Bioluminescence for TLC Detection - HPTLC Asia 2018 · HPTLC 2006 Wolfgang Kreiss. HPTLC 2006:...
Transcript of Bioluminescence for TLC Detection - HPTLC Asia 2018 · HPTLC 2006 Wolfgang Kreiss. HPTLC 2006:...
toxicity screening
bioactivity in mixtures
drug discovery
Bioluminescence forTLC Detection
HPTLC 2006Wolfgang Kreiss
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
Bioluminescence and TLC
! imaging of bioactivity on planar surfaces
! bioluminescence for TLC detection
! detection process
! application examples
! luminescent reporter gene cells
! bioluminescence coupling - TLC or HPLC
! benefits, limitations and current status
http://oceanexplorer.noaa.gov
sp. Myctophidae
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
Imaging of Biological Activity
****
sensor layer
bioactive samplediffusion
luminescentcells
CCDcamera
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
Spatial Resolution
lines per inch
5
10
15
20
1 inch
100 dotsper inch2
graphics forresolution testing
bioactivity imagefrom inkjet print
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
Luciferase
Flavinreductase
FMNH2
FMN
FMN
FNMH-OOH
FMNH-OH
FMNH-OH
RCHO
RCOOHFatty acidreductase
NAD(P) +AMP + PP
+
NAD(P)H +H + ATP+
NAD(P)+ NAD(P)H + H+
( )*
Substrateoxidized
Substratereduced O2
H 2O
O2
H 2O
FMNH2
Bioluminescence
coupling ofbioluminescenceto respiratory chainand ATP pool
close link to themetabolic status ofthe cell
luminescent cellsas biosensors
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
Principle of the BioTLC
! separate compound mixture by TLC
! evaporate solvent
! dip into bioluminescence kit
! evaluate by videoimaging
results:
TLC-plate
bioactive compounds
120 sec fordetection
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
imaging
BioTLC - Process
incubation
3 step procedure
TLC plate coating
specificcell absorption on plate
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
Toxicity Profiling of Waste Water
TLC-Bioluminescence screening - detection: Vibrio fischeri
decreased bioluminescence indicates toxic substance zones
samples from 15 days in succession
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
Natural Products Extracts
A - G : 100 µg natural products extractsnp : 200 ng 4-nitrophenol (tox-reference 1)bp : 20 ng 4-tert.-butylphenol (tox-reference 2)
TLC-technique: AMD detection: visual
A B C D E F Gnp bp np
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
Natural Products Extracts
A - G : 100 µg natural products extractsnp : 200 ng 4-nitrophenol (tox-reference 1)bp : 20 ng 4-tert.-butylphenol (tox-reference 2)
BIO-TLC detection: Vibrio fischeri
A B C D E F Gnp bp np
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
BioTLC of plant residuals
UV-scan
bioluminescencedetection
tea seed oil residual100 µg
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
Quantitation of Toxicity
B C Enp1 % 1,5 % 2 % 2,5 %2 %np np np np
quantitation by toxicity equivalentsbased on calibration with 4-nitrophenol (np)
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
BioTLC for USP-Methods
A - I : Matricaria chamomilla np : 4-nitrophenol
detection: bioluminescence Vibrio fischeri
A B C D E F G npH
06699
I npnp
detection: derivat. anisaldehyde / 366 nm
TLCby
CAMAG
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
A, B : Kava Kava sample 1C, D: Kava Kava sample 21 - 6: references; np = 4-nitrophenol
detection: Vibrio fischeri
A B C D 1 2 3 np4
plate 6653
np 5 6
BioTLC of Kava Kava
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
Toxicity in Process Water
TLC-Bioluminescence - detection: Vibrio fischeri
1 2 3 4 5 6 7 8
30 ng30 ng 1 µl 100 µl 100 µl 100 µl100 µl1 µl
treatment
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
Current state of BioTLC
! mature technique (Vibrio fischeri)
! sensor cells in research applications
! very fast bioassay (Vibrio fischeri)
! excellent fit with AMD technique
! high compatibility to standard TLC
! TLC kit available (BioLuminex / ChromaDex)
! detection equipment available (CAMAG)
Lampyris noctiluca
http://psteinmann.net/bilder_insekten
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
Bioluminescence: TLC or HPLC ?
pro TLC! single use separation system! simultaneous parallel detection! high productivity technology
pro HPLC! efficient correlation with MS data! quantitation! mainstream technology
Luciferase
Flavinreductase
FMNH2
FMN
FMN
FNMH-OOH
FMNH-OH
FMNH-OH
RCHO
RCOOHFatty acidreductase
NAD(P) +AMP + PP
+
NAD(P)H +H + ATP+
NAD(P)+ NAD(P)H + H+
( )*
Substrateoxidized
Substratereduced O2
H 2O
O2
H 2O
FMNH2
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
sample HPLC UV
NMR
MSmicrospotting
bioactivecomponent
start
HPLC-Bioluminescence-coupling
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
! rapid toxicity profilingsuccessful assignment of toxicity in compound mixtures
! versatile applicationwaste water analysis, food industries, regulatory affairs
! relevant resultsBioTLC employs accepted bioluminescence principles
! advanced technologyhigh productivity technique with easy set up
Benefits of BioTLC
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
Options for the BioTLC
! detection of specific bioactivity (specific cell types)
! luminescent cells e.g. yeast, insects, algae, ...
! multiplex detection (novel sensor cells or mixtures)
! regulation of bioluminescence (e.g. autoinducer)
! time dependent detection (fast/slow activity)
! standardization (kits, methods, camera systems)
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
Future developments
1 2 3
1: bioactivity reference; 2 and 3: natural products extracts
TLC-detection by reporter gene systems
HPTLC 2006: Bioluminescence for TLC Detection Wolfgang Kreiss
Acknowledgements
Microbiology
EberzBioluminescenceDetection
WeisemannAMD-TLC
KinastCAMAG
Reich
Verbitsky