Nelson M. Anaya Dr. Vinka Craver Dr. Teresa Kirschiling Dr. Jason Killgore

Transcription

1 Nelson M. Anaya Dr. Vinka Craver Dr. Teresa Kirschiling Dr. Jason Killgore

2 Introduction Silver nanoparticles (AgNps) One the most common nanomaterial used: 25% nanoproducts include Aug 2009 [nanotechproject.org] Applications Solar energy Chemical catalysis Excellent antimicrobial ability: >99% (E. coli) Regulations EPA is regulating antimicrobial nanomaterials that may pose unanticipated environmental risks 2

3 Introduction Imaging Effect of AgNPs To in situ image and quantify the impact of the exposure of AgNPs on the morphological and structural properties of microorganisms. Tools available are Sondy et al., 2004 SEM TEM AFM Ru Li et al.,

4 Problem Nanoparticles Exposure Traditional nanoparticle exposure process using SEM & TEM includes harsh additional conditions Drying Shaking Centrifugation Vacuum Filtration AFM Features High resolution Feasibility to analyze live microbiological samples in aqueous and physiological relevant environments Quantify changes Hydrodynamic conditions Cost Analysis of the time and cost for instrumentation of microscope techniques 4

5 Objective Determine the feasibility to image and quantify in situ the impact of the exposure of AgNPs on the morphological and structural properties of Escherichia coli, using an AFM-liquid cell under dynamic fluid and real time conditions. This presentation will focus on preliminary results Imaging of bacteria immobilized during dynamic fluid conditions 5

6 Methods Procedure Experiment design 1. Can we detect changes on bacteria using AFM-Flow cell? Exposure to silver 0.1 mg/l 2. Is there any limitation to inject the AgNps? Exposure to different flow rates 3. Test Scenarios Dynamic conditions Buffer solution Citrate-silver nanoparticles Buffer solution Static conditions Overnight exposure COMSOL modeling Nanoparticles characterization Bacteria immobilization Fluid conditions AFM MFP-3D SEM images Microplate reader study -size -zeta potential -Ion release -On glass slide -Buffer -Silver nanoparticles -Buffer -Contact mode - Pyrex-nitride triangular cantilever (PNP-TR-20, NanoWorld -Overnight exposure -Comparison at the same conditions 6

7 Methods-Immobilization Poly-L-Lysine (PLL) Why PLL? PLL immobilized bacteria without exert mechanical stress Due electrostatic interactions between the surface coated positively with the polymer and bacteria charged negatively This method can be used on cells with different shapes and sizes Does not produce cell deformation Does not modify the chemical composition of cells immobilization effectiveness depends on the ionic strength of the buffer solution

8 Methods-AFM AFM AFM is a very highresolution (nm) type of scanning probe microscopy. Depending of the image mode method, AFM produces topography and mechanical information of living cells Operation of the atomic force microscope (AFM) A flow cell secures the probe and positions the cantilever tip on the surface to be imaged while maintaining an enclosed and sealed environment

9 Methods AFM-Flow cell Real arrangement Schematic arrangement Storage container AFM-flow cell+ Chamber Syringe pump

10 Results Immobilization *10 6 bacteria immobilized / cm bacteria immobilized by scan size (20μm*20μm) Is the bacteria still live after immobilization? 4. Live/dead experiments after immobilization during 2h Green: Live bacteria Red Dead bacteria 3. 4 random areas were scanned for sampling Nanoparticles characterization -size nm -zeta potential mv -Ion release 1% -Shape: spherical

11 Results (Can we detect changes? ) During buffer solution 2min During silver nitrate 23 min After silver nitrate 55 min Height B/exposure (μm) Height A/exposure (μm)

12 Results ( is there any limitation to inject the AgNps?) Scenarios Fluid conditions Buffer solution 0.02 M 1h (20mL/h) Citrate-silver nanoparticles 10 mg/l 10 min (60mL/h)

13 Results (nanoparticles exposure) During buffer solution 10min After nanoparticles 90min During buffer solution 30 min Height B/exposure (μm) Height A/exposure (μm)

14 Discussion Static experiments at the same conditions using live and dead experiments showed that most of the cell membrane is undisturbed even for high concentrations of AgNPs. Less interaction between nanoparticles and bacteria in comparison with static exposure techniques

15 Conclusions AFM can be an useful tool for visualizing morphological changes in real time on bacteria under fluid conditions. However no considerable changes were observed for the experimental conditions used in this study. Several conditions such as a flow rate and contact time must be studied before nanoparticle exposure experiments to get a correct performance of the AFM-flow cell

16 Future works Force-distance analysis to quantify morphological changes produced by the interaction between AgNps and bacteria

17 Acknowledgments

18 18

19 20 min after NPs introduced 7 PM Day 1 10 AM day 2

20 Results (Buffer solution Inj.)

21 Results-Before AgNps injection

22 Results-during AgNps injection

23 Results-after AgNps injection

24 Results- Overnight exposure

25 %Co Series time (minutes)

26 After Overnight exposure w/o 18h After Overnight 17h