Advanced Biomaterials and Biodevicess

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10 Assembly of Polymers/Metal Nanoparticles and their Applications as Medical Devices Magdalena Stevanović Institute of Technical Sciences of the Serbian Academy of Sciences and Arts, Belgrade, Serbia Abstract Metallic nanoparticles have attracted much attention and have found applications in different fields such as medicine, pharmacy, controlled drug delivery, optics, electronics, and other areas. Among the most promising nanomaterials with anti- bacterial and antiviral properties are metallic nanoparticles (silver, gold, platinum, etc), which exhibit increased chemical activity due to their large surface to volume ratios, crystallographic surface structure and unique size-dependent optical, elec- trical and magnetic properties. However, it has been reported that bare metallic nanoparticles can be toxic. This supports the concept that this toxicity is associated to the presence of the bare metallic nanoparticle surface, while particles protected by an organic layer, i.e. polymer, are much more biocompatible, and thereby less toxic. Unrelated to the bare metallic surface, several recent studies indicate that, at a cellular level, metal nanoparticles interact with biological molecules within mammalian cells and can interfere with the antioxidant defense mechanism lead- ing to the generation of reactive oxygen species (ROS). Increase of ROS levels may result in significant damage to cell structures known as oxidative stress. This review article reports on obtaining metallic nanoparticles with special emphasis on obtaining silver nanoparticles, their incorporation within various polymer materials, physiochemical and biological properties of such obtained sys- tems as well as about their application as medical devices. Keywords: Metal nanoparticles, polymers, silver nanoparticles, medical devices *Corresponding author: [email protected]; magdalena.stevanovic@gmail .com Ashutosh Tiwari and Anis N. Nordin (eds.) Advanced Biomaterials and Biodevices, (343–366) 2014 © Scrivener Publishing LLC 343

344 Advanced Biomaterials and Biodevices 10.1 Introduction Nanomaterials are at the leading edge of the rapidly developing field of nanotechnology [1, 2]. Typically, though not exclusively, nanoparticles are defined as submicroscopic particles between 1 and 100 nm [3]. In recent years noble metal nanoparticles have been the subjects of focused researches due to their unique electronic, optical, mechanical, magnetic and chemical properties that are significantly different from those of bulk materials [1–5]. Their unique size-dependent properties make these materials superior and indispensable in many areas of human activity [5–10]. For example, intense research has led to a more comprehensive understanding of cancer at the genetic, molecular, and cellular levels pro- viding an avenue for methods of increasing antitumor efficacy of drugs while reducing systemic side effects. Nanoparticulate technology is of particular use in developing a new generation of more effective cancer therapies capable of overcoming the many biological, biophysical, and biomedical barriers that the body stages against a standard intervention. Nanoparticles show much promise in cancer therapy by selectively gain- ing access to tumor due to their small size and modifiability [8]. They are formulated out of a variety of substances and engineered to carry an array of substances in a controlled and targeted manner [8]. Discoveries in the past decade have demonstrated that unique properties of gold, silver and platinum nanoparticles, are strongly influenced by shape and size. This has motivated an increase in research on the synthesis routes that allow better control of shape and size for various nano-biotechnological applications. Currently metal nanoparticles (Figure 10.1) are used in various biomedical applications such as probes for electron microscopy to visualize cellular components, drug delivery (vehicle for delivering drugs, proteins, pep- tides, plasmids, DNAs, etc), detection, diagnosis and therapy (targeted and non-targeted), as antibacterial and antiviral agents, etc. (Table 10.1). However, it has been reported that bare metallic nanoparticles can be toxic. This supports the concept that this toxicity is associated to the pres- ence of the bare metallic nanoparticle surface. Also, at a cellular level, metal nanoparticles may interact with biological molecules and can inter- fere with the antioxidant defense mechanism leading to the generation of ROS. Particles protected by an organic layer, i.e. polymer, are much more biocompatible, and thereby less toxic. The main focus of this article is to provide an overview of design and properties of platinum, gold, and silver nanoparticles with special emphasis on obtaining silver nanoparticles, their incorporation within

Assembly of Polymers/Metal Nanoparticles 345 Gold Selenium Palladium Copper Platinum Metal nanoparticles Silver Iron Zink oxide Figure 10.1 Types of metal nanoparticles which are used in biomedical field. Table 10.1 Types of metal nanoparticles and their applications. Metal nanoparticles Applications Gold DNA labeling Biosensors Antiviral Antibacterial Drug delivery Cancer therapy Molecular imaging Diagnosis and therapy Palladium Biocatalysis Copper Antiviral Antibacterial Iron Molecular imaging Cancer therapy Zinc oxide Antiviral Antibacterial Cosmetics (Continued)

346 Advanced Biomaterials and Biodevices Table 10.1 (Cont.) Applications Metal nanoparticles Silver Medical devices Platinum Antiviral Selenium Antibacterial Cancer therapy Cancer therapy Antiviral Antibacterial various polymer materials, physiochemical and biological properties of such obtained systems as well as about their application as medical devices. 10.2 Platinum Nanoparticles Platinum-based therapeutic agents are widely used in medicine [11–20]. Platinum nanoparticles (nano-Pt) have been reported to possess anti- oxidant and anti-tumor activities. Platinum complexes, such as cisplatin, have been used for a decades to treat a number of maladies (any disorder or disease of the body, especially one that is chronic or deepseated) [11]. However, the use of platinum nanoparticles (PtNPs) in medicine is still in its nascent state [21]. Various approaches for making nanoparticles have been proposed in the literature (Figure 10.2). The most common method for the synthesis of platinum nanoparticles is by chemical reduction of metal salts, chief among these reduction agents are ethylene glycol and sodium borohydride [22, 23]. For example, Guo et al. synthesised PtNPs using borohydride as the reducing agent and citric acid as a stabilizer [24]. By varying the ratio of citric acid to the metal salt, they were able to form PtNPs ranging in size [24]. The morphology of PtNPs can be controlled by the precursor reduction conditions while employing supercritical fluid reactive deposition [25]. Herricks et al have described a scheme to gener- ate PtNPs with various size and shape [22]. In this method, polyethylene glycol serves as the reduction agent and solvent. Further modification of structure was obtained by changing the NaNO3/Pt ratio [22]. Additionally, platinum nanoparticles exhibit morphology (size and shape) dependent catalytic properties [26]. Other agents such as poly(N-vinyl-2-pyrrolidone) have been used in conjunction with NaBH4 reduction of H2PtCl6 6H2O

Assembly of Polymers/Metal Nanoparticles 347 Nanoparticle synthesis Botom up methods Top down methods (build up from smaller entities) (size reduction) Physicochemical Mechanical Explosion solvent/nonsolvent processes Aerosol processes Precipitation Condensation milling Ablation Vapor Sol/gel synthesis Chemical Sputtering deposition etching Reduction Metal salts Metal nanoparticles Figure 10.2 Various approaches for making nanoparticles. [27, 28]. The size of Pt nanoparticles can be produced using chemical rip- ening [29].The initial step of this multistep, multi-seed process starts with small individual platinum seeds in an water solution containing sodium citrate and L-ascorbic acid. The final diameter of the nanoparticles relies on the concentration of chloroplatinic acid and the initial seed size [29]. 10.3 Gold Nanoparticles Gold have fascinated scientists for ages and are now heavily utilized in chemistry, biology, engineering, and medicine [30–32]. These materials can be synthesized reproducibly, modified with apparently limitless chem- ical functional groups, and, in certain cases, characterized with atomic- level precision. Many examples of highly sensitive and selective assays based upon gold nanoconjugates have been described in the literature. Recently, focus has turned to therapeutic possibilities for such materials. Structures which behave as gene-regulating agents, drug carriers, imaging agents, and therapeutics have been designed and studied in the context of cells and many diseases [30–32]. These structures are not simply chosen as

348 Advanced Biomaterials and Biodevices alternatives to molecule-based systems, but rather for their new physico- chemical properties, which confer substantive advantages in cellular and medical applications. Depending on their morphology, degree of aglomeration, and local environment, gold nanoparticles can appear red, blue, or other colors (Figure 10.3). These visible colors reflect the underlying coherent oscilla- tions of conduction-band electrons, plasmons, upon irradiation with light Increasing particle size Increasing aspect ratio Figure 10.3 Photographs of aqueous solutions of gold nanospheres (upper panels) and gold nanorods (lower panels) as a function of increasing dimensions. Corresponding transmission electron microscopy images of the nanoparticles are shown (all scale bars 100 nm). [1, The reference 1 is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.]

Assembly of Polymers/Metal Nanoparticles 349 of appropriate wavelengths. These plasmons underlie the intense absorp- tion and elastic scattering of light, which in turn forms the basis for many biological sensing and imaging applications of gold nanoparticles [33–46]. The elastic light-scattering properties of gold nanoparticles are sufficient to detect individual nanoparticles in a visible light microscope with approxi- mately 10(2) nm spatial resolution. Jain et al have used Mie theory and discrete dipole approximation method to calculate absorption and scattering efficiencies and optical reso- nance wavelengths for three commonly used classes of nanoparticles: gold nanospheres, silica-gold nanoshells, and gold nanorods [47]. By increasing the size of gold nanospheres from 20 to 80 nm, the magnitude of extinction as well as the relative contribution of scattering to the extinction rapidly increases. Gold nanospheres in the size range commonly employed show an absorption cross-section 5 orders higher than conventional absorbing dyes, while the magnitude of light scattering by 80-nm gold nanospheres is 5 orders higher than the light emission from strongly fluorescing dyes. Jain et al have stated that the variation in the plasmon wavelength maximum of nanospheres, i.e., from approximately 520 to 550 nm, is too limited to be useful for in vivo applications. They have found that gold nanoshells have optical cross-sections comparable to and even higher with that of the nanospheres. Also gold nanorods show optical cross-sections comparable to nanospheres and nanoshells, however, at much smaller effective size. The growth of gold nanoparticles by reduction by citrate and ascorbic acid has been examined in detail by Kimling et al [48]. They have exam- ined growth of gold nanopaticles to explore the parameter space of reac- tion conditions. It is found that gold particles can be produced in a wide range of sizes, from 9 to 120 nm, with defined size distribution, following the earlier work of Turkevich and Frens [43, 49–50]. The reaction is initi- ated thermally or in comparison by UV irradiation, which results in simi- lar final products. The kinetics of the extinction spectra show the multiple steps of primary and secondary clustering leading to polycrystallites. Subrata et al have produced cubic gold nanoparticles under UV pho- toactivation by using a chiral reagent, 2-naphthol, under alkaline solution as a reductant for HAuCl(4) in CTAB micelle [51]. Prolonged irradia- tion helped the digestion of the primarily evolved spherical particles into smaller gold nanocubes, which then act as tiny cubic seeds, leading to the formation of larger nanocubes [51]. Alivisatos et al have described a strategy for the synthesis of ‘nanocrys- tal molecules’, in which discrete numbers of gold nanocrystals are orga- nized into spatially defined structures based on Watson-Crick base-pairing interactions [52]. They attached single-stranded DNA oligonucleotides of

350 Advanced Biomaterials and Biodevices defined length and sequence to individual nanocrystals, and these assemble into dimers and trimers on addition of a complementary single-stranded DNA template. They have anticipated that this approach should allow the construction of more complex two- and three-dimensional assemblies. Recently, Kang et al have conjugated gold nanoparticles with specific peptides and they were successful in selectively transporting them to the nuclei of cancer cells [53]. Confocal microscopy images of DNA double- strand breaks showed that localization of gold nanoparticles at the nucleus of a cancer cell damages the DNA. Gold nanoparticle dark-field imaging of live cells in real time revealed that the nuclear targeting of gold nanopar- ticles specifically induces cytokinesis arrest in cancer cells, where binucle- ate cell formation occurs after mitosis takes place. Flow cytometry results indicated that the failure to complete cell division led to programmed cell death (apoptosis) in cancer cells. These results show that gold nanopar- ticles localized at the nuclei of cancer cells have important implications in understanding the interaction between nanomaterials and living systems. Despite the great excitement about the potential uses of gold nanopar- ticles for medical diagnostics, as tracers, and for other biological applica- tions, researchers are increasingly aware that potential nanoparticle toxicity must be investigated before any in vivo applications of gold nanoparticles can move forward [33, 54, 55]. 10.4 Silver Nanoparticles Silver nanoparticles have characteristic optical, electrical, and thermal properties and are being integrated into products that range from photo- voltaics to biological and chemical sensors. Examples include conductive inks, pastes and fillers which utilize silver nanoparticles for their high elec- trical conductivity, stability, and low sintering temperatures. Additional applications include molecular diagnostics and photonic devices, which take advantage of the novel optical properties of these nanomaterials. An increasingly common application is the use of silver nanoparticles for anti- microbial coatings, and many textiles, keyboards, wound dressings, and biomedical devices now contain silver nanoparticles that continuously release a low level of silver ions to provide protection against bacteria. The literature describes different methods for obtaining silver nanopar- ticles, including chemical reduction, solid-state synthesis, sonochemical synthesis, in-situ radical polymerization, and spray pyrolysis [56]. Through the optimization of experimental conditions, it is possible to synthesize nanoparticles of different sizes and morphologies. Such optimization

Assembly of Polymers/Metal Nanoparticles 351 relates to concentrations of reactants, temperature, pH, reducing agents, different surfactants and reaction media, and these can significantly affect the stability of the resulting particles [57]. The application of silver nanoparticles in medicine can be broadly divided into diagnostic and therapeutic uses. Early diagnosis to any dis- ease condition is vital to ensure that early treatment is started and perhaps resulting in a better chance of cure. Lin et al reported silver nanoparticle based surface-enhanced raman spectroscopy (SERS) in non-invasive can- cer detection [58]. This approach is highly promising and may prove to be an indispensable tool for the future. The enhancement in the optical and photothermal properties of noble metal nanoparticles arises from resonant oscillation of their free electrons in the presence of light, also known as localized surface plasmon resonance (LSPR) [59, 60]. The plasmon resonance can either radiate light (Mie scat- tering), a process that finds great utility in optical and imaging fields, or be rapidly converted to heat (absorption); the latter mechanism of dissipation has opened up applications in several new areas. The ability to integrate metal nanoparticles into biological systems has had greatest impact in biol- ogy and biomedicine. In terms of therapeutics, one of the most commonly used application of silver nanoparticles is in wound healing. Compared with other silver compounds, many studies have demonstrated the superior efficacy of sil- ver nanoparticles in healing time, as well as achieving better cosmetic after healing. In the study of Kwan et al, it was shown that in wounds treated with silver nanoparticles, there was better collagen alignment after healing when compared to controls, which resulted in better mechanical strength [61]. 10.5 Assembly of Polymers/Silver Nanoparticles In nanoparticle-polymer composites, polymer can serve different pur- poses: as assembling the nanoparticles into clusters, serving as a matrix that includes ordering and anisotropic orientation of the nanoparticles, acting as a functional element, as organic protective layer, etc. Polymers can be tailored to serve either one of these functions or all. The most important requirement for the creation of polymer-nanopar- ticle composites is that the polymer and the protective organic layer on the surface of the nanoparticle are chemically compatible. This compa- tability enables inhomogeneous dispersion of the nanoparticles in the solid polymer matrix, which in certain systems may be viewed as a form of assembly [62].

352 Advanced Biomaterials and Biodevices Figure 10.4 PGA as capping agent of silver nanoparticles. The literature has described the synthesis of composites of sil- ver nanoparticles with polymers such as cellulose [63], polyurethane [64], poly(acrylamide) [65], chitosan [66], poly(e-caprolactone) [67], poly(styrene) [68], poly(methylmethacrylate) [69], montmorillonite [70], polyvinyl alcohol [71], etc. These have provided materials in the forms of films, scaffolds, fibers or grafts. The literature has also describes the obtaining of poly(l-lactide) and poly(lactide-co-glycolide) nanofibers containing AgNps using an electrospinning method [72], as well as the obtaining of poly(lactide-co-glycolide) /silver composite grafts by extrac- tion methods [73]. In the study of Stevanovic et al, polyglutamic acid (PGA) (Figure 10.4) was used as the organic layer (a capping agent) for silver nanoparticles obtained using saccharose as a reducing agent. PGA was chosen as the cap- ping agent to make the AgNps more biocompatibile and to protect them from agglomerating in the medium [74]. In general, stabilization of nanoparticles is achieved by adding cap- ping agents, which bind to the nanoparticle surface via covalent bonds or by chemical interaction. These capping agents are essential to prevent nanoparticle aggregation and increase the solubility of the nanosystem, and also can be used as a site for bioconjugation of the nanoparticle with important molecules. Different capping agents include biodegradable polymers, oligosaccharides and polysaccharides. PGA-capped AgNps (AgNpPGAs) were additionally encapsulated within poly(lactide-co-glycolide) spheres to ensure their release over an extended period of time, and therefore their extended antimicrobial effects. Sureshkumar et al have successfully developed a facile method to pre- pare a magnetic silver nanocomposite [63]. The 3-D nanofibrous structure of bacterial cellulose was homogenized with a ferric and ferrous mixture by a high speed blender. Magnetite nanoparticles were precipitated and

Assembly of Polymers/Metal Nanoparticles 353 incorporated into cellulose nanostructure by adjusting the homogenate to alkaline pH. The magnetic bacterial cellulose nanofiber soaked in dopa- mine solution will be coated with an adherent self-polymerized polydopa- mine layer. Since the polydopamine surface is very effective for reducing silver ion, silver nanoparticles were incorporated into the dopamine treated magnetic bacterial cellulose by soaking in silver nitrate solution. The mag- netic silver nanocomposite possesses a high antimicrobial activity against the model microbes Escherichia coli and Bacillus subtilis. It also has poten- tial as a mild fermentation medium sterilizing agent that a freshly prepared LB medium shows no appreciable contamination after incubating with Ag nanocomposite for 4 h and removed by an external magnet. As it was already mentioned above, silver nanoparticles could be strong bactericidal agents but they also can be cytotoxic. Embedding them in a polymer matrix may reduce their cytotoxic effect. In the study of Liu et al, silver nanoparticles in three average sizes were tested for their antibacterial activities and cytotoxicity. Nanocomposites from a new waterborne poly- etherurethane ionomer and silver nanoparticles were prepared without the use of any crosslinker. It was observed that the antibacterial activity of silver nanoparticles against Escherichia coli started at the effective con- centration of 0.1–1 ppm, while that against Staphylococcus aureus started at higher concentrations of 1–10 ppm. Cytotoxicity of silver nanoparticles was observed at the concentration of 10 ppm. Silver nanoparticles with smaller average size showed greater antibacterial activity as well as cyto- toxicity. The PEU synthesized in this study showed high tensile strength, and the addition of AgNPs at all sizes further increased its thermal stability. The delicate surface features of nanophases, however, were only observed in nanocomposites with either small- or medium-sized AgNPs. PEU-Ag nanocomposites had a strong bacteriostatic effect on the growth of E. coli and S. aureus. The proliferation of endothelial cells on PEU-Ag nanocom- posites was enhanced, whereas the platelet adhesion was reduced. The expression of endothelial nitric oxide synthase gene was upregulated on PEU-Ag containing small-sized AgNPs (30 ppm) or medium-sized AgNPs (60 ppm). This effect was not as remarkable in nanocomposites from large- sized AgNPs. Overall, nanocomposites from the PEU and 60 ppm of the medium-sized (5 nm) AgNPs showed the best biocompatibility and anti- bacterial activity. Addition of smaller or larger AgNPs did not produce as substantial an effect in PEU, especially for the larger AgNPs. Vimala et al have reported the preparation of semi interpenetrating hydrogel networks (SIHNs) based on cross-linked poly (acrylamide) pre- pared through an optimized rapid redox-solution polymerization with N,N -methylenebisacrylamide in presence of three different carbohydrate

354 Advanced Biomaterials and Biodevices polymers, namely gum acacia, carboxymethylcellulose and starch. They have obtained highly stable and uniformly distributed silver nanoparticles with hydrogel networks as nanoreactors via in situ reduction of silver nitrate using sodium borohydride as reducing agent. The formation of silver nanoparti- cles has been confirmed with ultraviolet visible spectroscopy, fourier trans- form infrared spectroscopy, X-ray diffraction analyses. Thermogravimetric analysis provides the amounts of silver nanoparticles exist in the hydrogel networks. Transmission electron microscopy results demonstrate that aca- cia employed hydrogels have regulated the silver nanoparticles size to 2–5 nm where as carboxymethylcellulose and starch composed hydrogel net- works result in a heterogeneous size from 2 to 20 nm. Potara et al have reported the formation of chitosan-coated silver nanoparticles of triangular shape in solution by synergistic action of chito- san and trisodium citrate in the presence of silver seeds and ascorbic acid [66]. It has been revealed that these anisotropic AgNps entrapped in bio- polymeric shells are particularly stable and can be successfully used as ver- satile plasmonic substrates for molecular sensing in solution. In particular, the binding of the probe molecule monolayer (para-aminothiophenol) at the surface of individual chitosan-coated silver nanoparticles was demon- strated both by LSPR shifts and SERS spectra. While the LSPR-shift assay is operational for signaling molecular binding events, the SERS allows iden- tifying the probe molecules and elucidating its orientation on the metal surface. The proof of concept for biosensing applications and dual func- tionality of plasmonic platform are evaluated through the combined LSPR- SERS detection of significant biological molecules, adenine. Potara et al have stated that the potential of chitosan-silver nanostructures to extend the standard approach of LSPR sensing by integrating SERS measurements and operate as dual plasmonic sensors would be very attractive for investi- gation of analytes in biological fluids. Nanofibers of poly(e-caprolactone) (PCL) which poses antimicrobial activity were prepared by electrospinning of a PCL solution with small amounts of silver-loaded zirconium phosphate nanoparticles for potential use in wound dressing applications [67]. These fibers have maintained the strong killing abilities of Ag + existed in the nanoAgZ against the tested bacteria strains and discoloration has not been observed for the nanofi- bers. The authors have tested the biocompatibility of nanofibers as poten- tial wound dressings, i.e. primary human dermal fibroblasts were cultured on the nanofibrous mats. The cultured cells were evaluated in terms of cell proliferation and morphology. The results indicated that the cells attached and proliferated as continuous layers on the nanoAgZ-containing nanofi- bers and maintained the healthy morphology of human dermal fibroblasts.

Assembly of Polymers/Metal Nanoparticles 355 A comparative study on the self-assembled nanostructured morphology and the rheological and mechanical properties of four different triblock copolymers, based on poly(styrene-block-butadiene-block-styrene) and poly(styrene-blockisoprene-block-styrene) matrices, and of their respec- tive nanocomposites with 1 wt% silver nanoparticles, is reported by Peponi and coworkers [68]. In order to obtain well-dispersed nanoparticles in the block copolymer matrix, dodecanethiol was used as surfactant. The block copolymer with the highest PS content shows the highest tensile modulus and tensile strength, but also the smallest elongation at break. When silver nanoparticles treated with surfactant were added to the block copolymer matrices, each system studied shows higher mechanical properties due to the good dispersion and the good interface of Ag nanoparticles in the matrices. Furthermore, it has been shown that semiempirical models such as Guth and Gold equation and Halpin-Tsai model can be used to predict the tensile modulus of the analyzed nanocomposites. Travan and coworkers have developed an antimicrobial non-cytotoxic coating for methacrylic thermosets (biomaterials for dental and orthopedic applications) by means of a nanocomposite material based on a lactose-mod- ified chitosan and antibacterial silver nanoparticles [69]. The authors have performed the in vitro tests for a biological characterization of the material which showed that the nanocomposite coating is effective in killing both bac- terial strains (gram+ and gram- strains) and that this material does not exert any significant cytotoxic effect towards tested cells (osteoblast-like cell-lines, primary human fibroblasts and adipose-derived stem cells), which are able to firmly attach and proliferate on the surface of the coating. It was stated that such biocompatible antimicrobial polymeric films containing silver nanoparticles may have good potential for surface modification of medical devices, especially for prosthetic applications in orthopedics and dentistry. Silver nanoparticles were synthesized into the interlamellar space of montmorillonite (MMT) by using the γ-irradiation technique in the absence of any reducing agent or heat treatment by Shameli and coworkers [70]. They have used silver nitrate and γ-irradiation as the silver precursor and physical reducing agent in MMT as a solid support. The MMT was suspended in the aqueous AgNO3 solution, and after the absorption of sil- ver ions, Ag+ was reduced using the γ-irradiation technique. The proper- ties of Ag/MMT nanocomposites and the diameters of silver nanoparticles were studied as a function of γ-irradiation doses. The interlamellar space limited particle growth and face-centered cubic silver nanoparticles with a mean diameter of about 20–30nm were produced. SEM images indicated that there were structure changes between the initial MMT and Ag/MMT nanocomposites under the increased doses of γ-irradiation. Furthermore,

356 Advanced Biomaterials and Biodevices energy dispersive X-ray fluorescence spectra for the MMT and Ag/MMT nanocomposites confirmed the presence of elemental compounds in MMT and silver nanoparticles. The results indicated that increasing the γ-irradiation dose enhanced the concentration of Ag-NPs. Also, the par- ticle size of the silver nanoparticles slowly increased when γ-irradiation dose of 1 to 20 kGy was applied. When the γ-irradiation dose increased from 20 to 40 kGy, the particle diameters decreased suddenly as a result of the induced fragmentation of Ag-NPs. The interactions between silver nanoparticles with the surface of MMT were weak due to the presence of van der Waals interactions. The synthesized Ag/MMT suspension was found to be stable more than three months without any sign of precipitation. Pragatheeswaran et al have used polyol process for the synthesis of silver nanoparticles embedded in polyvinyl alcohol [71]. Silver nanoparticles were deposited on glass plates by dip coating method and exposed to DC glow discharge plasma for 10 minutes and 60 minutes. Position of surface plasmon resonance peak changed after the samples were exposed to DC glow discharge plasma, which reveals that the DC glow discharge plasma can be used as a size controlling agent for the silver nanoparticles embedded in polyvinyl alcohol. Xing et al have used electrospining method for the preparation of sil- ver-containing poly(L-lactide-co-glycolide) nanofibrous scaffolds [72]. Nanofibrous scaffolds were prepared by electrospining a bend solution of poly(L-lactide-co-glycolide) and silver nanoparticles in 1,1,1,3,3,3,-hexa- fluoro-2-propanol. The resulting fibers ranged from 420 to 590 nm in diameter. To evaluate the possibility of using silver-containing PLGA as a tissue engineering scaffold, experiments on cell viability and antibacte- rial activity were carried out. As a result, PLGA nanofibrous scaffolds hav- ing silver nanoparticles of more than 0.5 wt% showed antibacterial effect against Staphylococcus aureus and Klebsiella pneumonia. Furthermore, silver-containing PLGA nanofibrous scaffolds showed viability, indicating their possible application in the field of tissue engineering. By incorporating silver ions into alginate fibres, highly absorbent algi- nate wound dressings with antimicrobial properties have been made by Qin et al [75]. They have shown that by incorporating fine particles of sil- ver sodium hydrogen zirconium phosphate, the silver-containing alginate fibres can maintain the white physical appearances while providing a sus- tained release of silver ions when in contact with wound exudates. Test results have proven that these fibres are highly effective against bacteria. Nanoparticles synthesized by the various methods have been used in diverse in vitro diagnostic applications [76–94]. Most of all metallic nanopar- ticles, gold and silver nanoparticles have been found to have broad spec- trum antimicrobial activity against human and animal pathogens [95–114].

Assembly of Polymers/Metal Nanoparticles 357 However there is still a lot of room for research. Particularly, recent findings indicate that the compound delays the wound-healing process and that sil- ver may have serious cytotoxic activity on various host cells. It is necessary to examine all available evidence about effects, often contradictory, of sil- ver on wound infection control and on wound healing trying to determine the practical therapeutic balance between antimicrobial activity and cellular toxicity [115–133]. The ultimate goal remains the choice of a product with a superior profile of infection control over host cell cytotoxicity. 10.6 Conclusion Owing to their unique properties, metallic nanoparticles find wide-range applications, from medicine and pharmaceutics to electronic industry, optics, etc. Presently there is great interest for the polymer-metallic nanoparticles systems in the field of biomedical applications based on the need for a system with a high antibacterial and antiviral activity against bacteria and viruses on contact, without the release of toxic biocides. This chapter reports on obtain- ing metallic nanoparticles such as platinium, gold and silver. It focuses on obtaining silver nanoparticles, their incorporation within various polymer materials, a physiochemical and biological property of such obtained sys- tems as well as about their application as medical devices. Acknowledgements M.S. acknowledges the support from the Ministry of Education, Science and Technological Development of the Republic of Serbia, under Grant No. III45004: Molecular designing of nanoparticles with controlled mor- phological and physicochemical characteristics and functional materials based on them. References 1. Vicky V. Mody, Rodney Siwale, Ajay Singh, and Hardik R. Mody, Introduction to metallic nanoparticles, J Pharm Bioallied Sci. 2010 Oct-Dec; 2(4): 282–289. 2. Salata O. Applications of nanoparticles in biology and medicine. J Nanobiotechnol. 2004, 2:3. 3. Praetorius NP, Mandal TK. Mandal, Engineered nanoparticles in cancer therapy. Recent Pat Drug Deliv Formul. 2007, 1:37–51.

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11 Combination of Molecular Imprinting and Nanotechnology: Beginning of a New Horizon Rashmi Madhuri*,1, Ekta Roy1, Kritika Gupta1 and Prashant K. Sharma*,2 1Department of Applied Chemistry, Indian School of Mines, Dhanbad, India 2Department of Applied Physics, Indian School of Mines, Dhanbad, India Abstract The molecular imprinting technology provides a distinctive prospect for the cre- ation of three-dimensional cavities, which mimic biological recognition. Over the last decade, substantial effort has been devoted from the micro to nanoscale to develop variety of polymeric formats that are compatible with molecular imprint- ing technology with the aim of emerging a variety of novel synthetic receptors. This advancement has offered considerable advantages, such as greater surface- to-volume ratio, accessibility to the maximum number of recognition sites, lower diffusion times to facilitate greater uptake and release of the template and over- all improved efficiency. In addition to these, there are also benefits related to the distinct differences in properties (optical, electrical, mechanical, etc.) demon- strated by nanomaterials when compared with their macroscopic counterparts. In response to this, a new generation of molecularly imprinted synthetic receptors has arisen over the past decade that display physical properties, which are often closer to those demonstrated by enzymes and antibodies, such as physical size, solubility, flexibility and recognition site accessibility. In this chapter, we like to focus on the recent development in the field of crafting recognition sites on nano- structured materials and/or designing imprinted materials at nanoscale and their consequences for the common mankind. Keywords: Molecularly imprinted polymer, nanotechnology, silica nanoparticles, core-shell nanoparticle, quantum dots, nanobeads, nanowires/fibers, carbon nanotubes (CNTs), TiO2 nanotubes, nanocomposite materials, thin film imprint- ing, nanosphere, imprinted nanogel, nano imprint lithography *Corresponding authors: [email protected], [email protected] Ashutosh Tiwari and Anis N. Nordin (eds.) Advanced Biomaterials and Biodevices, (367–422) 2014 © Scrivener Publishing LLC 367

368 Advanced Biomaterials and Biodevices 11.1 Introduction 11.1.1 What is “Imprinting”? After earshot the word “imprinting” the first thing came in our mind is what is “imprinting”? What is the difference between “imprinting” and “printing”? There is a major difference between these two; printing means making copy of something whereas, imprinting means making a copy of something onto the substrate and/or surface. In molecular imprinting technology, people attempt to make imprint of molecules (either micro- or macro-) in the polymer matrix, hence popularly known as “molecularly imprinted polymers (MIPs)”. The word or the innovation of imprinted materials came from our biological or natural phenomenon of “molecular recognition”. The term molecular recognition refers to the specific inter- action between two or more molecules through non-covalent bonding such as hydrogen bonding, metal coordination, hydrophobic forces, van der Waals forces, π-π interactions, halogen bonding and/or electrostatic interaction. The host and guest involved in molecular recognition exhibit molecular complementarity [1]. Molecular recognition plays an important role in biological systems and is observed in between receptor-ligand, antigen-antibody, DNA- protein, sugar-lectin, RNA-ribosome, etc. Molecular recognition can be subdivided into static molecular recognition and dynamic molecular rec- ognition (Figure 11.1). Static molecular recognition is likened to the inter- action between a key and a keyhole; it is a 1:1 type complexation reaction between a host molecule and a guest molecule to form a host-guest com- plex. To achieve advanced static molecular recognition, it is necessary to Figure 11.1 Schematic representation showing static and dynamic molecular recognition. [Source: Wikipedia page for molecular recognition].

Molecular Imprinting and Nanotechnology 369 make recognition sites that are specific for guest molecules. In the case of dynamic molecular recognition the binding of the first guest to the first binding site of a host affects the association constant of a second guest with a second binding site [2]. In the case of positive allosteric systems the binding of the first guest increases the association constant of the second guest. While for negative allosteric systems the binding of the first guest decreases the association constant with the second. The dynamic nature of this type of molecular recognition is particularly important since it provides a mechanism to reg- ulate binding in biological systems. Dynamic molecular recognition may enhance the ability to discriminate between several competing targets via the conformational proofreading mechanism. Dynamic molecular recog- nition is also being studied for application in highly functional chemical sensors and molecular devices. Origin of MIP is based on static molecular recognition i.e. specific host and guest interaction. So, molecular imprint- ing is, in fact, making an artificial tiny lock for a specific molecule that serve as miniature key. Like natural receptors the imprinted polymer (syn- thetic receptor) grabs specific molecule among several other chemicals, molecules or biomolecules. Many basic biological processes, from sens- ing of odors to signaling between nerve and muscle cells, rely on such host-guest, antibody-antigen or lock-and-key combinations. For decades, scientists trying to understand these interactions often play locksmith, searching for the right key to fit a particular receptor. Now, the elegance of molecular imprinting in nature has been spurring many scientists to build the locks themselves. They etch a material to create specific cavities, which in size, shape and functional groups, fit the target molecule. However, one of the greatest advantages of artificial receptors over naturally occurring ones is freedom of molecular design. Their frameworks are never restricted to proteins, and a variety of skeletons (e.g., carbon chains and fused aro- matic rings) can be used. Thus, the stability, flexibility, and other proper- ties are freely modulated according to need. Even functional groups that are not found in nature can be employed in these man-made compounds. Furthermore, when necessary, the activity to response towards outer stimuli (photo-irradiation, pH change, electric or magnetic field, and others) can be provided by using appropriate functional groups. The spectrum of func- tions is far wider than that of naturally occurring ones. To prepare a MIP, the basic needs are 1) template (any molecule, cell, organic or inorganic compound, to which MIP have to be prepared), 2) functional monomer(s) (one or more than one, having complementary functional groups to that of template molecule) 3) cross-linker(s) (to preserve the cavities inside the polymer matrix), 4) initiator (for initiation of polymerization reaction), 5)

370 Advanced Biomaterials and Biodevices a c assembly (binding) b 1. Add cross-linker 2. Polymerization (in porogenic solvent) template removal re-binding recognition site Figure 11.2 Schematic representation of synthesis of molecularly imprinted polymer. porogenic solvent (polar or non-polar medium for polymerization) and 6) extraction solvent (to extract the template from cross-linked polymer matrix) (Figure 11.2). There are two main methods for creating these spe- cialized polymers. The first is known as self-assembly, which involves the formation of polymer by combining all elements of the MIP and allowing the molecular interactions to form the cross-linked polymer with the tem- plate molecule bound. The second method of formation of MIPs involves covalently linking the imprint molecule to the monomer. After polym- erization, the monomer is cleaved from the template molecule [3]. The selectivity is greatly influenced by the kind and amount of cross-linking agent used in the synthesis of the imprinted polymer. The selectivity is also determined by the covalent and non-covalent interactions between the tar- get molecule and monomer functional groups. The careful choice of func- tional monomer is another important choice to provide complementary interactions with the template and substrates [4]. In an imprinted polymer, the cross-linker fulfills three major func- tions: First of all, the cross-linker is important in controlling the mor- phology of the polymer matrix, whether it is gel-type, macroporous or a microgel powder. Secondly, it serves to stabilize the imprinted bind- ing site. Finally, it imparts mechanical stability to the polymer matrix. From a polymerization point of view, high cross-link ratios are gener- ally preferred in order to access permanently porous materials and in

Molecular Imprinting and Nanotechnology 371 order to be able to generate materials with adequate mechanical stabil- ity. The self-assembly method has advantages in the fact that it forms a more natural binding site, and also offers additional flexibility in the types of monomers that can be polymerized. The covalent method has its advantages in generally offering a high yield of homogeneous bind- ing sites, but first requires the synthesis of a derivative imprint molecule and may not imitate the “natural” conditions that could be present else- where [5]. Over the recent years, interest in the technique of molecu- lar imprinting has increased rapidly, both in the academic community and in the industry. Consequently, significant progress has been made in developing polymerization methods that produce adequate MIP formats with rather good binding properties expecting an enhancement in the performance or in order to suit the desirable final application, such as beads, films or nanoparticles. One of the key issues that have limited the performance of MIPs in practical applications so far is the lack of simple and robust methods to synthesize MIPs in the optimum formats required by the application. Chronologically, the first polymerization method encountered for MIP was based on “bulk” or solution polymerization. This method is the most common technique used by groups working on imprinting especially due to its simplicity and versatility. It is used exclusively with organic solvents mainly with low dielectric constant and consists basically of mixing all the components (template, monomer, sol- vent and initiator) and subsequently polymerizing them. The resultant polymeric block is then pulverized, freed from the template, crushed and sieved to obtain particles of irregular shape and size between 20 and 50 μm. Depending on the target (template) type and the final application of the MIP, MIPs are appeared in different formats such as nano/micro spherical particles, nanowires and thin film or membranes. They are produced with different polymerization techniques like bulk, precipita- tion, emulsion, suspension, dispersion, gelation, and multi-step swelling polymerization. Most of investigators in the field of MIP are making MIP with heuristic techniques such as hierarchical imprinting method. For the first time the technique for MIP synthesis was done by Sellergren et al. [6] for imprinting small target molecules. With the same concept, Nematollahzadeh et al. [7] developed a general technique, so-called polymerization packed bed, to obtain a hierarchically structured high capacity protein imprinted porous polymer beads by using silica porous particles for protein recognition and capture. Recently, some rules were very popularized for the MIP synthesis and that are collectively called as “The MIP: Rule of six” [8].

372 Advanced Biomaterials and Biodevices 11.1.2 The MIP ‘Rule of Six’ 1. Never use the analyte as a template unless there is absolutely no alternative. 2. Make rational choices about which regions of an analyte are likely to command the best types of interaction in a low dielectric medium (organic solvent) and then incorporate these elements in an analog of the analyte molecule. 3. Select monomers that are likely to form strong interactions in the chosen solvent (e.g., Brönsted acids or bases/H-donors or acceptors/nonpolar groups, etc.) - this will increase capac- ity and influence homogeneity of the binding cavities. 4. Choose templates and monomers that will be soluble in the porogenic solvent to be used in the polymerization - this may seem obvious but it sometimes requires carrying out solubility tests. 5. Ensure as far as possible that the template-monomer mix- ture is stable and does not undergo side reactions under the polymerization conditions. 6. Consider the nature of the matrix from which the analyte will eventually be extracted when selecting the cross-linking monomer- a range of di- or tri-unsaturated cross-linking monomers (e.g., vinylic, acrylic, methacrylic, acrylamide, etc.) with varying chemistries are available to create the porous organic network material. Keeping in view the popularity and wide applications of MIP and/ or MIP-based materials, we like to basically focus on their demerits and improvement approach in this chapter. 11.1.3 Downsides of “The Imprinted Materials” According to a very famous quotation “some times the good things became bad for their selves”, just like that the major drawback of imprinted materi- als are the affinity of the imprinted cavities for the template which make their removal very hard. So, the major problem associated with MIP is washing of template from their binding sites. Because, if some template molecules there are remaining inside the MIPs, fewer cavities will be avail- able for rebinding, which decreases efficiency of imprinted material [9]. However, there are some other problem also ascends due to the incomplete removal of template like Figure 11.3:

Molecular Imprinting and Nanotechnology 373 Distortion of Adequate the binding removal points Incomplete Collapse of removal the cavity after removal Rupture of the cavity during removal Figure 11.3 Diagrammatic representation of the changes induced in the MIPs during the removal of the template [Co-opted from reference 9 with permission]. 1. Distortion of binding sites due to extreme treatment of poly- mer with washing solvent. 2. Collapse of binding site due to removal of template from polymer matrix. 3. Rupture of cavity during removal i.e. some part of polymer may get dissolved due to solvent used for washing. 11.1.4 How to Overcome the Problems To overcome these problems, recently, nanotechnologies and surface chem- istry are introduced into molecular imprinting strategy. Nanostructured, imprinted materials have a small dimension with extremely high surface-to- volume ratio, so that most of template molecules are situated at the surface and in the proximity of materials surface (Figure 11.4). Figure 11.4 illustrates the distribution of effective binding sites in the imprinted bulky materials and imprinted nanoparticles (NPs) after the extraction of templates is done [10]. We assume that these templates located within x-nanometers from the surface can be removed in the bulky materials with a scale of d, and the resultant imprinted sites can be accessed to target species. The effective vol- ume of imprinted materials that can rebind target species is [d3-(d-2x)3]. In general, the x value is very small for highly cross-linked bulky materials

374 Advanced Biomaterials and Biodevices Figure 11.4 The schematic illustration of the distribution of effective binding sites in the imprinted bulky materials and the nanosized, imprinted particles after the removal of templates is done. Redraw with permission from [10] Copyright 2007 ACS. although porogens or solvents are usually used in the imprinting process. If the imprinted materials with the same size are prepared in the form of nanostructure with a scale of 2x nm, all of templates can be completely removed from the highly cross-linked matrix, and these resultant sites are all effective for the binding of target species. In the case of nanosized par- ticles, most of imprinted sites are situated at the surface or in the prox- imity of surface. Therefore, the forms of imprinted materials are expected to greatly improve the binding capacity and kinetics and site accessibility of imprinted materials. Compared with the imprinted films and surface- imprinted materials, the imprinted nanomaterials have a higher affinity and sensitivity to target analyte, and a more homogeneous distribution of recognition sites. On the other hand, the low-dimensional nanostructures with imprinted sites have very regular shapes and sizes, and the tunable flexibility of shapes and sizes. The imprinted nanomaterials have also bet- ter dispersibility in analyte solutions and thus greatly reduce the resistance of mass transfer, exhibiting a fast binding kinetics [11]. In particular, novel nanostructure assembly technologies have achieved a wide success in building various nanodevices [12, 13]. The imprinted nanomaterials with well-defined morphologies can feasibly been installed onto the surface of devices in a required form for many applications in nanosensors and molecular detection. That’s why, several group of researchers are very gen- uinely involved in synthesis and application of imprinted nanomaterials. 11.2 Classification of Imprinted Nanomaterials Imprinted nanomaterials are basically classified in two broad categories: 1) Imprinting onto the nanostructures and 2) Imprinted nanostructures

Molecular Imprinting and Nanotechnology 375 i.e. synthesis of imprinted materials at the level of nanostructures. These two classes are based on modification protocols used in combination of imprinting and nanotechnology. In the first one, imprinting will be done onto the surface of nanomaterials viz., nanoparticles (Au, Ag, Pt, Zn, TiO2), nanotubes, nanowires, and/or magnetic nanoparticles, whereas, in another class imprinted materials are prepared at nanoscale. 11.2.1 Imprinting Onto the Nanostructure Surfaces 11.2.1.1 Imprinted Novel Metal Nanoparticles (NPs) Nanoparticles are the simplest form of structures with sizes in the nm range. In principle any collection of atoms bonded together with a structural radius of <100 nm can be considered a nanoparticle. These can include, e.g., fulle- rens, metal clusters (agglomerates of metal atoms), large molecules, such as proteins, and even hydrogen-bonded assemblies of water molecules, which exist in water at ambient temperatures. Most of methods for preparation of MIPs were bulk/precipitation polymerization to obtain bulky MIPs, which were traditional, and exhibited some disadvantages including incomplete template removal, small binding capacity, slow mass transfer, and irregular materials shape. The attempts to address these problems generally require that imprinted materials are prepared in the optimizing forms that con- trol templates to be situated at the surface or in the proximity of materials’ surface, providing the complete removal of templates, good accessibility to the target species, and low mass-transfer resistance. Grafting method for molecular imprinting on the surface of nanoparticles and simultaneously obtaining the regular/uniform morphology would be an option. In case of imprinting the most commonly used substrate nanoparticles are gold, silver, iron oxide, titanium oxide and silica oxide nanoparticles. Herein, we like to discuss the application of novel metal nanoparticles i.e. gold and silver NPs. Up to date, the molecular imprinting at the surface of various substrates remains a challenge. Recently, Ran et al. described a method to imprint molecular recog- nition sites into Au NPs composites [14]. The method includes the elec- tropolymerization of thioaniline-functionalized Au NPs in the presence of imprint substrates that exhibit affinity interactions with the thioaniline- functionalized Au NPs or with a co-added ligand associated with the elec- tropolymerizable NPs. The imprinted matrices are implemented for the sensing of explosives, herbicides, saccharides, and ions. π-Donor–acceptor interactions, ionic interactions and H-bonds, or ligand–substrate interac- tions are used to generate the imprinted sites. The coupling between the localized plasmon of the NPs and the surface plasmon wave of the support

376 Advanced Biomaterials and Biodevices is used to amplify the dielectric changes occurring in the NPs matrices upon the binding of the analytes to the imprinted sites, thus enabling the surface plasmon resonance (SPR) transduction of the sensing events. The imprinted Au NPs matrices demonstrate highly selective, stereoselective, and chiroselective sensing performance. In this way to modify the AuNPs, some authors also reported the appli- cation of gold nanoparticle in electropolymerization with template. Huang et al. reported a novel amperometric sensor for bisphenol A (BPA), using MIP and gold nanoparticle [15]. This layer is prepared by electro polymeri- sation of 2-aminothiophenol on gold nanoparticle modified glassy carbon electrode in the presence of BPA as template. Cyclic voltammetry (CV) is use to monitor the process. The properties of the layer were studied in the presence of Fe(CN)63-/Fe(CN)64- redox couples. The template and the non- binding molecules were removed by washing with H2SO4 (0.65 mol L-1) solution. The linear response range of the sensor was between 8.0×10–6– 6.0×10–2 mol L-1, with a detection limit of 1.38×10–7 mol L-1 (S/N = 3). Similarly, some other workers are also try to apply silver nanoparticles as MIP platform. Raghu et al. have been reported a novel electrochemical bio- sensor for the determination of pyrogallol (PG) and hydroquinone (HQ) based on the poly  l-arginine (poly(l-Arg))/carbon paste electrode (CPE) immobilized with horseradish peroxidase (HRP) and silver nanoparticles (AgNPs) through the sol–gel entrapment [16]. The electrochemical prop- erties of the biosensor were characterized by employing the electrochemi- cal techniques. The proposed biosensor showed a high sensitivity and fast response toward the determination of PG and HQ around 0.18 V. Under the optimized conditions, the anodic peak current of PG and HQ was lin- ear with the concentration range of 8 μM to 30×10−5 M and 1–150 μM. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 6.2 μM, 20 μM for PG and 0.57 μM, 1.92 μM for HQ respectively. The electrochemical impedance spectroscopy (EIS) studies have confirmed that the occurrence of electron transfer at HRP-SiSG/AgNPs/poly(l-Arg)/ CPE was faster. 11.2.1.2 Imprinted Magnetic Nanoparticle Magnetic nanoparticles (MNPs) are a class of engineered materials of <100nm that can be manipulated under the influence of an external mag- netic field. MNPs are commnonly composed of magnetic elements, such as iron cobalt, nickel and their oxides. In recent years, MNPs have been studied for biomedical and biotechnological applications, including tar- geted drug delivery, MRI contrast enhancement, biosensor and rapid

Molecular Imprinting and Nanotechnology 377 Figure 11.5 Photograph of Fe3O4@SiO2@MIPs suspended in standard solution in the absence (a) and presence (b) of an externally placed magnet [Co-opted from reference 17 with permission]. environmental and/or biological separation. For many of these applica- tions, surface modification of MNPs is a key challenge. In general, physi- cal/chemical adsorption or surface coating of specific ligands, depending on the specific applications, can accomplish surface modification. Now- a-days MNPs coated with MIP are very widely and progressively used in separation and targeted drug delivery technologies with the help of an external magnetic field. In this regard, Li et al. reported a synthesis protocol for magnetic molec- ularly imprinted polymers (m-MIPs) for the selective adsorption and sep- aration of dibenzothiophene (DBT) from oil solution [17]. The m-MIPs were characterized by Fourier transform infrared analysis, transmission electron microscopy, surface area and porosity analysis, and vibrating sample magnetometry (Figure 11.5). Batch mode adsorption studies were carried out to investigate the adsorption kinetics, adsorption isotherms and selective recognition. The adsorption kinetics was modeled with the pseudo first-order and pseudo-second-order kinetics, and the adsorption isotherms were fitted with Langmuir and Freundlich models. The m-MIPs can selectively recognize DBT over similar compounds. Static adsorption experiments showed that the m-MIPs display excellent recognition capacity, selective affinity for DBT, and superparamagnetism in presence of an external magnetic field. Similarly, Wang et al., have been also reported a Fe3O4 MNPs coated estrone-imprinted polymer with controlled size using a semi-covalent imprinting strategy [18]. In this protocol, the estrone-silica monomer complex was synthesized by the reaction 3-(triethoxysilyl) propyl isocya- nate with estrone, where the template was linked to the silica coating on the

378 Advanced Biomaterials and Biodevices (A) O O Fe3O4 TEOS Fe3O4 EstSi O Si O O Si O Fe3O4 NH DMSO NH H2O, H O C Fe3O4 O 4 12 3 CH3 O (B) OH OCH2CH3 O CH3 dibutyltin dilaurate, THF O CH3 +OCNH2CH2CH2C Si OCH2CH3 estrone OCH2CH3 O OCH2CH3 3-(triethoxysilyl) propyl OCN CH2CH2CH2 Si OCH2CH3 isocyante H OCH2CH3 EstSi Figure 11.6 (A) Multistep synthesis of estrone-imprinted MNPs. Fe3O4 MNPs (1) was prepared by co-precipitation method and the MNPs surface was then transformed to silica shell by a sol–gel process using tetraethyl orthosilicate to give Fe3O4@SiO2 MNPs (2). The Fe3O4@SiO2 MNPs reacted with a template-silica monomer complex to produce silica surface functionalized with estrone-imprinted polymer (3). After remove the template estrone by simple thermal reaction, estrone-imprinted polymer coated MNPs (4) were obtained. (B) Synthesis of template-silica monomer complex [Co-opted from reference 18 with permission]. iron oxide core via a thermally reversible bond. The removal of the tem- plate by a simple thermal reaction produced specific estrone recognition sites on the surface of silica shell. The resulting estrone-imprinted polymer coating Fe3O4 magnetic hybrid nanoparticles exhibit a much higher spe- cific recognition and saturation magnetization. The hybrid nanoparticles have been used for biochemical separation of estrone (Figure 11.6). In another prospect of MNPs, Kecili et al. fabricated super paramag- netic nanotraps. The nanoparticle comprises a superparamagnetic iron oxide nanoparticle core conjugated with trimethoxylsilyl propylmethacry- late and methacryloylamido serine, methacryloylamido histidine, methac- ryloylamido glutamic acid monomers, and p-nitrophenyl palmitate which is a substrate of lipase as a template molecule, which enables the creation of lipase active region [19]. The resulting hybrid superparamagnetic nano- traps are magnetically separable, highly active, and stable under harsh conditions. In this study, the advantages of high selectivity of molecular imprinting technique have used to get mimic lipase for the synthesis of methyl jasmonate and methyl oleate (Figure 11.7). Surface molecular imprinting, especially on the surface of silica-modi- fied MNPs, has been proposed as a promising strategy for protein recogni- tion and separation [20]. Inspired by the self-polymerization of dopamine,

Molecular Imprinting and Nanotechnology 379 Figure 11.7 Schematic representation of nanoshell based on p-nitrophenyl palmitate- template constructed onto the surface of iron oxide nanoparticles [Co-opted from reference 19]. Jia et al. synthesized a polydopamine-based molecular imprinted film coating on silica-Fe3O4 nanoparticles for recognition and separation of bovine hemoglobin. Herein, m-MIP having diameter (860nm) show a rela- tively high adsorption capacity (4.65 ± 0.38 mg g-1) and excellent selectivity towards bovine hemoglobin with a separation factor of 2.19. m-MIP with high saturation magnetization (10.33 emu g-1) makes it easy to separate the target protein from solution by an external magnetic field. After three continuous adsorption and elution processes, the adsorption capacity of m-MIP remained at 4.30 mg g-1. Similarly, a novel super paramagnetic surface molecularly imprinted Fe3O4@MIP nanoparticles for water-solu- ble pefloxacin mesylate were prepared via surface initiated atom transfer radical polymerization [21]. The Fe3O4@MIP exhibited high saturation magnetization of 41.4 emu/g leading to the fast separation. The adsorption behaviours indicated that the Fe3O4@MIP nanoparticles possessed specific recognition and high affinity towards template in aqueous media. Phase inversion method was also applied in some case to prepare m-MIP for different macromolecules like enzymes and protein. Lee et al., have reported a amylse imprinted m-MIP using phase inversion of poly (ethyl- ene-co-vinyl alcohol) solutions with 27−44 mol% ethylene in the presence of amylase [22]. The mean size of m-MIPs was found to be 100 nm and

380 Advanced Biomaterials and Biodevices the magnetization was 14.8 emu/g. The activities of both bound template and rebound enzyme was established by measuring glucose production via starch hydrolysis, at different temperatures, for MIPs with different com- positions (wt% polymer and mol% ethylene). The highest hydrolysis activ- ity of m-MIPs (obtained with 32 mol% ethylene) was found to be 1545.2 U/g. Compared to the conventional catalysis process, m-MIPs have the advantages of high surface area, suspension, easy removal from reaction, and rapid reload of enzyme. In the area of macromolecules separation and enrichment imprinting is in under-develped stage, due to several difficulties like structural changes, pH of the medium, solubility of macromolecules etc. Gao et al., have been reported imprinting of four proteins with different isoelectric point {bovine serum albumin (BSA, pI=4.9), bovine hemoglobin (BHb, pI=6.9), bovine pancreas ribonuclease A (RNase A, pI=9.4) and lysozyme (Lyz, pI=11.2)} onto the surface of MNPs with a uniform core-shell structure for the rec- ognition and enrichment of protein was developed [23]. The magnetic pro- tein-MIPs were synthesized by combining surface imprinting and sol-gel techniques. In comparison with the use of Lyz, BSA and RNase A as tem- plate proteins, BHb-imprinted Fe3O4 showed the best imprinting effect and the highest adsorption capacity among the four proteins. The as-prepared Fe3O4@BHb-MIPs NPs with a mean diameter of 230 nm were coated with an MIP shell that was 10 nm thick, which enabled the Fe3O4@BHb-MIPs to easily reach adsorption equilibrium. A high magnetic saturation value of 25.47 emu g-1 for Fe3O4@BHb-MIPs NPs was obtained, which endowed the adsorbent with the convenience of magnetic separation under an exter- nal magnetic field. The resultant Fe3O4@BHb-MIPs NPs could not only selectively extract a target protein from mixed proteins but also specifically capture the protein BHb from a real sample of bovine blood. In addition, dif- ferent batches of magnetic MIPs showed good reproducibility and reusabil- ity for at least six repeated cycles. Emulsion polyemrization techniques were also used for the fabrication of m-MIP. Another BHb-imprinted polysty- rene (PS) nanoparticles with magnetic susceptibility have been synthesized through multistage core-shell polymerization system using 3-aminophenyl- boronic acid (APBA) as functional and cross-linking monomers by Lin et al [24]. Super paramagnetic molecularly imprinted polystyrene nanospheres with poly (APBA) thin films have been synthesized and used for the first time for protein molecular imprinting in an aqueous solution. The magnetic susceptibility is imparted through the successful encapsulation of Fe3O4 nanoparticles. The imprinted super paramagnetic nanoparticles could eas- ily reach the adsorption equilibrium and achieve magnetic separation in an external magnetic field, thus avoiding some problems of the bulk polymer.

Molecular Imprinting and Nanotechnology 381 Gu et al., have reported chlorogenic acid imprinted MNPs via water- in-oil-in-water multiple emulsions suspension polymerization [25]. This kind of m-MIPs had the core–shell structure with the size of about 50 nm. Magnetic susceptibility was given by the successful encapsulation of Fe3O4 nanoparticles with a high encapsulation efficiency of 19.3 wt%. Super paramagnetic nanoparticles are of great current interest for bio- medical applications in both diagnostics and treatment. Lee et al., have reported Albumin, creatinine, lysozyme and urea-imprinted polymer nanoparticles from poly (ethylene-co-ethylene alcohol) via phase inver- sion, with both target molecules and hydrophobic magnetic nanoparticles mixed within the polymer solution [26]. The composite m-MIP were used for separation and sensing of template molecules (e.g., human serum albu- min) in real samples (urine). A Redox-active m-MIP nanospheres were first synthesized and func- tionalized with streptomycin templates for highly efficient electrochemical determination of streptomycin residues (STR) in food by coupling with bioelectrocatalytic reaction of enzymes for signal amplification by Liu et al [27]. The m-MIP nanospheres were synthesized by using Au(III)- promoted molecularly imprinted polymerization with STR templates on magnetic beads (Figure 11.8). Based on extraction of template molecules from them MIP surface, the imprints toward STR templates were formed. The assay was implemented magnetic bead Current ( A) Electrocatalytic nanogold particle reaction poly (OPD) reusage STR template 50R%emeothvaanl oPol tential vs.Ag/AgCl (V) Imprinting cavity Re5m0o%veatl hanol ITO electrode STR-GOX STR-GOX without analyte STR analyte strong signal mMIP-based sensor magnet weak signal Figure 11.8 Schematic illustration of nanogold-promoted magnetic molecularly imprinting polymer nanospheres for competitive-type electrochemical detection of streptomycin (STR) residues by coupling with bioelectrocatalytic reaction of glucose oxidase (GOX) for signal amplification [Co-opted from reference 27 with permission].

382 Advanced Biomaterials and Biodevices with a competitive-type assay format. Upon addition of streptomycin, the analyte competed with glucose oxidase-labeled streptomycin (GOX-STR) for molecular imprints on them MIP nanospheres. With the increasing streptomycin in the sample, the conjugation amount of GOX-STR on them MIP nanospheres decreased, leading to the change in the bioelectrocatalytic current relative to glucose system. Under optimal conditions, the catalytic current was proportional to STR level in the sample, and exhibited a dynamic range of 0.05–20ng mL-1 with a detection limit of 10pg mL-1 STR (at 3sB). Chen et al. reported the novel application of m-MIP in the separation of tetracycline antibiotics from egg and tissue samples [28]. The extraction and clean-up procedures were carried out in a single step by blending and stirring the sample, extraction solvent and polymers. The analytes can be transferred from the sample matrix to the polymers directly or through the extraction solvent as medium. When the extraction was complete, the polymers adsorbing the analytes were easily separated from the sample matrix by an adscititious magnet (Figure 11.9). The analytes eluted from the polymers were determined by liquid chromatography-tandem mass spectrometry. The recoveries ranging from 72.8% to 96.5% were obtained with relative standard deviations in the range of 2.9–12.3%. The limit of detection was less than 0.2 ng g−1. The feasibility of this method was vali- dated by analysis of incurred egg and tissue samples, and the results were compared with those obtained by the classical method in which solvent extraction, centrifugation, and subsequent clean-up and concentration by solid-phase extraction were applied. The proposed method reduced the complicacy of classical method and improved the reliability of method. 5% methanol Methanol Citrate buffer methanol or water (0.5% acetic acid) (pH 4) For egg Waste Waste Stirring For tissue Waste Eluate 5% methanol Condition Extraction Seperation Washing Elution Magnet Magnetic MIPs Sample matrices Figure 11.9 The extraction procedures for tetracycline by magnetic MIPs from egg and tissue samples [Co-opted from reference 28 with permission].

Molecular Imprinting and Nanotechnology 383 TEOS MPS NH3⋅H2O MBAAm, APS, TEMED Fe3O4 Fe3O4@SiO2 Modified-Fe3O4@SiO2 Fe3O4@SiO2@MIPs Lysozyme AAm MAA Phosphate buffer Rebinding protein Removal protein Sample Luminuo, NaCl Phosphate Phosphate CTMAB, solution buffer buffer K3[Fe(CN)6] 96-well plate Chemiluminescence analyzer Magnet Eluting Washing Loading Conditioning Figure 11.10 A schematic representation of Fe3O4@SiO2@MIPs preparation and CL detection of lysozyme [Co-opted from reference 29 with permission]. Molecular imprinting is an attractive technique for preparing mimics of natural, biological receptors. Nevertheless, the imprinting of macromol- ecule remains a challenge due to their bulkiness and sensitivity to denatur- ation. Jing et al., have presented a method for preparing multifunctional lysozyme-imprinted nanoparticles (magnetic susceptibility, molecular rec- ognition and environmental response) [29]. The magnetic susceptibility was imparted through the successful encapsulation of Fe3O4 nanoparticles. Selective lysozyme recognition depended on molecularly imprinted film. Moreover, it was also a hydrophilic stimuli-responsive polymer, which could undergo a reversible change of imprinted cavity in response to a small change in the environmental conditions. Thus, m-MIPs had high adsorp- tion capacity (0.11mg mL−1), controlled selectivity and direct magnetic separation (22.1emu g−1) in crude samples. After pre-concentration and purification with m-MIPs nanoparticles, a sensitive chemiluminescence method was developed for determination of lysozyme in human serum samples (Figure 11.10). The results indicated that the spiked recoveries were changed from 92.5 to 113.7%, and the RSD was lower than 11.8%. 2.1.3 Silica Nanoparticles Yuan et al. reported the MIP layer of estriol at the silica nanoparticles surface with selective recognition [30], where the methacryl groups of

384 Advanced Biomaterials and Biodevices Figure 11.11 Schematic illustration for the formation mechanism of estriol-MIPs [30]. functional monomer at the silica nanoparticles surface were acted as reac- tive sites to induce imprinting polymerization (Figure 11.11). The absorp- tion capacity results showed that the molecularly imprinted polymers had an excellent combining affinity, recognition selectivity and fast kinetics. Furthermore, the molecularly imprinted polymers were successfully used as absorbent of dispersive solid-phase extraction coupled with high-per- formance liquid chromatography to determinate trace estriol and estradiol in milk tablets. The high recoveries yielded of 89.1–93.5% were achieved with the relative standard deviations less than 9.4%. So, the molecularly imprinted polymers are the effective absorbents for the separation and enrichment of oestrogens in the complex matrices samples. Similarly, Peng et al. reported the preparation of metsulfuron-methyl (MSM) imprinted polymer layer-coated silica nanoparticles toward analy- sis of trace sulfonylurea herbicides in complicated matrices [31]. To induce the selective occurrence of surface polymerization, the polymerizable double bonds were first grafted at the surface of silica nanoparticles by the silylation using 3-(Methacryloxy) propyl trimethoxysilane (MPTS) (Figure 11.12). Afterwards, the metsulfuron-methyl templates were imprinted into the polymer-coating layer through the interaction with functional monomers (Methacrylic acid, MAA). The programmed heating led to the formation of uniform metsulfuron-methyl-imprinted polymer layer with controllable thickness, and further improved the reproducibility

Molecular Imprinting and Nanotechnology 385 (A) Modification SiO2 MPTS MPTS-SiO2 (B) Prearrangement MSM MAA Template-monomer complex (Template molicule) (Monomer) (C) Polymerization Cross-linker initiator MPTS-SiO2 Template-monomer complex Further aging Removal of MSM (Programed heating) Rebinding MSM SiO2@MSM-MIP Figure 11.12 Schematic representation of MPTS modification of silica nanoparticles (A), prearrangement of MSM and MAA (B), and preparation of SiO2@MSM–MIP nanoparticles (C) [Co-opted from reference 31 with permission]. of rebinding capacity. After removal of templates, recognition sites of met- sulfuron-methyl were exposed in the polymer layers. As a result, the maximum rebinding capacity was achieved with the use of optimal grafting ratio. There was also evidence indicating that the met- sulfuron-methyl-imprinted polymer nanoparticles compared with non- imprinted polymer nanoparticles had a higher selectivity and affinity to four structure-like sulfonylurea herbicides. Moreover, using the imprinted particles as dispersive solid phase extraction materials, the recoveries of four sulfonylurea herbicides determined by high performance liquid chro- matography (HPLC) were 80.2–99.5%, 83.8–102.4%, 77.8–93.3%, and 73.8–110.8% in the spiked soil, rice, soybean, and corn samples, respec- tively. These results show the possibility that the highly selective separa- tion and enrichment of trace sulfonylurea herbicides from soil and crop

386 Advanced Biomaterials and Biodevices pre-adsorption of positively charged template protein template self-assembly and subsequent surface removal graft copolymerization in highly dilute monomer solution of MAA, AAm, rebinding DMAEMA, and MBA Figure 11.13 Schematic illustration of the procedure for the imprinting of Lysozyme over vinyl and carboxyl group modified silica nanoparticles via surface graft copolymerization in highly dilute solution of functional and cross-linking monomers [Co-opted from reference 32 with permission]. samples can be achieved by the molecular imprinting modification at the surface of silica nanoparticles. Surface imprinting over nanosized support materials is particularly suitable for protein templates, considering the problems with mass trans- fer limitation and low binding capacity [32]. Recently, Chen et al. studied the synthesis process toward enhancement of the imprinting performance by examining the effect of several synthesis conditions. Interestingly, the feed crosslinking degree was found to have a great impact on the thick- ness of the formed imprinting polymer layers and the recognition prop- erties of the resulting imprinted materials (Figure 11.13). The imprinted particles with a crosslinking degree up to 50% showed the best imprint- ing effect. The imprinting factor achieved 2.89 and the specific binding reached 23.3 mg/g, which are greatly increased compared to those of the lowly cross-linked imprinted materials reported previously. Moreover, the relatively high crosslinking degree led to no significant retarding of the binding kinetics to the imprinted particles, and the saturated adsorption was reached within 10min. 11.2.1.4 Core-shell Nanoparticle Core–shell nanocrystals and/or nanoparticles (CSNPs) are a class of mate- rials, which have properties intermediate between those of small, individ- ual molecules and those of bulk, crystalline semiconductors (Figure 11.14). They are unique because of their easily modular properties, which are a result of their size. These nanocrystals are composed of a quantum dot core and a shell of a distinct semiconducting material. The core and the shell are

Molecular Imprinting and Nanotechnology 387 Figure 11.14 Schematic representation of core-shell nanoparticles. typically composed of type II–VI, IV–VI, and III–V semiconductors, with configurations such as CdS/ZnS, CdSe/ZnS, CdSe/CdS, and InAs/CdSe (typical notation is: core/shell). Organically passivated quantum dots have low fluorescence quantum yield due to surface related trap states. CSNPs address this problem because the shell increases quantum yield by pas- sivating the surface trap states. In addition, the shell provides protection against environmental changes, photo-oxidative degradation, and pro- vides another route for modularity. Precise control of the size, shape, and composition of the core and shell enable the emission wavelength to be tuned over a wider range of wavelengths than with either individual semi- conductor. These materials have found applications in biological systems and optics. Taking into account the recognition element for sensors linked to MIPs, a proliferation of interest has been witnessed by those who are interested in this subject [33]. Indeed, MIP nanoparticles are theme, which recently has come to light in the literature. Keeping this in mind, Gültekin et al. have proposed a novel thiol ligand-capping method with polymerizable methac- ryloylamidocysteine (MAC) attached to gold nanoparticles, reminiscent of a self-assembled monolayer. Furthermore, a surface shell by synthetic host polymers based on molecular imprinting method for recognition has been reconstructed. 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 the main participant of Bacillus cereus spores has been used as a template. Nanoshell sensors with templates produce a cavity that is selec- tive for DPA. The DPA can simultaneously chelate to Cr(III)-metal ion and fit into the shape-selective cavity. Thus, the interaction between Cr(III) ion and free coordination spheres has an effect on the binding ability of the

388 Advanced Biomaterials and Biodevices gold nanoparticles nanosensor. The interactions between DPA and MIP particles were studied observing fluorescence measurements. DPA addi- tion caused significant decreases in fluorescence intensity because they induced photoluminescence emission from Au nanoparticles through the specific binding to the recognition sites of the cross-linked nanoshell poly- mer matrix. The binding affinity of the DPA imprinted nanoparticles has been explored by using the Langmuir and Scatchard methods and the anal- ysis of the quenching results has been performed in terms of the Stern– Volmer equation. Combination of surface molecular imprinitng with sol-gel pro- cess was reported by Chen et al., for the preparation of core-shell mag- netic nanoparticle-based metronidazole-imprinted polymer. Chen et al. described imprinting onto the surface of magnetic nanoparticle using 3-aminoprophyltriethoxysilane as the functional monomer, and tetra- ethyl orthosilicate as the cross-linker [34]. The adsorbent can be applied to the solid phase extraction of metronidazole and exhibits high adsorption capacity, good selectivity, favourable reusability and the feature of magnetic separation. The binding performances of the adsorbent were evaluated by equilibrium rebinding experiments and Scatchard analysis. The material was successfully applied to solid phase extraction of metronidazole, fol- lowed by spectrophotometric determination in food samples. In another work, the use of core–shell imprinted nanoparticles for the selective rec- ognition of thifensulfuronmethyl (TFM) using an electrochemilumines- cence (ECL) method was reported by Li et al. The core–shell imprinted nanoparticles were first prepared by a surface monomer-directing strategy for imprinting TFM at the surface of 3- methacryloxypropyl trimethoxysi- lane modified silica particles [35]. Then, the ECL sensor was prepared by depositing the core–shell imprinted nanoparticles/chitosan composite film on the bare glassy carbon electrode surface and further removing silica cores from the composite film. The electrochemical and ECL behaviors of luminol at the sensor were investigated in the absence and presence of TFM. It was also found that the ECL intensity could be strikingly enhanced by the adsorbed TFM molecules in the composite film, which was about 2.7-fold as compared with the blank ECL intensity. Surface molecular imprinting, in particular over nanosized support materials, is very suitable for a template of bulky structure like protein [36]. Inspired by the surface template immobilization method reported previously, Fu et al. demonstrate an alternative strategy for enhanc- ing specific recognition of core-shell protein-imprinted nanoparticles through prefunctionalizing the cores with noncovalent template sorption groups. For proof of this concept, silica nanoparticles chosen as the core

Molecular Imprinting and Nanotechnology 389 materials were modified consecutively with 3-aminopropyltrimethoxysi- lane and maleic anhydride to introduce polymerizable double bonds and terminal carboxyl groups, hence capable of physically adsorbing the print protein. With lysozyme as a template, thin protein-imprinted shells were fabricated according to this newly developed approach for surface protein imprinting over nanoparticles. The rebinding experiments confirmed that the introduction of the carboxyl groups could remarkably improve the imprinting effect in relation to a significantly increased imprinting factor and specific rebinding capacity. Moreover, in contrast to the harsh template removal conditions required for the covalent template coupling approach, the template removal during the imprinted particle synthesis as well as desorption after rebinding could be mildly achieved via wash- ing with salt solution. Chemical nanosensors with a submicrometer core–shell composite design, based on a polymer core, a MIP shell for specific analyte recogni- tion, and an interlayer of gold nanoparticles for signal amplification, were also reported by Bompart et al. [37]. Surface-enhanced Raman scatter- ing (SERS) measurements on single nanosensors yield detection limits of 10−7 M for the  β-blocker propranolol, several orders of magnitude lower than on plain MIP spheres. Gültekin et al., have proposed a novel thiol ligand-capping method with polymerizable methacryloylamido-cysteine (MAC) attached to gold–silver nanoclusters, reminiscent of a self-assembled monolayer and have reconstructed surface shell by synthetic host polymers based on molecular imprinting method for recognition [38]. In this method, methacryloylamidoantipyrine–terbium (MAAP)2-Tb(III) has been used as a new metal-chelating monomer via metal coordination–chelation interactions and dipicolinic acid (DPA) which is main participant of Bacillus cereus spores used as a model. Nanoshell sensors with tem- plates give a cavity that is selective for DPA. The DPA can simultane- ously chelate to Tb(III) metal ion and fit into the shape-selective cavity. Thus, the interaction between Tb(III) ion and free coordination spheres has an effect on the bindingability of the gold–silver nanoclusters nano- sensor. The binding affinity of the DPA imprinted nanoclusters has been investigated by using the Langmuir and Scatchard methods, and the respective affinity constants determined were found to be 1.43×10–4 and 9.1×10–6 mol L-1. Recently Chen et al. have reported a sensitive and selective electro- chemical sensor for metronidazole (MNZ) [39]. The core-shell metro- nidazole-m-MIP was synthesized and then attached to the surface of magnetic glassy carbon electrode (MGCE) with the help of magnetic