Impedance Biosensors: Sustainable Nanomaterials

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1 Impedance Biosensors: Sustainable Nanomaterials Ian Ivar Suni Director, Materials Technology Center Professor, Chemistry and Biochemistry Professor, Mechanical Engineering and Energy Processes October 29, 2013

2 Outline» Electrochemical Impedance Spectroscopy (EIS): Academic studies of impedance biosensors.» Challenges to Development of Impedance Biosensors: Complexity of AC impedance detection methods. Non-specific adsorption (chemical interference). Protein immobilization onto conducive electrode. Stability Reproducibility Regeneration of recognition biomolecule Stability under electrical interrogation» Applications to Sustainability» Incorporation of Nanomaterials

3 Electrochemical Impedance Spectroscopy (EIS)

4 Nyquist Plot: Randles Equivalent Circuit R s C d R ct Z w R s : R ct : C d : Z w : Solution Resistance Charge Transfer Resistance Double Layer Capacitor Warburg impedance (Resistance to Mass Transfer, non-ideal)

5 -Z Im ( cm 2 ) Impedance Detection of Peanut Protein Ara h Z Re ( cm 2 ) 5 Hz Mouse monoclonal antibody immobilized at an Au electrode [Ara h 1] = 0, 0.02, 0.04, 0.08, 0.16 and 0.24 mg/ml Detection limit ~ 0.3 nm MW (monomer) = 64.5 kda

6 R ct ( -cm 2 ) C d (mf-cm -2 ) 2000 CNLS fit to Randles Equivalent Circuit Concentration of Ara h1 Protein (mg-ml -1 ) Concentration of Ara h1 Protein (mg-ml -1 ) R ct increases as the protein film becomes thicker j j 0 e t Capacitance decreases as the protein film becomes thicker, K d = 0.52 nm C A d t

7 Impedance Detection of Listeria monocytogenes Mouse monoclonal antibody immobilized at an Au electrode in 50 mm PBS and 5.0 mm K 3 Fe(CN) 6 /K 4 Fe(CN) 6 (A), and in filtered tomato extract (B). Detection limit ~ 5 CFU/ml

8 Commercial Development of Impedance Biosensors?? The major technical challenges to implementation are:» Greater complexity of AC impedance detection methods relative to DC electrochemistry. (Suh-Moon Park, Dept. of Chemistry, Pohang University of Science and Technology)» Non-specific adsorption: Proteins, cells, debris, small molecule organic species, ions, etc. may stick to the electrode surface.» Substrate material and linker chemistry for protein immobilization: Must have sufficient shelf life. Must be reproducible, or can be calibrated. Regeneration likely needed for sensor calibration. Must also be stable upon repeated electrical interrogation.

9 How to Prevent/Subtract Non-Specific Adsorption? Non-specific adsorption can be reduced or compensated for by:» Coating electrodes with bovine serum albumin (BSA)» Sample dilution» Control electrodes at which blank (control) antibodies are immobilized. Antibody of interest immobilized at measurement electrode. Blank antibody immobilized at control electrode.

10 Impedance Detection of Listeria monocytogenes Mouse monoclonal antibody to Listeria on an Au electrode tested in filtered tomato extract. Mouse monoclonal antibody to GAPDH on an Au electrode tested in filtered tomato extract.

11 Covalent Binding of Antibodies to an Au Electrode» Form organic film with carboxylate termination by immersing the Au surface into 11-mercaptoundecanoic acid (11-MUA), forming Au-S covalent bonds.» Carboxylate activation by EDC and NHSS.» React with amino groups on protein surface to form amide bond. B. Johnsson, S. Lofas and G. Lindquist, Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors, Anal. Biochem. 198, 268 (1991). 924 citations as of October 25, S S S S S S S S

12 Instability of Au-thiol Chemistry in Air

13 Degenerate Si Electrode for Impedance Biosensing Degenerate (highly doped) Si is an alternative substrate material, with improved storage stability and reproducibility. Proteins can be immobilized by:» Removal of Si oxide by HF or NH 4 F.» Photo-activated alkene insertion of bi-functional reagent (10-undecenoic acid) into Si-H bonds.» Amide bond formation using EDC and NHSS. r(au) = 2.2x10-6 -cm r(degenerate Si) = 5x10-3 -cm r(si) = 2.5x cm

14 -Z Im (k -cm 2 ) Impedance Detection of Peanut Protein Ara h Z Re (k -cm 2 ) Mouse monoclonal antibody immobilized at a degenerate Si electrode [Ara h 1] = 0, 0.005, 0.01, 0.015, 0.02, 0.04 and 0.08 mg/ml

15 Advantages of Degenerate Si as Biosensor Electrode» Degenerate Si is an electrical conductor, so electrochemistry is reversible without exposure to light.» Simpler electrical equivalent circuit than for n-type or p-type Si.» Si is directly beneath C in the periodic table, so Si-C bonds are strong, and the biosensor interface should be more stable. Si-C bonds (~520 kj/mole) are stronger than Au-S bonds (~ kj/mole).» Unlike C, Si surface preparation is simple and reproducible.» Si is easily incorporated into ULSI devices.

16 Antibody Regeneration atop Degenerate Si Mild refolding buffer that contains 1.0 M KSCN, but no HF or BSA. Interface gradually approaches capacitive behavior

17 Antibody Regeneration atop Degenerate Si Mild refolding buffer that contains 0.2 M KSCN, 0.1 M HF, and 0.1 M BSA. Interface evolves in opposite direction!!

18 Glucose Biosensors: DC Electrochemistry

19 Applications of Impedance Biosensors» Portable and/or implantable sensors applied to: Food Food allergens Food pathogens Mycotoxins (produced by fungi) Agriculture Atrazine and other pesticides Chlorpyrifos and other insecticides Avian influenza H5N1 virus Endocrine-disrupting chemicals (EDCs) Norfluoxetine and other pharmaceuticals (and their metabolites) Brominated flame retardants Other environmental toxins Microcystins and other toxins from cyanobacteria

20 Impedance Detection of Norfluoxetine, an EDC MW = Sheep polyclonal antibody immobilized at an Au electrode. Detection limit ~ 8.5 ng/ml, 28 nm

21 Impedance Detection of Norfluoxetine, an EDC Norfluoxetine Concentration (µg/ml) R s (Ω-cm 2 ) 19.4 (0.2) 19.9 (0.2) 21.6 (0.1) 21.9 (0.1) 21.8 (0.2) 21.8 (0.1) 21.9 (0.1) 22.5 (0.1) 23.3 (0.2) CPE-T ( µf cm -2 s n-1 ) n R ct (kω-cm 2 ) 7.21 (0.14) 9.91 (0.15) 12.4 (0.2) 14.7 (0.2) 16.1 (0.2) 16.9 (0.2) 17.4 (0.2) 17.8 (0.2) 18.0 (0.2) R ct much more sensitive to norfluoxetine binding than C d, R S

22 Impedance Biosensors: Incorporation of Nanomaterials» Nanomaterials are applied mainly for signal amplification, or for protein confinement: Substrate may be composed of or coated with nanomaterials- Au nanoparticles Carbon nanotubes or nanofibers Analyte may be conjugated with nanomaterials: Au nanoparticles CdS nanoparticles Horseradish peroxidase Proteins may be confined with nanopores: Nanoporous Al 2 O 3 membranes Nanoporous polycarbonate membranes

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24 Impedance Detection of Antibody with Au Nanoparticle Tagging Au

25 Impedance Biosensing in Polycarbonate Nanopores Pore diameter 15 nm Zero (Δ), 0.04 μg/ml ( ), 0.08 μg/ml (o), and 0.12 μg/ml ( ) peanut protein Ara h 1.

26 Impedance Biosensing in Polycarbonate Nanopores ZRe (Ω-cm 2 ) Pore diameter 30 nm Time(min) Zero ( ), 40 μm ( ), 70 μm ( ), 100 μm (x), and 200 μm (*) glucose.

27 Glucose-Galactose Receptor (GGR) Protein

28 Publications: Impedance Biosensors» R. Radhakrisnan and I.I. Suni, Electrochemical Immunosensor for Norfluoxetine, submitted to Sens. Actuat. B.» R. Radhakrisnan, G. Jahne, S.W. Rogers and I.I. Suni, Impedance Detection of Listeria monocytogenes, Electroanalysis 5, 2231 (2013).» **R. Singh and I.I. Suni, Minimizing Non-specific Adsorption in Protein Biosensors that Utilize Electrochemical Impedance Spectroscopy, J. Electrochem. Soc. 157, J334 (2010).» Y. Huang, M. Bell and I.I. Suni, Impedance Biosensor for Peanut Protein Ara h 1, Anal. Chem. 80, 9157 (2008).» **Y. Huang and I.I. Suni, Degenerate Si as an Electrode Material for Electrochemical Biosensors, J. Electrochem. Soc. 155, J350 (2008).

29 Publications: Impedance Biosensors» I.I. Suni, Impedance Methods for Electrochemical Sensors Using Nanomaterials, Trends Anal. Chem. 27, 604 (2008).» B. Tripathi, J. Wang, L.A. Luck and I.I. Suni, Nanobiosensor Design Utilizing a Periplasmic E. coli Receptor Protein Immobilized within Au/Polycarbonate Nanopores, Anal. Chem. 79, 1266 (2007).» **J. Wang, L.A. Luck and I.I. Suni, Immobilization of the Glucose/galactose Receptor (GGR) Protein onto an Au Electrode through a Genetically Engineered Cysteine Residue, Electrochem. Solid-State Lett. 10, J33 (2007).» J. Wang, J.A. Profitt, M.J. Pugia and I.I. Suni, Au Nanoparticle Conjugation for Impedance and Capacitance Signal Amplification in Biosensors, Anal. Chem. 78, 1769 (2006). **Selected for inclusion in the Virtual Journal of Biological Physics Research.

30 Conclusions» Numerous antigens can be detected by electrochemical impedance spectroscopy (EIS) at antibody-coated electrodes.» Non-specific adsorption (chemical interference) can be quantified/subtracted by the use of additional control electrodes.» Degenerate (highly doped) Si is an interesting alternative electrode material for impedance biosensors.» No fundamental technical limitations to impedance detection of antibody-antigen binding.» Numerous applications exist to Sustainability.» Nanomaterials often incorporated into impedance biosensors

31 Acknowledgements» Rajeswaran Radhakrishnan, Jianbin Wang, Rajdeep Singh, Clarkson University» Madhavi Pali, Southern Illinois University» Ying Huang, Postdoctoral Research Associate, Clarkson University (now in College of Chemistry and Materials Science, South-Central University for Nationalities Wuhan, Hubei China)

32 Acknowledgements» National Science Foundation o CTS , CCF , and ECCS » U.S. Army grant W911NF » New World Pharmaceuticals» Bayer HealthCare» Center for Advanced Materials Processing (CAMP) at Clarkson University