significant signal output after rinsing with buffer. This demonstrated that the only manual sample preparation required will be the original finger prick and loading a new trap device. 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 on chip. Attempted conjugation of anti-CD4 antibodies to carboxylate-modified europium chelate nanoparticles did not yield detectable binding to CD4 cells. A reliable conjugation and labeling protocol has been investigated in collaboration with Dr. Buck of Gauge Scientific and Dr. Jay Dickerson of the Vanderbilt Physics Department. The detection threshold using the Vanderbilt Reader portable TRF device was approximately 1:25 dilution from the original nanoparticle concentration (1% solids). These detection threshold test images are displayed in Figure 8. Figure 8: TRF images of cell traps. Europium nanoparticles were pumped into a trap array, and a TRF images were taken. This figure demonstrates that europium phosphorescence can be detected at dilutions as low as 1:25. Also, the resolution of the TRF image was high enough to illustrate the microscale structure of the trap array, which promises to aid diagnosis by allowing trap-by-trap image analysis instead of less precise macroscale analysis. Healthy and immuno-suppressed data were simulated by combining measurements from the FITC-labeled whole blood with accepted concentrations of CD4 lymphocytes. Cell size and signal intensity data were used to calculate the relative signal ratio in healthy versus diseased patients. The absolute quantification of CD4 cells per microliter of blood is a function of the total signal intensity in the Vanderbilt Reader and the fraction of CD4 cells captured during recirculation. The signal intensity of the europium-chelate nanoparticles was used to estimate a
significant signal output after rinsing with buffer. This demonstrated that the only manual sample preparation required will be the original finger prick and loading a new trap device. 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 on chip. Attempted conjugation of anti-CD4 antibodies to carboxylate-modified europium chelate nanoparticles did not yield detectable binding to CD4 cells. A reliable conjugation and labeling protocol has been investigated in collaboration with Dr. Buck of Gauge Scientific and Dr. Jay Dickerson of the Vanderbilt Physics Department. The detection threshold using the Vanderbilt Reader portable TRF device was approximately 1:25 dilution from the original nanoparticle concentration (1% solids). These detection threshold test images are displayed in Figure 8. Figure 8: TRF images of cell traps. Europium nanoparticles were pumped into a trap array, and a TRF images were taken. This figure demonstrates that europium phosphorescence can be detected at dilutions as low as 1:25. Also, the resolution of the TRF image was high enough to illustrate the microscale structure of the trap array, which promises to aid diagnosis by allowing trap-by-trap image analysis instead of less precise macroscale analysis. Healthy and immuno-suppressed data were simulated by combining measurements from the FITC-labeled whole blood with accepted concentrations of CD4 lymphocytes. Cell size and signal intensity data were used to calculate the relative signal ratio in healthy versus diseased patients. The absolute quantification of CD4 cells per microliter of blood is a function of the total signal intensity in the Vanderbilt Reader and the fraction of CD4 cells captured during recirculation. The signal intensity of the europium-chelate nanoparticles was used to estimate a