Sensing Superfund Chemicals with Recombinant Systems
Sapna K. Deo, S. Xu, D. Ghosh, X. Guan, A. Rothert, J. Feliciano, E. D’Angelo, Leonidas G. Bachas, and Sylvia Daunert Department of Chemistry and Department of Agricultural Sciences University of Kentucky Lexington, KY Molecular Recognition in Analytical Chemistry
• Proteins
• Cells
• High Throughput Screening • Whole Cell-Based Sensing Systems
Analyte No Analyte Signal
Reporter protein No Expression of Reporter Protein Arsenic Poisoning
• Applications • Agriculture • Treatment for diseases • Industrial uses
• Long exposure to low doses of arsenic • Skin hyperpigmentation and cancer • Other cancers • Inhibition of cellular enzymes
New Bangladesh Disaster: Wells that Pump Poison... New York Times November 10, 1998 Arsenic contamination in the USA
U. S. Geological Survey, Fact Sheet FS 063-00, May 2000 Arsenite Resistance in E. coli
O/P arsR arsD arsA arsB arsC Schematic Representation of the Antimonite/Arsenite Pump
- - AsO2 SbO2 Cytoplasm ADP ATP AsO - ATP 3- 2 AsO4 ADP ArsA ArsA ArsC
Periplasm Membrane ArsB
- - AsO2 SbO2 Fluorescent Reporter Proteins in Array Detection
Protein Excitation Emission
λ max λ max
GFP 395 (470) 509 EGFP 488 509 BFP 380 440 GFPuv 395 509 YFP 513 527 CFP 433 475 CobA 357 605 RFP 558 583 P H C 3 A Production of fluorescent A P
porphyrinoid compounds H3C N HN
oxidation NH N A A P A P H C 3 A A A P P P P NH HN H3C N HN UMT sirohydrochlorin
NH HN SAM P A NH HN H3C A A A A A UMT P HN P P P P H3C N SAM urogen III Dihydrosirohydrochlorin (Precorrin-2) NH N A CH3 A-CH2COOH A P-CH2CH2COOH
SAM - S-adenosyl-L-methionine P P
UMT- uroporphyrinogen methyltransferase III trimethylpyrrocorphin Construction of pSD601 plasmid
O/P arsR bla arsD PCR pRLUX arsR O/P luxAB arsR
+ pSD601 ampR cobA
cobA cobA
pISA417 PCR Time Study for Arsenite
10000
-5 1 x 10 M NaAsO2 8000
6000
4000
2000 Fluorescence (cps)
0
-2000 024681012 Time (h) ALA in Urogen Pathway
P A P A
COOH COOH A P A P COOH NH HN NH HN ALA PBG Urogen III HO O dehydratase deaminase synthase N NH HN NH HN H P A A A H N H N 2 2
ALA PBG A P P P HMB Urogen III ALA - δ-aminolevulinic acid PBG - Porphobilinogen HMB- Hydroxymethylbilane Urogen- Uroporphyrinogen Time Study of Arsenite with ALA
10000 NO ALA NO ALA 8000 1mM ALA
6000
4000
2000 Fluorescence (cps) 0
-2000 024681012 Time (h) Calibration Plot with ALA
45000
40000
35000
30000
25000
20000
15000
Fluorescence (cps) 10000
5000
0 -8 -7 -6 -5 -4 -3 log[sodium-m-arsenite] Selectivity Study
40
Induction time = 1 h 35 Analyte concentration = 5 x 10-6 M
(cps) 30
25
Intensity 20
15
10
Fluorescence 5
0 - - - 3- 2- - - Br Cl NO3 PO4 SO4 AsO2 SbO2 Field Challenges
• Conventional analytical techniques • Field-kits • Background signal • Viability of the cells • Freeze drying • Strips (β-galactosidase) Addressing Environmental Analysis with Self-Contained Kits
Lyophilized Reagents
“The bacteria are dim in tap water (top) but glow brightly when arsenic is present 0 0.1 0.2 0.5 1 (bottom).” Arsenite (µM)
Strip Sensors http://www.nature.com/nsu/030929/030929-7.html Centrifugal Microfluidic Platform for Micro-Total Analysis Systems
Low power and space requirements
Less reagent and sample consumption
Easy disposal
Short analysis time
Integrate washing, sample preparation, and calibration Prototype Compact Disc
7 6 5 4 3
1 2
1. Waste Reservoir 2. Optode 3-7. Solution Reservoirs Prototype CD for four simultaneous analyses Incorporation of Whole-cell Sensing System for Arsenite/Antimonite on the CD Platform
140
130
120
(Counts) 110
Fluorescence Intensity 100
0. y t i 0.7 s n 90 s l 6 l De
0.5 l Ce 0 a 15 c f 0.4 i 30 o 45 t 60 p Time (min) O Calibration Curves
Antimonite Arsenite 120 120 Detection Limit: 1 x 10-6 M Detection Limit: 1 x 10-6 M 100 100
80 80
60 60
40 40
20
Normalized Fluorescence 20 Normalized Fluorescence Normalized
0 0 -6.5 -5.5 -4.5 -3.5 -6.5 -5.5 -4.5 -3.5
log (Antimonite, M) log (Arsenite, M) Clc Operon and 3-chlorocatechol Pathway
clcR o/p clcA clcB clcD
O HOOC OH ClcA COOH ClcB ClcD COOH O COOH O COOH Cl Cl OH clcR clcA
clcB′ pSMM50R-B′
r amp lacZ Calibration Curves
5 (5 min induction) ) 5 3-clc
10 4 ×
3
2 4-clc 1 catechol
Light Intensity (counts Light Intensity 0 -10 -9 -8 -7 -6 -5 -4 -3 -2 log (concentration, M) Selectivity Study
1.5 1) 3-chlorocatechol; 2) 4-chlorocatechol; 3) 4-chlorobiphenyl ) 6 4-7) catechol, biphenyl, 2-chlorophenol, and 4-chlorophenol 1 10 ×
1.0
0.5 2
Light Intensity (counts Light Intensity 3 0 4-7 -10 -9 -8 -7 -5 -4 -3 -6 log (concentration, M) Challenges in Environmental Sample Analysis of Chlorocatechol
1. Extraction Method • Free Chlorocatechol Can be extracted by organic solvent O Fe • Bound Chlorocatechol O Cl OH Difficult to be extracted 2. Matrix Effect Selected Chemical Characteristics of Soils
Organi Electric cal Oxal atextractablee Soils car cbon onductivity pH P Al Fe -1 -1 (%) (µmhos•cm ) (mg kg ) Acid washed sand 0.02 12 6.1 8 32 54 Maury silt loam 3.3 53 5.0 704 1894 4126 Woolper 7.5 38 5.9 3203 3086 3203 Organic humus 15.3 1099 6.4 415 3575 1106
Optimized Protocol for Soil Analysis
Pellet (Intact, induced bacteria )
Supernatant 2 (Water + Bacteria) centrifuge Mixture 1 (10,000 ×g) (Soil + Water +Bacteria) Supernatant
Induction (2h), Precipitate (Discarded) centrifuge (400×g) (Soil) Results of Soils
HPLC Bacterial sensing system Sample Theoretical Experimental ± Theoretical Experimental ± matrix Value (mg•kg-1) SD (mg•kg-1) Value (mg•kg-1) SD (mg•kg-1) 0.5 0.49 ± 0.02 0.5 0.51 ± 0.02 Sand 2.0 2.03 ± 0.04 2.0 1.95 ± 0.08 10 9.75 ± 0.55 50 52.0 ± 2.6 Woolper 0.5 0.0 ± 0.0 0.5 0.52 ± 0.04 2.0 0.0 ± 0.0 2.0 1.90 ± 0.18 10 9.55 ± 0.75 50 54.0 ± 4.5 Maury 0.5 0.0 ± 0.0 0.5 0.43 ± 0.08 2.0 0.0 ± 0.0 2.0 2.24 ± 0.30 Organic 0.5 0.0 ± 0.0 0.5 0.43 ± 0.09 potting soil 2.0 0.0 ± 0.0 2.0 1.73 ± 0.28 Detection of PCBs Based on Dechlorination Followed by whole cell Sensing
Clx
Mg/K2Pd6 +H2O
Cly - +(X+Y)Cl+H2
PCB Dechlorination Reaction HPLC Chromatogram of Dechlorinated Product: Biphenyl Whole cell Sensing of Biphenyl Based on bph Operon from P. pseudoalcaligenes KF707
pE R1 A1A2(orf3)A3A4 B C
No biphenyl No Transcription
HOPD
O OH Transcription Biphenyl COOH pE Bphoperonof P. pseudoalcaligenes KF707
Biphenyl mol/L Increase in Light Intensity (%) lacZ pSD7001 1x 10-5 81.9 ± 9.5 ampR 1x 10-6 37 ± 6.2
1x 10-7 21 ± 4.8 Degradation Pathway of Hydroxylated-Biphenyl in the strain Pseudomonas azelaica HBP1
hbpR hbpC hbpA hbpD
OH OH COOH COOH + + NADH+H NAD O2 O H2O OH
OH OH COOH HbpC HbpD
O2 H2O HbpA Whole Cell-Based Sensing System for Hydroxylated PCBS
10000
7500
5000
2500 Relative Light Units
0 -9 -8 -7 -6 -5 -4 -3 -2 Log (2-hydroxy-3',4'-dichlorobiphenyl, M) Collaborators
Leonidas Bachas Marc Madou Barry Rosen Jan Roelof van der Meer
Acknowledgments
NIEHS-Superfund Basic Research Program the past and present members of the Daunert Group