Group 14: Lina Aboulmouna Peter DelNero Parker Gould Rosie Korman Chris Madison
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Transcript of Group 14: Lina Aboulmouna Peter DelNero Parker Gould Rosie Korman Chris Madison
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A POINT OF CARE DIAGNOSTIC DEVICE FOR MONITORING CD4 LEVELS IN HIV PATIENTS IN A RESOURCE POOR SETTINGGroup 14:Lina AboulmounaPeter DelNeroParker GouldRosie KormanChris MadisonStephen Schumacher
Advisor: Dr. Kevin Seale, BME
Vanderbilt UniversityVIIBRE/SyBBURENashville, TN
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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
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WHAT IS A CD4+ LYMPHOCYTE?
Note: Image not to scale
White blood cell
CD4
CD4
CD4 Antigen
Other surface markers
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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.
CD4+ count Stage Patient’s status
>500 cells/uL Stage 1 HIV-infected
200-500cells/uL Stage 2 HIV-infected
<200cells/uL Stage 3 AIDS
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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
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PRIMARY OBJECTIVE Create a working prototype that
accomplishes the specified goals and meets the performance criteria
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DIAGNOSTIC DEVICE DESIGN
Input
Outputs
PDMS
Glass
Buffer
Antibodies
BloodMixer
Pump
Filter Devic
e
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ANTIBODY CONJUGATION PROTOCOL1. 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
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CD4 DETECTION
Note: Image not to scale
White blood cell
CD4
CD4
Eu NP
ɑ-CD4 Antibody conjugated to Eu nanoparticle
Other surface markers Well
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1. Stepper Motor2. Alignment
Screws3. Set Screw4. Shaft Coupler5. Fluid Tubing6. PDMS Washer7. Thrust Bearing8. PDMS Device9. Polycarbonate
Base
1.
2.
4.
6.
9.8.7.
PERISTALTIC PUMP (HAND-CRANKABLE)
3.
5.
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PERISTALTIC PUMP (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
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Tunable pore size to selectively trap CD4+ cells
FILTERARRAY White blood
cellRed blood cell
Silicon
Pore
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Tunable pore size to selectively trap CD4+ cells
FILTERARRAY White blood
cellRed blood cell
Silicon
Pore
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Tunable pore size to selectively trap CD4+ cells
FILTERARRAY White blood
cellRed blood cell
Silicon
Pore
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Tunable pore size to selectively trap CD4+ cells
FILTERARRAY White blood
cellRed blood cell
Silicon
Pore
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Tunable pore size to selectively trap CD4+ cells
FILTERARRAY White blood
cellRed blood cell
Silicon
Pore
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Tunable pore size to selectively trap CD4+ cells
FILTERARRAY White blood
cellRed blood cell
Silicon
Pore
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Tunable pore size to selectively trap CD4+ cells
FILTERARRAY White blood
cellRed blood cell
Silicon
Pore
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Input
Outputs
PDMS
Filter
Glass
Filter with PDMS/Glass coverings
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TRF (TIME-RESOLVED FLUORESCENCE) IMAGINGIn a nutshell: Precisely timing a xenon excitation flash and image capture
with long lifetime fluorophores (europium nanoparticles). Reduces the impact of background fluorescence.
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TRF IMAGING WITH A CELL TRAP DEVICE
Possibly CD4+ cells, possibly EuNP aggregates
EuNP aggregate and water droplet
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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