDMS Outputs Filter Device Glass Pump Antibodies Buffer Blood Mixer

  8. Antibody conjugation protocol • Suspend antibodies and CMEUs (carboxylate-modified Europium nanoparticles) at 30 ugIgG/mg CMEU in the coating buffer: 10mM NaPO4 pH 8.0 • Allow the antibodies to coat the CMEUs for 1-2 hours, with gentle shaking. • After the coating, spin down the CMEUs, remove supernatant, and resuspend in blocking buffer: either 10 mg/ml BSA in buffer, or 5% PEG in buffer • Wash 2 or 3 times:  Spin down the CMEUs at 10,000-12000g, remove supernatant, resuspend in blocking buffer • Spin down CMEUs, remove supernatant, resuspend in Conjugate Dilution Buffer

  9. CD4 detection Eu NP ɑ-CD4 Antibody conjugated to Eu nanoparticle CD4 White blood cell CD4 Other surface markers Well Note: Image not to scale

  10. Peristaltic Pump (hand-crankable) 1. Stepper Motor Alignment Screws Set Screw Shaft Coupler Fluid Tubing PDMS Washer Thrust Bearing PDMS Device Polycarbonate Base 2. 3. 4. 5. 6. 7. 8. 9.

  11. Peristaltic Pump (hand-crankable) 1. 4. 2. 3. 5. 6. 7. 8. 9. Stepper Motor Alignment Screws Set Screw • Shaft Coupler • Fluid Tubing • PDMS Washer Thrust Bearing PDMS Device Polycarbonate Base

  12. FilterArray White blood cell Red blood cell Silicon Pore Tunable pore size to selectively trap CD4+ cells

  13. FilterArray White blood cell Red blood cell Silicon Pore Tunable pore size to selectively trap CD4+ cells

  14. FilterArray White blood cell Red blood cell Silicon Pore Tunable pore size to selectively trap CD4+ cells

  15. FilterArray White blood cell Red blood cell Silicon Pore Tunable pore size to selectively trap CD4+ cells

  16. FilterArray White blood cell Red blood cell Silicon Pore Tunable pore size to selectively trap CD4+ cells

  17. FilterArray White blood cell Red blood cell Silicon Pore Tunable pore size to selectively trap CD4+ cells

  18. FilterArray White blood cell Red blood cell Silicon Pore Tunable pore size to selectively trap CD4+ cells

  19. Input PDMS Filter Outputs Glass Filter with PDMS/Glass coverings

  20. TRF (Time-Resolved Fluorescence) Imaging In a nutshell: • Precisely timing a xenon excitation flash and image capture with long lifetime fluorophores(europium nanoparticles). • Reduces the impact of background fluorescence.

  21. TRF imaging with a cell trap device EuNP aggregate and water droplet Possibly CD4+ cells, possibly EuNP aggregates

  22. Goals • Convert DC pump to hand-cranked mechanical power generator mechanism • Integrate microfluidic platform with camera and pumps • Separation of white and red cells in whole blood sample • Fluorescent conjugation, labeling, and excitation of anti-CD4 antibodies • Digital image acquisition • Digital image analysis • Accomplish the primary objective

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