Folding-based Electrochemical Biosensors

Rebecca Y. Lai

Department of Chemistry

University of Nebraska-Lincoln

1-16-09

““Traditional” BiosensorTraditional” Biosensor

Measure changes in adsorbed mass, polarizability, sterics or charge

Specific Binding

Biosensor

SignalSpecific Binding

Biosensor

Specific Binding

Biosensor

Signal

Signaling linked to a binding-specific change in the physical properties of the biopolymer

Specific Binding

Biosensor

Non-specific Binding Signal

SignalSpecific Binding

Biosensor

Non-specific Binding Signal

Signal

Signal

Folding-basedFolding-based Biosensors: Biosensors: Signal Transduction MechanismSignal Transduction Mechanism

Low background, fewer electrochemically active

contaminants in biological systems

Low mass, volume, power

Low cost, mass production

Parallelization, adaptable to arraying strategies

Example: glucose meter

ElectrochemicalElectrochemical Biosensors Biosensors

Materials and MethodsMaterials and Methods

Biosensor Interrogation Method: Alternating Current Voltammetry

Length: 1 mmWidth: 0.88 mmArea : 0.88 mm2

Microfabricated gold electrode array

2 mm diameter

Area : 3.14 mm 2

Gold disk electrodes

Counter electrode

Ref electrodeWorking electrode

Electrochemical DNA Sensor (E-DNA)Electrochemical DNA Sensor (E-DNA)

eT

eT

eT

eT

Gold Electrode

+ target sequence

Denaturation /

Re-annealing

MB

5’-HS-(CH2)6- GCAGTAACAAGAATAAAACGCCACTGC -(CH2)7-NH-3’-MB

*Not to scale

E-DNA SensorE-DNA Sensor

Reagentless (direct detection of aqueous DNA)

Sensitive (pM detection limit) Rapid detection Reusable Selective Specific

AC Voltammograms

Methylene blue Leucomethylene blue+ 2e-

+ H+

E-DNA SensorE-DNA Sensor

Reusable and Reproducible Selective

E-DNA Sensor in a Microfluidic ChamberE-DNA Sensor in a Microfluidic Chamber

***In-situ sensor fabrication, hybridization and regeneration

Sensor Device Sensor Response

AptamersAptamers

Aptamers are DNA or RNA molecules selected for their ability to fold

into well-defined, three-dimensional structures and bind to specific

molecular targets (e.g. proteins, small molecules) with high affinity.

Electrochemical Aptamer-Based (E-AB) Sensor:Electrochemical Aptamer-Based (E-AB) Sensor:PDGF DetectionPDGF Detection

Reagentless Reusable Rapid Specific Selective Parallelizable Signal-on sensor

+ PDGF-BB

Sluggish Electron Transfer Efficient Electron Transfer

MB

5’-HS-(CH2)6- CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-(CH2)7-NH-3’-MB

*Not to scale

Sensor Response to PDGF-BB

In 50% Blood Serum

Physiological concentration of PDGF in human serum = 400 pM (normal) – 700 pM (cancerous)

Detection Limit: 50 pM

(the mass ratio of PDGF-BB to serum protein is ~ 1 : 25 Million)

Ligand-induced folding occurs when a favorable binding free energy

overcomes an unfavorable folding free energy, producing a folded complex.

Peptide or Protein-base Electrochemical BiosensorsPeptide or Protein-base Electrochemical Biosensors

Ligand-induced Folding (LIF)

Electronic Peptide-based Sensor (E-PB Sensor)Electronic Peptide-based Sensor (E-PB Sensor)

Peptide Probes

Naturally occurring peptide capable of LIF

Engineered peptide capable of LIF

et

+ target

- target

etMB

MB

* Not to scale

E-PB Sensor:E-PB Sensor:HIV DetectionHIV Detection

Protein of Interest

Surface Glycoprotein gp120Transmembrane Glycoprotein gp41Matrix Protein p17Capsid Protein p24

E-PB Sensor:E-PB Sensor:HIV DetectionHIV Detection

Proteins of Interest

Surface Glycoprotein gp120Transmembrance Glycoprotein gp41Matrix Protein p17Capsid Protein p24

p17 p24 gp41 gp120

% of patients for antigen

62 58 18 38

% of patients positive for antibody

60 92 100 34

HIV-1 p24 capsid protein

Highly antigenic

epitope sequence

EAAWDRVHP

Anti-p124 antibody (IgG)

(~150 kDa)

Response in earliest stages of AIDS

Anti-p24 concentration in serum may be tens to hundreds of nanomolar

Probe Target

Detection of Anti p24 AntibodiesDetection of Anti p24 Antibodies

Sensor Response to Anti-p24 Antibodies

Probe Sequence: HS-C11-EAAWDRVHP-K-MB

+ Ab

- Ab

Future Direction of Future Direction of E-PB Sensor Research E-PB Sensor Research (Immediate!)(Immediate!)

Improve Sensor Specificity

Test sensor / self-assembled monolayer (SAM) using surface

plasmon resonance (SPR)

Change / modify epitope if necessary

New immobilization methods to increase surface coverage

Improve Sensor Sensitivity and Dynamic Range

New immobilization methods

Incorporate thiolated polyethylene-glycol (PEG)

Improve Sensor Selectivity

Future Direction of Future Direction of E-PB Sensor ResearchE-PB Sensor Research

Azide terminated alkanethiol + alkyne modified peptide w/ MB

* Not to scale

N3 N3

OHOH OH OH

+ alkyne-peptide-MB

+ Cu(I)

OHOH OHOH

Advantages:

Flexibility in SAM choices

Step by step characterization by electrochemical methods and SPR

Site-specific modification/sensor fabrication

Click Chemistry

Future Direction of Future Direction of E-PB Sensor Research E-PB Sensor Research

System of Interest:

HIV Detection (p24, p17, gp41, gp120)

Biosensor Array: Mixed-platform Detection

E-PB Sensor

E-AB Sensor

E-DNA Sensor

Hybrid E-PB Sensor

Hybrid E-PB SensorHybrid E-PB Sensor

Peptide nucleic acid (PNA) stem + Peptide loop

et

+ Ab

- Ab

etMB

MB

* Not to scale

Peptide Loop

PNA Stem

p24 Probe Sequence: HS-C11-GAT-EAAWDRVHP-ATC-MB

Future Directions in Device FabricationFuture Directions in Device Fabrication

Microfabricated Devices

Too Pricy and Time Consuming!

gold-plated carbon electrode

2 mm diameter

Area : 3.14 mm 2

Disposable Sensor Strip

Handheld Potentiostat

E-DNA Sensor on a Gold-plated Screen-printed E-DNA Sensor on a Gold-plated Screen-printed Carbon ElectrodeCarbon Electrode

2 mm diameter Area : 3.14 mm 2

Sensor Device Gold-plated Carbon Electrode

-0.2 -0.3 -0.4 -0.5

2.0

2.5

3.0

3.5

4.0 Serum

A

C C

urre

nt / A

Potential / V

-0.2 -0.3 -0.4 -0.5

2.0

2.5

3.0

3.5

4.0

4.5

Buffer Solution

AC

Cur

rent

/ A

Potential / V

Before

Regenerated

Hybridization

Buffer 100%Serum

Disposable Sensor Strip:

Pros: cost effective, amenable to mass production

Cons: larger volume requirement

(50 µL in-situ, 10 µL ex-situ)

Future Work:

E-PB Sensor for HIV detection

Volume reduction

New electrode (array) design

Enhance sensor stability (6 months+ shelf life)

Future Directions in Sensor Strip FabricationFuture Directions in Sensor Strip Fabrication

Single use electrode (gold-plated carbon electrode )

2 mm diameter

Area : 3.14 mm 2

AcknowledgementAcknowledgement

University of Nebraska-Lincoln

Jennifer Gerasimov Dr. Weiwei Yang

Socrates Canete Andy Springer

THANKS!THANKS!

UC Santa Barbara