Tailored hydrogels for biosensor applications

Abstract

To date, hydrogels have gained attraction due to their inherent advantages such as wide selection of precursors and additives, low toxicity, easy shape control, and biological compatibility. Therefore, extensive studies are being conducted for their application in diverse fields. In this review, recent impressive studies on the use of tailored hydrogel materials for biosensor applications have been summarized. As the hydrogel precursors and the sensing mechanisms are wide and extensive, we have summarized chemical/biological sensors depending on the hydrogel precursors possessing biocompatible features. Hence, versatile biological/biochemical sensors using conventional hydrogels based on carbohydrates, polymers, DNA and peptides have been covered primarily. In addition, emerging conductive hydrogels possessing conducting additives such as graphene, conducting polymers, and nanocrystals have been introduced and their application in biosensors has been described extensively. Sensor operations are dependent on the changes in resistance and conductance, signal transduction through electrodes, sensor geometry, and interactions between sensing media and target analytes. As we primarily focused on the type of precursor materials and the sensor performance such as sensing mechanism, sensitivity, linear range, and selectivity was summarized and presented. A trend in the research fields of hydrogel-based elaborate sensors has also been briefly described. This article provides essential information for advanced research in related fields.

Introduction

Hydrogels are a class of polymeric materials with a three-dimensional networked structure, possessing a marginal amount of water. It is also defined as a certain type of substance, in which a liquid is dispersed microscopically inside a solid network [1], [2], [3], [4]. As hydrogels are highly compatible with most biological molecules and substrates, they have gained attraction in many research fields. To date, research activities using hydrogels as a major component in sensor systems have been extensively conducted [5], [6]. This seems reasonable because the need for versatile chemical/biochemical sensors has increased dramatically. In addition, various hydrogel precursors are available from the environment and industrial sectors as matrices for sensor geometries [7], [8], [9].

As the definition hydrogel is wide, numerous materials can be used as hydrogels. These materials include polymers, biocompatible materials such as proteins and carbohydrates, and inorganic and conductive materials [10]. In addition, various sensing mechanisms can be introduced for sensor development [11]. A change in resistance and conductance and electric charge transfer are the most widely used mechanisms and weak specific interactions such as hydrogen bonding are also efficient. Sensor geometries have also been flexible because the hydrogels are flexible and elastic [11]. Therefore, diverse strategies have been used for sensor development.

Hydrogels are attractive for sensor platforms owing to their inherent characteristics and structural advantages. Since the internal pore structure of hydrogels is interconnected and broad, the incorporation of foreign substances is relatively favorable [12], [13]. As the medium is generally in a liquid state, the addition of analyte to a sensing medium is convenient. Consequently, the entire hydrogel system is less influenced by the inflow of external components. That is, the liquid can act as a buffer system for the resulting sensor systems. [14]. In addition, a benign environment can be provided inside the hydrogel, which is excellent for sensor performance. As signal transduction and noise suppression can be achieved using those advantages, it has become possible to prepare elegant hydrogel materials and produce hyper-structured sensor systems for control over potential hazards [2], [13].

Therefore, it is important to understand the recent research trends regarding diverse biosensors using tailored hydrogels. In this article, a review and summary of recent reports in relevant research fields has been covered concisely. In the first part, the application of hydrogels depending on the hydrogel material type is described succinctly. Sensors using hydrogels composed of biomaterials and polymers are introduced and the addition of emerging conductive materials such as graphene, conductive polymers, nanocrystals, and inorganic components to the hydrogels for sensor applications is described subsequently. We have categorized novel sensors focused on the type of precursor materials, in addition, the sensing mechanism and sensor performance were also summarized clearly. Quantitative parameters such as detection limit and linear range are provided along with selectivity and remarkable information. In addition, recent research activities toward newly developed sensors such as mechanical, strain, and motional sensors have also been introduced briefly. This short article provides essential information for future research efforts toward unprecedented sensor systems.

Hydrogels have many advantages such as flexibility, biocompatibility, and chemical inertness, therefore, they can be employed for many purposes in diverse novel devices. Because lots of precursors are available, it is possible to select the best material for target applications. Among various target applications, chemical/biological sensors are very promising because precise and real-time monitoring of potential hazards is important in daily life. Even if hydrogels are generally soft and elastic, the internal structures are relatively durable because the solid frameworks are stable and interconnected [15]. In addition, hydrogels are mainly based on the polymer matrix and their mechanical properties are macroscopically analogous to those of polymer materials such as rubber elasticity, viscoelasticity, and creep and relaxation.

Among the advantages of hydrogels, the following properties are desirable in application for the development of electronic devices [16]. First, hydrogels are composed of flexible materials such as polymer, elastomer, biocompatible molecules, carbohydrates, and additives. Therefore, hydrogels are suitable for flexible/elastic devices. Second, hydrogels can provide benign environment for optimized operation of resulting devices. For example, a rapid inflow of external liquid or substance into the hydrogels can be avoided practically. The changes occurring in the internal space of hydrogels are relatively slow and mild. Third, signal transduction is accessible through the internal space of hydrogels, if appropriate sensing medium is successfully introduced. The use of hydrogels for sensors has been reported extensively [17], [18], [19]. The widest application field of hydrogel sensors is the biomedical sector such as drug delivery [17], [18].

Conductive hydrogels are also popular for sensor applications due to their functionalities [19]. These are a class of emerging materials encompassing conducting polymer, nanocrystals, carbon nanostructures, and conductive additives. A wide range of electrical conductivity can be tuned using several strategies, for example, selection of matrix and additive. Relatively high electrical conductivity promotes the signal transduction without incorporation of other components and suppression of noises is also achievable.

The use of biocompatible materials such as carbohydrates and peptides is very contradictory in terms of favorable interaction and signal fidelity. As they are naturally abundant and environmentally benign, they are popular for combination with hydrogel precursors. As the interaction between the biocompatible materials and target analytes are specific or weak, the influence of carbohydrates or peptides for sensor performance improvement is less appealing. However, if a selectivity is secured between sensing medium and target component, biocompatible materials seem attractive. As the signal generation derived from the specific or weak interaction between sensing medium and analyte is fluctuating, signal fidelity is always an important issue to be discussed during sensor performance investigation.

The introduction of inorganic components into the hydrogel-based sensor is a good choice. However, homogeneous and uniform dispersion of the added inorganic moieties is not always favorable. If a severe mixing step is required, the denaturation of hydrogel matrix would be a serious obstacle for sensor performance improvement. In spite of these limitations, inorganic additives are recommended for diverse sensor systems.

Before proceeding to the main section, it is important to summarize the impressive research works dealing with the theoretical background of hydrogel applications. Theoretical investigations on the effects of hydrogels in sensor systems have been reported [20], [21], [22]. Approximately a decade ago, the influence of the added intermediate hydrogel layer on biosensor signal transduction was reported [20]. Since then, mechanoelectrical transduction in polyelectrolyte hydrogel-based biomimetic sensors has been examined [21]. In this report, the predicted theoretical model and experimental results were observed to be in agreement. Thus, this study was useful for understanding the nature of signal transduction in complex hydrogels. The fundamental convection and diffusion behaviors in hydrogel sensors have also been studied theoretically [22]. The development of a scientific model to explain the responsive signal of a specific biosensor has been described [23]. Moreover, control over electrical properties was pursued, that is, the isoelectric point of a polyampholytic hydrogel was manipulated to improve the performance of the obtained biosensors [24].

Polymers are the most popular precursor materials for hydrogel preparation. Polymers are generally employed as matrix because they can provide stable platform, housing with chemical stability, and benign environment for sensing. Several polymers such as polyacrylamide (PAAm) and its derivatives [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], polyacrylic acid (PAAc) [37], [38], [39], polyethylene glycol (PEG) [40], [41], [42], [43], and poly-2-hydroxyethyl methacrylate (PHEMA) [44], [45] have been extensively employed for hydrogel production. Hydrophilic polymers are relatively favorable for these purposes than hydrophobic ones because interactions with water molecules and other components are prevalent. We introduced research activities focusing on the frequently used polymer precursors.

PAAm is a typical water-soluble polymer that is frequently used for the aggregation of solid residues in water purification facilities. It forms hydrogels easily by absorbing ample amount of water molecules. A decade ago, a biosensor for the continuous monitoring of glucose in blood was designed with a dimethylaminopropylacrylamide hydrogel using 3-phenylboronic acid and a tertiary amine [25]. The hydrogel was connected at the end of an optical fiber and the resulting sensor was tuned to improve the sensor system sensitivity and reproducibility. This study demonstrated the feasibility of measuring glucose in blood in the presence of interfering molecules. In contrast, a hydrogel was introduced on the surface of a glassy carbon electrode by casting a PAAm hydrogel film [26]. The electrochemical behavior of human cytochrome P450 2C9 incorporated in the PAAm hydrogel was monitored and applied for the determination of a toxic substance, bisphenol A. Recently, a practical approach for the detection of sulfion (S²⁻) was reported using an amine functional PAAm hydrogel with cross-linked fluorescein [27]. The generated sensor showed a volumetric response to the sulfions, enabling visual identification with the naked eye. The sensor showed a good linear relationship between 1−100 μM and a reproducibility of < 5 %. The use of PAAm-based molecularly imprinted polymers for monitoring protein crystallization has also been reported [28].

One of the most widely used PAAm derivatives is poly-N-isopropylacrylamine (PNI-PAAm). The properties of PNI-PAAm are expected to be similar to those of PAAm; therefore, the general behavior of hydrogels obtained with PNI-PAAm must be analogous to that of PAAm-based hydrogels. A series of creative research activities have been reported, where a carboxylated PNI-PAAm hydrogel film was attached to the metallic surface of a surface plasmon resonance optical setup. The hydrogel film served as a binding matrix and an optical waveguide. Compared with a conventional self-assembled monolayer, the hydrogel provided both improved resolution and elevated detection limit (10 pM) [29]. This study demonstrated the usefulness of hydrogels as extended three-dimensional biointerfaces. A similar concept was applied to biosensors using a hydrogel layer for the detection of an important hormone, 17beta-estradiol [30]. In another report, the PNI-PAAm hydrogel was introduced for immobilizing DNA on flat surfaces [31]. This strategy was proven effective because complete removal of DNA after measurement was possible, leading to reusability. A glucose sensor possessing temperature control ability was also reported using PNI-PAAm hydrogels [32]. In a subsequent study, PNI-PAAm-co-PAAm copolymer hydrogels were employed for the sequential detection of cholesterol and glucose [33]. The optimal detection conditions were suggested, while repeatability, sensitivity, and yield data were also provided. Stimuli-responsive hydrogels were prepared with PNI-PAAm-co-polymaleic acid copolymers with varying monomer ratios [34]. When the carboxylic groups in polymaleic acid were ionized, the resulting hydrophilic copolymer lost its thermosensitive property. On the contrary, if the carboxylic groups were protonated, the copolymer was thermosensitive at an appropriate pH. Adding nanocrystals to the PNI-PAAm hydrogels led to a dual DNA biosensor, enabling the detection of target DNA strands [35]. Recently, an interesting study was reported, in which six acrylamide monomers were used to produce a library of 21 hydrogel matrices [36]. A special set-up was made to allow the spectrophotometric measurement of iron-based complexes inside the hydrogels. Using the sensor, it was possible to identify the iron content in milk.

PAAc is a popular polyelectrolyte material used in many applications. A unique study has reported a sensor using a PAAc-co-polyacrylamido-methyl propane sulfonic acid copolymer hydrogel to determine the presence of transition metal ions [37]. In this work, the hydrogel was exposed to 11 different transition metal ions. The trend in the chemical responses of the hydrogels due to the formation of metal complexes was presented. A biosensor array was constructed using a polymer network composed of a reactive liquid crystal and enzyme-immobilized PAAc [38]. The sensor array showed a sensitive response to the urea molecule. This was an example of a lucrative and easy human urine detection method without using any complicated instruments. An easy technique was reported for the detection of Escherichia coli using electrospun PAAc/polyvinyl alcohol hydrogel nanofibers [39]. Since E. coli is sensitive to environmental pH, changes in pH as a result of the fermentation of sugar molecules could be monitored.

PEG has been extensively used for diverse biocompatible purposes. However, while PEG hydrogels have been applied for drug delivery and tissue engineering, their use for biosensors has been less explored. In a study, polydiacetylene (PDA) was incorporated into a PEG-hydrogel matrix by photopolymerization, as a color change occurs in PDA by external stimuli. The resulting hydrogel color changed from blue to red upon reaction with alpha-cyclodextrin [40]. A report regarding the use of peptide-cross-linked PEG-hydrogel for collagenase detection experimentally demonstrated the use of biodegradable hydrogels for point-of-care devices [41].

Recently, a novel method for monitoring nitrite ions, an important marker of inflammation, food safety, and water quality was described [42]. Fig. 1 shows a schematic diagram showing the detection method. First, the PEG-hydrogel was functionalized with an NHS group to attach the dyes. In this scheme, N-(1-naphthyl)ethylenediamine (1-Nap) was immobilized using an NHS group tethered to the hydrogel. The 1-Nap preloaded hydrogel color changed from pale yellow to red by a dropwise addition of a solution of sulfanilamide (SA) and nitrite ions. The color changed quickly and varied depending on nitrite ion concentration. The selectivity of the sensor and supplementary experiments were conducted with various anions, as shown in Fig. 2. It was remarkable that a significant color change occurred with as low as 1 mM nitrite ions, demonstrating the sensor specificity. In another case, a copolymer possessing a PEG backbone was introduced as a sensing medium for biosensors [43]. Graft copolymerization of PHEMA on the side chain of the PEG backbone produced a copolymer of PEG-PHEMA. For this copolymer, it was possible to vary the light radiation time; hence, a copolymer gradient was induced for application in biosensors.

PHEMA has a more flexible backbone; thus, it has been widely applied for less stiff hydrogels. A PHEMA hydrogel containing molecularly imprinted nanospheres was embedded as a coating for implantable biosensors [44]. The amperometric determination of nitrite followed a unique approach using carbon paste electrodes modified with PHEMA hydrogels [45]. In this study, the produced sensor could detect 1 μM to 1 mM nitrite in water samples, with a detection limit of 4.4 μM. In addition, zwitterionic hydrogels from polycarboxybetaine methacrylate (PCBMA) were designed and prepared to act as vesicles to avoid antifouling in complex media, such as human blood [46].

Table 1 summarizes the sensing media, target analytes, and performances of the sensors explained in the previous sections. Considering the information, it can be inferred that the sensors based on polymer hydrogels strongly dependent on the electrochemical and spectroscopic detection mechanisms. Electrochemical sensing mechanism is frequently employed because the polymer matrix is irresponsive to the electron transfer (oxidation/reduction) reactions of target analyte. Spectroscopic identification is also popular as measurements are relatively convenient compared with other techniques. In other cases, hydrogels are introduced as an intermediate layer to improve the sensor performance. In these studies, the role of added hydrogel layer is diverse depending on the purposes.

The combination of the inherent advantages of hydrogels with electrical conductivity can produce novel materials for sensor applications [47]. Conductive materials have been introduced by two representative methods, polymerization inside hydrogel precursors [48], [49], [50], [51], [52], [53], [54], [55] and mixing [56], [57], [58], [59], [60], [61], [62]. Conducting polymers such as polypyrrole (PPy) and polyaniline (PANi) are usually polymerized electrochemically in the presence of hydrogel precursors. On the contrary, other conductive additives such as graphene and carbon nanotube are introduced by conventional and advanced mixing techniques. Even if the conductive components are introduced using diverse methods, the signal transduction mechanism in the hydrogels is substantially similar. That is, a percolation threshold behavior was observed in the electrical conductivity variation. Once sufficient electrical paths are created, the electrical signal transport is facilitated through the paths. Above the loading at which the percolation threshold is observed, the signal becomes stable and suppression of noise is also accessible. When nanocrystals are introduced, the charge transport can be even accelerated depending on the nature of added nanocrystals [60], [61], [62].

The fabrication and application of conductive hydrogels are convenient because diverse conductive additives are produced and their properties are revealed [47]. Conducting polymers [48], [49], [50], [51], [52], [53], [54], [55], graphene and carbon nanomaterials [56], [57], [58], [59], and nanocrystals [60], [61], [62] were mainly introduced as conductive additives to obtain hydrogels. An advanced study showed that a biosensor based on nanostructured conductive hydrogels was constructed by the inkjet-printing method [63], [64].

Polypyrrole (PPy) polymerization in the presence of agarose hydrogel is shown in Fig. 3. Scanning electron microscopc images of the prepared conductive hydrogels as a function of PPy concentration is presented in Fig. 4. The surface morphology was irregular and macropores were apparent. An electrical conductivity of 10⁻¹ S/cm was achieved under various media, as shown in Fig. 5, because this was an important prerequisite for biosensor production.

Typical conducting polymers such as PPy, polyaniline, and polythiophene (PTh) are frequently used for hydrogels. In a study, PPy was embedded into the PHEMA matrix for preparing a glucose sensor [48]. A 3D networked PPy hydrogel was reported for the determination of ascorbic acid, dopamine, and uric acid [49]. The sensors showed very low detection limits. Porous carbon materials were prepared as supramolecular PPy hydrogels for sensitive acetaminophen detection [50]. Due to the high electrical conductivity and surface area, the sensor assembled with porous carbon showed a very low detection limit (1.2 nM). In a recent study, an agarose hydrogel was employed for recycling PPy sensing materials [51]. Fig. 6 shows the procedure for production of hydrogel sensors containing PPy nanotubes as a sensing material for dopamine detection. An agarose hydrogel containing PPy was placed on the Au electrode to generate a sensor device with an improved sensitivity of 100 nM; the PPy sensing material could be reused twice, a good example of the production of environment-friendly sensor devices. In addition, the hydrogel-based sensor provided a relatively better performance than the sensors with other geometries.

A 3D PANi hydrogel scaffold was produced for reliable xanthine detection [52]. The biosensor could be applied xanthine detection via in situ generated hydrogen peroxide and had a low detection limit (9.6 nM). An interesting skin-inspired conductive hydrogel was fabricated for epidermal sensors and diabetic foot wound dressings [53]. The conductive hydrogel was prepared from an assembly of Ag nanoparticles, PANi, and polyvinyl alcohol. The obtained hydrogels possessed desirable features and were used for monitoring human movements real time. As conducting polymers are easily combined with carbon nanostructures, the production of PANi/graphene oxide was demonstrated; the resulting composite hybrid was introduced into the PNI-PAAm matrix on a glassy carbon electrode [54]. In another study, a PANi-graphene oxide hybrid was incorporated into the PAAc hydrogel to produce a conductive hydrogel, and biosensors were produced by the immobilization of glucose oxidase (GOD) in the conductive hydrogel [55]. In particular, the sensor showed a fast response time of 1 s and detection limit of 25 μm.

Graphene is very competitive for sensor applications because of its physical/chemical stability, stable electrical properties, and fast charge transport. A reduced graphene oxide-based hydrogel was prepared as a support for biological oxygen demand (BOD) biosensor [56]. Notably, this sensor showed a low detection limit and long-term stability for up to 2 months. A useful apta-sensor was produced using a graphene oxide hydrogel for the detection of antibiotics [57]. It was demonstrated that this sensor showed a generic detection functionality for other molecules by replacing the sensing element. Hydrogels retaining 2D graphene were fabricated with stimuli-responsive swelling characteristics for signal transduction in biosensors and bioelectronics [58]. A resulting microfluidic sensor with a detection limit of 4 pg/mL was produced using a mesoporous nanostructured hydrogel with graphene additive for myoglobin quantification [59].

Nanocrystals are also very promising components for hydrogel functionalization, because they possess constant physical/chemical properties, durability, narrow size distribution, and wide selectivity. Nanocrystals such as Pt [60], Co₃O₄ [61], and NiCo₂O₄ [56] were used as graphene-based hydrogels for monitoring glucose. A 3D graphene hydrogel was decorated with NiCo₂O₄ nanoflowers by a simple hydrothermal method [62]. The obtained sensor showed a wide linear range of 0.005–10.95 mM for glucose detection and was applied for the determination of glucose in blood samples. As the addition of nanocrystals to various hydrogels is extensive and versatile, this topic will be covered in the following section.

Table 2 shows the sensing media, analytes, and performances of the sensors in section 2.2. In this section, the sensing mechanism is strongly dependent on the electrochemical charge transfer. This is because conducting polymers and additives are advantageous for charge transport through created conducting channels between components. However, compared to section 2.1, spectroscopic identification is less popular because interference factor might be induced by electron-photon interactions.

Carbohydrates are considered most desirable for hydrogel formation, because they are naturally abundant and environmentally benign. Carbohydrates such as oligosaccharides, polysaccharides, cellulose, chitosan, and dextran are suitable for hydrogel generation because the molecular units in these molecules are composed of hydrophilic moieties. Hence, they can sufficiently absorb water molecules inside the molecular network. Sometimes, they can transform their configuration to adjust to the environment. Therefore, carbohydrates are widely used for hydrogel preparation [65], [66], [67], [68], [69], [70], [71]. Compared with other hydrogel precursors, carbohydrates are less miscible with other components and show poor chemical/mechanical stability. This can be overcome by combination with other additives possessing robustness. On the other hand, production of conductive hydrogels is much difficult as signal transduction is frequently restricted by dominant organic moieties. Therefore, the usefulness of carbohydrates for sensor applications is limited.

The degradation of oligosaccharide hydrogel films is an effective direct criterion for monitoring alpha-amylase [65]. The formation of host-guest complexes with cross-linked beta-cyclodextrin (cyclic oligosaccharide) and analyte small molecules was translated for sensing small molecules [66]. Polysaccharides, such as starch, cellulose, and chitosan show diverse molecular structures depending on the packing geometry of the repeating monosaccharide units [67]. Hydrogels using chitosan and chitosan hybrids have been reported for applications in biosensors [68], [69], [70]. A study was conducted to fabricate an amperometric biosensor using laponite/chitosan hydrogels on a glassy carbon electrode for the measurement of lactate in beverages and dairy products [68]. Notably, the best hydrogel composition was investigated to improve the long-term stability of the prepared sensors. Mucin/chitosan hydrogels were introduced to biosensors for understanding the behavior of glucose oxidase (GOD) and oxalate oxidase in the hydrogel [69]. A biocompatible chitosan hydrogel with a specific recognition molecule for hydrogen peroxide was fabricated for an electrochemical sensor [70]. The sensor exhibited a fast response time (7 s) and a wide linear range (μM to mM). A dextran hydrogel was deposited on a gold sensor biochip for monitoring small drug molecules [71]. By using the hydrogel, it was possible to improve the sensitivity with a detection limit of 0.5 nM.

Hydrogels prepared with the precursors from seaweeds are also favorable for sensor applications [72], [73]. Agarose is a polysaccharide extracted from red seaweed. It is generally used for the electrophoresis matrix; therefore, it is suitable for biosensors [51]. A novel sensor system was designed using acetylcholinesterase immobilized in a hydrogel-agarose matrix with Au particles [72]. The biosensor could detect pesticides such as carbamates in food. The use of a magnetic sodium alginate hydrogel for the detection of thrombin was reported [73]. A chemiluminescence sensor system was based on a hydrogel functionalized with specific aptamers for thrombin.

Peptides and DNA are also an important class of biocompatible materials as they can also interact with most organic molecules and materials. DNAs [74], [75], [76], [77] and peptides [78], [79], [80], [81] are extensively used as auxiliary components for hydrogel formation. A study showed that the specific interactions between DNA/peptides and ions/small molecules have been a strategy for sensitive detection [74]. However, the fidelity of the sensing mechanism was relatively weak, because the signal generation was dependent on the specific interactions between DNA/peptide and target analytes. This feature was sometimes observed in the sensor systems based on the biocompatible materials. Therefore, extensive research activities are ongoing in many relevant fields. For example, heavy metal ions can be detected using DNA-functionalized hydrogels [75], [76]. The sensors could detect Hg²⁺ with an extremely low detection limit (0.04 pM) [75]. A DNA-based hydrogel capillary sensor was proposed for the sensitive determination of Pb²⁺ [76]. In this sensor, the capillary motion through the tube was controlled by a hydrogel film. The quantitative detection of Pb²⁺ was achieved by reading the physical movement in the capillary tube with the naked eye. The detection limit for this sensor was also relatively low (10 nM). The detection of fusion genes based on aptamer-based hydrogels has been reported for enzyme and label-free biosensors [77]. Encapsulation of enzyme bioreceptors along with quantum dots (QDs) to biosensing platforms composed of self-assembled peptide hydrogels has been reported [78]. The produced sensor was found to be effective for monitoring glucose and toxic compounds due to the synergistic effect induced by the combination of peptides and QDs. As mentioned earlier [41], peptide cross-linked hydrogel film degradation was employed as a direct monitoring indicator for matrix metalloproteinase-9, a neuroinflammation biomarker [79]. This method can be developed for early stage prediction and intervention of diseases. Self-assembling 3D peptide hydrogles attached with DNA-recognition motifs has been demonstrated as a diagnostic tool for human pathophysiology [80]. Peptide-derived hydrogels were incorporated into sensor systems for the separation of cell culture compartment and biosensing part [81]. The cells were cultured in the obtained hydrogels, and the released substance was detected subsequently.

The sensing media, target substances, and performances of the sensors in this section are summarized in Table 3. It is notable that the interactions between carbohydrate hydrogel and targeted bio-species are favorable, however, identification and exclusion of sensor signal becomes difficult in these cases. Therefore, the sensing mechanism is dominantly dependent on the electrochemical charge transfer in target analytes.

Inorganic materials are very competitive because wide range of properties can be provided by simple selection of materials. Therefore, inorganic components are actively introduced as a primary or secondary material for sensor preparation. A limited number of inorganic networks are useful for sensor platform preparation. These include siloxane or titanium oxide, which are fabricated using a simple sol-gel method. On the contrary, a wide selection is allowed if the inorganic materials are employed as additives. This strategy can be meaningful when homogeneous and uniform dispersion of the filler is achieved. If inorganic network or additive is effectively introduced into hydrogels, the sensors based on the produced hydrogels can show a very stable performance and signal transduction.

The use of inorganic components such as silica [82], titanium oxide [83], [84], nanoparticles and QDs [85], [86], [87], [88], [89], [90], [91], organosilicates [92], and ionic liquids [93], [94] is an attractive alternative strategy for functionalization and performance improvement of hydrogels, instead of developing new precursors. Each inorganic component possesses its own characteristics, consequently, they can be very competitive to meet a certain purpose and performance.

In a study, mesoporous silica was employed as a catalytic support to produce a glucose biosensor [82]. Fe₃O₄ nanoparticles functionalized with glucose oxidase (GOD) were embedded into mesoporous silica nanoparticles. The obtained nanoparticle fillers were mixed with PNI-PAAm polymeric matrix for the controllable detection of glucose. The sensor showed a linear response within 50−700 mg/dL with a detection limit of 8.33 mg/dL. Moreover, a mathematical equation was derived to prove the sensor performance. The addition of doped Au@silica nanoparticles into the TiO₂/chitosan matrix led to the production of biosensors for sensing organophosphorus pesticides [83]. The linear range and detection limit of the sensors monitoring the concentration of dichlovos and fenthion pesticides was measured quantitatively. It was impressive that the sensor could be used for commercial juice samples. The use of TiO₂ nanoparticles both as a photocatalyst and an additive for agarose hydrogel was reported [84]. It was revealed that both functions remained constant after embedding the nanoparticles in the hydrogel. The TiO₂ nanoparticles could be reused by a simple retrieval procedure as presented in Fig. 7.

The use of nanocrystals such as QDs [85], [86], [87], novel metal [88], [89], [90], and magnetic nanoparticles [91] have been extensively studied in many research fields. Approximately a decade ago, the addition of CdSe nanocrystals into an array of PEG-based hydrogels was demonstrated for a phenol sensor [85]. Enzymes were encapsulated into CdTe QD hydrogels and loaded with a biocatalyst and a signaling unit. The whole system was examined to obtain a versatile biosensing platform [86]. A challenging real-time sensing of Fe³⁺ was conducted using surface-engineered QDs in hydrogels [87]. A typical novel metal (Ag) nanoparticles have been extensively added to various hydrogels for many purposes. Due to the unique surface plasmon effect, it is considered one of the most efficient sensing media. A layer of chitosan hydrogel containing Ag nanoparticles was employed as a layer for the trichloroacetic acid sensor [88]. The sensing mechanism was dependent on the facilitated electron transfer induced by the interaction between Ag and trichloroacetic acid (TCA). The sensor system works properly in the range of 40−120 μM, but the improvement of the detection limit remains challenging. An interesting report has been published to demonstrate the in situ addition of Ag nanoparticles into PVA hydrogel without any reducing agent [89]. Using the Ag@PVA hydrogels, a dual mode “on and off” sensor was developed showing a sensor performance for Fe³⁺. Note that this study was expanded to real food samples. A study was conducted using Ag nanowire embedded PEG hydrogels for the differentiation of neuronal stem cells [90]. A microarray pattern was constructed on the flexible PET substrate surface. The results obtained in this work must be very lucrative for future therapy technology. Magnetic nanoparticles are also attractive because a limited number of candidate materials are accessible. Consequently, the incorporation of magnetic nanoparticles into hydrogels has drawn interest [91]. Recently, a colorimetric sensor was devised to detect aldehydes in aqueous solutions using PAAm hydrogels containing magnetic nanoparticles.

In addition, organosilicates such as laponite [92] were used for glucose sensors. The laponite hydrogel performance was greatly improved by polycation addition. Ionic liquids have recently been introduced into diverse hydrogel systems to examine their effect on the properties and performance of hydrogels and sensors [93], [94]. Table 4 also summarizes the notable features of the biosensors introduced in this section. Electrochemical sensing mechanism was prevalent as demonstrated in other sections, while photochemical identification was employed for the hydrogels containing inorganic nanocrystals.

We have discussed biosensors based on hydrogel materials. Recently, research activities regarding the previously mentioned topics have been actively pursued in many relevant fields. However, a conspicuous trend appears, that is, the emergence of stimuli-responsive and human motion sensors. Hydrogels are generally soft and elastic; consequently, the stiffness of the hydrogels can be altered by modifying precursors, additives, or preparation methods. Considering these features, it can be inferred that the hydrogels might be suitable for matrices and substrates for flexible, responsive, and human motion sensors. Therefore, in this section, a brief summary of the stimuli-responsive biosensors will be covered concisely. As the motion sensors, including strain and pressure sensors, are out of the general scope of this article, it will be discussed in a separate article.

There are many stimuli in the ambient environment, such as light, heat, pressure, pH, and pathogens. Stimuli-responsive materials are attractive because they can modify the structure, property, or geometric configuration depending on a change in physical parameters such as temperature and pressure, and chemical conditions such as pH and ionic character, electrical properties, and environment. As it is convenient to obtain biosensors by observing any visual changes in color, researches regarding colorimetric and fluorescence sensors are active [95], [96], [97], [98]. More than a decade ago, a stimuli-responsive hydrogel was used as an optical enzyme biosensor. The hydrogel was composed of Ag nanoparticles and polymer matrix. The results showed that a dramatic change in the localized surface plasmon resonance absorbance was induced by the designed reaction, leading to a very low detection limit (10 pM) [95]. A real-time colorimetric sensor array was designed using a simple method instead of a complicated fabrication process [96]. Distinctive color change patterns were categorized to extract the underlying information. This sensor could be used for the detection of multiple analytes. Owing to the relatively high performance of the sensor array, the ammonia sensor showed an excellent detection limit of 0.3 ppm. A fluorescence sensor was developed for monitoring warfare nerve agents such as sarin [97]. The rapid quenching of fluorescence in 6,7-dihydroxycoumarin was an indicator for naked eyes under UV exposure. To date, microRNAs have been detected using a large-scale instrument. However, a convenient label-free polygonal-plate fluorescence biosensor was proposed for the on-site detection of microRNAs [98].

Different types of sensors have been reported using photonic crystals [99], [100], [101], [102], [103], as these structures can be manipulated by both regular and ordered 3-D array of microparticles. A change in optical properties is induced after the penetration of light through the photonic crystal structures. Therefore, these structures have also been widely used for sensor applications. A representative study was reported regarding the introduction of a 2D polystyrene crystalline colloidal array in a polymer hydrogel [99]. In a sensor using a colloidal array, the particle spacing changed from 917 to 824 nm depending on the analyte (glucose) concentration. Consequently, the sensor color changed from red to orange, finally to cyan. A similar concept was applied for sensing urea [100]. Stimuli-responsive photonic crystal hydrogel microbeads were incorporated into flexible sensors for the recognition of changes to the naked eye [101]. The strategy was expanded to produce user-friendly devices for control over the environment and healthcare hazards. Molecularly imprinted photonic crystals in hydrogels were employed for the detection of antibiotics in milk [102], [103].

Hydrogels also respond to thermal stimuli and pathogens. A thermo-responsive memory hydrogel was introduced on a glassy carbon electrode to obtain sensors for bovine serum albumin detection [104]. A virus-imprinted hydrogel was applied to a diffraction grating sensor using imprint lithography [105]. Monitoring changes in the electrical properties of hydrogels is also a promising strategy for biosensor fabrication [106], [107].