Spectroscopic Detection of Organophosphorus Agents. Dr. Roger M. Leblanc Department of Chemistry Coral Gables, FL

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1 pectroscopic Detection of rganophosphorus Agents Dr. Roger M. Leblanc Department of Chemistry Coral Gables, FL

2 Report Documentation age Form Approved MB No ublic reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. end comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters ervices, Directorate for Information perations and Reports, 1215 Jefferson Davis Highway, uite 1204, Arlington VA Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it does not display a currently valid MB control number. 1. RERT DATE 15 NV RERT TYE N/A 3. DATE CVERED - 4. TITLE AND UBTITLE pectroscopic Detection of rganophosphorus Agents 5a. CNTRACT NUMBER 5b. GRANT NUMBER 5c. RGRAM ELEMENT NUMBER 6. AUTHR() 5d. RJECT NUMBER 5e. TAK NUMBER 5f. WRK UNIT NUMBER 7. ERFRMING RGANIZATIN NAME() AND ADDRE(E) Department of Chemistry Coral Gables, FL 8. ERFRMING RGANIZATIN RERT NUMBER 9. NRING/MNITRING AGENCY NAME() AND ADDRE(E) 10. NR/MNITR ACRNYM() 12. DITRIBUTIN/AVAILABILITY TATEMENT Approved for public release, distribution unlimited 11. NR/MNITR RERT NUMBER() 13. ULEMENTARY NTE ee also ADM001849, 2004 cientific Conference on Chemical and Biological Defense Research. Held in Hunt Valley, Maryland on November 2004., The original document contains color images. 14. ABTRACT 15. UBJECT TERM 16. ECURITY CLAIFICATIN F: 17. LIMITATIN F ABTRACT UU a. RERT unclassified b. ABTRACT unclassified c. THI AGE unclassified 18. NUMBER F AGE 26 19a. NAME F RENIBLE ERN tandard Form 298 (Rev. 8-98) rescribed by ANI td Z39-18

3 Insecticides and pesticides Nerve gases 2 N Diethyl p-nitrophenyl phosphate (araoxon) F hosphonofluoridic acid, methyl-, 1,2,2-trimethylpropyl ester (oman) 2 N Diethyl para-nitrophenol thiophosphate (arathion) F Methylphosphonofluoridic acid 1-methyl-ethyl ester (arin) F N Di-isopropyl fluoro-phosphate (DF) -Ethyl -(2-(diisopropylamino)ethyl)methylphosphonothioate (VX)

4 rganophosphorus Hydrolase Et N 2 H201 H230 H57 H55 M1 H H254 X 2 H257 M2 X1 araoxon H201 H230 H57 H55 M1 H H H254 X 2 M2 H257 X1 Et N 2 Et Et Et Et H + H N 2 Isolated from seudomonas diminuta Metalloenzyme (zinc at active site) N 2 mechanism of hydrolysis

5 Quantum Dot Characteristics Quantum dots ize dependent emission High quantum yield Broad excitation spectra Narrow emission band Chemical versatility Core-shell quantum dots Higher quantum yield Chemical and photophysical stability Low toxicity nm Excited with same wavelength (365 nm)

6 ynthesis of Quantum Dots n-trioctylphosphine oxide (T) Tetradecylphosphonic acid Cd TGA-Cde (or Te) H-CH 2 -CH (TGA) T-Cde (or Te) 310 o C e or Te in Trioctylphosphine 250 o C, growth temperature Adam-eng, Z. and eng, X. J. Am. Chem. oc. 2001, 123, Chan, W. C. W. and Nie,. cience 1998, 281,

7 ensing device 1: Cde composite films : paraoxon detection Layer-by by-layer technique (electrostatic interaction) Quartz slide Composite film Chitosan Cde--CH CH 2 -CH quantum dots rganophosphorus hydrolase (H) Constantine, C. A.; Gattás-Asfura, K. M.; Mello,. V.; Crespo, G.; Rastogi,V.; Cheng, T.-C.; DeFrank, J. J. and Leblanc, R. M. Langmuir 2003, 19,

8 ptical properties of the quantum dots in aqueous solution : Intensity, cps Absorbance, a.u Avg. size: 3.4 nm λ, nm 0.0 Monitoring growth of the composite film on the quartz slide : (1-7 7 = # layers of quantum dots) Absorbance a.u Absorbance Number of bilayers Intensity, cps Last layer (H) λ, nm λ, nm Constantine, C. A.; Gattás-Asfura, K. M.; Mello,. V.; Crespo, G.; Rastogi,V.; Cheng, T.-C.; DeFrank, J. J. and Leblanc, R. M. Langmuir 2003, 19,

9 Response of film upon exposure to paraoxon solution Absorbance, a.u min M 1-7 = 0, 0.5, 1, 2, 5, 10, and 15 min Intensity, cps a c d e f b g h Blank 10-9 M 10-4 M min. λ, nm λ, nm N 2 + H N 2 + Et ET Et Epifluorescence images of film 895µm 713 µm Before exposure to paraoxon After exposure to paraoxon Constantine, C. A.; Gattás-Asfura, K. M.; Mello,. V.; Crespo, G.; Rastogi,V.; Cheng, T.-C.; DeFrank, J. J. and Leblanc, R. M. Langmuir 2003, 19,

10 Conclusion Quantum dots were successfully immobilized into composite films through utilization of the Layer-by by- Layer technique and electrostatic interactions The convenient quantum dot-h composite film fabricated was highly selective and sensitive towards organophosphorus compounds (e.g. paraoxon)

11 ensing device 2: Formation of H/QDs bioconjugate Zn Cde H H + NH NH 3 + NH 3 - H + NH 3 - NH 3 + H NH NH + 3 H 3 N - Zn Cde NH 3 NH + 3 H 3 N H + H 3 N - + H 3 N - H 3 N - H 3 N - + H 3 N + H 3 N - + H 3 N H H ( = Active ite of H ) Formation of H/QDs bioconjugation

12 ynthesis of Cde(Zn) QDs Cd + e TDA, T T, 250 ºC Cde QDs Cde + (TM) 2 + Zn(C 2 H 5 ) 2 Cde(Zn) ) QDs Zn Cde H CH 2 C Zn Cde

13 L spectra of H/QDs Normalized L Intensity H : QDs =10:1 0 M 4.2 x 10-8 M 1.5 x 10-7 M 1.1 x 10-6 M 2.6 x 10-6 M 4.2 x 10-6 M 2.6 x 10-5 M 4.2 x 10-5 M Wavelength (nm) hotoluminescence spectra of 10:1 molar ratio H/QDs bioconjugate in different concentrations of paraoxon. amples were excited at 350 nm.

14 L spectra of pure QDs Normalized L Intensity QDs only 0 M 4.2 x 10-8 M 1.5 x 10-7 M 1.1 x 10-6 M 2.6 x 10-6 M 4.2 x 10-6 M 2.6 x 10-5 M 4.2 x 10-5 M Wavelength (nm) hotoluminescence spectra of pure QDs in different concentrations s of paraoxon. amples were excited at 350 nm.

15 Mechanism of L quenching N 2 H 2 N 2 H econdary tructure Change H QDs QDs Quenching H + H N 2

16 Michaelis-Menten Menten Equation k E + +1 k +2 k -1 E E + e 0 [E] s 0 - [E] [E] teady state: d[e]/ [E]/dt = k +1 (e 0 [E])[] k -1 [E] k +2 [E] = 0 ( [] = s 0 - [E] ) [E] = k +1 e [] 0 k -1 + k +2 + k +1 [] e 0 s 0 [E] = ( s 0 >> e 0 > [E] ) ( k -1 + k +2 ) / k +1 + s 0 Q = k q [E] ] = k q e 0 s 0 ( k -1 + k +2 ) / k +1 + s 0

17 Mechanism of L quenching Relative L intensity percentage of (a) 10:1 (molar ratio) and (b)( 100:1 (molar ratio) H/QDs bioconjugates as a function of paraoxon concentration. 40 (a) 40 (b) 100(F 0 -F n )/F 0 (%) Hyperbolic Fit: Y = X / (4.321 x X) 100(F 0 -F n )/F 0 (%) Hyperbolic fit: Y = X / ( x X) x x x x x x x x10-5 [araoxon] (M) [araoxon] (M) F 0 : L intensity at 573 nm in the absence of paraoxon F n : L intensity at 573 nm in the presence of paraoxon

18 Conclusion The H and Cde(Zn) ) QDs can form stable bioconjugate through electrostatic interaction CD spectra indicate a secondary structure change of H in the presence of paraoxon The intensity of photoluminescence of H/QDs bioconjugate was quenched in the presence of paraoxon The secondary structure change of H was responsible for the observed L quenching Increasing the molar ratio of H over QDs will not substantially increase the sensitivity of H/QDs biosensor

19 ensing device 3: H covalently bound to solid support N C 7-isothiocyanato-4-methylcoumarin (1) i NH 2 Et 2 N Cl 70ºC, 10 h i N C H ph 9.6, 10 h i NH NH Enzyme 1 1 ph 9.6, 10 h i NH NH Enzyme

20 Imaging ellipsometry

21 nm 2-D image of the silanized slide 3-D image of the silanized slide nm 2-D image of covalently attached H 3-D image of covalently attached H

22 ATR-IR spectrum of the secondary structure of H Frequency band position (cm -1 ) Assignment anti-parallel ß-sheet or pleated turn ß-sheet α-helix Absorbance amide I ß-sheet ß-sheet α-helix 1534 amide II Wavenumber / cm -1

23 UV-Vis spectra for paraoxon analysis Absorbance, a.u 0.5 A, A.U Time, min A, A.U. time, min λ, nm

24 Fluorescence spectra of H-coumarin sensing system in presence of paraoxon solution Fluorescence intensity, a. u initial fluorescence 5 x 10-9 M paraoxon 5 x 10-8 M 5 x 10-7 M 1 x 10-6 M 1 x 10-5 M recovery of fluorescence Absorbance, a.u Fluorescence, c.p.s λ, nm λ, nm

25 Conclusion H was successfully bound to a silanized quartz substrate This bio-functional surface successfully detects the presence of paraoxon aqueous solution

26 Group Members h.d. graduate students Celeste A. Constantine Kerim M. Gattás-Asfura Xiaojun Ji Changqing Li David Naistat Jianmin Xu Liang Zhao Jiayin Zheng Undergraduate students Gema Crespo hilip Deaola Andrea Escobar Michael J. nedaker Daniel A. Thimann anjeev irpal enior Research Assoc. Jhony rbulescu

27 Acknowledgement U.. Army Research ffice (DAAD ) 0131)