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Week Eight Progress

During the eighth week of the hydrogel restructuring module, testing trials were conducted to determine the therapeutic release rates of variant hydrogel densities, including sample of: high-density, low-density, intermediate-density, and a layered sample of both high and low densities.
The testing of the hydrogel layers was conducted by the injection of the fluorescent signal protein, FITC-BSA, within each sample. The hydrogel samples containing FITC-BSA were constructed at three-six hour intervals, then tested concurrently through the spectrophotometer. The testing phase was completed twice during week eight. During the first testing phase, the employment of tap water in the construction of the samples had produced hydrogel samples that were not uniform in consistency. As such, a second trial was conducted in which pure water was utilized to account for the mistakes of the first testing phase. The second trial proceeded to the spectrophotometer phase of testing. The results of the spectrophotometer were obtained with the intention to create a standard curve that will exhibit a hydrogel modulation with the ideal therapeutic release, as evidenced by a slow decrease in fluorescence intensity. Moreover, FITC-BSA has a maximum absorbance value of around 0.9 a.u. when measured at a  wavelength of approximately 495 nm. Nonetheless, FITC-BSA will rapidly lose fluorescence when exposed to light at 495 nm or higher. As such, when placed in the spectrophotometer, the hydrogel samples constructed at 8:00 PM on May 18, 2017 and 2:00 AM on May 19. 2017 were measured at a wavelength of 227 nm, while the sample created at 8:00 AM on May 19, 2017 were measured at 231 nm. The results of the spectrophotometer testing is delineated in figure 1. 


Figure 1: Results of spectrophotometer readings
The following results were utilized to generate a graph delineating the loss of FITC-BSA particles within the hydrogel structures, thus paralleling the therapeutic release of the prototype drug, as illustrated in figure 2: 

Figure 2: Graphed results of spectrophotometer test

The graphed results of the spectrophotometry delineate the release of FITC-BSA over three-six hour intervals. In order to determine the percentage of FITC-BSA that was released over these intervals, a standard curve will be generated. This curve will show the fluorescent intensity of FITC-BSA as it relates to concentration, and this will be used to determine the concentration of FITC-BSA in each of the samples tested above. By comparing the concentrations in the samples with the initial concentrations in the gel, the percentage of FITC-BSA released over time can be calculated. As such, the ideal structure of the hydrogel adhesive was determined to be a layered hydrogel structure, consisting of both high and low density hydrogels. As the initial objective was to create a hydrogel adhesive of two variant densities, the objective could not have been fulfilled until variant densities were individually tested for absorbance with a spectrophotometer. As such, the an absorbance curve could be generated for individual densities before determining a hydrogel modulation which would offer an ideal therapeutic release. The results of the testing may be improved by the utilization of more precise measuring tools. As such, a re-testing phase will be conducted to account for the errors delineated by the graphed results, at present. During the new testing phase, experimental methods will be improved. Case in point: the test tubes containing the supernatant of the hydrogel solution, will be covered in aluminum foil before they are tested in the spectrophotometer. As the group was unaware of the fact that FITC-BSA is a light sensitive substance, the act of leaving the test tubes of the past testing phase exposed to light may have substantially altered the results. Consequently, the mistake will corrected during the next testing phase. Moreover, during the past testing phase, new hydrogel samples were constructed during the four, six-hour time intervals. Subsequently, the supernatant of each test tube was transferred to the Eppendorf tubes. During the new testing phase, one hydrogel sample will be created for each density during a single time. Following, the supernatant of the hydrogel samples will be transferred at four, six-hour time intervals. The results of the spectrophotometer testing were comprehended and justified by a clear understanding of the sources of experimental error, and will be accounted for during the next testing phase, to occur during week nine. 




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