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)

312-320.

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http://www.textbookofbacteriology.net/Anthrax.html.

Esposito, A. P.; Talley, C. E.; Huser, T.; Hollars, C. W.; Schaldach, C. M.; Lane, S. M., Analysis of single bacterial

spores by micro-Raman Spectroscopy, Appl Spectrosc 2003, 57, 868-871.

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Thiele, H.; Motzkus, H.-W.; Popp, J. Anal Chem; 2006; pp 2163-2170.

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Size, Shape, and Dielectric Environment. J. Phys. Chem. B. 2003, 107, 668-677.

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chemical and biological agent simulants, IEEE Sens. J. 2005, 5, 665-670.

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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