1 msc: f-elements, prof. j.-c. bünzli, 2008 analyte specific biochemical reaction labeled antibody...
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1MSc: f-Elements, Prof. J.-C. Bünzli, 2008
Analyte
Specificbiochemicalreaction
Labeledantibody
Reporter:Lanthanidechelate
SA
LuminescentpropertiesŒ
Linkinggroup
signal
5.7 Luminescent biochemical sensors
Chapter 5 Selected applications
2MSc: f-Elements, Prof. J.-C. Bünzli, 2008
Time resolved heterogeneous immunoassays
Immobilizedhormone
LnIII
ChelateLabelled antibody
Step 1
H3O+ Step 2
micelle
Chapter 5 Selected applications
3MSc: f-Elements, Prof. J.-C. Bünzli, 2008
-NTA TOPO
CF3
O-
O
O P
O (CH2 CH2 O)nHCCH2C
Triton X-100
Delfia® technology
Production of the in situ luminescent chelate:
NCN
S
HH N
N
N
COO-
COO-
COO-
COO-
Eu3+
protein chain
Linking the 1st chelateon the protein chain
Chapter 5 Selected applications
4MSc: f-Elements, Prof. J.-C. Bünzli, 2008
Q(Eu) = 70 %
Luminescentmicelle
CF3
O O
OOEu
OPO
P
CF3
OO CF3
O(CH2
CH 2O) n
H
C
CH2
C
O(CH
2CH
2O)nH
C
CH2
C
O(CH 2
CH 2O) n
H
CCH2
C
Chapter 5 Selected applications
5MSc: f-Elements, Prof. J.-C. Bünzli, 2008
Human immunoglobulin IgE• Related to various allergies• lowest concentration among the five immunoglobulins• detection limit for radioimmunoassay: 40-300 pg/ml in 2-5 daysDevelopment of an Eu-based luminescent immunoassay:
J. Yuang, G. Wang, H. Kimura, K. MatsumotoAnal. Biochem. 1997, 254, 283
SO2Cl
O
O
C3F7
O
O
C3F7
SO2Cl
O
O
C3F7
O
O
C3F7
SO2Cl
O
O
C3F7
O
O
C3F7
Time resolved homogeneous immunoassays
Chapter 5 Selected applications
6MSc: f-Elements, Prof. J.-C. Bünzli, 2008
Step 1: Labeling of streptamycin SA
SASA +
Eu(BHHCT)
or of the BSA-SA conjugate:
SA:Eu(BHHCT)1: 21
BSA:SA:Eu(BHHCT)1:0.9:46
BSA SA
Chapter 5 Selected applications
7MSc: f-Elements, Prof. J.-C. Bünzli, 2008
Step 2:
IgE coating biotylinatedanti IgE antibody
Step 3: TRS measurement
1010.10.010.001 1010.10.010.001
[IgE]
Iem
1010.10.010.001 1010.10.010.001
[IgE]
Iem
UI
Detection limit: 1.5x10-3 IU/ml 4 pg/ml 10-18 mol 6x105 molecules
Time: 4-5 hours
Chapter 5 Selected applications
8MSc: f-Elements, Prof. J.-C. Bünzli, 2008
Time resolved homogeneous immunoassays
Energy transfer
Antigen
Energy acceptor
Monoclonalantibody B
Eu
Monoclonalantibody A
N N
N NNN
N N
OHN
UV Excitation Emission660 nm
LRET: Luminescent resonance energy transfer
Chapter 5 Selected applications
9MSc: f-Elements, Prof. J.-C. Bünzli, 2008
Hybridation detection
Energyacceptor
h
UV Excitation Energy transfer
5’ end of DNA strands
-N
O
N
HO
N
N
N
O
NO
PCCTAGAGTGG3'
CO2-
CO2-
CO2-
NN
SO3--O3S
ON
H
O
3'GGATCTCACCP
Eu
Chapter 5 Selected applications
10MSc: f-Elements, Prof. J.-C. Bünzli, 2008
5.8 Imaging of cancerous cells
Chapter 5 Selected applications
Four cell lines tested:
Human breast adenocarcinoma MCF-7Human cervical adenocarcinoma HeLaHuman T leukaemia Jurkatnon-malignant epithelial HaCat (Human keratinocyte)
One mAb 5D10 antibody used as diagnostic tool
Ln complex
Ln complex
Ln complex
Ln complex
11
Suspension cells Adherent cells
5D10 Hybridoma HeLa (CCL-2)
Jurkat (TIB152) MCF-7 (HTB-22)
100 m
Cervical carcinoma
Breast carcinomaT leukemia
Human cell lines
Chapter 5 Selected applications
12
Ln coordination
Water solubilityCoupling
R1 =CO2H
ttp “cavities”
ttp9-coordination
Ligand design
NN N
NR3 R3
N
R1
N
R1
R2 R2
R4 R4
Shifting excitationwavelength
Chapter 5 Selected applications
13
O
OO
NN N
N
N
HOO
N
OHO
O
O
OO
O
H2LC2
A.-S. Chauvin, S. Comby, Song Bo, C. Vandevyver, J.-C.G. Bünzli
Chem. Eur. J. 2008, 14(6), 1726
Chapter 5 Selected applications