Mass spectrometry data shows that the Zn-BDPA probes can be detected within the samples following serial trypsinization and small molecule extraction. Following serial trypsinization and static dosing for 2 or 6 hours, Zn-BDPA probes can be seen interacting with the cells in the various regions within the three-dimensional cell culture model system. Mass spectra indicate that the probes are selectively binding in the necrotic core in high abundance. However, like previous laboratory results the Zn-BDPA probes can also been seen at moderate levels in the outer and intermediate regions. We hypothesize that this result occurs because the probe moves into the spheroid but has no method that allows it to move out if free and unbound. Dynamic dosing of the spheroids with the Zn-BDPA probes can possibly solve the problem of probes being seen at high levels in all regions of the spheroid. Dynamic dosing introduces the component of flow which gives the Zn-BDPA probes a place to move if they are free and unbound. This setup should provide us with more information of where the apoptotic cells are located within the spheroid regions. A proof of concept study was completed to show that the Amino TyrBDPA probe was able to penetration the spheroid and bind to the apoptotic cells. The probe (50 μM in 250μL of media) was placed into the insert along with the spheroids. Media was then flowed at a rate of 2μL/min through the channel beneath the insert and the free and unbound probe was able to diffuse through the membrane of the insert and ultimately be discarded. Mass spectrometry data of dynamically dosed spheroids (seen in Figure 6) show the Amino Tyr-BDPA probe binding in high abundance (5.16E8) in the necrotic core of the spheroids. Lower abundance of probe was detected in the intermediate and outer regions of the spheroids (5.74E6 and 1.12E4 respectively). These results indicate that more apoptotic cells are located within the core region of the spheroid than the outer and intermediate regions. This directly correlates to the cell viability results where more viable are located. Dynamic dosing provides an avenue for the Zn-BDPA probes to bind to apoptotic cells in a way that static dosing does not. The element of flow within dynamic dosing allows for the probes to bind only to phosphotidylserine exposed on the outer leaflet of apoptotic cells and the free and unbound probe to move through the spheroid and out of the micro-fluidic device.
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