Characterization of oxidoreductase-redox polymer electrostatic film assembly on gold by surface plasmon resonance spectroscopy and Fourier transform infrared-external reflection spectroscopy

Aleksandr L. Simonian, Alexander Revzin, James R. Wild, Jerry Elkind, Michael V. Pishko

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

The electrostatic assembly of nanocomposite thin films consisting of alternating layers of an organometallic redox polymer (RP) and oxidoreductase enzymes, glucose oxidase (GOX), lactate oxidase (LOX) and pyruvate oxidase (PYX), was investigated. Multilayer nanostructures were fabricated on gold surfaces by the deposition of an anionic self-assembled monolayer of 11-mercaptoundecanoic acid, followed by the electrostatic attachment of a cationic RP, poly(vinylpyridine Os(bis-bipyridine)2Cl-co-allylamine) (PVP-Os-AA), and anionic oxidoreductase enzymes. Surface plasmon resonance (SPR) spectroscopy, Fourier transform infrared external reflection spectroscopy (FT-IR-ERS) and electrochemistry were employed to characterize the assembly of these nanocomposite films. The surface concentration of GOX was found to be 2.4ng/mm2 for the first enzyme layer and 1.96ng/mm2 for the second enzyme layer, while values of 10.7 and 1.3ng/mm2 were obtained for PYX and LOX, respectively. The apparent affinity constant for GOX adsorption was found to be 8×107M-1. FT-IR-ERS was used to verify the incorporation of GOX and its conformational stability inside of these nanocomposite thin films. An SPR instrument with a flow-through cell was modified by additions of Ag/AgCl reference and Pt counter electrodes, with the gold-coated SPR surface film serving as the working electrode. This enabled real-time observation of the assembly of sensing components and immediate, in situ electrochemical verification of substrate-dependent current upon the addition of enzyme to the multilayer structure. A glucose-dependant amperometric response with sensitivity of 0.197μA/cm2/mM for a linear range of 1-10mM of glucose was obtained. The SPR and FT-IR-ERS studies also showed no desorption of polymer or enzyme from the nanocomposite RP-GOX structure when stored in aqueous environment occurred over the period of 3 weeks, suggesting that decreasing substrate sensitivity with time was due to loss of enzymatic activity rather than loss of film compounds from the nanostructure.

Original languageEnglish (US)
Pages (from-to)201-212
Number of pages12
JournalAnalytica Chimica Acta
Volume466
Issue number2
DOIs
StatePublished - Aug 27 2002
Externally publishedYes

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Surface Plasmon Resonance
Surface plasmon resonance
Fourier Transform Infrared Spectroscopy
Glucose Oxidase
Static Electricity
Gold
Fourier transform
Oxidation-Reduction
Nanocomposites
Electrostatics
Spectrum Analysis
Fourier transforms
Oxidoreductases
Polymers
glucose
lactate 2-monooxygenase
polymer
gold
spectroscopy
Spectroscopy

Keywords

  • Biosensor
  • Electrostatic film assembly
  • Enzyme electrode
  • Oxidoreductase
  • Redox polymer

ASJC Scopus subject areas

  • Biochemistry
  • Analytical Chemistry
  • Spectroscopy
  • Environmental Chemistry

Cite this

Characterization of oxidoreductase-redox polymer electrostatic film assembly on gold by surface plasmon resonance spectroscopy and Fourier transform infrared-external reflection spectroscopy. / Simonian, Aleksandr L.; Revzin, Alexander; Wild, James R.; Elkind, Jerry; Pishko, Michael V.

In: Analytica Chimica Acta, Vol. 466, No. 2, 27.08.2002, p. 201-212.

Research output: Contribution to journalArticle

Simonian, Aleksandr L. ; Revzin, Alexander ; Wild, James R. ; Elkind, Jerry ; Pishko, Michael V. / Characterization of oxidoreductase-redox polymer electrostatic film assembly on gold by surface plasmon resonance spectroscopy and Fourier transform infrared-external reflection spectroscopy. In: Analytica Chimica Acta. 2002 ; Vol. 466, No. 2. pp. 201-212.
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AU - Elkind, Jerry

AU - Pishko, Michael V.

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N2 - The electrostatic assembly of nanocomposite thin films consisting of alternating layers of an organometallic redox polymer (RP) and oxidoreductase enzymes, glucose oxidase (GOX), lactate oxidase (LOX) and pyruvate oxidase (PYX), was investigated. Multilayer nanostructures were fabricated on gold surfaces by the deposition of an anionic self-assembled monolayer of 11-mercaptoundecanoic acid, followed by the electrostatic attachment of a cationic RP, poly(vinylpyridine Os(bis-bipyridine)2Cl-co-allylamine) (PVP-Os-AA), and anionic oxidoreductase enzymes. Surface plasmon resonance (SPR) spectroscopy, Fourier transform infrared external reflection spectroscopy (FT-IR-ERS) and electrochemistry were employed to characterize the assembly of these nanocomposite films. The surface concentration of GOX was found to be 2.4ng/mm2 for the first enzyme layer and 1.96ng/mm2 for the second enzyme layer, while values of 10.7 and 1.3ng/mm2 were obtained for PYX and LOX, respectively. The apparent affinity constant for GOX adsorption was found to be 8×107M-1. FT-IR-ERS was used to verify the incorporation of GOX and its conformational stability inside of these nanocomposite thin films. An SPR instrument with a flow-through cell was modified by additions of Ag/AgCl reference and Pt counter electrodes, with the gold-coated SPR surface film serving as the working electrode. This enabled real-time observation of the assembly of sensing components and immediate, in situ electrochemical verification of substrate-dependent current upon the addition of enzyme to the multilayer structure. A glucose-dependant amperometric response with sensitivity of 0.197μA/cm2/mM for a linear range of 1-10mM of glucose was obtained. The SPR and FT-IR-ERS studies also showed no desorption of polymer or enzyme from the nanocomposite RP-GOX structure when stored in aqueous environment occurred over the period of 3 weeks, suggesting that decreasing substrate sensitivity with time was due to loss of enzymatic activity rather than loss of film compounds from the nanostructure.

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KW - Enzyme electrode

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