Adhesion Forces during Coagulation as Evaluated by Atomic Force Microscopy
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Presented at the American Society of Microbiology, Washington D.C., May 2003
![Adhesion Forces during Coagulation as Evaluated by Atomic Force Microscopy Ajay Kashi 1 , Anneta Razatos 2 , Absar Alum 3 and Morteza Abbaszadegan 4 1 Graduate Student, Department of Civil and Environmental Engineering, Arizona State University, Tempe, AZ 2 Associate Professor, Department of Chemical and Materials Engineering, Arizona State University, Tempe, AZ 3 Faculty Research Associate, Department of Civil and Environmental Engineering, Arizona State University, Tempe, AZ 4 Associate Professor, Department of Civil and Environmental Engineering and Director, National Science Foundation Water Quality Center, Arizona State University, Tempe, AZ ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],N-210 Contact Information Ajay Kashi 1 , Anneta Razatos 2 and Morteza Abbaszadegan 3 1 Department of Civil and Environmental Engineering Arizona State University, Tempe, AZ 85287-5306 E-mail: ajay.kashi@asu.edu, Phone: (480) 965-7978 2 Department of Chemical and Materials Engineering Arizona State University, Tempe, AZ 85287-6006 E-mail: anneta.razatos@asu.edu, Phone: (480) 965-0874 3 Department of Civil and Environmental Engineering Arizona State University, Tempe, AZ 85287-5306 E-mail: abbaszadegan@asu.edu, Phone: (480) 965-3868 MATERIALS AND METHOD Figure 4 . Immobilization of cells on a planar surface. POLYETHYLENEIMMINE Coated Tip Bacterial Lawn on Tip CELLS + GLUTARALDEHYDE FIXED CELLS Figure 5 . Immobilization of cells on the cantilever tip. POLYETHYLENEIMMINE CELLS + GLUTARALDEHYDE FIXED CELLS COATED GLASS POLYETHYLENEIMMINE Planar surface (Glass Plate) Bacteria Cantilever with Silicon Nitride Tip Figure 3 . Configuration to study Bacterial Adhesion using AFM. Laser Photodiode Detector Cantilever with Silicon Nitride Tip Planar Surface Figure 1 . Key Elements of Atomic Force Microscope C D Tip Deflection vs. Separation A B C D E Approach Retraction E A A. No Interaction B B. Attractive Interaction E. Pull-Off Force due to binding C. Constant Compliance Region D. Binding During Retraction Figure 2 . Operation of AFM in Force Mode. Substrate is advanced and then retracted away from stationary AFM cantilever. Cantilever deflection is recorded as a function of distance of separation resulting in approach and retraction curves. Figure 6. AFM Image of Immobilized E. coli on a planar surface in a liquid medium. Figure 7. SEM Micrographs of a clean AFM cantilever tip and immobilized E. coli on a cantilever tip. Cantilever length:- 100 - 200 μ m Nominal tip Radius of curvature:- 20 to 60nm As a control, bacteria-coated planar surface are imaged by the AFM and bacteria-coated cantilevers are imaged by scanning electron microscopy (SEM) following every AFM force measurement to ensure the presence of confluent bacterial cell lawns on the surface and cantilever tips following throughout the measurement. EXPERIMENTS Control experiment was conducted to determine bacteria-bacteria interactions in a physiological buffer solution. During the AFM operation, as the surface approached the cantilever, the cantilever deflected towards the surface when the relative distance of separation was about 30nm. During retraction, the cantilever deflected due to binding forces between bacteria on tip and on surface. These deflection curves during approach and retraction indicate an attractive interaction between bacterial cells, which may be due to van der waals forces (weak adhesive forces). Another control experiment was conducted to determine bacteria-bacteria interactions in an electrolyte (NaCl) plus buffer solution. During AFM operation, the cantilever deflected towards the surface when the relative distance of separation was about 70nm. The binding force was also greater compared to the previous experiment, as indicated by the retraction curves. So from the deflection curves, it is clear that the addition of an electrolyte (NaCl) reduces repulsive electrostatic interactions between bacterial cells and hence attractive interactions dominate. Approach Retraction Approach Retraction Approach Retraction 35nm 45nm 55nm 12mg/l 18mg/l 24mg/l Bacteria-Bacteria interactions were determined in different concentrations of Alum in a buffer solution. For 12mg/l of alum, during the AFM operation, as the surface approached the tip, the cantilever deflected towards the surface when the relative distance of separation between bacterial cells was about 35nm. But with 18mg/l and 24 mg/l of alum, the cantilever deflected when the relative distance of separation was about 45nm and 55nm respectively. So, the approach curves indicate that as the concentration of alum is increased, attractive interactions between bacterial cells are taking place at a greater distance of separation between them. Even during the retraction, cantilever deflections are increasing due to greater binding forces between bacterial cells with the increase in alum concentration. Hence the addition of alum increases bacterial adhesion due to greater attractive interactions between them. CONCLUSIONS Control studies (Experiments with PBS and NaCl) demonstrate that physiochemical interactions play a dominant role in bacterial adhesion. Alum coagulant reduces repulsive electrostatic interactions such that attractive forces (primarily van der waals) become stronger over greater distance of separation. AFM-methodology makes it possible to optimize coagulation conditions by providing quantitative data (force versus distance of separation curves. Microbes Microbial Lawn Other Microbial cells commonly found in water 1. Inorganic Particles Microbes 3. Microbe-coated cantilever probing sediment-coated substrate Sediment-coated cantilever probing sediment-coated substrate Inorganic Particle Inorganic Particles 2. FUTURE WORK ACKNOWLEDGEMENTS Funding Agency - National Science Foundation Water Quality Center, Arizona State University, Tempe, Arizona. Post Doctoral Research Associate - Dr. Laura Palmer Students - Jay Schwartz (Undergraduate) and Rong Kou (Doctoral) RESULTS AND DISCUSSION Force values for Bacteria – Bacteria Interaction in Different Concentrations of Alum -1.77 ± 0.2 -0.77 ± 0.02 -0.70 ± 0.06 Force in (nN) 24 18 12 Alum Conc. in (mg/l) The force values for cells interaction in different concentrations of alum demonstrates that alum coagulant reduces repulsive electrostatic interactions such that attractive interactions dominate over greater distance of separation between them. Force, F = K * Δ X, Where, K = 0.06nN/nM (Spring Constant) & Δ X = Tip Deflection for Approach curve in nm. Force values for Bacteria-Bacteria interaction in PBS and in PBS + NaCl. Experiment in PBS only Experiment in PBS + NaCl -0.35 ±0.06nN -0.45 ±0.02nN The Negative sign indicates downward deflection of the cantilever due to attractive interactions between bacteria on the AFM Cantilever tip and bacteria on the planar surface. The deflection curves during the approach for both the experiments were converted in to force curves. For cells interaction in PBS, the force value was 0.35nN whereas in PBS plus NaCl it was 0.45nN. These force values indicate that physiochemical forces such as van der waals forces, attractive and repulsive electrostatic interactions play a dominant role in bacterial adhesion.](https://image.slidesharecdn.com/asmposterpresentation-12743356206064-phpapp01/85/Adhesion-Forces-during-Coagulation-as-Evaluated-by-Atomic-Force-Microscopy-1-320.jpg)
Presented at the American Society of Microbiology, Washington D.C., May 2003
