Gel Diffusion Experiment

1. Gel Diffusion Experiment STEM ED/CHM Nanotechnology 2015 Presented by Jennifer Welborn

2. Learning Goals In this activity, nanotech participants will: • See how food dyes and gelatin are used to model the delivery of nanoscale medicines to cells in the human body • Measure diffusion distances of 3 different colors of food dye by: Eye, photo image on a computer, ADI software (Analyzing Digital Images)

3. Diffusion and Teaching Standards This lab has content which is applicable to various disciplines/standards • Physical Science/Chemistry: particle motion theory • Biology: passive transport; cellular structure, etc. • Ecology/Environmental Science: environmental effects on living systems • Math: rates; proportions, data collection, measurement, precision/accuracy

4. Diffusion Diffusion– movement of a substance from a region of higher concentration to a region of lower concentration. Diffusion continues until equilibrium--- the concentration of a substance is equal throughout a space https://www.youtube.com/watch?v=n5nubvwJJQM https://www.youtube.com/watch?v=pjg7ryrDxdg https://www.youtube.com/watch?v=qBig2wevHhw

5. Diffusion and Cells • Dissolved particles that are small or non-polar can diffuse through the cell membranes. • The process of diffusion is one of the ways in which substances like oxygen, carbon dioxide and water move into and out of cells. Carbon dioxide from the environment diffuses into plant cells

6. Background For Lab Activity • The delivery of nanoscale medicines to cells in the human body requires diffusion through organs, tissues and cell membranes • This activity will explore the affect of particle size (molecular weight) on diffusion rates • Understanding molecular diffusion through human tissues is important for designing effective drug delivery systems

7. A Model for Nanomedicine Delivery • Measuring the diffusion of dyes in gelatin is a model for the transport of drugs in the extra-vascular space • Gelatin: biological polymeric material with similar properties to the connective extracellular matrix in tumor tissue • Dyes are similar in molecular weight and transport properties to chemotherapeutics

8. Some Uses of Nanoscale Particles in Medicine • Youtube video made by the Center for Hierarchical Manufactoring at UMASS, Amherst: • http://www.youtube.com/watch?v=bUvi5eQhPTc • 5:40-7:40 shows some of the uses of nano-scale particles in medicine.

9. Nanotechnology for Targeted Cancer Therapy • https://www.youtube.com/watch?v=RBjWwlnq3cA • 5-minutes

10. Experiment Overview • Gelatin will be cut into cylindrical disks, placed in Petri dishes and colored solutions will be added to the outer ring • The distance that the dye particles diffuse into the gelatin disks will be measured over time • The diffusion of the dyes will be compared to model the effect of molecular weight on movement of molecules in tumors

11. Collect materials Petri Dishes Food Dye Syringes/10 ml graduated cylinders Paper Cups Plain Gelatin Crisco/Petroleum Jelly Baking Pan Biscuit cutter Prepare Gel Disks Determine amount of water needed to fill up a pan to a depth of 1 cm. Dissolve gel into cold water (2Pks/Cup/200 ml) Microwave for 90 Sec. Pour into pan which has been coated with petroleum jelly and let set. Lab Prep

12. Gel Disks Cut disks--bisquit cutter Thin coating of Petroleum jelly on inside bottom of Petri dish Put gel disk –top side down and centered- on bottom of dish Gently press disk to secure Adding Dye Mix dyes in cups Inject one color/petri dish No dye on top of gel No seepage under gel Do not move dishes after dye inserted Lab Procedure

13. Important Details For Procedure • Make the dye solutions according to directions. • Inject dye towards the outside of the petri dish, not towards the gel. • Photograph the gel: same time, same distance, same ambient lighting, flash off, cover off petri dish, same sequence. Keep camera parallel to gel (do not tilt) to avoid parallax.

14. Data Collection • Method 1-- By eye: measure (in mm) the distance each dye has diffused for each time interval. Record data in a data table or use excel spreadsheet • Method 2--Using a digital camera: take photos of each petri dish at the same time each day, 8:45 and 4:45, from the same height and angle

15. Data Collection3 Food Dyes Start 4 hours Diffusion is first visible

16. Gel Diffusion Analysis

17. Gel Diffusion AnalysisMethod 1: Determining Rate of Diffusion by Eye

18. Create a graph by hand, in excel, plotly or other graphing program

19. https://plot.ly/

21. Diffusion AnalysisMethod 2: Using a Digital Camera • Group Pictures by Color in date/time order 7-9-1600 7-10-0800 7-10-1600 7-11-0800

22. Pick one color to start Load the first morning shot Windows Photo Gallery or other image program

23. Using the magnifier, expand the photo Using a mm ruler, measure from the edge of the gel disk to the inner most edge of the diffusion for each color.

24. Calculate the diffusion distances for each dye and for each time period: • --Gel diameter measurement (mm) on the computer screen/65 mm = multiplier. • --Gel diffusion distance (mm) on screen x multiplier = actual distance. • Record calculated diffusion distances for each color and time period in a data table or spread sheet.

25. Calculate Mean Percentage of Diffusion For the last time period measured and for each color of dye, calculate and record the mean percentage of diffusion Use: total distance traveled by dye in mm / 32.5 x 100 = ________% Record the mean percentage of diffusion for each color in your data table or spread sheet

26. Diffusion Analysis Method 3: Using ADI (Analyzing Digital Images Software • Download DEW software from: http://umassk12.net/adi/ • Click on Analyzing Digital Images

28. Open a picture, then trim the photo to increase processing time

29. Click on the drop down menu

30. Choose Full Image at Selected Resolution Then click on trim and use image

31. Choose this option

32. Draw a line across the diagonal of the petri dish Record petri dish diameter and units Then, click done

33. Select line tool option

34. Click on the blue and red adjustment tools to help you place the blue and red dots at The beginning and end of the line Draw a line from the edge of the gel to where the diffusion of dye molecules appears to end Note length of line Zoom in to see diffusion line and edge of gel more clearly

35. QUALITATIVE OBSERVATION OF DIFFUSION You can also use ADI software to see a qualitative graph of the diffusion of the yellow dye molecules at a particular time. You can compare the qualitative graph with the quantitative measurements. A qualitative graph also helps to see that diffusion is a dynamic process with a trend in movement but no clear end point.

36. Draw a line across the Gel going through the diagonal Choose line tool option

37. Choose graph colors option

38. This graph shows the intensities of red, green and blue pixels along the line drawn across the gel. Notice that around 20/100 the lines level off, indicating edge of diffusion

39. If you turn off all colors but green, you can more easily see that around both 20 and 80 is where the diffusion of the dye molecules tapers off. So, diffusion of the yellow dye particles at this time interval is about 20/100, or .20. Compare this with 1.09 (diffusion distance)/6.03 (gel diameter) = .18

40. Questions to consider • Which dyes diffused the fastest? • Does fast diffusion mean greater or poorer retention? • How could diffusion and retention be optimized? This is an important consideration for the delivery of nanoscale medication

41. Molecular Weights of The Food Colorings • Red 40 • Molecular Formula: C18H14N2Na2O8S2 Molecular Weight: 496.421900 [g/mol]

42. Red 3 • Molecular Formula: C20H6I4Na2O5 Molecular Weight: 879.856060 [g/mol]

43. Yellow 5 • Molecular Formula: C16H9N4Na3O9S2 Molecular Weight: 534.363370 [g/mol

44. Blue 1 • Molecular Formula: C37H34N2Na2O9S3 Molecular Weight: 792.848400 [g/mol]