(SERS) cellular imaging. biosynthesized gold. Surface-enhanced Raman spectroscopy. nanoparticles. of intracellularly
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1 Surface-enhanced Raman spectroscopy (SERS) cellular imaging of intracellularly biosynthesized gold nanoparticles Rebecca Halvorson Lahr Peter Vikesland Virginia Tech Civil and Environmental Engineering SNO, November 5, 2013
2 Research motives for mechanistic study of AuNP biosynthesis Optimize processes through mechanistic understanding Green nanoparticle synthesis Metal recovery from aqueous wastes SERS cellular imaging Pseudokirchneriella subcapitata How do algae biosynthesize AuNP? Why do algae biosynthesize AuNP? Mechanism proposed by Focsan, M., et al Nanotechnology, 22, Can we control the size, shape, and production of biosynthesized AuNP?
3 Experimental setup #3) Characterize with UV-Vis spectroscopy ESEM, EDX, TEM cross sectioning, SERS spectral mapping #2) Incubate under light on shaker table at 23 C Algae mm HAuCl 4 Algae mm HAuCl 4 UV-Vis 4 μm ESEM 200 ml 150 ml 100 ml 50 ml Au3+ Au 3+ Au 3+ #1) Mix algae, OECD/OCDE media, and HAuCl 4 1 μm TEM 260 cm -1 peak map Raman spectroscopy Pseudokirchneriella subcapitata
4 Raman spectra record unique molecular vibrations for any polarizable analyte Halvorson & Vikesland 2010
5 Raman Intensity Raman Intensity Raman Surface-enhanced spectra record Raman unique spectroscopy molecular (SERS) vibrations detects for molecular any polarizable vibrations of analyte molecules that interact with AuNP Raman signal is enhanced by when a laser interacts with SPR of nanoscale Au or Ag AuNP AuNP nm AuNP + 5 μg/l MC-RR -- 5 μg/l MC-RR Halvorson & Vikesland 2010 Raman shift (cm -1 ) Raman shift (cm -1 )
6 SERS Intensity AuNP biosynthesized by algae are surface enhanced Raman spectroscopy (SERS) active Nanoparticle sizes from ESEM and TEM Spheres nm, plentiful aggregates 600 nm Spheres, polygons, rods, cones nm, less aggregates Spheres 20 nm, eggs 200 nm 40 nm, very few aggregates No particles
7 SERS Intensity AuNP biosynthesized by algae are surface enhanced Raman spectroscopy (SERS) active Au(0)-Cl - vibrations Optical Image: Algae mm HAuCl 4 Surface 9 μm 0.3 μm deep 0.6 μm deep 7560 spectra transformed into a single image for each depth Compiled SERS spectral maps tracking intensity of entire spectrum through x-y-z of algae 0.9 μm deep 1.2 μm deep
8 Data reduction using principal component analysis (PCA) and cluster analysis Percent of Variability in Data Explained (%) SERS intensity Cluster analysis partitions spectra into a predefined number of groups of similar spectra PCA highlights variation in the data, rather than the features that remain constant Reduce 441 spectra to 1 spectrum that highlights ~90% of variability across the cell Principal Component (number) Raman Shift (cm -1 ) PC1 Loadings Most Intense Cluster
9 Absolute Raman Intensity Cluster analysis demonstrates the intracellular nature of biosynthesized AuNP Wavenumber (cm -1 ) False color image Colors match spectra RNA Glutathione Nitrate reductase NAD+ Hydroxyquinoline Chlorophyll a & carotenoids Glutaraldehyde (fixing agent) Saccharides Primary metabolites Ascorbic acid, riboflavin Amino acids DNA
10 Absolute Raman Intensity Cluster analysis demonstrates the intracellular nature of biosynthesized AuNP Au(0) Cl,C,N, or S Key RNA False color image Colors match spectra Au(0) CN - Occurs in SERS hotspots Wavenumber (cm -1 )
11 Absolute Raman Intensity Cluster analysis demonstrates the intracellular nature of biosynthesized AuNP Key Glutathione Wavenumber (cm -1 ) False color image Colors match spectra Au(0) CN - H Protects against oxidative stress Precursor to metal binding peptide phytochelatin
12 Absolute Raman Intensity Cluster analysis demonstrates the intracellular nature of biosynthesized AuNP Au(0) Cl,C,N, or S Key Glutathione Nitrate reductase NAD+ Hydroxyquinoline False color image Colors match spectra Au(0) CN Wavenumber (cm -1 ) Mechanism for fungus proposed by Duran 2005, demonstrated in situ by Kumar 2007
13 Absolute Raman Intensity Cluster analysis demonstrates the intracellular nature of biosynthesized AuNP Au(0) Cl,C,N, or S Key Chlorophyll a & carotenoids False color image Colors match spectra Mechanism for cyanobacteria proposed by Focsan et al Au(0) CN Wavenumber (cm -1 )
14 Cluster analysis demonstrates spectral differences specific to each condition Chlorophyll a & carotenoids
15 Experimental data correlates well with hypothesized mechanisms Cyanobacteria (Synechocystis sp. PCC 6803) proposed by Focsan et al Nanotechnology. Importance of glutathione as an intracellular nanoparticle coating Fungus (Rhizopus oryzae) proposed by Das et al ACS Nano Fungus (Fusarium oxysporum) proposed by Duran et al Journal of Nanobiotechnology
16 Conclusions and future outlook SERS spectral imaging provides locations, sizes, and local molecular environments of noble metal nanostructures within transparent or translucent matrixes 3D SERS cellular imaging requires PCA or cluster analysis to quickly infer the most important aspects of the large datasets Understanding the interactions between AuNP and their local environment will improve overall understanding of these systems such that they may be engineered to best fit the needs of society H 0.15 mm HAuCl mm HAuCl 4
17 Thank you! Dr. Peter Vikesland Kathy Lowe Steve McCartney Matt Chan Weinan Leng Matt Hull Algae mm HAuCl 4 9 μm
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21 Principal component analysis (PCA) Creates a new set of variables known as principal components (PCs) PCs highlight the variation in the data set, rather than features that remain constant Each PC is a unique linear combination of the original variables PCs are arranged in order of significance Examples Jarvis 2004 SERS to distinguish between E. coli and B. subtilis Used 2 PCs to differentiate Jarvis 2006 SERS to distinguish between 9 strains of Bacillus Used 7 PCs to describe 98.7% of variance Pearman 2006 SERS to distinguish between 2 endospores, 2 chemical agent simulants, & 2 toxin simulants used 12 partial least squares discriminate analysis variables (similar to PCA) Guicheteau 2009 SERS and normal Raman to identify components of bacterial mixtures used 15 PCs to describe 90% of the variance
22 Cluster analysis demonstrates the intracellular nature of biosynthesized AuNP
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