Imprinted Polymer Layer Research Articles

Adsorption and specific recognition of DNA by using imprinted polymer layers grafted onto ionic liquid functionalized magnetic microspheres

A molecularly imprinted polymer based on silica modified Fe3O4 microsphere and ionic liquid was prepared for the adsorption and specific recognition of DNA. Water-compatible imidazolium ionic liquids were introduced on the surface of silica-coated Fe3O4 microsphere, then used as the adsorbents, againsting DNA from salmon testes (stDNA) by combining immobilized template and surface imprinting techniques. The imprinted polymer layers were grafted onto ionic liquid functionalized microspheres through sol-gel approach using N-3-(3-triethoxysilylpropyl)-3-aminopropyl imidazolium chloride and N-3-(3-triethoxysilylpropyl)-3-mexyl imidazolium chloride as the functional monomers, and tetraethoxysilane as the cross-linker. Because both imidazolium ionic liquid and DNA are soluble in water, it is mandatory to implement an imprinted process in an aqueous medium to well maintain the integrity of the conformation. Fourier transform infrared spectrometry, thermogravimetric analysis, transmission electron microscopy, X-ray diffraction and vibrating sample magnetometry were employed to characterize the materials. The nanomaterials with multiple imprinting sites and biocompatible cavities exhibit high specific adsorption capacity of DNA (162 mg·g−1) and excellent rebinding selectivity (imprinting factor up to 6.6) against non-imprinted molecules at pH value of 5.10. Moreover, it is feasible to strip DNA from the highly cross-linked polymer network by using aqueous NaCl (1.0 M). The nanomaterials are cap able of capturing DNA from calf whole blood, which might provide a viable tool for DNA preconcentration, separation and recognition.

Surface-imprinted magnetic nanoparticles for the selective enrichment and fast separation of fluoroquinolones in human serum.

Surface enrofloxacin-imprinted magnetic nanoparticles were prepared for the selective recognition and fast separation of fluoroquinolones in human serum by surface-initiated reversible addition fragmentation chain transfer polymerization. The surface morphology and imprinted behavior were investigated and optimized. The living/controlled nature of reversible addition-fragmentation chain transfer polymerization reaction allowed the successful construction of well-defined imprinted polymer layer outside the Fe3 O4 core. Such molecularly imprinted polymers exhibited superparamagnetic properties and specific recognition toward fluoroquinolones. Combined with reversed-phase high-performance liquid chromatography, the prepared molecularly imprinted polymers were used for the selective enrichment and analysis of fluoroquinolones in human serum samples. The recoveries of four fluoroquinolones were 86.8-95.3% with relative standard deviations of 2.0-6.8% (n=3). Such magnetic molecularly imprinted polymers have great prospects in the separation and enrichment of trace analysts in complex biological samples.

Molecularly Imprinted Membrane Electrospray Ionization for Direct Sample Analyses.

Typically dealing with practical samples with very complex matrices, ambient ionization mass spectrometry suffers from low detection sensitivity. In this study, molecular imprinting technology was explored and integrated with the membrane electrospray ionization (MESI) method for direct sample analyses. By enriching targeted analytes on molecularly imprinted membranes (MIMs), improvement (by 10- to 50-fold) in the limit of quantitation could be achieved, compared to conventional nanoelectrospray ionization methods or other ambient ionization methods. MIMs were prepared by cross-linking a synthesized molecularly imprinted polymer layer onto a polyvinylidene difluoride (PVDF) membrane. The characteristics of MIM in recognizing target analytes were investigated and verified. Experiments showed that MIM-ESI could provide satisfactory performances for direct quantification of targeted analytes in complex samples using mass spectroscopy (MS), and the quantitative performance of this methodology was validated. With the capability of target enrichment, the uses of MIM-ESI MS in different application fields were also demonstrated, including food safety, quantification of drug concentrations in blood, pesticide residues in soil, and antibiotic residues in milk.

Micro-structured interdigital capacitors with synthetic antibody receptors for ABO blood-group typing

Interdigital capacitors coated with surface imprinted polymer layers can be designed for ABO-blood group typing. Measurements with 10mm IDCs in deionized water suggested a change in dielectric properties by erythrocyte adhesion which leads to increase in capacitance. In hypotonic conditions, reduction in resistance was monitored as the erythrocytes interacted with imprinted polymer layers, subsequently the cells burst and the ions were enriched near interdigital electrodes. A clear differentiation among all ABO-blood groups was achieved even for A-subgroups, with selectivity factor as high as four i.e. in case of blood group B typing. The basis of these findings are selective interactions between erythrocyte antigens with imprinted sensor coatings. Under isotonic conditions, inclusion of erythrocytes into cavities leads to increase in resistance, because of closed pores in the polymer layers hinder ion diffusion. An enhancement of this sensor signal was observed by a factor of 10 through using smaller IDCs electrode width i.e. 5μm. Reversibility was improved by using different concentrations of sodium chloride for washing. Optimal frequency was found to be 10kHz for the measurements to be done. Capacitive measurements confirmed that sensor layers can be reused even after 300h of operation for blood group typing.

Preparation and characterization of novel thermosensitive magnetic molecularly imprinted polymers for selective recognition of norfloxacin

Novel norfloxacin (NOR) thermosensitive magnetic molecularly imprinted polymers (T-MMIPs) were prepared with functional monomer methacrylic acid (MAA), temperature-response monomer N-isopropylacrylamide (NIPAM) and cross-linking agent N,N′-methylenebisacrylamide (MBA) by the surface imprinting technique. The silica layer and imprinted polymers layer coated on the surface of Fe3O4 nanoparticles, forming double-shell structure. The morphology and composition of the thermosensitive magnetic molecularly imprinted polymers were investigated by transmission electronmicroscope (TEM), Fourier transform infrared spectrometry (FT-IR), thermogravimetic (TGA), vibrating sample magnetometer (VSM), powder X-ray diffraction (XRD) and nitrogen adsorption analysis. The phase behavior and thermosensitivity of T-MMIPs were studied by swelling experiments and adsorption tests at different temperatures. And the adsorption performance of T-MMIPs at 35 °C were evaluated by adsorption experiments, including kinetic, isotherm and selectivity tests. The maximum capacity of T-MMIPs at 35 °C was 52.85 mg g−1. In selective recognition tests, the T-MMIPs showed the highest selectivity for NOR among four components while the T-MNIPs showed similarly adsorption for all components. The prepared T-MMIPs have great potential in the detection and separation of norfloxacin due to the good temperature response, adsorption capacity, selectivity and reusability.

Preparation of Magnetic Molecularly Imprinted Polymer for Melamine and its application in milk sample analysis by HPLC

In this work, a simple and selective method was successfully developed for the determination of melamine (MEL) in milk sample using magnetic surface molecularly imprinted polymer (MMIPs) as sorbent. The MMIPs has been prepared by using MEL as template molecule, methacrylic acid (MAA) as functional monomer and Fe3O4@ SiO2 as magnetic support. The polymer was characterized by means of fourier transform infrared(FT-IR) analysis, X-ray diffraction (XRD) analysis, thermogravimetric Analysis (TGA) ,scanning electron microscope(SEM) and vibrating sample magnetometer (VSM).The results demonstrated that an imprinted polymer layer was successfully coated onto the surface of modified Fe3O4 nanomaterials, resulting in a good magnetic responsibility. The UV spectrophotometry was used to demonstrate the interaction between MEL and MAA. It was found that one MELwas entrapped by two MAA molecules. Binding selectivity, adsorption capacity and the reusability for MMIPs were also evaluated in details. Results indicated the prepared MMIPs exhibited high adsorption capacity, fast binding ability and high selectivity for melamine. Detection limits for melamine were within the range of 0.15-3.00 μg/mL, which could meet the analysis of trace melamine in milk. Calibration curve was established with R of 0.9997. The limit of detection (LOD) of this new method was 15 μg/L. The obtained results revealed that the extraction recoveries of melamine from real samples were in the range of 85.6% to 104.2%. This work proposed a sensitive, rapid, reliable and convenient approach for the determination of trace melamine in complicated milk sample.

Surface imprinted bacterial cellulose nanofibers for cytochrome c purification

Molecularly imprinted polymers possessing synthetic recognition sites present great potential for various application areas, which require specific recognition of molecules. However various molecularly imprinted polymers (MIPs) reported previously have low affinity capacity and low binding kinetics due to weak interactions between template molecule and functional monomer in aqueous media and also high mass transfer resistance. The synthesis of MIPs onto nanomaterial surface via metal ion coordination interactions serves as an effective solution. In this work, we prepared thin molecularly imprinted polymer layer on bacterial cellulose nanofiber surface using Cu2+ chelating monomer, N-Methacryloyl-(L)-histidinemethylester (MAH) as a functional monomer orienting cytochrome c molecules. The conditions of polymerization and adsorption were optimized to obtain best affinity performance. Due to unique properties of bacterial cellulose nanofibers such as high surface-to-volume ratio and macro-porosity, the resultant cytochrome c imprinted nanofibers (Cyt c-MIP NFs) reducing diffusional resistances could achieve high recognition capacity and excellent binding kinetics. Cyt c-MIP NFs were also successfully applied to capture template proteins from real sample. This novel procedure presents simple, cheap and stable fabrication strategy for efficient separation of proteins.

Preparation of a Two-Dimensional Ion-Imprinted Polymer Based on a Graphene Oxide/SiO₂ Composite for the Selective Adsorption of Nickel Ions.

In the present work, a novel two-dimensional (2D) nickel ion-imprinted polymer (RAFT-IIP) has been successfully synthesized based on the graphene oxide/SiO2 composite by reversible addition-fragmentation chain-transfer (RAFT) polymerization. The imprinted materials obtained are characterized by Fourier transmission infrared spectrometry (FT-IR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). The results show that the thermal stability of the graphene oxide/SiO2 composite is obviously higher than that of graphene oxide. RAFT-IIP possesses an excellent 2D homogeneous imprinted polymer layer, which is a well-preserved unique structure of graphene oxide/SiO2. Owing to the intrinsic advantages of RAFT polymerization and 2D imprinted material, RAFT-IIP demonstrate a superior specific adsorption capacity (81.73 mg/g) and faster adsorption kinetics (30 min) for Ni(II) in comparison to the ion-imprinted polymer prepared by traditional radical polymerization and based on the common carbon material. Furthermore, the adsorption isotherm and selectivity toward Ni(II) onto RAFT-IIP and nonimprinted polymer (NIP) are investigated, indicating that RAFT-IIP has splendid recognizing ability and a nearly 3 times larger adsorption capacity than that of NIP (30.94 mg/g). Moreover, a three-level Box-Behnken experimental design with three factors combining the response surface method is utilized to optimize the desorption process. The optimal conditions for the desorption of Ni(II) from RAFT-IIP are as follows: an HCl-type eluent, an eluent concentration of 2.0 mol/L, and an eluent volume of 10 mL.

Preparation of lysozyme imprinted magnetic nanoparticles via surface graft copolymerization

Molecular imprinting as a facile and promising separation technique has received considerable attention because of their high selectivity for target molecules. In this study, we imprinted lysozyme (Lys) on the surface of core–shell magnetic nanoparticles via surface imprinting. The magnetic supports were functionalized with maleic acid and then coated with imprinted polymer layers. The structure and morphology of the resulting magnetic imprinted nanoparticles were characterized by transmission electron microscopy, scanning electron microscope, dynamic light scatting, vibrating sample magnetometer, and thermogravimetric analysis. Binding experiments were carried out to evaluate the properties of magnetic molecularly imprinted polymers (magnetic MIPs) and magnetic non-molecularly imprinted polymers (magnetic NIPs). The protein adsorption results showed that the magnetic MIPs had significant specific recognition toward the template protein and could be easily separated from solution by an external magnetic field. Moreover, the MIPs exhibited fast kinetics for the rebinding of the target protein due to the thin-imprinted layer and showed good reusability by four adsorption–desorption cycles. Therefore, the surface imprinting approach combined with magnetic nanoparticles provided an easy and fast method for the specific recognition of Lys.

Molecularly imprinted polymer layers using Navicula sp. frustule as core material for selective recognition of lysozyme

Surface molecular imprinting onto supported materials is particularly suitable for bulky structure template like protein. Inspired by this method, diatom frustules (Navicula sp. frustules) were used for the first time as supported material due to its unique properties of moderate surface area, reproducible with exactness in vast numbers and biocompatible, in order to address the limitation of nanoparticle as core material with low adsorption capacity. The molecular imprinting on Navicula sp. frustules was synthesized using polyacrylamide incorporate with two charged monomers and LYZ as template. The effect of synthesis conditions such as molar ratio of monomers and feed crosslinking degree were examined. With the optimal crosslinking degree (50%) and molar ratio of monomers (1/0.5/0.5), the LYZ-imprinted frustules exhibited enhancement in both rebinding capacity (124±1.09mgg−1) and imprinting factor (2.99), without showing significant decrease in adsorption kinetics (within 15min) when compared to work of other research groups. Additionally, template removal during LYZ-imprinted frustules synthesis as well as desorption after rebinding was achieved via mildly washing with salt solution. The outstanding recognition, rebinding and desorption performance suggested that Navicula sp. frustules are potential core material for surface molecular imprinting in the application of medical diagnostics.

Preparation and applications of 4-methyl imidazole magnetic surface molecularly imprinted polymers

The magnetic surface molecularly imprinted polymers (MIPs) with specific recognition of 4-methyl imidazole (4-MI) were prepared by using 4-MI as template molecule, methacrylic acid (MAA) as functional monomer and Fe3O4 as magnetic fluid. The polymers were characterized by of Fourier transform infrared spectrometer (FT-IR) analysis, X-ray diffraction (XRD) analysis, transmission electron microscopy (TEM) and vibrating sample magnetometer (VSM). The results demonstrated that an imprinted polymer layer was successfully coated onto the surface of modified Fe3O4 nanomaterials, resulting in a narrow diameter distribution and good magnetic responsibility. The ultraviolet (UV) spectrophotometry was used to demonstrate the interaction between 4-MI and MAA. It was found that one 4-MI molecule was entrapped by one MAA molecule, which was the main existing form of subject and object. By UV spectrophotometric method to study the adsorption performance of magnetic molecularly imprinted polymers, the specific adsorption equilibrium and selectivity were evaluated by batch rebinding studies. The Scatchard analysis showed that there were two kinds of binding sites in the Fe3O4 @ (4-MI-MIP). The corresponding maximum adsorption capacities of 4-MI onto Fe3O4 @ (4-MI-MIP) were 40.31 mg/g and 23.07 mg/g, and the dissociation constants were 64.85 mg/L and 30.41 mg/L, respectively. The kinetic experimental data were correlated with second-order kinetic model. The magnetic molecularly imprinted polymers were used for the adsorption of 4-methyl imidazole in environmental water samples, and good results were obtained.

Enhanced surface imprinting of lysozyme over a new kind of magnetic chitosan submicrospheres

Surface protein imprinting over nano- or micron-sized substrates is an effective approach for improving the biomacromolecule mass transfer and rebinding capacity. For achieving high recognition performance, it is necessary to introduce certain functional groups onto the surface of the support materials which can interact with the template protein. Herein, we report a surface protein imprinting approach using a new kind of core–shell magnetic chitosan submicrospheres as the supports. The surface of these magnetic chitosan particles is tethered with uncross-linked chitosan chains, hence bearing plenty of amino and hydroxyl groups, where a large amount of functional ligands can be readily coupled for capturing of the protein template. With lysozyme as a model print protein, the magnetic supports were functionalized with maleic acid and then coated with imprinted polymer layers. The resulting imprinted microspheres show significantly selective rebinding for lysozyme. In particular, they exhibit a specific rebinding capacity about three times higher than achieved with our previous lysozyme-imprinted particles synthesized in similar way but with maleic acid modified silica nanoparticles as the supports. This can be attributed to the much higher template binding capacity to the modified magnetic chitosan submicrospheres. Also, the resultant imprinted particles can be easily collected by a magnet. Therefore, such kind of chitosan submicrospheres may be a versatile carrier for constructing high-capacity and magnetically recyclable surface protein-imprinted particles.

Rapid and molecular selective electrochemical sensing of phthalates in aqueous solution.

Reported research work presents real time non-invasive detection of phthalates in spiked aqueous samples by employing electrochemical impedance spectroscopy (EIS) technique incorporating a novel interdigital capacitive sensor with multiple sensing thin film gold micro-electrodes fabricated on native silicon dioxide layer grown on semiconducting single crystal silicon wafer. The sensing surface was functionalized by a self-assembled monolayer of 3-aminopropyltrietoxysilane (APTES) with embedded molecular imprinted polymer (MIP) to introduce selectivity for the di(2-ethylhexyl) phthalate (DEHP) molecule. Various concentrations (1-100 ppm) of DEHP in deionized MilliQ water were tested using the functionalized sensing surface to capture the analyte. Frequency response analyzer (FRA) algorithm was used to obtain impedance spectra so as to determine sample conductance and capacitance for evaluation of phthalate concentration in the sample solution. Spectrum analysis algorithm interpreted the experimentally obtained impedance spectra by applying complex nonlinear least square (CNLS) curve fitting in order to obtain electrochemical equivalent circuit and corresponding circuit parameters describing the kinetics of the electrochemical cell. Principal component analysis was applied to deduce the effects of surface immobilized molecular imprinted polymer layer on the evaluated circuit parameters and its electrical response. The results obtained by the testing system were validated using commercially available high performance liquid chromatography diode array detector system.

Molecularly imprinted polymeric sensings layers grafted from aryl diazonium‐modified surfaces for electroanalytical applications. A mini review

We review our strategy to attach polymerization initiators to electrode surfaces by electroreduction of aryl diazonium salts. The ~5 nm‐thick aryl layers efficiently initiate the growth of crosslinked polymer grafts prepared in the presence of template molecules. We address the important parameters in obtaining highly sensitive molecularly imprinted vinylic polymer layers grafted on working electrodes such as glassy carbon, gold and indium tin oxide. Square wave voltammetry is the preferred electroanalytical tool while the limit of detection (LOD) of analytes can be decreased down to subnanomolar detection by synthesizing ultrathin layers in the 20–30 nm thickness. Further improvement can be achieved with molecularly imprinted polymer layers with embedded gold nanoparticles. Rebinding solvent is demonstrated to be another important parameter for lowering LOD. This work highlights XPS as a unique surface sensitive technique for monitoring the chemical composition changes occurring at the electrode surface during the building of the high performance sensing layers as well as for evidencing the reversible complexation of the template molecule by the molecularly imprinted recognition sites. Copyright © 2014 John Wiley & Sons, Ltd.

Preparation and Adsorption Property of Glutathione Magnetic Molecularly Imprinted Polymers

The aim of this work was to produce a kind of magnetic molecular imprinted polymers (MMIPs) with core-shell structure and good magnetic separation performance. Compared with the preparation of traditional MIPs, a novel process was adopted to prepare MMIPs with good simplicity, flexibility and selectivity, in which Fe3O4 NPs were firstly prepared by a reverse microemulsion method, and subsequently directly added to the mixed solution to undergo the polymerization reaction without washing and drying. This method avoids availably the agglomeration and oxidation of Fe3O4 NPs. The MMIPs for glutathione (GSH) specific recognition (GSH-MMIPs), with core-shell structure, were prepared via one-step method polymerization, in which GSH as template molecule, N-vinylpyrrolidone (NVP) and acrylamide (AM) as functional monomers, N,N'-methylene bisacrylamide as crosslinker, H2O2-Vc as initiator and Fe3O4 nanoparticles (NPs) modified with γ-methacryloxypropyltrimethoxysilane (KH-570) as magnetic carrier. The as-prepared polymers were characterized by using of X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy and vibrating sample magnetometer (VSM). The results demonstrated that an imprinted polymer layer was successfully coated onto the surface of modified Fe3O4 NPs, resulting in a narrow diameter distribution and good magnetic responsibility. The specific adsorption equilibrium and selectivity were evaluated by batch rebinding studies. The Scatchard analysis showed that there were two kinds of binding sites in the GSH-MMIPs, the dissociation constants were 8.786×10 mol/L and 5.424×10 mol/L, and the corresponding maximum adsorption capacities of GSH onto GSH-MMIPs were 49.195 mg/g and 155.003 mg/g, respectively. The selectivity of the GSH-MMIPs was also evaluated to determine the rebinding capability of GSH and its analogues, compared with that of the magnetic non imprinted polymers (MNIPs) with similar chemical components. The GSH-MMIPs with high imprinting factor of 3.99 indicated that it was of good selectivity. This protocol was further employed for the extraction and separation of GSH from an aqueous solution.

Preparation and Application of Imprinted Electrochemical Sensor Based on Dopamine Self-Polymerization

An imprinted polymer layer was easily modified on the Au electrode surface by spontaneous oxidative polymerization of dopamine in the presence of theophylline to fabricate a novel imprinted electrochemical sensor. The results of scanning electron microscopy and cyclic voltammetry measurements demonstrated the successfully preparation of the imprinted sensor. The effects of ratio between theophylline and dopamine, polymerization pH and time were optimized. The prepared sensor exhibited specific adsorption, selective recognition, and sensitive detection capacities toward theophylline. The linear response to theophylline ranged from 2.0 × 10−7-1.6 × 10−4 mol L−1 with a detection limit of 1.0 × 10−7 mol L−1. Moreover, the sensor could be applied in the detection of theophylline in real samples with satisfactory results. It can be reasonably expected that this simple, novel, and efficient strategy for imprinted sensor preparation based on dopamine self-polymerization provides a promising platform for other molecule recognition and detection.

Grating-structured metallic microsprings.

We fabricate grating-structured metallic microsprings with well-defined helical angles and diameters, which are self-rolled from strained nanomembranes patterned with gratings. The grating structures on the metal membrane, replicated from the imprinted polymer layer beneath, give rise to the controlled rolling direction after selective etching of the underlying sacrificial layer. The rolling direction of the grating-structured thin metal film is always perpendicular to the long side edge of gratings, offering a good way to roll up strained strips into well controlled three-dimensional (3D) microsprings simply by altering the dimension and orientation of the structured strips. The mechanical elasticity of these grating-structured metallic microsprings is verified for the potential application as a flow rate sensor. Our work may stimulate rigorous synthesis of highly functional and complex 3D helical micro and nanostructures, and hint a broad range of applications such as environmental sensors, micro-/nanoscale robots, metamaterials, etc.

Specific recognition of bovine serum albumin using superparamagnetic molecularly imprinted nanomaterials prepared by two-stage core-shell sol-gel polymerization.

A novel type of uniform magnetic imprinted nanomaterial for the recognition of bovine serum albumin (BSA) was prepared by anchoring MIP shells on the surface of silica deposited Fe3O4 NPs via a surface imprinting process and two-stage core-shell sol-gel polymerization. The resulting magnetic nanomaterials were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, and a vibrating sample magnetometer (VSM). The measurements indicated that the as-synthesized nanospheres exhibited good dispersion, high crystallinity, and satisfactory superparamagnetic properties. Moreover, the obtained Fe3O4@BSA-MIPs had a high saturation magnetization (43.82 emu g-1), which allowed them to be easily separated from solution by means of an external magnetic field. The thickness of the imprinted polymer layer was approximately 5 nm, which would be effective for the mass transport between the solution and the surface of Fe3O4@BSA-MIPs. The kinetic adsorption experiment showed that the imprinted nanomaterials could reach equilibrium within 15 min and be well described by the second-order kinetics model, indicating chemical adsorption might be the rate-limiting step. Meanwhile, the imprinting factor and selectivity coefficient of the Fe3O4@BSA-MIPs were as high as 16.4 and 4.65, displaying excellent selectivity towards BSA. In addition, the resulting imprinted polymers were without obvious deterioration after ten adsorption-desorption cycles and different batches of which exhibited excellent reproducibility. Successful application in the selective separation and enrichment of BSA from a bovine blood sample and good recovery after a reasonably mild elution suggested that the Fe3O4@BSA-MIPs could specifically capture BSA from a real complex matrix and had potential value in practical applications.

Core–shell molecularly imprinted polymer nanoparticles with assistant recognition polymer chains for effective recognition and enrichment of natural low-abundance protein

Core–shell molecular imprinting of nanomaterials overcomes difficulties with template transfer and achieves higher binding capacities for macromolecular imprinting, which are more important to the imprinting of natural low-abundance proteins from cell extracts. In the present study, a novel strategy of preparing core–shell nanostructured molecularly imprinted polymers (MIPs) was developed that combined the core–shell approach with assistant recognition polymer chains (ARPCs). Vinyl-modified silica nanoparticles were used as support and ARPCs were used as additional functional monomers. Immunoglobulin heavy chain binding protein (BiP) from the endoplasmic reticulum (ER) was chosen as the model protein. The cloned template protein BiP was selectively assembled with ARPCs from their library, which contained numerous limited-length polymer chains with randomly distributed recognition and immobilization sites. The resulting complex was copolymerized onto the surface of vinyl-modified silica nanoparticles under low concentrations of the monomers. After template removal, core–shell-structured nanoparticles with a thin imprinted polymer layer were produced. The particles demonstrated considerably high adsorption capacity, fast adsorption kinetics and selective binding affinities toward the template BiP. Furthermore, the synthesized MIP nanoparticles successfully isolated cloned protein BiP from protein mixtures and highly enriched BiP from an ER extract containing thousands of kinds of proteins. The enrichment reached 115-fold and the binding capacity was 5.4μgg−1, which were higher than those achieved by using traditional MIP microspheres. The advantageous properties of MIP nanoparticles hold promise for further practical applications in biology, such as protein analysis and purification.

Sensors based on cytochrome P450 and CYP mimicking systems

Cytochrome P450 enzymes (CYPs) act on more than 90 percent of all drugs currently on the market. The catalytic cycle requires electron supply to the heme iron in the presence of oxygen. Electrochemistry allows to characterise the reaction mechanism of these redox enzymes by observing the electron transfer in real time. According to the number of publications on protein electrochemistry CYP has the third position after glucose oxidase and cytochrome c. CYP based enzyme electrodes for the quantification of drugs, metabolites or pesticides have been developed using different iso-enzymes. A crucial step in the sensor development is the efficiency of coupling the biocatalytic systems with the electrode is.In the 1970s the direct electron transfer of heme and heme peptides called microperoxidases (MPs) was used as model of oxidoreductases. They exhibit a broad substrate spectrum including hydroxylation of selected aromatic substrates, demethylation and epoxidation by means of hydrogen peroxide. It overlaps with that of P450 making heme and MPs to alternate recognition elements in biosensors for the detection of typical CYP substrates. In these enzyme electrodes the signal is generated by the conversion of all substrates thus representing in complex media an overall parameter. By combining the biocatalytic substrate conversion with selective binding to a molecularly imprinted polymer layer the specificity has been improved.Here we discuss different approaches of biosensors based on CYP, microperoxidases and catalytically active MIPs and discuss their potential as recognition elements in biosensors. The performance of these sensors and their further development are discussed.