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

Outline

Objectives Introduction to AFM force measurements Experimental Results and Discussion Conclusion and future work

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.

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.

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

Possible Configuration to Study Bacterial Adhesionby AFM

Planar surface(Glass Plate)

Bacteria

Cantilever with Silicon Nitride Tip

MATERIALS AND METHODS

CELLS

+ GLUTARALDEHYDE

FIXED CELLS

POLYETHYLENEIMMINECOATED GLASS

POLYETHYLENEIMMINE

POLYETHYLENEIMMINE

MODIFIED AFM CANTILEVERS

CELLS

+ GLUTARALDEHYDE

FIXED CELLS

POLYETHYLENEIMMINECoated Si3N4 TipsBacterial Lawn on Si3N4 Tip

AFM Image of Immobilized E. coli and SEM Micrograph of AFM cantilever tip

Lipid A

Glucose

KDO

Heptose

N-acetyl Glucosamine

Galactose

Legend

D21

LIPOPOLYSACCHARIDE STRUCTURE OF E. coli K-12

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

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.

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

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

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

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).

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.

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)