Elsevier

Sensors and Actuators B: Chemical

Volume 203, November 2014, Pages 327-332
Sensors and Actuators B: Chemical

Binding and potential-triggered release of l-glutamate with molecularly imprinted polypyrrole in neutral pH solutions

https://doi.org/10.1016/j.snb.2014.06.030Get rights and content

Highlights

  • •

    We investigate binding of l-glutamate (Glu) in molecularly imprinted polypyrrole (MIPPy).

  • •

    Immunofluorescence microscopy analysis using specific anti-Glu antibodies is applied to confirm binding.

  • •

    Electrochemical quartz microbalance measurements are applied to quantify binding interactions.

  • •

    Potential-regulated trafficking of Glu by MIPPy is demonstrated.

Abstract

Molecularly imprinted polymers (MIPs) are interesting for potential-regulated trafficking of molecules. In this study, we investigate the binding properties of l-glutamate (Glu) in molecularly imprinted polypyrrole (MIPPy) in neutral pH solutions. We prepared MIPPy by electrochemically depositing Glu-doped polypyrrole and subsequently overoxidizing the polymer. By means of immunofluorescence microscopy analysis of MIPPy using specific anti-Glu antibodies, we demonstrate binding of Glu in MIPPy. Visual imaging of the samples allows us to easily discern between selective and non-selective interactions. Significant fluorescence is observed from MIPPy incubated in solutions containing Glu, while control MIPPy samples imprinted with Cl− and subsequently exposed to Glu do not yield detectable fluorescence signals. To quantify the binding interactions, we use electrochemical quartz microbalance (EQCM) measurements. Glu uptake by MIPPy occurs in the absence of an applied electrical potential. From the change in mass of MIPPy versus the Glu concentration in solution, we determine a binding free energy of −6.0 ± 0.2 kJ/mol. Based on these results, the binding interactions are discussed in terms of hydrogen bonding combined with electrostatic interactions between the polymer and the neurotransmitter. Glu release from MIPPy is triggered at low potentials (−0.3 V (Ag/AgCl)). These properties make MIPPy interesting for applications such as drug delivery and chemical implants.

Introduction

Molecularly imprinted polymers (MIPs) are being widely investigated in the literature for applications related to sensing, catalysis, and drug delivery [1], [2], [3], [4], [5]. MIPs have great potential for the development of sophisticated biomedical devices, such as chemical implants, which regulate biomolecular concentrations in solution via selective uptake and release processes. The reliability of MIP functionality, however, is often limited by factors such as the poor accessibility of binding sites within the polymer matrix [6], heterogeneous [7] or non-selective binding [1], [3], interactions of the anion with polar solvents such as water [8], and the poor long-term stability for molecular recognition [9]. For this reason, materials which meet the strict demands for molecular recognition applications are required.

Electrochemically synthesized MIPs offer much potential for realizing flexible and novel applications [10], [11], [12]. Polypyrrole (PPy) is a stable, electroactive polymer which was widely investigated in the 1980s and 1990s and has recently re-emerged as a highly relevant material for MIP applications [10] due to its stability and interesting redox properties. PPy can be easily synthesized electrochemically. During synthesis, anions from the electrolyte are incorporated into the polymer backbone, resulting in conductive, doped polymer films which adhere to electrode surfaces [13]. The dopant anion influences the PPy properties [14] and can be chosen to achieve desired functionalities [15]. Deore et al. [16] initially showed that overoxidizing PPy results in molecular selectivity for the templating ion. Since then molecularly imprinted PPy (MIPPy) has been investigated for the potential-regulated, selective uptake of cationic amino acids, such as l-Glu [17] and l-aspartate [18], in low pH solutions. Recently, Mehdini et al. [19] demonstrated a novel synthesis route allowing for increased surface area for improved recognition of ascorbic acid by MIPPy in aqueous solutions.

As l-Glu is the most abundant neurotransmitter in the central nervous system, the selective regulation of l-Glu concentrations in physiological pH solutions would be highly interesting in the fields of biology and medicine. This, however, has been difficult to realize to date. We recently demonstrated the proof-of-concept of potential-regulated enantioselective uptake and release, i.e. molecular trafficking, of anionic l-Glu from MIPPy in neutral pH solutions [20].

Selective uptake of amino acids by MIPPy has been attributed to electrostatic interactions between the charged molecule and electrode combined with shape recognition [17], [18], [20], [21]. The exact mechanisms, however, have not been confirmed. Generally, molecular recognition occurs due to specificity, e.g. shape recognition, between the target molecule and binding site combined with non-covalent interactions, e.g. electrostatic, hydrogen bonding or hydrophobic interactions [22], [23]. In the case of molecular recognition with MIPs, interactions have been additionally attributed to Van der Waals and charge-transfer interactions [1]. While shape recognition has been clearly demonstrated for the uptake of amino acids with MIPPy, the nature of the binding interactions remains unclear. In order to develop molecular trafficking devices, where the target molecule is subsequently released via an optical or electrical trigger, it is of key importance to identify and quantify these interactions.

In this study, we investigate the binding interactions of Glu in MIPPy in neutral pH solutions. A protocol for the fabrication of MIPPy is described. Immunofluorescence microscopy using a specific anti-Glu antibody is applied to confirm binding of the neurotransmitter in MIPPy. Electrochemical quartz microbalance (EQCM) measurements are used to quantify the binding energy between the neurotransmitter and the polymer. The results from the fluorescence studies and EQCM data indicate that uptake of Glu by MIPPy is dominated by hydrogen bonding combined with electrostatic interactions. Release of Glu from MIPPy is triggered by applying a low negative potential to the MIPPy electrode. Finally, potential-regulated trafficking of Glu with MIPPy is demonstrated.

Section snippets

Materials

Pyrrole (Py) (99%, extra pure and 98% FCC) was purchased from Acros Organics and from Sigma-Aldrich, and glutamic acid monosodium salt monohydrate (NaGlu) and sodium chloride, both ≥99% from Sigma-Aldrich. Water was purified by a Milli-Q water system (R = 18.2 × 106 Ω cm). Py was vacuum distilled (10−2 to 10−3 bar) before use and stored under Ar atmosphere at −10 °C. All other chemicals were used as-received.

Electrochemical synthesis of Glu-templated PPy

Py (0.4 M) and NaGlu (0.5 M) or NaCl (0.5 M) were added to Milli-Q water (R = 18 × 106 Ω cm) and mixed

Synthesis of Glu-templated MIPPy

The morphology and adhesion characteristics of electrochemically synthesized PPy strongly depend on the deposition parameters, such as potential, pH, temperature, and counterion [13], [15], [26]. For reproducible synthesis procedures, polymer films are homogeneous and smooth [13]. Unwanted oxidation of the PPy layer during synthesis is a major factor limiting the reproducibility and has been attributed to water oxidation [27], particularly due to replacement of the counteranion by hydroxyl

Conclusions

We demonstrate the uptake and electrically triggered release of Glu with MIPPy in neutral pH solutions. Binding of Glu in MIPPy was confirmed with immunofluorescence microscopy of MIPPy using anti-Glu antibodies and EQCM. Using electrochemical quartz microbalance measurements, we determined the binding energy of Glu in MIPPy of ΔG = −6.0 ± 0.2 kJ/mol. Based on the results from the fluorescence analysis and EQCM data, we attribute the uptake mechanism to hydrogen bonding combined with electrostatic

Acknowledgements

The authors would like to thank the German Ministry of Education and Research (BMBF, project number 16SV3889) and the EWE Research Group “Dünnschichtphotovoltaik” by the EWE AG, Oldenburg, for financial support.

Ivan Chernov received his Ph.D. at the Institute of Problems of Chemical Physics of the Russian Academy of Sciences in Russia. He worked as a Post Doc in the Institute of Physics, Carl von Ossietzky University of Oldenburg. His research interests focus on problems in chemical physics related to thin-film semiconductors.

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    Ivan Chernov received his Ph.D. at the Institute of Problems of Chemical Physics of the Russian Academy of Sciences in Russia. He worked as a Post Doc in the Institute of Physics, Carl von Ossietzky University of Oldenburg. His research interests focus on problems in chemical physics related to thin-film semiconductors.

    Helena Greb is a Ph.D. student in neuroscience at Carl von Ossietzky University Oldenburg, Germany. Her current research interests focus on neuronal mechanisms of signal processing within the retina, studied through molecular, biochemical and physiological techniques, as well as the development of an autonomous neurochemical implant, including uptake/release of glutamate from molecular-imprinted polypyrrole and detection of glutamate within the polymers via histological techniques.

    Ulrike Janssen-Bienhold received her Ph.D. in neurobiology at the Department of Biology at the University of Bremen in 1988. She is currently working as a Professor for molecular and cellular neurobiology at the Department for Neuroscience at the University of Oldenburg, Germany. Her current research interests are the molecular processes and signalling mechanisms modulating and regulating electrical synapses (gap junctions) between retinal neurons and to unravel how electrical coupling affects vision in vertebrates.

    Jürgen Parisi received his Ph.D. in experimental physics at the University of Tübingen, Germany, in 1982. He is a Full Professor in the Department of Physics at the University of Oldenburg. His current research interests are condensed matter physics, materials science, and complementary structural, spatial, temporal, and spectral high-resolution measuring techniques.

    Reto Weiler received his Ph.D. in biology at the Ludwig Maximilian University, Munich, in 1977. He is a Full Professor of Neurobiology at the Carl von Ossietzky University of Oldenburg, Head of the Neuroscience Research Center and Rector of the Hanse-Wissenschaftskolleg, Institute for Advanced Study, Delmenhorst. His research interests are the elucidation of the neuronal network of the retina at the cellular and molecular level. Currently, he is particularly interested in the role of gap junctions.

    Elizabeth von Hauff received her Ph.D. in experimental physics at the Carl von Ossietzky University of Oldenburg, Germany, in 2005. She is an associate professor in the Department of Physics and Astronomy at the Vrije Universiteit Amsterdam. Her research interests are understanding optical and electrical phenomena in molecular and nanostructured materials for sensing and energy applications.

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    These authors are contributed equally.

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