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The detection problem in biomarker analysis of biological
fluids Mike Thompson
Department of Chemistry and Institute for Biomaterials and Biomedical Engineering,
University of Toronto
International Centre of Biodynamics
Bucuresti, Romania July, 2010
Clinical and Biomarker Targets Province of Ontario
• 1 billion dollars annually for hospital and central lab assays (22 bd Provincial Budget)
• Many assays involve magnetic bead ELISA
• High level of automation but chemistry is often “old”
• Virtually no introduction of lab-on-a-chip or sensor technology
• Blood, urine and tissue are extremely difficult matrices
Label Free Detection Methods
• Transverse wave acoustic physics in the FIA liquid-phase mode –protein small-molecule interactions, neuron cell behavior, nucleic acid damage by oxidants
• Electromagnetic detection based on the propagation of ultra high frequency (1 GHz) acoustic physics
• Kelvin current detection in scanning format and time-dependant measurements over nucleic acids, proteins and neurons on substrates such as ITO
Topics
• Transverse wave acoustic physics as a sensor detection strategy - examples
• Ultra high frequency electromagnetic physics• Model probe attachment to EMPAS surface• Linker chemistry and minimization of the
pervasive NSB biosensor problem • Applications – preliminary work on the detection
of ovarian cancer and HIV in serum• Application – collaboration with UK MOD• Outline of work on scanning Kelvin detection
Viscous Liquid
Liquid
L, L
Rm Lm Cm
C0
Biolayer
Measure:fs – Energy storageRm – Energy dissipation
Frequency response for Tat-30 binding
0 2000 4000 60009000300
9000350
9000400
9000450
9000500
9000550
9000600
9000650
9000700
9000750
Tat-30
TAR
neutravidinfr
eque
ncy
(Hz)
time(s)
0 1000 2000 3000 4000
-10
-5
0
5
10
15
20
25
30
35
Tat-25
Tat-22
Tat-18
Tat-12
freq
uenc
y ch
ange
(Hz)
time(s)
Neuron culture in acoustic vibrational fields (TSM)
CO2
in
CO2
out
growth medium and or drugs in
growth medium and or drugs out
Microscopic image of neurons (N-38)
Metabolic line over 48hrs
2ml chamber for neuron growth
Cellular OscillationsCOHERENT SYNCHRONOUS SIGNAL OF 2 MINUTES PERIODICITY!
EMPAS System Layout
Criteria for Protein or Aptamer Probe Attachment to Device
• Molecular assembly for reproducible surface – BUT?
• Si dioxide - silanization chemistry• High receptor site packing density• Capability for steric control of density• Simple bi-functionality allowing 100%
reaction with probe• Minimize or eliminate NSB in biological
fluids – blood, serum, urine
General Probe Model
• Develop a new generation of linkers onto which thiol-containing biomolecules could immobilize in a subsequent step for the purpose of fabricating EMPAS biosensing interface– Biotin-avidin was chosen as a model system in order to test the
viability of our biosensor – Chemically modified biotin to yield a thiol group on its tail
13
Alkyltrichlorosilane Linkers
• Trichlorosilyl tail shows strong affinity to quartz crystal– Forms a strong Si-O bond on the surface of quartz crystal
• The Head function can be modified to immobilize target biomolecules
14
SiCl
Cl Cl
Thiosulfonate Chemistry
• Thiosulfonate was chosen as the head function – Known to react chemoselectively with thiols to form disulfide
bonds
15
Gamblin, D. P.; Garnier, P.; Ward, S. J.; Oldham, N. J.; Fairbanks, A. J., and Davis, B. G. Org. Biomol. Chem. 2003, 1(21), 3642-3644.
S SR
O
O
R' + R'' SH R S S R'' + S
O
HO
R'
Trichlorosilyl Undecenyl Benzene ThioSulfonate (TUBTS)
• Synthesis
16
TUBTS SAM Formation: Time Trial
17
Cleaned quartz crystal TUBTS SAM
OH OHOH OHSi
OSiO O
S
SiO
SiO
S
O O
TUBTSPhMe, rt, time
OO
SS
OO
SS
OO
SS
OO
Biotinthiol Immobilization: Time Trial
18
TUBTS SAM
SiO
SiO O
S
SiO
SiO
S
O O
BiotinthiolMeOH, rt, time
OO
SS
OO
SS
OO
SS
OO
SiO
SiO O
S
SiO
SiOO O
S S S
Biotinthiol Biotinthiol Biotinthiol Biotinthiol
Biotinthiol functionalized TUBTS SAM
XPS analysis for biotinthiol immobilization on TUBTS SAMs at various time
19
XPS peak profile for N
N signal is unique to biotinthiol
S
NHHN
SH
O
Biotinthiol
Chemoselectivity of TUBTS SAM
20
+ N. R.
OSiO
SS
OO
SiO
S
NHHN
O
BiotinOH
O
S
NHHN
O
BiotinolOH
S
NHHN
O
BiotinamineNH2
An Example of EMPAS measurement
824.215
824.22
824.225
824.23
824.235
824.24
0 500 1000 1500 2000 2500 3000
Time (sec.)
Fre
qu
en
cy (
MH
z)
21
Injection of 0.1 mg/mL avidin solution (50 µL)
Frequency shift of 17900 Hz
EMPAS measurements for TUBTS SAM
22
Specific to non-specific ratio – 1.5:1 Acceptable reproducibility
OEG-TUBTS
23
Synthesis
OEG-TUBTS SAM
24
OEG-TUBTS SAM
SiO
O
O
SiO O
O
O
O
S
SiO
SiO
S
O
O
O
O
O O
BiotinthiolMeOH (Et3N), rt, 2 h
OO
O
SS
OO
O
SS
OO
O
SS
OO
SiO
O
O
SiO O
O
O
O
SiO
SiO
O
O
O
O
O O
O O O
Biotinthiol Biotinthiol Biotinthiol Biotinthiol
Biotinthiol functionalized OEG-TUBTS SAM
S S S S
EMPAS measurements for OEG-TUBTS SAM
25
Specific to non-specific ratio – 1.75:1 High reproducibility
Incorporation of diluent• Next Step: Incorporation of a diluent molecule in our
system – A diluent - a shorter molecule used to space out the linker
within the SAM• Provides greater space for the analyte to interact with the
biosensing element
• Also attempted the biotinthiol immobilization under aqueous conditions
26
7-OEG
27
Synthesis
OOH O
OF
SiCl
ClCl
7-OEG59%
O
F
F2 steps
OEG-TUBTS/7-OEG SAM
28
OEG-TUBTS/7-OEG SAM
SiO
O
O
SiO O
O
O
O
SiO
SiO
S
O
O
O
O
O O
OO
O
SO
O
O
FF
F
O
FF
F
SS
Cleaned quartz crystal
OH OHOH OH
OEG-TUBTS/7-OEG
PhMe, rt, time
EMPAS measurements for OEG-TUBTS/7-OEG SAM
29
Specific to non-specific ratio – 2:1 High reproducibility Immobilization under aqueous condition is possible
OEG-TUBTS/7-OEG SAM formation on quartz crystal: Time Trial
0
20
40
60
80
100
0 30 60 90 120 150 180 210 240
Time (min)
Co
nta
ct A
ng
els
(deg
ree)
30
CAM and XPS values both continued to change after 120 min Indicated that the silanization process was not complete by 120 min
OEG-TUBTS/7-OEG SAM formation on quartz crystal: Time Trial (cont’d)
31
Closer look at the %F and %S in XPS analysis
Sulfur unique to OEG-TUBTS Fluorine unique to 7-OEG
Possible multilayer formation Dramatically decrease the biosensing
performance of our surface
Decreased the silanization time to 60 min to avoid multilayer formation
EMPAS measurements for OEG-TUBTS/7-OEG SAM with reduced silanization time
32
Specific to non-specific ratio – 15:1 High reproducibility
Conclusions for work on linker• Successfully prepared SAMs onto piezoelectric quartz crystals with new
thiosulfonate-based linkers
• Chemoselectively immobilized biotinthiol under aqueous conditions in a single, straightforward, reliable and coupling-free manner
• With OEG-TUBTS/7-OEG system, we demonstrated a 15-fold difference in signal response of EMPAS between specific and non-specific measurements for avidin interaction
• Same chemistry for device in goat serum spiked with avidin gives a 6-fold signal ratio – best we have ever observed
33
And what we have learned
• Proteins adsorb to hydrophilic and hydrophobic surfaces just about equally
• Modified optically flat surfaces with SAMs in place produce high NSB
• For steric reasons you need a receptor functioning in tandem with a surface diluent
• The linker chain length must be about 5 C longer than the diluent
• PEG functionality does reduce NSB very significantly• Receptor exclusion volume plays a crucial role
Ovarian CancerOverview
• Most serious gynaecologic cancer with ≈ 1700 deaths every year in Canada
• Cancer patients develop a mechanism to evade and suppress the immune system
• Ovarian cancer cells have reduced expression in signal transducing zeta chain molecules (e.g. CD3-zeta) reduced expression of T-cell receptor molecules ( and ) suppressed T-cell activation and proliferation reduced cytokine production and proliferative response
Ovarian Cancer
Cause
• Proteomic studies revealed an early pregnancy factor (EPF) in the serum and urine of pregnant women during the 1st and 2nd trimesters
• This EPF has been identified as a heat shock protein 10 (HSP10)
• Cancer cells were found to produce HSP10 and release it to the cytoplasm, extracellular ascites and peripheral blood
• HSP10 was associated with the reduction of T-cell CD3-zeta
expression and immunosuppression
5) On-line Detection of HSP10: TSM Response
Possible indication of aptamer conformational change upon HSP10 binding
Detection of HIV Antibodies in Blood
• Screening test for HIV takes 3 drops of blood and effected in 2.5 minutes
• Commercial kits available in several countries such as China, India and Canada
• Confirmatory test for HIV requires positive detection of 10 Ab in blood
• Confirmation uses electrophoresis and blotting, 3 days and is costly
Towards Multiplexed HIV Ab Detection Using Acoustic Wave
Physics
• Develop flow-through label-free EMPAS electromagnetic system for diagnostic assays
• Attachment of probe (antigen/peptide) to device surface
• Surface chemistry to maximize analytical signal and minimize response for NSB (serum-blood?)
• Design engineer multiplexed system• Extend to replace ELISA approach to diagnostics
Clean quartz crystal
2) NaI, acetone, rt, 1.5h
O
Si
O
SiO
O
O
SiO
O
SiO
OOH OH OH OH
OO
I I
1) CATD/HTS (50/50 v/v)toluene, rt, 2h
PBS buffer only
Injection of antibody solution
Return to buffer only flow
(rinse-off)
Frequency shift
Collaboration with UK MOD Porton Down
• MOD has developed rapid response SPR system for detection of bacteria/viruses
• Similar to diagnostics – based on Ab/aptamer probes on gold substrate
• Serious issue with interference of particles/non-specific binders
• Developed long-chain, PEG thiol linker
Principle of Scanning Kelvin nanoprobe
Lord KelvinLord Kelvin The original apparatus of Lord Kelvin The original apparatus of Lord Kelvin
The Scanning Kelvin Nanoprobe is Based on the Measurement of the Local Work Function
Evacuum
1
1
2
2----
++++
1
2
eV
1
2
1 2 1
+
V0= -Vd
Two metals are separated by a distance d
At electrical contact, equalization of Fermi levels, surface charging, electron flow
Inclusion of a backing potential V0, null-field condition achieved when V0 = -V
Block Diagram of the Scanning Kelvin Nanoprobe
Vibrationpiezo
tipsampleinsulator Topography control
piezo
XY-scantable
Sample voltage
power supply
piezo driver signal generator2kHz
lock-in amp. 1
lock-in amp. 2
signal generator100kHz
NI BNC-2120 interface
CPD signal
topography signal
shielded cable
PC with LabViewNI PCI 6160 DAQ BoardC-842.20 DC Motor Controller
motorscontrol
Sum
circuit
Charge amplifier
pie
zo d
rive
r
DNA Microarrays
Array map showing the exact position of duplicates and the number of mismatches
Surface potential image of the scanned oligonucleotide microarray
Protein Microarrays
Image of Rabbit IgG protein microarray (35 spots in a 7x5 grid) showing the dependency of work function level on the
protein abundance in different spots
SKN
Mike Thompson Research Group 2010
• Jack Sheng Sumra Bokhari
• Sonia Sheikh Dr. Larisa Cheran
• Shilin Cheung Alin Cheran
• Elaine Chak Miguel Neves
• Kiril Fedorov Timothy Chung
• Pat Benvenuto
• Dr. Chris Blaszykowski