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

Aptamer MIPs: BioFET in serum Tamboli et al., submitted

• Detection of low levels of PSA in serum

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