**2.2 Recent development of biosensor researches**

Electrochemical biosensors are the oldest and most widely available group in the solid-state chemical sensor field. Electrochemical sensors provide a crucial analytical tool as demand for

**Figure 2.** Illustration of enzyme immobilization methods

sensitive, rapid, and selective determination of analytes increases. Over the past two decades we have witnessed a tremendous amount of activity in the area of biosensors. Enzyme based electrodes require the immobilization of an enzyme onto an electrode surface for the quanti‐ fication of an analyte and hold a leading position among biosensor systems presently available. Biosensors have found promising applications in various fields such as biotechnology, food and agriculture product processing, health care, medicine and pollution monitoring. Recent development has focused on improving the immobilization and stability of the enzymes. There have been a number of recent review articles that have focused on the development of various materials, techniques, and applications of biosensors [19-24]. Since there have been a wealth of biosensor developments in the past years, new approaches and materials for enzyme based sensors have been primarily focused. Strategies for incorporating materials to enhance speed, sensitivity, and stability of these sensors has been of particular interest. Major advancements in biosensors revolve around immobilization and interface capabilities of the biological material with the electrode. The use of polymer and nanomaterials has provided a means for increasing the signal response from these types of sensors. Moreover, the combination of various nanomaterials into composites in order to explore their synergistic effects has become an interesting area of research. The ability to incorporate biomaterials with the potential for direct electron transfer is another growing research area in this field [25-31]. In general, we believe that the field of electrochemical sensors will focus on the incorporation and interaction of unique materials, both nano and biological, in the coming years.

## **3. Polymers coating in biosensors**

Enzyme immobilization using supports have been of great interest for many researches. Various supporting films on electrode surface have been developed to immobilize proteins or enzymes and many polymeric materials were used for enzyme immobilizations. Polystyrene (PS) membrane is a very promising support for the immobilization of enzymes due to its excellent biocompatibility, no toxicity, high affinity, strong adsorption ability, low molecules permeability, physical rigidity and the chemical inertness in biological processes. Its molecular structure is shown in Fig. 3. It is popular for the immobilization in enzyme-linked immuno‐ sorbent assays (ELIA) by adsorption, however, and few methods for immobilizing proteins on the PS surface by covalent bonding have been proposed, because the complicated multistep methods must be employed for introduction of functional groups that react with proteins and their procedures are tedious and time consuming. In order to solve the difficulty of introduction of functional groups, we adopted polymaleimidostyrene (PMS) to introduce maleimide group in the bulk of PS, and the coating of enzyme containing PS membrane on the electrode surface under mild conditions opens up enormous possibilities for the immobiliza‐ tion of biomolecules [32-40].

**Figure 3.** Molecular structure of polystyrene

sensitive, rapid, and selective determination of analytes increases. Over the past two decades we have witnessed a tremendous amount of activity in the area of biosensors. Enzyme based electrodes require the immobilization of an enzyme onto an electrode surface for the quanti‐ fication of an analyte and hold a leading position among biosensor systems presently available. Biosensors have found promising applications in various fields such as biotechnology, food and agriculture product processing, health care, medicine and pollution monitoring. Recent development has focused on improving the immobilization and stability of the enzymes. There have been a number of recent review articles that have focused on the development of various

self -assembly cross-linking

entrapment

encapsulation

adsorption

70 State of the Art in Biosensors - General Aspects

covalet binding

micelle

**Figure 2.** Illustration of enzyme immobilization methods

The interferents and biofouling are two major problems which can affect the performance of a biosensor. Interference from electroactive substances is especially problematic when electrochemical measurements are being made in vivo. Biocompatible membranes are preferable both as a selective barrier as well as for enhancing biocompatibility within electro‐ chemical biosensors [41]. The cellulose acetate layer permits only small molecules, such as hydrogen peroxide to reach the electrode, eliminating many electrochemically-active com‐ pounds that could interfere with the measurement. Fig. 4 depicts the molecular structure of cellulose.

**Figure 4.** Molecular structure of cellulose

Nafion, a perfluorinated sulfonated cation exchanger (see Fig. 5), has been widely used as an electrode modifier due to the chemical inertness, thermal stability, mechanical strength, and antifouling properties. Nafion coated electrodes have been applied in the analysis of phenol, for the determination of parathion [42, 43]. Nafion has also been widely utilised as a coating material. The polymer displays the advantages of being chemically inert and easily cast from solution. The polymer is anionic and upon casting forms a structure with hydrophilic channels contained within a hydrophobic matrix. Films formed from this material are reasonably robust, show strong exclusion of anionic interferents and display enhanced biocompatibility.

As functional materials, chitin and chitosan offer a unique set of characteristics: biocompati‐ bility, biodegradability to harmless products, nontoxicity, physiological inertness, antibacte‐ rial properties, heavy metal ions chelation, high affinity to proteins, gel forming properties and hydrophilicity, remarkable affinity to proteins, availability of reactive functional groups for direct reactions with enzymes and for chemical modifications, mechanical stability and rigidity, and ease of preparation in different geometrical configurations that provide the system with permeability and surface area suitable for a chosen biotransformation [44]. Owing to these characteristics, chitin and chitosan offer a unique set of these characteristics and are predicted to be widely exploited in the near future especially in enzyme immobilization supports. The most distinguishing chitosan properties are its biodegradability and biocom‐ patibility, which makes it a green polymer. The increasing importance of materials from renewable sources has put chitosans in the spotlight, especially due to their biological

**Figure 5.** Molecular structure of Nafion

The interferents and biofouling are two major problems which can affect the performance of a biosensor. Interference from electroactive substances is especially problematic when electrochemical measurements are being made in vivo. Biocompatible membranes are preferable both as a selective barrier as well as for enhancing biocompatibility within electro‐ chemical biosensors [41]. The cellulose acetate layer permits only small molecules, such as hydrogen peroxide to reach the electrode, eliminating many electrochemically-active com‐ pounds that could interfere with the measurement. Fig. 4 depicts the molecular structure of

O

Nafion, a perfluorinated sulfonated cation exchanger (see Fig. 5), has been widely used as an electrode modifier due to the chemical inertness, thermal stability, mechanical strength, and antifouling properties. Nafion coated electrodes have been applied in the analysis of phenol, for the determination of parathion [42, 43]. Nafion has also been widely utilised as a coating material. The polymer displays the advantages of being chemically inert and easily cast from solution. The polymer is anionic and upon casting forms a structure with hydrophilic channels contained within a hydrophobic matrix. Films formed from this material are reasonably robust,

show strong exclusion of anionic interferents and display enhanced biocompatibility.

As functional materials, chitin and chitosan offer a unique set of characteristics: biocompati‐ bility, biodegradability to harmless products, nontoxicity, physiological inertness, antibacte‐ rial properties, heavy metal ions chelation, high affinity to proteins, gel forming properties and hydrophilicity, remarkable affinity to proteins, availability of reactive functional groups for direct reactions with enzymes and for chemical modifications, mechanical stability and rigidity, and ease of preparation in different geometrical configurations that provide the system with permeability and surface area suitable for a chosen biotransformation [44]. Owing to these characteristics, chitin and chitosan offer a unique set of these characteristics and are predicted to be widely exploited in the near future especially in enzyme immobilization supports. The most distinguishing chitosan properties are its biodegradability and biocom‐ patibility, which makes it a green polymer. The increasing importance of materials from renewable sources has put chitosans in the spotlight, especially due to their biological

HO

O

O

n

OH

OH

O

OH

OH

cellulose.

HO

**Figure 4.** Molecular structure of cellulose

O

72 State of the Art in Biosensors - General Aspects

**Figure 6.** Molecular structure of chitin

properties, which have been exploited in many applications [45, 46]. The molecular structure of chitin and chitosan are shown in Fig. 6 and Fig. 7, respectively.

## **4. Polymaleimidostyrene in biosensors**

Polymers are becoming inseparable from biomolecule immobilization strategies and biosensor platforms. Their original role as electrical insulators has been progressively substituted by their electrical conductiveabilities, which opens a new and broad scope of applications in both the physical adsorption and chemical coupling methods, protein molecules are immobilized on the

**Figure 7.** Molecular structure of chitosan

surface with random orientations and are likely to be denatured. In order to increase the lifetime stability of enzyme electrode, it is necessary that there should be a strong and an efficient bonding between the enzymes and immobilizing material. Hence, covalent binding of enzyme on a stabilizer or on the transducer is an efficient method of immobilization. Recently, we have developedanadvanceddesignandpreparationofenzyme-basedamperometricbiosensorsusing enzyme reverse micelle membrane, as well as the functional structure and principle. Particular emphasis is directed to the discussion and exploration of electrochemical biosensors based on novel functional polymer, polymaleimidostyrene (PMS), as effective immobilization stabilizer assupport.Itcanbeexpectedtobeacommonmethodfortheimmobilizationofenzymestofabricate various bioelectrochemical sensors [32-40].

#### **4.1. Synthesis, structure and properties**

PMSwhichisapolymerizationofN-4-vinylphenylmaleimide(N-VPMI)isanewtypeofpolymer mixible with polystyrene (PS). It was synthesized by Prof. Hagiwara's group in 1991 [34]. The compound was prepared by a modified method for synthesis of N-phenylmaleimide (N-PMI).N-VPMIpurifiedbyrecrystallizationwasusedasamonomerafterthoroughlydriedbelow 20 ℃ under reduced pressure. Polymerization was started by the addition of a tetrahydrofur‐ an(THF) solutionofinitiatortothemonomer solutionatadeterminedtemperaturewithstirring. The synthesis scheme and the molecular structure of PMS are depicted in Scheme. 1.

PMS possesses two polymerizable carbon-carbon double bonds with different reactivities, one of which is the vinylene group of the maleimide moiety and the other the vinyl group of the styrene moiety. The vinylene groups of a maleimide moiety react easily with sulphydryl or amino groups of enzyme by covalent bonds which prevent the unfolding of enzyme. PMS is a very effective, important and useful reagent to immobilize enzyme strongly via covalent bond, because high density of maleimide groups of PMS can catch not only exposed SH groups but also buried SH groups forming enzyme micelles [35−36]. To model the enzyme micelle structure, an illustration is displayed in Fig. 8. The hydrophobic PMS groups are outside the structure shielding the hydrophilic enzyme inside the interior. Therefore, the structure is named as reverse micelle. The reverse micelle structure tends to evolve to a lower-energy configuration under equilibrious conditions.

**Scheme 1.** Synthesis of polymaleimidostyrene

surface with random orientations and are likely to be denatured. In order to increase the lifetime stability of enzyme electrode, it is necessary that there should be a strong and an efficient bonding between the enzymes and immobilizing material. Hence, covalent binding of enzyme on a stabilizer or on the transducer is an efficient method of immobilization. Recently, we have developedanadvanceddesignandpreparationofenzyme-basedamperometricbiosensorsusing enzyme reverse micelle membrane, as well as the functional structure and principle. Particular emphasis is directed to the discussion and exploration of electrochemical biosensors based on novel functional polymer, polymaleimidostyrene (PMS), as effective immobilization stabilizer assupport.Itcanbeexpectedtobeacommonmethodfortheimmobilizationofenzymestofabricate

O

O

OH

NH2

OH

NH2

HO

O

n

OH

PMSwhichisapolymerizationofN-4-vinylphenylmaleimide(N-VPMI)isanewtypeofpolymer mixible with polystyrene (PS). It was synthesized by Prof. Hagiwara's group in 1991 [34]. The compound was prepared by a modified method for synthesis of N-phenylmaleimide (N-PMI).N-VPMIpurifiedbyrecrystallizationwasusedasamonomerafterthoroughlydriedbelow 20 ℃ under reduced pressure. Polymerization was started by the addition of a tetrahydrofur‐ an(THF) solutionofinitiatortothemonomer solutionatadeterminedtemperaturewithstirring.

PMS possesses two polymerizable carbon-carbon double bonds with different reactivities, one of which is the vinylene group of the maleimide moiety and the other the vinyl group of the styrene moiety. The vinylene groups of a maleimide moiety react easily with sulphydryl or amino groups of enzyme by covalent bonds which prevent the unfolding of enzyme. PMS is a very effective, important and useful reagent to immobilize enzyme strongly via covalent bond, because high density of maleimide groups of PMS can catch not only exposed SH groups but also buried SH groups forming enzyme micelles [35−36]. To model the enzyme micelle structure, an illustration is displayed in Fig. 8. The hydrophobic PMS groups are outside the structure shielding the hydrophilic enzyme inside the interior. Therefore, the structure is named as reverse micelle. The reverse micelle structure tends to evolve to a lower-energy

The synthesis scheme and the molecular structure of PMS are depicted in Scheme. 1.

various bioelectrochemical sensors [32-40].

O

NH2

**Figure 7.** Molecular structure of chitosan

O

HO

OH

74 State of the Art in Biosensors - General Aspects

HO

HO

**4.1. Synthesis, structure and properties**

configuration under equilibrious conditions.

**Figure 8.** Illustration of enzyme reserve micelle in polystyrene film

Although, it should be mentioned that the free enzyme exists in the outer surface of micelle may lose its activity due to the hydrophobility of chloroform reagent, which was used to dissolve PS and PMS, thus there is partly loss of enzyme activity during the immobilization, the strong adsorption ability of PS membrane makes the enzyme micelle based biosensors particularly attractive for on-line analytical systems which need high stability and good endurability due to the long-time and continuous determinations in a flow system during experimentation.

Furthermore, PMS exhibits a strong affinity specific to a variety of hydrophobic materials such as polystyrene, polyethylene and polyetheretherketone due to the styrene moiety. Then PMS is considered to be an ideal stabilizer for both covalent bonding enzyme and hydrophobic affinity to PS film [30−32]. The application of PMS as a convenient immobilization reagent is summarized in table 1. PMS appears to be effective and promising for the maintenance of biological activity as well as long time stability.

#### **4.2. Application of PMS to biosensors**

It has been found that the urease reverse micelle membrane exhibits good sensitivity after stored in a phosphate buffer solution (0.1 M, PH 5.5) for one month compared with its initial sensitivity. The ideal immobilization process should be easy, quick, and enzyme friendly, result in high loading surface. In our previous study, enzyme micelle membrane, which is onestep immobilization, has been proved to be an excellent immobilization method to preserve enzyme conformation resulting in good activity and stability. To form enzyme micelle, hydrophilic PMS covalently bonded-enzyme (via both exposed and buried sulfhydryl groups and amino groups of enzyme and maleimide groups of PMS) was dispersed in hydrophobic PS solution. PMS-bonded enzyme aggregated to form micelle with enzyme inside the structure and PMS outside. The enzyme micelles were immobilized on the surface of glassy carbon electrode (GCE) utilizing PS, which has admirable properties of biocompatibility and strong adsorption ability. PMS also possess good biocompatibility, the PMS-bonded enzyme exhibits good activity due to PMS is a significantly excellent stabilizer for enzyme. On the other hand, it is amazing that the free enzyme exists in the inner part of micelle enhanced the stability of enzyme. The applications of PMS as a function polymer have been published [34-40] and were summarized in table 1.
