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Polymer Materials in Biosensors

K. E. Geckeler and B. Mt~ller Institut fur Organische Chemie der Universitfit, W-7400 Tt~bingen, FRG

Fundamentals and application examples of polymeric materials in different types of biosensors are presented and discussed in view of their molecular structure and biosensor design and construction. The role of a series of polymers with respect to their typical application and their specific properties, like sensitivity and stability, is highlighted. Future trends of polymer materials for biosensors in the area of medical and environmental applications are outlined. 18

iosensors are small, special sensor devices for the concentration determination of compounds with biological relevance on a molecular basis. Polymer materials are essential components of biosensors because most of them cannot be constructed at all without them. Generally, biosensors are composed of three units: the receptor as the biospecific component, the transducer, which converts the measured physical effect, and the electronic component which combines the different parts [1-4]. The basis of biosensors is reactions between the immobilized species and the molecules to be sensed which set up a physical signal. During the function of a biosensor, the following processes can be discerned: specific recognition of the analyte, transformation of the physicochemical parameter, which is a result of the interaction with the receptor, into a signal, and signal intensification and processing. According to the functional principle, biosensors are subdivided into several classes: bioaffinity sensors (alteration of the electron density), metabolism sensors (substrate consumption), coupled and hybrid systems, and biomimetic sensors [5-8]. A considerable number of different polymer materials have been investigated in view of their application in biosensors. At present, biosensots for about 100 different analytes have been described, however, only about a dozen have been commercialized [2]. The most important part of a biosensor in terms of the material component is based on organic polymers. Their use in biosensor devices serves to enhance the stability, biocompatibility, and selectivity. The application of such materials includes the use as supports for enzymes, antibodies, microorganisms, organelles, dyes [1-3, 9, 10], and as components for the construction of membranes in the transducer of field effect transistors, thermistors, optical signal transformers, and piezoelectrical crystals [2, 3]. Naturwissenschaften80, 18-24 (1993) @Springer-Verlag1993

er is the biocompatibility of the polymer materials used in biosensors [14, 15]. They play an increasing role in conjunction with artificial organs [16]. Dependent on their designed function, they can be found in different application forms like polymeric gels, functional polymers, electrically conducting polymers [17], redox mediators [4, 8, 18, 19], or optical fibers [4, 8]. A number of polymers used in biosensors are listed in Table 1.

Polymeri r Membrane

Receptor

i

I Transducer

i

Polymer

Field effect transistors

Electroactive polymers

Optical fibers

matrices

Mediators

Table 1. Polymer materials used in biosensors Name Polyacetylene Poly(acrylamide)

Repeat unit

Function

-c~=cH-cH-cn2c=o I

Ref.

Redox polymer [17] Polymer gel [4, 5, 7, 8]

NH2

Fig. 1. Application of polymers as receptors and transducers in biosensors

Function of Polymer Materials The applicability of polymer materials in the different components of biosensors is illustrated schematically in Fig. 1. They are mainly applied in the preparation of membranes (enzyme membranes, redox membranes, etc.). On the one hand, they are used in receptors in the form of a polymer matrix, redox polymer, or mediator, and on the other hand, they are introduced into transducers as ion-selective membranes (field effect transistors) and as optical fibers in optoelectronic sensors. The most extensive use is found in designing the receptors where different methods of immobilization are applied in order to attach the biologically active compound onto the surface of the receptor. Such compounds include enzymes, inhibitors, antigens, antibodies, cells, microorganisms, and other substrates [111. The following advantages can be attributed to the application of polymer materials in biosensors: • • • • • • •

stability enhancement sensitivity enhancement reduction of measuring time reusability of the enzyme no contamination of the analyte solution possibility of a continuous process low-cost production

Organic polymers essentially meet the required criteria of the physical and chemical properties for such materials [12]. Their features are partial hydrophobicity and hydrophilicity as well as hygroscopicity and electrical conductivity [13]. An important aspect to consid-

Polyaniline

-@~=@=,H-

Redox polymer [401

Polyethylene

-c~2-cH2-

Membrane

[4, 5,

7, 81 Poly(ethyleneimine) Poly(ethylenephthalate)

-CH2-eH2-NH-

-O-CH2CH20-C

@ ~

Poly(methacrylate) -CCCH3/-CH2c=o I

c-

~

Membrane

[41]

Enzyme membrane

[8]

Enzyme membrane

[22]

O-R

Poly(oxyethylene)- <~>-C-(OCH2CH21ferrocenes ~e o Mediator

[4, 7, 8, 42]

Polypropylene

[4, 51

-CH-CH2-

Membrane

CH 3

Polypyrrole

~

Redox polymer [301

Polystyrene

©

-CH-CH2-

Optical fiber

[s]

-cF~-CF2-

Membrane

[7]

@-..~(~./_

Redox polymer [17]

~o

Enzyme membrane

Poly(tetrafluoroethylene) Polythiophene Polytyramine

~

[261

CII2CH2NH 2

Polyurethane

-C-NH-~-NH-C-O-R-O- Enzyme g membrane [22] Poly(vinyl alcohol) -CH-CH2Polymer matrix [21] OH

Poly(vinyl butyral)

=?.-c.2-cHO

Poly(vinyl chloride)

\

/

Polymer gel

[22]

0

CH2CH2CH3

-C,H-CHzcl

Poly(vinyl pyrroli- -7H-C"2done) Q~.o

Polymer matrix [24] Enzyme membrane

[43]

19

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•:

mm

,.

l =

_..

//////// ADSORPTION

GS, CROSS-LINKING

==

me

//////// CROSS-LINKED ADSORPTION

-,

COVALENT FIXATION

I®/EL ENTRAPMENT

Fig. 2. Immobilization methods for biomolecules on polymer materials

Polymers for Immobilization Various methods are used for the immobilization of enzymes on polymeric materials [20]. A selection is schematically depicted in Fig. 2. In biosensors, gel entrapment and the covalent fixation onto modified, water-insoluble polymer supports play a dominant role. By using adsorption, the biomolecule is attached to the polymer by weak interactions like van-der-Waals forces, dipole-dipole interactions, or hydrogen bonding [8]. The advantage is an immobilization procedure without any reagent, and consequently, there is no danger of contaminating the solution to be analyzed by excess or cleaved components. However, the disadvantage is that a change in the reaction conditions (pH, temperature, etc.) can result in a weakening of the interaction forces followed by a diffusion-driven cleavage of the biomolecule. The entrapment of biologically active substances in polymeric gels is a very gentle method of immobilization and prevents the biomolecules from diffusing away from the reaction sites. With regard to the mild conditions it is comparable to the adsorption methods. The inclusion of biomolecules in the polymeric matrix is performed by light, X-rays, or the addition of water [4, 21]. 20

////////

ENCAPSULATION

The main representatives of the gel polymer group are poly(vinyl alcohol) (PVA), poly(vinyl chloride) (PVC), poly(acrylamide) (PAM), and polyurethane (PU). They are characterized by simple and universal handling properties and a very broad application field. PVA and PVC can be used for amperometric and potentiometric biosensors [4, 5, 7, 8]. PAM is applied analogously. Interestingly, a very high loading yield of enzyme in the polymer matrix was obtained by X-ray radiation of a mixture of PVA and enzyme [21]. However, the general use of this method is restricted by the radiation sensitivity of enzymes. Polyurethanes are generally used if the biomolecules are attached to the polymer by using bifunctional reagents like glutardialdehyde or diisocyanates [22, 23]. Figure 3 shows the percent distribution of the different polymer types for the immobilization by gel entrapment applications of biomolecules. Based on its universal applicability, poly(vinyl chloride) and poly(acrylamide) occupy major positions with 35 and 25 %. This method of immobilization is investigated further in view of its application regarding enzyme glucose oxidase (GOD) in Fig. 4. To this end, frequently nylon (23 %), for dialysis membranes, and ferrocene derivatives (20 %), for enzyme membranes, are exploited. Maximum sensitivity values (detection limits of the substrate) of polymers used in glucose sensors are summarized in Table 2 in order to demonstrate the influence of polymer materials. For comparison, Table 3

Table 2. Maximum sensitivity of glucose sensors depending on the polymer material applied in the membrane

Other

pu . - " f ~ ~

PVC

10 ~

Name

Sensitivity [retool/l]

Ref.

Collagen Polyurethane Organic conducting salta Poly(vinyl chloride) Poly(vinyl alcohol) Mediator chemically modified electrode Polypyrrole

0.01 0.05

[44] [22]

0.50 0.50 0.60

[45] [24] [21]

1.00 5.00

[36] [30]

5~

25 PAM

15 PVA

Fig. 3. Immobilization by gel entrapment: percentage of different polymers. PAM poly(acrylamide), PVA poly(vinyl alcohol), PVC poly(vinyl chloride), FU polyurethane

ai.e., TCNQ/NMP + = tetracyano-p-quinonedimethane and Nmethylphenazinium (for comparison) Table 3. Major advantages of some polymers in biosensors

I

I

I

I

I

[

0

5

10

15

20

25

Fig. 4. Percentage of different polymers for the immobilization of glucose oxidase (GOD). P-Fer poly(oxyethylene) ferrocenes, PAn polyaniline, PAM poly(acrylamide), PPy polypyrrole, PU polyurethane, PVA poly(vinl alcohol)

shows an evaluation of the advantages of the most common polymers for immobilized G O D in glucose sensors.

Advantages of Polymer Matrices As described above, the use of polymers essentially improves the properties and facilitates the construction of biosensors. The application of PVC membranes in urease sensors shows a significant decrease in the measuring time and an enlargement of the linear measuring area. The main advantage, however, is the variety of construction possibilities, e.g., the possibility of designing flow-through sensors for flow-injection analyses [24]. PVA has found major application as a basic material in glucose sensors. There, it is cross-linked with multifunctional agents (e.g., triisocyanate) or by

Polymer

Sensitivity

Stability

Measuring time

Polyurethane Poly(vinyl chloride) Poly(vinyl alcohol) Polypyrrole Mediator chemically modified electrode

+++ ++ ++ -

++ +++ ++ +

+++ ++ + ++

+

++

+

radiation with X-rays, following a mechanism via polymer radicals, in the presence of the enzyme glucose oxidase [25]. A significant reduction in the measuring time as well as a considerable sensitivity enhancement is thus observed. Depending on the radiation sensitivity, if other enzymes are used, allyl methacrylate is added as an additional agent to accelerate the crosslinking process [21]. The application of polyurethane to enzyme immobilization on silicium chips and field effect transistors in glucose and urease sensors is very advantageous and considerably extends the long-term stability (more than 10 months) [22].

Immobilization by Covalent Attachment Two different methods of covalent attachment are available: the direct polymer-analogous reaction of the biomolecule with a functionalized polymer support or the preparation by copolymerization of a suitable monomer. It is essential that only groups which do not contribute to the biological activity of the biomolecule are involved in the attachment reaction. The different stages of the attachment reaction include the activation of the polymeric support, the coupling reaction, and the removal of excess reactant. An example of a typical coupling process is the reaction of isocyanates with activated hydrogen atoms according to the following equation: 21

-NCO + HX--+-NH-CO-X. A variety of functional groups exist both in synthetic polymers and in biomolecules. For example, hydroxyl groups of compounds introduced into polyurethanes and amino groups of enzymes can be utilized for this purpose [22]. Similar to the polymer gels, the advantages of this immobilization technique include a reduction in the measuring time and enhanced sensitivity and stability of the corresponding biosensors. If the biomolecule is already immobilized in a polymer gel, additional crosslinking leads to a higher enzyme density of the membrane. However, a potential loss of enzyme activity should be taken into consideration in this case. Typical representatives of this concept are poly(vinyl alcohol)/GOD [21], polyurethane/diisocyanate/GOD [22], and poly(acrylamide) [25] materials or immunosensors like polytyramine/anti-IgG, where the linkage is attained between the amino group of the polymer and the carboxyl function of the antibody [26]. In Fig. 5 the measuring times of a series of polymers are compared. As conventional hydrogen ion glass electrodes (GE) have been used for the preparation of enzyme pH electrodes by either entrapping the enzyme within poly(acrylamide) gels around the electrode or as a liquid layer trapped within a cellophane membrane, they are also included for comparison [25]. This value is about four to five times higher than those of membranes based on organic polymers. The long-term stability of several polymers used in biosensors, which is dependent on the enzyme loading and the immobilization applied, is compared in Fig. 6. The salient examples are PVC and PU membranes with a stability in the range of 10 to 12 months.

400 lIE

300"

Time (s) 200

100"

HCHE PVC

Coil.

Polymer Fig. 5. Influence of the polymer type on the measuring time of glucose sensors. PU polyurethane, PVC poly(vinyl chloride), Coll. collagen, PPy polypyrrole, PVA poly(vinylalcohol), MCME mediator chemically modified electrode, GE glass electrode (enzyme pH electrode)

~ ~ e

i Nylon diat r chemical modified electrode

Polymer

0

2

4

6

8

Stability (months)

Use of Redox Polymers To enhance the selectivity, the concept of the ion-gate membrane was developed. The permeability for ions is in this case regulated by the redox state of the membrane material. This group includes polymers which are conducting in the oxidized form but insulating in the reduced form [4, 8]. Heteroaromatic and aromatic-type materials like polyaniline, polypyrrole, and polythiophene as well as linear polymers like polyacetylene are suitable for this application [17,27-29]. Preparatively, the biomolecules are added to the monomer and then the mixture is electropolymerized [8, 30]. Redox polymers are applied in I on-Selective Field Fffect Transistors (ISFETs), Enzyme-Sensitive Field Fffect Transistors (ESFETs), and in amperometric electrodes [31-34]. Another possibility for the electron transfer in such systems is the use of redox mediators. These include polymer-bound ferrocenes [19] and 22

10 12 14

Fig. 6. Maximum stability of glucose sensors depending on different polymer materials. PVC poly(vinyl chloride) also the application of conducting organic salts [7]. Among the methods used to prepare mediator chemically modified electrodes (MCME), as described in the literature [35], the most successful was that based on ferrocene derivatives [36]. The prepolymer or polymer chain (spacer) of these derivatives can be easily varied and adjusted to the desired conditions. Also, electrodes constructed with such materials on the basis of polycarbonate dialysis membranes are characterized by small electrochemical interferences, long lifetimes, and short measuring times. The electroactive polymers, which are most in use, are shown in Fig. 7. Among these, polypyrrole and polyaniline play the leading role with more than 30 % and 15 %, respectively, due to their versatile applicability.

10,1t ~ 1

[] PPy [] PAn [] Pth [] PAr [] PAc [] Other

i 7. H0~

Fig. 7. Electroactive polymer materials in enzyme membranes: statistical distribution. PPy polypyrrole, FAn polyaniline, Pth polythiophene, PAr polyaromates, PAc polyacetylene

Q

Enzyme

•

Indicator

dye

Fig. 8. Scheme of an intrinsic optical enzyme sensor Another advantage, in addition to those already mentioned, is the high enzyme loading density of the receptor. The reason for this is that electropolymerization is used for immobilization [30].

nent use must be considered. As a result, optimized materials can be mass produced at low cost.

Future Trends Polymers in Optical Biosensors The principle of optical biosensors is based on the detection of substrates using UV/VIS and infrared spectroscopy, fluorescence, phosphorescence, bio- and chemiluminescence as well as Rayleigh and Raman scattering. Here, the function of the polymer is also to immobilize the biomolecules in the receptor. The ideal biosensor requires molecules with biospecific recognition which are immobilized on the transducer surface of the biosensor. In the same way, macromolecules like polystyrene are used for optical fibers which are needed to build intrinsic optical enzyme sensors. The scheme of such a sensor is shown in Fig. 8. Furthermore, polymers are required for the immobilization of indicator molecules on the surface of optical fibers. In this type of sensor, the determination of substrate is made by ellipsometry, and fluorescence and reflection spectroscopy [8, 37]. As shown by the data presented here, the main task is optimum macromolecular immobilization of the biomolecules on the membrane. This means that a high enzyme activity in a very thin layer and a high enzyme loading of the polymeric material result in optimal sensitivity, stability of function, and short measuring periods. Other applications of polymers in biosensors attempt to improve their physical properties and to develop multi-analyte devices. The main focus is both the extension of the long-term stability, which is determined by the enzyme activity and concentration, and the optimization of the measuring sensitivity. In addition, economic aspects and the possibility of perma-

One of the main obstacles that currently inhibit rapid progress in biosensor research is the lack of polymer materials with the suitable properties. Enormous efforts in materials research are needed to meet the technological challenge and for an early breakthrough. Significant progress must be made also in in-vivo research with major emphasis on biocompatibility. New trends, e.g., the substitution of the labile biological component by artificial equivalents like synzymes, can essentially improve the stability and reusability of biosensors. Synzymes are artificial enzymes which consist of stable, synthetic polymers with functional moieties that mimic the natural enzyme activity [12, 38]. Poly(ethyleneimine)-based materials exhibit a broad spectrum of applicability in this context [38, 39]. The durability was reported to be extended from less than 1 week using a natural enzyme to at least 6 months with the artificial enzyme [38]. The synthesis and application of such artificial systems represent an interesting challenge for interdisciplinary-oriented polymer chemists to meet the needs for biosensor application. The development of tailor-made polymers will lead to considerable miniaturization of the biosensor architecture and to increased efficiency and optimized performance in terms of selectivity, sensitivity, and stability. In addition, we anticipate that the spectrum of substances to be determined will be impressively enlarged, thus fulfilling the dramatically increasing demands in biotechnology as well as in biomedical and environmental analysis. 23

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24

24. 25. 26. 27.

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