1 The Use of the Atomic Force Microscope to Study Biological Membranes Brandy Perkins Materials Science Engineering REU Program Summer 2001 Purdue University

Introduction Conducted research in the laboratory of Professor R.P. Andres Conducted research in the laboratory of Professor R.P. Andres Carbon nanotubes and commercial cantilevers Carbon nanotubes and commercial cantilevers Goal Goal  To be able to use a carbon nanotube enhanced probe to look at a cell membrane through an atomic force microscope (AFM)

Topics of Discussion What is the Atomic Force Microscope What is the Atomic Force Microscope What are carbon nanotubes and how do we use them What are carbon nanotubes and how do we use them Obtaining tapping mode images Obtaining tapping mode images Derivatizing the tips of carbon nanotubes Derivatizing the tips of carbon nanotubes The big picture of the research The big picture of the research Next step ! Next step ! My experiences and accomplishments My experiences and accomplishments Acknowledgements Acknowledgements

What is the Atomic Force Microscope ? The Atomic Force Microscope (AFM) is an amazing instrument presently being used to solve problems in the areas of electronics, telecommunications, biomedical, chemical, automotive,aerospace, and energy industries. The Atomic Force Microscope (AFM) is an amazing instrument presently being used to solve problems in the areas of electronics, telecommunications, biomedical, chemical, automotive,aerospace, and energy industries. Some of the materials it can work with include ceramics, glasses, synthetic and biological membranes, and semiconductors. Some of the materials it can work with include ceramics, glasses, synthetic and biological membranes, and semiconductors.

The AFM Family Tree Lateral Force Microscope (LFM) Magnetic Force Microscope (MFM) Atomic Force Microscope (AFM) Scanning Tunneling Microscope (STM) Scanning Probe Microscope

Technically Speaking Can examine any rigid surface either in air or with the specimen immersed in a liquid Can examine any rigid surface either in air or with the specimen immersed in a liquid Can resolve single atoms Can resolve single atoms Can be used to examine rough surfaces Can be used to examine rough surfaces By using different probes and scanning modes one can find the spot their looking for within 1 micron By using different probes and scanning modes one can find the spot their looking for within 1 micron Can produce 3-D images and quantitative data analysis integrated and interpreted in the context of your problem Can produce 3-D images and quantitative data analysis integrated and interpreted in the context of your problem  feature size, surface roughness and area, cross-section plots With a custom adapter attached, SEM tubes micro-toned blocks, metallurgical mounts, and other odd shapes and sizes can be accommodated  Up to 1.5” thick and 42”wide The location of interest can be quickly found and documented using the built-in optical microscope  with a magnification up to 2000X

Major Modes of Operation 1.Contact Mode The tip scans the sample in close contact with the surfaceThe tip scans the sample in close contact with the surface PROBLEMPROBLEM 2.Non-Contact Mode The tip doesn’t touch the surface of the sampleThe tip doesn’t touch the surface of the sample Oscillates A above the sample surfaceOscillates A above the sample surface PROBLEMPROBLEM 3.Tapping Mode A vertically oscillating tip is placed in contact with the surface and then liftedA vertically oscillating tip is placed in contact with the surface and then lifted Oscillates at ,000 taps per secondOscillates at ,000 taps per second

Cantilevers vs. Carbon Nanotubes A critical component to the AFM A critical component to the AFM Determines the force applied to the sample Determines the force applied to the sample Fabricated from silicon Fabricated from silicon Size Size  100 – 200 microns long  10 – 40 microns wide  0.3 – 2 microns thick Why do we use carbon nanotube probes ? Why do we use carbon nanotube probes ?  They are long and slender  They buckle  Has a small-end diameter  Have versatile carboxylic acid tips  Amine groups

Imaging Forces between the tip and the sample cause the cantilever to bend and deflect Forces between the tip and the sample cause the cantilever to bend and deflect By way of detector we are able to measure the change in amplitude and derive a topographical map of the sample surface By way of detector we are able to measure the change in amplitude and derive a topographical map of the sample surface Signals sent by the detector to the computer Signals sent by the detector to the computer Height images Height images  Based on changes in a constant height mode Phase images Phase images  The difference in phase between the driving amplitude and the tip oscillation

I can Prove it ! Hypothesis Hypothesis  Carbon nanotubes can enhance imaging done with commercial cantilevers in the tapping mode of an AFM Control Control  Stiff Cantilevers without carbon nanotubes attached Experiment Experiment  Use a stiff cantilever to image a silicon sample without a carbon nanotube attached  Use a stiff cantilever to image a silicon sample with a nanotube attached Result Result  Nanotubes do enhance images taken using a stiff cantilever in the tapping mode of an Atomic Force Microscope

Stiff Cantilever Without a Nanotube

Stiff Cantilever With a Nanotube

Soft Cantilever Without a Nanotube Future research Future research  Obtain an image with a soft cantilever with a carbon nanotube attached

Derivatization Problem Problem  Carboxylic tips rupturing the surface of living cell membranes Hypothesis Hypothesis  chemically change the molecules at the tips of the carbon nanotubes Experiment Experiment  Using the AFM and a benzyl amine solution  Using apparatus to maintain a bubble of water and a dodecylamine solution  The cantilever and a jar of solution  Using the optical bench and a dodecylamine solution Possible Result Possible Result  One could possibly probe the surface without rupturing the surface carboxylic tip

Recipe 1 st solution 1 st solution  EDC  1-ethyl-3-(3- dimethylaminopropyl) carbodimidehydrochloride  Etherbenzylamine  MES  2-[N-morpholine] ethanesulphonic acid ethanesulphonic acid 2 nd solution  EDC  1-ethyl-3-(3- dimethylaminopropyl) carbodimidehydrochloride  MES  2-[N-morpholine] ethanesulphonic acid  Docecylamine

Separatory Funnel Experiment Never got to the nanotube and cantilever stage Never got to the nanotube and cantilever stage Placed the solution in a separatory funnel Placed the solution in a separatory funnel The solution ran out of the funnel and through a foot of opaque tubing and a glass pipette The solution ran out of the funnel and through a foot of opaque tubing and a glass pipette The bubble was maintain at the tip of the pipette The bubble was maintain at the tip of the pipette Results Results  Was able to maintain a stable bubble with an ethanol and water mixture  Was not able to maintain a stable bubble with the dodecylamine mixture Conclusion Conclusion  Would not allow a nanotube to be chemically changed Funnel Tubing Pipette Bubble

The Jar Theory Used a shortened nanotube Used a shortened nanotube Filled a jar semi full of solution Filled a jar semi full of solution Placed a cantilever on an alligator clip supported by a thick layer of foil Placed a cantilever on an alligator clip supported by a thick layer of foil Methods Methods  Slowly lowered the cantilever into the solution  Used a glass pipette to bring the solution up to the cantilever Results Results  Nanotubes were lost

Using the Optical Microscope Modifications Modifications  Gold cantilever, shortened nanotube Placed a stiff cantilever with a nanotube on a piece of tape in an alligator clip in the optical bench Placed a stiff cantilever with a nanotube on a piece of tape in an alligator clip in the optical bench Hung over a glass slide containing dodeclyamine solution Hung over a glass slide containing dodeclyamine solution Cantilever slowly lowered into the solution Cantilever slowly lowered into the solution Results Results  Test the time – 5 sec.,1 min.,15 min., 30 min.,1 hour 30 min.,1 hour Conclusion Conclusion  Could be used as a method to derivatize the tips of carbon nanotubes

The Big Picture If we are able to successfully probe a membrane surface with a nanotube then possibly we can configure a map of the proteins located on it. If we are able to successfully probe a membrane surface with a nanotube then possibly we can configure a map of the proteins located on it. Developments in this area could lead to advancements in Cancer research. Developments in this area could lead to advancements in Cancer research.

Next Step ! Continue to use carbon nanotubes as a modification for probing sample surfaces Continue to use carbon nanotubes as a modification for probing sample surfaces To probe the surface of a bilayer membrane To probe the surface of a bilayer membrane To probe the surface of a living cell membrane To probe the surface of a living cell membrane

My Experience and Accomplishments Able to operate an Atomic Force Microscope in tapping mode Able to operate an Atomic Force Microscope in tapping mode Able to attach carbon nanotubes to hydrophobic gold plated tapping and contact cantilevers Able to attach carbon nanotubes to hydrophobic gold plated tapping and contact cantilevers Obtained images from stiff cantilevers with and without carbon nanotubes attached Obtained images from stiff cantilevers with and without carbon nanotubes attached Furthered proved that with a carbon nanotube one can obtain clearer images Furthered proved that with a carbon nanotube one can obtain clearer images Developed a possible method for Derivatizing carbon nanotube tips Developed a possible method for Derivatizing carbon nanotube tips

Acknowledgements Luis Roman Luis Roman Venu Santhanam Venu Santhanam Professor R.P.Andres Professor R.P.Andres Dr. E. Slamovich Dr. E. Slamovich The cluster group The cluster group Purdue University Purdue University