Molecular Recognition: Perspective and a New Approach.

This perspective presents an overview of approaches to the preparation of molecular recognition agents for chemical sensing. These approaches include chemical synthesis, using catalysts from biological systems, partitioning, aptamers, antibodies and molecularly imprinted polymers. The latter three approaches are general in that they can be applied with a large number of analytes, both proteins and smaller molecules like drugs and hormones. Aptamers and antibodies bind analytes rapidly while molecularly imprinted polymers bind much more slowly. Most molecularly imprinted polymers, formed by polymerizing in the presence of a template, contain a high level of covalent crosslinker that causes the polymer to form a separate phase. This results in a material that is rigid with low affinity for analyte and slow binding kinetics. Our approach to templating is to use predominantly or exclusively noncovalent crosslinks. This results in soluble templated polymers that bind analyte rapidly with high affinity. The biggest challenge of this approach is that the chains are tangled when the templated polymer is dissolved in water, blocking access to binding sites.

Stimuli-Responsive Templated Polymer as a Target Receptor for a Conformation-based Electrochemical Sensing Platform.

The use of highly crosslinked molecularly imprinted polymers as a synthetic target receptor has the limitations of restricted accessibility to the binding sites resulting in slow response time. Moreover, such artificial receptors often require additional transduction mechanisms to translate target binding events into measurable signals. Here, we propose the development of a single-chain stimuli-responsive templated polymer, without using any covalent interchain crosslinkers, as a target recognition element. The synthesized polymer chain exhibits preferential binding with the target molecule with which the polymer is templated. Moreover, upon specific target recognition, the polymer undergoes conformation change induced by its particular stimuli responsiveness, namely the target binding event. Such templated single-chain polymers can be attached to the electrode surface to implement a label-free electrochemical sensing platform. A target analyte, 4-nitrophenol (4-NP), was used as a template to synthesize a poly-N-isopropylacrylamide (PNIPAM)-based copolymer chain which was anchored to the electrode to be used as a selective receptor for 4-NP. The electrode surface chemistry analysis and the electrochemical impedance study reveal that the polymer concentration, the interchain interactions, and the Hofmeister effect play a major role in influencing the rate of polymer grafting as well as the morphology of the polymers grafted to the electrode. We also show that the specific binding between 4-NP and the copolymer results in a substantial change in the charge transfer kinetics at the electrode signifying the polymer conformation change.

Application of Voltage in Dynamic Light Scattering Particle Size Analysis.

Dynamic light scattering (DLS) is a common method for characterizing the size distribution of polymers, proteins, and other nano- and microparticles. Modern instrumentation permits measurement of particle size as a function of time and/or temperature, but currently there is no simple method for performing DLS particle size distribution measurements in the presence of applied voltage. The ability to perform such measurements would be useful in the development of electroactive, stimuli-responsive polymers for applications such as sensing, soft robotics, and energy storage. Here, a technique using applied voltage coupled with DLS and a temperature ramp to observe changes in aggregation and particle size in thermoresponsive polymers with and without electroactive monomers is presented. The changes in aggregation behavior observed in these experiments were only possible through the combined application of voltage and temperature control. To obtain these results, a potentiostat was connected to a modified cuvette in order to apply voltage to a solution. Changes in polymer particle size were monitored using DLS in the presence of constant voltage. Simultaneously, current data were produced, which could be compared with particle size data, to understand the relationship between current and particle behavior. The polymer poly(N-isopropylacrylamide) (pNIPAM) served as a test polymer for this technique, as pNIPAM's response to temperature is well-studied. Changes in the lower-critical solution temperature (LCST) aggregation behavior of pNIPAM and poly(N-isopropylacrylamide)-block-poly(ferrocenylmethyl methacrylate), an electrochemically active block-copolymer, in the presence of applied voltage are observed. Understanding the mechanisms behind such changes will be important when trying to achieve reversible polymer structures in the presence of applied voltage.

NON-ENZYMATIC ELECTROCHEMICAL DETECTION OF GLUTAMATE USING TEMPLATED POLYMER-BASED TARGET RECEPTORS.

We present a novel electrochemical biosensing platform for the detection of neurotransmitter glutamate using templated polymer-based target receptors. Our sensing approach demonstrates, for the first time, a non-enzymatic approach without the need of glutamate oxidase, leading to a more specific and rapid response. The proposed detection principle is based on the following two claims: (1) our templated polymer-based receptor results in specific molecular recognition of the target glutamate and, (2) upon target binding, the polymer undergoes a conformation change which can then be measured via electrochemical techniques. This sensing platform has the potential to provide direct monitoring of a variety of non-electroactive species and to eliminate the incorporation of enzymes thereby providing a simpler and more robust alternative to enzyme-based sensors.

A Single-chain Templated Polymer-based Target Receptor as a New Platform for Label-free Selective Electrochemical Sensing.

We report a novel single-chain polymer-based chemical receptor that can be used for the label-free electrochemical detection of an analyte with high selectivity. The polymer was developed using poly-N-isopropylacrylamide (pNIPAM) as a backbone structure in addition to other functional monomers that are used to imprint the template molecule 4-nitrophenol. The polymer also contains a redox reporting monomers (ferrocene) which create a change in the electrochemical signal upon molecular recognition. We hypothesize that the analyte binding to the receptor causes the polymer conformation change from the extended to the collapsed phase. After anchoring the polymer-based receptors onto the surfaces of the gold electrode, when exposed to the analyte, the changes in the electrochemical signals were observed which confirmed the selective target binding as well as the polymer conformation change as a result.

Rapid, High Affinity Binding by a Fluorescein Templated Copolymer Combining Covalent, Hydrophobic, and Acid⁻Base Noncovalent Crosslinks.

A new type of biomimetic templated copolymer has been prepared by reverse addition fragmentation chain transfer polymerization (RAFT) in dioxane. The initial formulation includes the template fluorescein, N-isopropylacrylamide (NIPAM, 84 mol %), methacrylic acid (MAA, 5-mol %), 4-vinylpyridine (4-VP, 9 mmol %), and N,N′-methylenebis(acrylamide) (MBA, 2 mol %). PolyNIPAM is a thermosensitive polymer that comes out of aqueous solution above its lower critical solution temperature forming hydrophobic ‘crosslinks’. MAA and 4-VP interact in dioxane forming acid⁻base crosslinks. The excess 4-VP serves as a recognition monomer organizing around the template fluorescein to form a binding site that is held in place by the noncovalent and covalent crosslinks. The MBA is a covalent crosslinker. The RAFT agent in the resulting copolylmer was reduced to a thiol and attached to gold nanoparticles. The gold nanoparticle bound copolymer binds fluorescein completely in less than two seconds with an affinity constant greater than 108 M−1. A reference copolymer prepared with the same monomers by the same procedure binds fluorescein much more weakly.