1. 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

2. P ROBLEM S TATEMENT 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. W HAT IS A CD4+ L YMPHOCYTE ? Note: Image not to scale White blood cell CD4 CD4 Antigen Other surface markers

4. CD4+ COUNTS AND S TAGE OF HIV I NFECTION 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. CD4+ countStagePatient’s status >500 cells/uLStage 1HIV-infected 200-500cells/uLStage 2HIV-infected <200cells/uLStage 3AIDS

5. P ERFORMANCE C RITERIA 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. P RIMARY OBJECTIVE Create a working prototype that accomplishes the specified goals and meets the performance criteria

7. D IAGNOSTIC D EVICE D ESIGN Input Outputs PDMS Glass Buffer Antibodies Blood Mixer Pump Filter Device

8. A NTIBODY CONJUGATION PROTOCOL 1. Suspend antibodies and CMEUs (carboxylate- modified Europium nanoparticles) at 30 ug IgG/mg CMEU in the coating buffer: 10mM NaPO4 pH 8.0 2. Allow the antibodies to coat the CMEUs for 1-2 hours, with gentle shaking. 3. 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 4. Wash 2 or 3 times: Spin down the CMEUs at 10,000-12000g, remove supernatant, resuspend in blocking buffer 5. Spin down CMEUs, remove supernatant, resuspend in Conjugate Dilution Buffer

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

10. 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 1. 2. 4. 6. 9. 8. 7. P ERISTALTIC P UMP ( HAND - CRANKABLE ) 3. 5.

11. P ERISTALTIC P UMP ( HAND - CRANKABLE ) 1.Stepper Motor 2.Alignment Screws 3.Set Screw 1. 2. 4. 6. 9. 8. 7. 3. 5. 7.Thrust Bearing 8.PDMS Device 9.Polycarbonate Base 4.Shaft Coupler 5.Fluid Tubing 6.PDMS Washer

12. Tunable pore size to selectively trap CD4+ cells F ILTER A RRAY White blood cell Red blood cell Silicon Pore

13. Tunable pore size to selectively trap CD4+ cells F ILTER A RRAY White blood cell Red blood cell Silicon Pore

14. Tunable pore size to selectively trap CD4+ cells F ILTER A RRAY White blood cell Red blood cell Silicon Pore

15. Tunable pore size to selectively trap CD4+ cells F ILTER A RRAY White blood cell Red blood cell Silicon Pore

16. Tunable pore size to selectively trap CD4+ cells F ILTER A RRAY White blood cell Red blood cell Silicon Pore

17. Tunable pore size to selectively trap CD4+ cells F ILTER A RRAY White blood cell Red blood cell Silicon Pore

18. Tunable pore size to selectively trap CD4+ cells F ILTER A RRAY White blood cell Red blood cell Silicon Pore

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

20. TRF (T IME -R ESOLVED F LUORESCENCE ) I MAGING 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 Possibly CD4+ cells, possibly EuNP aggregates EuNP aggregate and water droplet

22. G OALS 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