Post on 10-Jul-2018
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…