Electrochemical Sensing of Cancer Biomarkers - … Sensing of Cancer Biomarkers ... post-translational modification that attaches glycans ... Aptamer on polypyrrole Specificity study:
Post on 15-Mar-2018
222 Views
Preview:
Transcript
Electrochemical Sensing of Cancer Biomarkers
Pedro Estrela Coordinator of the Marie Curie ITN PROSENSE Department of Electronic & Electrical Engineering University of Bath, UK
DNA Aptamers
Aptamer Self-Assembled Monolayers (PSA)
Binary SAM
Sulfo-betaine moiety
Aptamer within Molecular Imprinted Polymers (PSA)
Aptamer on polypyrrole
PEG – ANTA/Cu2+ (AMACR)
Direct linking (PSA)
microRNAs
PNA-AuNP
Contents
• DNA aptamers are single-stranded DNA that can bind to their targets with
high affinity and specificity by undergoing conformational changes
• DNA aptamers have a number of advantages over antibodies, in
particular with regards to their lower cost, easy manipulation and potential
for controlled chemical attachment to electrodes
PSA
DNA Aptamers
• Different DNA aptamers have different secondary structures
PSA aptamer
Single stem and loop
AMACR aptamer
Multiple stem and loop
Thrombin aptamer
Quadruplex
DNA Aptamers
A. Direct immobilisation (binary SAM, thiol chemistry)
10-3 10-2 10-1 100 101 102 103 104
0
-5
-10
-15
-20 PSA --- Background
ΔRct (%
)
[PSA] [ng/mL]
60 ng/mL • Detection via electrochemical impedance
spectroscopy (EIS) the presence of redox
markers
Aptamer SAMs
Glycosylation – post-translational modification that attaches glycans (carbohydrate chains) to proteins, lipids, or other organic molecules Glycoprofiling – determining the glycan composition of the protein, cell, tissue, etc Aberrant glycosylation – characteristic for tumorigenesis, indication of cancer → studying structure of the oncomarker, rather than its level (PSA)
Lectins – proteins that react specifically with glycosidic residues of other molecules, act as a biorecognition element
Electrochemical glycoprofiling of PCa biomarkers
Prostate Specific Antigen (PSA)
PSA + glycosilated PSA
Jolly et al., Biosens Bioelectron 79 (2016) 313
DNA aptamers / Lectins
0.01 0.1 1 10 100
0
20
40
60
80
100
Aptamer-PSA-Antibody Aptamer-PSA-Lectin
chem
ilum
ines
cenc
e
[PSA] (ng/mL)
0.01 0.1 1 105
10
15
20
25
30 1:50 ratio of Aptamer to MCH
ΔRct
(%)
[PSA] [ng/mL]
B. Immobilisation on self assembled gold nanoparticles
10 pg/mL
Aptamer SAMs
0 1 2 30
1
2
3 Aptasensor 100 µM HSA
-Z ''
(kΩ
)
Z ' (kΩ)
• High non-specific binding with mercapto-hexanol (MCH) based SAM
ca. 15%
• Alternative: Use of antifouling surface chemistry (Sulfo betaine moiety) S
S HN
N
SO3
OH MeMe
Aptamer SAMs
C. Use of Antifouling surface chemistry (Sulfo-betaine moiety)
Aptamer SAMs Jolly et al., Sens. Actuators B 209 (2015) 306
Aptamer SAMs C. Use of Antifouling surface chemistry (Sulfo-betaine moiety)
Jolly et al., Sens. Actuators B 209 (2015) 306
0 20 40 60 80 1000
20
40
60
80
100
-Z ''
(kΩ
)
Z ' (kΩ)
Aptasensor 100 µM HSA
MCH Sulfo-betaine
0
5
10
15
20
∆R
ct (%
)10-1 100 101 102 103 1040
-10
-20
-30
-40
∆R
ct (%
)
[PSA] (ng/mL)
• Reduction of non-specific binding of HSA to less than 2%
• Increased sensitivity due to combined effect of linker length and sulfo-betaine
Detection down to 1 ng/mL (60 times lower than MCH-based surface chemistry)
Aptamer SAMs Jolly et al., Sens. Actuators B 209 (2015) 306
• Hybrid DNA aptamer / molecular imprinted polymer
Advantages: • DNA aptamers used for controlled surface
chemistry • Resistant to stringent fabrication process:
polymerisation, washing with 5% SDS and 5% acetic acid
Aptamer MIPs Jolly et al., Biosens. Bioelectron. 75 (2016) 188
Detection down to 1 pg/mL (1000 times lower than betaine-based surface chemistry)
• Increased sensitivity which can be attributed to imprinting effects
• Potentially minimise nuclease degradation
Aptamer MIPs Jolly et al., Biosens. Bioelectron. 75 (2016) 188
In mammalian cells, the enzyme is responsible for converting (2R)-
methylacyl-CoA esters to their (2S)-methylacyl-CoA epimers
Biomarker for prostate cancer with high sensitivity of 77.8% and specificity
of 80.6%
It is still a tissue biomarker but studies have shown its presence in blood in
the range of µg/mL and fg/mL in urine samples
Have high potential to complement PSA screening in identifying patients
with clinically significant prostate cancer, especially those with
intermediate PSA levels
AMACR (α-methylacyl-CoA racemase)
NH
O
O
O
HN
NO
O
OOO
O
Cu2+
HN
N
N
NH
Gold
DNA aptamer as bioreceptor
-0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.41
2
3
4
5
6
7
8
9
10
Cur
rent
(µA)
Voltage (V vs. Ag/AgCl)
ANTA ANTA + Cu2+
ANTA + Cu2+ + Aptamer
Square Wave Voltammetry
Aptamer on polypyrrole Jolly et al., submitted
ANTA-Copper complex as
redox marker
Polyethylene glycol for antifouling
properties
Polypyrrole as sensor surface
NH
O
O
O
HN
NO
O
OOO
O
Cu2+
HN
N
N
NH
Gold
NH
O
O
O
HN
NO
O
OOO
O
Cu2+
HN
N
N
NH
Gold
AMACR
Capture of AMACR
Aptamer on polypyrrole
10-1710-1610-1510-1410-1310-1210-1110-10 10-9 10-8 10-7 10-60
5
10
15
20
25
30
35
40
ΔI/I 0(%
)
AMACR [M]
Jolly et al., submitted
-0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4
2
3
4
5
6
7
8
Cur
rent
(µA
)
Voltage (V)
Aptasensor (PEG) Blank 4% HSA
Polyethylene glycol (PEG) based surface chemistry
< 3% change in signal on incubation with 4 % HSA for 30 min
Aptamer on polypyrrole Specificity study: 4% Human Serum Albumin (HSA)
Jolly et al., submitted
AMACR PSAPSA-ACT hK2
0
5
10
15
20
25
30
35
40
ΔI/I 0(%
)
Negligible signal change with other prostate cancer biomarkers (all proteins used were of same concentration 100 nM)
Aptamer on polypyrrole Specificity study: other prostate cancer biomarkers
Jolly et al., submitted
10-16 10-15 10-14 10-13 10-12 10-11 10-10 10-9 10-8 10-7
0
2
4
6
8
10
12
14
16
18
20
ΔI/I 0(%
)AMACR [M]
• Potential to develop multiplexed platform
• Copper can be replaced with other metal ions
(nickel, zinc, etc.)
Detection in human plasma samples
• Detection via square wave voltammetry • Broad range from 0.1 fM to 10 nM • Detection limit down to 1.4 fM
Aptamer on polypyrrole Jolly et al., submitted
Functionalisation of polypyrrole with carboxylate groups
NH2 NH2 NH2 NH2
Electrochemical deposition of amine terminated aptamers
Capture of PSA
• One-step easy and fast deposition of probes bearing amines
• Detection method: EIS without any redox marker
Aptamer on polypyrrole (direct)
0 10 20 30 40 50 60 700
10
20
30
40
50
60
70 PSA Aptamer PSA 10 µg/mL
-Z''(
kΩ)
Z' (kΩ)
ca. 123%
0 5 10 15 20 25 30 35 400
5
10
15
20
25
30
35
40 Random DNA sequence PSA 10 µg/mL
-Z''(
kΩ)
Z'(kΩ)
• Preliminary experiments with PSA aptamers
• Also being used for DNA/DNA hybridisation
• Negligible signal change with a random DNA sequence
Aptamer on polypyrrole (direct)
• Small (18-25 nt long) non-coding RNAs that are
involved in regulation of gene expression (post
transcriptional regulation)
• Increasing reports on role of miRNAs in oncogenic
processes such as proliferation, apoptosis,
differentiation and development of androgen
independence
• Consequently, studies show that the altered levels of miRNA in blood can act like finger
prints of cancer (diagnosis, prognosis and also the stage of the cancer)
• Different miRNAs are associated with different diseases and also published for essentially
all cancer forms including prostate cancer
microRNAs
PNA
DNA-DNA interactions: • due to charge screening / counterion condensation, change in net charge
upon hybridisation is small • formation of duplex “thickens” DNA layer, increasing electrostatic barrier to
[Fe(CN)6]3-/4- in-between DNA sites
PNA-DNA interactions: • initial probe layer has no electrostatic barrier • hybridisation with DNA results in large increase in electrostatic barrier
(a) (b)
Electrode surface
3’ 3’
OH
OH
O
H
OH
O
H
OH
OH
O
H
OH
O
H
OH
OH
3’
3’ 3’ 3’
5’ 5’ 5’
Electrode surface
3’ 3’
OH
OH
O
H
OH
O
H
OH
OH
O
H
OH
O
H
OH
OH
3’
3’ 3’ 3’
5’ 5’ 5’
(c)
3’
OH
3’
5’
Capture PNA probe (neutral)
Spacer molecule
Complementary strand (negatively charged) to capture PNA
Positively charged gold nanoparticles (self-assembled polyethylenimine)
Electrode surface
3’ 3’ 3’
OH
OH
O
H
OH
O
H
OH
OH
O
H
OH
O
H
OH
O
H
EIS: PNA with AuNPs
• Electrochemical Impedance Spectroscopy (EIS) was used in the presence of redox marker to confirm the concept
• PNA creates physical barrier to negatively charged redox couple in solution: [Fe(CN)6]3-/4-
Electrode surface
3’ 3’ 3’
OH
OH
O
H
OH
O
H
OH
OH
O
H
OH
O
H
OH
O
H
0 2 4 6 8 10 12 140
2
4
6
8
10
12
14
-Z"
(kΩ
)
Z' (kΩ)
PNA
EIS: PNA with AuNPs
Jolly et al., submitted
• Electrochemical Impedance Spectroscopy (EIS) was used in the presence of redox marker to confirm the concept
• PNA creates physical barrier to negatively charged redox couple in solution: [Fe(CN)6]3-/4- • Charge transfer resistance (Rct) significantly increased with target miRNA by increasing the
electrostatic barrier
Electrode surface
3’ 3’
OH
OH
O
H
OH
O
H
OH
OH
O
H
OH
O
H
OH
OH
3’
3’ 3’ 3’
5’ 5’ 5’
0 2 4 6 8 10 12 140
2
4
6
8
10
12
14 PNA 100 nM complementary miRNA
-Z"
(kΩ
)
Z' (kΩ)
EIS: PNA with AuNPs
Jolly et al., submitted
Electrode surface
3’ 3’
OH
OH
O
H
OH
O
H
OH
OH
O
H
OH
O
H
OH
OH
3’
3’ 3’ 3’
5’ 5’ 5’
• Electrochemical Impedance Spectroscopy (EIS) was used in the presence of redox marker to confirm the concept
• PNA creates physical barrier to negatively charged redox couple in solution: [Fe(CN)6]3-/4- • Charge transfer resistance (Rct) significantly increased with target miRNA by increasing the
electrostatic barrier • Charge transfer resistance (Rct) significantly decreased with AuNPs
0 2 4 6 8 10 12 140
2
4
6
8
10
12
14 PNA 100 nM complementary miRNA 100 nM complementary miRNA + AuNPs
-Z"
(kΩ
)
Z' (kΩ)
EIS: PNA with AuNPs
Jolly et al., submitted
CONTROLS
• AuNPs do not interact with SAM (red curve) (~2%) • Negligible interactions with non-complementary DNA (1.08%) and also with BSA (Bovine
Serum Albumin, 2.3%)
BSA
Electrode surface
3’ 3’ 3’
OH
OH
O
H
OH
O
H
OH
OH
O
H
OH
O
H
OH
O
H
0 2 4 6 8 10 12 140
2
4
6
8
10
12
14
-Z"
(kΩ
)
Z' (kΩ)
PNA PNA + AuNPs
EIS: PNA with AuNPs
Jolly et al., submitted
Z’ : Real Part of impedance Z’’ : Imaginary part of impedance ǀZǀ2 : ((Z’)2 +(Z’’)2)
𝐶𝐶 =−𝑍𝐶𝐶
ω 𝑍 2
𝐶𝐶𝐶 =−𝑍𝐶
ω 𝑍 2
• Impedance measurements without redox markers
• Very high impedance is observed
0 200 400 600 800 1000 1200 14000
500
1000
1500
2000
2500
3000
3500 PNA PNA + miRNA (100 nM) Attachment of AuNPs
- Z ''
(kΩ
)
Z ' (kΩ)
Non-Faradaic EIS: PNA with AuNPs
C* ≡ -1/jωZ
Jolly et al., submitted
Monitoring non-Faradaic processes
Cole - Cole plot
0.0 0.1 0.2 0.3 0.4 0.50.00
0.05
0.10
0.15
-C''
(µF)
C' (µF)
PNA PNA + miRNA (100 nM) Attachment of AuNPs
0 200 400 600 800 1000 1200 14000
500
1000
1500
2000
2500
3000
3500 PNA PNA + miRNA (100 nM) Attachment of AuNPs
- Z ''
(kx(
03A9
))
Z ' (kx(03A9))
Non-Faradaic EIS: PNA with AuNPs Nyquist plot
Jolly et al., submitted
10-16 10-15 10-14 10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-60
5
10
15
20
25
30
35
40
45
50
55
% C
hang
e in
Cap
acita
nce
miRNA [M]
• Potential detection down to 1fM of complementary miRNA strand
6% change
Non-Faradaic EIS: PNA with AuNPs
Jolly et al., submitted
100 nM non complementary miRNA target
gold nanoparticles
Control experiments output
• Around 1.5% capacitance change with just AuNPs was observed
• With non-complementary miRNA (100 nM), around 2% change was recorded
• With 100 nM of miRNA sequence with 2 mismatch, around 2.5% change was recorded
• With 1 mismatch sequence (100 nM), around 20% change was observed
0.0 0.1 0.2 0.3 0.40.00
0.05
0.10
0.15
0.20 PNA PNA + non-comp miRNA
C' (µF)
-C''
(µF)
0.0 0.1 0.2 0.3 0.40.00
0.05
0.10
0.15
0.20
-C''
(µF)
C' (µF)
PNA PNA + AuNPs
Non-Faradaic EIS: PNA with AuNPs
Jolly et al., submitted
Electrode surface
3’ 3’
OH
OH
O
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
3’
3’ 3’ 3’
5’ 5’ 5’
Electrode surface
3’ 3’
OH
OH
O
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
3’
3’ 3’ 3’
5’ 5’ 5’
Fe SH
Fe
Fe
Amperometric: PNA with AuNPs
Jolly et al., submitted
• Square wave voltammetry was used to monitor ferrocene peaks for different concentrations of miRNA
Dose Response • Provision of dual detection technique
10-16 10-15 10-14 10-13 10-12 10-11 10-10 10-9 10-80
1
2
3
4
5
6
7
8
9
10
Cur
rent
(µA)
comp miRNA [M]
Control experiments output
• Around 1 µA peak current with just AuNPs
• With non-complementary miRNA (100 nM), around 1.2 µA
• With 100 nM of miRNA sequence with 2 mismatch, around 1.6 µA was recorded
• With 1 mismatch (100 nM) sequence, around 7 µA was observed
Amperometric: PNA with AuNPs
Jolly et al., submitted
Antibodies Antibody fragments
Peptides Affimers
DNA aptamers MIPs
Lectins
DNA, PNA, LNA
The near-future…
fPSA PSA
AMACR HER2
…
glycosilation
miRNAs
i-STAT®, Abbott
Potentiometric Impedimetric Amperometric
p.estrela@bath.ac.uk go.bath.ac.uk/biosensors
Acknowledgments
Marie Curie Initial Training Network “Cancer Diagnosis: Parallel Sensing of
Prostate Cancer Biomarkers” (PROSENSE)
www.prosense-itn.eu
Biosensors Group: Pawan Jolly, Anna Miodek
Dept Pharmacy & Pharmacology, Uni Bath: Matthew Lloyd
Cardiff University: Vibha Tamboli, Chris Allender, Jenna Bowen
University of São Paulo: Marina Batistuti, Marcelo Mulato
Slovak Academy of Sciences: Peter Kasák, Jan Tkáč
National Taiwan University: Deng-Kai Yang, Lin-Chi Chen
Latest issue of Essays in Biochemistry
Covering an introduction to biosensors, discussion of analytical biosensors, and applications of biosensors, including in the biomedical field Guest edited by Pedro Estrela, University of Bath, U.K. Available at essays.biochemistry.org/content/60/1
PROSENSE Conference on Prostate Cancer Diagnosis Bath, 12-13 September 2016 Deadline for abstracts: 31 July 2016
BioNanoScience: topic Issue on Prostate Cancer Diagnosis Deadline for papers: 23 September 2016
Follow links on http://go.bath.ac.uk/biosensors
top related