Vasanth
V. Balasubramanian, and B. Moudgil
Microbial Adhesion and Removal from Bio‐Surfaces
‐ Adsorption Density of Microbes on Flat Surfaces ‐
Center for Particulate and Surfactant Systems (CPaSS)Fall 2019 IAB Meeting
Columbia University, New York, NYAugust 6‐7, 2019
Overview of the PresentationResearch Team:Vasanthakumar Balasubramanian (Vasanth), Brij Moudgil, J. Carnali, C. Walsh, and P. Chandar. Goal: Establish techniques for adsorption density measurements on lowsurface area surfaces
Industrial Relevance: Unilever, Ecolab, Colgate‐Palmolive, Johnson & Johnson, P&G etc…
In this presentation
Microbes as sensors for determining polymer adsorption density on Vitroskin
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Technical Information:Cell viability can be used as a detection parameter. For a given polymer, cell viability depends on free polymer concentration in solution (indirect Г (adsorption density) measurement). Fluorescence‐based protocol may also be used for enumeration of viable microbes on surfaces and in solution (direct Г measurement).
Desorption
Addition of microbes to supernatant Enumeration
Adsorption of microbes
Microbesas sensors
Current Scenario… 3
High Specific Surface Area Systems (e.g., fine particles)1. Indirect (depletion) method – UV absorption, fluorescence, colorimetric methods, radioisotope‐labeled fluorescently labeled molecules, TOC
2. Direct method – Radiolabeling, Fluorescence
Low Specific Surface Area Systems (flat surfaces)1. Indirect (depletion) method ‐ requires new approaches.2. Direct method – Radiolabeling, Fluorescence, SPR, OWLS, DPI, ATR‐FTIR, SE, QCM‐D, AFM
Promising techniques ‐measurements on biosurfaces‐ QCM ‐ AFM‐ Microbes as sensors
Background: Microbial removal from surfaces using engineered particlesdemonstrated.
Adsorption density measurements required to develop quantitativecorrelations.
4Quartz Crystal Microbalance (QCM)
The change in QCM frequencydetermines the mass of analyteadsorbed in ng/cm2.Sauerbrey equation
Microbes immobilized using antibodies/ DNA/protein as bioreceptor (Quartz surface)
Bacillus thuringiensis 107 CFU – 60mg/cm2
Bacillus anthracis 107 CFU – 6mg/cm2
E.coli 108 CFU ‐ 10μg/cm2
Δm=−C′Δf
QCM sensing system
Frontiers in Bioengineering and Biotechnology (2018) Vol. 6 p‐158
5Atomic Force Microscopy (AFM)
Atomic force microscopy (AFM) was used to determine the adhesion forcesbetween bacteria and substrate in water and to gain insight into the nano‐scalesurface morphology. Rastering ‐ Tapping mode
Parameters E. coliCell length/μm 2.3 ± 0.5
Cell width/μm 1.3 ± 0.1Cell height/nm 237 ± 28
Cell envelop thickness/nm 22 ± 4
Pilithickness/nm 4.8 ± 0.8
Surface topographies of microbes on the substrate can be measured in tapping mode. Г calculated from thickness and surface coverage.
Scientific Reports volume5, 16857 (2015)
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Part ‐ I Microbes as sensors for determining polymeradsorption density on Vitroskin
Promising techniques ‐measurements on biosurfaces‐ QCM ‐ AFM‐ Microbes as sensors
7Illustrative example: Cationic polymer adsorption density on Vitroskin using E.coli as a biosensor
Initial polymer concentration
(ppm)
Final polymerConcentration
(ppm)
Adsorption density on VitroskinμM/cm2
(Depletion method)
5 3 20 (not reliable)10 ~6 40
Sensitivity: Viable cell detection limit > 104/mlReliability of viable cell measurement: ± 0.5 log
CPaSS Approach:‐ Cell viability used as a detection parameter.‐ Agar Plate method to determine cell viability.‐ For a given polymer, cell viability depends on free polymer concentration in solution.
Polymer adsorption density on Vitroskin can be determined based on cell viability measurements.
Calibration curve
Reliable region
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Part – II CPaSS approach: Fluorescence‐based protocolfor enumeration of viable microbes on surfaces and insolution.
(invention disclosure submitted)
CPaSS Resazurin Fluorescence Dye Assay
PrestoBlue® uses the metabolically active resazurin, which is weakly fluorescent and is irreversibly reduced to highly fluorescent resorufin.
Resazurin and resorufin freely diffuse through cell membranes, so readings are taken from the bulk solution.
Resorufin Excitation and EmissionPrestoBlue® Mechanism
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• Potential for utilization as a reporter for enumeration from surfaces.
NAD(P)H
NAD(P)H
Non‐Fluorescent Fluorescent
Non‐Fluorescent
Data Extraction
0E+0
2E+5
4E+5
6E+5
8E+5
1E+6
1E+6
1E+6
0:00:00 2:24:00 4:48:00 7:12:00 9:36:00 12:00:00 14:24:00 16:48:00
Fluorescence (AU)
Time (HH:MM:SS)
Building a Calibration Curve
LOG7 LOG6
LOG5
LOG4
LOG3
0
100
200
300
400
500
600
1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 1.E+08
Time to Peak (min)
Initial Inoculum Size (# of cells)
Time to Peak vs. Initial Inoculum Size
Since the independent variable isinoculum size, the dependentvariable becomes lag time.
Each curve represents a different initial inoculum size.
A unique lag phase exists for each inoculum size. By plotting the lag time, a LOG – linear curve is generated.
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Advantages: Lower cell count limit extended due to higher fluorescence sensitivity‐ Reliable up to 1E+2 as compared to Agar plate method (1E+4)
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y = ‐42.92ln(x) + 757.58R² = 0.93
y = ‐48.75ln(x) + 902.27R² = 0.88
0
100
200
300
400
500
600
700
800
900
1E+2 1E+3 1E+4 1E+5 1E+6 1E+7 1E+8
Time to Peak (min)
Initital Inoculum Size (# of cells)
E. coli Calibration Curves
Log. (in solution)Log. (on vitroskin)Solution Inoculation (n=8)
Vitro‐Skin® Inoculation (n=12)
E.coli calibration curves in solution and at Vitroskin
Initial inoculationCFU/cm2
Adsorption density on VitroskinCFU/cm2 pg/cm2
1E+4 1E+3.2 11E+5 1E+3.8 11E+6 1E+4.7 10
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Microbes as sensors is a promising approach for adsorption
measurements (depletion as well as direct methods).
QCM and AFM may be used for direct adsorption density
measurements. (QCM ‐ limitation of substrates).
Viability of AFM and QCM for microbial adsorption density need
to be established.
Summary and Future work
Challenges in the adsorption of biomolecules on solid surfaces:
very small amounts of adsorbed material to be detected
inherent difficulties to sense at an interface without interference from
the solution.
concomitance of several molecular phenomena (adsorption, desorption,
conformational changes, and rearrangements)
Thank you Acknowledgment
This material is based upon work supported by the National Science Foundationunder Grant No. 1362060 and by CPaSS industry members.
Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the
National Science Foundation/Sponsors.
Disclaimer
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