Biosensors Based on Electrogenerated Chemiluminescence (ECL) Detection

ECL Determination of Immobilized DNA and CRP Protein on Au (111) Electrodes Using Ru(bpy)3

2+ Labels

Wujian Miao

Outline of Talk

•Introduction and General Principles

•Annihilation and Coreactant ECL

•DNA Hybridization and C-Reactive Protein (CRP)

•Ru(bpy)32+ Entrapped Microspheres

•Conclusions

Photoluminescence

Es

LUMO

HOMO

1R0

(Ground State)

1R1

(Excited State)

+ hv

1R0

1R1(R*)

E = hv = hc/λ = 4.13×10-15 (eV s)×3.00×108 (m/s)/λ (m)

VISIBLE REGION

ULTRA-VIOLET

(UV)

INFRA-RED(IR)

λ

3.2 eV > Es > 1.6 eV

390 nm 780 nm

Wavelength

(DPA)

FluorescenceEmission Spectrum

of DPA In Cyclohexane

9, 10-Diphenylanthracene

1R1

1R0

3.0 eV

1.7 eV3R1

λ ~ 410 nm

(R*)

Es

Photoluminescenceis a process

that using light to generate light

Chemiluminescence (CL)— Cool light

Reactionsthat produce light

without heatare called

Chemiluminescent (CL) Reactions

YES

NO

A-

(RED)

+ +

DD+

(OX)

A*Excited State

A,Ground State

Chemiluminescence--Mechanism

hν

e e

e

A D

A- D+

DA*hν

Cathode Anode

- +ECL

e e

e

A D

A-D+

D

Cathode Anode

-+

A

Eco Ea

o

Eao - Ec

o - 0.1 > ES(Es ~ 2 to 3 eV)

Criterion for excited state formation

ECL

Ea0 = 1.30 V

Ec0 = -1.85 V

In MeCN at Pt

Ea0 –Ec

0 - 0.1 = 1.30 –(-1.85) –0.1 = 3.05 eV > Es (3.0 eV)

(DPA)

Ring Disk Teflon

9,10-diphenylanthracencein MeCN at Rotating Ring Disk Electrode

Es = 3.0 eVl ~ 410 nm

Ea (+) (disk)

Ec (-)(ring)

Disk Ring(DPA)

Two Types of ECL

D+ + A- A + D*

D + hv

1.Annihilation ECL (A = D = DPA)

+

_

tE

(Two Electrodes) (One Electrode with an AC Potential)

Two Types of ECL

D+ + Int D* + Product

(Where Int is formed on Oxidation)

D + hv

2. Coreactant ECL(D + Coreactant)

E t

+

_

N

N

N

N

N

Ru

2+

Ru(bpy)32+

Coreactant ECL

(CH3CH2CH2)3N:

TPrA

(TriPropyl Amine)

(As coreactant)

This System Gives the Most Efficient ECL Known So Far.

IECL & ICV ~ E Profiles forRu(bpy)3

2+(1 mM) /TPrA (10 mM) System

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0-0.4

-0.2

0.0

0.2

0.4

CV

ECL

E (V va Ag/AgCl)

I CV(m

A)

-0.6

-0.3

0.0

0.3

0.6

TPrA and Ru(bpy)32+ Oxidation

Light Emission

1 2 3 4 50

1

2

3

4

5 [TPrA ] = 0 .10 M , pH = 7

Log

[IEC

L, nA

]

L og [Ru(bpy)3

2+, pM ]

[Ru(bpy)32+] 8 IECL

ECL Mechanism

TPrA.

-H+

TPrA

TPrA.+- e- (1)

- e-

Ru(bpy)33+

Ru(bpy)32+

(2) *Ru(bpy)32+

(3)

TPrA = Tripropylamine*Ru(bpy)3

2+ = Excited Ru(bpy)32+

Ele

ctro

de

0 ~ 1.4 V

(Light)

ECL Detector

(λmax ~ 620 nm)

W. Miao, J-P, Choi, A.J. Bard, J. Am. Chem. Soc., 124 (48), 14478 -14485, 2002.

ECL Analysis Advantages

•No Light Source Required•No Scattered Light•No Interference From Luminescent

Impurities •High Sensitivity, Selectivity and Stability•Reactions Are Localized and Controllable

--Good Spatial and Temporal Resolution

DNA Antibody-Antigen

ECL Applications

ECL

DNA Double Helical Structure

Unknown DNA(Target DNA)

Known DNA(Probe DNA)

Immobilizationlayer

Substrate(Electrode)

+Hybridization

ECL Detection •Large IECLà Complementary

•Small IECLà Base-pair mismatched

•IECL ~ 0 à Non-complementaryRu(bpy)32+

Label

ds-DNA

ECL Detection with Sandwich Type Immunoassay

Substrate(Electrode)

Immobilization layer

Antibody Antigen

ECL label--Ru(bpy)32+

ECL Experimental Set-up

Interface

Potentiostat

PMThv

Electrolyte Solution0.10 M TPrA/LiClO4/Tris, pH 8

WE

RE

AE

Coreactant--Tripropylamine

Immobilization of ssDNA onto Electrode

SH

C

S C

S C

(Amino-ssDNA)

H3C NC

NHN

CH3H3C

Cl_

1-Ethyl-3-(3-dimethylaminopropyl) Carbodimide Hydrochloride

EDC/EDAC

N

N

CH3

EDC + 1-methyl-imidazole/HCl Buffer, pH 7

S C

S C

OH

O P

OH

O

OH2N

OHO P

OH

OO

HN

OHO P

OH

OO

HN

1-methyl-imidazole

OH

O

OH

O

O OH

O

O

AuAu

Immobilization of Antibody onto Electrode

S C OH

O

AuAu

Mixed Thiols (10:1 v/v)

HS COOH15

HSCOOH

1

S C OH

O

Avidin

(EDC + NHS)

S C NH-Avidin

O

S C

ONH-Avidin

Biotinylated Antibody

Au

Avidin layer

Antibody

N-OH

O

O

NHS (N-Hydroxysuccinimide)

Avidin— Biotin Interaction(Non-Covalent Binding)

+

Avidin— BiotinConjugate

Ka-- Affinity Constant (Biological Reaction)

Ka ~ 1×1015 M

Avidin

NHHN

S

O

COOH

Biotin

Surface Coverage Verification ofSAMs and/or Avidin Layer on Electrode

S C

S C

OH

O

OH

O

Au

S C NH-Avidin

O

S C

ONH-Avidin

S C

S C

NH-Avidin

O

NH-AvidinO

Au

AvidinEDC

BiotinylatedFluorescein

(Fluorescein Biotin)

Avidin layer

Fluorescent Imaging

Fluorescent Images(Excited at ~490 nm, Monitored at ~520 nm)

Au/S(CH2)2CO-NH-AvidinßFluorescein Biotin(Exposure time: 3s)

Dark— Au/SAMs only

Dark— Au/SAMs only

Au/S(CH2)nCO-NH-Avidin ßFluorescein Biotin (n=2 & 15,

Exposure time: 30s)

ECL Tags Used for DNA and Antibody Labeling

N

N

N

N

N

N

Ru

OP

OCN

N

2+

Ru(bpy)32+

Phosphoramiditefor DNA Labeling

N

N

N

N

N

N

Ru

O

O

N

O

O

Ru(bpy)32+ NHS Ester

for Antibody Labeling

Available from IGEN, Inc.

Modification of ssDNA with Ru-TagOH

O POH

OHO(5')

(3')

OP

OCN

N

[Ru(bpy)32+]

O POH

OHO(5') (3')

OPO

[Ru(bpy)32+]

OH

O

ssDNA

Ru-Phosphoramidite

ssDNA tagged with Ru(bpy)32+

Exp. Conditions:

(1) Pure ssDNA(2) Excess of Ru-Tag

(~100 times to DNA)(3) In CH3CN (+ ~H2O for a long DNA sequence)(4) R.T., 30 ~ 60 min

with shaking in the dark(5) Gel filtration/separation

Modification of Antibody with Ru-Tag

NH2O

O

N

O

O

[Ru(bpy)32+]

NH

O

[Ru(bpy)32+]

Ru-NHS Ester Antibody

Antibody Tagged with Ru(bpy)32+

N-OH

O

O

NHS

(1) Ru-NHS pre-dissolved in DMSO(2) In PBS buffer, pH ~7.2

(3) R.T. for ~ 1hr with shaking in the dark(4) Gel filtration/separation

1. How to separate the newly formed complexes from the starting material Ru(bpy)3

2+ tag?

2. What is the composition of the complexes?

Principle of Gel Filtration

Small molecule

Large molecule

Gel filtration resin

Small molecules are“included” and elute last

Large molecules are“excluded” and elute first

Verification of DNA-Ru Tag Formation

ssDNA: 5’-CTCCA AATGT AGGAG CTATC GTT-3’ (23-mer)

240 280 320 360 400 440 480

259 (0.836)

245 (0.268)

287 (0.747)

457 (0.137)

257 (0.972)278 (0.908)

457 (0.089)

Wavelength (nm)

Abs

orba

nce

ssDNA (23-mer)

Ru Tag (13.3 µM)

ssDNA-Ru Tag

ssDNA:Ru Tag = 1:2

Biotinylation of Antibody WithSulfo-NHS-LC-Biotin

NH2

NH

Sulfo-NHS-LC-BiotinAntibody

HNHN

S

O

NH

O

O

HNHN

S

O

ONH

O

O

N

O

O

NaO 3S

Biotinylated Antibody

N-OH

O

O NHS

(1) ~ 12 fold excess of biotin(2) PBS buffer, pH ~ 7.2(3) R.T. for ~ 30' with shaking(4) Gel filtration/separation

Inert Gas Spraying Reduces Non-specific Adsorption of Ru(bpy)32+

Au/Si pre-cleaned withUV (10’)— MeOH (20’)

Au/Si Immersed in1 mM Ru(bpy)32+ (60’)

ECL carried out in0.10 M TPrA/LiClO4

/Tris buffer, pH 8

Scan rate: 50 mV/s0

5

10

15

20

25

30

35

00.20.40.60.811.2

I EC

L (nA

)

E (V vs Ag/AgCl)

a

b

c

Electrode washed with

(a) water + ethanol

(b) as (a) + "T-Duster" Spraying

(c) as (a) + N2 Spraying

Strategies for the Reduction of ssDNA-Ru Tag Non-specific Adsorption

SCOOH

SCOOH

SCOOH

SCOOH

SC

NH

NH 2-c-ssDNA/EDAC

Au Au

O

SC

NH

O

SC

OH

O

SC

NH

O

"pinhole"

ssDNA

"pinhole"

(Free -COOH)

SCOOH

SCOOH

SCOOH

SCOOH

SC

NHOH

NH 2CH 2CH 2OH/EDAC

BSA

Au Au

O

SC

NHOH

O

SC

NHOH

O

SC

NHOH

O

BSA"pinhole"

BSA: Bovine Serum Albumin

ECL Detection of DNA Hybridization

ssDNA-Ru Tag: Ru-5’-CTCCA AATGT AGGAG CTATC GTT-3’-Ru (23-mer)c-ssDNA: 5’-Amino-AACGA TAGCT CCTAC ATTTG GAG-3’ (23-mer)Non-c-ss-DNA: 5’-Amino-TTAACACCTTAGCGACGGCTAGT-3’ (23-mer)

-100

-50

0

50

100

-80

-40

0

40

80

00.20.40.60.81

I CV

(nA

)

IEC

L (nA

)

E(V vs Ag/AgCl)

ECL

CV

Non-complementary Hybridization(~ 11 % of specific adsorption)

Complementary DNA Hybridization

ssDNAderived

from the

Bacillus anthracis

(4 base-pair matches)

C Reactive Protein (CRP)

• An acute phase reactant.• Released by the body in

response to acute injury, infection, inflammatory stimuli.

• Silent Predictor of Heart Disease.

*CRP Structure, http://www.keele.ac.uk/depts/ph/research/px/home.html

*

0 5 10 15

0

500

1000

1500

2000

2500

3000

[CRP] vs Time

Kushner: Hosp. Pract. 1990: 25, 218

Acute phasesitimulation

% C

hang

e in

Pla

sma

Leve

l

Days

Correlation between [CRP] andthe risk of coronary heart disease

For apparently healthy people: Mean value of CRP:~1.9 µg/mL.

Highest Risk3.9 - 15.05

High Risk2.0 - 3.84

Moderate Risk1.2 - 1.93

Low risk0.7 - 1.12

Lowest Risk< 0.71

Risk of Coronary Heart DiseaseCRP (µg/mL)Quintile

ECL Detection of C Reactive Protein

When cCRP = 23.5 µg/mL

-100

-50

0

50

100

-400

-200

0

200

400

00.20.40.60.811.2

I CV

(nA

)IE

CL

(nA)

E(V vs Ag/AgCl)

ECL

CV

Non-specific adsorption(1.5% of specific adsorption)

Correlation Between cCRP and IECL

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

3 0 0

3 5 0

4 0 0

0 5 1 0 1 5 2 0 2 5

I ECL (n

A)

cC R P

( µ g / m L )

Determination of [CRP] in Human Serum and Plasma

0

50

100

150

200

-8 -6 -4 -2 0 2 4 6 8

cCRP

Added (µg/mL)

I EC

L (nA

)

a

b

(a) Citrated human Serum(b) Citrated and lyophilized human plama

6.7 µg/mL

5.7 µg/mL

(Standard Addition Method)

So far,......

•General principles of ECL•Attachment of receptor to electrode•Characterization•Labeling and binding of analyte•DNA and CRP Detection

ECL Based Biosensors –Promising

Limitations of Currently Used Method

O POH

OHO(5') (3')

OPO

[Ru(bpy)32+]

OH

O

ssDNA tagged with Ru(bpy)32+

NH

O

[Ru(bpy)32+]

Antibody Tagged with Ru(bpy)32+

1.Only one or few ECL Labels can be attached to t-DNA or antibodies à Low detection limit2. Two different ECL labels for DNA and antibody labeling

New Strategies…

YY Y

Y+

YMagnetic bead

ECL label loadedpolystyrene bead

Antigen

Antibody

Magnet

ssDNA(a)

(b)

Schematic diagram of (a) DNA hybridization, and (b) sandwich type immunoassay

Beads DissolutionIn MeCN

Anodic Coreactant(TPrA) ECL Detection

3.0 2.5 2.0 1.5 1.0 0.5 0.0-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

E (V vs Ag)

-200

-100

0

100

200ECL

CV

iEC

L (nA)i C

V (

mA

)

ECL Detection of DNA hybridization

SEM Images of c-DNA Hybridization

ECL Labels

• High ECL efficiency• Sufficiently soluble in organic solvents, but

completely insoluble in aqueous solutions

N

N

N

N

N

N

Ru

FF

FF

F

FF

F F

F

F

FF

F F

F

F F

FF

B

2

Ruthenium(II) tris(2,2'-bipyridine) tetrakis(pentafluorophenyl) borate

Ru(bpy)3[B(C6F5)4]2— Ru(bpy)3(Brookhart)2

How to Effectively Load ECL Labelsinto Polystyrene Beads?

COOH

COOHHOOC

COOH

ECL label

Organic solvents

COOH

HOOC

COOH

COOH

Carboxylate-modified polystyrene beads

Swellen beads containing ECL labels

or transfer to

an aq.phase

COOH

HOOC

COOH

COOH

EDAC/NHS

Dry under vacuum

Avidin

Fluorescent Images

After Ru(bpy)32+ Loading (a) + Avidin ß Fluorescein Biotin

Amplification factor:7×109 Ru(bpy)3

2+/1×105 ssDNA70,000 Ru(bpy)3

2+/ssDNA

Loading capacity:7×109 molecules/bead

ECL Detection of DNA Hybridization Using Ru(bpy)3

2+ Entrapped Microspheres

-16 -15 -14 -13 -12 -11 -10 -9 -8 -71.0

2.5

3.0

3.5

4.0 2.8 µm MB, MB/PSB = 4

t-ss-DNA 2-bp-m-ss-DNA nc-ss-DNA

Log [ct-ss-DNA

(M)]

Inte

grat

ed E

CL

Inte

nsity

[IE

CL(

µA)x

Tim

e(s)

]

Towards a Single Biomolecule Detection

Biotin-Ru/PS -Biotin

DNA Hybridization

M

Y Y

+

Immunoassays

Conclusions

•Biosensors based on ECL are promising.•The new strategy could have the ECL

signal amplified by 4 to 5 orders of magnitude.

•Ru(bpy)32+ entrapped microspheres can be

used to label both DNA and Proteins.•Single biomolecule ECL detection could

be achievable.

Acknowledgements

Prof. Allen J Bard

SupportMURI - DAAD 19-99-1-0207 (DOD, UT)

IGEN, Inc.

Questions & Answers

Why Biosensors?(DNA Probe Assays and Immunoassays)

A wide range of applications in the areas of, e.g.,

•Clinical Diagnostics•Forensic Chemistry•Environmental Investigations•Pharmaceutical Studies•Biological Warfare Agent Detections

Why ECL Based Biosensors?

ECLà Electrochemical + Spectroscopic Methods

•Very sensitive (~10 pM Ru(bpy)32+ in

TPrA)•No background ECL •No light source required•Reactions are localized and controllable•Relatively simple instrumentation

Technology Transfer: Biochips

ECL

TPrA(Tripropylamine)Coreactant

Potential Scanning from 0 to 1.4 V vs Ag/AgCl

Dose ECL Emission Need Direct Oxidation of Ru(bpy)32+?

Ele

ctro

de TPrA TPrAH+-H+

e

TPrA-H+

TPrA

.+

.

Ru(bpy)32+P1

Ru(bpy)3+ Ru(bpy)3

2+*

TPrA

+H+

hvRu(bpy)3

2+

A Route for the Generation of Ru(bpy)32+*

in DNA/Protein-Ru(II)/TPrA System

1. W. Miao, J-P, Choi, A. J. Bard, J. Am. Chem. Soc., 124 (48), 14478 -14485, 2002 2. W. Miao, A. J. Bard, Anal. Chem., 75, 5825-5834, 2003

Carboxylated Polystyrene Beads

(PS, d ~10 µm)

Magnetic Beads(MB, d ~1 µm)

Synthesis of the ECL LabelsRu(bpy)3Cl2 (aq) + 2Li[B(C6H5)4].nEt2O (n = 2-3)

Ru(bpy)3[B(C6F5)4]2 ( )

"Lithium Brookhart Salt"

+ 2LiCl + 2nEt2O

(a) Washedwith water

(b) Recrystallized from MeCN/water soln

Ru(bpy)3[B(C6F5)4]2 (Dried under vacuum)

How Many Ru(II)/PSB Can Be Detected?(In 0.50 mL 0.10 M TPrA-0.055 M TFAA-0.10 M (TBA)BF4-1% H2O, 10 µm PSB, Pt)

0 20 40 60 80 100 120

0

20

40

60

80

100

120

140

160

Loading capacity: ~7.5x109 Ru(bpy)

3[B(C

6F

5)]

2/bead

* With background ECL subtraction

~0.5 nM Ru(bpy)3

2+

Number of Beads loaded with Ru(bpy)3[B(C

6F

5)

4]2

Inte

grat

ed E

CL

Inte

nsity

[IE

CL(

nA)x

Tim

e(s)

]*

NC

N NH+

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC)

Cl-

O

O

NHO

N-hydroxysuccinimide (NHS)

R1 N C N R2 + R3 C

O

OH

R1 N C

H

N R2

OCR3

O

H+

(EDAC)

EDAC reacts with carboxylic acid group and activates the carboxyl group, allowing it to be coupled to the amino group (R4HN2) in the reaction mixture.

R1 N C

H

N R2

OCR3

O R4NH2NHR4CR3

OC

O

R1HN NHR2+

(Urea)

Carbodiimide Reaction

H2N

NH2

N+Br-

Ethidium bromide

The detection limit of Ethidium Bromide stained DNA using an UV transiluminator is generally accepted to be

approximately 1 ng. ~0.1 µM dsDNA (with 25-mer)

Gold Crystal Structure

90.00090.00090.000407.82407.82407.82

? / °ß / °a / °c /pmb /pma /pmCell parameters:

ccp (cubic close-packed)Structure:

Fm-3m (Space group number: 225)Space group:

1. Increasing the size of PSB2. Producing “hollow polymeric beads”

capable of encapsulating of solution-phase or solid-state Ru(bpy)3

2+

3. Loading ECL labels during the polymerization process of bead formation

Increasing Ru(bpy)32+ Loading Capacities

Hollow Polymeric Beads

SiO2

Amino functionlized

silica bead

-NH2 -COOH

CarboxylatedPSB

EDAC

NHS

SiO2

PS layer

Melting

Etching

x% HF

Hollow BeadECL labels

Org. Soln.

ECL labels

Using Different ECL Labels

N

N

NN

NN

R uN

NN

N

NN

R u

NN

N

N

N

NRu

(A ) (B )

(C )

Ph

Ph

Ph

Ph

Ph Ph2+ 2+

Ph

Ph

Ph

Ph

Ph

Ph

2+

R uthenium (II)4,7-diphenyl-1,10-phenanthroline

R uthenium (II) 4 ,4 '-diphenyl-2 ,2 '-bipyrid ineR uthenium (II) tris(2,2 '-b ipyridine)

EC L Label EC L Efficiency

R u(bpy)32+ 5%

R u(dp-bpy)32+ 14%

R u(dp-phen-)32+

24%

ECL Detection of DNAand Antibody/Antigen

•DNA: Synthesized DNA Sequences•Proteins: Human serum albumin

(HAS)/Anti-HAS; E Coli/Anti-E Coli; CRP/Anti-CRP; p16INK4A gene/Anti-p16INK4A gene (a predictive factor in many cancers)…

•Real samples: Human Serum/Blood…