Electrochemical Sensors for Biomolecules

1. Electrochemical Sensors for Biomolecules Sophia Robinson October 29, 2014 CHE480

2. Overview • What is an electrochemical biosensor? • Components of an electrochemical biosensor • Biosensor requirements for non-specialist market • How do biosensors work? • Types of electrochemical devices • Typical recognition elements • Surface architecture • Examples of biosensors: • Enzyme biosensor • Enzyme labeled DNA biosensor

3. Electrochemical Biosensors Quantitatively produce an electrical signal related to the concentration of a biological analyte using electrochemical techniques for biological processes Reaction investigated generates a measurable current (amperometric), measurable potential (potentiometric), or measurably alters the conductive properties of a medium between electrodes (conductometric). Electrochemical biosensors require a reference electrode (often the Ag/AgCl reference electrode is used)

4. Elements of a Biosensor Sensors (Basel) 2009, 8(3), 1400-1458

5. Simplified Graphic of How Biosensors Work www.micruxfluidic.com

6. Constructing a biosensor for the non-specialist market • Biocatalyst must be highly specific for the purpose of the analysis, stable under normal storage conditions • Reaction should be independent as possible of physical parameters such as pH and temperature. • Response should be accurate, precise, and reproducible. • Biosensor should provide-real-time analysis of analytes from human samples • Complete biosensor should be cheap, small, portable and capable of being used by semi-skilled operators.

7. Typical Recognition Elements • Biosensors must be highly selectively of the analyte • Use biological recognition elements on sensor substrate with a specific binding affinity for analyte: • Enzymes (most common) • Nucleic Acids • Antibodies • Whole Cells • Receptors • Surface architecture of the sensor must suppress any non-specific interaction

8. Surface Architecture of biosensors • Electrochemical biosensors have suffered from a lack of surface architectures allowing high enough sensitivity and unique identification of the response with the desired biochemical event. • Recent emphasis on nanotechnology , which minimizes dimensions of electrochemical sensor elements to sizes that allow for increased signal-to-noice ratio • Carbon Nanotubes • Nanorods • Nanoparticles • Nanowires

9. Electrochemical Biosensor Surface Materials • The material used for the biosensor surface depends on the measurement technique • Common materials: • glass, other oxide surfaces, gold, glass carbon, graphite and indium tin oxide. • Conducting polymers such as polyaniline or polystyrene are also useful

10. Amperometric devices • Continuously measure the current resulting from the oxidation or reduction of an electroactive species in a biochemical reaction • Voltammetry is a form of amperometry in which the current is measured during controlled variations of the potential

11. Potentiometric Devices • Measure the accumulation of charge at the working electrode and relate it to a reference electrode • Relationship between the concentration and the potential is goverened by the Nernst equation. Ecell= Ecell – (RT/nF)lnQ

12. Enzyme Biosensors • Enzyme based electrodes function by immobilizing an enzyme onto the electrode surface to capture the desired analyte • Subsequent quantification of the analyte is accomplished via electrical signal From engineering.purdue.edu

13. Enzyme Biosensors • Glucose Oxidase (GOx) • Operate based on amperometric detection of hydrogen peroxide www.worthington-biochem.com

14. Application 1: Enzyme Biosensor • Hrapovic et al. improved upon a previously developed glass carbon (GC) electrode and a carbon fiber microelectrode (CFM) • Increased the electroactivity for hydrogen peroxide via the combination of each electrode with platinum nanoparticles and carbon nanotubes. • Glucose oxidase (GOx) served as the enzyme model

15. Single-Wall Carbon Nanotubes (SWCNTs) • Highly hydrophobic • Difficult to adhere metals to carbon nanotubes • Hrapovic et al. depositied platinum nanoparticles onto Nafion-modified carbon nanotubes • Nafion:perfluorosulfonated and negatively charged polymer electrolyte • Platinum nanoparticles deposited via charged interactions

16. Linear sweep + cyclic voltammetry • Four types of electrodes: • Bare GC • GC modified by CNT • GC modifed by Pt nanoparticles • GC modified by CNT and Pt nanoparticles • Most significant electrocatalytic activity for oxygen reduction observed for CNT + Pt nanoparticle modified GC

17. Estimation of Active Surface Area of Electrodes • Cyclic Voltammetry of GC electrodes in 20 mM Fe(CN)64- and 0.2 M KCl at 20 mV/s versus Ag/AgCl reference electrode • Well-defined oxidation and reduction peaks due to Fe3+/Fe2+ redox couple noticeable at +0.30 V and +0.17 V in forward and reverse scans, respectively. • The CNT + Ptnano-modified GC electrode had highest electroactive surface area

18. Cyclic Voltammograms

19. Electrocatalytic activity toward H2O2

20. Sensitivity toward Glucose

21. Application 1: Summary of results • Repeated use of electrodes did not affect long term stability • New approach to deposit platinum nanoparticles onto nafion-solubilized SWCNT • Of the 4 electrodes, only the CNT + Pt nanoparticle + GOx modified GC or CFM electrode sensitive toward glucose • 3 second response time • 0.5 μM detection limit

22. DNA Hybridization biosensors • Used to detect medical issues ranging from pathogenesis to neurodegenerative diseases • High sensitivity of enzyme labeled DNA sensing techniques • Desired amplification for detection of low target levels possible • Base pairing recognition event of DNA detected by enzyme labels captured following hybridization • Convert hybridization event to useful electrical signals

23. Application 2: Enzyme labeled Genosensor • Crucial step in design of genosensor is immobilization of single-stranded DNA probes onto electrode surface • Sensitivity and reproducibility of genosensor determined by immobilization method • Immobilization method examples: physical adsorption on carbon surfaces, covalent attachment to activated surfaces, avidin/biotin interaction to attach biotinylated probes on electrode surface • Biotin/avidin approach allows for a sensing phase with more strands of DNA than by direct adsorption

24. Application 2: Enzyme Labeled Genosensor • Hernandez-Santos et al. developed an enzymatic genosensor based on screen-printed carbon electrode surface coated with streptavidin for identification of nucleic acid determinants exclusively present on the genome of the pathogen Streptococcus pneumonia • Goal is to be used to diagnose human infectious pulmonary disease

25. Construction of genosensor and detection of target

26. Construction of genosensor • Sensing phase formed by adding 3’-biotinylated oligonucleotide probes that target pneumolysin (ply), autolysin (lytA), and cellwall protein (psA) genes. • Sensing phase hybridized with FITC-labeled oligonucleotide • Enzyme capture with rabbit IgG anti-FITC conjugated to alkaline phosphatase in presence of 3-indoxyl phosphate (3-IP) for electrochemical analytical signal detection • Enzymatic reaction conducted to yield insoluble indigo blue then stopped with sulfuric acid to yield hydrosoluble indigo carmine (IC).

27. Hybridization with complementary and nocomplementary targets

28. Detection of Mutations by mismatched targets

29. Application 2: Summary of Results • Successfully formed sensitive and reproducible enzymatic genosensors on streptavidin-modified-SPCEs. • Use of biotinylated probes results in better orientation and enahnced absorption of ss on the geno-sensning phase • Leaves probes accessible to react with their complementary targets • Probe reaction with noncomplementary targets is easily detectable

30. References • 1. Dorothee Grieshaber, R. M., Janos Voros, Erik Reimhult, Electrochemical Biosensors - Sensor Principles and Architectures. Sensors (Basel) 2008,8 (3), 1400-1458. • 2. Danielle W. Kimmel, G. L., Mike E. Meschievitz, David E. Cliffel, Electrochemical Sensors and Biosensors. Analytical Chemistry 2012,84, 685-707. • 3. Sabuhudin Hrapovic, Y. L., Keith B. Male, John H. T. Luong, Electrochemical Biosensing Platforms Using Platinum Nanoparticles and Carbon Nanotubes. Analytical Chemistry 2004,76, 1083-1088. • 4. Bakker, E., Electrochemical Sensors. Analytical Chemistry 2004,76, 3285-3298. • 5. Qin, E. B. a. Y., Electrochemical Sensors. Analytical Chemistry 2006,78, 3965-3983. • 6. David Hernandez-Santos, M. D.-G., Maria Begona Gonzalez-Garcia, Agustin Costa-Garcia, Enzymatic Genosensor on Streptavidin-Modified Screen-Printed Carbon Electrodes. Analytical Chemistry 2004,76, 6887-6893.