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

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

W HAT IS A CD4+ L YMPHOCYTE ? Note: Image not to scale White blood cell CD4 CD4 Antigen Other surface markers

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 cells/uLStage 2HIV-infected <200cells/uLStage 3AIDS

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

P RIMARY OBJECTIVE Create a working prototype that accomplishes the specified goals and meets the performance criteria

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

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 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, g, remove supernatant, resuspend in blocking buffer 5. Spin down CMEUs, remove supernatant, resuspend in Conjugate Dilution Buffer

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

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 P ERISTALTIC P UMP ( HAND - CRANKABLE ) 3. 5.

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

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

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

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

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

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

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

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

Input Outputs PDMS Filter Glass Filter with PDMS/Glass coverings

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.

TRF IMAGING WITH A CELL TRAP DEVICE Possibly CD4+ cells, possibly EuNP aggregates EuNP aggregate and water droplet

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