Gel Diffusion Gel Diffusion ExperimentExperiment

STEM ED/CHMSTEM ED/CHM

Nanotechnology 2012Nanotechnology 2012

Presented by Jennifer Welborn Presented by Jennifer Welborn

Learning GoalsLearning Goals

In this activity, nanotech participants will:In this activity, nanotech participants will: See how food dyes and gelatin are See how food dyes and gelatin are

used to model the delivery of nanoscale used to model the delivery of nanoscale medicines to cells in the human bodymedicines to cells in the human body

Measure diffusion distances of 3 Measure diffusion distances of 3 different colors of food dye by: Eye, different colors of food dye by: Eye, photo image on a computer, ADI photo image on a computer, ADI software (Analyzing Digital Images)software (Analyzing Digital Images)

Diffusion and Teaching StandardsThis 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

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

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

Background For Lab ActivityBackground For Lab Activity

The delivery of nanoscale medicines The delivery of nanoscale medicines to cells in the human body requires to cells in the human body requires diffusion through tissues, organs and diffusion through tissues, organs and cell membranescell membranes

This activity will explore the affect of This activity will explore the affect of particle size on diffusion ratesparticle size on diffusion rates

Understanding molecular diffusion Understanding molecular diffusion through human tissues is important through human tissues is important for designing effective drug delivery for designing effective drug delivery systems systems

Background ContinuedBackground Continued

Measuring the diffusion of dyes in Measuring the diffusion of dyes in gelatin is a model for the transport of gelatin is a model for the transport of drugs in the extra-vascular space drugs in the extra-vascular space

Gelatin is a biological polymeric Gelatin is a biological polymeric material with similar properties to material with similar properties to the connective extracellular matrix in the connective extracellular matrix in tumor tissuetumor tissue

Dyes are similar in molecular weight Dyes are similar in molecular weight and transport properties to and transport properties to chemotherapeuticschemotherapeutics

Experiment OverviewExperiment Overview

The diffusion of the dyes will be The diffusion of the dyes will be compared to model the effect of compared to model the effect of molecular weight on movement of molecular weight on movement of molecules in tumors molecules in tumors

Gelatin will be cut into cylindrical disks, Gelatin will be cut into cylindrical disks, placed in petri dishes and colored placed in petri dishes and colored solutions will be added to the outer ringsolutions will be added to the outer ring

The distance that the dye particles The distance that the dye particles penetrate into the gelatin disks will be penetrate into the gelatin disks will be measured over timemeasured over time

Lab PrepLab Prep

Collect materialsCollect materials– Petri DishesPetri Dishes– Food DyeFood Dye– Syringes/10 ml Syringes/10 ml

graduated cylindersgraduated cylinders– Paper CupsPaper Cups– Plain GelatinPlain Gelatin– Crisco/Petroleum JellyCrisco/Petroleum Jelly– Baking PanBaking Pan– Biscuit cutterBiscuit cutter

Prepare Gel DisksPrepare Gel Disks– Determine amount of Determine amount of

water needed to fill up a water needed to fill up a pan to a depth of 1 cm. pan to a depth of 1 cm.

– Dissolve gel into cold Dissolve gel into cold water (2Pks/Cup/200 ml)water (2Pks/Cup/200 ml)

– Microwave for 90 Sec.Microwave for 90 Sec.– Pour into pan which has Pour into pan which has

been coated with been coated with petroleum jelly and let petroleum jelly and let set.set.

Lab ProcedureLab Procedure

Gel DisksGel Disks– Cut disks in pan Cut disks in pan

w/metal bisquit w/metal bisquit cuttercutter

– Remove excessRemove excess– Move gel disk from Move gel disk from

pan to petri dishpan to petri dish– Top side down!Top side down!– Centered as best Centered as best

cancan

Adding DyeAdding Dye– Mix dyes in cupsMix dyes in cups– Inject one Inject one

color/petri dishcolor/petri dish– No dye on top of gelNo dye on top of gel– No seepage under No seepage under

gelgel– Do not move dishes Do not move dishes

after dye insertedafter dye inserted– Note volume of dye Note volume of dye

addedadded

Important Details For ProcedureImportant Details For Procedure Make the red and yellow solutions fairly Make the red and yellow solutions fairly

concentrated. Blue should not be concentrated- it concentrated. Blue should not be concentrated- it should be strong, but translucent. should be strong, but translucent.

Inject dye towards the outside of the petri dish, Inject dye towards the outside of the petri dish, not towards the gel. not towards the gel.

Use an even number of milliliters for the volume Use an even number of milliliters for the volume of dye. of dye.

The initial level of dye should not exceed ¾ of the The initial level of dye should not exceed ¾ of the way (7 mm) up the gel. way (7 mm) up the gel.

Photograph the gel: same time, same distance, Photograph the gel: same time, same distance, same sequence. Keep camera parallel to gel (do same sequence. Keep camera parallel to gel (do not tilt) to avoid parallax. not tilt) to avoid parallax.

Data CollectionData Collection

Method 1-- By eyeMethod 1-- By eye: measure (in mm) : measure (in mm) the distance each dye has diffused for the distance each dye has diffused for each time interval. Record data in a each time interval. Record data in a data table or use excel spreadsheet data table or use excel spreadsheet

Method 2--Using a digital cameraMethod 2--Using a digital camera: take : take photos of each petri dish at the same photos of each petri dish at the same time each day, 8:45 and 4:45, from the time each day, 8:45 and 4:45, from the same height and angle same height and angle

Data CollectionData Collection3 Food Dyes3 Food Dyes

Start

4 hours

Diffusion is first visible

Gel Diffusion AnalysisGel Diffusion Analysis

Gel Diffusion AnalysisGel Diffusion AnalysisMethod 1: Determining Rate of Method 1: Determining Rate of

Diffusion by Eye Diffusion by Eye

– Use graph paper to plot distance (mm) Use graph paper to plot distance (mm) vs time (hours) for each color of dyevs time (hours) for each color of dye

– The rate is the slope of the line. During The rate is the slope of the line. During the relatively short diffusion time (as in the relatively short diffusion time (as in this lab), the relationship between this lab), the relationship between distance and time is somewhat linear. A distance and time is somewhat linear. A line of best fit may not have a y-line of best fit may not have a y-intercept of 0 due to error. intercept of 0 due to error.

Sample Graph of Diffusion Sample Graph of Diffusion Measurements Made by EyeMeasurements Made by Eye

y = 0.2057x + 1.0715

0

2

4

6

8

10

12

14

16

18

0 20 40 60 80

Time

Dis

tan

ce

(m

m)

Diffusion AnalysisDiffusion AnalysisMethod 2: Using a Digital CameraMethod 2: Using a Digital Camera

Group Pictures by Color in date/time Group Pictures by Color in date/time

orderorder

7-9-1600 7-10-0800 7-10-1600 7-11-0800

Pick one color to startPick one color to start Load the first morning shotLoad the first morning shot

– Windows Photo Gallery or other image Windows Photo Gallery or other image programprogram

Using the magnifier, expand the Using the magnifier, expand the photophoto

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

Calculate the diffusion distances for Calculate the diffusion distances for each dye and for each time period: each dye and for each time period:

--Gel diameter measurement (mm) on the --Gel diameter measurement (mm) on the computer screen/65 mm = multiplier. computer screen/65 mm = multiplier.

--Gel diffusion distance (mm) on screen x --Gel diffusion distance (mm) on screen x multiplier = actual distance.multiplier = actual distance.

Record calculated diffusion distances Record calculated diffusion distances for each color and time period in a for each color and time period in a data table or spread sheet. data table or spread sheet.

Sample Spread Sheet Entry

When finished, your table might When finished, your table might look something like thislook something like this

Create a graph by hand or in excel Create a graph by hand or in excel

Calculate Mean Percentage of Diffusion

For the last time period measured and for each colorof 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

Diffusion Analysis

Method 3: Using ADI (Analyzing Digital Images Software Download DEW software from: http://umassk12.net/adi/

Click on Analyzing Digital Images

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

Click on the drop down menu

Choose Full Image at Selected Resolution

Then click on trim and use image

Choose this option

Draw a line across the diagonal of the petri dish

Record petri dish diameter and

units

Then, clickdone

Select line tool option

Zoom in to see diffusion lineand edge of gel more clearly

Note length of line

Draw a line from the edgeof the gel to where the diffusion of dye molecules appears to end

Click on the blue and red adjustment tools to help you place the blue and red dots atThe beginning and end of the line

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.

Draw a line across theGel going through the diagonal

Choose line tool option

Choose graph colors option

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

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 tapersoff. 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

Questions to considerQuestions to consider

Which dyes diffused the fastest? Which dyes diffused the fastest? Does fast diffusion mean greater or Does fast diffusion mean greater or

poorer retention?poorer retention? How could diffusion and retention be How could diffusion and retention be

optimized? This is an important optimized? This is an important consideration for the delivery of consideration for the delivery of nanoscale medication nanoscale medication