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

Thanks everyone, for listening to me!!

And a special thanks to Mihaela

mikethom@chem.utoronto.ca