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

5. Carbon dioxide from the environment diffuses into plant cells
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:
5:40-7:40 shows some of the uses of nano-scale particles in medicine.

9. Nanotechnology for Targeted Cancer Therapy
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. Lab Prep Collect materials Prepare Gel Disks 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.

12. Lab Procedure Gel Disks Adding Dye 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

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 Collection 3 Food Dyes
Start
4 hours
Diffusion is first visible

16. Gel Diffusion Analysis

17. Gel Diffusion Analysis Method 1: Determining Rate of Diffusion by Eye

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

21. Diffusion Analysis Method 2: Using a Digital Camera
Group Pictures by Color in date/time order

22. Load the first morning shot
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. Method 3: Using ADI (Analyzing Digital Images Software
Diffusion Analysis
Method 3: Using ADI (Analyzing Digital Images Software
Download DEW software from:
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. The beginning and end of the line
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. Gel going through the diagonal
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 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: [g/mol]

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

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

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