Chemical Design of Functional Polymer Structures for Biosensors - MDPI

polymers Review

Chemical Design of Functional Polymer Structures for Biosensors: From Nanoscale to Macroscale Kyoung Min Lee 1,2,† , Kyung Ho Kim 1,† , Hyeonseok Yoon 1,3, * 1 2 3

* †

ID

and Hyungwoo Kim 1,3, *

ID

Department of Polymer Engineering, Graduate School, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 61186, Korea; [email protected] (K.M.L.); [email protected] (K.H.K.) Department of Materials Science and Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Korea School of Polymer Science and Engineering, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 61186, Korea Correspondence: [email protected] (H.Y.); [email protected] (H.K.); Tel.: +82-062-530-1778 (H.Y.); +82-062-530-1775 (H.K.) These authors contributed equally to this work.





Received: 20 April 2018; Accepted: 14 May 2018; Published: 21 May 2018

Abstract: Over the past decades, biosensors, a class of physicochemical detectors sensitive to biological analytes, have drawn increasing interest, particularly in light of growing concerns about human health. Functional polymeric materials have been widely researched for sensing applications because of their structural versatility and significant progress that has been made concerning their chemistry, as well as in the field of nanotechnology. Polymeric nanoparticles are conventionally used in sensing applications due to large surface area, which allows rapid and sensitive detection. On the macroscale, hydrogels are crucial materials for biosensing applications, being used in many wearable or implantable devices as a biocompatible platform. The performance of both hydrogels and nanoparticles, including sensitivity, response time, or reversibility, can be significantly altered and optimized by changing their chemical structures; this has encouraged us to overview and classify chemical design strategies. Here, we have organized this review into two main sections concerning the use of nanoparticles and hydrogels (as polymeric structures) for biosensors and described chemical approaches in relevant subcategories, which act as a guide for general synthetic strategies. Keywords: functional polymers; nanoparticles; hydrogels; chemical structures; biosensor

1. Introduction Polymers have advantages over metallic and ceramic materials for biosensing applications, such as mild synthetic conditions, scalable and large-area processing, chemical and structural diversity, flexibility, low operating temperature, and biocompatibility. Progress in nanotechnology has provided opportunities to design and fabricate various polymer nanostructures for biosensing applications. Several key requirements of polymeric materials are necessary to expand their applications. The most important parameters for sensing performance are sensitivity, response/recovery time, and reversibility/reproducibility of response, which are strongly dependent on the chemical structure and size of the polymers [1,2]. In addition, selectivity toward a specific target and the prevention of non-specific binding or adsorption are prerequisites for precise detection. Nanoparticles are of great importance in sensing applications because of their large, effective surface area, which increases the rate of response, as well as the sensitivity. In the past few decades, rapid progress in nanotechnology has occurred, and this has stimulated the development of nanoparticles. Thus, we can now not only optimize the chemical or physical properties of the particles

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on demand but also manipulate the nanostructure or morphology, achieving an optimized sensing system. For in vivo biosensing applications, the toxicity of nanoparticles is a critical factor to consider. Nanoparticles can be engineered with various surface functional groups and bioconjugates to enhance their biocompatibility [3]. On the other hand, stimuli-responsive polymeric materials have been investigated on a large scale for practical use [4]. In particular, interest in hydrogels has increased, and they have been frequently used in healthcare devices [5]. Hydrogels confine a considerable amount of water, which provides biocompatibility as well as biodegradability. In addition, the hydrogel-based, macroscale materials are known to be elastic yet durable, and transparent, all necessary properties of prosthetic devices. Furthermore, these materials are functionally alterable, allowing the preparation of optimized and bespoke materials for application in wearable or even implantable devices [6]. In this review paper, we focus on the design principles and chemical strategies for the preparation of polymeric materials from the perspective of synthetic chemistry and materials science using recent, notable examples. These materials have been designed to achieve the desired properties in the biosensing applications and would lead to a prosthetic polymer-based nervous system in the future (e.g., artificial human skin). 2. Nanostructure: Nanoparticles With the rapid progress in nanotechnology, a variety of nanostructures have been developed for sensing applications. Compared to their bulk counterparts, nanoparticles have enhanced the effective surface area and small dimensions, which result in increased sensitivity and faster response times for the nanoparticle-based sensors. Control over the morphology and microstructures of the nanoparticles in biosensor applications is also crucial [7,8]. It is well known that the major chemical/physical properties of the nanoparticles depend on the morphology and microstructures [9–11]. Furthermore, the biocompatibility of the nanoparticles is also affected by the morphological characteristics. As a typical example, Jang and co-workers reported that poly(3,4-ethylenedioxythiophene) (PEDOT) nanostructures of different shapes (aspect ratios) exhibited aspect-ratio dependent cell viability and cytotoxicity (Figure 1) [12]. Briefly, the cytotoxicity and apoptosis of the PEDOT nanoparticles increased with their decreasing aspect ratio for both cell lines, and the formation of reactive oxygen species in cells treated with the PEDOT nanoparticles also depended on the shape and concentration of the nanoparticles. Therefore, for nanoparticle biosensing applications, the parameters affecting biocompatibility must be considered, as well as developing a level of biofunctionality to interact passively or actively with the surrounding biological species/processes. Compared to inorganic semiconductors and metals, polymers are structurally unstable at the nanoscale, which makes it difficult to prepare polymer nanoparticles with controlled sizes and morphologies. Nevertheless, diverse methods have been developed to produce polymer nanostructures, such as the so-called template and template-free approaches. The templates encompass porous membranes, micelles, vesicles, and macromolecules. For example, Feng group has successfully fabricated mesoporous conducting polymer nanosheets using block copolymer templates [13–15]. On the other hand, template-free approaches utilize the self-assembly of molecules or nanoscale building blocks in the absence of any templates. These methods for fabricating polymer nanostructures have been well summarized and described in several review articles [16,17]. Importantly, most polymer nanoparticles require additional synthetic procedures when their inherent chemical structure is not appropriate for biosensor applications. The preparation of polymers with functionalities that yield antifouling behavior and selectivity in biosensing applications can be accomplished through physical and chemical routes, such as the incorporation of dopants into polymers, direct polymerization of monomers with functional groups, and post-polymerization modification. The following subsection addresses the strategies that have been used to design and prepare polymer nanostructures for biosensing applications.

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Figure 1. (a) Transmission electron microscope (TEM) images of poly(3,4-ethylenedioxythiophene)

Figure 1. (a) Transmission electron microscope (TEM) images of poly(3,4-ethylenedioxythiophene) (PEDOT) nanomaterials of three different shapes: (i) PEDOT-1; (ii) PEDOT-2; and (iii) PEDOT-3; (b) (PEDOT) nanomaterials of three different shapes: (i) PEDOT-1; (ii) PEDOT-2; and (iii) PEDOT-3; Viability of fibroblast (IMR90) and macrophage (J774A.1) cells in the presence of PEDOT (b) Viability of fibroblast (IMR90) and macrophage (J774A.1) cells in the presence of PEDOT nanomaterials, which was determined by the amount of ATP in the cells. IMR90 cells were incubated nanomaterials, which was determined the(iii) amount of ATP infor the(ii)cells. cells were with PEDOT nanomaterials for (i) 24by and 48 h; J774A.1 cells 24 andIMR90 (iv) 48 h. Values incubated with PEDOT nanomaterials for (i) 24(SD), andand (iii)each 48 h; J774A.1 cells for (ii) 24inand (iv) 48* h. expressed as the mean ± standard deviation experiment was performed triplicate. Values expressed the meandifference ± standard deviation andtoeach experiment was performed Statisticallyassignificant from control (SD), exposed PEDOT nanomaterials (p < 0.05).in Reproduced withsignificant permissiondifference from [12]. Copyright 2010, John Wiley & Sons. nanomaterials (p < 0.05). triplicate. * Statistically from control exposed to PEDOT Reproduced with permission from [12]. Copyright 2010, John Wiley & Sons. 2.1. Physical Doping

2.1. Physical Functional Doping components can be doped into polymers, allowing further chemical functionalization of the polymers under a controlled environment. In particular, conducting polymers have

Functional components can be doped into polymers, allowing further chemical functionalization electronic/electrical properties that depend on the nature and incorporated status of the dopant ions of the [8]. polymers under a controlled environment. In particular, conducting polymers have Importantly, appropriate chemical functionalities can be incorporated into the polymers via the electronic/electrical properties on theinnature and incorporated status of the dopant ionsis[8]. doping process. Dopantsthat candepend be trapped the polymer, and the doping/dedoping process Importantly, appropriate chemical functionalities can bethat incorporated viaaffect the doping reversibly controlled. It should also be noted the natureinto of the polymers dopant can the of trapped conducting polymers. Wallace anddoping/dedoping co-workers reported the influence of process.biocompatibility Dopants can be in the polymer, and the process is reversibly biological compounds as the dopant on the biocompatibility and biofunctionality of PEDOT. They controlled. It should also be noted that the nature of the dopant can affect the biocompatibility of found that dopants such asand dextran sulfate, chondroitin sulfate, and alginate significantly affected as conducting polymers. Wallace co-workers reported the influence of biological compounds the biologically relevant properties the polymer (e.g.,ofnanoroughness, wettability, the dopant on the biocompatibility andof biofunctionality PEDOT. Theymodulus, found that dopantsand such electrical impedance) and in turn biological interactions (e.g., cell adhesion) [18]. as dextran sulfate, chondroitin sulfate, and alginate significantly affected the biologically relevant A good example of physical doping in biosensing has been demonstrated by Park and Jang [19]. properties of the polymer (e.g., nanoroughness, modulus, wettability, and electrical impedance) and A dopamine field-effect transistor (FET) biosensor was fabricated using dopamine-receptorin turn biological interactions (e.g., cell adhesion) [18]. A good example of physical doping in biosensing has been demonstrated by Park and Jang [19]. A dopamine field-effect transistor (FET) biosensor was fabricated using dopamine-receptor-containing nanovesicle-immobilized carboxylated PEDOT (CPEDOT) nanotubes (DRNCNs) as a channel,

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4 ofas 34 a carboxylated PEDOT (CPEDOT) nanotubes (DRNCNs) channel, where the nanovesicles acted as a gate-potential modulator. Figure 2 shows the fabrication Polymers 2018, 10, x FOR PEER REVIEW 4 of 34 process for the DRNCN FET sensor substrate. First, the CPEDOT nanotubes were attached to an where the nanovesicles acted as a gate-potential modulator. Figure 2 shows the fabrication process for containing nanovesicle-immobilized carboxylated PEDOTcoupling (CPEDOT) nanotubes (DRNCNs) as a group interdigitated microelectrode array (IMA) via covalent with the surface amino-silane the DRNCN FET sensor substrate.acted First,asthe CPEDOT nanotubes were attached to an interdigitated channel, where the nanovesicles a gate-potential modulator. Figure 2 shows the fabrication of the IMA and then, importantly, the nanovesicles were immobilized on the CPEDOT nanotubes microelectrode array (IMA)FET via sensor covalent coupling the surface amino-silane group to of an the IMA process for of thepolyDRNCN substrate. First,with the CPEDOT nanotubes were attached with the aid D-lysine (PDL), which is a synthetic amino acid widely used as a coating to and then, importantly, the nanovesicles were immobilized onwith the the CPEDOT nanotubes with interdigitated microelectrode array (IMA) via covalent coupling surface amino-silane groupthe aid improve cell attachment because of its positive charge [20,21]. The resultant liquid-ion gated DRNCN of the IMA and then, which importantly, the nanovesicles on as theaCPEDOT of polyD -lysine (PDL), is a synthetic aminowere acidimmobilized widely used coating nanotubes to improve cell FETwith sensor showed high sensitivity (minimum detectable level of as used low as asa 10 pM) toand high the aid of polyD -lysine (PDL), which is a synthetic amino acid widely coating attachment because of its positive charge [20,21]. The resultant liquid-ion gated DRNCN FET sensor selectivity, even in human serum, as seen in Figure 3. improve cell attachment because of its positive charge [20,21]. The resultant liquid-ion gated DRNCN showed high sensitivity (minimum detectable level of as low as 10 pM) and high selectivity, even in FET sensor showed high sensitivity (minimum detectable level of as low as 10 pM) and high human serum, as seen in Figure 3.

selectivity, even in human serum, as seen in Figure 3.

Figure Schematic illustration ofofconstruction steps for dopamine-receptor-containing nanovesicleFigure 2.2. 2. Schematic illustration of construction for dopamine-receptor-containing Figure Schematic illustration construction steps forsteps dopamine-receptor-containing nanovesicleimmobilized carboxylated PEDOT (CPEDOT) nanotubes (DRNCNs) geometry. (DRNCNs) Reproduced with nanovesicle-immobilized carboxylated PEDOT (CPEDOT) nanotubes geometry. immobilized carboxylated PEDOT (CPEDOT) nanotubes (DRNCNs) geometry. Reproduced with permission from [19]. Copyright 2014, Nature Publishing Group. Reproduced from [19]. 2014, Nature Publishing Group. permissionwith frompermission [19]. Copyright 2014,Copyright Nature Publishing Group.

Figure 3. (a) Real-time responses with normalized current changes (ΔI/I0) and (b) calibration curves of DRNCN toward various dopamine concentrations (S indicates the normalized current change). (c) Figure 3. 3. (a)(a) Real-time responses with normalized current changes (∆I/I (b)(b) calibration curves 0 ) 0and Selective responses of the dopamine biosensor using DRNCN toward non-target neurotransmitters Figure Real-time responses with normalized current changes (ΔI/I ) and calibration curves of of DRNCN toward various dopamine concentrations (S indicates the normalized current change). (PBS, 1 mM Serotonin, and 1dopamine mM Epinephrine) and dopamine (10 pM dopamine). Reproduced DRNCN toward various concentrations (S indicates the normalized currentwith change). (c) permission [19]. Copyright 2014, Nature Publishing Group. (c) Selective responses of the dopamine biosensor using DRNCN toward non-target neurotransmitters

Selective responses of the dopamine biosensor using DRNCN toward non-target neurotransmitters (PBS, 1 mM Serotonin, and 1 mM Epinephrine) and dopamine (10(10 pMpM dopamine). Reproduced with (PBS, 1 mM Serotonin, and 1 mM Epinephrine) and dopamine dopamine). Reproduced with There [19]. are many candidate dopants that can affect the biological interactions of conducting permission Copyright 2014, Nature Publishing Group. permission [19]. Copyright 2014, Nature Publishing Group. polymers, such as glycosaminoglycans (e.g., chondroitin sulfate, heparin/heparin sulfate, and dextran sulfate) [22–27]. Those macromolecules all are negatively charged and, thus, can effectively There dopants that affect thethe biological conducting There aremany manycandidate dopants that can affect biological interactions of conducting serve asare the dopant incandidate conducting polymers. Incan addition, most polymers areinteractions amenable to of physical polymers, such as glycosaminoglycans (e.g., chondroitin sulfate, sulfate, and dextran polymers, such as glycosaminoglycans (e.g., chondroitin sulfate, sulfate, and doping, and a number of functional dopants are available, includingheparin/heparin peptidesheparin/heparin and proteins. However, unfortunately, undesirable dedoping canare occur during sensing, which limits the applications of sulfate) [22–27]. Those macromolecules all negatively and,charged thus, can effectively serve as the dextran sulfate) [22–27]. Those macromolecules all arecharged negatively and, thus, can effectively physical in biosensing. dopant in polymers. In addition, most amenable toare physical doping, and a serve as conducting thedoping dopant in conducting polymers. In polymers addition, are most polymers amenable to physical

number ofand functional dopants are available, including peptides and proteins. However, unfortunately, doping, a number of functional dopants are available, including peptides and proteins. However, undesirable dedoping can occur during can sensing, limits the applications physical dopingof unfortunately, undesirable dedoping occurwhich during sensing, which limitsofthe applications inphysical biosensing. doping in biosensing.

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2.2. Chemical Structure Modification of Monomers The most straightforward route to functional polymers for specific applications involves direct Polymers 2018, 10, x FOR PEER REVIEW 5 of 34 polymerization of monomers with the desired chemical structures and properties. Many commercial 2.2. Chemical Structurefrom Modification of Monomers monomers are available chemical manufacturers, derivatives of which can be synthesized via appropriate chemical reactions. From both quantitative andfor qualitative points of view, thedirect direct use The most straightforward route to functional polymers specific applications involves of monomers with more than a functionality (f-monomers) allows easy control on the incorporation polymerization of monomers with the desired chemical structures and properties. Many commercial monomers are available from manufacturers, of the which can be synthesized viaof the of chemical functionalities into chemical homopolymer chains.derivatives However, important properties appropriate chemical From bothstructures, quantitative andas qualitative the direct use resulting polymers dependreactions. on the chemical such polarity,points intra-oforview, interchain interactions, of monomers withadditional more than aeffort functionality control on the incorporation and reactivity. Thus, should(f-monomers) be devotedallows to theeasy polymerization of f-monomers to of chemical functionalities into homopolymer chains. However, the important properties of the control the size and morphology of the resulting nanoparticles. In some cases, f-monomers may resulting polymers depend on the chemical structures, such as polarity, intra- or interchain prove interactions, incompatible with the polymerization conditions required to achieve high-molecular-weight and reactivity. Thus, additional effort should be devoted to the polymerization of fpolymers and to form well-defined nanoparticles with sizes and morphologies. Furthermore, monomers to control the size and morphology of controlled the resulting nanoparticles. In some cases, fthe reaction yieldmay of the f-monomer maywith be too to produceconditions an appreciable quantity of the polymer monomers prove incompatible the low polymerization required to achieve highmolecular-weight and to form nanoparticles controlled sizes and limits for use, which reduces polymers cost-effectiveness and well-defined efficiency. As a result, thiswith approach significantly morphologies. Furthermore, the reaction yield of the f-monomer may be too low to produce an the flexibility in nanostructure design enabled by the broad range of polymerization techniques. appreciable quantity of the polymer for use, which reduces cost-effectiveness and efficiency. As a Thiophene may be referred as the most prominent unit of derivatization to prepare result, this approach significantly limits the flexibility in nanostructure design enabled by the broad f-monomers. There are many thiophene derivatives, including 3,4-ethylenedioxythiophene range of polymerization techniques. (EDOT) [28]. Polythiophene has attractive electrical and optical properties as a π-conjugated polymer. Thiophene may be referred as the most prominent unit of derivatization to prepare f-monomers. In particular, alkyl thiophenes have been extensively for device applications such as FETs, There are many thiophene derivatives, includingexploited 3,4-ethylenedioxythiophene (EDOT) [28]. light-emitting diodes, sensors. A recent article, byasMancuso and Gabriele, describes the Polythiophene hasand attractive electrical andreview optical properties a π-conjugated polymer.well In particular, synthetic strategies forhave various thiophene exploited derivatives [29]. Although EDOT well-known thiophene alkyl thiophenes been extensively for device applications such is asaFETs, light-emitting diodes, and no sensors. A recent reviewfor article, by Mancuso and Gabriele, wellEDOT describes synthetic derivative, it has functional groups further modification. Therefore, hasthe also been further strategies for various thiophene derivatives Although EDOT a well-known thiophene modified to contain functional groups such as [29]. hydroxyl, ether, andisester groups. Figure 4 shows derivative, it has no functional groups for further modification. Therefore, EDOT has also been synthetic procedures for the synthesis of several EDOT derivatives. In addition to the possibilities for further modified to contain functional groups such as hydroxyl, ether, and ester groups. Figure 4 further functionalization, the derivatization of the monomer leads to different properties compared to shows synthetic procedures for the synthesis of several EDOT derivatives. In addition to the the original monomer. Advances in polymerization techniques, such as controlled/living radical possibilities for further functionalization, the derivatization of the monomer leads to different polymerization have enabled diverse polymerization reactions that yield highly functionalized properties compared to the original monomer. Advances in polymerization techniques, such as polymeric materials. However, there is stillhave a broad range of side-chain functionalities thatyield cannot be controlled/living radical polymerization enabled diverse polymerization reactions that highly functionalized However, there is still a broad range of side-chain directly incorporated into polymeric polymers materials. by existing polymerization techniques. Such functional groups functionalities that prevent cannot be directly incorporated into or polymers by existinginpolymerization may either completely controlled polymerization may participate side reactions that techniques. functional groups may either completely controlledmost polymerization may may lead to loss Such of control in the polymerization reaction.prevent Consequently, thiopheneorderivatives participate in side reactions that may lead to loss of control in the polymerization reaction. have been polymerized so far in the form of polymer films rather than nanostructures: commonly, Consequently, most thiophene derivatives have been polymerized so far in the form of polymer films polythiophene derivatives can be electrochemically depositedderivatives as a thin film appropriate dopant rather than nanostructures: commonly, polythiophene canwith be an electrochemically and then receptor molecules are covalently immobilized to the polymer. Such problems are similarly deposited as a thin film with an appropriate dopant and then receptor molecules are covalently foundimmobilized in other kinds of f-monomers. to the polymer. Such problems are similarly found in other kinds of f-monomers.

Figure 4. Synthetic schemes of several 3,4-ethylenedioxythiophene (EDOT) derivatives such as

Figure 4. Synthetic schemes of several 3,4-ethylenedioxythiophene (EDOT) derivatives such as hydroxymethylated EDOT, sulfonatoalkoxy-substituted EDOT, and alkoxy-substituted EDOT [28]. hydroxymethylated EDOT, sulfonatoalkoxy-substituted EDOT, and alkoxy-substituted EDOT [28].

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2.3. Copolymerization To circumvent the problematic issues occurring in the polymerization of f-monomers alone, functional groups can be introduced into polymers through copolymerization. In other words, f-monomers can be introduced to the polymerization process of monomers with no functionality (n-monomers) to yield copolymers, where the degree of functionalization is controlled by adjusting the molar ratio of f-monomer to n-monomer. Similar to the homopolymerization of f-monomers, there are several issues that should be considered carefully for the successful fabrication of polymer nanostructures. As a detailed example, while the most monomers are hydrophobic, functional groups that enable subsequent chemical reactions are mostly hydrophilic. Therefore, there should be precise interfacial control between the different monomers during the fabrication of nanostructures. Additionally, as mentioned earlier, the major properties of the polymer nanoparticles are affected by the introduction of f-monomers, which may affect the sensing performance depending on the degree of functionalization. For these reasons, most researchers have prepared polymer films for sensing applications using copolymerization strategies. Foot and co-workers developed a potentiometric urea biosensor using a conductive thiophene copolymer, poly(3-hexylthiophene-co-3-thiopheneacetic acid) (P(3HT-3TAA)) [30]. Urease was covalently linked to the surface carboxyl group of the P(3HT-3TAA) film and the resultant urease-immobilized P(3HT-3TAA) film electrode had a detectable concentration range of about 1-5 mM (the normal level of urea in blood serum is 1.3–3.5 mM). Travas-Sejdic used pyrrole and thiophene derivative monomers, such as carboxylic acid-bearing pyrrole and thiophene phenylenes, to immobilize receptors on the resulting polymer film [31]. Oligonucleotides were readily attached to the pyrrole-/thiophene-based termonomers using carbodiimide chemistry, and subsequent electrochemical polymerization resulted in conductive polymer thin films, which allowed selective DNA detection. Despite the difficulties in fabricating copolymer nanostructures, several works have successfully demonstrated that copolymerization can be used for the preparation of transducer polymer nanoparticles. As a notable example, Jang and Yoon developed for the first time a soft template approach to fabricate one-dimensional polymer nanostructures [32,33]. Electrically conductive polypyrrole (PPy) and PEDOT nanostructures were prepared using a reverse cylindrical micelle template comprised of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) [9–11,34]. The cylindrical reverse micelle was robust as a template and, thus, sustainable during the chemical oxidation polymerization. As a result, the copolymerization of pyrrole or EDOT monomers with carboxyl-group-bearing counterpart monomers was successfully carried out without any template deformation. The degree of carboxyl-group functionalization in the final product was also tunable by adjusting the concentration of the carboxylated monomer. Representatively, carboxylic-acid-functionalized PPy (CPPy) nanotubes for biosensing were prepared by the AOT cylindrical micelle templating [35]. Pyrrole-3-carboxylic acid (P3CA) was copolymerized with pyrrole on the cylindrical micelle template, yielding well-defined nanotubes, as shown by the scanning electron microscope (SEM) and transmission electron microscope (TEM) images in Figure 5a. The introduction of the carboxyl group to the nanotubes and its availability for further functionalization were confirmed by covalently attaching pyrene molecules to the nanotube surface (see the confocal laser scanning microscopy (CLSM) images in Figure 5a). The surfaces of the nanotubes were modified with ethylenediamine, and, subsequently, pyreneacetic acid was chemically anchored to the nanotubes via amide bond formation. In addition, the chemical functionality of the nanotubes was quantitatively tuned by adjusting the P3CA-to-pyrrole molar ratio (CPNT-1, 1:15; CPNT-2, 1:30). Figure 5b shows the X-ray photoelectron spectra (XPS) of the C1s spectra of the nanotubes in CPNT-1 and CPNT-2. The intensity of the carboxylic acid group component at 288.7 eV is clearly dependent on the P3CA-to-pyrrole molar ratio.

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Figure 5. Carboxylic-acid-functionalized PPy (CPPy) nanotubes with different chemical Figure 5. Carboxylic-acid-functionalized PPy (CPPy) nanotubes with different chemical functionalities. functionalities. (a) (i,ii) SEM and TEM (inset) images and (iii,iv) CLSM images (λexc = 458 nm), where (a) (i,ii) SEM and TEM (inset) images and (iii,iv) CLSM images (λexc = 458 nm), where pyreneacetic pyreneacetic acid was covalently conjugated to the nanotubes: (i,iii) CPNT-1 and (ii,iv) CPNT-2; (b) acid was covalently conjugated to the nanotubes: (i,iii) CPNT-1 and (ii,iv) CPNT-2; (b) XPS C1s spectra XPS C1s spectra (the arrow indicates the C1s component that originates from carboxylic acid group). (the arrow indicates the C1s component that originates from carboxylic acid group). Reproduced with Reproduced withCopyright permission2008, fromJohn [35].Wiley Copyright 2008, John Wiley & Sons. permission from [35]. & Sons.

It is important to note that the surface chemical functionality of the resultant CPPy nanotubes It is important to note that the nanotubes surface chemical functionality of the resultant CPPyasnanotubes allows the anchoring of the themselves to an electrode substrate, well as the allows the anchoring the nanotubes themselves to Figure an electrode substrate, well assteps the for immobilization of of bioreceptors on the nanotubes. 6a illustrates the as reaction immobilization of bioreceptors on the nanotubes. Figure 6a illustrates the reaction steps for constructing constructing a CPPy-nanotube-based sensing platform. First, the surface of the microelectrode a CPPy-nanotube-based sensing platform. First, the surface of the microelectrode substrate was substrate was modified with primary amino groups of an amino-silane and then the carboxyl group modified with primary amino groups of an amino-silane and then the carboxyl group of of the nanotubes were covalently linked with the surface amino group of the substrate throughthe amide nanotubes covalently linked with the surface amino group ofas thethe substrate through bond bond were formation. Amine-terminated thrombin aptamers receptor wereamide subsequently formation. Amine-terminated thrombin as the were subsequently immobilized immobilized on the surface of the aptamers nanotubes via receptor the same amide bond chemistry. Polymer on the surface of the nanotubes via the same amide bond chemistry. Polymer nanostructures not nanostructures are not suitable to the conventional lithographic process for the are fabrication suitable to the conventional lithographic process for the fabrication microelectrode sensing substrates microelectrode sensing substrates because they are highly vulnerable to the chemicals and heat used because theythe are lithographic highly vulnerable to the chemicals heat usedofduring the lithographic process. during process. The covalentand anchoring a polymer transducer onto the The microelectrode covalent anchoring of a polymer transducer onto the microelectrode substrate was possible not for substrate was possible not just for the one-dimensional CPPy nanotubes but also just the for CPPy the one-dimensional CPPy nanotubes but also anchoring for the CPPy nanoparticles [36]. Therefore, nanoparticles [36]. Therefore, the covalent approach is an excellent alternative to the covalent anchoring approach is an excellent alternative to construct polymer nanostructures-based construct polymer nanostructures-based microelectrode sensing platforms. Figure 6b shows the microelectrode sensing platforms. Figure 6b substrate, shows theand CPPy-nanotube-anchored microelectrode FET CPPy-nanotube-anchored microelectrode Figure 6c represents a liquid-ion-gated substrate, Figure based 6c represents liquid-ion-gated FET sensing platform based on theThe CPPy sensingand platform on the aCPPy nanotubes-anchored microelectrode substrate. CPPy nanotubes-anchored microelectrode substrate. The CPPy nanotubes anchored on the electrode nanotubes anchored on the electrode substrate provided stable transistor behavior in the liquid phase, substrate transistor behaviorresponses in the liquid phase, finallyspecies, resulting in sensitive and finallyprovided resulting stable in sensitive and selective toward the target thrombin (Figure 6d,e). selective responses toward the target species, thrombin (Figure 6d,e). Similar approaches had been used Similar approaches had been used for other FET sensors, such as human olfactory receptor-based for other FET sensors, human olfactory receptor-based bioelectronic noses [37], enzyme-based bioelectronic nosessuch [37],asenzyme-based glucose sensors [38], and heparin-based protein sensors [39]. glucose sensors [38], and heparin-based protein sensors [39]. The copolymerization strategy provides The copolymerization strategy provides effective control of the chemical functionality of polymer effective control of both the chemical functionality of polymer nanostructures, both qualitatively nanostructures, qualitatively and quantitatively, and, thus, may be widely applicable toand various quantitatively, and, thus, may be widely applicable to various types of materials and devices. types of materials and devices.

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Figure 6. (a) Schematic illustration of reaction steps for the fabrication of sensor platforms based on Figure 6. (a) Schematic illustration of reaction steps for the fabrication of sensor platforms based on CPPy nanotubes; (b) Scanning Electron Microscope (SEM) image of CPPy nanotubes that are CPPy nanotubes; (b) Scanning Electron Microscope (SEM) image of CPPy nanotubes that are deposited deposited on the interdigitated microelectrode substrate; (c) Schematic representation of a CPPy on the interdigitated microelectrode substrate; (c) Schematic representation of a CPPy nanotube sensor nanotube sensor platform with a field-effect transistor (FET) configuration: the source (S), drain (D), platform with a field-effect transistor (FET) configuration: the source (S), drain (D), and liquid-ion and liquid-ion gate (G) are labelled; (d) Typical real-time responses of CPPy nanotube FET sensors at gate (G) are labelled; (d) Typical real-time responses of CPPy nanotube FET sensors at a constant V SD a constantT;Vbovine SD (thrombin, T; bovine serum albumin, BSA, B), where a control experiment was (thrombin, serum albumin, BSA, B), where a control experiment was performed by using performed by CPNT-1 with no thrombin aptamers attached; Calibration curves of CPPy CPNT-1 with nousing thrombin aptamers attached; (e) Calibration curves of(e)CPPy nanotube FET sensors. nanotube FET sensors. Reproduced with permission from [35]. Copyright 2008, John Wiley & Sons. Reproduced with permission from [35]. Copyright 2008, John Wiley & Sons.

2.4. Post-Polymerization Modification 2.4. Post-Polymerization Modification A variety of functional polymers have been prepared by implementing small-molecule chemical A variety of functional polymers have been prepared by implementing small-molecule chemical reactions to homopolymers [40–42]. Diverse chemical strategies have been utilized for the postreactions to homopolymers [40–42]. Diverse chemical strategies have been utilized for the polymerization modification, where versatile functionalities have been introduced into polymer post-polymerization modification, where versatile functionalities have been introduced into polymer chains by conjugating the polymer with appropriate chemical and biological species. Fundamentally, chains by conjugating the polymer with appropriate chemical and biological species. Fundamentally, the reaction sites of f-monomers for propagation are screened by bulky functional groups and, thus, the reaction sites of f-monomers for propagation are screened by bulky functional groups and, the controlled polymerization of the monomers suffers from steric hindrance. Such problems can be thus, the controlled polymerization of the monomers suffers from steric hindrance. Such problems removed by using the post-polymerization modification strategy. Chemical strategies for the postcan be removed by using the post-polymerization modification strategy. Chemical strategies for polymerization functionalization have been summarized in several literatures [43,44]. In particular, the post-polymerization functionalization have been summarized in several literatures [43,44]. Klok and co-workers well described in detail the chemical strategies available for post-polymerization In particular, Klok and co-workers well described in detail the chemical strategies available for mechanism [42,44]. The chemical reactions can be categorized into several classes, as listed in Table post-polymerization mechanism [42,44]. The chemical reactions can be categorized into several 1, such as thiol-ene (radical thiol) addition, thiol-disulfide exchange, epoxides/anhydrides/isocyanates, classes, as listed in Table 1, such as thiol-ene (radical thiol) addition, thiol-disulfide exchange, ketones/aldehydes, active esters, Diels–Alder cycloaddition, and Michael addition. It is important to epoxides/anhydrides/isocyanates, ketones/aldehydes, active esters, Diels–Alder cycloaddition, note that the chemical reactions requiring harmful catalysts, high temperatures, and other toxic and Michael addition. It is important to note that the chemical reactions requiring harmful catalysts, reagents are not suitable for preparing biosensing materials. high temperatures, and other toxic reagents are not suitable for preparing biosensing materials. Table 1. Representative post-polymerization reactions that can be used for the functionalization of polymers [42,44].

Classification Thiol-ene addition: The antiMarkovnikov addition of thiols to alkenes is facilitated by a radical source or by UV irradiation.

Reaction scheme

Refs.

[45,46]

removed by using the post-polymerization modification strategy. Chemical strategies for the postpolymerization functionalization have been summarized in several literatures [43,44]. In particular, Klok and co-workers well described in detail the chemical strategies available for post-polymerization mechanism [42,44]. The chemical reactions can be categorized into several classes, as listed in Table 1, such as thiol-ene (radical thiol) addition, thiol-disulfide exchange, epoxides/anhydrides/isocyanates, ketones/aldehydes, active esters, Diels–Alder cycloaddition, and Michael addition. It is important to 9 of 34 Polymers 2018, 10, 551 note that the chemical reactions requiring harmful catalysts, high temperatures, and other toxic reagents are not suitable for preparing biosensing materials. Table 1. Representative post-polymerization reactions that can be used for the functionalization of Table[42,44]. 1. Representative post-polymerization reactions that can be used for the functionalization of polymers polymers [42,44]. Classification Classification

Reaction scheme scheme Reaction

Refs. Refs.

Thiol-ene addition: The antiThiol-ene addition: Theaddition anti-Markovnikov Markovnikov of thiols addition to of thiols to alkenes is facilitated by a radical source or alkenes is10, facilitated by a radical source Polymers 2018, x FOR PEER REVIEW UV irradiation. Polymers 2018, 10,by x FOR PEER REVIEW Polymers 2018, 10, x FOR PEER REVIEW Polymers 2018, 10, 10, xx FOR FOR PEER PEER REVIEW REVIEW Polymers UVPEER irradiation. Polymers 2018, 2018, or 10, by x FOR REVIEW

[45,46] [45,46] 99 of 34 of 34 9 of of 3434 999 of of 34 34

Thiol-disulfide exchange: This type of Thiol-disulfide exchange: This type Thiol-disulfide exchange: This type ofof Thiol-disulfide exchange: This type of

Thiol-disulfide exchange: This type of is Thiol-disulfide exchange: This type reaction reaction is frequently found in biological reaction frequently found in biological reaction is is frequently found inof biological reaction is frequently found in biological reaction is frequently found in biological frequently found in biological systems. Disulfides as systems. Disulfides as pyridyl disulfide systems. Disulfides pyridyl disulfide systems. Disulfides asas pyridyl disulfide systems. Disulfides as pyridyl disulfide pyridyl disulfide are readily exchanged in high systems. Disulfides as pyridyl disulfide are readily exchanged in high yields with are readily exchanged high yields with are readily exchanged inin high yields with yields with thiol compounds. are exchanged in are readily readily exchanged in high high yields yields with with

[47,48] [47,48] [47,48] [47,48] [47,48] [47,48]

thiol compounds. thiol compounds. thiol compounds. thiol thiol compounds. compounds.

Epoxides, anhydrides, isocyanates: These Epoxides, anhydrides, isocyanates: These

Epoxides, anhydrides, isocyanates: These Epoxides, anhydrides, isocyanates: These Epoxides, anhydrides, isocyanates: These are a class Epoxides, anhydrides, isocyanates: These are aa class of reactive groups, that are, are a class of reactive groups, that are, are class of reactive groups, that are, of reactive groups, that are, importantly, tolerant are aa class of reactive groups, that are, are class of reactive groups, that are, importantly, tolerant toward radicaltoward radical-based polymerization methods. importantly, tolerant toward radicalimportantly, tolerant toward radicalimportantly, tolerant toward radicalimportantly, tolerant toward radicalbased polymerization methods. based polymerization methods. based polymerization methods. based based polymerization polymerization methods. methods.

[49–51] [49–51] [49–51] [49–51] [49–51] [49–51]

Ketones and aldehydes: These can Ketones and aldehydes: These can Ketones and aldehydes: These can Ketones and aldehydes: These can

Ketones and aldehydes: can react Ketones and aldehydes: These canThese selectively selectively react with primary amines, selectively react with primary amines, selectively react with primary amines, selectively react with primary amines, selectively react with primary amines, with primary amines, alkoxyamines, and hydrazines, alkoxyamines, and hydrazines, alkoxyamines, and hydrazines, alkoxyamines, and hydrazines, alkoxyamines, and hydrazines, producing imines, and alkoxyamines, andoximes, hydrazines, producing imines, oximes, and producing imines, oximes, and producing imines, oximes, and hydrozones, respectively. producing imines, oximes, and

[52,53] [52,53] [52,53] [52,53] [52,53] [52,53]

producing imines, oximes, and hydrozones, respectively. hydrozones, respectively. hydrozones, respectively. hydrozones, hydrozones, respectively. respectively.

Active esters: The reaction of active ester Active esters: The reaction active ester Active esters: The reaction ofof active ester Active esters: The reaction of active ester

Active esters: The reaction ofproceeds active ester Active esters: The reaction ofcan active ester groups groups with amines groups with amines can proceeds groups with amines can proceeds groups with amines can proceeds groups with amines can proceeds with amines can proceeds selectively evenofin the selectively even in the presence selectively even the presence selectively even inin the presence ofof selectively even in presence of presence of weaker nucleophiles, such as alcohols. selectively even in the the presence of weaker nucleophiles, such as alcohols.

[54–56] [54–56] [54–56] [54–56] [54–56] [54–56]

Diels–Alder cycloaddition: A diene and aa a Diels–Alder cycloaddition: diene and Diels–Alder cycloaddition: AA diene and Diels–Alder cycloaddition: A diene Diels–Alder cycloaddition: A make diene and and aa substituted alkene can Diels–Aldersubstituted cycloaddition: A diene and a substituted substituted alkene can make substituted alkene can make alkene can make substituted alkene canwhich make alkene can make cycloaddition reaction, cycloaddition reaction, is cycloaddition reaction, which cycloaddition reaction, which is is cycloaddition reaction, which is cycloaddition which isreaction, reversible.which is reversible. reversible. reversible. reversible. reversible.

[57,58] [57,58] [57,58] [57,58] [57,58] [57,58]

Michael addition: Thiols undergo Michael addition: Thiols undergo Michael addition: Thiols undergo Michael Thiols undergo Michael addition: addition: Thiols undergo Michael-type addition to activated Michael-type addition to activated Michael-type addition to activated Michael addition: Thiols undergo Michael-type Michael-type addition to activated Michael-type addition torapidly activated alkenes, which proceeds in additionalkenes, to activated alkenes, whichrapidly proceeds alkenes, which proceeds rapidly in alkenes, which proceeds rapidly inrapidly which proceeds in alkenes, which proceeds rapidly in in aqueous media under mild conditions. aqueous media under mild conditions. aqueous media under mild conditions. aqueous media under mild conditions. aqueous aqueous media media under under mild mild conditions. conditions.

[59–61] [59–61] [59–61] [59–61] [59–61] [59–61]

weaker nucleophiles, such as alcohols. weaker nucleophiles, such alcohols. weaker nucleophiles, such asas alcohols. weaker weaker nucleophiles, nucleophiles, such such as as alcohols. alcohols.

Post-polymerization strategies areareapplicable forforthethefunctionalization ofofvarious Post-polymerizationstrategies strategiesare applicablefor functionalizationof variouspolymer polymer Post-polymerization strategies are applicable for the functionalization functionalization of various various polymer Post-polymerization Post-polymerization strategies are applicable applicable for the the functionalization of Representatively, various polymer polymer nanostructures, as well as the modification of surfaces/interfaces of interest. nanostructures,as wellas themodification modificationof surfaces/interfacesof interest.Representatively, Representatively, nanostructures, asaswell well asasthe the modification ofofsurfaces/interfaces surfaces/interfaces ofofinterest. interest. Representatively, nanostructures, nanostructures, ascan well as the modification of surfaces/interfaces of interest. Representatively, Post-polymerization strategies are applicable for the functionalization ofproperties various for polymer polymer brushes be introduced to the surface of a material to tailor the surface polymer brushes can introduced the surface amaterial material tailor the surface properties polymer brushes can bebe introduced toto the surface ofof toto tailor the surface properties forfor polymer brushes can be introduced to the surface of aaa material to tailor the surface properties for polymer brushes can be introduced to the surface of material to tailor the surface properties for nanostructures, as well as the applications modification ofA surfaces/interfaces of interest. Representatively, biomedical and bioanalytical [62]. convenient way to control the functionality of aa a biomedical and bioanalytical applications [62]. convenient way control the functionality biomedical and bioanalytical applications [62]. AA convenient way toto control the functionality ofof biomedical and bioanalytical applications [62]. A way control the of biomedical and bioanalytical applications [62]. A convenient convenient way to to control the functionality functionality of aa polymer encompassing structural flexibility, chemical/biological affinity, and biocompatibility polymer brushes can be introduced to the surface of a material to tailor the surface properties polymerencompassing encompassingstructural structuralflexibility, flexibility,chemical/biological chemical/biologicalaffinity, affinity,and andbiocompatibility biocompatibility for polymer encompassing structural flexibility, chemical/biological affinity, and biocompatibility polymer polymer encompassing structural flexibility, chemical/biological affinity, and biocompatibility involves the modification of the polymer surface with other functional polymers via surface-initiated biomedical and bioanalytical applications [62]. A convenient way to control the functionality of involves the modification the polymer surface with other functional polymers via surface-initiated involves the modification ofof the polymer surface with other functional polymers via surface-initiated involves the of the surface with other via involves the modification modification ofefficient the polymer polymer surface with other functional functional polymers polymers via surface-initiated surface-initiated polymerization. The most strategies forfor thethe surface-initiated polymerization involve atom polymerization. The most efficient strategies surface-initiated polymerization involve atom polymerization. The most efficient strategies strategies for the the surface-initiated polymerization polymerization involve atom a polymer encompassing structural flexibility, affinity, andinvolve biocompatibility polymerization. The efficient for atom polymerization. The most most efficient strategies for chemical/biological the surface-initiated surface-initiated polymerization involve atom transfer radical polymerization (ATRP), nitroxide-mediated polymerization (NMP), photoinifertertransfer radical polymerization (ATRP), nitroxide-mediated polymerization (NMP), photoinifertertransfer radical polymerization (ATRP), nitroxide-mediated polymerization (NMP), photoiniferterphotoiniferterinvolves theradical modification of (ATRP), the polymer surface polymerization with other (NMP), functional polymers via transfer polymerization nitroxide-mediated transfer radical polymerization (ATRP), nitroxide-mediated polymerization (NMP),chain photoinifertermediated polymerization (PIMP), and reversible addition-fragmentation transfer mediated polymerization polymerization (PIMP), (PIMP), and reversible addition-fragmentation chain transfer mediated polymerization (PIMP), and reversible addition-fragmentation chain transfer mediated and reversible addition-fragmentation chain surface-initiated polymerization. The most efficient strategies for the surface-initiated polymerization mediated polymerization (PIMP), and reversible addition-fragmentation chain transfer transfer polymerization (RAFT) [63]. Figure 7 represents typical examples of each of the surface-initiated polymerization (RAFT) [63]. Figure 7 represents typical examples of each of the surface-initiated polymerization (RAFT) (RAFT) [63]. [63]. Figure Figure 77 represents represents typical typical examples examples of of each of of the surface-initiated surface-initiated polymerization polymerization (RAFT) [63].polymerization Figure 7 represents typical examples of each each of the the surface-initiated involve atom transfer radical (ATRP), nitroxide-mediated polymerization (NMP), controlled radical polymerization techniques, where thethe initiators were attached to thethe substrates controlled radical polymerization techniques, where initiators were attached substrates controlled radical polymerization techniques, where the initiators initiators were attached toto the substrates controlled radical polymerization techniques, where the were attached to controlledsilane-based radical polymerization techniques, where the initiators were attached to the the substrates substrates through bonds [62,64]. First, ATRP has been widely used as a prominent surfacethroughsilane-based silane-basedbonds bonds[62,64]. [62,64].First, First,ATRP ATRPhas hasbeen beenwidely widelyused usedas prominentsurfacesurfacethrough silane-based [62,64]. First, ATRP has widely used asasa aprominent prominent surfacethrough through polymerization silane-based bonds bonds [62,64]. First, ATRP hasinbeen been widely used and, as aathus, prominent surfaceinitiated method. The ATRP proceeds a mild condition is is applicable toto initiated polymerization method. The ATRP proceeds in a mild condition and, thus, applicable initiated polymerization method. The ATRP proceeds in a mild condition and, thus, is applicable to initiated polymerization method. The ATRP in condition and, thus, is to initiatedbiological polymerization method.The Thesurface-initiated ATRP proceeds proceeds ATRP in aa mild mild condition and, thus, is applicable applicable to various environments. has generated various polymer brushes, various biological environments. The surface-initiated ATRP has generated various polymer brushes, various biological environments. The surface-initiated ATRP has generated various polymer brushes, various biological environments. The ATRP has generated polymer brushes, variousare biological environments. The surface-initiated surface-initiated ATRP has generated various various polymer brushes, which ofofimportance inina arange ofofbioapplications [65]. Nonspecific adsorption ofofbiological which are importance range bioapplications [65]. Nonspecific adsorption biological which are of importance importance in aa range range of bioapplications bioapplications [65]. Nonspecific adsorption of biological biological which are of in of [65]. Nonspecific adsorption of which are of importance in a range of bioapplications [65]. Nonspecific adsorption of biological

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photoiniferter-mediated polymerization (PIMP), and reversible addition-fragmentation chain transfer polymerization (RAFT) [63]. Figure 7 represents typical examples of each of the surface-initiated controlled radical polymerization techniques, where the initiators were attached to the substrates through silane-based bonds [62,64]. First, ATRP has been widely used as a prominent surface-initiated polymerization method. The ATRP proceeds in a mild condition and, thus, is applicable to various biological environments. The surface-initiated ATRP has generated various polymer brushes, which are of importance in a range of bioapplications [65]. Nonspecific adsorption of biological species, such as proteins, cells, and bacteria, is mostly triggered by the chemical structure, topography, and the flexibility of the material near the surface. Importantly, nonspecific adsorption causes blood coagulation, complement activation, inflammation, biodegradation, and infection, subsequently resulting in device failure. It has been found that polymer brushes can suppress such nonspecific protein adsorption and cell adhesion [66]. In particular, poly(ethylene glycol) (PEG) and its derivatives, such as poly(oligo(ethylene glycol) methacrylate) [67–70], have been widely used to prevent undesirable bio-fouling in many applications. Other kinds of polymer grafts have been created from a range of neutral and zwitterionic polymers [71,72], including poly(2-hydroxyethyl methacrylate) [73], poly(2-methacryloyloxyethyl phosphorylcholine) [74–77], poly(sulfobetaine methacrylate) [78–81], and poly(carboxybetaine methacrylate) [82,83]. Recently, zwitterionic groups such as phosphorylcholine, sulfobetaine, and carboxybetaine have been considered to be good alternatives to conventional PEG groups owing to their enhanced antifouling effects, biocompatibility, and stability. Surface-initiated ATRP generally includes two steps. Firstly, the low-molecular-weight initiators are covalently grafted onto the surface of interest. Thiol-metal and silane-based bonds can be easily formed onto metal or metal oxide surfaces to attach the initiator. However, it is significantly more challenging to introduce the initiator on a polymeric substrate than an inorganic substrate. There are several strategies for the introduction of the initiator onto polymeric substrates. For examples, Zhao et al. covalently attached an ATRP initiator, 2-bromoisobutyryl bromide, to the hydroxyl group of hydroxymethyl 3,4-ethylenedioxythiophene (EDOT–OH) monomer via an esterification reaction [84]. Polymer brushes consisting of poly((oligo(ethylene glycol) methacrylate) and zwitterionic poly([2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide) were successfully grafted from PEDOT by ATRP. Despite the advantages of ATRP, its wide application is hindered by certain limitations. First, ATRP proceeds in the presence of a transition metal catalyst; these catalysts are generally toxic. Therefore, the catalyst must be removed prior to use in bioapplications. Secondly, acidic monomers that can react or complex with the transition metal catalyst are not amenable for ATRP. Similar to ATRP, NMP takes advantage of the reversible activation–deactivation of propagating polymer chains by a nitroxide radical. RAFT is based on reversible chain transfer with a combination of a conventional free radical initiator and a chain transfer agent. PIMP utilizes a somewhat unique class of unconventional initiators, so called iniferters that can act as initiators, chain transfer agents, and terminators for controlled polymerization. The rate of polymerization in PIMP depends on the intensity of the ultraviolet light (UV) source. The surface-initiated polymerization approach by photoirradiation allows spatial and temporal control on light exposure, providing various ways for more complicated, multidimensional, and larger-area surface/interface engineering without being limited to specific types of monomers. In addition, the use of a UV light source removes the need for transition metal catalysts that may be detrimental to the biocompatibility of the resulting polymers. These controlled/living radical polymerization methods have the advantage of simple experimental setup, mild reaction conditions, tolerance toward diverse functional groups, and compatibility with both aqueous and organic media, thus serving as powerful tools for surface and interface engineering. However, it should be noted that the elimination or minimization of biofouling alone is not sufficient for biosensing applications. In addition, appropriate receptors should be immobilized on the transducer polymer in a controlled fashion for the specific recognition of target species.

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Figure of of thethe surface-initiated controlled radical polymerization techniques: atom Figure 7. 7.Typical Typicalexamples examples surface-initiated controlled radical polymerization techniques: transfer radical polymerization (ATRP) of styrene, nitroxide-mediated polymerization (NMP) of atom transfer radical polymerization (ATRP) of styrene, nitroxide-mediated polymerization (NMP) styrene, reversible addition-fragmentation chain transfer polymerization (RAFT) of vinylbenzene of styrene, reversible addition-fragmentation chain transfer polymerization (RAFT) of vinylbenzene chloride, and photoiniferter-mediated polymerization (PIMP) (PIMP) of of acrylic acrylic acid acid [62,64]. [62,64].

3. 3. Macrostructure: Macrostructure: Hydrogels Hydrogels Hydrogels past decades decades as as promising promising materials materials in in industry industry and and Hydrogels have have gained gained interest interest over over the the past daily life. They have been used as forms of hydrophilic matrices for soil conditioning, diapers, and daily life. They have been used as forms of hydrophilic matrices for soil conditioning, diapers, contact lenses and and havehave broadened the applications of nanoscience or nanoengineering withwith the aid and contact lenses broadened the applications of nanoscience or nanoengineering the of advanced analytical and processing technologies. Recently, these materials have been extensively aid of advanced analytical and processing technologies. Recently, these materials have been extensively investigated forbiosensor biosensor applications in wearable or healthcare devices. Here, we investigated for applications suchsuch as in as wearable or healthcare devices. Here, we summarize summarize the designfor principles for macrostructured hydrogel biosensors. This chapter is divided the design principles macrostructured hydrogel biosensors. This chapter is divided into three into three subcategories of covalent chemistry, non-covalent chemistry, and miscellaneous subcategories of covalent chemistry, non-covalent chemistry, and miscellaneous approaches. approaches. 3.1. Covalent Chemistry for Hydrogels 3.1. Covalent Chemistry for Hydrogels 3.1.1. Radical Polymerization 3.1.1.Radical Radicalpolymerization Polymerization is the most potent method to fabricate hydrogels among the covalent approaches. monomers suchpotent as acrylamide (AAm), acrylic acid (AA), methacrylic acid Radical Hydrophilic polymerization is the most method to fabricate hydrogels among the covalent (MAA), vinylHydrophilic acetate (VA; the monomer for poly(vinyl alcohol), PVA), N,N-dimethylacrylamide (DMA), approaches. monomers such as acrylamide (AAm), acrylic acid (AA), methacrylic acid N-isopropylacrylamide (NIPAM), and 2-hydroxyethyl methacrylate (HEMA) have been widely used (MAA), vinyl acetate (VA; the monomer for poly(vinyl alcohol), PVA), N,N-dimethylacrylamide and polymerized by radical initiation (Figure Other monomers having water-soluble groups (DMA), N-isopropylacrylamide (NIPAM), and8a). 2-hydroxyethyl methacrylate (HEMA) have been (e.g., carboxylate, sulfonate, quaternary ammonium, and 8a). phosphate groups, orhaving zwitterions) are also widely used and polymerized by radical initiation (Figure Other monomers water-soluble used in (e.g., hydrogel-based (Figure 8b). Interestingly, bio-extractable monomers groups carboxylate,materials sulfonate, quaternary ammonium,hydrophilic, and phosphate groups, or zwitterions) (e.g., Tulipalin β-pinene) canmaterials also be polymerized make hydrogel polymers (Figure 8c) [85]. are also used A in and hydrogel-based (Figure 8b).toInterestingly, hydrophilic, bio-extractable For cross-linking reactions, bior trifunctional cross-linkers are incorporated during polymerization to monomers (e.g., Tulipalin A and β-pinene) can also be polymerized to make hydrogel polymers establish8c) entangled structures (Figure (Figure [85]. For network cross-linking reactions, bi- 8d). or trifunctional cross-linkers are incorporated during polymerization to establish entangled network structures (Figure 8d).

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(a) commercial vinyl monomers O

O

O

O

N H NIPAM

Polymers 2018, 10, x 2FOR PEER NH OHREVIEW OH AAm AA MAA (a) commercial vinyl monomers (b) water-soluble O Ogroups

(b) water-soluble groups (c) bio-extractables O O NaO O SO 3 O NO O H sulf onate T ulipalin A (c) bio-extractables O (d)Ocross-linkers

OH

NaO

O T ulipalin Aspacer

O

NaON

OH

O OPO 32-

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HEMA O N

O

O N H

VA phosphonate DMA

OH N

SO3

HEMAzwiterion

O O

OPO 32phosphonate

N H

O

N

SO3 zwiterion

n

O pinene

OH NaO O

O

bifunctional cross-linker (d) cross-linkers

O O

OH

O

DMA

O

O

quaternary ammonium n

O R O

VA

O

O N

O

N OH N H O MAA quaternary NIPAM ammonium

NH 2 OH SO3 N AAmH sulfAA onate

O

O

O O

O

O

O

O OH O R

O O n

trifunctional pinene cross-linker R = Et, Me

O O

n

O

O

Figure 8.O(a–d) Chemical structures of: (a) select vinyl monomers; (b) vinyl monomers that contain O Figure typical 8. (a–d) Chemical structures Oof:O (a) vinyl monomers; (b) vinyl monomers that trifunctional O select spacer water-soluble groups; (c) bioextractable molecules with polymerizable groups and cross-linker O contain polymerization typical water-soluble groups; (c) bioextractable molecules with polymerizable groups and R = Et, Me cross-linkers. bifunctional processes; cross-linker and (d) bi- and trifunctional

polymerization processes; and (d) bi- and trifunctional cross-linkers. The monomeric vinyl species mentioned above aremonomers; polymerized by water-soluble initiators Figure 8. (a–d) Chemical structures of: (a) select vinyl (b) vinyl monomers that contain such as persulfates (e.g., ammonium persulfate, APS; potassium persulfate, KPS) or acetophenone typical water-soluble groups; (c) bioextractable molecules with polymerizable groups and The monomeric vinyl species mentioned above are polymerized by water-soluble initiators ® series), which initiate polymerization after exposure to heat or light. derivatives (e.g., IRGACURE polymerization processes; and (d) bi- and trifunctional cross-linkers. such as Redox persulfates (e.g., ammonium persulfate, APS; potassium persulfate, KPS) or acetophenone reactions also form radicals from the persulfates, initiating polymerization in the presence of ® The monomeric vinyl species mentioned above arepolymerization polymerized by water-soluble initiators suchor light. derivatives (e.g., IRGACURE series), whichofinitiate after exposure to heat a redox promoter [86–89]. A combination KPS–TEMED (N,N,N,N-tetramethylethylenediamine) as persulfate, APS; also potassium persulfate, KPS)radicals or acetophenone haspersulfates been widely used,ammonium and other from combinations have been shown to generate (Figure 9a). Redox reactions also (e.g., form radicals the persulfates, initiating polymerization in the presence of ® series), which initiate polymerization after exposure to heat or light. derivatives (e.g., IRGACURE particular,[86–89]. redox-initiated polymerization quite useful for(N,N,N,N-tetramethylethylenediamine) labeling hydrogel materials. Lee et al. a redoxIn promoter A combination of is KPS–TEMED Redox reactions also form radicals from the persulfates, polymerization in the presence of demonstrated a stimuli-responsive hydrogel film labeledinitiating with a rhodamine-based probe [90]. In this has been widely used, and other combinations have also been shown to generate radicals (Figure 9a). asystem, redox the promoter [86–89]. aAsecondary combination of KPS–TEMED probe contains amine group, which (N,N,N,N-tetramethylethylenediamine) plays a dual role in the film: as a redox In particular, redox-initiated polymerization quite labeling materials. Lee et al. has been widely and other combinations have beenfor shown to generate radicals (Figure The 9a). promoter for theused, polymerization of AAm is and as also auseful detection probe for hydrogel fluorescent sensing. demonstrated a stimuli-responsive hydrogel film labeled with and ahydrogel rhodamine-based probe [90]. In particular, redox-initiated polymerization is quite useful for labeling materials. Lee et al. transparent hydrogel film was fabricated easily via one-step synthesis used for the selective and demonstrated a stimuli-responsive hydrogel labeled withofa essential rhodamine-based Inin this In this system, probe secondary amine group, which metal playsions aprobe dual role the film: reversiblethe detection ofcontains Al3+, an ionathat inhibitsfilm the indigestion and is[90]. suspected system, the probe contains aand secondary amine group,and which a dual role in the as a redoxsensing. to be a cause offor Parkinson’s Alzheimer’s (Figure as a redox promoter the polymerization of disease AAm as plays a9b). detection probe forfilm: fluorescent promoter for the polymerization of AAm and as a detection probe for fluorescent sensing. The The transparent hydrogel film was fabricated easily via one-step synthesis and used for the selective transparent hydrogel film was fabricated easily via one-step synthesis and used for the selective and and reversible detection of Al3+ , an ion that inhibits the indigestion of essential metal ions and is reversible detection of Al3+, an ion that inhibits the indigestion of essential metal ions and is suspected suspected toabe a cause of Parkinson’s and Alzheimer’s disease to be cause of Parkinson’s and Alzheimer’s disease (Figure 9b). (Figure 9b).

Figure 9. (a) Schematic description of the potassium persulfate-N,N,N,N-tetramethylethylenediamine (KPS–TEMED) redox system for radical initiation; (b) Stimuli-responsive hydrogel film having a

Figure 9. (a) Schematic description of the potassium persulfate-N,N,N,N-tetramethylethylenediamine

Figure 9. (a) Schematic description of the potassium persulfate-N,N,N,N-tetramethylethylenediamine (KPS–TEMED) redox system for radical initiation; (b) Stimuli-responsive hydrogel film having a (KPS–TEMED) redox system for radical initiation; (b) Stimuli-responsive hydrogel film having a rhodamine-based probe that induces the redox reaction for radical initiation and selectively and reversibly detects Al3+ .

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rhodamine-based probe that induces the redox reaction for radical initiation and selectively and reversibly detects Al3+.

The copolymerization of vinyl monomers significantly alters the overall physical properties and broadens the applications of the final product [91–93]. AA or MAA have been extensively The copolymerization of vinyl monomers significantly alters the overall physical properties and employed with monomers because the[91–93]. carboxylic AAbeen or MAA can employed be used as a broadens theother applications of the final product AA oracid MAAinhave extensively pH-sensitive bidentate ligand. Yetisen et al. fabricated a photonic-crystal pH sensor from a with other monomers because the carboxylic acid in AA or MAA can be used as a pH-sensitive hydrogel matrix, which was prepared by copolymerizing HEMA and MAA in the presence bidentate ligand. Yetisen et al. fabricated a photonic-crystal pH sensor from a hydrogel matrix, which of was dimethylacrylate prepared by copolymerizing HEMA and MAA in the presence of ethylene dimethylacrylate ethylene (EGDMA, bifunctional cross-linker). After UV polymerization, the hydrogel (EGDMA, bifunctional cross-linker). After UV polymerization, the hydrogel was doped resulting with matrix was doped with silver ions, which were further reduced to silvermatrix nanoparticles, silver ions, which were further reduced to silver nanoparticles, resulting in one-dimensional photonic in one-dimensional photonic crystal flakes. The free-standing flakes showed a color change while crystal flakes. The free-standing flakes showed a color change while shifting the diffraction shifting the diffraction wavelength in response to pH change (Figure 10a) [81]. Similarly, Jia et al. wavelength in response to pH change (Figure 10a) [81]. Similarly, Jia et al. prepared a photonic-crystal prepared a photonic-crystal hydrogel by incorporating iron oxide nanoparticles into a hydrogel matrix hydrogel by incorporating iron oxide nanoparticles into a hydrogel matrix of AAm, AA, and N,Nof AAm, AA, and N,N-methylenebisacrylamide The colloidal hydrogel sensor a methylenebisacrylamide (MBAm). The colloidal(MBAm). hydrogel sensor displayed a complete color displayed change complete color change in response to pH or ions [82]. Furthermore, exploiting the coordination ability in response to pH or ions [82]. Furthermore, exploiting the coordination ability of AA, Lu et al. of AA, Lu et al. developed elastic nanocomposite hydrogels without covalent cross-linking. During developed elastic nanocomposite hydrogels without covalent cross-linking. During radical radical copolymerization of DMA andaluminum AA, aluminum oxide nanoparticles induced non-covalent copolymerization of DMA and AA, oxide nanoparticles induced non-covalent crosslinking via which gave riserise to to a three-dimensional colloidal arrayarray of of cross-linking viasecondary secondaryinteractions, interactions, which gave a three-dimensional colloidal nanocomposites that can be used to monitor the strain of materials via color change and can be nanocomposites that can be used to monitor the strain of materials via color change and can be applied for intraocular pressure tonometry blood analysis(Figure (Figure10b) 10b)[83]. [83]. The The acid acid group group was applied for intraocular pressure tonometry or or blood gasgas analysis was also used for the selective detection of paracetamol, an analgesic drug, in an electrochemical also used for the selective detection of paracetamol, an analgesic drug, in an electrochemical sensor. sensor. Copolymerization of AA, AAm, and MBAm afforded a hydrogel matrix containing both Copolymerization of AA, AAm, and MBAm afforded a hydrogel matrix containing both acidic and acidic and basic groups, which prevented the denaturation of enzyme (polyphenol oxidase, PPO) basic incorporated groups, which enzyme (polyphenol oxidase, PPO)voltammetry incorporated in in prevented the matrix. the Thedenaturation oxidation of of paracetamol was monitored by cyclic the matrix. of paracetamol was monitored by cyclic voltammetry (Figure 10c) [94]. (FigureThe 10c)oxidation [94]. (a)

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Figure 10. (a) Chemical structure of hydrogel sensor comprised of methacrylic acid (MAA), 2-

Figure 10. (a) Chemical structure of hydrogel sensor comprised of methacrylic acid (MAA), hydroxyethyl methacrylate (HEMA), and ethylene dimethylacrylate (EGDMA) where silver ions are 2-hydroxyethyl methacrylate (HEMA), and ethylene dimethylacrylate (EGDMA) where silver reduced to form nanoparticles; (b,c) Schematic description of (b) non-covalent cross-linking of ions are reduced to form nanoparticles; (b,c) Schematic description of (b) non-covalent cross-linking of poly(N,N-dimethylacrylamide-co-acrylic acid) by aluminum oxide nanoparticles, and (c) enzyme-incorporated hydrogel matrix that selectively oxidizes paracetamol and senses it electrochemically.

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poly(N,N-dimethylacrylamide-co-acrylic acid) by aluminum oxide nanoparticles, and (c) enzymePolymers 2018, 10, 551 14 of 34 incorporated hydrogel matrix that selectively oxidizes paracetamol and senses it electrochemically.

The acid acid group group has has been been also also used bio-conjugation by by forming forming ester ester or or The used for for post-polymerization post-polymerization bio-conjugation amide linkages. Zhang et al. prepared microgels containing AA and introduced H O -sensitive amide linkages. Zhang et al. prepared microgels containing AA and introduced H22O22-sensitive ferrocenyl groups groups via via Steglich Steglich esterification esterificationusing usinga awater-soluble water-solublecarbodiimide carbodiimide (EDC) ferrocenyl (EDC) andand a 4-a 4-dimethylaminopyridine (DMAP) catalyst. Furthermore, they deposited microgels between dimethylaminopyridine (DMAP) catalyst. Furthermore, they deposited microgels between two two transparent plates plates to to build build optical optical devices, devices, etalons etalons that that exhibited exhibited optical optical change change in in response response to to transparent peroxides (Figure (Figure 11a) 11a)[95]. [95].Kowalczyk Kowalczyk et attached al. attached single-stranded (ssDNA) peroxides et al. single-stranded DNADNA (ssDNA) onto aonto cross-a cross-linked hydrogel matrix containing AA repeating units. They pre-modified acid groups in linked hydrogel matrix containing AA repeating units. They pre-modified acid groups in the matrix the matrix with N-hydroxysuccinimide (NHS)antoactive yield an active that sequentially reacted with with N-hydroxysuccinimide (NHS) to yield ester thatester sequentially reacted with amineamine-functionalized ssDNA in sequence under mild conditions (Figure 11b) [96]. functionalized ssDNA in sequence under mild conditions (Figure 11b) [96]. (a)

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Figure 11. (a,b) covalent Figure 11. (a,b) Schemes Schemes for for post-polymerization post-polymerization modification modification of of hydrogel hydrogel matrices matrices via via covalent bonds: bonds: (a) (a) Steglich Steglich esterification esterification using using carbodiimide carbodiimide (EDC) (EDC) and and 4-dimethylaminopyridine 4-dimethylaminopyridine (DMAP) (DMAP) to to introduce ferrocenyl groups or (b) active ester formation using N-hydroxysuccinimide (NHS) to introduce ferrocenyl groups or (b) active ester formation using N-hydroxysuccinimide (NHS) to tether tether single-stranded DNA (ssDNA). single-stranded DNA (ssDNA).

Figure 12 shows other comonomers that impart hydrogel sensors with new functionalities or Figure 12 shows other comonomers that impart hydrogel sensors with new functionalities optimize physical properties. Miller group used allylamine to add a biotin anchoring group that or optimize physical properties. Miller group used allylamine to add a biotin anchoring group selectively recognizes the protein avidin via antigen–antibody interactions. They prepared that selectively recognizes the protein avidin via antigen–antibody interactions. They prepared functionalized microgel particles and demonstrated a label-free sensor array using the specific functionalized microgel particles and demonstrated a label-free sensor array using the specific interaction (Figure 12a) [97]. Song et al. also used the monomer for the encapsulation of gold interaction (Figure 12a) [97]. Song et al. also used the monomer for the encapsulation of gold nanoparticles and further developed multi-stimuli-responsive hydrogel valves that regulate the flow nanoparticles and further developed multi-stimuli-responsive hydrogel valves that regulate the flow of water [98]. In addition, various functional monomers have been designed and polymerized. Zhang of water [98]. In addition, various functional monomers have been designed and polymerized. et al. synthesized allyl-substituted mannose as a comonomer where allyl groups played a role as a Zhang et al. synthesized allyl-substituted mannose as a comonomer where allyl groups played a polymerizable group and mannosyl group as an anchor for lectin concanavalin (Con A) via lectin– role as a polymerizable group and mannosyl group as an anchor for lectin concanavalin (Con A) carbohydrate interactions (Figure 12b). A hydrogel matrix including the monomer was polymerized via lectin–carbohydrate interactions (Figure 12b). A hydrogel matrix including the monomer was and further processed to the photonic crystal that optically sensed Con A by the change in the polymerized and further processed to the photonic crystal that optically sensed Con A by the diameter of the Debye ring [99]. Multi-functional monomers were synthesized by Hamilton et al. The change in the diameter of the Debye ring [99]. Multi-functional monomers were synthesized monomer consists of a polymerizable fluorescent signal transducer and dipicolylamine receptor. The by Hamilton et al. The monomer consists of a polymerizable fluorescent signal transducer and monomer was non-fluorescent because of photoinduced electron transfer (PET) but emitted dipicolylamine receptor. The monomer was non-fluorescent because of photoinduced electron transfer fluorescence after the coordination of Zn2+ (Figure 12c) [100]. Gong et al. synthesized a functional (PET) but emitted fluorescence after the coordination of Zn2+ (Figure 12c) [100]. Gong et al. synthesized monomer based on 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dye containing a a functional monomer based on 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dye containing polymerizable allyl group. They copolymerized the monomer with NIPAM by living radical a polymerizable allyl group. They copolymerized the monomer with NIPAM by living radical polymerization, i.e., reversible addition-fragmentation transfer (RAFT) polymerization. The resulting polymerization, i.e., reversible addition-fragmentation transfer (RAFT) polymerization. The resulting polymer formed a hydrogel because of the lower critical solution temperature (LCST) behavior in polymer formed a hydrogel because of the lower critical solution temperature (LCST) behavior in accordance with temperature change (Figure 12d) [101]. accordance with temperature change (Figure 12d) [101].

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Figure 12. 12. (a) of of thethe biotin-containing hydrogel for label-free sensor array;array; (b,c) Figure (a)Synthetic Syntheticprocedures procedures biotin-containing hydrogel for label-free sensor Chemical structures of (b) thethe biocompatible monomer containing the (b,c) Chemical structures of (b) biocompatible monomer containinga amonosaccharide monosaccharide and and (c) (c) the multi-functional monomer containing a chromophore and anchoring group; (d) RAFT multi-functional monomer containing a chromophore and anchoring group; (d) RAFT polymerization polymerization of N-isopropylacrylamide (NIPAM) andmonomer. the fluorescent monomer. The resulting of N-isopropylacrylamide (NIPAM) and the fluorescent The resulting polymer shows polymer shows fluorescence in response to temperature change. fluorescence in response to temperature change.

3.1.2. Other Covalent Reactions 3.1.2. Other Covalent Reactions In addition to radical polymerization, other chemical reactions that form diverse chemical In addition to radical polymerization, other chemical reactions that form diverse chemical linkages linkages have been used for the preparation of hydrogels. Because radical polymerization is suitable have been used for the preparation of hydrogels. Because radical polymerization is suitable for for the construction of various polymer backbone structures, other bond-making reactions have been the construction of various polymer backbone structures, other bond-making reactions have been adopted for post-polymerization modification or cross-linking reaction via substitution or addition adopted for post-polymerization modification or cross-linking reaction via substitution or addition reactions. Among the reactions, condensation reactions are primarily used such as imine reactions. Among the reactions, condensation reactions are primarily used such as imine condensation. condensation. Imine bonds, specifically Schiff bases, are reversibly formed covalent bonds and can Imine bonds, specifically Schiff bases, are reversibly formed covalent bonds and can be easily controlled be easily controlled by an acid-catalyzed condensation reaction, eliminating a water molecule in the by an acid-catalyzed condensation reaction, eliminating a water molecule in the process. Strano group process. Strano group used this bond to fabricate a sensor array containing single-walled carbon used this bond to fabricate a sensor array containing single-walled carbon nanotubes (SWCNT) that nanotubes (SWCNT) that served as a point-of-care (POC) device for the detection of troponins, served as a point-of-care (POC) device for the detection of troponins, standard indicators of acute standard indicators of acute myocardial infarction or heart attack [102]. To achieve this, they myocardial infarction or heart attack [102]. To achieve this, they dispersed SWCNTs in a solution of dispersed SWCNTs in a solution of chitosan and printed the suspension using a microarray printer, chitosan and printed the suspension using a microarray printer, which was then sequentially covered which was then sequentially covered by drops of a glutaraldehyde solution that cross-linked the by drops of a glutaraldehyde solution that cross-linked the printed pattern (Figure 13a). Because printed pattern (Figure 13a). Because of the robustness of the cross-linked structure, more of the robustness of the cross-linked structure, more functionalities could be introduced, such as a functionalities could be introduced, such as a Ni(II) complex and histidine (His)-tagged antibody, to Ni(II) complex and histidine (His)-tagged antibody, to the SWCNT substrate, finally yielding the POC the SWCNT substrate, finally yielding the POC device. Besides natural polymers, other synthetic device. Besides natural polymers, other synthetic applied polymers also did not hinder the electrical applied polymers also did not hinder the electrical performance of the nanotubes and could be used performance of the nanotubes and could be used in field-effect transistors as demonstrated by Loi in field-effect transistors as demonstrated by Loi group [103–105], which would be further extended group [103–105], which would be further extended to bio-sensing fields. Another interesting example to bio-sensing fields. Another interesting example had been reported by Zhang group [106]. They had been reported by Zhang group [106]. They exhibited multi-stimuli-responsive hydrogels based on exhibited multi-stimuli-responsive hydrogels based on cellulose. After periodate oxidation, the cellulose. After periodate oxidation, the aldehyde-containing cellulose was cross-linked by a functional aldehyde-containing cellulose was cross-linked by a functional linker. The linker contained two linker. The linker contained two amine groups for the formation of Schiff bases with the oxidized amine groups for the formation of Schiff bases with the oxidized cellulose and a disulfide bond for cellulose and a disulfide bond for redox-responsive behavior. Thus, the resulting hydrogel showed a redox-responsive behavior. Thus, the resulting hydrogel showed a reversible transformation of the reversible transformation of the shape by both pH change and redox reaction (Figure 13b). shape by both pH change and redox reaction (Figure 13b).

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Figure 13. (a) Synthetic procedures for the single-walled carbon nanotubes (SWCNT) sensor. Chitosan Figure 13. (a) Synthetic procedures for the single-walled carbon nanotubes (SWCNT) sensor. Chitosan is cross-linked by glutaraldehyde forming imine bonds in the presence of SWCNTs. The CNT network is cross-linked by glutaraldehyde forming imine bonds in the presence of SWCNTs. The CNT network was further functionalized with succinic anhydride to give a carboxylic acid group that could be was further functionalized with succinic anhydride to give a carboxylic acid group that could be conjugated with His-tagged antibody using Ni(II) complexation. Reprinted with permission from conjugated with His-tagged antibody using Ni(II) complexation. Reprinted with permission from [103]. [103]. Copyright (2014) John Wiley & Sons; (b) Depiction of redox-responsive cross-linker that Copyright (2014) John Wiley & Sons; (b) Depiction of redox-responsive cross-linker that provided a provided a dual signal-responsive hydrogel. The hydrogel showed reversible transformation by dual signal-responsive hydrogel. The hydrogel showed reversible transformation by redox reaction or redox reaction or pH because of disulfide group and imine groups respectively. Reprinted with pH because of disulfide group and imine groups respectively. Reprinted with permission from [106]. permission from [106]. Copyright (2017) American Chemical Society. Copyright (2017) American Chemical Society.

Acetal groups are typically obtained from the condensation of an aldehyde and two equivalent AcetalLiu groups typically obtained from theof condensation of an aldehyde two equivalent alcohols. et al.are demonstrated that this type reaction is efficient for the and preparation of selfalcohols. Liu et al. demonstrated that this type of reaction is efficient for the preparation of self-healing, healing, elastic hydrogels [107]. In this reaction, the gel network consisted of PVA and elastic hydrogels [107]. In this reaction, thewere gel network of PVA and poly(vinylpyrrolidinone) poly(vinylpyrrolidinone) (PVP), which joined consisted together via ketal condensation (Figure 14a). (PVP), which were joined together via ketal condensation (Figure 14a). Simultaneously Simultaneously they incorporated cellulose nanofibers with Fe3+, which formed the second, they hard 3+ , which formed the second, hard network through ionic incorporated cellulose nanofibers with Fe network through ionic bonding. Taking advantage of the synergistic effect from the soft and hard bonding. advantage effect from the soft hard segments, theyBoronic were segments,Taking they were able to of usethe thesynergistic double network hydrogel as aand wearable strain sensor. able useundergoes the doublea network hydrogel as a to wearable strain sensor. Boronic acid also undergoes acidtoalso condensation reaction form a boronate linkage. Nishiyabu et al. used this areaction condensation reaction to form a boronate linkage. Nishiyabu et al. used this reaction to formAll a to form a multi-component, stimuli-responsive, polymer film (Figure 14b) [108]. multi-component, stimuli-responsive, polymer film (Figure 14b) [108]. All components such as the components such as the cross-linkers, receptors, and signal transducers containing boronic acid cross-linkers, andthe signal containing boronic groups were to the groups werereceptors, attached to PVAtransducers simultaneously, resulting in aacid transparent film attached that selectively 2+ by emitting green PVA simultaneously, resulting in a transparent film that selectively detected Zn 2+ detected Zn by emitting green fluorescence. The condensation reaction of an amino acid affords fluorescence. The condensation reaction of an amino biocompatible and[109]. biodegradable biocompatible and biodegradable poly(amino acids)acid as a affords subclass of polypeptides Figure 14c 2+ shows the synthetic procedures of a colorimetric chemosensor for Cu demonstrated by Zhang et al. [110]. The polyimide was obtained from an acid-catalyzed condensation reaction of aspartic acid,

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i

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poly(amino acids) as a subclass of polypeptides [109]. Figure 14c shows the synthetic procedures of a colorimetric chemosensor for Cu2+ demonstrated by Zhang et al. [110]. The polyimide was obtained Polymers 2018, 10, x FOR PEER REVIEW 17 of 34 from an acid-catalyzed condensation reaction of aspartic acid, which was further cross-linked through which was further cross-linked through amide bonds with hydrogel ethylenediamine. fabricated a amide bonds with ethylenediamine. They fabricated a fibrous film basedThey on this chemistry fibrous hydrogel filmpresence based onofthis could detect presence the target ion by the and could detect the the chemistry target ion and by the naked eye the because of theofcolorimetric response naked eye because of the colorimetric response of the material. of the material.

Figure poly(vinyl Figure 14. 14. (a) (a) Schematic Schematic description description of of the the cross-linked cross-linked polymer polymer network network that that consist consist of of poly(vinyl alcohol) alcohol) and and poly(vinylpyrrolidinone) poly(vinylpyrrolidinone) (PVP); (PVP); (b) (b) Chemical Chemical structure structure of of the the transparent transparent boronate boronate hydrogel film containing receptor and chromophore for the selective sensing of Zn(II) ions; (c) Acidhydrogel film containing receptor and chromophore for the selective sensing of Zn(II) ions; catalyzed polymerization of aspartic acid and subsequent cross-linking reaction using a 1,2-a (c) Acid-catalyzed polymerization of aspartic acid and subsequent cross-linking reaction using diaminoethane linker by forming amide bonds. 1,2-diaminoethane linker by forming amide bonds.

Other reactions are suitable for the preparation of hydrogels. Jiang group demonstrated the use Other reactions are suitable for the preparation of hydrogels. Jiang group demonstrated the use of of thiol-yne reactions for cross-linking reactions [111]. The addition reaction is photo-clickable and thiol-yne reactions for cross-linking reactions [111]. The addition reaction is photo-clickable and may may further undergo a subsequent thiol-ene reaction, which is irreversible, unlike the thiol-ene further undergo a subsequent thiol-ene reaction, which is irreversible, unlike the thiol-ene reaction. reaction. They synthesized a multifunctional, hyperbranched polymer bearing peripheral acetylene They synthesized a multifunctional, hyperbranched polymer bearing peripheral acetylene groups. groups. Through a photo-cross-linking reaction with thiol-containing polyhedral oligomeric Through a photo-cross-linking reaction with thiol-containing polyhedral oligomeric silsesquioxane silsesquioxane (POSS), they prepared organic–inorganic hybrid hydrogels. The gels could be (POSS), they prepared organic–inorganic hybrid hydrogels. The gels could be patterned during patterned during UV irradiation; furthermore, residual functionalities on the patterned matrices UV irradiation; furthermore, residual functionalities on the patterned matrices further facilitated further facilitated diverse post-cross-linking reactions, for example, thiol-based click reactions or ynediverse post-cross-linking reactions, for example, thiol-based click reactions or yne-based click based click reactions (Figure 15a). Another notable example is the Diels–Alder reaction reported by reactions (Figure 15a). Another notable example is the Diels–Alder reaction reported by Yu et al. Yu et al. (Figure 15b) [112]. Here, hyaluronic acid (HA) was pre-functionalized with furan moieties (Figure 15b) [112]. Here, hyaluronic acid (HA) was pre-functionalized with furan moieties by forming by forming amide bonds and then cross-linked with bis-maleimide-functionalized PEG (MAL–PEGamide bonds and then cross-linked with bis-maleimide-functionalized PEG (MAL–PEG-MAL) to MAL) to afford biomedical HA-PEG hydrogels. Simple tuning of the ratio between furyl and afford biomedical HA-PEG hydrogels. Simple tuning of the ratio between furyl and maleimide maleimide moieties significantly changed the mechanical properties, and the resulting gels showed moieties significantly changed the mechanical properties, and the resulting gels showed cell cell encapsulation viability, which could shed light on mimicking articular cartilages in terms of encapsulation viability, which could shed light on mimicking articular cartilages in terms of tissue tissue engineering applications. engineering applications.

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(a)

(a)

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Figure 15. 15. (a) polymer with terminal alkynes andand the Figure (a)Chemical Chemicalstructures structuresofofthe thehyperbranched hyperbranched polymer with terminal alkynes functionalized polyhedral oligomeric silsesquioxane (POSS) as macro-monomers for thiol-yne crossthe functionalized polyhedral oligomeric silsesquioxane (POSS) as macro-monomers for thiol-yne linking reaction. Reprinted with with permission from from [111].[111]. Copyright 2014,2014, JohnJohn Wiley & Sons; (b) cross-linking reaction. Reprinted permission Copyright Wiley & Sons; Synthetic procedure for hyaluronic acid-poly(ethylene glycol) (HA-PEG) hydrogel via Diels–Alder (b) Synthetic procedure for hyaluronic acid-poly(ethylene glycol) (HA-PEG) hydrogel via Diels–Alder click reaction. reaction. HA multiple furan moieties andand PEG waswas tethered withwith two click HAwas wasfunctionalized functionalizedwith with multiple furan moieties PEG tethered maleimides. two maleimides.

3.2. Non-Covalent Chemistry for Hydrogels 3.2. Non-Covalent Chemistry for Hydrogels 3.2.1. Coordination Coordination Bonds Bonds 3.2.1. Non-covalent interactions interactions facilitate facilitate facile facile preparation preparation or or orthogonal orthogonal synthetic synthetic methodologies, methodologies, Non-covalent giving rise to synergetic physical properties in hydrogel materials. Coordination bonds or strong strong giving rise to synergetic physical properties in hydrogel materials. Coordination bonds or secondary bonds including hydrogen bonding or hydrophobic interaction are extensively used for secondary bonds including hydrogen bonding or hydrophobic interaction are extensively used for this this purpose. Among metal coordination a key position in the fabrication diverse purpose. Among them, them, metal coordination holds a holds key position in the fabrication of diverse of polymeric polymeric hydrogels that typically include polysaccharides, for instance, alginate or κ-carrageenan. hydrogels that typically include polysaccharides, for instance, alginate or κ-carrageenan. Such dynamic Such dynamic coordination allows forand easyimparts synthesis imparts the stimuli hydrogels with stimuli coordination bonding allowsbonding for easy synthesis theand hydrogels with responsiveness, responsiveness, as well [113]. as reversibility [113]. Ding group sophisticatedly designed an alginate-based as well as reversibility Ding group sophisticatedly designed an alginate-based hydrogel in hydrogel in which a hierarchical structure was formed, containing an ultrahigh amount of water, >99 which a hierarchical structure was formed, containing an ultrahigh amount of water, >99 wt % [114]. wt.particular, % [114]. In particular, SiO2 nanofibers played a pivotal role, while the alginate formed a threeIn SiO 2 nanofibers played a pivotal role, while the alginate formed a three-dimensional, 3+ , aswith dimensional, fibrillar framework through coordination bonds Al 3+,inasFigure shown16. in Figure 16. The fibrillar framework through coordination bonds with Al shown The resulting resulting hydrogel was elastic and injectable, and further capable of showing pressure-sensitive hydrogel was elastic and injectable, and further capable of showing pressure-sensitive conductivity. conductivity.

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16. (a) Figure 16. (a) Schematic Schematic description description of of the synthetic steps; (b) an optical photograph showing a nanofibrous network (NFH) with an ultrahigh water content of 99.8 wt %; (c–e) schematics of the structural levels levels of of the the hydrated hydratednanofibrous nanofibrousnetworks: networks: alginate/SiO alginate/SiO22 composite three structural composite nanofibers, nanofibers, 3+;; (f–h) crosslinks through through Al Al3+ alginate gels, and ionic crosslinks (f–h) microscopic microscopic structure structure of NFHs at various magnifications demonstrating demonstratingthethe hierarchical nanofibrous architecture; (i) tunneling scanning magnifications hierarchical nanofibrous cellularcellular architecture; (i) scanning electron microscopy–energy dispersive spectroscopy (STEM–EDS) images of a single nanofiber with tunneling electron microscopy–energy dispersive spectroscopy (STEM–EDS) images of a single corresponding mapping imagesmapping of Si, O, and Al, respectively; therespectively; four levels of nanofiber withelemental corresponding elemental images of Si, O, and(j)Al, (j)hierarchy the four of relevant structures in three structures dimensions. Reprinted with permission Copyright levels of hierarchy of relevant in three dimensions. Reprintedfrom with[114]. permission from (2017) [114]. John Wiley(2017) & Sons. Copyright John Wiley & Sons.

Another interesting interestingexample exampleis is multi-functional epidermal sensor reported by et Liao et al. Another thethe multi-functional epidermal sensor reported by Liao al. [115]. [115]. The sensor was fabricated based on a hydrogel comprised of multiple non-covalent bonds (e.g., The sensor was fabricated based on a hydrogel comprised of multiple non-covalent bonds coordination bonds, hydrogen bonds, and π–π stacking,), which are used as a design concept to build (e.g., coordination bonds, hydrogen bonds, and π–π stacking,), which are used as a design concept thebuild entirethe structure maximize desirable properties. reversibleThe coordination between to entire and structure and the maximize the desirableThe properties. reversiblebond coordination the hydroxyl group and borate is responsible for the hydrogel matrix, leading to the self-healing bond between the hydroxyl group and borate is responsible for the hydrogel matrix, leading to the properties ofproperties the sensor;offurthermore, the inclusionthe of carbon nanotubes fornanotubes electrical for conductivity self-healing the sensor; furthermore, inclusion of carbon electrical and polydopamine for epidermal adhesion allows the strain of the hydrogel material on the skin to conductivity and polydopamine for epidermal adhesion allows the strain of the hydrogel material be determined. Therefore, the multifunctional, wearable sensor monitors the change in strain arising on the skin to be determined. Therefore, the multifunctional, wearable sensor monitors the change in from human (Figure 17). (Figure 17). strain arising motion from human motion

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Figure 17. 17. Schematic Schematic representation representation of of the the fabrication fabrication of of the the healable, healable, adhesive, adhesive, wearable, wearable, and and soft soft Figure human-motion sensors. sensors. By a delicate delicate conformal conformal coating coating of of (a) (a) conductive conductive functionalized functionalized SWCNT SWCNT human-motion networksthrough throughdynamic dynamicsupramolecular supramolecularcross-linking, cross-linking,(b) (b)the theconductive, conductive,healable, healable,and andadhesive adhesive networks hybrid network network hydrogels hydrogels are are prepared. prepared. The The human-motion human-motion sensors sensors assembled assembled from from the the hybrid hybrid hybrid network hydrogels hydrogels could could be be (c,d) (c,d) reversibly reversibly self-healed, self-healed, and and (e) (e) self-adhered self-adhered on on the the wrist wrist during during network human–machine interactions interactions and and healthcare healthcare monitoring. monitoring. Reprinted human–machine Reprinted with permission from [115]. Copyright Copyright(2017) (2017)John JohnWiley Wiley&&Sons. Sons.

3.2.1. Supramolecular Supramolecular Chemistry Chemistry 3.2.2. Self-assembly provides provides an an efficient efficient approach approach to to form form aamacroscopic, macroscopic, well-ordered, well-ordered, polymeric polymeric Self-assembly structure that that brings brings about about other other significant significant physical physical or orchemical chemicalproperties. properties. Small Small molecules molecules or or structure polymers are autonomously areare mostly dependent on polymers autonomously assembled assembled under undercontrolled controlledconditions conditionsthat that mostly dependent concentration or or temperature, thus microscopic, on concentration temperature, thusforming formingthe theassembled assembledstructures structures that that contain microscopic, crystalline domains. The resulting hierarchical structures are subject to hydrogen bonding, π–π stacking, electrostatic interactions, or van der Waals interactions. As an example, Das group

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crystalline domains. The resulting hierarchical structures are subject to hydrogen bonding, π–π stacking, electrostatic interactions, van der Waals interactions. As an example, synthesized a small-molecule hydrogelatoror containing fluorescent pyrene, phenylalanine, and Das group synthesized a small-molecule hydrogelator containing fluorescent pyrene, phenylalanine, phenylboronic acid receptor moieties (Figure 18a). The gelator showed thermo-reversible gelation and phenylboronic receptor moieties (Figure 18a). Theingelator showed [116]. thermo-reversible gelation properties in wateracid and detected glucose by changes fluorescence Ma et al. prepared properties in water and detected glucose by changes in fluorescence [116]. Ma et al. prepared supramolecular hydrogels and investigated a reversible sol-gel transition induced by external stimuli supramolecular hydrogels and investigated a reversible sol-gel transition induced by external [117]. The small-molecule gelator was synthesized from a benzimidazole derivative after stimuli [117]. The small-molecule gelator was synthesized from a benzimidazole derivative after functionalization with hydrazide, and further formed a hydrogel network through metal functionalization hydrazide, andinfurther hydrogel network through coordination coordination withwith terbium(III) ions water formed (Figure a18b). The resulting hydrogelmetal showed not only with terbium(III) ions inbut water (Figure 18b). The resulting hydrogel only sensitized sensitized luminescence also thermo-reversibility in response to heat showed and pH. not A multi-component luminescence thermo-reversibility response to heat andconsists pH. A multi-component hydrogel hydrogel also but wasalso demonstrated by Yang etinal. [118]. The system of a cationic organogelator also wasanionic, demonstrated by Yang et al.dye. [118]. systemhierarchical consists ofstructure a cationic and an low-molecular-weight TheThe resulting canorganogelator discriminate and an anionic, low-molecular-weight dye. The resulting hierarchical structure canpreserved discriminate adenosine-phosphates. The addition of monoor diphosphates such as AMP or ADP the adenosine-phosphates. The addition of monoor diphosphates such as AMP or ADP preserved the structure, although ion exchange occurred. However, in the case of the triphosphate ATP, structure, although ion exchange occurred. However, in the case of the triphosphate ATP, the structure collapsed rapidly, allowing the efficient detection of triphosphate by the naked eyestructure (Figure collapsed rapidly, allowing the efficient detection of triphosphate by the naked eye (Figure 18c). 18c). (a)

signal transducer O

O

H N

N H

O

O

O

N H

receptor B(OH)2

(b) N O H2N NH (c)

Tb3+

N coordination site

N N

N

SO3Na signal transducer H N

O

+ O N Cl

N H

N H

H N O

matrix

Figure 18. 18. Chemical phenylalanine-based hydrogelator containing pyrene pyrene Figure Chemical structures structures of: of: (a) (a) the the phenylalanine-based hydrogelator containing fluorophore and phenylboronic acid receptor; (b) the benzimidazole-based gelator containing fluorophore and phenylboronic acid receptor; (b) the benzimidazole-based gelator containing aa hydrazide group; group; and (c) multicomponent-gelator multicomponent-gelator with cationic gelator gelator and and an an anionic anionic methyl methyl hydrazide and (c) with aa cationic orange dye. orange dye.

The crystallization of polymeric materials also induces dynamic physical cross-linking in threeThe crystallization of polymeric materials also induces dynamic physical cross-linking in dimensional networks, resulting in responsiveness to external stimuli by enhancing the mechanical three-dimensional networks, resulting in responsiveness to external stimuli by enhancing the properties of the hydrogel network. Zhang group incorporated cellulose nanofibers (CNF) in situ into mechanical properties of the hydrogel network. Zhang group incorporated cellulose nanofibers cellulose hydrogels. The polysaccharide fibers showed concentration-dependent gelation after loose (CNF) in situ into cellulose hydrogels. The polysaccharide fibers showed concentration-dependent chemical cross-linking, which further progressed to induce crystalline, micro-fibrillated structures gelation after loose chemical cross-linking, which further progressed to induce crystalline, inside the gel by physical interactions. Therefore, they fabricated anisotropic, mechano-responsive micro-fibrillated structures inside the gel by physical interactions. Therefore, they fabricated hydrogels via a bottom-up approach, which switched on and off polarized light on the application of anisotropic, mechano-responsive hydrogels via a bottom-up approach, which switched on and off an external force (Figure 19) [119]. polarized light on the application of an external force (Figure 19) [119].

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Figure 19. 19. (a–c) cellulose hierarchical hierarchical networks networks and and the the orientation orientation within within Figure (a–c) Schematic Schematic diagram diagram of of cellulose deformed cellulose hydrogel: (a) construction of loosely chemically cross-linked cellulose network as deformed cellulose hydrogel: (a) construction of loosely chemically cross-linked cellulose network as framework through throughreacting reacting cellulose by adding tiny amount of cross-linker, (b) formation of framework cellulose by adding tiny amount of cross-linker, (b) formation of cellulose cellulose submicrobundles and nanofibers, and (c) synchronized orientation of both and physical and submicrobundles and nanofibers, and (c) synchronized orientation of both physical chemical chemical networks underforce; external (d–f) Representative atomic force microscopy height images. networks under external (d–f)force; Representative atomic force microscopy height images. Reprinted Reprinted with permission from [119]. Copyright (2017) American Chemical Society. with permission from [119]. Copyright (2017) American Chemical Society.

3.3. Miscellaneous 3.3. Miscellaneous Approaches Approaches 3.3.1. Additives Additives are essential, low-molecular-weight components that are incorporated into polymeric matrices to alter alter the the performance performance of of the the entire entire material. material. The The inclusion of additives enhances processability (typically having a plasticizing effect) and can render the processed materials highly functional (e.g., biodegradable, antimicrobial, fragrant, or durable under extreme conditions), as well as reducing the cost of production and satisfying the design requirements [120–122]. Such additives can be used in polymer hydrogels. Lee group investigated an alginate hydrogel matrix containing a pyrocatechol-violet-baseddye dyethat thatsenses sensesHF HFgas gas (top, Figure dye, consisting a binding pyrocatechol-violet-based (top, Figure 20).20). TheThe dye, consisting of aof binding site site and a signal transducer, selectively dissociated in the presence HFhydrogel, in the hydrogel, which and a signal transducer, selectively dissociated in the presence of HF inofthe which resulted resulted in ainchange in color detectable by the eye [123]. additives Inorganic bring additives about in a change color detectable by the naked eye naked [123]. Inorganic aboutbring synergetic synergetic effects in hydrogels. Wu group developed a self-healable ionic skin based on a hydrogel effects in hydrogels. Wu group developed a self-healable ionic skin based on a hydrogel containing containing calcium crystals. carbonateThe crystals. Thenot mineral not onlydynamic induced dynamic cross-linking reaction calcium carbonate mineral only induced cross-linking reaction through through chelation with the carboxylate groups of poly(acrylic (PAA) and alginate, butionic also chelation with the carboxylate groups of poly(acrylic acid) (PAA) acid) and alginate, but also affected affected ionic conductivity as a charge carrier. Thus, the artificial skin was sensitive to external conductivity as a charge carrier. Thus, the artificial skin was sensitive to external pressure, for example, pressure, for or example, touch or even thewater pressure from(bottom, a small Figure water droplet (bottom, Figure gentle touch even thegentle pressure from a small droplet 20) [124]. 20) [124].

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Figure 20. 20. (top) (top) Chemical Chemical structure structure of of the the pyrocatechol pyrocatechol violet-based violet-based dye dye incorporated incorporated in in the the alginate alginate Figure hydrogel, which changed color from dark green to yellow after dissociation in response to hydrogel, which changed color from dark green to yellow after dissociation in response to HF HF gas. gas. (bottom) (a) (a) Schematic Schematic structure structure of of the the mineral mineral hydrogel. hydrogel. (b) (bottom) (b) SEM SEM image image of of the the freeze-dried freeze-dried hydrogel. hydrogel. (c) Mechanical Mechanical behavior Various shapes of of thethe hydrogel. (e) The hydrogel film (c) behavior of of the thehydrogel. hydrogel.(d)(d) Various shapes hydrogel. (e) The hydrogel attached to a prosthetic finger. Reproduced with permission from [124]. Copyright 2017, John Wiley film attached to a prosthetic finger. Reproduced with permission from [124]. Copyright 2017, & Sons. John Wiley & Sons.

3.3.2. Molecular Molecular Imprinting Imprinting 3.3.2. Molecular imprinting imprinting has has attracted attracted great great attention attention over over the the last last few few decades. decades. This This approach approach can can Molecular yield artificial artificial receptors receptors that that feature feature specific specific recognition recognition for for target target molecules, molecules, mimicking mimicking biological biological yield systems such as antibody–antigen or enzyme–substrate. The tailor-made binding sites in a systems such as antibody–antigen or enzyme–substrate. The tailor-made binding sites in a molecularly molecularly imprinted polymer (MIP) formed by: (i)with complexation templates; (ii) crossimprinted polymer (MIP) are formed by:are (i) complexation templates; with (ii) cross-linking reactions linking reactions in the presence of the templates; and (iii) subsequent removal of the templates in the presence of the templates; and (iii) subsequent removal of the templates (Figure 21) [125]. (Figure The MIP then target selectively target molecules or analogous even bioThe MIP 21) then[125]. selectively recognizes smallrecognizes molecules or even small bio-macromolecules macromolecules to thecan templates. This can of be materials applied toincluding various not kinds of to the templates. analogous This technique be applied to technique various kinds only materials including not only cross-linked powders [126] but also fibers [127] or inorganic materials cross-linked powders [126] but also fibers [127] or inorganic materials [128]. In particular, a hydrogel [128]. In particular, hydrogel used as a matrix forEL-Sharif binding target moleculesa[129]. EL-Sharif et was used as a matrixafor bindingwas target molecules [129]. et al. fabricated thin-film MIP for fabricated a thin-film MIP for a quartz crystal microbalance (QCM) for the protein detection. aal.quartz crystal microbalance (QCM) sensor for protein detection. Theysensor prepared hydrogel MIP They prepared the hydrogel MIP film using N-hydroxymethylacrylamide (NHMA) and MBAm in film using N-hydroxymethylacrylamide (NHMA) and MBAm in the presence of target proteins and the presence target proteins and by deposited the film QCM crystals by spin-casting. The film deposited theof film on QCM crystals spin-casting. Theon film sensor displayed selective recognition sensor displayed selective recognition through rebinding tests of protein analytes. through rebinding tests of protein analytes.

materials including not only cross-linked powders [126] but also fibers [127] or inorganic materials [128]. In particular, a hydrogel was used as a matrix for binding target molecules [129]. EL-Sharif et al. fabricated a thin-film MIP for a quartz crystal microbalance (QCM) sensor for protein detection. They prepared the hydrogel MIP film using N-hydroxymethylacrylamide (NHMA) and MBAm in the presence of target proteins and deposited the film on QCM crystals by spin-casting. The film Polymers 2018, 10, 551 24 of 34 sensor displayed selective recognition through rebinding tests of protein analytes.

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Figure 21. 21. (a–c) (a–c) Depiction Depiction of of the the preparation preparation of of aa molecularly molecularly imprinted imprinted polymer polymer including including three three Figure steps: complexation (a); cross-linking (b); and the removal of template molecules (c). Reprinted with steps: complexation (a); cross-linking (b); and the removal of template molecules (c). Reprinted with permission from from [125]. [125]. Copyright Copyright 2014, 2014, John John Wiley Wiley& &Sons. Sons. permission

3.3.3. Multilayered Multilayered Structure Structure 3.3.3. Macroscopic responses responses can can emerge emerge from from the the structure structure without without losing losing the the hydrogel hydrogel properties properties Macroscopic when hydrogels that display independent physical properties are integrated into a designed structure. when hydrogels that display independent physical properties are integrated into a designed structure. Recently, Kim group fabricated a hydrogel actuator that consists of two hydrogel layers with different Recently, Kim group fabricated a hydrogel actuator that consists of two hydrogel layers with cross-linking densities. densities. Superabsorbent PAA was used theused hydrogels. bilayeredThe hydrogel was different cross-linking Superabsorbent PAAfor was for theThe hydrogels. bilayered fabricated via sequential in situ radical polymerization; the only difference was the cross-linking hydrogel was fabricated via sequential in situ radical polymerization; the only difference was the density. Hence, the bilayer showed actuationactuation under physiological conditions. When cross-linking density. Hence, the bilayerreversible showed reversible under physiological conditions. swelling, the hydrogel bilayer bent in water because of subtle differences in the cross-linking density, When swelling, the hydrogel bilayer bent in water because of subtle differences in the cross-linking which was markedly dependent on theon pH. the bilayered material waswas used as an density, which was markedly dependent theFurthermore, pH. Furthermore, the bilayered material used as underwater electronic circuit switch (Figure 22) [130]. These kinds of materials have the potential for an underwater electronic circuit switch (Figure 22) [130]. These kinds of materials have the potential autonomous, biomimetic applications, where materials areare required to to notnot only recognize changes in for autonomous, biomimetic applications, where materials required only recognize changes the surroundings but also provide feedback and adapt themselves to the new environment. in the surroundings but also provide feedback and adapt themselves to the new environment.

Figure Figure 22. 22. Schematic Schematic description description of of the the fabrication fabrication of of bilayered bilayered hydrogels hydrogels that that show show heterogeneous heterogeneous deformation. The hydrogel bilayers were made from poly(acrylic acid) (PAA) via deformation. The hydrogel bilayers were made from poly(acrylic acid) (PAA) via sequential sequential in in situ situ polymerization thethe cross-linking density. TheThe linear bilayer curled into ainto round shape polymerizationwhile whilecontrolling controlling cross-linking density. linear bilayer curled a round under conditions, and theand patch-type bilayerbilayer could be usedbeasused the underwater switch.switch. shape aqueous under aqueous conditions, the patch-type could as the underwater

3.3.4. 3.3.4. Electrospinning Electrospinning Thundat grouphave have demonstrated a photoelectrochemical sensoronbased on nanofibers hydrogel Thundat group demonstrated a photoelectrochemical sensor based hydrogel nanofibers [131]. The nanofibers were deposited, via electrospinning, as an active layer and were [131]. The nanofibers were deposited, via electrospinning, as an active layer and were pH-sensitive pH-sensitive presence acid). of poly(acrylic acid). Furthermore, theadevice generated a because of thebecause presenceofofthe poly(acrylic Furthermore, the device generated photocurrent when

irradiated with light (i.e., light addressable potentiometric sensor, LAPS) (Figure 23). In principle, with pH change, the fibers swell to different extents, and, thus, the device gives the corresponding current as a signal, which can be used to detect extracellular acidification.

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photocurrent when irradiated with light (i.e., light addressable potentiometric sensor, LAPS) (Figure 23). In principle, with pH change, the fibers swell to different extents, and, thus, the device gives the Polymers 2018, x FOR PEER Polymers 2018, 10,10, xcurrent FOR PEER 2525 ofof 3434 corresponding asREVIEW aREVIEW signal, which can be used to detect extracellular acidification.

Figure Explanatory illustration ofthe thepH-sensitive pH-sensitive hydrogel-nanofiber-integrated lightFigure 23.23. Explanatory illustration hydrogel-nanofiber-integrated light23. Explanatory illustration of theof pH-sensitive hydrogel-nanofiber-integrated light-addressable addressable potentiometric sensor (NF-LAPS). The pH-sensitive hydrogel was fabricated top addressable potentiometric sensor (NF-LAPS). The pH-sensitive hydrogel was onon top ofof a2 a potentiometric sensor (NF-LAPS). The pH-sensitive hydrogel was fabricated onfabricated top of a p-type SiO p-typeSiO SiO 2 substrate anactive active layer (themeasured nanofiber layer measured Thechange change p-type substrate asasan layer (the nanofiber layer measured bybySEM). The inin substrate as2 an active layer (the nanofiber layer by SEM). The change inSEM). photocurrent in the photocurrent the setup was detected while thepH pHwas wasvaried. varied.Reprinted Reprinted withpermission permission from photocurrent ininthe setup was while the with from setup was detected while the pHdetected was varied. Reprinted with permission from [131]. Copyright (2016) [131]. Copyright (2016) American Chemical Society. [131]. Copyright (2016) American Chemical Society. American Chemical Society.

3.3.5.Hydrothermal HydrothermalProcess Process 3.3.5. 3.3.5. Hydrothermal Process Thehydrothermal hydrothermalprocess processhas hasbeen beenwidely widelystudied studiedand andused usedto prepare3D 3Dstacked stackedstructures. structures. The The hydrothermal process has been widely studied and used totoprepare prepare 3D stacked structures. Yan reported asynthetic synthetic procedure 3D hydrogel from graphiticcarbon carbonnitrides, nitrides,aa agraphene graphene Yan etet al.al. reported procedure ofof 3D hydrogel from graphitic carbon nitrides, graphene Yan et al. reported aa synthetic procedure of 3D hydrogel from graphitic ◦in analog,by byheating heatingatat at200 200 presence amphiphilic ionic liquids (ILs);this thisallows allows thecontrol control analog, °C°C presence ofofamphiphilic ionic liquids (ILs); analog, by heating 200 Cinthe inthe the presence of amphiphilic ionic liquids (ILs); this the allows the of the morphology, size, colloidal stability, or solubility of the nanomaterial. During the process, the of the morphology, size, colloidal stability, or solubility of the nanomaterial. During the process, the control of the morphology, size, colloidal stability, or solubility of the nanomaterial. During the carbonnitrides nitrides were exfoliated theILs ILs andgelled, gelled, thusforming forming thehydrogel hydrogel network. The carbon were exfoliated bybythe the network. The process, the carbon nitrides were exfoliated byand the ILs and thus gelled, thus forming the hydrogel network. hydrogel was further used to prepare a film on an electrode by layer-by-layer deposition, forming hydrogel was further used to prepare a film on an electrode by layer-by-layer deposition, forming aa The hydrogel was further used to prepare a film on an electrode by layer-by-layer deposition, forming chemiresistive sensor that sensitively detected hydrogen sulfide (Figure 24) [132]. chemiresistive sensor that sensitively detected hydrogen sulfide (Figure 24) [132]. a chemiresistive sensor that sensitively detected hydrogen sulfide (Figure 24) [132].

Figure (a,b) Schematic ofofhydrogel hydrogel formation: (a) the gelation occurs through Figure24. (a,b)Schematic Schematicof hydrogelformation: formation:(a) (a)the thegelation gelationoccurs occursthrough throughIL-assisted IL-assisted Figure 24.24.(a,b) IL-assisted stabilization of the exfoliated layers and (b) the ionic liquid drives the exfoliation of bulk stabilizationofofthe theexfoliated exfoliatedlayers layersand and(b) (b)the theionic ionicliquid liquiddrives drivesthe theexfoliation exfoliationofofbulk bulkcarbon carbon stabilization carbon nitride, whereas the interactions with the nanosheets prevent restacking. Reprinted with permission nitride, whereas the interactions with the nanosheets prevent restacking. Reprinted with permission nitride, whereas the interactions with the nanosheets prevent restacking. Reprinted with permission from [132]. Copyright 2017, John Wiley & Sons. from [132]. Copyright 2017, John Wiley Sons. from [132]. Copyright 2017, John Wiley && Sons.

The chemical approaches discussed above including covalent chemistry, non-covalent chemistry, The chemical approaches discussed above including covalent chemistry, non-covalent chemistry, including covalent chemistry, chemistry, andother otherprocessing processingmethods methodsare areclassified the type reaction and functional groups, and reaction and functional groups, asas other processing methods are classifiedbybythe thetype typeofof of reaction and functional groups, summarized ininTable 2.2.2. assummarized summarized inTable Table

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Table 2. Chemical approaches for the preparation of hydrogels with relevant references.

Covalent Chemistry

Type of Reaction

Functional Group or Materials

Refs.

Radical polymerization Radical copolymerization Esterification 1 Amidation 1 Imine condensation 1 Ketal formation 1 Boron esterification Thiol-yne Diels–Alder

Vinyl monomers Vinyl monomers Carboxylic acid–alcohol Carboxylic acid–amine Imine–aldehydes Diol–ketone Boronic acid–diol Thiol–terminal alkyne Furan–maleimide

[85–90,98,99] [91–94,101] [95] [96,97,106,110] [102,106] [107] [98] [111] [112]

Carboxylic acid–metal Sodium borate–diol Small molecule Polymers

[114,133–135] [115] [116–118] [119]

Synthetic dye CaCO3 crystal Acrylamides PAA PAA Graphene

[123] [124] [129] [130] [131] [132,136,137]

Metal coordination Non-covalent Chemistry Self-assembly/crystallization Additive processing Miscellaneous

Molecular imprinting Multilayered structure Electrospinning Hydrothermal method 1

Including post-polymerization modification.

4. Summary and Perspectives Compared with metals and ceramics, polymers are relatively unstable in the nanometer regime because of the nature of covalent bonds. Thus, it is not still easy to fabricate polymer nanostructures with well-defined sizes and morphologies for specific applications. In that sense, considerable attention should be paid to the functionalization of polymer nanostructures compared to the inorganic nanostructures. In addition to physical doping, many chemical routes to functionalize the polymer nanostructures for biosensing applications have been addressed in this article, which is categorized into the chemical modification of monomers, copolymerization, and post-polymerization modification. The pros and cons of each strategy should be considered to achieve the best structure and properties of polymers for biosensing applications. Nanostructures provide a high surface-to-volume ratio and small dimensions, allowing fabrication of miniaturized, highly sensitive sensors. However, it is hard to control the structure and properties of the nanostructured polymers during polymerization compared to their bulk counterparts. The use of modified monomers or comonomers before polymerization can deform the morphology of the resulting products because the interfacial tension between the monomer and polymerization medium is changed. The chemical reactivity of the modified monomers is also changed compared to non-modified, non-functionalized monomers. Therefore, much effort is required to determine the most important polymerization parameters that control the polymer properties. After polymerization, control over the morphology of the nanostructures is not required. Although the morphology is preserved without significant change, the polymer properties may be considerably altered during the post-polymerization modification reaction. The careful design of reaction procedures for post-polymerization should have no negative effects on the desired properties of the polymer. Judicious choice of experimental variables such as temperature, reaction medium, and chemicals should be made depending on the polymer type. The macrostructures of the polymers have also been widely researched. Among them, hydrogels have attracted significant attention because of various advantages including 3D porous network, outstanding biocompatibility, good liquid permeability, chemical diversity, and facile modification, which make hydrogels ideal candidates for biosensing materials. In particular, the 3D porous matrices provide a larger contact area toward target molecules in water, and the embedding of powerful sensing

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tools enhances the sensing abilities of the materials. The chemical structures of hydrogel biosensors are prepared by a variety of synthetic methodologies, which have been addressed in this review in detail. Radical polymerization is a conventional yet superior method to prepare hydrogel materials together with other covalent-bond-making reactions, which play an important role under certain conditions for post-polymerization modification. Non-covalent chemistry or other miscellaneous processing approaches that result in cross-linked structures have also been introduced. Compared with molecular biosensors, hydrogel sensors tend to show a slow response time that would hinder real-time sensing because of the mass transfer of target materials inside the hydrogel matrices. Another drawback of hydrogels is their poor mechanical properties. However, these limitations can be overcome by engineering and optimizing the structure of hydrogels, which further facilitates the development of tissue engineering or wearable devices. In particular, from the perspective of biosensing, many devices that can sensitively monitor biosignals (e.g., breath, pulse, sweat, and strain) have been successfully developed based on hydrogel materials. Furthermore, new designs for hydrogels (e.g., double-network hydrogels, dynamic bonding-assisted hydrogels, and nanocomposite-reinforced hydrogels) have been extensively investigated over the last decade, and new chemical reactions such as head-to-tail depolymerization [138–140] or self-propagating reactions [141,142] can be further integrated into hydrogels, giving rise to an autonomous response system. In the future, hydrogels could be used as a platform for theragnosis, a combination of diagnosis and therapeutics, which is an emerging concept in biomedicine. Such hydrogel sensors would not only find early symptoms of diseases, but also visualize or release drug molecules simultaneously in response to the external changes including pH, temperature, light, and magnetic or electrical fields. Author Contributions: H.Y. and H.K. organized the structure of the paper; K.M.L. and K.H.K. contributed to survey literature; all authors participated in writing the paper. Acknowledgments: This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT, Ministry of Science and ICT) (No. 2017R1C1B5017785, 2018R1A2A2A05020201). This study was financially supported by Chonnam National University (Grant number: 2017-2779). Conflicts of Interest: The authors declare no conflict of interest.

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