1. Folding-based Electrochemical Biosensors
Rebecca Y. Lai
Department of Chemistry
University of Nebraska-Lincoln

2. “Traditional” Biosensor
Specific Binding
Biosensor
Signal
Measure changes in adsorbed mass,
polarizability, sterics or charge

3. Folding-based Biosensors: Signal Transduction Mechanism
Specific Binding
Biosensor
Non -
specific Binding
Signal
Signaling linked to a binding-specific change
in the physical properties of the biopolymer

4. Electrochemical Biosensors
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

5. Microfabricated gold electrode array
Materials and Methods
Biosensor Interrogation Method: Alternating Current Voltammetry
Counter electrode
Ref electrode
Working electrode
Length: 1 mm
Width: 0.88 mm
Area : mm2
Microfabricated gold electrode array
2 mm diameter
Area : 3.14 mm 2
Gold disk electrodes

6. Electrochemical DNA Sensor (E-DNA)
Gold Electrode
+ target sequence
Denaturation /
Re-annealing MB
*Not to scale
5’-HS-(CH2)6- GCAGTAACAAGAATAAAACGCCACTGC -(CH2)7-NH-3’-MB

7. Methylene blue Leucomethylene blue
E-DNA Sensor
AC Voltammograms
Reagentless (direct detection of aqueous DNA)
Sensitive (pM detection limit)
Rapid detection
Reusable
Selective
Specific
Methylene blue Leucomethylene blue
+ 2e-
+ H+

8. Reusable and Reproducible
E-DNA Sensor
Reusable and Reproducible
Selective

9. E-DNA Sensor in a Microfluidic Chamber
***In-situ sensor fabrication, hybridization and regeneration
Sensor Device
Sensor Response

10. Aptamers
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.

11. Electrochemical Aptamer-Based (E-AB) Sensor: PDGF Detection
+ PDGF-BB
Sluggish Electron Transfer
Efficient Electron Transfer MB
Reagentless
Reusable
Rapid
Specific
Selective
Parallelizable
Signal-on sensor
*Not to scale
5’-HS-(CH2)6- CAGGCTACGGCACGTAGAGCATCA
CCATGATCCTG-(CH2)7-NH-3’-MB

12. 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)

13. Peptide or Protein-base Electrochemical Biosensors
Ligand-induced Folding (LIF)
Ligand-induced folding occurs when a favorable binding free energy overcomes an unfavorable folding free energy, producing a folded complex.

14. Electronic Peptide-based Sensor (E-PB Sensor)et
+ target
- target MB
* Not to scale
Peptide Probes
Naturally occurring peptide capable of LIF
Engineered peptide capable of LIF

15. E-PB Sensor: HIV Detection
Protein of Interest
Surface Glycoprotein
gp120
Transmembrane Glycoprotein
gp41
Matrix Protein
p17
Capsid Protein
p24 15

16. E-PB Sensor: HIV Detection
Proteins of Interest
Surface Glycoprotein gp120
Transmembrance Glycoprotein gp41
Matrix Protein p17
Capsid Protein p24
p17
p24
gp41
gp120
% of patients for antigen 62 58 18 38
% of patients positive for antibody 60 92
100 34

17. Detection of Anti p24 Antibodies
Probe
Target
Anti-p124 antibody (IgG)
(~150 kDa)
Response in earliest stages of AIDS
Anti-p24 concentration in serum may be tens to hundreds of nanomolar
HIV-1 p24 capsid protein
Highly antigenic
epitope sequence
EAAWDRVHP

18. Sensor Response to Anti-p24 Antibodies
+ Ab
- Ab
Probe Sequence: HS-C11-EAAWDRVHP-K-MB

19. Future Direction of E-PB Sensor Research (Immediate!)
New immobilization methods to increase surface coverage
Improve Sensor Sensitivity and Dynamic Range
Improve Sensor Specificity
Test sensor / self-assembled monolayer (SAM) using surface plasmon resonance (SPR)
Change / modify epitope if necessary
New immobilization methods
Incorporate thiolated polyethylene-glycol (PEG)
Improve Sensor Selectivity

20. Future Direction of E-PB Sensor Research
Click Chemistry
Azide terminated alkanethiol + alkyne modified peptide w/ MB
* Not to scale N3 OH
+ alkyne-peptide-MB
+ Cu(I)
Advantages:
Flexibility in SAM choices
Step by step characterization by electrochemical methods and SPR
Site-specific modification/sensor fabrication

21. Future Direction of 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

22. Peptide nucleic acid (PNA) stem + Peptide loop
Hybrid E-PB Sensor et
+ Ab
- Ab MB
* Not to scale
Peptide Loop
PNA Stem
Peptide nucleic acid (PNA) stem + Peptide loop
p24 Probe Sequence: HS-C11-GAT-EAAWDRVHP-ATC-MB

23. Future 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

24. E-DNA Sensor on a Gold-plated Screen-printed Carbon Electrode
2 mm diameter
Area : 3.14 mm 2
Sensor Device
Gold-plated Carbon Electrode
Before
Regenerated
Hybridization
Buffer
100%Serum

25. Future Directions in Sensor Strip Fabrication
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)
Single use electrode (gold-plated carbon electrode )
2 mm diameter
Area : 3.14 mm 2

26. THANKS! Acknowledgement University of Nebraska-Lincoln
Jennifer Gerasimov Dr. Weiwei Yang
Socrates Canete Andy Springer
UC Santa Barbara
THANKS!