Hybrid Material for Protein Sensing Based on Electrosynthesized MIP on a Mannose Terminated Self‐Assembled Monolayer

A novel strategy to prepare a surface confined molecularly imprinted polymer (MIP) film directly on a transducer surface for protein sensing is achieved by combining interaction with a natural binding receptor and binding to a fully synthetic MIP. A thiolated oligoethyleneglycol (OEG)/mannose conjugate is first self‐assembled on the transducer surface. Then the carbohydrate binding protein, concanavalin A (ConA), is “vectorially” immobilized as a submonolayer on the underlying mannose modified surface. Afterwards, an ultrathin polyscopoletin film with the thickness comparable to that of the protein is electrodeposited on the top. This architecture ensures that the target is confined to the film surface. The resulting functional material shows an approximately 20‐fold higher affinity than that obtained from the mannose self‐assembled monolayer. This result shows a synergism between multivalent binding of the natural sugar ligand and the non‐covalent interactions of the target within the MIP cavities. Recognition capability of the film is characterized by a real‐time measurement using quartz crystal microbalance. In comparison to the non‐imprinted film, the imprinted film reveals 8.6 times higher binding capacity towards ConA. High discrimination towards the target protein's homologues shows size and shape specificity of the imprint.     

Nanosphere Lithography as a Versatile Method to Generate Surface‐Imprinted Polymer Films for Selective Protein Recognition

A versatile approach based on nanosphere lithography is proposed to generate surface‐imprinted polymers for selective protein recognition. A layer of 750 nm diameter latex bead‐protein conjugate is deposited onto the surface of gold‐coated quartz crystals followed by the electrosynthesis of a poly(3,4‐ethylenedioxythiophene)/poly(styrenesulfonate) (PEDOT/PSS) film with thicknesses on the order of the bead radius. The removal of the polymer bead‐protein conjugates, facilitated by using a cleavable protein‐nanosphere linkage is shown to result in 2D arrays of periodic complementary size cavities. Here it is demonstrated by nanogravimetric measurements that the imprinting proceeds further at molecular level and the protein (avidin) coating of the beads generates selective recognition sites for avidin on the surface of the PEDOT/PSS film. The binding capacity of such surface‐imprinted polymer films is ca. 6.5 times higher than that of films imprinted with unmodified beads. They also exhibit excellent selectivity against analogues of avidin, i.e., extravidin, streptavidin, and neutravidin, the latter being in fact undetectable. This methodology, if coupled with properly oriented conjugation of the macromolecular template to the nanoparticles, offers the possibility of site‐directed imprinting.     

Molecularly Imprinted Polymer Grafted Porous Au‐Paper Electrode for an Microfluidic Electro‐Analytical Origami Device

Molecular imprinting technique is introduced into microfluidic paper‐based analytical devices (μ‐PADs) through electropolymerization of molecular imprinted polymer (MIP) in a novel Au nanoparticle (AuNP) modified paper working electrode (Au‐PWE). This is fabricated through the growth of a AuNP layer on the surfaces of cellulose fibers in the PWE. Due to the porous morphology of paper as well as the high specific surface area and conductivity of the resulting AuNP layer on the cellulose fibers, the effective surface area and the sensitivity of the Au‐PWE is enhanced remarkably. Based on this novel MIP‐Au‐PWE and the principle of origami, a microfluidic MIP‐based electro‐analytical origami device (μ‐MEOD), comprised of one auxiliary pad surrounded by four sample tabs, is developed for the detection of D‐glutamic acid in a linear range from 1.2 nM to 125.0 nM with a low detection limit of 0.2 nM. The selectivity, reproducibility, and stability of this μ‐MEOD are investigated. This μ‐MEOD would provide a new platform for high‐throughput, sensitive, specific, and multiplex assay as well as point‐of‐care diagnosis in public health, environmental monitoring, and the developing world.     

Hot Spot‐Localized Artificial Antibodies for Label‐Free Plasmonic Biosensing

The development of biomolecular imprinting over the last decade has raised promising perspectives in replacing natural antibodies with artificial antibodies. A significant number of reports have been dedicated to imprinting of organic and inorganic nanostructures, but very few were performed on nanomaterials with a transduction function. Herein, a relatively fast and efficient plasmonic hot spot‐localized surface imprinting of gold nanorods using reversible template immobilization and siloxane copolymerization is described. The technique enables a fine control of the imprinting process at the nanometer scale and provides a nanobiosensor with high selectivity and reusability. Proof of concept is established by the detection of neutrophil gelatinase‐associated lipocalin (NGAL), a biomarker for acute kidney injury, using localized surface plasmon resonance spectroscopy. The work represents a valuable step towards plasmonic nanobiosensors with synthetic antibodies for label‐free and cost‐efficient diagnostic assays. It is expected that this novel class of surface imprinted plasmonic nanomaterials will open up new possibilities in advancing biomedical applications of plasmonic nanostructures.     

A Molecularly Imprinted Copolymer Designed for Enantioselective Recognition of Glutamic Acid

A newly designed molecularly imprinted polymer (MIP) material was developed and successfully used as recognition element to fabricate a capacitive sensor for enantioselective recognition of glutamic acid (Glu). The MIP with a well‐defined structure was synthesized on a gold electrode in one step by electrochemical copolymerization of o‐phenylenediamine (o‐PD) and dopamine (DA) in the presence of template molecule Glu. The resulting MIP material was characterized with a potentiostatic frequency scan method, cyclic voltammetry, capacitance measurements, atomic force microscopy, and X‐ray photoelectron spectroscopy. The structure and recognition behaviour of the copolymer film to template molecule depended on its composition. The optimal composition was at the o‐PD to DA molar ratio of 3:2. With a potentiostatic time scan method the copolymer displayed high enantioselectivity and sensitivity to the stereoselective rebinding of L ‐ or D ‐Glu to their corresponding artificial receptor due to the exact definition of the imprint cavity. The capacitance response of the sensor for L ‐Glu or D ‐Glu was proportional to their concentration in the range of 16.7 to 250 μM . The enantiometric selectivity coefficients for L ‐Glu and D ‐Glu imprinted films against their respective enantiomers are 24 and 15, respectively. The resulting MIP capacitive sensors showed good reproducibility, stability and repeatability. This strategy opened a convenient way for preparation of enantioselective MIPs and recognition of enantiotropic molecules.     

Magnetic Molecularly Imprinted Polymer Nanocomposites via Surface‐Initiated RAFT Polymerization

A general protocol to synthesize superparamagnetic molecularly imprinted polymer particles, using a RAFT‐mediated approach, is described. S‐ propranolol‐imprinted composites were obtained by functionalizing commercially available amino‐modified Fe₃O₄ nanoparticles with a trithiocarbonate agent and subsequently by polymerizing thin molecularly imprinted layers. Different parameters were optimized and their effect on both nanomorphology and imprinting behaviour was studied. Optimum conditions allowed the synthesis of 40 nm composite particles with a 7 nm MIP shell, exhibiting superparamagnetic properties and specific molecular recognition of S‐ propranolol. The possibility of fine‐tuning the surface properties of the particles is demonstrated by using the “living” nature of active RAFT fragments present on the surface of the composites to further functionalize the particles with ethylene glycol methacrylate phosphate polymer brushes.     

Vectorially Imprinted Hybrid Nanofilm for Acetylcholinesterase Recognition

Effective recognition of enzymatically active tetrameric acetylcholinesterase (AChE) is accomplished by a hybrid nanofilm composed of a propidium‐terminated self‐assembled monolayer (Prop‐SAM) which binds AChE via its peripheral anionic site (PAS) and an ultrathin electrosynthesized molecularly imprinted polymer (MIP) cover layer of a novel carboxylate‐modified derivative of 3,4‐propylenedioxythiophene. The rebinding of the AChE to the MIP/Prop‐SAM nanofilm covered electrode is detected by measuring in situ the enzymatic activity. The oxidative current of the released thiocholine is dependent on the AChE concentration from ≈0.04 × 10^?6 to 0.4 × 10^?6m . An imprinting factor of 9.9 is obtained for the hybrid MIP, which is among the best values reported for protein imprinting. The dissociation constant characterizing the strength of the MIP‐AChE binding is 4.2 × 10^?7m indicating the dominant role of the PAS‐Prop‐SAM interaction, while the benefit of the MIP nanofilm covering the Prop‐SAM layer is the effective suppression of the cross‐reactivity toward competing proteins as compared with the Prop‐SAM. The threefold selectivity gain provided by i) the “shape‐specific” MIP filter, ii) the propidium‐SAM, iii) signal generation only by the AChE bound to the nanofilm shows promise for assessing AChE activity levels in cerebrospinal fluid.     

Ultrasensitive Specific Stimulant Assay Based on Molecularly Imprinted Photonic Hydrogels

Taking theophylline and (1R,2S)‐(−)‐ephedrine as template molecules, two imprinted photonic‐hydrogel films are prepared by a combination of colloidal‐crystal and molecular‐imprinting techniques. This paper shows a new approach for rapid and handy stimulant detection with high sensitivity and specificity. One film is proposed for analogous molecule assay, another one for chiral recognition. The key point of this approach is that the imprinted photonic polymer (IPP) consists of a three‐dimensional (3D), highly‐ordered and interconnected macroporous array with a thin hydrogel wall, where nanocavities complementary to analytes in shape and binding sites are distributed. This special, bicontinuous, hierarchical structure enables this polymer to report quickly, easily, sensitively and directly a molecular recognition event without any transducers and treatments for analytes (label‐free). The inherent affinity of the nanocavities, deriving from molecular imprinting, makes these sensors highly specific to analytes, even if in a competitive environment. Their sensitive and specific responses to stimulants in buffer are determined by Bragg diffractive shifts due to the lattice change of their 3D ordered macroporous arrays resulting from their preferential rebinding to the target molecules. The measurements show that the prepared hydrogel films exhibit high sensitivity in such a 0.1 fM concentration of analytes and specificity even in a competitive urinous buffer. The reported method provides a rapid and handy approach for stimulant assay and drug analysis in athletic sports.     

Molecularly Imprinted Silver‐Halide Reflection Holograms for Label‐Free Opto‐Chemical Sensing

Hierarchical structuring of materials offers exciting opportunities to construct functional devices that exploit the ordering at different length scales to impart key functional properties. Herein, multiple processes are combined to create complex materials organized at the molecular, nano, and microscales for selective detection of testosterone by label‐free opto‐chemical sensing. Molecular imprinting is used to construct molecular scale analyte‐selective cavities. Microphase separation produces a porous polymer film within which sensitized silver halide nanocolloids are dispersed by a process of infusion and controled precipitation, then converted to periodic layers of silver nanoparticles by holographic patterning followed by chemical development. Testosterone binding is followed via wavelength changes of the holographic reflection peak as a function of testosterone concentration and incubation time. Polymer cross‐linking and film porosity are optimized with respect to the needs of both molecular recognition and hologram quality. The silver halide infusion step does not destroy the molecular selectivity of the molecularly imprinted polymers (MIP). Selective, label‐free sensing of testosterone is possible at concentrations down to 1 μm . The approach is generic and should be applicable to many types of molecules and conventional MIP formulations, individually or in multiplexed arrays.     

Polymer Films Composed of Surface‐Bound Nanofilaments with a High Aspect Ratio,Molecularly Imprinted with Small Molecules and Proteins

Hierarchically nanostructured materials that combine two or more levels of structuring and that exhibit a combination of useful features have gained considerable interest over recent years. Here, the generation of surface‐bound nanofilaments with a high aspect ratio by nanomolding on a nanoporous template surface is described. The filaments, at the same time, carry molecularly imprinted binding sites. The dye fluorescein and the protein myoglobin are used as model templates for imprinting. The surfaces exhibit specific binding as revealed by fluorescence microscopy. The wetting properties of the surfaces depend on the dimensions of the nanofilaments and on the nature of the polymer. It is believed that these materials can potentially be useful for applications in biosensors and biochips.     

Surface Imprinting in Layer‐by‐Layer Nanostructured Films

In this Full Paper, we develop a novel approach for the generation of stable molecularly imprinted sites in polymeric films by combining the layer‐by‐layer (LbL) technique and photochemical crosslinking of the layered structure. After photo‐crosslinking, the imprinted films show high reproducibility and rapid loading and unloading of imprinted sites by the template molecules. Moreover, the competitive adsorption of template molecules and redox labels into the imprinted film using electrochemical methods indicates that the imprinted film has higher affinity for template molecules. We believe this approach may have some advantages over traditional ways of preparing imprinted sites in polymer matrices and it may open a new avenue for the functionalization of LbL films.     

Ultrathin Polydopamine Films with Phospholipid Nanodiscs Containing a Glycophorin A Domain

Cellular membranes have long served as an inspiration for nanomaterial research. The preparation of ultrathin polydopamine (PDA) films with integrated protein pores containing phospholipids and an embedded domain of a membrane protein glycophorin A as simplified cell membrane mimics is reported. Large area, ultrathin PDA films are obtained by electropolymerization on gold surfaces with 10–18 nm thickness and dimensions of up to 2.5 cm². The films are transferred from gold to various other substrates such as nylon mesh, silicon, or substrates containing holes in the micrometer range, and they remain intact even after transfer. The novel transfer technique gives access to freestanding PDA films that remain stable even at the air interfaces with elastic moduli of ≈6–12 GPa, which are higher than any other PDA films reported before. As the PDA film thickness is within the range of cellular membranes, monodisperse protein nanopores, so‐called “nanodiscs,” are integrated as functional entities. These nanodisc‐containing PDA films can serve as semi‐permeable films, in which the embedded pores control material transport. In the future, these simplified cell membrane mimics may offer structural investigations of the embedded membrane proteins to receive an improved understanding of protein‐mediated transport processes in cellular membranes.     

Solid‐Phase Synthesis of Molecularly Imprinted Polymer Nanoparticles with a Reusable Template–“Plastic Antibodies”

Molecularly imprinted polymers (MIPs) are generic alternatives to antibodies in sensors, diagnostics, and separations. To displace biomolecules without radical changes in infrastructure in device manufacture, MIPs should share their characteristics (solubility, size, specificity and affinity, localized binding domain) whilst maintaining the advantages of MIPs (low‐cost, short development time, and high stability) hence the interest in MIP nanoparticles. Herein, a reusable solid‐phase template approach is reported (fully compatible with automation) for the synthesis of MIP nanoparticles and their precise manufacture using a prototype automated UV photochemical reactor. Batches of nanoparticles (30–400 nm) with narrow size distributions imprinted with: melamine (d = 60 nm, K_d = 6.3 × 10^?8 M ), vancomycin (d = 250 nm, K_d = 3.4 × 10^?9 M ), a peptide (d = 350 nm, K_d = 4.8 × 10^?8 M ) and proteins have been produced. The instrument uses a column packed with glass beads, bearing the template. Process parameters are under computer control, requiring minimal manual intervention. For the first time, the reliable re‐use of molecular templates is demonstrated in the synthesis of MIPs (≥30 batches of nanoMIPs without loss of performance). NanoMIPs are produced template‐free and the solid‐phase acts both as template and affinity separation medium.     

Conducting‐Polymer Microcontainers: Controlled Syntheses and Potential Applications

We have demonstrated the controlled synthesis of conducting‐polymer microcontainers through the electrochemical generation of surfactant (i.e., β‐naphthalenesulfonic acid, β‐NSA)‐stabilized H₂ gas bubbles on the working electrode, followed by electrochemical polymerization of pyrrole around the wall of the “soap‐bubble” template. It was noticed that the density, shape, and wall thickness of the polypyrrole microcontainers thus prepared could be regulated by controlling the electrochemical potential applied for the generation of H₂ gas and the experimental conditions (e.g., the surfactant concentration, number of the cyclic voltammetric scanning) for the electropolymerization of pyrrole. By pre‐patterning the working electrode surface with non‐conducting polymers using microcontact printing (μCP) or plasma patterning, we have also produced conducting‐polymer microcontainers in a patterned fashion. Furthermore, potential applications of the patterned and non‐patterned conducting‐polymer microcontainers have been demonstrated; for example, through the encapsulation of appropriate fluorescence‐labeled molecules (e.g., fluorescein cadaverin) into the conducting‐polymer microcontainers by sealing their opened mouths with sequential electropolymerization of pyrrole. The resulting closed microcontainers could then be used for controlled releases.     

Adsorption and Direct Electron Transfer from Hemoglobin into a Three‐Dimensionally Ordered Macroporous Gold Film

Application of protein‐based, direct electron communication in bioelectronic devices, biosensors, or biofuel cells usually requires high stability and function density of the immobilized proteins or enzymes. Traditional methods have been used to increase the function density using multilayer immobilization techniques at the expense of losing stability and electron‐communication rate, that is, generally only protein molecules near the electrode surface are electroactive. In order to overcome the above problems, a three‐dimensional, ordered, macroporous gold film electrode is synthesized electrochemically by an inverted colloidal crystal template technique. The uniform, three‐dimensional macroporous gold provides superior conductivity, high stability, and large surface area. Its interconnected macroporous structure, containing gold nanoparticles, significantly enhances the amount of adsorbed hemoglobin (Hb) molecules at the monolayer level and also provides a good microenvironment for retaining the biological activity of the adsorbed protein, as confirmed by electrochemical and attenuated total reflection Fourier‐transform infrared spectroscopy. Therefore, direct electron transfer between the adsorbed Hb and the electrode is achieved. Adsorption of Hb on the macroporous gold film electrode is monitored using electrochemical impedance spectroscopy. The saturated adsorption amount, Γ, of the Hb is determined to be 6.55×10^–10 mol cm^–2 with a surface coverage of 88.1 %. The electrochemical behavior and the adsorption mechanism of Hb on the macroporous gold film electrode are discussed on the basis of the experimental results.     

Selective Artificial Receptors Based on Micropatterned Surface‐Imprinted Polymers for Label‐Free Detection of Proteins by SPR Imaging

A novel concept to generate micropatterned surface‐imprinted polymers (SIPs) for protein recognition by using standard photolithographic technology is introduced. Avidin‐imprinted poly(3,4‐ethylenedioxythiophene)/poly(styrenesulfonate) (PEDOT/PSS) conducting polymer microbands are prepared directly on surface plasmon resonance (SPR) chips, which enable convenient label‐free monitoring of the binding events. The novel surface‐imprinted microstructures bind avidin, the template protein, with dissociation constants in the submicromolar range (125 nM ). The SIPs have an avidin binding capacity approximately one order of magnitude higher than the corresponding nonimprinted polymers and are able to discriminate among functional homologues of avidin, i.e., neutravidin, extravidin, and streptavidin.     

Design of Catalytically Active Cylindrical and Macroporous Gold Microelectrodes

Porous gold structures with a well‐defined pore size and thickness are obtained through electrochemical deposition of gold in a colloidal crystal template synthesized by the Langmuir–Blodgett (LB) technique. Cylindrical gold wires were used as substrates for the LB deposition of successive monolayers of silica particles of various sizes, and the electrodeposition of gold through this inorganic template led to a homogeneous, porous metal structure. These materials were characterized through typical electrochemical experiments and showed high surface‐to‐volume ratios with promising features for their further use in miniaturized electrocatalytic devices.     

Nanoimprint Lithography and the Role of Viscoelasticity in the Generation of Residual Stress in Model Polystyrene Patterns

Understanding polymer deformation during the nanoimprinting process is key to achieving robust polymer nanostructures. Information regarding this process can be extracted from monitoring the decay of the imprinted polymer patterns during thermal annealing. In the present work, the effect of both the molar mass and the imprinting temperature on the pattern decay behavior during thermal annealing is investigated. Previously, it was found that the decay rate is fastest for a highly entangled polymer due to the elastic recovery caused by the residual stress created during the imprinting process. The present paper demonstrates that this residual stress level can be modified through control of the imprinting temperature. These results are contrasted with those for an unentangled polymer over a similar range of imprinting temperatures, where it is found that the pattern decay is controlled by simple Newtonian flow. In particular, the pattern decay is well described by surface‐tension‐driven viscous flow, and no imprinting‐temperature effect is observed during thermal annealing. It is shown that the stability of the film against pattern decay can be optimized for moderately entangled polymer films. This effect is attributed to the competition between the effect of increased viscosity with increasing molar mass and increased residual stresses with entanglements. These observations provide guidance for the optimization of imprinting process in terms of selection of molar mass and processing temperatures.     

Gold Nanoparticle–Colloidal Carbon Nanosphere Hybrid Material: Preparation,Characterization, and Application for an Amplified Electrochemical Immunoassay

A rapid microwave‐hydrothermal method has been developed to prepare monodisperse colloidal carbon nanospheres from glucose solution, and gold nanoparticles (AuNPs) are successfully assembled on the surface of the colloidal carbon nanospheres by a self‐assembly approach. The resulting AuNP/colloidal carbon nanosphere hybrid material (AuNP/C) has been characterized and is expected to offer a promising template for biomolecule immobilization and biosensor fabrication because of its satisfactory chemical stability and the good biocompatibility of AuNPs. Herein, as an example, it is demonstrated that the as‐prepared AuNP/C hybrid material can be conjugated with horseradish peroxidase‐labeled antibody (HRP‐Ab₂) to fabricate HRP‐Ab₂‐AuNP/C bioconjugates, which can then be used as a label for the sensitive detection of protein. The amperometric immunosensor fabricated on a carbon nanotube‐modified glass carbon electrode was very effective for antibody immobilization. The approach provided a linear response range between 0.01 and 250 ng mL^?1 with a detection limit of 5.6 pg mL^?1. The developed assay method was versatile, offered enhanced performances, and could be easily extended to other protein detection as well as DNA analysis.     

Design of thermal imprinting system with uniform residual thickness

A new thermal imprinting system for the printed circuit boards (PCBs) with both large areas and fine conducting lines was developed adopting hot airs with a high pressure. Several small nickel stamps were used to cover the large area, and the stamps were replicated from an electroforming process, in addition, a vacuum jig was utilized to avoid bubbles captured in resins or imprinted interfaces. Stefan’s equation was used to estimate residual thicknesses of the imprinted resins, and effects of imprinting conditions on the residual thickness were investigated from numerical analyses to confirm process profiles and specifications of the developed equipment. The results show that the developed imprinting system can remarkably improve the uniformity of the residual thickness after imprinting, as compared with those of the conventional press, in spite of the thickness difference between the used stamps.