Optimization of Coagulation Process by Evaluating Adhesion Forces using Atomic Force Microscopy
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Transcript of Optimization of Coagulation Process by Evaluating Adhesion Forces using Atomic Force Microscopy
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Optimization of Coagulation process by Evaluating Adhesion forces using Atomic Force Microscopy
Ajay Kashi1, Anneta Razatos2 and Morteza Abbaszadegan1
1Department of Civil & Environmental Engineering2Department of Chemical and Materials Engineering
UAS
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Outline
Objectives Introduction to AFM force measurements Experimental Results and Discussion Conclusion and future work
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Objectives
Use Atomic Force microscopy (AFM) to directly measure the forces of interaction between biological and inorganic colloidal particles during coagulation.
Correlate force measurements to 1) real time coagulation studies 2)Size and shape of the particles.
Develop basic understanding of the interaction forces to Evaluate Bacterial Adhesion during Coagulation.
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Advantages of AFM Technique
Currently the only technique to measure interactions between bacteria and colloidal particles.
Sensitive enough to detect forces in the nN range.
All measurements are carried out in a physiological buffer solution.
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AC
DB
E
1) Line A
2) Line B
3) Line C
4) Line D
5) Lines E & F
AFM Force Measurement
A, B & C - Approach D & E - Retraction
Distance of Separation (nm)
Can
tilev
er D
efle
ctio
n (n
m)
Z
X
F
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Possible Configuration to Study Bacterial Adhesionby AFM
Planar surface(Glass Plate)
Bacteria
Cantilever with Silicon Nitride Tip
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MATERIALS AND METHODS
CELLS
+ GLUTARALDEHYDE
FIXED CELLS
POLYETHYLENEIMMINECOATED GLASS
POLYETHYLENEIMMINE
POLYETHYLENEIMMINE
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MODIFIED AFM CANTILEVERS
CELLS
+ GLUTARALDEHYDE
FIXED CELLS
POLYETHYLENEIMMINECoated Si3N4 TipsBacterial Lawn on Si3N4 Tip
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AFM Image of Immobilized E. coli and SEM Micrograph of AFM cantilever tip
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Lipid A
Glucose
KDO
Heptose
N-acetyl Glucosamine
Galactose
Legend
D21
LIPOPOLYSACCHARIDE STRUCTURE OF E. coli K-12
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Control ExperimentsBacteria-Bacteria interaction in PBS
-25
-20
-15
-10
-5
0
5
10
15
0 10 20 30 40 50 60 70 80
Relative Distance of Separation (nm)
Tip
Def
lect
ion
(n
m)
Approach
Retraction
Bacteria-Bacteria interaction in PBS + NaCl
-25
-20-15
-10-5
0
510
15
0 10 20 30 40 50 60 70 80
Relative Distance of Separation (nm)
Tip
Defl
ecti
on
(nm
)
Approach
Retraction
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Results and Discussion
Force Plot for Bacteria-Bacteria interaction in PBS & in PBS+NaCl
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0 10 20 30 40 50
Relative Distance of Separation (nm)
Fo
rce (
nN
)
PBS+NaCl
PBS only
Force, F = k x ΔX Spring Constant of Cantilever, k = 0.06nN/nM
ΔX = Tip Deflection for the Approach curve.
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Results and Discussion
-0.45 ± 0.02 -0.35 ± 0.06
Experiment in PBS+NaClExperiment in PBS only
E. coli bacteria on tip and on glass surface
E. coli bacteria on tip and on glass surface
Configuration
Experiment
Force Values in nN
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Bacteria-Bacteria Interactions in Different Concentrations of Alum + PBS
0 10 20 30 40 50 60 70 80
Relative Distance of Separation (nm)
Tip
De
fle
cti
on
s (
nm
)w
ith
5n
m o
ffs
ets
Approach
Retraction
Approach
Retraction
Approach Retraction
35nm
45nm
55nm
12mg/l
18mg/l
24mg/l
Experiment #2
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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)
241812Alum Conc. in (mg/l)
Force Plots for bacteria-bacteria Interaction in Various concentrations of Alum in PBS
-2.5
-2
-1.5
-1
-0.5
0
0.5
0 10 20 30 40 50 60
Relative Distance of Separation (nm)
Fo
rce (
nN
)
12 mg/l
18 mg/l
24 mg/l
Results and Discussion
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Conclusions
Control Studies (Experiments with 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.
The AFM-methodology makes it possible to optimize coagulation conditions by providing quantitative data (force versus distance of separation curves).
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Future Work
Microbes
Microbial Lawn
Other Microbial cells commonly found in water
1.
Inorganic Particles
Microbes
2.
Microbe-coated cantilever probing sediment-coated substrate
Sediment-coated cantilever probing sediment-coated substrate
Inorganic Particle
Inorganic Particles
3.
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Control Experiments in PBS
It is a Physiological Buffer Solution. So the working environment (medium) can be varied without causing much stress to the cells.
Any bacteria in PBS is in Isotonic Conditions ( Bacteria is not under any stress due to Osmotic pressure conditions)