Advanced Biomaterials and Biodevicess

390 Advanced Biomaterials and Biodevices force in order to prepare m-MIP/MGCE sensor [39]. The two very popu- lar techniques CV and EIS have been applied to investigate the perfor- mance of the obtained imprinted sensor. Various factors, known to affect the response behavior of the MMIP/MGCE electrode, were studied and optimized. The imprinted sensor exhibits high recognition ability and affinity for MNZ in comparison with the non-imprinted one. In addition, the MMIP/MGCE also shows good stability and acceptable reproduc- ibility for the determination of MNZ. Under the optimal experimental conditions, the current response of the electrochemical sensor was linear to MNZ concentrations in the range from 5.0×10–8 to 1.0×10–6 M, with the detection limit of 1.6×10–8 M. The method was successfully applied to the analysis of MNZ in milk samples and honey samples with acceptable recoveries of 93.5% to 102.2%. Similarly, Ma et al., prepared a core–shell molecularly imprinted polymers of 17β-estradiol on the surface of silica nanoparticles (SiO2@ E2-MIPs) [40]. The sorption capacity of the SiO2@E2-MIPs were nearly 5 times that of the NIPs, and it only took 25 min to achieve the sorption equilibrium. It indicated that the SiO2@E2-MIPs exhibited a high selectiv- ity, large adsorption capacity and fast kinetics. When the SiO2@E2-MIPs were used as dispersive solid-phase extraction absorbents to selectively enrich and determine estrogens in duck feed, the average recoveries of E2 and estriol (E3) were higher than 96.74% and 72.07%, respectively, and the relative standard deviations (RSD) of E2 and E3 were less than 1.61% and 3.28%, respectively. The study provides an effective method for the separa- tion and enrichment of estrogens in the complex matrix samples by the SiO2@E2-MIPs. In general, SiO2 based core shell nanoparticle are more preferable and reported in a lot, but some core-shell gold nanoparticle are also reported as a substrate for imprinting. In this year 2013, Xue et al. explored a surface- imprinted core-shell AuNPs for the highly selective detection of BPA by SERS [41]. A triethoxysilane-template complex (BPA-Si) was synthesized and then utilized to fabricate a MIP layer on the AuNPs via a sol-gel pro- cess (Figure 11.15). The imprinted BPA molecules were removed by a simple thermal treatment to generate the imprint-removed material, MIP-ir-AuNPs, with the desired recognition sites that could selectively rebind the BPA molecules. The morphological and polymeric characteristics of MIP- ir-AuNPs were investigated by transmission electron microscopy and Fourier-transform infrared spectroscopy. The results demonstrated that the MIP-ir-AuNPs were fabricated with a 2nm MIP shell layer

Molecular Imprinting and Nanotechnology 391 Imprinting BPA-Si TEOS Remove MIP-AuNPs Raman Probe template MIP-ir-AuNPs Portable Raman Rebind spectrometer MIP-ir-AuNPs incubated in BPA 600 1000 1400 Raman shift (cm–1) Figure 11.15 Schematic illustration for fabricating MIP-ir-AuNPs and using MIP-ir- AuNPs for selective detection of BPA using a small portable Raman spectrometer [41]. within which abundant amine groups were generated. The rebinding kinetics study showed that the MIP-ir-AuNPs could reach the equi- librium adsorption for BPA within 10min owning to the advantage of ultrathin core-shell nanostructure. Moreover, a linear relationship between SERS intensity and the concentration of BPA on the MIP-ir- AuNPs was observed in the range of 0.5 –23 22.8 mg/L, with a LOD of 0.12 mg/L. Similarly Du et al. also reported a core-shell composite of AuNPs and SiO2 molecularly imprinted polymers (AuNPs@SiO2-MIPs) through sol– gel technique and applied as a molecular recognition element to construct an electrochemical sensor for determination of dopamine [42]. Compared with previous imprinting recognition, the main advantages of this strat- egy lie in the introduction and combination of AuNPs and biocompatible porous sol–gel material (SiO2) (Figure 11.16). The template molecules were firstly adsorbed at the AuNPs surface due to their excellent affinity, and subsequently they were further assembled onto the polymer membrane through hydrogen bonds and p–p interactions formed between template molecules and silane monomers. CV was carried out to extract dopamine molecules from the imprinted membrane, and as a result, dopamine could be rapidly and effectively removed. The prepared AuNPs@SiO2-MIPs sen- sor exhibited not only high selectivity toward dopamine in comparison to other interferents, but also a wide linear range over dopamine concentra- tion from 4.8×10–8 to 5.0×10–5 M with a detection limit of 2.0×10–8 M (S/ N=3). Moreover, the new electrochemical sensor was successfully applied to the dopamine detection in dopamine hydrochloride injection and human urine sample.

392 Advanced Biomaterials and Biodevices I/A PTMOS, TMOS DPV GCE GCE E/V Gold nanoparticles PVP Template (DA) Au@SiO2-MIPs (before elution) Figure 11.16 Preparation procedure of the Au@SiO2-MIPs [Co-opted from reference 42 with permission]. 11.2.1.5 Quantum Dots A quantum dot (QD) is a semiconductor whose excitons are confined in all three spatial dimensions. The electronic properties of these materials are intermediate between those of bulk semiconductors and of discrete mol- ecules. QDs were discovered in the early 1980s by Alexei Ekimov [43] in a glass matrix and Louis E. Brus in colloidal solutions and the term coined “quantum dot” by Mark Reed [44]. Researchers have studied applications for quantum dots in transistors, solar cells, LEDs, and diode lasers. They have also investigated QDs as agents for medical imaging and as possible qubits in quantum computing. QDs are semiconductors whose electronic characteristics are closely related to the size and shape of the individual crystal. Size and band gap are inversely related in quantum dots. For example, in fluorescent dye appli- cations, emission frequencies increase as the size of the QDs decreases, resulting in a color shift from red to blue in the light emitted (Figure 11.17). Excitation and emission of the QDs are therefore highly tunable. Because the size of the crystals can be controlled during synthesis, the conductive properties can be carefully controlled. When a beam of light hits semiconductors QDs, some of their elec- trons are excited into higher energy states. As the electrons fall back to the ground state, they emit photons of light in a colour that is characteristic of the material. When the electrons in a semiconductor are excited, they prefer to dwell at a certain fixed distance from the positive charges they leave behind, called the exciton radius. For the semiconductors used to make fluorescent QDs, this radius is around 5–10 nanometres. If the entire

Molecular Imprinting and Nanotechnology 393 Figure 11.17 Quantum dots showing quantum confinement effect. crystal’s size is less than the exciton radius, however, an effect called quan- tum confinement comes into play, and shifts the colour of the emitted light towards shorter wavelengths. For crystals of this size, more energy than normal is required to force electrons out of the ground state. So when the electrons return to the ground state, the photons they release have more energy and therefore a shorter wavelength than normal. There is a simple linear relationship between crystal size and colour: the smaller the size, the shorter the wavelength. For cadmium selenide one of the most popular materials for making fluo- rescent QDs varying the size of the crystals over a range of 2–6 nanometres covers the entire visible spectrum. The advantages of highly selective MIPs combined with the high sensitivity of fluorescence sensing of QDs can be used to reduce the LOD and analyze trace substances in samples. Zhang et al reported newly designed QDs based MIP-coated composite for selec- tive recognition of the template cytochrome c (Cyt) [45]. The composites were synthesized by sol–gel reaction (imprinting process). The imprinting process resulted in an increased affinity of the composites toward the cor- responding template (Figure 11.18). The fluorescence of MIP-coated QDs was stronger quenched by the template versus that of non-imprinted polymer (NIP)-coated QDs, which indicated the composites could recognize the corresponding template. The results of specific experiments further exhibited the rec- ognition ability of the composites. Under optimum conditions, the lin- ear range for Cyt is from 0.97μM to 24μM, and the detection limit is 0.41μM. The new composites integrated the high selectivity of molec- ular imprinting technology and fluorescence property of QDs and could convert the specific interactions between imprinted cavities and

394 Advanced Biomaterials and Biodevices Figure 11.18 Preparative procedures for the fabrication of fluorescent MIP-coated CdTe QDs composites [Co-opted from reference 45 with permission]. corresponding template to the obvious changes of fluorescence signal. Therefore, a simple and selective sensing system for protein recognition has been realized. Recently Zhang et al. again reported a MIP-based fluorescent artificial receptor anchoring MIP on the surface of denatured bovine serum albu- min (dBSA) modified CdTe QDs using the surface molecular imprinting process for three different proteins [46]. The approach combined the mer- its of molecular imprinting technology and the fluorescent property of the CdTe QDs. The dBSA was used not only to modify the surface defects of the CdTe QDs, but also as assistant monomer to create effective recog- nition sites (Figure 11.19). Three different proteins, namely Lyz, Cyt and methylated bovine serum albumin (mBSA), were tested as the template molecules and then the receptors were synthesized by sol–gel reaction (imprinting process). The results of fluorescence and binding experiments demonstrated the recognition performance of the receptors toward the corresponding template. Under optimum conditions, the linear range for Lyz was from 1.4×10–8 to 8.5×10–6 M, and the detection limit was 6.8 nM. Moreover, the new artificial receptors were applied to separate and detect

Molecular Imprinting and Nanotechnology 395 CdTe CdTe dBSA TEOS CdTe Remove Template CdTe NH3⋅H2O Ribind EtO EtO Si NH2 Template EtO Figure 11.19 Preparative procedures for the fabrication of MIP-coated CdTe QDs as fluorescent artificial receptor [Co-opted from reference 46 with permission]. Lyz in real samples. This fluorescent artificial receptor may serve as a start- ing point in the design of highly effective synthetic fluorescent receptor for recognition of target protein. An improved imprinted film-based electrochemical sensor for urea recognition was developed using CdS QDs doped chitosan as the func- tional matrix [47]. The microstructure and composition of the imprinted films depicted by scanning electron microscopy (SEM), attenuated total reflection infrared (ATR–IR), X-ray diffraction (XRD), and electrochemi- cal impedance spectroscopy (EIS) indicated the fabricated feasibility of the nanoparticle doped films via in situ electrodeposition. Differential pulse voltammetric responses under the optimal fabrication conditions showed that the sensitivity of CdS QDs–MIP electrochemical sensor was enhanced from the favorable electron transfer and magnified surface area of CdS QDs with a short adsorption equilibrium time (7 min), wide linear range (5.0×10–12 to 4.0×10–10 M and 5.0×10–10 to 7.0×10–8M), and low detection limit (1.0×10–12M). Meanwhile, the fabricated sensor showed excellent specific recognition to template molecule among the structural similarities and coexistence substances. Furthermore, the proposed sensor was applied to determine the urea in human blood serum samples based on its good reproducibility and stability, and the acceptable recovery implied its feasi- bility for practical application. Following slightly different pathways than other ones Chen et al. reported grafting of molecularly imprinted film with diphenolic acid (DPA) as dummy template molecule on the surface of Mn-doped ZnS QDs to develop a selective and sensitive sensor for rapid determination of tetra- bromobisphenol A (TBBPA) in water and soils [48]. The obtained diphe- nolic acid-MIP-QDs sensor has distinguished selectivity and high binding affinity to TBBPA (Figure 11.20). The fluorescence quenching fractions of the sensor presented a satisfactory linearity with the concentrations of TBBPA in the range of 0.1–100 μM, and its limit of detection can reach

396 Advanced Biomaterials and Biodevices TEOS Remove of template QDs QDs Recognition of analyte QDs QDs Figure 11.20 Schematic procedures for the preparation of DPA-MIP-QDs sensor [48]. 0.015 μM. The sensor has been successfully applied to determine the TBBPA in water and soil samples, and the average recoveries of the TBBPA at various spiking levels ranged from 80.2% to 96.5% with relative standard deviation below 8.0%. The results provided a clue to develop sensors for rapid determination of hazardous materials from complex matrixes. Liu et al. also reported a novel dual-function material by anchoring a MIP layer on CdTe/ZnS QDs using a sol–gel with surface imprinting [49]. The material exhibited highly selective and sensitive determination of rac- topamine through spectrofluorometry and solid-phase extraction coupled with HPLC. A series of adsorption experiments revealed that the material showed high selectivity, good adsorption capacity and a fast mass transfer rate. Fluorescence from the MIP-coated QDs was more strongly quenched by ractopamine than that of the non-imprinted polymer, which indicated that the MIP-coated QDs acted as a fluorescence sensing material could recognize ractopamine (Figure 11.21). In addition, the MIP-coated QDs as a sorbent was also shown to be promising for Solid Phase Extraction coupled with HPLC for the determination of trace ractopamine in feeding stuffs and pork samples. Under optimal conditions, the spectrofluorome- try and Solid Phase Extraction-HPLC methods using the MIP-coated QDs had linear ranges of 5.00×10–10–3.55×10–7 and 1.50×10–10–8.90×10–8 mol L-1, respectively, with limits of detection of 1.47×10–10 and 8.30×10–11 mol L-1, the relative standard deviations for six repeat experiments of ractopa- mine (2.90×10–9 mol L-1) were below 2.83% and 7.11%. Fan et al. reported for the first time a chemical method to prepare gra- phene quantum dots (GQDs) from Graphene Oxide [50]. Water soluble and surface unmodified GQDs, serving as a novel, effective and simple

Molecular Imprinting and Nanotechnology 397 L-cycteine CdTe/ZnS QDs TEOS APTES RAC Rebinding template Remove template Figure 11.21 Schematic illustration showing the preparation of MIP-coated CdTe/ZnS QDs [49]. (a) hν Exc Exc hν TNT LUMO Quenching (b) 1 2 HOMO GQDs TNT GQDs TNT Resonance Energy Transfer Charge Transfer Figure 11.22 (a) Schematic of the FRET-based GQDs sensor for detection of TNT. (b) Quenching mechanism through (1) resonance energy transfer from the GQDs donor to TNT acceptor and (2) charge transfer from the excited GQDs to TNT [Co-opted from reference 50 with permission]. fluorescent sensing platform for ultrasensitive detection of 2,4,6-trini- trotoluene (TNT) in solution by fluorescence resonance energy transfer (FRET) quenching (Figure 11.22). The fluorescent GQD scan specifically bind TNT species by the p–p stacking interaction between GQDs and aro- matic rings. The resultant TNT bound at the GQDs surface can strongly suppress the fluorescence emission by the FRET from GQDs donor to the irradiative TNT acceptor through intermolecular polar–polar interactions at spatial proximity. The unmodified GQDs can sensitively detect down to 0.495 ppm (2.2 mM) TNT with the use of only 1mL of GQDs solu- tion. The simple FRET-based GQDs reported here exhibit high and stable

398 Advanced Biomaterials and Biodevices fluorescence. Eliminating further treatment or modification, this method simplifies and shortens the experimental process. 11.2.1.6 Nanobeads Rajabi et al. reported a voltammetric sensor for selective recognition and sensitive determination of mercury ions using glassy carbon electrode (GCE) modified with a novel ion imprinted polymeric nanobeads (IIP) and multi- wall carbon nanotubes (MWCNTs) [51]. The ion-imprinted polymers were prepared by dissolving the certain amount of mercury chloride and 5, 10, 15, 20-tetrakis (3-hydroxyphenyl) porphyrin, in the presence of methacrylic acid and ethyleneglycol-dimethacrylate, using 2,2-azobisisobutyronitrile as initiator. The differential pulse anodic stripping voltammetric technique was employed to investigate the performance of the GC–IIP–MWCNTs modi- fied electrode for determination of hazardous mercury ions. The designed modified electrode was shown a linear response in the range of 1×10–8– 7.0×10–4M of Hg2+ ion with a detection limit of 5.0 nM. It was found that the peak currents of the modified electrode for Hg2+ ions were at maximum value in acetate buffer. The pre-concentration potential and accumulation time were optimized to be -1.0 V and 100 s, respectively. 11.2.1.7 Nanowires/Fibers A nanowire is a nanostructure, with the diameter of the order of a nano- meter (10−9 meters). Alternatively, nanowires can be defined as structures that have a thickness or diameter constrained to tens of nanometers or less and an unconstrained length. At these scales, quantum mechanical effects are important which coined the term “quantum wires”. Many different types of nanowires exist, including metallic (e.g., Ni, Pt, Au), semiconduct- ing (e.g., Si, InP, GaN, etc.), and insulating (e.g., SiO2, TiO2). First of all, Li et al. reported a convenient imprinting method for the preparation of magnetic molecularly imprinted nanowires within the pores of nanoporous alumina membrane in the year 2006 [52]. The tem- plate molecule (theophylline) was immobilized on the pore walls of a nanoporous alumina membrane. The nanopores were then filled with a pre-polymerization mixture containing the superparamagnetic MnFe2O4 nanocrystallites. After polymerization, the alumina membrane was subse- quently removed by chemical dissolution, leaving behind magnetic poly- mer nanowires that contain theophylline-binding sites uniquely residing at the surface and have a saturated magnetization (MS) of 1.97 emu/g. The resulting magnetic imprinted polymer nanowires were capable of binding theophylline more strongly than the non-imprinted nanowires.

Molecular Imprinting and Nanotechnology 399 BHb DA extraction polymerization rebinding SiNW Figure 11.23 Schematic of preparation of MIP for Bovine Hemoglobin (BHb) recognition [53]. Preparations of nanowire and/or fibers are less popular in the field of imprinting compared to other nanostructures. In 2012, Chen et al. employed silicon nanowires as the reinforcement material in protein molecular imprinting with dopamine as the monomer and BHb as the template molecule (Figure 11.23). In the experiments, the imprinted nanowires showed fast adsorption kinetics (took up 75% of the equilibrium amount during only 5min), significant selectivity and large binding capacity (213.7 mg g-1) for the template protein. Furthermore, the stability and regeneration were also investigated, which indicated that the imprinted nanowires had outstanding reusability. Electrospinning method was also employed for the preparation of imprinted nanofibers. For example, Kim et al. prepared an estrone imprinted polyimide nanofiber mat using an electrospinning method [54]. The diamine monomer–template complex was synthesized by the reaction of the diamine monomer having an isocyanate group and estrone (template) having a phenol moiety, in which the template was attached to the monomer via a thermally reversible urethane bond. A poly(amic acid) was synthesized by polymerization of the diamines (1:19 mole ratio of the diamine monomer– template complex to 4,4 -oxydianiline) and pyromellitic dianhydride in N,N-dimethylformamide and the reaction solution was used for electrospin- ning (Figure 11.24). The poly(amic acid) fibers were thermally imidized and then heated in 1,4-dioxane in the presence of water to remove the template molecules. The imprinted polyimide nanofibers showed the specific recogni- tion ability and fast kinetic adsorption for estrone. 11.2.1.8 Carbon Nanotubes (CNTs) Carbon nanotubes can be visualized as a sheet of graphite that has been rolled into a tube. Unlike diamond, where a 3-D diamond cubic crystal

400 Advanced Biomaterials and Biodevices Figure 11.24 FE-SEM images of the imprinted (a, b) and control (c, d) electrospun nanofibermats with different concentration of monomers used during their synthesis [Co-opted from reference 54 with permission]. structure is formed with each carbon atom having four nearest neighbors arranged in a tetrahedron, graphite is formed as a 2-D sheet of carbon atoms arranged in a hexagonal array. In this case, each carbon atom has three nearest neighbors. ‘Rolling’ sheets of graphite into cylinders forms carbon nanotubes. The properties of nanotubes depend on atomic arrange- ment (how the sheets of graphite are ‘rolled’), the diameter and length of the tubes, and the morphology, or nano structure [55]. The two main types of CNTs are single-walled CNTs (SWCNTs) and multi-walled car- bon nanotubes (MWCNTs). SWCNTs are sp2-hybridized carbon in a hexagonal honeycomb structure that is rolled into hollow tube morphol- ogy. MWCNTs are multiple concentric tubes encircling one another [56]. SWCNTs can be classified as either semi-conducting or metallic allotropes, depending on the chirality. The distinction of semiconducting or metallic is important for their use in different sensors but the physical separation of allotropes has proven to be one of the more difficult challenges to over- come. In MWCNTs, a single metallic layer results in the entire nanotube displaying metallic behavior. Due to cheap and easier synthesis procedure, MWCNTs are more widely used as compared to SWCNTs. MWCNTs were employed as medium for electron transfer and the electro-catalyst to enhance the sensitivity of the electrochemical detec- tion in electrochemical sensor. The poor solubility of carbon nanotubes in organic solvents restricts them to be used as drug delivery agents into living systems in drug therapy. Hence many modification approaches like

Molecular Imprinting and Nanotechnology 401 physical, chemical or combined have been exploited for their homoge- neous dispersion in common solvents to improve their solubility. Murray et al. first reported the chemical modification of a carbon substrate mate- rial in 1970s [57]. The modification protocol was generally achieved by attaching specific molecule or entity, which imparts chemical specificity to the substrate material. These chemical modification routes are mainly classified into two types namely surface modification and bulk modifica- tion. The surface modification includes electrochemical-induced method, polymer grafting and metal nanoparticle deposition. The later includes chemical reduction of diazonium salts with reducing agent, thermally acti- vated covalent modification, microwaves assisted modification and ball milling modification. In comparison surface modification is easy in opera- tion and produce more authentic results. That’s why, major group of scien- tists working in molecular imprinting are involved in surface modification of CNTs. Usually, MWCNTs are used either in the fabrication of ceramic or paste electrode or as a substrate for polymerization. Recently, Tong et al. has been reported a monolithic molecular imprinting sensor based on ceramic carbon electrode (CCE) [58]. It was fabricated by thoroughly mix- ing MWCNTs, MIP, graphite powder, and silicon alkoxide, and then pack- ing the resulting complex mixture of components firmly into the electrode cavity of a Teflon sleeve (Figure 11.25). The incorporated MWCNT@MIP in CCEs functioned as recognition element for cholesterol determination. Physisorption MWCNTs DMAc/LiCI Imprinted cavities Cholesteroyl Chitin TDI Chitin-Chol chloroformate Removal of template MIP shell MWCNT@MIP Graphite powder Figure 11.25 Schematic of synthesis of MWCNT@MIP and preparation of ceramic carbon materials by doping MWCNT@MIP [Co-opted from reference 58 with permission].

402 Advanced Biomaterials and Biodevices The MWCNT@MIP-CCEs was tested in the presence or absence of choles- terol by cyclic voltammetry and linear sweep voltammetry. The cholesterol sensor has excellent sensitivity with a linear range of 10 to 300 nM and a detection limit of 1 nM (S/N=3). The monolithic molecular imprinting sensor exhibits good stability, high sensitivity, and user-friendly reusability for cholesterol determination. This study shows that CCE is a promising matrix for MIP sensors. Prasad et al., have reported some recent works based the electrochemical synthesis of molecularly imprinted polymeric nano-materials for different template molecules i.e. aspartic acid, methionine in combinaton with multi- walled carbon nanotubes for enantioselective analysis.  Enantioselective analysis of enantiomers were validated in real samples that suggested practicability of the proposed sensor for the evaluation of this bioactive molecule as a disease biomarker in clinical settings, without any cross- reactivity and false-positives [59]. Molecular imprinted polymer (MIP) is a good matrix that exhibit satisfactory recognition ability when integrated onto sensing transducer. We report on the preparation of such material by graft-polymerizing MIP on the surface of carbon nanotube (CNT) as using MIP as a probe material for chemical sensor fabrication. The MIP is characterized by Fourier transform infrared analysis and UV-Vis analysis. The batch binding analysis is carried out to analyze selective recognition of MIP towards serine (amino acid). SEM images showed the structure of MIP on CNT surface. The serine-imprinted polymer grafted on CNT possessed higher binding capacity for serine than non-imprinted polymer (NIP) grafted on CNT. An insulin-imprinted polymer was synthesized over the surface of vinyl group functionalized multiwalled carbon nanotubes, using phos- photidylcholine-containing functional monomer and cross-linker was also developed by Prasad et al [60]. Phosphotidylcholine is a major component of all biological membrane; its incorporation in polymer backbone assures water-compatibility, bio-compatibility and specificity to molecularly imprinted nanomaterials, without any cross-reactivity or interferences from biological sample matrices. An electrochemical sen- sor fabricated by modifying multiwalled carbon nanotubes-molecularly imprinted polymer onto the pencil graphite electrode,wasused for trace level detection of insulin in aqueous, blood serum, and pharmaceuti- cal samples (LOD 0.0186 nmol L−1, S/N=3), by differential pulse anodic stripping voltammetry. In another approach, a sensitive electrochemical sensor was fabricated for allopurinol (AP) based on immobilization of MIP onto the surface of MWCNTs which later on casted on glassy carbon electrode (GCE) [61].

Molecular Imprinting and Nanotechnology 403 HH O OO O Acid treatment Polymerization on MWCNT surface Cast on GCE Remove template Template GCE MWCNT MIP network Figure 11.26 The simplified sketch for the construction of MIPCNT on GCE [61]. The near equilibrium time to adsorb AP on the surface of electrode is about 9min (Figure 11.26). The modified electrode was used to detect the concentration of AP with a linear range and detection limit (S/N=3) of 0.01–1.0μM and 6.88 nM, respectively. Finally, the modified electrode was successfully applied to determine AP in the human serum sample and two brand tablets. 11.2.1.9 TiO2 Nanotubes During the recent decades, fascinating inorganic semiconductor titanium dioxide (TiO2) has attracted extensive attention in the photocatalytic area for decomposition of organic compounds, sterilization, cancer treatment, etc. due to its good stability and photochemical activity. Recently, Wang et al. proposed a MIP thin film for photoelectrochemical (PEC) sensing of lindane molecules constructed by electropolymerizing o-phenylene- diamine (o-PD) monomer and lindane template molecule on titanium dioxide nanotubes [62]. The resulting PEC sensors were characterized by scanning electron microscopy, ultraviolet (UV)-vis spectra and electro- chemical impedance spectra (Figure 11.27). Under visible light irradia- tion, MIP film can generate the photoelectric transition from the highest occupied molecular orbital to the lowest unoccupied molecular orbital, delivering the excited electrons to the conduction band of titanium diox- ide nanotubes. Simultaneously, it is believed that a positive charged hole (H+) of MIP that took part in oxidation process was consumed to promote the amplifying photocurrent response. The MIP-based PEC sensor had an excellent specificity and could be successfully applied to the recognition and detection of lindane, indicating a promising application in handling with organochlorine pesticide.

404 Advanced Biomaterials and Biodevices Anodization Electro- Lindane (a) (b) Polymerization removed (c) (d) e– PoPD Photocurrent Lindane h+ Time TiO2NTS (e) (g) (f) Figure 11.27 Schematic illustration for fabrication (a, b, c, d and e) and detection mechanism (f and g) of the PEC sensor [Co-opted from reference 62 with permission]. 11.2.1.10 Nanocomposite Materials A nanocomposite is a multiphase solid material where one of the phases has one, two or three dimensions of less than 100 nanometers (nm), or structures having nano-scale repeat distances between the different phases that make up the material. In the broadest sense this definition can include porous media, colloids, gels and copolymers, but is more usually taken to mean the solid combination of a bulk matrix and nano-dimensional phase(s) differing in properties due to dissimilarities in structure and chemistry. The mechanical, electrical, thermal, optical, electrochemical, catalytic properties of the nanocomposite will differ markedly from that of the component materials. Size limits for these effects have been proposed, <5 nm for catalytic activity, <20 nm for making a hard magnetic mate- rial soft, <50 nm for refractive index changes, and <100 nm for achiev- ing superparamagnetism, mechanical strengthening or restricting matrix dislocation movement. Nanocomposites are found in nature, for example in the structure of the abalone shell and bone. In mechanical terms, nano- composites differ from conventional composite materials due to the excep- tionally high surface to volume ratio of the reinforcing phase and/or its exceptionally high aspect ratio. The reinforcing material can be made up of particles (e.g. minerals), sheets (e.g. exfoliated clay stacks) or fibres (e.g. carbon nanotubes or electrospun fibres). The area of the interface between the matrix and reinforcement phase(s) is typically an order of magnitude greater than for conventional composite materials. This large amount of reinforcement surface area means that a relatively small amount of nanoscale reinforcement can have an observable effect on the macroscale

Molecular Imprinting and Nanotechnology 405 properties of the composite. For example, adding carbon nanotubes improves the electrical and thermal conductivity. Other kinds of nanopar- ticulates may result in enhanced optical properties, dielectric properties, heat resistance or mechanical properties such as stiffness, strength and resistance to wear and damage. In general, the nano reinforcement is dis- persed into the matrix during processing. The percentage by weight (called mass fraction) of the nanoparticulates introduced can remain very low (on the order of 0.5% to 5%) due to the low filler percolation threshold, espe- cially for the most commonly used non-spherical, high aspect ratio fillers (e.g. nanometer-thin platelets, such as clays, or nanometer-diameter cylin- ders, such as carbon nanotubes). Zhang et al. reported a synthesis protocol to prepare novel composite imprinted material, incorporating MWCNTs layer, melamine as a tem- plate, MAA as a functional monomer, and EGDMA as a cross-linker by a surface imprinting technique (Figure 11.28) [63]. The imprinted/MWCNTs sorbent was characterized by a scanning elec- tron microscope (SEM). Adsorption dynamics and a Scatchard adsorp- tion model were employed to evaluate the adsorption process. The results showed that the imprinted/MWCNTs sorbent displayed a rapid dynamic adsorption and a high adsorption capacity of 79.9 μmol g-1 toward melamine. Applied as a sorbent, the imprinted/MWCNTs sorbent was used for the determination of melamine in a real sample by online solid- phase extraction-HPLC. An enrichment ratio of 563-fold, detection limit (S/N=3) of 0.3 μg L-1, and quantification limit of 4.5 μg L-1 was achieved during the experiment. Acid-functionalized CNTs Polyacrylic acid-functionalized CNTs COOH AIBN, NαOH Methacrylic acid Vinyl group functionalized CNTs HCI (NH4)2S2O8 H Crude CNTs COOH C H C COOH O N CH2 COCI CO NH2 n SOCI2 n n NN Acrylamide (CNTs) (CNTs) (CNTs) H2N N NH2 Methacrylic acid Imprinted/CNTs sorbent (melamine) n Extraction n Ethylene glycol dimethacrylate Rebind 2, 2 -Azobisisobutyronitile (CNTs) (CNTs) Figure 11.28 Schematic Representation of Preparation of the imprinted/MWCNTs Sorbent [63].

406 Advanced Biomaterials and Biodevices MWCNTs-AuNPs Chitosan Pre-polymer Rebind Misture Remove GCE MWCNTs-AuNPs Chitosan Monomer Tyramine Figure 11.29 The preparation of the molecularly imprinted tyramine-sensing electrode [64]. Some nanoparticle-MWCNTs composites are also reported in the lit- erature viz., Huang et al. developed a novel sensitive molecularly imprinted electrochemical sensor for selective detection of tyramine by combination of MWCNT-AuNP composites and chitosan [64]. MWCNT-AuNP com- posites were introduced for the enhancement of electronic transmission and sensitivity. Chitosan acts as a bridge for the imprinted layer and the MWCNT-AuNP composites. The MIP was synthesized using tyramine as the template molecule, silicic acid tetracthyl ester and triethoxyphe- nylsilane as the functional monomers (Figure 11.29). The molecularly imprinted film displayed excellent selectivity towards tyramine. Under the optimum conditions, the current response had a linear relationship with the concentration of tyramine in the range of 1.08×10–7 to 1×10–5 mol/L, with a limit of detection 5.7×10−8 mol/L. The proposed sensor exhibited excellent repeatability, which was better than the result from previous lit- erature. The relative standard deviation (RSD) of the repeated experiments for tyramine (5 mmol/L) was 7.0%. Determination of tyramine in real samples showed good recovery. The author also reported a molecularly imprinted electrochemical sen- sor based on gold electrode decorated by β-cyclodextrin (CD) incorpo- rated MWCNTs, AuNPs-polyamide amine dendrimer nanocomposites (Au-PAMAM) and chitosan derivative (CSDT) for selective and convenient determination of chlortetracycline (CTC) [65] and oxytetracycline (OTC) [66]. The electrochemical sensor was fabricated by stepwise modification of cyclodextrin-CNTs composites (CD-MWCNTs) and Au-PAMAM onto the gold electrode (Figure 11.30). The layer of MIPs was the outer layer of the electrochemical sensor. Herein, CSDT acted as functional monomer, and CTC or OTC as the template molecule. CV and amperometry were used to characterize the electrochemical behavior of the developed sensor. The linear range of the molecularly imprinted sensor was from 9.00×10–8 to 5.00×10–5 mol/L (from 0.0464 mg/kg to 25.767 mg/kg), with the LOD

Molecular Imprinting and Nanotechnology 407 CD-MWCNTs Au-PAMAM CTC-MIP Rebinding Removal Gold electrode CD MWCNTs PAMAM AuNPs CTC CSDT Figure 11.30 The preparation of the CD-MWCNTs/Au-PAMAM/MIPs/gold electrode [65]. of 4.954×10–8 mol/L (0.0255 mg/kg, S/N=3). The developed sensor showed high selectivity and excellent stability toward CTC. The linear range was found to be from 3.0×10–8 to 8.0×10–5 mol/L, with a LOD of 2.7×10–8 mol/L (S/N=3) for OTC. The results from real sample analysis confirmed the applicability of the sensor to quantitative analysis. QDs-based MIP composite nanospheres were successfully prepared via a facile and versatile ultrasonication-assisted encapsulation method [67]. Unlike the hydrogen-bond-based MIPs, these so-prepared QDs- MIP composite nanospheres, relying on the interaction including van der Waals forces and hydrophobic forces, demonstrated excellent selec- tivity in aqueous media. Their small particle sizes and carboxyl-enriched polymer matrixes give rise to their good dispersibility and stability in aqueous solution, and faster adsorption and desorption kinetics, which further make them extensively applicable for chemical/biological sen- sors in aqueous media. Based on the fluorescence quenching via tem- plate analytes (diazinon) rebinding into the recognition cavities in the polymer matrixes, the QDs-MIP nanospheres were successfully applied to the direct fluorescence quantification of diazinon, independent of extracting templates from the MIP nanospheres, as well as further com- plicated and time-consuming assays. This novel method can selectively and sensitively detect down to 50 ng/mL of diazinon in water, and a lin- ear relationship has been obtained covering the concentration range of 50–600 ng/mL. The present studies provide a new and general strategy to fabricate other multifunctional (luminescent and magnetic) inorganic- organic MIP nanocomposites with highly selective recognition ability in aqueous media and are pretty desirable for biomedical/chemical sensing applications.

408 Advanced Biomaterials and Biodevices Polymeric chemiresistor are a class of chemical sensor that has promise for being practical in situ sensor of volatile organic compound (VOC) in various environmental monitoring [68]. Chemiresistors are manufactured by dissolving a chemically sensitive polymer in an appropriate solvent and mixing the dissolve polymer with conductive carbon particle. The resulting ink is then deposed and dried onto thin film (1). When exposed to various gases, the polymer within the composite film sorbs the vapor and swells reversibility. This swelling can change the distance between the conductive carbon Particles and thus induces a resistance change in the composite film which can be measured (2). Molecular imprinting is a technique to intro- duce molecular recognition sites for a specific analyte in a synthetic polymer for selective separation or concentration of target molecules (3)  Firstly, the nano-sized imprinted polymer for ethanol was synthesized in the presence of methacrylic acid (MAA) as a monomer and divinyl benzene (DVB) as a cross linker and AIBN as an initiator. The applied chemiresistor ink was a mixture of a known concentration of polymeric adhesive, carbon nano- tube, MIP particles and organic solvent. A little amount of the described ink was deposed between the electrodes and waited to dry. In order to test the sensor, a known concentration of ethanol was added in the designed sampling system. It was observed that the sorption of the target gas in the composite layer, deposited between the electrodes, increased considerably the sensor resistance. MIPs as a recognition element for sensors are increasingly of inter- est and MIP nanoclusters have started to appear in the literature [69]. Gültekin et al. proposed a novel thiol ligand-capping method with polymerizable methacryloylamidocysteine (MAC) attached to gold– silver nanoclusters, reminiscent of a self-assembled monolayer and have reconstructed surface shell by synthetic host polymers based on molec- ular imprinting method for recognition. In this method, methacryloyl iminodiacetic acid-chrome (MAIDA-Cr(III)) has been used as a new metal-chelating monomer via metal coordination–chelation interactions and dipicolinic acid (DPA) which is a main participant of Bacillus spores has been used as a template. Nanoshell sensors with templates give a cavity that is selective for DPA. The DPA can simultaneously chelate to Cr(III) metal ion and fit into the shape-selective cavity. Thus, the inter- action between Cr(III) ion and free coordination spheres has an effect on the binding ability of the gold–silver nanoclusters nanosensor. The binding affinity of the DPA imprinted nanoclusters has been investigated by using the Langmuir and Scatchard methods and determined affinity constants were found as 18×106 mol L−1 and 9×106 mol L−1, respectively. 4-Nonylphenol (4-NP) was reported to affect the health of wildlife and

Molecular Imprinting and Nanotechnology 409 Gold TiO2-NH2 AuNPs Cysteamine MIP with After remove electrode template template Figure 11.31 The simplified sketch for the fabrication process of the electrode [70]. humans through altering endocrine function. Huang et al. developed a novel electrochemical sensor for sensitive and fast determination of 4-NP [70]. TiO2-NPs and AuNPs were introduced for the enhancement of electron conduction and sensitivity. 4-NP-imprinted functionalized AuNPs composites with specific binding sites for 4-NP was modified on electrode (Figure 11.31). Rebinding experiments were carried out to determine the specific binding capacity and selective recognition. The linear range was over the range from 4.80×10−4 to 9.50×10−7 mol L−1, with the detection limit of 3.20×10−7 mol L−1 (S/N=3). The sensor was success- fully employed to detect 4-NP in real samples. In another work, the preparation of CNTs functionalized with MIPs for advanced removal of estrone has been reported by Gao et al [71]. CNTs@Est-MIPs nanocomposites with a well-defined core–shell struc- ture were obtained using a semi-covalent imprinting strategy, which employed a thermally reversible covalent bond at the surface of silica- coated CNTs for a large-scale production (Figure 11.32). The adsorption properties were demonstrated by equilibrium rebinding experiments and Scatchard analysis. The results demonstrate that the imprinted nanocomposites possess favourable selectivity, high capacity and fast kinetics for template molecule uptake, yielding an adsorption capacity of 113.5μmol/g. The synthetic process is quite simple, and the different batches of synthesized CNTs@Est-MIPs nanocomposites showed good reproducibility in template binding. The feasibility of removing estro- genic compounds from environmental water using the CNTs@Est-MIPs nanocomposites was demonstrated using water samples spiked with estrone.

410 Advanced Biomaterials and Biodevices Si Si TEOS Ext-Si NH Remove template NH H OC Rebind O 12 CH3 3 O Figure 11.32 Scheme for the synthesis of (A) CNTs@Est-MIPs. The surface of purified CNTs was converted to a silica shell by a sol–gel process using TEOS and APTES in the presence of CTAB to give core@shell CNTs@SiO2 (1). The CNTs@SiO2 reacted with Est– Si to produce a silica surface functionalized with estrone-imprinted polymer (2). After removing estrone by a simple thermal reaction, estrone-imprinted polymer coated CNTs were obtained (3) [71]. 11.2.2 Thin Film Imprinting A thin film is a layer of material ranging from fractions of a nanometer (monolayer) to several micrometers in thickness. A familiar application of thin films is the household mirror, which typically has a thin metal coating on the back of a sheet of glass to form a reflective interface. The process of silvering was once commonly used to produce mirrors. Several imprinted thin film of nanometer thickness were also reported in the literature. Recently, screen-printed electrodes were modified with MWCNTs and molecularly imprinted membranes (MIM) were directly synthesized on these modified electrodes using the in situ thermal polymerization tech- niques [72]. The sensing system was easily established by connecting the screen-printed electrode modified with MWCNT-MIM to an electro- chemical analyzer through an electrode slot. The signal for the determi- nation of ractopamine was recorded using differential pulse voltammetry (DPV) and the optimization for the experimental conditions was also con- ducted. The results showed that the response of the sensor to concentration of ractopamine displayed a linear correlation over a range from 20 nM to 200 nM with a detection limit of 6 nM, demonstrating favourable sensitiv- ity and selectivity for the detection of ractopamine. The recoveries reached 87.7–96.9% based on pig urine samples. TiO2 nano-thin films with imprinted (R)- and (S)-enantiomers of pro- pranolol, 1,1’-bi-naphthol, and 2-(4-isobutylphenyl)-propionic acid were fabricated by Mizutani et al. on quartz plates by spin-coating their solu- tions with Ti(O-nBu)4 in a toluene-ethanol mixture (1:1, v/v) [73]. After template removal, the imprinted films showed better binding for original

Molecular Imprinting and Nanotechnology 411 templates than to the corresponding enantiomers. The assessment of tem- plate incorporation, template removal, and re-binding was conducted through UV-vis measurements. Significant enhancement of enantiose- lectivity was achieved by optimization of the film thickness and by heat- treatment of the imprinted films. After subtraction of non-specific binding, the optimized films provided chiral recognition with the enantioselectivity of almost 100% for (R)-propranolol and 95% for (S)-propranolol. Using tribenuron-methyl as a template and N,O-bismethacryloyl etha- nolamine as a functional cross linking monomer, a molecularly imprinted nanowire membrane was prepared over an anodic alumina oxide mem- brane by Liu et al [74]. The nanowire fabric of the imprinted membrane was established with a scanning electron microscope and a transmission electron microscope. However, the nonimprinted particulate membrane is formed in the absence of a template. Scatchard analysis showed that an equal class of binding sites were formed in the imprinted nanowire membrane and the dissociation constant and the maximum numbers of these bind- ing sites were estimated to be 1.44×10−5 M and 22.7 μmol/g, respectively. The permeation experiments throughout the imprinted membrane and the nonimprinted one were carried out in a solution containing the template and its competitive analogs. These results demonstrated that the molecu- larly imprinted nanowire membrane exhibited higher transport selectivity for the template tribenuron-methyl than its analogs, chlorimuron-ethyl, thifensulfuron-methyl and  N-(4-bromophenyl carbamoyl)-5-chloro-1H- benzo[d] imidazole-2-carboxamide. But the nonimprinted granular mem- brane had no permselectivity for the four substrates. A useful molecular imprinted film based on chitosan/nafion/nano- silver/poly quercetin compound was prepared by a compound elec- trochemical method at paraffin-impregnated graphite electrode for clenbuterol (CLB) sensing, which was characterized by means of field emission scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy spectra (XPS), infrared spectra (IR) and electrochemical techniques to confirm the formation [75]. The molecular imprinted film modified electrode was successfully applied to the determination of CLB with a reliable result. In optimum conditions, CLB at concentrations of 0.3–50.0 μM could be determined with a detection limit of 0.01 μM (3r). Determination of CLB in practical samples of pork liver showed good recovery. Lu et al. reported a technique of forming nanofilms of poly- 3-aminophenylboronic acid (pAPBA) on the surfaces of polystyrene (PS) microbeads for proteins (papain and trypsin) in aqueous [76]. Papain was chosen as a model to study the feasibility of the technique and trypsin as an extension. The results show that pAPBA formed nanofilms (60–100 nm in

412 Advanced Biomaterials and Biodevices thickness) on the surfaces of PS microbeads. The specific surface area of the papain-imprinted beads was about 180m2 g-1 and its pore size was 31 nm. These imprinted microbeads exhibit high recognition specificity and fast mass transfer kinetics. The specificity of these imprinted beads mainly originates from the spatial effect of imprinted sites, because the protein- imprinted sites were located at, or close to, the surface, the imprinted beads have good site accessibility toward the template molecules. The facility of the imprinting protocol and the high recognition properties of imprinted microbeads make the approach an attractive solution to problems in the field of biotechnology. 11.3 Imprinted Materials at Nanoscale 11.3.1 Imprinted Nanoparticle Recently Kobra et al., reported the synthesis of nanoparticles of MIPs were by precipitation polymerization method using glucose as a template mole- cule [77]. Experimental data based on uniform design were analyzed using artificial neural network to find the optimal condition. The results showed that the binding ability of nanoparticles of MIPs prepared under optimum condition was much higher than that of the corresponding non-imprinted nanoparticles (NIPs). Behbahani et al., describes the preparation of new Pb(II)-imprinted nanostructured polymeric particles using 2-vinylpyridine as a functional monomer, ethylene glycol dimethacrylate as the cross-linker, 2,2’- azo- bisisobutyronitrile as the initiator, diphenylcarbazone as the ligand, ace- tonitril as the solvent, and Pb(NO3)2 as the template ion, through bulk polymerisation technique. The prepared ion-imprinted nanostructured polymer particles have an increased selectivity toward Pb(II) ions over a range of competing metal ions with the same charge and similar ionic radius. This ion-imprinted polymer is an efficient solid phase for extrac- tion and preconcentration of lead ions in complex matrixes [78]. Forouzani et al., have been reported nalidixic acid imprinted uniformly sized polymers in the nanometer range by precipitation polymerization using methacrylic acid (MAA) and methyl methacrylate (MMA) as func- tional monomers at different mole ratios [79]. The effect of combination of MAA-to-MMA on the morphology, binding, recognition and release behaviors of the final particles were studied. The produced polymers were characterized by differential scanning calorimetry and their morphology was precisely examined by scanning electron microscopy. A very uniform

Molecular Imprinting and Nanotechnology 413 imprinted nanospheres with diameter of 120- 180 nm are obtained. Among the MIP nanospheres the MIPs using combination of MAA and MMA showed nanospheres with lowest mean diameter (120 nm) and the highest selectivity factor (9.7). Moreover, release experiments showed the controlled release of Nalidixic acid in longtime period. In another work, Cu2+-mediated salbutamol-imprinted polymer nanoparticles, synthesis via precipitation polymerization, were reported by Alizadeh et al., which later on mixed with graphite powder and n-eicosane in order to fabricate a modified carbon paste electrode [80]. This electrode was then applied for indirect differential pulse voltammetry determination of salbutamol. In the presence of Cu2+ ions, the formed Cu2+–salbutamol complex was adsorbed in to the pre-designed cavities of the MIP particles, situated on the electrode surface. Since the electrochemical signal of sal- butamol was intrinsically small, the oxidation peak of the participant Cu2+, after reduction step, was recorded and used as an indication of salbutamol amount, adsorbed in the electrode. Different variables influencing the sen- sor performance were studied and the best conditions were chosen for the determination purpose. Folic acid has been used as a template to generate molecularly imprinted polymers (MIPs), both thin films and nanoparticles [81]. Systematic stud- ies on binding behavior include using two different polymer systems, namely methacrylates and acrylate-vinyl pyrrolidone copolymers. Both yield sensor characteristics with lower limits of detection of 1–30 ppm with QCM (quartz crystal microbalance), whereas the non-imprinted polymers do not generate any signals. For methacrylate-based systems, switching from thin films to MIP nanoparticles increases sensitivity by a factor of 3.0 and selectivity toward metabolites (leucovorin and anhydroleucovo- rin) from broadband to specific. In contrast to this, in poly vinyl pyrrol- idone based materials going from thin films to MIP nanoparticles does not increase sensitivity, but selectivity: thin films yield selectivity factors of 2–3 between folic acid and its metabolites, whereas nanoparticles do not show any response toward the latter and thus can be regarded specific. Hence, not only the heterocyclic backbone, but also the carboxylic groups of the folic acid molecule play fundamental role in detection. Vinyl pyrrolidone thus is the more suitable monomer than methacrylic acid to ensure these properties. In another work, Tahir alizadeh et al. reported the synthesis of nanopar- ticles of promethazine-imprinted polymers by the ultrasonic assisted sus- pension polymerization in silicon oil [82]. The MIP particles were then embedded in a carbon paste (CP) electrode in order to prepare the MIP (nano)-CP electrode. This electrode showed higher response to analyte,

414 Advanced Biomaterials and Biodevices compared to the carbon paste electrode, modified with non-imprinted polymer (NIP (nano)-CP). It response ranges of 4×10–12–1×10–10 M and 1×10–9–1×10–7 M with the sensitivities of 31.7 and 0.17 μA nM-1, respec- tively. The lower detection limit of the sensor was calculated equal to 2.8×10–12M (S/N). The sensor was applied for promethazine (PMZ) deter- mination in plasma samples without applying any sample pre-treatment. Similarly, the author has also reported a potentiometric sensor based on the nano-sized molecularly imprinted polymer (MIP) for the determina- tion of promethazine [83]. This MIP nanoparticles were prepared by two method micro emulsion polymerisation and suspension polymerisation regarded as nano-MIP (1) and nano-MIP (2). The nano-MIP (2) based sensor showed higher selectivity and sensitivity, compared to the nano- MIP (1) based electrode. Both electrodes demonstrated a response time of 5s, a high performance and a satisfactory long-term stability. The elec- trodes were applied for PMZ determination in syrup and serum samples. Asadi et al. used novel synthetic conditions of precipitation polymer- ization to obtain nanosized cyproterone molecularly imprinted polymers for application in the design of new drug delivery systems [84]. The scan- ning electron microscopy images and Brunauer–Emmett– Teller analy- sis showed that MIP prepared by acetonitrile exhibited particles at the nanoscale with a high degree of monodispersity, specific surface area of 246 m2 g−1, and pore volume of 1.24 cm3 g−1. Controlled release of cyproter- one from nanoparticles was investigated through in vitro dissolution tests and by measuring the absorbance by HPLC-UV. The pH dissolution media employed in controlled release studies were 1.0 at 37 °C for 5 h and then at pH 6.8 using the pH change method. Results show that MIPs have a better ability to control the cyproterone release in a physiological medium compared to the NIPs. Imprinted nanoparticles were also employed as packening material in solid phase extraction technique. A new, simple, rapid, and sensitive solid- phase extraction with MIP for determination of Carbamazepine in bio- logical samples was developed by Akbari-adergani et al [85]. These nano particle polymers were synthesized via a non-covalent molecular imprint- ing approach through precipitation polymerization method using MAA as a functional monomer, ethylene glycol dimethacrylate (EGDMA) as a cross-linker agent, carbamazepine as a target template molecule and AIBN as an initiator. The optimal conditions for solid phase extraction technique consisted of conditioning the cartridge using a pH=3.0 water, loading 5.0 ml of the sample under basic aqueous conditions, clean-up using 2×2ml acetonitrile and elution with 3.0 ml methanol. After optimization of solid phase extraction technique procedure, an aliquot of extracted template was

Molecular Imprinting and Nanotechnology 415 injected to the ACE, 5μm 250×4.6 mm analytical column with the mobile phase as the same as elution solvent. This method was used for extraction of carbamazepine from an anticonvulsant aqueous solution and the results revealed an extraction recovery of more than 90%. HPLC chromatograms show an efficient clean up, which supports the potential of MI-solid phase extraction technique for clean-up of trace amounts of carbamazepine from the drug formulation. Ivanova-Mitseva et al. reported for the first time the synthesis of cubic MIP with fluorescent core structure [86]. For the first time cubic organic NPs are reported. Cubic NPs were an unexpected result and the origin of this morphology could be the subject of future study, however this shape may be an advantage where close packing of NP-based material is required, such as in dense coatings. Fluorescent-core cubic MIPNPs were prepared in an innovative concept. They demonstrated excellent selectivity and affin- ity after just 10min incubation time. Advantages such as precise control of the number of the fluorescent labels per particle and its polymer shielding are reported for the first time as a new technique. NPs prepared in this way are promising materials to replace antibodies in sensors and immunoas- says and in drug delivery and diagnostics. 11.3.2 Nanosphere Esfandyari-Manesh et al. reported a synthetic condition of precipitation polymerization to obtain uniformly sized molecularly imprinted nano- spheres of dipyridamole for application in the design of new drug deliv- ery systems [87]. In addition, the morphology, drug release, and binding properties of MIPs were studied, and the effects of morphology on other properties were investigated. The MIPs prepared by acetonitrile/chloro- form (19:1, v/v) were uniformly sized nanospheres with an average mean diameter of approximately 88 nm at a wetted state, 50 nm at a dry state, and a polydispersity index of 0.062. The imprinted nanospheres showed excellent binding properties and had 62.7% of template binding compared with 17.1% of its blank polymer. The imprinted nanospheres with 67.5 (mg template/of polymer) of binding capacity had better imprinting efficiency than the 50.5% of binding capacity shown by irregularly shaped MIP par- ticles that were prepared by chloroform. The molecular binding abilities of imprinted nanospheres in human serum were evaluated by HPLC analysis (binding about 77% of dipyridamole). Results from release experiments of MIPs showed a very slow, controlled, and satisfactory release of dipyr- idamole. The loaded drug was released up to 99% in 17 days for nano- spheres and 22 days for irregularly shaped particles.

416 Advanced Biomaterials and Biodevices APTMS NH2 dry Toluene NH4OH H2N NH2 N UO22+ TEOS Ethanol 110°C, dry N2 CH 35°C, 4h H2N NH2HCHO, CH3COOH Hema egdma 4-VP AIBN Silica NH2 nano particles APTMS- Silica Quinoline-8-OL-APTMS-Silica H2N N N O CH2 HN HN H2C N UO2 HO NO HN N CH2 Figure 11.33 Schematic representation of uranyl ion nanosurface imprinting [88]. Imprinted polymer nanospheres for uranium were prepared by com- plexing uranyl ion on to quinoline-8-ol functionalized 3-aminoprop- yltrimethoxysilane (APTMS) modified silica nanoparticles (Tetraethyl orthosilicate, TEOS) followed by surface imprinting with 4-VP (4-vinyl pyridine), HEMA (2-hydroxy ethyl methacrylate) and EGDMA as the functional monomers and cross linking agent respectively with AIBN as initiator and 2-methoxyethanol as the porogen [88]. Non-imprinted poly- mer material was also prepared under similar conditions omitting uranyl ion. The above materials were used for solid phase extraction of uranium. Recent realization that its chemical toxicity is dominant than radiation hazards makes decontamination a relevant topic for environmental point of view; particularly in the light of projected global thrust for uranium fuel based atomic power plants. The material offers high retention capacity of 97.1μmol g−1 for 10mg L−1 of uranium that does not require tedious grind- ing and sieving steps, is water compatible and works in the pH range of 5–7, making it ideal for possible use in decontamination of polluted natu- ral water samples or front end effluents of nuclear power reactors. 11.3.3 Comparative Study Between Micro- and Nano-imprnted Materials Alizadeh et al. reported a comparative study between micro- and nano- sized imprinted nanopaticle, containing atenolol selective sites synthesized by the methods of bulk polymerization, precipitation polymerization and suspen- sion polymerization in silicon oil [89]. Then, the MIP particles were used as the carrier elements in a bulk liquid membrane (BLM). Atenolol transport capabilities of different MIPs were compared with those of their relevant non imprinted polymers (NIP). It was shown that both nano- and micro-sized MIPs, obtained from suspension polymerization in silicon oil and precipi- tation polymerization, respectively, could transport atenolol more effective

Molecular Imprinting and Nanotechnology 417 than the relevant NIPs. However, the nano-sized MIP was better than the micro-sized MIP for transportation of atenolol. Furthermore, the bulky MIP was not proper atenolol carrier in the BLM, since its transportation characteristic was similar to that of its relevant NIP. Moreover, the selectiv- ity of the BLM containing different kinds of the MIPs obeyed the order of nano-MIP > micro-MIP > bulky MIP. The nano-MIP was adopted as the best atenolol carrier among the tested MIP-based carriers and then the effect of different factors on its transportation efficiency was evaluated. A kinetic model was proposed for the transportation of atenolol through the nano- MIP based BLM. It was found that the extraction of atenolol from the source to the membrane control the separation rate. 11.3.4 Imprinted Nanogel Cakir et al., proposed a new approach for the synthesis of MIPs (synthetic antibodies) as soluble nanogels with sizes close to the size of real antibodies [90]. To imprint a molecular memory in particles consisting of only a few polymer chains, an initiator carrying multiple iniferter moieties is used. This allows for the simultaneous initiation of several polymer chains, and yields molecularly imprinted nanogels (17 nm, molecular weight (MW) = 97 kDa) with good affinity and selectivity for the target. Molecularly imprinted hydrogel nanospheres as devices for the con- trolled/sustained release of 5-fluororacil in biological fluids were synthe- sized employing one-pot precipitation technique as the polymerization method. MAA as a functional monomer and EGDMA as a cross-linker were used in polymeric feed [91]. Morphological and hydrophilic prop- erties were determined by scanning electron microscopy and water con- tent measurement, and recognition and selectivity properties of spherical molecularly imprinted polymers were compared with the spherical non- imprinted polymers, both in organic (acetonitrile) and water media. Finally, in vitro release studies were performed in plasma simulating fluids. The interactions between the template and the functional monomer are a key to the formation of cavities in the imprinted nanogels with high molecular recognition properties [92]. Nanogels with enzyme-like activity for the Kemp elimination have been synthesized using 4-vinylpyridine as the functional monomer and indole as the template. The weak hydrogen bond interaction in the complex is shown to be able to induce very distinc- tive features in the cavities of the imprinted nanogels. The percentage of initiator used in the polymerisation, ranging from 1% to 3%, although it does not have a substantial effect on the catalytic rate, reduces considerably the imprinting efficiency. The alteration of the template/monomer ratio

418 Advanced Biomaterials and Biodevices is also investigated, and the data show that there is considerable loss of imprinting efficiency. In terms of substrate selectivity, a number of experi- ments have been performed using 5-Cl-benzisoxazole as substrate ana- logue, as well as 5-nitro-indole as template analogue for the preparation of a different set of nanogels. All the kinetic data demonstrate that the chemi- cal structure of the template is key to the molecular recognition properties of the imprinted nanogels that are closely tailored and able to differentiate among small structural changes. 11.3.5 Nano Imprint Lithography Nanoimprint lithography (NIL) can generate well defined nanostruc- tures with high efficiency and at very low cost. Molecular imprinting is a “bottom-up” technique creating a polymer layer exhibiting structures with a molecular selectivity [93]. Such polymer structures may be employed as molecular recognition sites for sensing applications. Herein, the authors combine NIL with MIP and they are able to obtain micro- and nanopat- terns of polymer with features down to 100 nm that show high molecular selectivity. 11.4 Conclusions & Future Outlook The current status, challenges, and highlighted applications of MIPs have been described in this book chapter. Major work has been performed to resolve the problems associated with the development of MIPs during last few years. Owing to their high selectivity, high sensitivity, low cost, and ease of preparation, MIPs have been extensively utilized as chromatographic media, sensors, and artificial antibodies to detect various compounds in environmental, bioanalytical, pharmaceutical and food samples. Although remarkable achievements have been attained in molecular imprinting after the combination of nanotechnology with it, still there are substan- tial development challenges and opportunities are required. Herein, we try to summarize some important exploration initiatives which are still required and that are as follows: (1) to explain the molecular imprinting and recognition mechanism at the molecular level with the aid of advanced equipment and computational chemistry; (2) to transfer the imprinting process from organic phase to aqueous phase, reaching the level of natu- ral molecular recognition; (3) to design and synthesize new monomers in order to imprint those molecules without functional groups, broadening the application field of MIP; (4) to exploit new polymerization methods

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12 Prussian Blue and Analogues: Biosensing Applications in Health Care Salazar P1,2,*, Martín M1,3, O’Neill RD4, Lorenzo-Luis P5, Roche R1,2 and González-Mora JL1 1Neurochemistry and Neuroimaging group, Faculty of Medicine, University of La Laguna, Tenerife, Spain 2Informática y Equipamiento Médico de Canarias S.A., Tenerife, Spain 3Atlántica Biomédica S.L., Tenerife, Spain 4UCD School of Chemistry and Chemical Biology, University College Dublin, Ireland 5Department of Inorganic Chemistry, Faculty of Chemistry, University of La Laguna, Tenerife, Spain Abstract Prussian Blue (PB), Fe4[Fe(CN)6]3, belongs to a transition metal hexacyano- metallate family. Its electrochemical properties were revealed in 1978 when Neff reported the successful deposition of a thin layer on platinum foil. After that, numerous publications have appeared exploring its electrocatalytic properties and its applications in biomedical science. During the last decade, a great number of studies involving PB have appeared, using different biosensor substrates and dif- ferent oxidase enzymes. Together with the facile modification of the electrode sub- strate and the low cost of production, this has led to an on-going replacement of the more common enzymatic detection method involving horseradish peroxidase. Its high electrocatalytic activity and low operating overpotential have contributed to the diversification of its use in enzyme-based biosensors and immunosensors. Based on these results, it is clear that PB and its analogues will have important roles in the future development of biomedical devices for next generation health- care strategies. Keywords: Prussian blue, biosensors, immunosensors, health care *Corresponding author: [email protected] Ashutosh Tiwari and Anis N. Nordin (eds.) Advanced Biomaterials and Biodevices, (423–450) 2014 © Scrivener Publishing LLC 423

424 Advanced Biomaterials and Biodevices 12.1 Introduction According to the International Union of Pure and Applied Chemistry (IUPAC) a biosensor is defined as a self-contained integrated device, which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element, which is retained in direct spatial contact with a transduction element Figure 12.1 [1]. In electrochemical approaches to biosensor design, the chemical reactions produce or consume ions or electrons which in turn cause some change in the electrical properties of the solution and/or transducer surface. There is a growing demand for new analytical devices for health applications, espe- cially highly selective and non-invasive methods. In this context, biosensors are ideally small and portable devices, allowing the selective quantifica- tion of chemical and biochemical analytes. Today, they are replacing other, more sophisticated, techniques such as chromatographic and spectroscopic methods, and biosensors are already an important tool in health-care applications [2–3]. Taking account of the general definition of health care (diagnosis, treatment, and prevention of disease, illness, injury, and other physical and mental impairments), biosensors may be applied from preven- tion and early diagnosis to the progression and monitoring of treatment. Another important advantage in biosensor design is that different trans- duction elements may be used such as electrical, optical, mass, thermal, etc. [1]. Nevertheless, electrochemical biosensors (which will be discussed in this chapter) are particularly attractive due to their many advantages over other detection methods. These benefits include high sensitivity and selec- tivity, low cost, real-time output, simplicity of starting materials, possibility to develop user-friendly and wireless integrated devices, and ready-to-use biosensors. The last two advantages enable even non-qualified patients to measure and control their own metabolite levels at home. Enzyme Electroactive substance Electrode Electrical signal Cell Heat Thermistor Antibody Mass change Piezoelectric Light Photon counter Micro- organism pH, pK, pNH4 Ion selective electrode Recognizing Transducer materials Figure 12.1 Schematic display of different biosensor configurations, illustrating different recognition materials being coupled to different signal transducers.

Prussian Blue and Analogues 425 Undoubtedly, biosensors for detecting glucose have received more attention than other devices due to the significance of diabetes as a global health problem, which has generated considerable interest in the development of an efficient glucose biosensor. In 2000 it was estimated that about 171 million people worldwide were diabetic and that this value will reach 370 million in 2030 [4]. It is not surprising, therefore, that there are a great number of commercial devices for determining glucose [5]. Nevertheless, it is also possible to find commercial biosen- sors for other metabolites, such as cholesterol, lactate, urea, creatine, uric acid, ascorbic acid, choline and glutamate, related to emerging health issues [2–3]. Although biosensors emerged in the early 1960s, when Clark and Lyons [6] coupled an enzyme (glucose oxidase) to an amperometric electrode for detecting O2, and Prussian Blue (PB) electrochemical prop- erties have been known since the late 1970s [7], the first work on biosen- sors involving the use of a PB-modified electrode was not reported until 1994 [8]. Due to its high activity and selectivity towards H2O2 reduction, PB has been called an “artificial enzyme peroxidase” [9–10]. During the last decades, a great number of studies involving PB have appeared using different biosensor substrates (carbon paste, screen-printed electrodes, glassy carbon, etc.) and oxidase enzymes (glucose oxidase, lactate oxi- dase, glutamate oxidase, etc.) Figure 12.1 [11]. Together with the facile modification of the electrode substrate and the low cost of production, this has led to an on-going replacement of the more common enzymatic detection method involving horseradish peroxidase, which is more expensive, with inferior temporal stability and is more complicated than PB-modified substrates. In addition, other useful PB properties such as non-toxicity, high electrocatalytic activity and low operating overpoten- tial have contributed to the diversification of its use in enzyme-based biosensors and label-free immunosensors. More recently, new applications for PB and its analogues [12] have appeared in the literature for a variety of important analytes in biomedi- cal fields, mainly due to their electrocatalytic properties. In this way, new applications for the determination of biomolecules [12] such as dopa- mine, epinephrine, norepinephrine, morphine, cysteine, methionine, thiocholine and other thiols, ascorbic acid, nitric oxide, nitrite, iso- prenaline, and vitamin B-6 have already been described (however, in the present chapter we discuss only biosensing and immunosensing applica- tions). Based on these results, it is clear that PB and its analogues will have important roles in the future development of biomedical devices for next generation health-care strategies.

426 Advanced Biomaterials and Biodevices 12.2 General Aspects of Prussian Blue and Other Hexacyanoferrates 12.2.1 Overview PB can be considered as the oldest known hexacyanometallate compound, and has attracted the attention of many scientists seeking an understanding of its formation, composition, structure and physical properties. The link- ing of two different metal ions by the cyanide ligand is the basis of these properties. In fact, the question of cyanide–isocyanide isomerism is as old as the discussion of its structure [13, 14], and since then the electronic interactions between two metals across a cyanide bridge have proven to be a fertile area of research [11, 15–18]. 12.2.2 Chemical and Structure of Prussian Blue and Its Analogues The cyanide ligand (CN–) is a reactive ligand in important organometallic catalytic reactions, and is an ancillary ligand in coordination and bioinorganic chemistry. Like carbon monoxide, the cyanide ion can function as a π-acid ligand, but because of its negative charge, the cyanide ion can also form strong σ-bonds. This behavior allows CN– to stabilize both high and low oxidation Glucose Gluconolactone O2 Glucose oxidase (GOx) H2O2 OH– Prussian Blue (PB) Transducer Figure 12.2 Detection scheme for a glucose biosensor based on a Prussian Blue (PB) modified electrode. Glucose is converted to gluconolactone, catalyzed by glucose oxidase (GOx) immobilized on the electrode surface. Secondary to this reaction is the production of hydrogen peroxide that can be reduced amperometrically at low applied overpotentials, electrocatalyzed by the PB.

Prussian Blue and Analogues 427 Figure 12.3 One-eighth of the unit cell of KFe[Fe(CN)6] soluble Prussian blue. The K+ ions and the remaining interstitial or zeolitic water in the cubic sites have been omitted for clarity from the scheme. states of metals. The cyanide ion can bind to metals in both terminal and bridging M-CN-M´ modes; the bridges are commonly linear, and are pres- ent in many polymeric metal types of cyanide [19], and in particular in PB [20]. Depending on the specific conditions of the preparation, several meth- ods have typically been used to prepare these cyanide complexes. Addition of [Fe(CN)6]3- to Fe2+aq gives the deep blue complex Turnbull´s Blue (TB), while if [Fe(CN)6]4- is added to aqueous Fe3+, the deep blue complex PB is produced [14, 19]. Both PB and TB are hydrated salts of formula Fe4III[FeII(CN)6]3·xH2O (x∼14), and related to them is KFe[Fe(CN)6] – soluble PB [11]. As depicted in Figure 12.3, the zeolite-like structure possesses extended lattices containing cubic arrangements of Fen+ centres linked by CN– bridges; each Fen+ (high- and low-spin) is an octahedral anionic building block [Fen(CN)6]n-6 with a cubic unit cell of 10.2 Å along the FeIII-NC-FeII- CN-FeIII-sequence [13, 14]. The selective diffusion of low molecular weight molecules (such as O2 and H2O2) and some ions with small hydrated radius (such as Cs+, K+ and NH4+) is due to its channel diameter of about 3.2 Å [11]. Consequently, the degree of hydration, as well as the size of ion, are basic factors for the diffu- sion of ions through the channels of the PB lattices [15]. 12.2.3 pH Stability and Deposition Method The chemical literature reports that to achieve a regular structure of electro-deposited PB, two main factors have to be considered: the pH of

428 Advanced Biomaterials and Biodevices the initial growing solution and the deposition potentials [11, 12]. In this way, pH stability of PB film seems to be dependent also on the different modes of deposition of the PB layer. For this reason, the solution pH is a critical point not only during deposition, but also during its applica- tions in real samples. The reason for this behavior has been ascribed to the strong interaction between the Fe3+ from PB and hydroxide ions (OH−) which form Fe(OH)3 at pH higher than 6.4, thus leading to the solubili- zation of the PB film [11, 15]. For the second factor, the applied poten- tial should not be lower than 0.2 V vs SCE, where ferricyanide ions are intensively reduced (vide infra). Different strategies have been described in order to obtain stable PB films, such as galvanostatic or cyclic voltam- metric methods, chemical deposition or PB microparticle synthesis. In this context, cyclic voltammetric methods and a heat-treatment step are commonly used to activate and stabilize the PB film, respectively [11]. 12.3 Prussian Blue: Hydrogen Peroxide Electrocatalysis Although H2O2 is electrochemically ambivalent in that it can be either oxidized to molecular oxygen or reduced to hydroxide ions, depending on the applied potential used [21], the former (anodic) mode of electro- activity has been by far the more common approach for the detection of enzyme-generated H2O2 in first-generation biosensors [22]. However, an intrinsic problem associated with the relatively high applied anodic poten- tials needed to oxidize H2O2 efficiently on most electrode materials (0.4 to 0.7 V vs SCE [23, 24]) is that many substances, including ascorbic and uric acids, in the biosensor target medium (blood, fat, neural tissues, etc.) also oxidize at these potentials, thus interfering with the biosensor signal. In recent decades, one strategy being explored to limit this interference is the use of PB and its analogues to electrocatalytically reduce H2O2 at mild applied potentials (~0 V vs SCE). In 1984, Itaya et al. [7] showed that the reduced form of PB (Prussian White, PW; see Figure 12.4) displayed catalytic activity for the reduction of O2 and H2O2. The zeolite structure of PB, with its small channel diam- eter (see Section 12.2.2), allows the diffusion of low molecular weight mol- ecules (such as O2 and H2O2) through the crystal structure [11]. Nowadays its electrochemical behavior is well understood with cyclic voltammo- grams (CVs) of PB-modified electrodes showing two quasi-reversible redox couples [11, 25] (Figure 12.4). The first peak pair corresponds to the interconversion of PW and PB forms, and the second pair from PB to

Prussian Blue and Analogues 429 (PW) K4Fe(II)4[Fe(II)(CN)6]3 + H2O2 PW PB PG ↓ ↑+4e– + 4K+ – 4K+ – 4OH– (PB) Fe(III)4[Fe(II)(CN)6]3 ↑ ↓–3e– + 3CI– (PG) Fe(III)4[Fe(III)(CN)6]3CI3 0.00 0.25 0.50 0.75 1.00 E vs SCE (V) Figure12.4 CV and redox reactions associated with the interconversion of surface- bound PB to the fully oxidized form, Prussian Green (PG; Eo ≈ 0.85 V vs SCE), and a more reduced form, Prussian White (PW; Eo ≈ 0.1 V vs SCE). In biosensor applications, PB-modified electrodes are often poised at ~0 V vs SCE, a potential where the PW form predominates which can electrocatalytically reduce H2O2 to hydroxide ions. Prussian Green (PG). Also shown in Figure 12.4 are the electron transfer reactions in the presence of potassium chloride as supporting electrolyte, with corresponding formal electrode potentials at ~0.1 V and ~0.85 V vs SCE, respectively [12]. Electrochemical properties such as electrode potential, sensitivity, sta- bility and electron transfer rate constants of the PB/PW and PB/PG conver- sions depend on deposition method, pH, nature and concentration of the supporting electrolyte, etc. As illustrated in Figure 12.4, these reduction and oxidation reactions involve diffusion of cationic and anionic species, respectively, through the PB matrix, and the size of the ionic constituents of the solution medium exerts a major influence on the electrochemical properties of the PB layer [16, 17]. The main drawback of PB as an electrocatalyst for peroxide reduction is its gradual degradation in solutions with pH values close to neutrality [26]. PB is unstable in alkaline solutions, so OH– formed during peroxide reduc- tion (Figure 12.4) can cause a loss of electrocatalytic activity. Because of the prevalence of neutral media in the context of sensor applications in biologi- cal environments, several strategies have been used to improve the stabil- ity of PB films in this pH region. For example, enhanced stability has been achieved by coating electro-deposited PB with a cast Nafion layer [15], and by deposition of PB in the presence of a variety of surfactants [16, 17]. A recent paper by Araminaitė et al. [26] has revealed details of the mech- anism and pH dependence of H2O2 catalytic activity of electro-deposited PB. The results were interpreted within the framework of a 2-step reaction

430 Advanced Biomaterials and Biodevices mechanism, involving dissociative adsorption of H2O2 with the formation of OH radicals, followed by 1-electron reduction of these radicals to OH–. At a higher concentration of H2O2, and especially at a higher pH (pH 7.3), the second process appears rate limiting. The analytical implications are that a linear dependence of cathodic current on H2O2 concentration should be observed within the narrow peroxide concentration range associated with biosensor applications in neutral solutions, as has been reported in practice [17, 15]. In a parallel paper [27], electrocatalytic reduction of H2O2 at electrodes modified by electro-deposited layers of PB were studied with an in-situ Raman spectro-electrochemical technique. During the cathodic reduc- tion of H2O2, PW appeared to turn partially into its oxidized form (PB; see Figure 12.4) even at electrode potentials corresponding to the reduced form of a modifier. The ratio of PB/PW within the modifier layer was shown to depend on H2O2 concentration, indicating that electrocataly- sis proceeds within the modifier layer rather than at an outer modifier– electrolyte interface. In contrast, electrooxidation of AA did not affect the in-situ Raman spectra, indicating an outer interface as the most probable site for AA oxidation. More recently, PB and its analogues have been combined with a variety of novel materials for electrocatalytic detection of H2O2. For example, a PB composite with graphene oxide and chitosan (Chi) gave a detection limit of 100 nM H2O2 [28] and cobalt hexacyanoferate nanoparticles (CoHCF/ NPs) modified with carbon nanotubes (CNT) showed a synergic effect toward H2O2 detection [29]. In this way Han et al., 2013, reported a com- posite of CoHCF and platinum nanoparticles on carbon nanotubes pro- vided a sensor with a linear response up to 1.25 mM H2O2, also with a detection limit of 100 nM, and a fast response time (< 2 s) [30]. Controlled synthesis of mixed nickel-iron hexacyanoferrate nanoparticles (~35 nm average size) has been shown to be an excellent material for selective elec- troanalytical applications for H2O2 and glutathione sensing [31]. A number of these novel, mostly nanoparticle-based materials, have been exploited to develop sensitive and selective devices for electroanalysis, including glu- cose biosensors [32, 33] and immunosensors [34]. 12.4 Prussian Blue: Biosensor Applications During the last two decades some authors have suggested the use of electro- catalytic films to detect H2O2in biosensing applications [8, 10]. Based on this approach, Karyakin and Chaplin [8] proposed to modify the transduction

Prussian Blue and Analogues 431 element with a thin film of PB, allowing the detection of the H2O2 gener- ated enzymatically by GOx at a potential close to 0 V vs SCE. That year, Jaffari and Turner [35] presented a patent in the UK (later extended to an international patent) for an amperometric biosensor for the determination of blood glucose using a PB-modified graphite electrode [36]. After that, PB-modified biosensors for other molecules such as lactate, sucrose, galac- tose, cholesterol, choline, oxalate, lysine, acetylcholine, ethanol, glutamate and NADH have been reported in the literature [11, 12]. Although the first PB-modified transducers were carbon paste, glassy carbon and platinum electrodes, recently screen-printed electrodes (SPEs) have been used because they are inexpensive, simple and quick to prepare, versatile, and are the most economical method for large-scale production and for the assembling of spot-test kits for clinical and environmental analysis. First reports were based on the chemical synthesis of PB and sub- sequent bulk modification of the carbon ink by PB microparticles [37] or the in-situ modification of glassy carbon or graphite powder with PB [38]. Another method, proposed by Ricci et al. [39], involved the direct chemi- cal synthesis of PB onto SPEs, by placing a drop of precursor solution onto the working electrode area. An important advance in the context of the electro-deposition of PB (and other hexacyanoferrates) was the addition a cationic surfactant such as CTAB (acetyl-trimethyl-ammonium-bromide), BZTC (benzethonium chloride) or CPC (cetylpyridinium chloride). With this approach, a significantly enhanced film growth, efficient charge trans- fer kinetics, and high stability and sensitivity toward H2O2 detection have been reported for SPEs [16, 17, 40, 41]. Nowadays, Dropsens SL (Oviedo, Spain) commercializes screen- printed carbon electrodes (SPCEs) modified with PB (Figure 12.5), Counter Reference Electrode Electrode (Carbon) (Silver) Working Electrode W.E. (Prussian connection Blue/Carbon) C.E. connection R.E. 5m connection Figure 12.5 The structure of commercial Prussian Blue-modified screen printed electrode commercialized by Dropsens SA, Spain, including a scanning electron micrograph on the right.

432 Advanced Biomaterials and Biodevices Glucose Gluconolactone Glucose oxidase H2O2 OH– (GOx) O2 Prussian Blue (PB) Transducer Figure 12.6 Detection scheme for a Prussian Blue (PB)-modified electrode in immunosensing. Glucose oxidase-labeled antibody converts glucose to gluconolactone on the electrode surface. Secondary to this reaction is the production of hydrogen peroxide that can be reduced amperometrically at low applied overpotentials, electrocatalyzed by the PB. being recommended for the development of enzymatic biosensors based on oxidases, for working with microvolumes and for decentralized assays. 12.4.1 Prussian Blue and Analogues Enzyme System Due to the electrocatalytic properties of PB toward the reduction of H2O2 at mild applied potentials (see Figure 12.6) most of the enzymes employed with this approach have been oxido-reductases [11]. However, the recent discovery of more generic electrocatalytic properties of PB for other com- pounds [12] has led to new classes of enzymes being incorporated into biosensors, such as hydrolases (e.g., acetylcholinesterase and butyrylcho- linesterase) (see Section 12.4.1.8). 12.4.1.1 Glucose Oxidase Glucose oxidase enzyme (GOx) (EC 1.1.3.4) is an oxido-reductase that catalyses the oxidation of glucose to H2O2 and D-glucono-δ-lactone. In cells, it aids in breaking the sugar down into its metabolites. It is highly selective for β-D-glucose and does not act on α-D-glucose. GOx, which is often extracted from Aspergillus niger, is widely used for the determination

Prussian Blue and Analogues 433 of free glucose in body fluids, and in the chemical, pharmaceutical, food, beverage, biotechnology and other industries. Glucose biosensors based on PB have been successfully applied to blood, serum, saliva and urine samples. First data were presented by Deng et al. [42], where the serum samples obtained from healthy and diabetic persons were diluted 1/50 in phosphate buffered saline and showed a good agree- ment with the reference method. In 2003, Wang et al. [43] presented a glu- cose biosensor based on Chi/PB film and compared their results obtained in whole-blood samples with those obtained by a spectrophotometric method; results from 100 samples were in excellent agreement, with a cor- relation coefficient of >0.99. In recent years, Salazar et al. [44, 45] have been working on PB-modified carbon fiber microelectrodes (CFEs) to detect enzyme-generated H2O2 at low applied potentials as an alternative to first- and second-generation bio- sensors used for physiological applications. Thanks to this approach, the glucose biosensor reached very low dimensions (~10 μm diameter) and displayed excellent in-vitro and in-vivo responses based on criteria rele- vant to applications in neuroscience. Using last approach, Roche et al. [46] studied different aspects of the relationship between oxygen and glucose supplies during neurovascular coupling by detecting the temporal and spa- tial characteristic of hemoglobin states and extracellular glucose concen- tration, combining the use of glucose PB-modified microbiosensors with 2-dimension optical imaging techniques. 12.4.1.2 Lactate Oxidase Lactate oxidase (LOx, EC 1.13.12.4) is classified as a flavoenzyme, which is an enzyme containing a flavin nucleotide (FMN or FAD). LOx is a member of the FMN-containing enzymes which catalyze the oxidation of α-hydroxyacids without the formation of any intermediates. Historically, lactate was considered a dead-end metabolite of glycolysis or a sign of hypoxia and anaerobic energy metabolism. However, a body of evidence has been accumulated to indicate that large amounts of lactate can be pro- duced in many tissues under fully aerobic conditions, including neural activations [47]. In 2001, Garjonyte et al. [48] presented a PB-modified GCE where LOx was immobilized in Nafion. Biosensors operated at –50 mV (vs Ag/ AgCl, 0.1 M KCl) in a flow injection analysis (FIA) system showed a linear range up to 0.8 mM with a detection limit of <1 μM. However, biosen- sor stability and reproducibility were limited. Using a similar approach, Lowinsohn and Bertotti [49] developed a lactate biosensor and measured

434 Advanced Biomaterials and Biodevices the lactate concentration in blood, demonstrating that PB-modified bio- sensors were suitable for monitoring changes in the lactate levels during physical exercise. Recently, Salazar et al. [50] presented a lactate microbiosensor with low dimensions (~10 μm diameter, 250 μm length) to allow its use in neuroscience applications. CFEs were modified with PB for the detection of enzyme-generated H2O2 at a low applied potential. In this way, theses authors electrodeposited PB in presence of the surfactant BZTC in order to improve its stability and sensitivity toward H2O2 (see above). This lactate microbiosensor design displayed a sensitivity of 42 nA mM-1 cm-2 with a detection limit of ~6 μM and a linear range up to 0.6 mM. Furthermore, the linear range was extended up to 1.2 mM with an additional Nafion film. Finally, the microbiosensor response was checked under physiological and electrical stimulation conditions in rat brain and exhibited good results for in-vivo applications. 12.4.1.3 Cholesterol Oxidase Cholesterol oxidase (ChOx, EC 1.1.3.6) is a monomeric flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en- 3-one using O2 as electron acceptor. Two forms of the enzyme are known, one containing the cofactor non-covalently bound to the protein and one in which the cofactor is covalently linked to a histidine residue. It is the most commonly studied enzyme for the construction of biosensors for cholesterol assessment in biological samples. Preliminary determination of cholesterol is clinically important because abnormal concentration is related to disorders such as hypercholesterolemia, high blood pressure, type-2 Diabetes, peripheral vascular diseases, stroke and coronary diseases. In 2003 Li et al. [51] reported a cholesterol biosensor prepared by immo- bilizing ChOx in a silica sol-gel matrix on the top of a PB-modified elec- trode. The ChOx in the sol-gel layer maintains its activity for a long time (35 days half-life). Biosensors gave a detection limit of ~0.2 μM and were free of the most common interference effects. Finally, the authors deter- mined dissociated cholesterol in serum with excellent results. Later, Tan et al. [52] developed an amperometric cholesterol biosensor based on CNTs and a sol-gel Chi/SiO2 organic/inorganic hybrid material. The biosensor exhibited high sensitivity, good reproducibility and selec- tivity, and long-term stability. The authors compared the free cholesterol concentration in human serum obtained with their cholesterol biosen- sor against a spectrophotometric method, and found a good correlation between the two approaches.

Prussian Blue and Analogues 435 In 2013 Liu et al. [53] reported a novel cholesterol biosensor based on a hydrophobic ionic liquid (IL)/aqueous solution interface on a PB-modified GCE. According the authors the hydrophobic IL thin film played a sig- nal amplification role because it not only partitioned the cholesterol from the aqueous solution, but also served as an immobilization matrix for the ChOx. The fabricated IL-ChOx/PB/GCE exhibited a linear response to cholesterol in the range of 0.01–0.40 mM with a detection limit of ~4 μM. 12.4.1.4 Alcohol Oxidase Alcohol oxidase (AOx, EC 1.13.13) is an oligomeric enzyme consisting of eight identical sub-units arranged in a quasi-cubic arrangement, each containing a strongly bound cofactor, FAD molecule. It is produced by methylotrophic yeasts (e.g. Hansenula, Pichia, Candida) in subcellular microbodies known as peroxisomes. AOx is the first enzyme involved in the methanol oxidation path- way of methylotrophic yeasts and although its physiological role is the oxidation of methanol, it is also able to oxidise other short-chain alcohols, such as ethanol, propanol and butanol. AOx is thus responsible for the oxidation of low molec- ular weight alcohols to the corresponding aldehyde, using O2 as the electron acceptor. Due to the strong oxidizing character of O2, the oxidation of alcohols by AOx is irreversible. The detection and quantification of alcohols with high sensitivity, selec- tivity and accuracy is required in many different areas. Accurate and rapid measurement of ethanol is very important in clinical and forensic analysis in order to analyze human body fluids, e.g. blood, serum, saliva, urine, breath and sweat, among others. The first alcohol biosensors based on PB were reported by Karyakin et al. in 1996 [54], where they immobilized AOx within a Nafion layer onto PB-modified GCEs. Different alcohols were checked such as methanol, ethanol, n-propanol, i-propanol and i-butanol. The authors found that alcohol sensitivity decreased with increasing carbon chain length and was higher for primary than secondary alcohols. The detection limits for meth- anol and ethanol were 1 and 50 μM, respectively. Recently, Costa et al. in 2012 [55] presented a comparative study of different alcohol biosensors based on SPCEs modified with three differ- ent mediators (Prussian Blue, ferrocyanide and Co-phthalocyanine). In addition, AOx from three different yeasts (Hansenula sp., Pichia pastoris and Candida boidinii) were employed also. The authors found the high- est sensitivity value with PB-modified SPCEs and AOx from Hansenula, although the background currents were very high which seriously affected the reproducibility.

436 Advanced Biomaterials and Biodevices 12.4.1.5 NADH Oxidase NADH oxidase (EC 1.6.99.3) is a dimeric flavoprotein and carries out oxidoreduction reactions. NADHOx is very active at room temperature, catalyzing the proton transfer from NADH to an electron acceptor, such as FAD, ferricyanide, oxygen and others. Moreover, the enzyme is able to catalyze the electron transfer from NADH to various other electron accep- tors such as PB, Methylene blue, cytochrome c, p-nitroblue tetrazolium, 2,6-dichloroindophenol, even in the absence of flavin shuttles. NADH plays a central role in mitochondrial respiratory metabolism, stimulating the energy production in all living cells (notably, brain, heart and muscles). NADH detection is of a great importance because it is produced in reac- tions catalyzed by more than 250 dehydrogenases. In 2007 Raoi et al. [56] developed a biosensor for the determination of reduced NADH using a recombinant enzyme NADHOx from Thermus thermophilus covalently immobilized on PB bulk-modified SPEs. FIA was selected to optimize the biosensor configuration and other analytical parameters such as cofactor (FMN) concentration, flow rate, buffer types, pH dependence, response time and operational stability. The biosensor showed the highest response at pH 5.0, for which the detection and quan- tification limits were 0.1 and 0.4 μM, respectively, with a linear working range between 1 and 400 μM. Finally, the proposed biosensor was stable for 2 months. Another interesting approach was presented by Gurban et al. in 2008 [57], where NADH oxidase was immobilized on PB-modified SPEs. The amperometric detection of NADH was performed at +0.25 V vs Ag/AgCl. Two different approaches were employed: either adding FMN to the reac- tion medium or immobilized on the biosensor. The optimal configuration was obtained when FMN was entrapped with NADHOx in the biocata- lytic layer using a sol–gel matrix, and displayed sensitivity, linear range and detection limits of 4.6 mA M−1 cm−2, 1.6 mM and 1.2 μM, respectively. Finally, biosensors showed good long-term and operational stability. 12.4.1.6 Diamine Oxidase Diamine oxidase (DaOx, DAO, EC 1.4.3.6) catalyzes the degradation of histamine and other biogenic amines. The enzyme belongs to the class of copper-containing amine oxidases which catalyze the oxidative deamina- tion of primary amines by O2 to form aldehydes, NH3 and H2O2. These cop- per amine oxidases are characterized by possessing the active-site cofactor topa quinone, formed post-translationally by modification of a conserved tyrosine residue. Biogenic amines (histamine, putrescine, cadaverine,

Prussian Blue and Analogues 437 spermine) are volatile amines that are produced as a result of the break- down of amino acids. Histamine has been identified as the causative agent of the disease Scombrotoxicosis or scombroid poisoning, which can, in severe cases, cause symptoms such as headache, nausea, vomiting, diar- rhoea, itching, oral burning sensation, red rash and hypotension. Biogenic amines may also be considered as carcinogens because of their ability to react with nitrites to form potentially carcinogenic nitrosamines. In 2010 Piermarini et al. [58] presented a simple and rapid method for the analysis of biogenic amines in human saliva by using DaOx immo- bilized on a PB-modified SPE. The biosensor response was investigated for different amines such as putrescine, cadaverine, spermine, histamine, etc. The results obtained during the evaluation of saliva showed that the developed electrochemical biosensor can be considered a valid point-of- care testing method for the determination of salivary polyamines, as well as being suitable for biomedical studies. 12.4.1.7 Choline Oxidase Choline oxidase (ChlOx, EC 1.1.3.17) is an enzyme that catalyzes the oxi- dation of choline to generate glycine betaine via betaine aldehyde with H2O2 generation. The enzyme acts on the CH-OH groups of donor with O2 as electron acceptor and FAD as cofactor. Choline and its metabolites are needed for three main physiological purposes such as structural integrity for cell membranes, cholinergic neurotransmission and a major source for methyl groups via its metabolite, betaine. On the other hand, choline, as a marker of cholinergic activity in brain tissue, is very important in biologi- cal and clinical analysis, especially in the clinical detection of neurodegen- erative disorders. In 2006, Shi et al. [59] reported an amperometric choline biosensor based on the immobilization of ChlOx in a layer-by-layer (LBL) multilayer film on a PB-modified Pt electrode. The authors suggested that the high sensitivity and fast response time observed may be due to the efficacy of the enzyme immobilization and to the ultrathin nature of the LBL film, in which mass-transport problems were minimized. The analytical values of choline in serum samples obtained by this choline biosensor agreed satis- factorily with those by a spectrophotometric method. Finally, the choline biosensor retained ~86% of its initial current response to choline after ca. 2 months. In 2012, Zhang et al. [60] developed an electrochemical approach for the detection of choline based on PB-modified iron phosphate nanostruc- tures (PB–FePO4). These nanostructures showed a good catalysis toward

438 Advanced Biomaterials and Biodevices the electro-reduction of H2O2, and allowed the construction of an ampero- metric choline biosensor immobilizing ChlOx on the PB–FePO4 nano- structures. The biosensor exhibited a rapid response (ca. 2 s), low detection limit (0.4 μM), wide linear range (2 μM to 3.2 mM), high sensitivity (∼75 μA mM−1 cm−2), as well as good stability and repeatability. Also, the com- mon interfering species, such as ascorbic acid, uric acid and 4-acetamido- phenol did not cause observable interference. During recent decades, organophosphorus (OP) compounds have been received much attention due to their harmful effects on human health. Therefore, the development of fast and sensitive detection methods has become more urgent. In 2009, Sajjadi et al. [61], developed a PB-modified SPEs coupled with ChlOx for detection of paraoxon as inhibitor. The con- centration of H2O2 produced by ChlOx was electrochemically determined by the PB-modified electrode poised at −50 mV versus the internal screen- printed Ag pseudo-reference electrode. The decrease in current caused by the addition of inhibitor was used for evaluation of paraoxon concentra- tion. For an incubation time of 5 min, the biosensor response was linear from 0.1 to 1 μM of paraoxon with a detection limit of 0.1 μM. 12.4.1.8 Acetilcholinesterase and Butyrylcholinesterase Acetylcholinesterase (AChE, EC 3.1.1.7) is a serine protease that hydro- lyzes the neurotransmitter acetylcholine, and belongs to the carboxyles- terase family. The active site of AChE comprises two subsites – the anionic site and the esteratic subsite. The structure and mechanism of action of AChE have been elucidated from the crystal structure of the enzyme. AChE is found at mainly neuromuscular junctions and cholinergic brain synapses, where its activity serves to terminate synaptic transmission. Butyrylcholinesterase (BuChE, EC 3.1.1.8) is a non-specific cholinester- ase enzyme that hydrolyses many different choline esters. In humans, it is found primarily in the liver and is encoded by the BCHE gene. It is very similar to the neuronal acetylcholinesterase. Recently, biosensor tech- niques based on the inhibition of AChE and BChE activity by OPs and toxins have gained considerable attention due to the advantages of sim- plicity, rapidity, reliability and low cost devices. In addition, the challenges of biohazards and bioterrorism, especially the need for early detection of nerve agents have contributed to the use of such approaches. In 2010, Sun and Wang [62] developed a novel acetylcholinesterase AChE biosensor based on dual-layer membranes (a Chi membrane and a PB membrane) modifying a GCE. Before biosensor operation, the Chi enzyme membrane was quickly fixed on the surface of PB/GCE with an

Prussian Blue and Analogues 439 O-ring to prepare an amperometric GCE/PB-AChE sensor for OP pesti- cides. The proposed biosensor exhibited extreme sensitivity to OP pesti- cides compared to the other kinds of AChE biosensor and provided good results for dichlorvos, omethoate, trichlorfon and phoxim. In 2012, Zhang et al. [63] demonstrated a facile procedure to efficiently prepare PB nanocubes/reduced graphene oxide (PBNCs/rGO) nano- composite by directly mixing Fe3+ and Fe(CN)63- in the presence of GO in a polyethyleneimine aqueous solution. Later, thanks to the high elec- trocatalytic activity of PBNCs/rGO towards the oxidation of thiocholine, this nanocomposite was employed to develop a novel acetylcholinesterase (AChE) biosensor for detection of OP pesticides. Recently, Arduini et al. [64] immobilized BChE onto PB-modified SPEs, and nerve agent detection was performed by measuring the residual activ- ity of the enzyme. The optimized biosensor was tested with two common nerve agent standard solutions (sarin and VX), and showed detection lim- its of 12 and 14 ppb (10% of inhibition), respectively. The enzymatic inhibi- tion was also checked by exposing the biosensors to sarin in gas phase at a concentration of 0.1 mg m−3. Finally, Arduini et al. [65], have reported a portable prototype for nerve agent detection based on an electrochemical AChE biosensor; tests with paraoxon gave satisfactory results. 12.5 Prussian Blue: Immunosensor Applications Radioimmunoassay (RIA), enzyme-linked immunoassays (ELISA) and fluoroimmunoassay (FISA) have been successfully used for great num- bers of applications, such as detection of polypeptide hormones (insulin, glucagon), detection of steroid and amino acid/fatty acid-derived hor- mones (aldosterone cortisol, melatonin), detection of therapeutic agents (amikacin, chloramphenicol, gentamicin), drugs of abuse (amphetamines, barbiturates, canabinoids, cocaine, opiates) and disease markers (thyroid disease, cancer, hypercalcemia and bone disease, hirsutism, virilism, infer- tility). However, these methods involve complicated, time-consuming assay processes, need specially equipped personnel and sophisticated instru- mentation. Moreover, implementation of Point of Care (POC) testing, which is very important especially for developing countries where access to medical and analytical resources is limited, is difficult with conventional immunoassays because rather large and expensive equipment is necessary. Therefore, new techniques for simple, rapid and reliable detection of dis- ease markers are strongly desirable. Recently, electrochemical immunosen- sors, which combine the high efficiency of enzyme catalysis, specificity of