Bacillus thuringiensis Detection and Characterization by Normal Raman and SERS
at logaritmic and stationary growth phases
State of the Art Challenges and Technology Transfer
This project was supported by the U.S. Department of Homeland Security under Award Number 2008-ST-061-ED0001. The views and conclusions contained in this document are
those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the U.S. Department of Homeland Security.
Based on the current status of world wide antiterrorism efforts there is a need to develop
effective standoff detection techniques for biological agents. Using spectroscopic techniques
the target of this study, Bt, will provide a molecular identification of the strand. These gram-
positive bacteria are recognized for their toxicity on larvae and are used commercially as
insecticides. B. thuringiensis was chosen due to its similarity with B. anthracis which has a
potential of being used during terrorist attacks. Both of these bacteria form spores which
are able to tolerate extreme environments and make them suitable for transport before or
during a biological attack.
Abstract
Conclusions
Spectroscopic techniques such as Normal Raman (NR) and Surface Enhanced Raman Spectroscopy (SERS)
are considered fast, in situ alternative methods for identification for microorganisms. These techniques
provide important information about the spectroscopic signatures of cellular components of in vitro or in vivo
organisms. The techniques have significant benefits for Industrial Microbiology, Food Microbiology and
biological warfare agents detection. The proposed method of this work is the use of vibrational Raman
techniques as NR and SERS and to detect bioaerosol particles of Bacillus thuringiensis (Bt) employing a fast
and simple synthesis of silver colloids based on reduction of silver nitrate with hydroxylamine hydrochloride
and sodium citrate including pH changes to modified the surface charge of the nanoparticles (NP) to study the
interaction of the NP and the bacteria.
Methodology
Growth curve Washing procedure
Bacillus thuringiensis preparation
Normal Raman Spectroscopy (NRS) and Surface Enhanced Raman
Spectroscopy (SERS) can be used as quick methods for liquid bacterial
detection in suspension and as bioaerosol particles with great interest on
standoff detection.
References
Synthesis of metallic nanoparticles for SERS
experimentsHydroxylamine nanoparticles
Biological sample preparation for Raman experiments
ResultsSynthesis of Ag-NP for SERS experiments
Hydroxylamine nanoparticles
0
0.5
1
1.5
2
2.5
3
3.5
0 3 6 9 12 15 18 21
Ab
so
rban
ce
Time (h)
Growth curve of Bt
Objective: Behavior of the surface hydroxylamine reduced Ag-NP at different pH
Results: Max absorbance: 3-11 pH: very stable Ag-NP
Changes of silver-hydroxilamine nanoparticles at ~ 427 nm
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
pH
Ab
so
rban
ce
Influence of pH on the plasmon band of silver-hydroxilamine
nanoparticles
0
0.5
1
1.5
2
2.5
330 530 730 930 1130
Wavelength/nm
Ab
so
rba
nc
e
pH5
pH3
pH1
pH7
pH9
pH11
pH13
488 nm
514 nm
427 nm
884 nm
Raman spectra of Bt @ 5 and 15 h using hydroxylamine reduced Ag NP
Citrate nanoparticlesCitrate nanoparticles
Objective: Behavior of the surface citrate reduced Ag-NP at different pH
Result: Max absorbance: 7-9 pH
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1 2 3 4 5 6 7 8 9 10 11
Ab
s m
ax
pH
0
0.2
0.4
0.6
0.8
1
1.2
300 500 700
Ab
s
wavenumber (nm)
pH 1
pH 2
pH 3
pH 4
pH 5
pH normal
pH 7
pH 8
pH 9
pH 10
pH 11
Maximum absorbance of the citrate nanoparticles at 1-11 pH rangeUV-Vis spectra of the citrate nanoparticles at different pH
Raman experimentsHydroxylamine reduced Ag-NP Citrate reduced Ag-NP
VR=0.02532 nm
(60-80mW, 10x, 3adq t)
0
20000
40000
60000
80000
100000
100 600 1100 1600
Inte
nsi
ty/a
u
Raman shift/cm-1
Bt 5 hrs_5mins Bt _1 in AgNP
Bt 5 hrs_1hr Bt _1 in AgNP
Bt 5 hrs_24 hrs Bt _1 in AgNP
Bt 5 hrs_1
10000
15000
20000
25000
30000
35000
40000
250 750 1250 1750
Inte
nsi
ty/a
u
Raman shift/cm-1
Bt 15 hrs_1
Bt 15 hrs_1 + AgNP mix 5min
Bt 15 hrs_1 + AgNP mix 1 hra
Bt 15 hrs_1 + AgNP mix 24 hrs
0
2000
4000
6000
8000
10000
12000
14000
16000
300 550 800 1050 1300 1550 1800
Inte
nsi
ty
Raman shift/cm-1
Bt 5 hrs_1
Bt 5hrs_1 hydroxilamine nP
Bt 5hrs_1 citrate nP
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
300 800 1300 1800
Inte
nsi
ty/
au
Raman shift/ cm-1
Bt + Np pH 5
Bt + Np pH 7
Bt + Np pH 9
Bt
Assignment SERSpH
5.82
SERSpH 7.0
SERSpH
9SERSC-O-C ring
deformation guanine
(nucleic acid)
730
958C=C
deformation1146 1149 1149
Amide 3 1220amide 2 1331 1350 1351=C-O-C=
unsaturated fatty acids in
lipids
1383 1388 1390
CH2
deformation1459 1457 1434
ring stretching (adenine, guanine)
1504 1508 1508
Amide 1 1652 1641 1645
Band assignments of Bt experiments using
hydroxylamine reduced Ag NP at different pH
Assignment NR Bt SERS5 mins
SERS1 hra
SERS24 hrs
SS stretch cysteine 513 518
C-S methionine 656 657 656
C-O-C ring deformation guanine (nucleic acid)
731 730 731 732
921 959 959 960
C=C deformation 1131 1146 1144 1145
Amide 3 1219 1222
amide 2 1336 1330 1332
=C-O-C= unsaturated fatty acids in lipids
1389 1389 1386 1389
CH2 deformation 1473 1461 1462 1461
ring stretching (adenine, guanine)
1508 1512 1506
Amide 1 1641 1657 1655
Band assignments (Raman shifts) of the Bt experiments ~15 hrs growth using hydroxylamine nanoparticles at
different hours
Raman spectra of Bt @ 15 hrs using 5, 7 and 9 pH ‘s of hydroxylamine Nps
Future experiments
Best results:- Hydroxylamine reduced Ag NP- 24 h after mix Bt and NP: best signal- 532nm, 10s, 3 acq, 60-80 mW- VR = 0.02
W.R. Premasiri, D.T. Moir, M.S. Kempler, N. Krieger, G. Jones, L.D. Ziegler, Journal of Physical Chemistry 109 (2005)
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spores by micro-Raman Spectroscopy, Appl Spectrosc 2003, 57, 868-871.
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Enhanced Raman Scattering (SERS) of bacteria, J. Phys. Chem. B 2005, 109, 312-320.
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0
2000
4000
6000
8000
10000
12000
14000
16000
300 550 800 1050 1300 1550 1800
Inte
nsi
ty
Raman shift/cm-1
Bt 5hrs 5_31 citrate Np
Bt 5hrs_1 citrate nP
Bt 5hrs 7_02 citrate Np
Bt 5hrs 9 citrate Np
Raman spectra of Bt @ 15 hrs using 5, 7 and 9 pH ‘s of citrate Nps
Filter
Filter LensLens
Aerolized
sample
Laser
Metallic
Nanoparticle
suspension
solution
Detector
Spectrograph
Mirror
Computer
Experimental Raman set up for bioaerosol detection
González-Sosa, R.1 ;Félix H.2 ;Hernández-Rivera, S.P.2 ;Soto, K.3 ;Ríos-Velázquez, C.3
1Industrial Biotechnology, Biology Department, University of Puerto Rico-Mayaguez Campus2ALERT-DHS Center of Excellence, Chemistry Department, University of Puerto Rico-Mayaguez Campus
3Microbial Biotechnology and Bioprospecting lab., Biology Department, University of Puerto Rico –Mayaguez Campus
Changes of pH in colloidal
nanoparticles
NaCl 0.8%
One sample of 40-50 mL was centrifuged for 20 minutes at 6,000 rpm. A bacterial pellet was formed at the bottom of the microtube, while LB
broth media formed the supernatant and was removed using a micropipette. In order to remove the remaining LB broth from the pellets,
bacteria was washed with 5 mL of NaCl 0.80% w/v. Bacteria were centrifuged for 20 minutes at 6,000 rpm and the NaCl supernatant was
removed with a micropipette. This procedure should be repeated twice. Then, the bacterial pellet is resuspended in NaCl 0.8% w/v to obtain a
solution containing the bacterial endospore cells.
48 hours
32°Celsius24 hours – 5 mL LB Broth OD (Absorbance)
50 mL- 15 hours
Characterization in UV-VIS and SERSThe SERS experiments will be performed in
solution and on solid substrates. For
experiments in solution sodium chloride will
be used as a SERS active substrate. For
SERS analysis, 200 µL of the silver colloids,
25uL of Bacillus thuriengiesis and 25uL of
NaCl 0.8% to get a VR=0.02 to be analyzed
will be combined in a vortex vial and
experiments will be run at different times until 30-120 minutes
N2 for
degasification
10.44 mg NH2OH HCl
1.67X10-3 M
115 mL of
Ultrapure H2O
25 mL from degassed
ultrapure H2O
11.98 mg NaOH
3.33X10-3 M
42.47 mg AgNO3 0.10M
Drop wise
Stirring for 30 min
N2 for
degasification
50 ml of a 10-3 M AgNO3
Aqueous solution was heated
1 ml of a 1% itrsodium Cctrate (C6H5O7Na3) solution
To boiling for 1 hour and then was allowed to cool down