Some new aspects in biosensors

Reviews in Molecular Biotechnology 82 Ž2002. 303᎐323

Some new aspects in biosensors Shaojun DongU , Xu Chen State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China

Abstract This paper reviews recent advances in biosensors contributed mainly by our laboratory. The biosensors, based on the new immobilization materials ᎏ sol᎐gel organic᎐inorganic hybrid materials, cryohydrogel Žor organohydrogel. and bilayer lipid membranes, are presented. The analytical performances of the biosensors are discussed. Applications of the biosensors in extreme environment are emphasized. A new generation of biosensors ᎏ surface plasmon resonance biosensors and capacitance biosensors, are also described. 䊚 2002 Published by Elsevier Science B.V. Keywords: Biosensors; Immobilization materials; Analytical performance

1. Introduction Since Leland C. Clark fabricated the first enzyme electrode, biosensors have been an attractive and popular field. Such devices are based on incorporating some kind of biological element in a sensing layer intimately connected with a transducer. Due to its simplicity, high sensitivity and potential ability for real-time and on-site analysis, biosensors have been widely applied in various fields including industrial process, clinical detection, environmental control and so on. Great efforts have been made by different groups to improve the selectivity and sensitivity of the sensing layer, to explore new concepts in transduction modes, and to miniaturize both the U

Corresponding author. E-mail address: [email protected] ŽS. Dong..

probes and related smart-signal processing systems. Rapid advances in biosensors have been achieved over the past few years, such as rapid growth of DNA sensors, introduction of advanced sensing materials, application of quartz-based piezoelectric oscillators and surface acoustic wave-detectors. New generation of biosensors combining of new bioreceptors with the evergrowing number of transducers is emerging. This review focuses on recent development of biosensors in our laboratory.

2. Amperometric biosensors based on sol–gelderived organic–inorganic hybrid materials Electrochemical sensors, especially amperometric biosensors, hold a leading position among various biosensors and many research papers have

1389-0352r02r$ - see front matter 䊚 2002 Published by Elsevier Science B.V. PII: S 1 3 8 9 - 0 3 5 2 Ž 0 1 . 0 0 0 4 8 - 4

304

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323

been devoted to them. A very important factor in enzyme-based amperometric biosensors development is the immobilization of enzymes. Commonly used immobilization protocols include physical entrapment Žbehind semi-permeable membranes or within a polymeric film., covalent binding or cross-linking using multi-functional reagents, and non-covalent schemes such as surface adsorption or mixing within the bulk of composite electrode materials. While offering an effective interface, some of these procedures are tedious, result in poor stability and perturbed function, require expensive reagents, or environmentally unattractive solvents. New immobilization schemes and advanced sensing materials are highly desired for improving the analytical capabilities of biosensing devices, and for meeting the challenges posed by complex environmental and clinical samples ŽWang, 1999.. Sol᎐gel-derived glasses have emerged in recent years as a new class of material well suited for the immobilization of biomolecules ŽDave et al., 1994; Lev et al., 1995.. This inorganic material is particularly attractive for the fabrication of biosensors since it possesses physical rigidity, chemical inertness, high photochemical and thermal stability, excellent optical transparency, and experiences negligible swelling in aqueous and organic solvents. But its cracking is still a major problem in this field. Recently, our group synthesized a selfgelatinizable grafting copolymer of polyŽvinyl alcohol. and 4-vinylpyridine ŽPVA-g-PVP. ŽLi et al., 1998. and hybridized this copolymer into silica sol, and successfully developed an organic᎐ inorganic hybrid thin film ŽWang et al., 1998.. The sol᎐gelrhydrogel hybrid material combines sol᎐gel rigidity with the biocompatibility of hydrogel and overcomes both shortcomings Žthe cracking of the silica glass and the swelling of the hydrogel in the solution.. Fourier-transform infrared ŽFTIR. spectroscopy, scanning electron micrograph ŽSEM. and quartz crystal microbalance ŽQCM. have been employed to characterize the hybrid thin film. The results demonstrate that the hybrid film contains a large amount of hydroxyl groups and hydrogen bond, and two components form interpenetrating network. In addition, enzyme leaching was not observed when enzyme

molecules were encapsulated in the composite film. Several high-stable amperometric biosensors based on this hybrid material were constructed in our laboratory. 2.1. Amperometric glucose biosensor based on sol᎐gel organic᎐inorganic hybrid material Glucose oxidase ŽGOD. as a model enzyme was firstly immobilized in sol᎐gelrcopolymer hybrid matrix to develop amperometric glucose biosensor ŽWang et al., 1998.. Fig. 1 shows a current᎐time plot of the enzyme electrode on successive step changes of glucose concentration. The sensor exhibits rapid response Ž11 s. due to fast diffusion of the substrate molecule in the thin hybrid film. On the other hand, the enzyme electrode containing 1% Žwrw. GOD also has a high sensitivity Ž600 nA mMy1 .. Because large quantities of hydroxyl groups in the hydrogel may stabilize the activity of the enzyme ŽDong and Guo, 1994b., the hybrid matrix can provide good biocompatible microenvironment and retain the activity of the enzyme to a large extent. Moreover, the biosensor has good operational and storage stability. The enzyme electrode was measured intermittently Ževery 2᎐3 days., and no apparent change of the response to 0.4 mM glucose after 150 days was found. The glucose biosensor was used to practically

Fig. 1. Typical steady-state responses of the enzyme electrode on increasing the concentration of glucose in 0.4 mM steps. Potential, 1050 mV; electrolyte, pH 7.5 PBS; enzyme-loading, 1.0% Žwrw. ŽWang et al., 1998..

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323

detect human plasma samples. To avoid the influence of chemical interferences, redox mediator tetrathiafulvalene ŽTTF. and Nafion film were introduced and a TTFrhybrid-GODrNafion electrode was constructed. The contents of glucose in blood were assayed by using the mediated enzyme electrode. The results are satisfactory and agree closely with those measured by the spectrophotometric method in the hospital. 2.2. Amperometric hydrogen peroxide biosensors based on sol᎐gel organic᎐inorganic hybrid material Hydrogen peroxide is the product of the reactions catalyzed by a large number of oxidases, and it is essential in chemical, biological, clinical and many other fields, so the determination of hydrogen peroxide is practically important. Several mediated amperometric H 2 O 2 biosensors, based on immobilization of horseradish peroxidase ŽHRP. in the sol᎐gelrcopolymer hybrid matrix, have been fabricated. Table 1 gives the analytical performances of four H 2 O 2 biosensors fabricated. Firstly, potassium hexacyanoferrate ŽII. ŽWang et al., 2000b. was used as a mediator because of its high electron transfer efficiency. The sensor exhibits fastest response and widest linear scope among the four H 2 O 2 sensors. Moreover, high K mapp of the sensor indicates the electrode has a high affinity for H 2 O 2 . Because hydrogen perox-

305

ide is often used as a preservative agent in milk, the sensor was used to assay milk samples from different sources. The results, calculated from the calibration curve, agree well with those measured by the standard spectrophotometric method. Sensors by using mediator in solution are not desirable in situ monitoring and in vivo analysis. So it would be preferable to immobilize a mediator on the electrode surface. We constructed a reagentless H 2 O 2 biosensor ŽChen et al., 2001a.. TTF, as a mediator, was adsorbed on the electrode surface, and then sol᎐gelrcopolymer film including HRP was coated on the TTF-modified electrode. The biosensor shows good selectivity in the presence of 0.4 mM H 2 O 2 . Addition of glucose, sucrose, acetic acid and citric acid in the same concentration as that of H 2 O 2 separately did not cause any observable interference. However, addition of 0.4 mM ascorbic acid and 0.1 mM sulfide reduced the biosensor response by about 10 and 27%. Moreover, water-soluble dyes, methylene green ŽMG. was immobilized on the surface of Nafion-modified electrode by the electrostatic force between MGq and the negatively charged sulfonic acid groups in Nafion polymer. A hydrogen peroxide biosensor by coating a sol᎐gelperoxidase layer onto a Nafion᎐methylene green modified electrode was fabricated ŽWang and Dong, 2000b.. However, when the enzyme elec-

Table 1 Analytical performances of four different hydrogen peroxide biosensors based on immobilization HRP in sol᎐gelrcopolymer hybrid materials Mediator

Existing model

Sensitivity Ž␮A mMy1 .

Response time Žs.

K4 FeŽCN.6

Dissolved in buffer solution Adsorbed on surface of the electrode Adsorbed in Nafion polymer by electrostatic force Adsorbed in FIOHMa membrane by electrostatic force

15

10

TTF Methylene green

Meldola’s blue

a

6.69

15

13.5

20

75

25

Functionalized inorganic᎐organic hybrid material.

Line range ŽmM. 0.1᎐3.4 y1.3 0.5᎐1.6

y0.6

Km app ŽmM.

Reference

4.6

Wang et al., 2000b

1.5

Chen et al., 2001a

2.1

Wang and Dong, 2000b

᎐

Zhang et al., 1999

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323

306

trode was used three times a day, the response of the sensor decreased to 91% of its initial response after 1 week due to the release of MG from the film. Another method for immobilization of dyes on the surface of electrode is the use of organic modification of sol᎐gel derived matrices, which introduce reactive functional groups that can be subsequently used for anchoring of molecular recognition species on pre-prepared xerogel ŽLev et al., 1995.. A sulfonated sol᎐gel derived membrane was prepared by oxidation of thiol groups in 3-mercaptopropyltrimethoxysilane. Similar to Nafion-modified electrode, meldola’s blue ŽMDB. was incorporated into the membrane. The functionalized inorganic᎐organic hybrid material ŽFIOHM. derived by sol᎐gel was developed for construction of mediated amperometric hydrogen peroxide biosensor ŽZhang et al., 1999.. Cyclic voltammograms of MDB incorporated within FIOHM exhibit more reversible electrochemical characteristics than those of the solution species. The stability of incorporated MDB is very good. No noticeable decrease in Faradic current was observed when the electrode was immersed in 0.1 M phosphate buffer for 10 h. This is helpful to extend the lifetime of the biosensor. 2.3. Amperometric tyrosinase-based biosensors based on silic sol᎐gel hybrid film From our previous results, sol᎐gel organic᎐ inorganic hybrid materials are a kind of good matrix that can effectively immobilize of enzymes and maintain its activity to a large extent. Therefore, we immobilized tyrosinase in sol᎐gel thin film and constructed an am perom etric

tyrosinase-based biosensor in aqueous phase ŽWang et al., 2000c.. The analytical performances of the biosensor for five phenolic compounds were tested. Table 2 lists the response characteristics of the biosensor including response time, sensitivity, linear range and detection limit for several phenolic compounds. Response characteristics vary with the substitution group of phenolic compounds. Among the five phenolic compounds, o-cresol and o-aminophenol do not yield any signal, while the other compounds give a fast response within 20 s. Their sensitivities follow the order as: catechol ) p-cresol ) phenol. Moreover, organic᎐phase tyrosinase enzyme electrode based on a functionalized sol᎐gel composite was also fabricated for phenolic determination in organic media ŽWang and Dong, 2000a.. The composition of the sol᎐gel matrix was optimized. In view of high sensitivity and good stability, polyŽvinyl alcohol. and 3-aminopropyltrimethoxysilane were used as composition of the sol᎐gel hybrid film in this work. The specific apparent activity of tyrosinase immobilized in the matrix was determined to be 35% of the soluble enzyme with spectrophotometry. The resulting sensor exhibits sensitive response to several substrates in chloroform containing 0.1 M TBAP saturated with 0.01 M phosphate buffer ŽpH 7.0.. This is convenient to detect phenolic compounds in organic solvents. 2.4. A hydrogen peroxide biosensor based on sol᎐gel-deri¨ ed glasses doped with Eastman AQ polymer As mentioned above, the biosensors are based on sol᎐gelrcopolymer hybrid matrices. Similar to above construction, we have also used a poly

Table 2 Response characteristics of the tyrosinase biosensor to several phenolic compounds ŽWang et al., 2000c. Compound

Catechol p-Cresol o-Cresol Phenol o-Aminophenol a

Response time Žs. 16 20 ᎐ 17 ᎐

Sensitivity Ž␮A mMy1 .

Linear range Ž␮M.

Ra

Detection limit Ž␮M.

59.6 39.4 0 23.1 0

0.1᎐100 0.1᎐230 ᎐ 0.2᎐160 ᎐

0.994 0.999 ᎐ 0.999 ᎐

0.04 0.05 ᎐ 0.1 ᎐

Correlation coefficient of the linear range.

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323

Žester sulfonic acid.-Eastman AQ 55D doped sol᎐gel-derived glasses as a matrix for the immobilization of enzyme ŽChen et al., 2001b.. It is well known that poly Žester sulfonic acid.-Eastman AQ is a typical ionomer, which is composed of a hydrophobic polyester backbone and sulfonated exchange sites, and shows strong cation exchange ability due to the sulphonate groups in the polymer network ŽGennett and Purdy, 1990.. Moreover, Eastman AQ film has good biocompatibility ŽFortier et al., 1990; Wang et al., 1991.. Hence, sol᎐gel-derived glasses combined with Eastman AQ 55D can not only form good film and retain high enzyme activity, but also immobilize mediators firmly through the electrostatic interaction and greatly improve the performance of biosensor. HRP was selected as a model enzyme with thionine ŽTH. as a mediator. The linear range of the sensor spans the concentration of H 2 O 2 from 2 ␮m to 0.4 mM and the sensitivity of the biosensor is 11.36 ␮A mMy1 . Compared with the biosensor in the absence of Eastman AQ 55D polymer, the sensitivity of this biosensor has been greatly enhanced due to strong interaction between THq and the sulphonate groups in the polymer network. On the other hand, the biosensor is more sensitive than that of H 2 O 2 biosensor by glutaraldehyde cross-linked method ŽLiu et al., 1996. because both Eastman AQ and sol᎐gel matrix could maintain the activity of enzyme to a larger extent ŽDave et al., 1994; Wang et al., 1991.. Moreover, TH and HRP can be together immobilized within the same film and contact directly with each other, so that there is shorter distance between mediator molecules and the enzyme active center than those where the mediator layer was sandwiched between the enzyme layer and the electrode. Hence, the mediator molecules can more efficiently mediate the electrons from the base electrode to the enzyme. The sensor has a detection limit of 5 = 10y7 M at a signal to noise ratio of 3.

3. Electrochemical biosensors in extreme environment Over the past 30 years, growing interest in

307

biosensor development has resulted in increasingly widespread applications. The reported biosensors are usually used in neutral aqueous solution. However, in the practical application the detected substrates sometimes exist in extreme environment. In order to meet the specific requirement, different kinds of biosensors have been developed ŽWang and Lu, 1998; Karyakin et al., 1996; Zhang et al., 1994.. In addition, indirect biosensing strategy is also employed to determine inhibitor ŽWang et al., 1993; Stancik et al., 1995. and trace water ŽWang and Reviejo, 1993.. These largely expand the application scope of biosensors. Here, several special electrochemical biosensors developed in our laboratory were described. 3.1. Hydrogen peroxide biosensors in acid medium Usually amperometric biosensors for hydrogen peroxide are operated in neutral and weak alkaline medium. However, in fermentation industry, food industry and environmental analyses, the samples containing H 2 O 2 exhibit weak acidity. Therefore, the detection of H 2 O 2 in acid medium is important and expedient. Recently, a very stable peroxidase-soybean peroxidase ŽSBP. has become commercially available ŽVreeke et al., 1995.. It can maintain its activity in broad pH range, which makes it possible to sensing H 2 O 2 in acid medium. According to this idea, we purified a kind of SBP from Chinese soybean seed coat by successive aqueous two-phase partition, chromatography on Mono-Q fast flow and butylsuperose fast flow. The activity of soybean peroxidase is 200 pyrogallol U mgy1 , which is much higher than that of Sigma product Ž130 pyrogallol U mgy1 .. The peroxidase maintains its activity in a broad pH range from 3 to 10 and exhibits notable thermostability ŽT1r2 is 20 h at 60 ⬚C.. The enzyme, for the first time, was used to construct acid-stable hydrogen peroxide biosensors ŽWang et al., 1999 .. Sol᎐gelrcopolymer hybrid material was used as the immobilization matrix, and methylene blue as a mediator was dissolved in buffer solution. The largest response of the sensor to H 2 O 2 arrived at pH 5.0. The resulted sensor exhibits a fast response Ž5 s. and high sensitivity Ž27.5 A

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323

308

Fig. 2. FIA peaks for 1 mM Ž1., 2 mM Ž2., 4 mM Ž3. and 8 mM Ž4. H 2 O 2 at the sol᎐gel biosensor Ža., and FIA peaks for 8 mM H 2 O 2 for nine successive injections Žb.. Experimental conditions: carrier solution, 0.02 M biphthalate buffer ŽpH 5.0. containing 0.2 mM MB; operating potential, y200 mV; flow rate, 1.05 ml miny1 ŽWang et al., 1999..

mMy1 .. The sensor has also good thermostability. The immobilized enzyme lost about 6% of its activity at 65 ⬚C after 2 h of operation. Moreover, the performance of the sensor was investigated using flow injection analysis ŽFIA.. Fig. 2a shows the current responses of the biosensor to different concentrations of H 2 O 2 , Fig. 2b gives the peaks for nine consecutive injections of 8 mM H 2 O 2 after the electrode has immersed in biphthalate buffer solution for 4 days. The enzyme electrode did not swell in the flowing solution after continuous operation, which implies high stability of the sol᎐gel thin film. Good reproducibility of enzyme electrode means that the biosensor can be used in on-line detection for H 2 O 2 in acid medium. In order to further improve the acid-stability of

the biosensor, recently we encapsulated soybean peroxidase in a graft copolymer of poly Žvinyl alcohol. and 4-vinylpyridine ŽWang et al., 2001.. The hydrogen peroxide biosensor shows high acid-stability and good long-term stability at pH 3.0. Table 3 compares the main characteristics of three different hydrogen peroxide sensors. Obviously, the sensors based on SBP have higher acid-stability than that based on HRP, which may be explained by UV᎐vis spectrometric experiments of HRP and SBP at pH 3.0. SBP can take a firm hold on its prosthetic group at pH 3.0, while the porphyrin group of HRP falls off the active center at the same condition. Moreover, the SBP-based biosensors have higher detection upper limits than those of HRP-based biosensors ŽLi et al., 1996b, 1998.. Therefore, these SBPbased biosensors can be used to measure a system with high H 2 O 2 concentration. 3.2. Electrochemical biosensors in pure organic phase The applications for biosensors are currently limited to the measurement of analytes soluble in aqueous solutions to prevent denaturation of the protein. The enzymatic assays in organic phase have gained considerable interest ŽHall et al., 1988; Schubert et al., 1991; Wang, 1993; Wang and Lin, 1993.. Organic phase biocatalytic sensing possesses distinct advantages, including monitoring of hydrophobic substrates, elimination of microbial contamination, reduction of side reactions, enhanced thermostability, and relative ease of enzyme immobilization based on their insolubility in organic solvents. Strictly, it is impossible

Table 3 Analytical performances of three different hydrogen peroxide biosensors Enzyme electrode

Mediator

Acid stability

Optimum pH

HydrogelrSBP

K 4 FeŽCN. 6

3.0

Sol᎐gelrSBP

Methylene blue K 3 FeŽCN. 6

Extremely stable Stable

5.0

Not stable

7.0

HydrogelrHRP

Sensitivity Ž␮A mMy1 .

Response time Žs.

Linear range ŽmM.

Thermost ability at 60 ⬚C

Reference

5.2

40

0.01, y6.2

Good

Wang et al., 2001

27.5

5

0.02, y2.6

Good

Wang et al., 1999

20

0.01, y0.3

Bad

Li et al., 1998

5.75

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323

for enzymes to retain catalytic activity in a waterfree environment. Enzymes on the organic phase enzyme electrodes ŽOPEEs. must retain a thin aqueous film, essential for their catalytically active conformation in organic media. However, in organic solvent, especially water-miscible solvent, this ‘essential’ layer is easily disturbed or even lost, and the enzyme is deactivated. In order to provide and retain the essential hydration layer for enzymes immobilized Žgenerally adsorbed. on OPEEs, water has been added to the organic solvents prior to organic phase analysis. Therefore, the OPEEs previously reported ŽHall et al., 1988; Schubert et al., 1991; Wang, 1993; Wang and Lin, 1993. were not operated in absolute nonaqueous media, which causes inconvenience and problems in many actual operations, especially for determination and monitoring in situ or on line. In view of the special requirements for OPEEs, we developed a new material and an immobilization method, and so solved the dehydration problem simply and effectively. A kind of polyhydroxyl cellulose was prepared by mixture of polyŽvinyl alcohol. and carboxymethyl hydroxyethyl cellulose, the aqueous solution of which can be frozen to produce a hydrogel. This cryohydrogel has a semi-interpenetrating network, relatively high mechanical strength, and retains its water molecules even in organic solvents. This matrix provides a water-containing microenvironment for the enzyme and allows it to function in pure Žwater-free. organic solvents. Consequently, a new kind of OPEE ᎏ a pure organic-phase amperometric enzyme electrode based on immobilization of enzymes in cryohydrogel ᎏ was developed. 3.2.1. Pure organic-phase horseradish peroxidase electrodes The monitoring of hydrogen peroxide presented in organic media, is of practical importance in clinical, food, pharmaceutical, and environmental fields. HRP is most frequently used to catalyze the reduction of hydrogen peroxide. A reagentless hydrogen peroxide biosensor was developed, based on coimmobilization of a soluble mediator-potassium hexacyanoferrate ŽII. and HRP on a graphite electrode using a cryohydro-

309

gel ŽDong and Guo, 1994a,b.. A sensitive response of the enzyme electrode was obtained in the vicinity of 0.0 V. A stable base current is obtained after an equilibration time of 5᎐15 min, which is much more rapid than that previously described Ž1᎐1.5 h. ŽSchubert et al., 1991. and has a shorter response time of 0.2᎐5 min. The useful measuring range is up to 2.5 mM and has a detection limit of 5 = 10y7 M. Comparing the response of the enzyme electrode in chloroform and in acetonitrile, we can see that the enzyme electrode has an even shorter response time Žwithin 10 s. and a wider measuring range Žup to 7.5 mM. in acetonitrile than in chloroform, whereas the sensitivity decreases in acetonitrile possible because of the solubility of H 2 O 2 in different solvents. The H 2 O 2 standard solution is much more miscible with acetonitrile than with chloroform. Therefore, in acetonitrile, equilibration is more readily reached after injection of H 2 O 2 , and the response time is shorter. The improved measuring range and decreased sensitivity could be interpreted in the same way. In the chloroform background solution the perconcentration effect of H 2 O 2 in the water phase inside the hydrogel contributes to the low detection limit and higher sensitivity in chloroform. The cryohydrogel enzyme electrode was used daily for 2 months, and the electrode retained a sensitivity of 60% of its initial value. The direct electron transfer of HRP on a graphite electrode in aqueous solution is well known. Hence, we studied the response of a cryohydrogel-immobilized HRP graphite electrode in pure chloroform and pure chlorobenzene ŽDong and Guo, 1995.. The apparent direct electron transfer between a spectrographic graphite electrode and immobilized HRP was obtained. HRP was also immobilized on platinum and glassy carbon electrodes with a cryohydrogel. These modified electrodes show much larger responses to H 2 O 2 , which imply that direct electron transfer between the electrodes and HRP could achieve. Using this enzyme electrode, a stable base current is obtained within 10 min, and the response time is 0.5᎐2 min. The useful measuring range is up to 5.0 mM and has a detection limit of 1.3= 10y6 M in chloroform. Corresponding values are

310

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323

7.0 mM and 2.5= 10y7 M in chlorobenzene. The enzyme electrode was used intermittently for a month without obvious deterioration in the sensing characteristics. 3.2.2. Tyrosinase-based enzyme electrodes in pure organic phase Tyrosinase is catalytically active in a number of organic solvents. A cryohydrogel-immobilized tyrosinase electrode was constructed in our laboratory ŽDeng and Dong, 1995.. The characteristics of this modified electrode were discussed and different phenolic substrates were determined in different pure organic solvent. The effect of solvent on the OPEE can be divided into three aspects: Ž1. the effect of solvent hydrophobicity on the state of the enzyme layer; Ž2. the effect of the solution on the catalytic activity of the enzyme; and Ž3. the effect of solvent viscosity and the solubility of the conducting electrolyte on mass transport properties. In an organic solvent, the state and amount of water contained in the material depend on the characteristic of the solvent, especially the hydrophobicity. The more hydrophobic the solvent, the more water the cryohydrogel retains, the longer time the enzyme electrode can be successively used, and the easier the enzyme layer is to rehydrate. The recovery of enzymatic activity varies with solvents. After the enzyme electrode is used in chlorobenzene, chloroform, octanol, 1-butanol and ethanol, respectively, the activity is recovered fully by dipping in water. However, its recovery is only 30% in acetonitrile and 2-propanol, 50% in benzene, and 85% in toluene. Once the electrode has been used in N, N⬘-dimethylformamide ŽDMF., the activity is never recovered. Recently, a new generation of organic-phase biosensors based on enzyme inhibition has been developed for monitoring pesticides and related compounds in organic media. This enlarges the practical applications of biosensors. Moreover, the study of the mechanism of enzyme inhibition in the organic phase will enhance the understanding of organic-phase enzymology. In our laboratory, thiourea, 2-mercaptoethanol, and benzoic acid are determined by exploiting their noxious effect on tyrosinase immobilized in cryohydrogel in pure

Fig. 3. Calibration plots for inhibitors with a tyrosinase electrode in chloroform using phenol as substrate. ŽA. Benzoic acid; ŽB. thiourea; and ŽC. 2-mercaptoethanol ŽDeng and Dong, 1996..

water-immiscible solvents ŽDeng and Dong, 1996.. Fast and sensitive responses are observed for the three inhibitors. The steady-state response of the electrode to phenol is obtained within one and a half minutes. The response decreases dramatically with successive additions of inhibitors. For 2-mercaptoethanol, response time for inhibition is less than 20 s. The inhibitory effect of benzoic acid is very strong, and the response time is within one and a half minutes. Thiourea exhibits a relatively slow inhibition. The response time is between 2 and 3 min. The plots of the fractional inhibition against the concentration of the three inhibitors in pure chloroform are shown in Fig. 3. The Y value rises linearly with an increase in inhibitor concentration and then starts to level off. The hyperbolic nature of the plots indicates that the inhibitor only binds to one active site of the tyrosinase and that all the bindingractive sites are equivalent in the interaction between the enzyme and the inhibitor. The detection limits of three inhibitors are 0.05, 0.1 and 0.5 ␮M for benzoic acid, 2-mercaptoethanol, and thiourea, respectively, in chloroform. 3.2.3. Organic phase enzyme electrodes based on organohydrogel HRP and tyrosinase OPEEs based on cryohydrogel have been applied in various water-free organic solvents. However, due to the high hy-

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323

drophilicity of the cryohydrogel, the cryoimmobilization method has been demonstrated to be feasible mostly to water-soluble analytes, e.g. hydrogen peroxide and phenols, and unworkable for hydrophobic substances, e.g. organic peroxides, bilirubin and cholesterol. Therefore, we used a universal organic solvent ŽDMF. with mixture of polyhydroxyl cellulose and further prepared an organohydrogel. Besides possessing advantages of cryohydrogel, the organohydrogel has high partition coefficients for both hydrophilic and hydrophobic analytes to obtain high measuring sensitivity. Three enzymes ŽHRP, tyrosinase and bilirubin oxidase. were immobilized in organohydrogel to demonstrate the feasibility and workability of the OPEEs ŽGuo and Dong, 1997.. With immobilization of HRP in DMF organohydrogel, these organic peroxides have been determined mediatorlessly in pure organic solvents. Fig. 4 shows the typical response of an HRP᎐DMF organohydrogel-modified electrode polarized at y200 mV in chloroform to successive additions of 2-butanone peroxide and tert-butyl hydroperoxide. As can be seen from Fig. 4, the HRP organohydrogel electrode shows sensitive responses to organic peroxides. Table 4 gives response parameters of substrates at the tyrosinase organohydrogel electrode ŽTOE. and the tyrosinase cryohydrogel electrode ŽTCE. in pure chloroform. The TOEs have higher sensitivity for phenolic compounds than TCEs, which are attributed that the DMF organohydrogel has a high partition coefficient for phenolic compounds and these compounds can be extracted and preconcentrated in the enzyme membrane. For practical application, this investigation achieves the ultimate evolution of the detection medium in biosensing ᎏ from water buffer to

311

Fig. 4. Steady-state response of 2-butanone peroxide Žb1. and tert-butyl hydroperoxide Žb2. at the HRP᎐DMF organohydrogel-modified electrode Žb1, b2. and the HRP-free organohydrogel-modified electrode Ža. in chloroform. Applied potential: y200 mV; each injection used 0.5 mM organic peroxides ŽGuo and Dong, 1997..

waterroil mixtures, to organic solvents saturated with water, to anhydrous organic solvents, and finally to anhydrous dimethylformamide, the universal organic solvent. Meanwhile, the analytes vary from water-soluble to poorly soluble and to insoluble and hydrophobic compounds. Furthermore, the DMF organohydrogel immobilization makes it possible to construct biosensors of many types: mediatorless, with a mediator, and a type of detecting the electroactive product of an enzyme reaction. Therefore, biosensing can be applied in various situations Žaqueous buffer, waterroil mixtures, and anhydrous organic solvents. and for analytes of different physicochemical properties. 3.3. Amperometric quantification of polar organic sol¨ ents based on tyrosinase biosensors There is no absolute need for the analyte to be a substrate of the enzyme reaction. Wang re-

Table 4 Comparison of response parameters of substrates at tyrosinase organohydrogel electrode ŽTOE. and tyrosinase cryohydrogel electrode ŽTCE. in chloroform ŽGuo and Dong, 1997. Substrate

Catechol p-Cresol Phenol

TCE

TOE

Km app ŽmM.

Sensitivity ŽnA mMy1 .

Km app ŽmM.

Sensitivity ŽnA mMy1 .

0.06 3.5 0.58

23.7 3.9 10.9

0.15 0.506 0.167

55.0 23.1 40.5

312

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323

ported a novel application of OPEEs to measure trace water in nonaqueous media ŽWang and Reviejo, 1993., since small amounts of water are known to enhance enzyme activity in organic solvents. Recently we demonstrated that amperometric tyrosinase biosensors could be exploited to monitor organic solvent ŽZhang et al., 2000; Wang et al., 2000a.. After the response of the tyrosinase biosensor in phosphate buffer containing certain concentration phenol substrate was reached a stable state, injections of a polar organic solvent into the solution caused the drop of response current. The current decrease is proportional to the volume content of organic solvent, therefore the electrochemically inert polar organic solvents can be amperometrically quantified. The factors influencing on the biosensor response to organic solvent were studied and its mechanism was described in Fig. 5. This unique application is based on partition equilibrium of the substrate probe between the enzyme membrane and the water᎐organic solvent media. The solubility of phenols in organic solvents is much greater than in water, and the injected organic solvents quickly hydrate and concentrate in the solution, which causes the decrease of the substrate concentration in the solution. Because of the dynamic balance of the substrate between water᎐organic solvent media and the enzyme

membrane, the phenols in the enzyme membrane diffuse toward the solution. This brings about the decrease of the substrate concentration in the enzyme membrane, and accordingly, the response of the sensor decreases. This mechanism has been proved by in situ steady-state amperometry-quartz crystal microbalance. The sensitivity for a certain organic solvent is dependent on the kind and concentration of the substrate probe and the hydrophobicity of the immobilization matrix; accordingly sensitive detection can be obtained by optimization of the immobilization matrix and the substrate probe. At the condition of fixing immobilization matrix and substrate probe, the more hydrophilic the organic solvent is, the wider the linear range for it is. Increasing the concentration of the substrate probe can improve the detection limit. Fast response was obtained for the tested organic solvents. The response time for all the organic solvents used in this study is less than 2 min, which means that partition equilibrium can be established within 2 min. Table 5 shows the detection limits for several common organic solvents obtained with the biosensor. Adopting a sensitive substrate probe and increasing the concentration of the substrate probe as discussed above can further improve these values. Moreover, the main advantage of the biosensor re-

Fig. 5. Schematic description of the mechanism of the sol᎐gel tyrosinase biosensor for organic solvent ŽWang et al., 2000a..

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323 Table 5 Typical detection limits for several common organic solvents obtained with the biosensor ŽZhang et al., 2000. a

Solvent

Detection limit Ž% vrv.

Methanol Ethanol n-Butanol Acetonitrile Acetone THF

0.12 0.11 0.056 0.042 0.040 0.075

a

The data were calculated at SrN of 3 from the response curves obtained in the presence of 39.2 ␮M p-cresol in 0.2 M KNO 3 q 0.1 M phosphate buffer ŽpH 6.9..

ported here can be used to quantify some organic solvents such as acetone, acetonitrile, tetrahydrofuran, etc. for which no specific enzyme has been found to construct a specific biosensor.

4. Bilayer lipid membranes (BLMs)-based electrochemical biosensors Biomembranes are the basic structure of nature’s sensor and molecular devices and its study is of great importance in the exploration of the life activity. BLMs are the most widely used model systems for biomembrane studies. Modified BLMs can be employed to imbed a host of compounds such as enzymes, antibodies, protein complexes, ionophores and redox species for the detection of their counterparts, respectively, such as substrates, antigens, hormones, ions and electron donors or acceptors. Compared with all other immobilization matrixes, BLMs provide the nature environment for embedding biomolecules. The functions of biomembranes are mediated by specific modifiers, which assume their active conformations only in the lipid bilayer environment. Furthermore, the presence of the lipid bilayer greatly reduces the background noise Žinterferences . and effectively excludes hydrophilic electroactive compounds from reaching the detecting surface causing undesired reactions ŽTien, et al., 1998.. Hence, BLM system offers a wider opportunity for the biosensor development ŽOttova and Tien, 1997..

313

4.1. Ion sensor A novel kind of Kq sensor with valinomycin-incorporated bilayers supported on a gold electrode consisting of self-assembled monolayers ŽSAMs. of alkanethiol and a lipid monolayer, has been fabricated successfully in our laboratory ŽDing et al., 1997.. When alkali ions were presented, valinomycin can interact with them to form a valinomycin᎐alkali ion complex. This causes the change of the membrane potential of the lipid. By measuring the response potential, we can know the intensity of the valinomycin᎐alkali ions interactions and finally measure the concentration of Kq ions. The lipid monolayer was deposited on the alkylated surface of the first alkanethiol monolayer through three different methods, such as the Langmuir᎐Blodgett ŽLB. technique, painted method and painted-frozen method. The response of Kq sensors produced by a painted or painted-frozen lipid monolayer on an alkanethiol alkylated gold electrode is larger than that by the LB method, which is due to the difference in fluidity of the three kinds of bilayers. Selectivity coefficient K K q , Naq, K K q , Liq, K K q , Ca 2q and K K q , Mg 2q are 10y4 , 10y4 , 2 = 10y5 and 3 = 10y5 M, respectively, and there is no obvious difference among different fabricating methods. A linear response toward Kq ion was found in the range from 10y5 to 10y1 M with a detection limit of 10y6 M. The sensor has a slope of 60 mV per decade. Moreover, the lifetime of the sensor was improved obviously for at least two months at about y10 ⬚C. In addition, monensin was incorporated in alkanethiolrphospholipid bilayers by the same method and exhibited similar behaviors for alkali ions ŽLi et al., 1996a.. So the novel immobilized method suggests the possibility to make a novel kind of practical biosensor. 4.2. Enzyme sensor Recently an amperometric glucose biosensor based on lipid film was constructed in our laboratory ŽWu et al., 2000b.. A small electroactive molecules ᎏ tetrathiafulvalene ŽTTF. was incorporated in cast dimyristoylphosphatidylglycerol ŽDMPG. film as an electron transfer mediator,

314

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323

and glucose oxidase was entrapped in the grafted polymer ŽPVA-g-PVP. covered on the lipid film. The cyclic voltammetric experiment shows that TTF was firmly embedded in lipid film and a reversible electrochemical behavior was observed. The potential difference of the peaks is 60 mV, which is almost the same as the electrochemical behavior of TTF on a bare glassy carbon electrode ŽWang et al., 1998.. In addition, the peak current i p , is proportional to the square root of the scan rate ␯ 1r2 , which illustrates that the electrode reaction is controlled by the diffusion of TTF. After glucose was added into the buffer solution, an enhancement in oxidation current of TTF was observed and the cyclic voltammogram exhibits a well-defined sigmoidal form, which is characteristic of electrocatalysis. This illustrates that the enzyme electrode can catalyze the oxidation of glucose efficiently. The sensor has fast response Ž20 s., and the linear range of glucose concentration spans between 0.2 and 10 mM with a detection limit of 2 = 10y5 M. The reproducibility of the sensor was examined and the relative standard deviation is 2.4% Ž n s 8. for the same electrode. Moreover, the effect of substances that might interfere with the response of the enzyme electrode was studied. Uric acid and pacetamidophenol did not cause any observable interference, and only ascorbic acid interfered slightly. It is obvious that the lipid film on the surface of the electrode can greatly reduce the interference and effectively exclude hydrophilic electroactive material from reaching the detecting surface. Especially, as a kind of anionic lipid, DMPG can reduce the anion response of ascorbic acid effectively ŽSnejdarkova et al., 1993.. The enzyme electrode shows good storage stability Ž30 days., which also demonstrates that the electron transfer mediator can be embedded firmly in the cast lipid film. In general in living organisms, proteins must remain in a lipid matrix to retain their native structure and activity. So studying the structure and function of the membrane-bound proteins is of great importance. Previously it was of failure to embed GOD in the cast DMPG film ŽWu et al., 2000b.. Here, we developed a facile approach to immobilize protein for biosensor preparation: the

supported bilayer lipid membranes were selfassembled on a glassy carbon ŽGC. electrode ŽWu et al., 2001.. HPR was immobilized into the dimyristoylphosphatidylcholine ŽDMPC. to fabricate a kind of mediator-free H 2 O 2 biosensor. After HRP was incorporated into the supported bilayer lipid membranes Žs-BLM., the enzyme electrode exhibits a sensitive and fast response to H 2 O 2 without any mediator. This indicates a fast diffusion process and a high activity of HRP in the enzyme-membrane system. The linear range of H 2 O 2 concentration spans between 0.5 and 18 mM with a detection limit of 1.7= 10y4 M. The good reproducibility and storage stability of the sensor were obtained. These results demonstrate that the high activity of HRP was maintained because a biological environment was supplied by s-BLM on the surface of the GC electrode. This also demonstrates that the s-BLM is an ideal choice to immobilize enzyme on GC electrode for constructing a third-generation biosensor. 4.3. Study of ion-channel beha¨ ior of supported bilayer lipid membranes The ion channel is a very important system in biological activities, e.g. conversion of extracellar events into intracellar signals via hormones, transfer of information in the nervous system, and others. Study of ion-channel behavior of biomembranes is of very signification. Because the supported bilayer lipid membrane has been proved to be very useful and easy to work in the field of membrane research and has solved the shortcomings of the conventional BLM, s-BLM on solid substrates will be an ideal model for investigating the ion channel behavior of biomembranes ŽCornell et al., 1997.. Therefore, we selected a synthetic lipid-didodecyldimethylammonium bromide ŽDDAB. and made supported lipid membrane on glassy carbon electrode ŽWu et al., 2000a.. From electrochemical impedance experiments, it was demonstrated that the lipid layers on the GC electrode were bilayer lipid membranes. Perchlorate anion was chosen as the stimulus and rutheniumŽII. complex cation as the marker ions. Ion channel of behavior of

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323

315

5. Surface plasmon resonance (SPR) biosensors

Fig. 6. Cyclic voltammograms of 0.5 mM rutheniumŽII. complex cation at the bare GC electrode with 5 mM NaCl as a supporting electrolyte Ža. and at the GC electrode coated with BLM in supporting electrolytes: Žb. 5 mM NaCl, Žc. 5 mM NaClO4 , Žd. 5 mM NaCl. Scan rate, 50 mV sy1 ŽWu et al., 2000a..

the supported bilayer membranes was studied with cyclic voltammeric method ŽFig. 6.. In the absence of perchlorate anions, the channels were in a closed state due to the regular alignment of the lipid quaternary ammonium. The channels were open in the presence of perchlorate anions, because perchlorate anions could form ion association compounds with the lipid quaternary ammonium cations in the bilayer membranes ŽSugawara et al., 1987. and change the regular alignment of the lipid; thus, the ion channels in the membranes were formed and open. The reversible open᎐closed processes could be repeated many times. Furthermore, the ion channel behavior was concentration-dependent and time-dependent. The intensity of peak current in cyclic voltammetry increased with the concentration of perchlorate anions. The peak current of the rutheniumŽII. complex cation increased with time and reached steady state after 80 min.

Transducer is an import component of a biosensor. In biosensors mentioned above, a conventional transducer ᎐ amperometric electrode was used. New generations of biosensors based on novel and promising transducers are emerging, SPR is the one and begin to use in biosensors ŽWeimar, 2000.. SPR is an optical phenomenon, which is sensitive to changes in the optical property of the medium close to a metal surface. The commercially available BIAcore apparatus ŽPharmacia Biosensor AB, Uppsala, Sweden., based on SPR principle, is designed to measure biospecific interaction both qualitatively and quantitatively in real time, having rapid and label-free detection ŽLoefaas et al., 1991.. This technique has been widely studied in the fields of kinetics analysis, concentration measurements of biomolecules and molecule recognition involving protein᎐protein, DNA᎐DNA, receptor᎐ligand interactions, etc. ŽMalmqvist, 1993; Corr et al., 1994; Rao et al., 1999.. Here, the recent works on SPR biosensors from our laboratory are described. Antibodies play an important role in the body’s immune system, and hold a firm place as powerful diagnostic and research tools. Therefore, the rapid detection of activity and analysis of binding properties of antibodies are crucial in the evaluation of their performance in different applications. SPR biosensor is a useful tool for this purpose and studies on kinetic binding properties of antibodies have also been published ŽTaremi et al., 1996.. However, there are only a few reports on activity detection of antibodies in their native form. We developed a SPR biosensor to determine the antibody activity in a native form without any purification of the immobilized antigen protein ŽPei et al., 2000.. The interaction of mouse IgG and sheep anti-muse IgG polyclonal antibody was chosen as a model system and was investigated in real time. The factors, including solution pH, ionic strength, protein concentration, influencing on electrostatic adsorption of mouse IgG protein onto the carboxylated dextran-coated sensor chip surface, were studied. The procedures of MIgG protein immobilization and immune reaction were monitored in real time.

316

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323

The regeneration effect by using the different elution reagents was also investigated. 0.1 M glycine᎐HCl buffer ŽpH 2.0. has a better elution effect and retains reaction activity well and was used to desorb antibody bound in our experiments. The detection time for each binding and elution cycle is only 12᎐14 min and the same MIgG immobilized surface can be used for 100 cycles with only 0.38% loss per regeneration in reactivity. Similar to above system, the activity of anti-human serum albumin antibody was well detected using SPR biosensor ŽCui et al., 2000.. These results show that the surface plasmon resonance biosensor is a rapid, simple, sensitive, accurate and reliable detection technique for real-time immunoassay of antibody activity. The assay allows antibodies to be detected and studied in their native form without any purification. Moreover, the sensor does not suffer significantly from non-specific adsorption of non-analyte molecules in crude samples due to the hydrophilic nature of the dextran matrix on sensor surface and flowing system for sample handling. It is particularly useful for the detection of substrates of interest in crude samples. Because the assay is based on a general principle, when different special ligands are immobilized on a sensor surface, this technique can be used for rapid determination of many substances of interest such as proteins, viruses, toxins and other small molecules in crude samples. Moreover, SPR technique was also used to characterize and monitor the formation of multilayer films from solution in real time continuously ŽPei et al., 2001a.. The assembly of alternating DNA and positively charged poly Ždimethyldiallylammonium chloride. ŽPDDA. multilayer films by electrostatic layer-by-layer adsorption has been studied. After the C1 chip surface Ža flat ᎐COOH layer on the surface . was coated with polyŽethylenimine. ŽPEI. and the baseline was stable, DNA and PDDA solution were alternatively injected across the surface. The results show that each injection of DNA and PDDA caused the SPR response ŽRU. increasing for a certain value. For DNA, the response increased about 2963 " 467 RU Ž0.2963⬚. for each injection. For PDDA, the response increased about 1914 " 255 RU

Ž0.1914⬚.. By plotting the response of the total DNA or PDDA on surface after each DNA or PDDA injection against the number of layers, a good linear relationship can be obtained. As the increase of SPR response means the increase of mass of DNA and PDDA on the sensor chip surface, we can conclude that the uniform multilayer of DNA᎐PDDA has been constructed on PEI coated surface. The kinetics of the adsorption of DNA on PDDA surface was also studied by real-time SPR technique, and the rate constant measured was calculated using the Langmuir model Ž k obs s Ž1.28" 0.08. = 10y2 sy1 .. However, SPR method has a major disadvantage for bioanalytical applications. It is difficult to use for detection of low concentration or low molecular mass analytes. The detection limit is ca. 1᎐10 nM for a 20-kDa macromolecule and is even higher for smaller molecules ŽKooyman et al., 1988.. Many strategies have been proposed to enhance the response signal ŽSevers and Schasfoort, 1993; Lyon et al., 1998; Wink et al., 1998.. We present a novel strategy for improving the sensitivity of SPR immunosensing using a streptavidin᎐biotinylated protein complex ŽPei et al., 2001b.. The process of enhanced immunoassay of the analyte Žantigen. is represented in Fig. 7. A capture antibody was firstly immobilized on the sensor chip surface by the amine couple method. Then, injections of an antigen sample and a biotinylated detecting antibody Žbio-Ab. were completed, respectively. The bound amount of the detecting antibody is related to the analyte. Finally, the streptavidin᎐biotinylated protein complex was added to amplify considerably the response signal and improve dramatically the detection sensitivity. This amplification strategy is based on the high-affinity interaction of biotin and streptavidin ŽWilchek and Bayer, 1988; Ghafouri and Thompson, 1999.. Each streptavidin has four equivalent sites for biotin binding. The tetravalency of strepavidin for biotin allows the construction of a molecular complex between streptavidin and biotin labeled proteins when one molecule of this protein is labeled by several molecules of biotin. This complex can be formed in a crosslinking network of molecules so that only a few binding events of the analyte at the sensing sur-

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323

Fig. 7. Schematic diagram of enhanced immunoassay of hIgG ŽPei et al., 2001b..

face may lead to a detectable surface mass due to the large molecular size of the complex. The results show that the amplification strategy causes a dramatic improvement of the detection sensitivity. hIgG protein could be detected in the range of 0.005᎐10 ␮g mly1 . Moreover, the method can be generally applicable to the enhanced assay of other biomolecules and other transduction means, such as the quartz crystal microbalance or impedance spectroscopy. Recently we combined SPR with electrochemical method to monitor the electrochemical growth process of the conducting polymerᎏpolyaniline ŽPAn. ŽKang et al., 2001b.. The results show that SPR is sensitive to the change in the conductivity of PAn film. Upon oxidation or reduction, PAn film can change its conductivity by several orders of magnitude, which thus leads to a large change of SPR response due to the change of the film dielectric property. Based on the SPR response to the change of the optical dielectric property of the PAn film by enzymatic catalytic reaction, a novel electrochemical SPR H 2 O 2 biosensor was presented ŽKang et al., 2001a.. HRP was chosen as a model enzyme and was immobilized in an electropolymerized film of polyŽ1,2-diaminobenzene. grown on the top of the PAn base film, while PAn acted as an effective redox enzyme mediator to transduce and amplify the chemical information between PAn and HRP into the SPR optical signal.

317

In the presence of H 2 O 2 , the reaction of HRP with H 2 O 2 is followed by oxidation of the PAn film with the oxidized form of the enzyme, so that the PAn film is switched from the reduced state to the oxidized state. This switch results in a change in the imaginary part of dielectric constant of the PAn film and leads to a large change of SPR signal Žshown in Fig. 8.. It was noted that the potential switching between the reduced and the oxidized forms of PAn causes a much larger change of the reflectivity at the fixed angle 62.6⬚ Žmarked ⌬ R in Fig. 8. relative to shift of the resonance minimum Žmarked ⌬␪ in Fig. 8.. We attempted to record the change of reflectance with time at the fixed angle 63.8⬚ Žcorresponded to the resonance minimum of the oxidized state of PAn film. with increasing concentration of H 2 O 2 in electrochemical SPR cell, and simultaneously the potential change was recorded by chronpotentiometry to monitor the redox transformation of the film. From Fig. 9, it was seen that the change of reflectance is dependent on H 2 O 2 concentration. When a lower concentration of H 2 O 2 is added, a smaller transformation rate is obtained. For a higher concentration as 0.5 mM H 2 O 2 , the transformation is completed in less than 100 s. The slopes of the SPR kinetic curves in the linear regions reflect the rate of this transformation, and thus can be used to produce calibration curve of the relationship between H 2 O 2 concentration and the slope of the curve. Here the linear relationship was obtained by use of the change rate rather than the time for complete transformation. This allowed measuring low levels of H 2 O 2 in less than 100 s with the SPR biosensor. The detect limit of 10 ␮M H 2 O 2 was obtained. In addition, this biosensor can be reused by electrochemically reduction of the PAn film to its reduced state. In a set of experiments as shown in Fig. 9, the sensor was washed between each measurement and re-reduced at 0 V vs. AgrAgCl. The results show good reproducibility, and no detectable loss in activity in 2 h under continuous operations. This method provides a new route to the fabrication of SPR biosensor because the other oxidoreductases can be used as an immobilization enzyme to transform the oxidation states of PAn film. This work also demon-

318

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323

6. Capacitive biosensors

Fig. 8. SPR spectra of Ž1. the bare gold electrode Žas a comparison., Ž2. the reduced PAn film Žafter holding potential at 0 V for 5 min. and Ž3. oxidized PAn film Žafter holding potential at 0.9 V for 5 min. ŽKang et al., 2001a..

strated that a larger SPR signal can be obtained in this SPR measurement than that in the direct binding assay of SPR.

The emergence of novel transducers opens new scopes for the development of biosensors. Recently, a capacitive transducer has been developed as a highly sensitive approach ŽBataillard et al., 1988; Billard et al., 1991; Berggren and Johansson, 1997., which is based on the theory of the electrical double-layer. A metal electrode immersed in an electrolyte solution can generally be described as resembling a capacitor in its ability to store charge. In most cases, the capacitance is measured at the metalrsolution interface in the electrochemical system. Ions and dipoles are ordered outside a metal electrode in such a way that charges in the metal are balanced, thereby forming the electrical double layer. Since capacitive measurements give information about the metal᎐solution interface, a chemical modification of this structure will lead to a change in capacitance. Compared with other electrochemical methods Žamperometry, conductance, potential, etc.., the capacitance measurement is made for some special purposes: Ži. membrane thick-

Fig. 9. Time course of reflectivity in 0.1 M citrater0.2 M phosphatesr0.1 M Na 2 SO4 buffer solution ŽpH 5. with various H 2 O 2 concentrations. The angle was fixed at 63.8⬚; between each measurement, the sensor was washed and reproduced at the potential of 0 V vs. AgrAgCl ŽKang et al., 2001a..

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323

ness measurements; Žii. investigation of membrane formation processes; Žiii. suitable for studying the insulating material, such as the antibody᎐antigen, biotin᎐avidin, enzyme᎐substrate interaction; Živ. suitable for detection of small molecules at very low detection limits ŽBontidean et al., 1998.. 6.1. A kind of potassium sensor based on capacitance measurement of mimic membrane There are many methods reported for detecting potassium ion for its leading role in membrane transport ŽBuhlmann et al., 1998; Steinem et al., ¨ 1998.. However, there is a need for fast, easy, and reliable methods for the detection of potassium ion. Previously, we have constructed a potassium ion sensor based on supported bilayers and the change of the membrane potential of the lipid membranes was detected in the presence of potassium ion ŽDing et al., 1997.. Recently, a new potassium ion sensor based on capacitance mea-

319

surement of mimic membrane has been developed in our laboratory ŽCheng et al., 2001a.. It is known that valinomycin is a neutral carrier and uncharged. While valinomycin interacts with alkali ions, a valinomycin᎐alkali ion complex would be formed. It means that the carrier᎐ion complex is the only charged component in the membrane ŽSteinem et al., 1998.. The binding of metal ions to the initially neutral layer increases the relative permittivity of the layer, which leads to the increase of the capacitance. So the total capacitance of the membranes should increase with the addition of Kq ions. Fig. 10 shows a typical recording of a trace of capacitance change as a function of different concentrations of Kq injected to a stirred buffer solution of 0.01 M Tris᎐HCl. A minimum concentration of 5.0= 10y8 M was required to induce the significant signal change compared to the background capacitance. As can be seen, the electrode modified with valinomycin-incorporated membrane shows a much larger response to Kq.

Fig. 10. Capacitance change of the AurMErDODBq valinomycin electrode vs. Kq concentration. Ža. 5.0= 10y7 M, Žb. 5.0 =10y6 M, Žc. 5.0 =10y5 M, Žd. 5.0 =10y4 M, Že. 5.0= 10y3 M. The measurements were performed in 0.01 M Tris᎐HCl ŽpH 8.28. buffer solution ŽCheng et al., 2001a..

320

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323

However, the response is much lower for potassium ion in the absence of valinomycin in the membrane Ždata not shown here.. In addition, it is obvious that the steady-state capacitance was reached in a short time between each step. In the concentration range 5.0= 10y7 ᎐5.0= 10y3 M, the steady-state capacitance increases linearly with the increase of potassium ion concentration Žthe inset of Fig. 10.. The detection limit obtained was 5.0= 10y8 M, which is lower than that of other electrochemical methods Žusual 10y6 M.. Obviously, the capacitance measurement is sensitive and simple for detection of potassium ions compared to other methods. It is reasonable to expect that this capacitance meter will be widely used in other systems as well. 6.2. Capaciti¨ e detection of glucose using molecularly imprinted polymers In most cases, detection of glucose is based on enzymatic redox reactions. However, enzyme electrodes have their inherent problems, such as the high-cost and short lifetime of enzymes. Recently, a QCM biosensor for glucose was developed using molecularly imprinted polymers ŽMalitesta et al., 1999.. It offers an easy way for the preparation of biomimetic sensors. It is usually difficult for most of the electrochemical sensors to detect glucose because glucose does not show any electroactivity on bare electrodes in the usual potential window, which means another suitable electrical signal should be found to replace the usual current signal. As pointed out above, capacitive measurement is a very sensitive method for material adsorption from solution to the electrode surface. Consequently, we developed a novel glucose biosensor based on capacitive detection using molecularly imprinted polymers ŽCheng et al., 2001b.. The sensitive layer was prepared by electropolymerization of o-phenylenediamine on a gold electrode in the presence of the template Žglucose.. The uncovered areas of the layer surface were plugged with 1dodecanethiol to make the layer dense and insulating. The template molecules and the nonbound thiol were removed from the layer surface on electrode by washing with distilled water. When

glucose bounds to the imprinted sites, there will be an additional layer decreasing the capacitance further. The binding between glucose and imprint sites is therefore detected directly. The physical basis for the response is thought to arise from displacement of the polar water further out from the electrode surface, replacing it with a much less polar molecule. The change in capacitance versus the concentration of glucose was found to give a linear relationship between 0.1 and 20 mM with a correlation coefficient of 0.9943, and the sensor had a detection limit of 0.05 mM. The values of capacitance were obtained at a frequency of 10 Hz. Moreover, the effect of substances that might interfere with the response of the imprinted polymers was also studied. The ascorbic acid was firstly chosen because it usually interferes with glucose detection in the real sample. When ascorbic acid was added to give the normal blood concentration, it did not cause any observable interference for 2 mM glucose. However, fructose caused a very small capacitance change Žless than 7%. under the same conditions, which is more similar to glucose structure.

7. Conclusion and prospects Biosensors have been widely studied and developed over the past 30 years. But the number of commercially available biosensors is still low. There is huge challenge to create biosensors with superior performance for reliable and continuous use. From our point of view, future development of biosensors focuses on the following: Ža. New immobilization schemes and advanced sensing materials are highly desired for improving the analytical capabilities of biosensing devices, and for meeting the challenges posed by complex environmental and clinical samples. In particular, the application of nanomaterial in biosensors will bring amazing results. Žb. Use of novel and promising transducers such as electrochemical luminescence or optoelectronic devices may open the new scopes for the sensitive detection of trace substrates.

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323

Žc . Miniaturization, microfabrication and multi-sensor arrays are becoming trends in the development of biosensors. In particular, biochip has attracted much attention at present. Žd. On line, in real time and in vivo measurement is the ultimate application goal of biosensors. The development of the biosensors in extreme environment is the need for practical application. Že. Advances in the mimic technology will accelerate the process of realization of large-scale production of biosensors. In conclusion, as stated many improvements can be imagined to make these analytical tools widely available, and the huge amount of interdisciplinary knowledge accumulated since the first description of an enzyme electrode make us truly optimistic for the future of biosensors and bioelectronics ŽBlum and Coulet, 1991..

Acknowledgements This work was supported by the National Natural Science Foundation of China Žnos. 29835120, 29875028.. References Bataillard, P., Gardies, F., Jaffrezic-Renault, N., Martelet, C., Colin, B., Mandrand, B., 1988. Direct detection of immunospecies by capacitance measurements. Anal. Chem. 60, 2374᎐2379. Berggren, C., Johansson, G., 1997. Capacitance measurements of antibody᎐antigen interactions in a flow system. Anal. Chem. 69, 3651᎐3657. Billard, V., Martelet, C., Binder, P., Therasse, J., 1991. Toxin detection using capacitance measurements on immunospecies grafted onto a semiconductor substrate. Anal. Chim. Acta 249, 367᎐372. Blum, L.J., Coulet, P.R., 1991. Biosensor Principles and Applications. New York, Marcel Dekker, p. 343. Bontidean, I., Berggren, C., Johansson, G., Csoregi, E., Matti¨ asson, B., Lloyd, J.R., Jakeman, K.J., Brown, N.L., 1998. Detection of heavy metal ions at femtomolar levels using protein-based biosensors. Anal. Chem. 70, 4162᎐4169. Buhlmann, P., Aoki, H., Xiao, K.P., Amemiya, S., Tohda, K., ¨ Umezawa, Y., 1998. Chemical sensing with chemically modified electrodes that mimic gating at biomembranes incorporating ion-channel receptors. Electroanalysis 10, 1149᎐1158.

321

Chen, X., Wang, B., Dong, S., 2001a. Amperometric biosensor for hydrogen peroxide based on sol᎐gelrhydrogel composite thin film. Electroanalysis 13, 1149᎐1152. Chen, X., Zhang, J., Wang, B., Cheng, G., Dong, S., 2001b. A reagentless hydrogen peroxide biosensor based on sol᎐gelderived glasses doped with Eastman AQ polymer. Anal. Chim. Acta 434, 255᎐260. Cheng, Z., Luo, L., Wu, Z., Wang, E., Yang, X., 2001a. A new kind of potassium sensor based on capacitance measurement of mimic membrane. Electroanalysis 13, 68᎐71. Cheng, Z., Wang, E., Yang, X., 2001b. Capacitive detection of glucose using molecularly imprinted polymers. Biosens. Bioelectron. 16, 179᎐185. Cornell, B.A., Braach-Maksvytis, V.L.B., King, L.G., Osman, P.D.J., Raguse, B., Wieczorek, L., Pace, R.J., 1997. A biosensor that uses ion-channel switches. Nature 387, 580᎐583. Corr, M., Slanetz, A.E., Boyd, L.F., Jelonek, M.T., Khilko, S., Al-Ramadi, B.K., Kim, Y.S., Maher, S.E., Bothwell, A.L.M., Margulies, D.H., 1994. T cell receptor-MHC class I peptide interactions: affinity, kinetics and specificity. Science 265, 946᎐949. Cui, X., Pei, R., Yang, X., 2000. Detection of anti-human serum albumin antibody using surface plasmon resonance biosensor. Chinese J. Anal. Chem. 28, 950᎐955. Dave, B.C., Dunn, B., Valentine, J.S., Zink, J.I., 1994. Sol-gel encapsulation methods for biosensors. Anal. Chem. 66, 1120A᎐1127A. Deng, Q., Dong, S., 1995. Construction of a tyrosinase-based biosensor in pure organic phase. Anal. Chem. 67, 1357᎐1360. Deng, Q., Dong, S., 1996. Amperometric biosensor for tyrosinase inhibitors in a pure organic phase. Analyst 121, 1979᎐1982. Ding, L., Li, J., Wang, E., Dong, S., 1997. Kq sensors based on supported alkanethiolrphospholipid bilayers. Thin Solid Films 293, 153᎐158. Dong, S., Guo, Y., 1994a. A novel enzyme electrode for the water-free organic phase. J. Electroanal. Chem. 375, 405᎐407. Dong, S., Guo, Y., 1994b. Organic phase enzyme electrode operated in water-free solvents. Anal. Chem. 66, 3895᎐3899. Dong, S., Guo, Y., 1995. An organic-phase enzyme electrode based on an apparent direct electron transfer between a graphite electrode and immobilized horseradish peroxidase, J. Chem. Soc. Chem. Commun. 483᎐484. Fortier, G., Beliveau, R., Leblond, E., Belanger, D., 1990. ´ ´ Development of biosensors based on immobilization of enzymes in Eastman AQ polymer coated with a layer of Nafion. Anal. Lett. 23, 1607᎐1619. Gennett, T., Purdy, W.C., 1990. Voltammetric determination of the ion-exchange behavior of polyŽester sulfonic acid. anionomers in acetonitrile. Anal. Chem. 62, 2155᎐2158. Ghafouri, S., Thompson, M., 1999. Interfacial properties of biotin conjugate-avidin complexes studied by acoustic wave sensor. Langmuir 15, 564᎐572.

322

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323

Guo, Y., Dong, S., 1997. Organic phase enzyme electrodes based on organohydrogel. Anal. Chem. 69, 1904᎐1908. Hall, G., Best, D., Turner, A.P.F., 1988. The determination of p-cresol in chloroform with an enzyme electrode used in the organic phase. Anal. Chim. Acta 213, 113᎐119. Kang, X., Cheng, G., Dong, S., 2001a. A novel electrochemical SPR biosensor. Electrochem. Commun. 3, 489᎐493. Kang, X., Jing, Y., Cheng G., Dong, S., 2001b. In situ analysis of electropolymerization process of aniline by combining electrochemistry with surface plasmon resonance, Anal. Chem., revised. Karyakin, A.A., Karyakina, E.E., Gorton, L., Bobrova, O.A., Lukachova, L.V., Gladilin, A.K., Levashov, A.V., 1996. Improvement of electrochemical biosensors using enzyme immobilization from water᎐organic mixtures with a high content of organic solvent. Anal. Chem. 68, 4335᎐4341. Kooyman, R.P.H., Kolkman, H., Van Gent, J., Greve, J., 1988. Surface plasmon resonance immunosensors: sensitivity considerations. Anal. Chim. Acta 213, 35᎐45. Lev, O., Tsionsky, M., Rabinovich, L., Glezer, V., Samapath, S., Pankratov, I., Gun, J., 1995. Organically modified sol᎐gel sensors. Anal. Chem. 67, 22A᎐30A. Li, B., Niu, L., Kou, W., Deng, Q., Cheng, G., Dong, S., 1998. Synthesis of a self-gelatinizable grafting copolymer of polyŽvinyl alcohol. for construction of an amperometric peroxidase electrode. Anal. Biochem. 256, 130᎐132. Li, J., Ding, L., Wang, E., Dong, S., 1996a. The ion selectivity of monensin incorporated phospholipidralkanethiol bilayers. J. Electroanal. Chem. 414, 17᎐21. Li, J., Tan, S.N., Ge, H., 1996b. Silica sol᎐gel immobilized amperometric biosensor for hydrogen peroxide. Anal. Chim. Acta 335, 137᎐145. Liu, H., Ying, T., Sun, K., Qi, D., 1996. A reagentless biosensor highly sensitive to hydrogen peroxide based on new methylene blue N dispersed in Nafion gel as the electron shuttle. J. Electroanal. Chem. 417, 59᎐64. Loefaas, S., Malmqvist, M., Roennberg, I., Stenberg, E., Liedberg, B., Lundstroem, I., 1991. Bioanalysis with surface plasmon resonance. Sens. Actuators B 5, 79᎐84. Lyon, L.A., Musick, M.D., Natan, M.J., 1998. Colloidal Au-enhanced surface plasmon resonance immunosensing. Anal. Chem. 70, 5177᎐5183. Malitesta, C., Losito, I., Zambonin, P.G., 1999. Molecularly imprinted electrosynthesized polymers: new materials for biomimetic sensors. Anal. Chem. 71, 1366᎐1370. Malmqvist, M., 1993. Biospecific interaction analysis using biosensor technology. Nature 361, 186᎐187. Ottova, A.L., Tien, H.T., 1997. Self-assembled bilayer lipid membranes: from mimicking biomembranes to practical applications. Bioelectrochem. Bioenerg. 42, 141᎐152. Pei, R., Cui, X., Yang, X., Wang, E., 2000. Real-time immunoassay of antibody activity in serum by surface plasmon resonance biosensor. Talanta 53, 481᎐488. Pei, R., Cui, X., Yang, X., Wang, E., 2001a. Electrostatic layer-by-layer assembly of polycation and DNA multilayer films by real-time surface plasmon resonance technique. Chin. J. Chem. 19, 433᎐435.

Pei, R., Yang, X., Wang, E., 2001b. Enhanced surface plasmon resonance immunosensing using a streptavidin-biotinylated protein complex. Analyst 126, 4᎐6. Rao, J., Yan, L., Xu, B., Whitesides, G.M., 1999. Using surface plasmon resonance to study the binding of vancomycin and its dimer to self-assembled monolayers presenting D᎐Ala᎐D᎐Ala. J. Am. Chem. Soc. 121, 2629᎐2630. Schubert, F., Saini, S., Turner, A.P.F., Scheller, F., 1991. Mediated amperometric enzyme electrode incorporating peroxidase for the determination of hydrogen peroxide in organic solvents. Anal. Chim. Acta 245, 133᎐138. Severs, A.H., Schasfoort, R.B.M., 1993. Enhanced surface plasmon resonance inhibition test ŽESPRIT. using latex particles. Biosens. Bioelectron. 8, 365᎐370. Snejdarkova, M., Rehak, M., Otto, M., 1993. Design of a glucose minisensor based on streptavidin-glucose oxidase complex coupling with self-assembled biotinylated phospholipid membrane on solid support. Anal. Chem. 65, 665᎐668. Stancik, L., Macholan, L., Scheller, F., 1995. Biosensing of tyrosinase inhibitors in nonaqueous solvents. Electroanalysis 7, 649᎐651. Steinem, C., Janshoff, A., Dem Bruch, K., Reihs, K., Goossens, J., Galla, H.-J., 1998. Valinomycin-mediated transport of alkali cations through solid supported membranes. Bioelectrochem. Bioenerg. 45, 17᎐26. Sugawara, M., Kojima, K., Sazawa, H., Umezawa, Y., 1987. Ion-channel sensors. Anal. Chem. 59, 2842᎐2846. Taremi, S.S., Prosise, W.W., Rajan, N., O’Donnell, R.A., Le, H.V., 1996. Human interleukin 4 receptor complex: neutralization effect of two monoclonal antibodies. Biochemistry 35, 2322᎐2331. Tien, H.T., Barish, R.H., Gu, L.-Q., Ottova, A.L., 1998. Supported bilayer lipid membranes as ion and molecular probes. Anal. Sci. 14, 3᎐18. Vreeke, M.S., Yong, K.T., Heller, A., 1995. A thermostable hydrogen peroxide sensor based on ‘wiring’ of soybean peroxidase. Anal. Chem. 67, 4247᎐4249. Wang, B., Dong, S., 2000a. Organic-phase enzyme electrode for phenolic determination based on a functionalized sol᎐gel composite. J. Electroanal. Chem. 487, 45᎐50. Wang, B., Dong, S., 2000b. Sol᎐gel-derived amperometric biosensor for hydrogen peroxide based on methylene green incorporated in Nafion film. Talanta 51, 565᎐572. Wang, B., Li, B., Deng, Q., Dong, S., 1998. Amperometric glucose biosensor based on sol᎐gel organic᎐inorganic hybrid material. Anal. Chem. 70, 3170᎐3174. Wang, B., Li, B., Wang, Z., Xu, G., Wang, Q., Dong, S., 1999. Sol᎐gel thin-film immobilized soybean peroxidase biosensor for the amperometric determination of hydrogen peroxide in acid medium. Anal. Chem. 71, 1735᎐1739. Wang, B., Zhang, J., Cheng, G., Dong, S., 2000a. Amperometric biosensor for inert organic solvents based on a sol᎐gel hybrid material, J. Chem. Soc. Chem. Commun. 2123᎐4. Wang, B., Zhang, J., Cheng, G., Dong, S., 2000b. Amperometric enzyme electrode for the determination of hydrogen

S. Dong, X. Chen r Re¨ iews in Molecular Biotechnology 82 (2002) 303᎐323 peroxide based on sol᎐gelrhydrogel composite film. Anal. Chim. Acta 407, 111᎐118. Wang, B., Zhang, J., Dong, S., 2000c. Silica sol᎐gel composite film as an encapsulation matrix for the construction of an amperometric tyrosinase-based biosensor. Biosens. Bioelectron. 15, 397᎐402. Wang, B., Li, B., Dong, S., 2001. Acid-stable amperometric soybean peroxidase biosensor based on a grafting copolymer of polyvinyl alcohol and 4-vinylpyridine. Electroanalysis 13, 555᎐558. Wang, J., 1993. Organic-phase biosensorsᎏnew tools for flow analysis: a short review. Talanta 40, 1905᎐1909. Wang, J., 1999. Sol᎐gel materials for electrochemical biosensors. Anal. Chim. Acta 399, 21᎐27. Wang, J., Lin, Y., 1993. On-line organic phase enzyme detector. Anal. Chim. Acta 271, 53᎐58. Wang, J., Lu, F., 1998. Oxygen-rich oxidase enzyme electrodes for operation in oxygen free solutions. J. Am. Chem. Soc. 120, 1048᎐1050. Wang, J., Reviejo, A.J., 1993. Organic-phase enzyme electrode for the determination of trace water in nonaqueous media. Anal. Chem. 65, 845᎐847. Wang, J., Dempsey, E., Eremenko, A., 1993. Organic-phase biosensing of enzyme inhibitors. Anal. Chim. Acta 279, 203᎐208. Wang, J., Leech, D., Ozsoz, M., Martinez, S., Smyth, M.R., 1991. One-step fabrication of glucose sensors based on entrapment of glucose oxidase within poly Žester-sulfonic acid. coatings. Anal. Chim. Acta 245, 139᎐143. Weimar, T., 2000. Recent trends in the application of evanescent wave biosensors. Angew. Chem. Int. Ed. Engl. 39, 1219᎐1221.

323

Wilchek, M., Bayer, E.A., 1988. The avidin᎐biotin complex in bioanalytical applications. Anal. Biochem. 171, 1᎐32. Wink, T., Van Zuilen, S.J., Bult, A., Van Bennekom, W.P., 1998. Liposome-mediated enhancement of the sensitivity in immunoassays of proteins and peptides in surface plasmon resonance spectrometry. Anal. Chem. 70, 827᎐832. Wu, Z., Tang, J., Cheng, Z., Yang, X., Wang, E., 2000a. Ion channel behavior of supported bilayer lipid membranes on a glassy carbon electrode. Anal. Chem. 72, 6030᎐6033. Wu, Z., Wang, B., Dong, S., Wang, E., 2000b. Amperometric glucose biosensor based on lipid film. Biosens. Bioelectron. 15, 143᎐147. Wu, Z., Wang, B., Cheng, Z., Yang, X., Dong, S., Wang, E., 2001. A facile approach to immobilize protein for biosensor: self-assembled supported bilayer lipid membranes on glassy carbon electrode. Biosens. Bioelectron. 16, 47᎐52. Zhang, J., Lin, B., Wang, Z., Cheng, G., Dong, S., 1999. Functionalized inorganic᎐organic composite material derivated by sol᎐gel for construction of mediated amperometric hydrogen peroxide biosensor. Anal. Chim. Acta 388, 71᎐78. Zhang, J., Wang, B., Xu, B., Cheng, G., Dong, S., 2000. Amperometric quantification of polar organic solvents based on a tyrosinase biosensor. Anal. Chem. 72, 3455᎐3460. Zhang, Y., Hu, Y., Wilson, G.S., Moatti-Sirat, D., Poitout, V., Reach, G., 1994. Elimination of the acetaminophen interference in an implantable glucose sensor. Anal. Chem. 66, 1183᎐1188.