Manuel Alvarez-lcaza and Ursula Bilitewski Gesellschaflfur Biotechnolcgische Forschuna mbH DepartmentsfEnzyme Technolcgy Mascheder Weg 1 D-W-3300 Braunschweig. Germany ~
~~
~
Biosensors are analytical devices based on the combination ofa biological component with a suitable transducer. The biological component is in an immobilized form, generally in proximity to the transducer. It may catalyze chemical reactions (enzymes, microorganisms, or o r ganelles) or specifically bind the analyte (antibodies or receptors). The transducer monitors the biochemical reaction (Le., either products or cosubstrates of the catalyzed chemical reaction) or the formation of complexes. Because of the specificity of the biochemical reaction, biosensor systems can be used in complex media such as blood, serum, urine, food, or fermentation broth-generally with minimum sample pretreatment. When the concept of trapping a biochemical layer between mem0003~27W~W65-525A1$04.W0,0 0 1993 Amercan Chemcal Society
branes was combined with electrodes and introduced more than 30 years ago ( J ) , it was thought to be the beginning of an analytical revolution. Efforts were foeused on the exploration of the various combinations of biological components with measuring principles (Table I) and, in the past 10 years, thousands of publications have appeared. However, only a limited number of these devices have been applied to real
samples, and very few are wmmercially available. A prerequisite for commercial pmduction is that the biosensor must be fabricated at least on a mediumbatch scale, with appropriate quality aasurance (QA). In most cases. biosensor systems are developed to solve specific research problems. Product engineering is not included as part of development; the biosensors can be produced only by research scientists and thus they are not suitable for large-scale production.
Additionally, not all experimental parameters d e c t i n g sensor response are completely understood. Therefore, they a r e not carefully controlled, and large deviations in the characteristic features of the sensors occur. This prevents guaranteed applicability and documented QA such as that provided by Good Manufacturing Practices (2). Several commercially available biosensor systems are presented in Table I1 (3).Most of these devices were developed for medical applications, mainly for glucose or lactate determinations in blood or serum, and are based on the Combination of suitable enzymes with electrodes. The development of biosensors based on enzyme reactions has matured sufficiently t o justify a discussion about their mass production. In this REPORT we will summarize the theoretical and practical aspects of enzyme electrodes as well as the possible steps that can be taken toward biosensor mass production.
Theoretical aspects Potentiometric devices. For analytical purposes, the two basic elec-
ANALYTICAL CHEMISTRY, VOL. 65, NO. 11, JUNE 1,1993
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REPOR7
I. wain biological components ano measuring principles :::;used .... in biosensor systems ,...
? ::
I awe
Surfaceplasmon resonator Grating coupler, interferometer Piezoelectric device absorption, fluorescence, C02 electrodes
trochemical methods are potentiometry and amperometry. Potentiometric measurements involve determination of the potential between two electrodes when there is no current flowing between them. The electrodes can be simple metal wires whose surfaces are modified to make them selective for a particular ion. They a r e dipped directly into the sample or separated h m the sample by a membrane or a porous plug and are placed in an electrolyte solution of defined composition. Generally, one electrode is the reference electrode and the other is the indicator (or working) electrcde. The most common potentiometric devices are pH electrodes and other ion-selective electrodes. The potential of these electrodes ( E ) is dependent on the activity (concentration) of a defmed ion (uJ, a s described by the Nernst equation, if only a single ion is relevant and if electrochemical equilibrium between t h e solution and the electrode is obtained
E = E o+ -m -ln~, ZF
(’)
Eo is the standard potential, R is the gas constant, F is the Faraday constant, I is the number of electrons transferred between each molecule of the analyte and the electrode, and T is the temperature. The potential difference with respect to the reference electrode is dependent on all potential differences t h a t appear a t t h e various phase boundaries of t h e electrochemical setup, including the potential of the reference electrode itself and differences between electrolytes, separated by membranes or porous plugs (4). Reproducible fabrication of POtentiometric measuring systems de-
pends on the reproducible fabrication of reference electrodes and on the junctions between electrolytes. An important variation of the systems used to determine ion concentrations are the ion-sensitive field effect transistors (ISFETs), which a r e composed of a n ion-selective membrane built directly over the insulation of the transistor gate (5). The device is similar to a conventional MOSFET (metal oxide semiconductor FETJ except t h a t t h e metal contact at the gate is removed to expose the underlying modified insulatbr to the solution. Ampemmetric devices. Amperometry is based on the oxidation or reduction of a n electroactive compound a t a n electrode while a constant potential is applied to this electrode with respect t o a second electrode. The resulting current is measured by using either these two electrodes (working and counter electrodes) or a three-electrode arrangement (working, counter, and reference electrodes) to comDensate for the potential drop caused by passage of the current through the solution. The measured current I is a direct measurement of the electrochemical reaction rate (oxidation or reduction rate of the analyte a t the electrode), as dewxibed by Faraday’s law (Equation 2)
dn I=zFdt
(2)
where d d d t is the oxidation or reduction rate (in mol s-l). Because of the heterogeneous nature of the process, the reaction rate depends on the rate of electron transfer a t the surface of the electrode and on the mass transport of the analyte to the surface. The rate of electron transfer
526 A * ANALYTICALCHEMISTRY, VOL. 65, NO. 11, JUNE 1,1993
can be accelerated by increasing the potential difference between t h e electrodes. When the reaction a t the surface of the electrode is fast, because of the increased potential, a maximum overall rate of reaction is reached. This fast reaction, known as the reversible case, is limited by the maximum rate of mass transport. This maximum occurs when the concentration of analyte a t the surface of the electrode is zero. The total rate of mass transport t o t h e electrode surface depends on the bulk concentration of analyte, on the area of the electrode, and on diffusion and convection conditions (6).For example, in a quiescent solution, after a potential between t h e electrodes is poised, the current (1)decreases with time (t) because of the slow spread of the diffusion layer out into the bulk solution combined with a decrease of the concentration gradient. The current change is described by the Cottrel equation
where C is the concentration of the electroactive compound (in mol ~ m - ~ ) , A is the electrode area (cm’), and D is t h e diffusion coefficient of t h e electroactive compound in the solution (em*8-9. Under these conditions, the current should become zero aRer a relatively long time. Nevertheless, this process stops because of random convection in the solution; even after a very long time, low currents can be observed. I n most practical situations, forced convection is provided by moving the electrcde with respect to the liquid or vice versa. A stagnant layer, which has a thickness (L) t h a t depends on the relative movement of the electrode and the liquid, is formed on the surface of the electrode (7). Consequently, mass transport to the electrode surface is controlled by diffusion through this layer. This approach is convenient because a steady state is reached in a relatively s h o r t time and because t h e final value of the current is different from zero and depends on the analyte concentration, described by the following equation
I
=
zFA-C, D L
(4
where c, is the analyte concentration in the bulk solution. Equation 4 can be obtained from Fick’s l a w s ( s t e a d y s t a t e ) . T h e
boundary conditions assume uniform analyte distribution in the bulk s o h tion up to the stagnant layer, and a t the surface of the electrode assume the condition of mass transport control, C = 0 a t x = - L (Figure 1). In many practical situations, mass transport to the electrode is best attained by placing a diffusion-limiting membrane over t h e electrode surface rather than depending on the stagnant layer, because its thickness
can be easily controlled only in the case of laminar flow. I n other practical situations, it may be preferable to increase mass transport. This result can be obtained by using microelectrodes (8). For these electrodes diffusion takes place radially through a sphere centered in the electrode. The conditions for this geometry are not described by the Cottrel equation, and the current does not tend to approach zero ~
Table II. Commerclally available biosensor systems c0mp.w
Y0d.l
rrulvt. Glucose
Genetics International, U I(.
ExacTech
Pmfgeratewerk Medingen GmbH, Freitai. Germany
ESAT 6660-2
Glucose Lactate
Metertech 11% Nan Kang Taiw.
M W l m
wwon
Prlnclpl.
Disposable mediated enzyme elect& Enzyme
Blood
Glucose
Glucosestnp
Blood
Glucose Lactate Ethanol Lactose
Enzyme electrode
Blwd, serum,
~
plasma
electrode
Taiwan
SUNOW
Galactose Methanol Starch Glucose Enzyme Laclate electrode Glucose Enzyme
Blood, plasma,
serum
Blood, plasma
electrode
TOA Electronics
FGA-1 Glucose Gluccse
Enzyme
electrode + flow injec-
Biotechdogy
tion analysis
Glucose
En'iL
Fwd, medidna
Lactate
Em me
Food, biotech-
Electrdux Fermen- Electmlux tatlon Getinge AB, Gelinge, Sweden
Glucose
Biotechnalogy Enzvnm riactor + How injection analysis
Sigma. Russia
EXAN
Glucose
Cusivit, Nantes,
MC2 Mu1
Glucose
Enzyme electrode Electm-
Microzym-L
FranrS
sensor
sucrose
eictctrode
'
with time and is not affeded by convection in the bulk solution. Microelectrodes have opened many new possibilities in electrochemistry and may have a positive effect on the biosensors field. Suitable electrode materials for working and counter electrodes for amperometry are conductive, inert materials such a s noble metals, graphite and other modified forms of carbon, and conducting polymers. AgIAgC1 is the most common reference electrode. Fundamentals of enzymatic reactions. Enzymes, proteins with molecular weights ranging from 12,000 Da up to 1,000,000 Da, act as specific catalysts for chemical reactions. Their specificity is determined mainly by the 3D structure near the active site, and they can be used for analytical purposes by taking advantage of changes i n t h e i r activity caused by the presence of substrates, inhibitors, or activators. The activity is related to the conversion rate of the substrate. Under standard wnditions (25 "C, optimal pH) it is reported in units representing the amount of enzyme required for the conversion of 1p o l of substrate in 1min. Enzymes can be classified into six groups, depending on their mode of action. From an analytical perspective, the most important classes are the oxidoreductases, which catalyze the oxidation of wmpounds using oxygen or NAD, and the hydrolases, which catalyze the hydrolysis of compounds.
%%he
Chemioal
enzyme sensor
Enzyme
elect&
Microbial
elmrcde
Microbial electrode
Sewage and
Mimbial reactor + Os electrode
Municipal and industrial wastewater
lmmunolwic surfacapasmon
Bi0molecular
resonance
wastewater
interactions
coicantration profile of i n electmhemically active compound at
an amperometric electrode.
ANALYTICAL ChcmiS;TRY, VOL. 65.NO. 11. JUNE 1.1993
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REPORT The general form of an enzymatic reaction is (9)
E
+ S + ES; ES+ E + P
(5)
where E is the enzyme, S is the substrate, ES is the enzyme-substrate complex, and P i s the pmduct. When S is the only limiting substrate, the reaction rate is limited by the decomposition of t h e enzyme-subs t r a t e complex, leading t o t h e Michaelis-Menten equation
w h e r e u,, i s t h e r e a c t i o n r a t e , is the maximum reac(mol s-l), V tion rate, [SI is the substrate concentration, and K, is the MichaelisMenten constant (concentration for which u, = 0.5 VmJ. Vmmxdepends on the amount of enzyme or the enzyme activity. This parameter can be determined by measuring the initial reaction rate a t high substrate concentrations ([SI>> K,). At low substrate concentrations ([SI L, where t h e analyte concentration is constant; a diffusion-limiting region, between x = 0 and x = L, where a Dure diffusion Drocess occurs: and the region wheie the enzyme is immobilized and a diffusion reaction process takes place (x c 0). To keep the model simple, the following approximations are made. The Dartition coefficients for t h e three-regions are considered equal to unity. The SUDD~Yof cosubstrate for the enzyme is&nsidered to be plentiful, which implies that for low substrate concentrations the enzymatic reaction rate depends linearly on the substrate (Equation 7). The enzyme is distributed uniformly for x c 0. Boundary conditions a r e established a t the two interfaces of the diffusion-limiting layer and very
Enzyme layer
Diffusion
control laver
Bulk solut
+ 0, +
Gluconic acid + H,O, (9) This reaction can be monitored by the electrochemical reduction of 0,, the oxidation of H,O,, or changes in pH. The hydrolysis of urea catalyzed by urease Urea
+ H,O + NH; + HCO;
+ OH-
(10)
can be monitored by pH measurements, NHZ-selective electrodes, or conductivity measurements. In all enzyme electrodes, the enzyme is immobilized. The enzymatic reaction takes place only in a region separated from the bulk solution. The enzyme substrate must reach the enzyme by convection or diffusion. The
Figure 2. Concentration profile of an enzyme substrate at an enzyme membrane that includes a diffusion-limiting region of the thickness L. The intermediate concentration, c,, of me substrate is defined by Equation 13. : ;. . :;': . ,
528 A * ANALYTICAL CHEMISTRY, VOL. 65, NO. 11. JUNE 1,1993
'
deep within t h e enzyme region, where the substrate concentration should reach zero. The interface a t x = 0 is particularly interesting because several characteristics of the sensors depend on the concentration of substrate located here. Although this boundary condition is established because of the continuity of the mass flow between the regions, it is convenient to use the concentration a t this interface c, a s an intermediate variable to express the 801~tions of the differential equations a t both sides of the interface. Using c,, it is possible to express the concentration profile within the enzyme region by
where De is the diffusion coefficient in the zone with enzyme and a is defined as in Equation 8. The concentration profile within the enzyme region is described by a simple exponential decay (Equation 11).This simple form results from the boundary conditions chosen, but they a r e not applicable i n every problem. Specifically, the condition of zero concentration a s x + is difficult to apply in many cases because the region with enzyme has only a finite thickness. A condition in which no flow is allowed beyond x c -d, where d is the thickness of the enzyme region, h a s t o he implemented. This leads to an expression that is complicated in mathematical language but describes a physical situation similar to that described by exponential decay ( 1 0 , l l ) . The sensor response is controlled by mass transport if the enzymatic reaction is faster than the diffusion process-that is, if the concentration cL is 0 or a t least negligible. The value of c, is small if the substrate concentration reaches zero at least at a distance x = -d inside the region with enzyme. From Equation 11,this is possible when the dimensionless parameter 0' = a dZ/De= V,,.dZ/ K,D, is large. The parameter o represents the ratio between the maximum rate of reaction and the rate of diffusion (maximum Thide modulus). The concentration profile in the pure diffusion region can also be written by using the intermediate variable c,
--
X
c(x)= -(ce L
- Ci) + ci; O < X C L (12)
The boundary condition a t x = 0 can be used to determine the value of ci
as a function of e,. The flow moving into the region with enzyme can be calculated by using Fick’s first law applied to the profile described by Equation 11 and evaluated at x = 0. The flow out of the pure difFusion region is the difference (c, - C J multiplied by the mass transfer coefficient for this region DJL, where D, is the diffusion coefficient. The condition for both flows to be equal is defined when
ci
=
CiJ
(13)
1+”*D. The analyte concentration c, is the maximum concentration within the enzyme region. Therefore, if c. > De where 0. is the diffusion coefficient in the stagnant layer) and application of an additional membrane of thickness L on top of the enzyme layer results in a range of ratios of De to D,. In the rest of this section we will discuss the need for the application of this additional mem-
brane with respect to reproducible fabrication of enzyme electrodes. Probably the main problem in the mass production of biosensors, especially disposable sensors, is reproducible fabrication (reproducible sensitivity and linear range) of numerous sensors, because there are variations in time, pH, temperature, and during transport and storage. The most fragile part of the sensor is its biological component. Hence, we have to analyze the sensor’s performance when variations in this biological component a r e produced. From this analysis, we can decide if the incorporation of a membrane on top of t h e sensor will improve its performance. Variations in t h e sensitivity of sensors with a n d without membranes are shown in Figures 3 and 4. We assumed that for stationary conditions, the sensor signal depends on product generation. This signal should be equal to the flow of substrate into the sensor. By using the value of ci (Equation 13)to express the difference (c, - ci), the flow density moving into the diffusion membrane j , can be expressed (Fick’s law) as
and for the sensor without a membrane, j,
ir= f i c B
(16)
Sensitivity (S)is obtained by dividing the output signal (substrate flow) by the input signal (the analyte concentration in the bulk). The variation in sensitivity is represented by the derivative of the sensitivity with respect to the parameter a, which represents the enzyme kinetics. For the sensor with membrane
and for the sensor without a membrane
To make a fair comparison, because the sensitivity of the two types of sensors is different, we divide by the sensitivity to obtain t h e relative variation of the sensitivity with respect to the variation of the enzyme activity. For the sensor with a membrane
as
and for the sensor without membrane
as.
Figure 3. Influenceof a on the concentration profile of an enzyme substrate at an enzyme electrode covered with a membrane that acts as a diffusion barrier.
The two equations are very similar, but the one describing the situation for the sensor covered with a diffusion membrane contains a n extra term [l + (L/D,)(D.,a)”*I-’. All the variables must be positive values; therefore, this term can be only 51. The limiting case when this term equals one is for a n infinitely fast diffusion across the membrane (0.+ -) or for a membrane of zero thickness ( L = O), which is equivalent to the case without a membrane. Or, for the uninteresting situations, there is no enzyme activity (a= 0 ) or t h e substrate cannot diffuse into the region where the enzyme is immobilized (0.= 0). Therefore, for all real cases the use of a membrane on top of the sensor reduces the relative variation of the sensor’s sensitivity with respect to variations of the enzyme activity. The extra effort involved during sensor production to generate and affii this membrane to
ANALYTICAL CHEMISTRY, VOL. 65. NO. 11, JUNE 1,1993 * 529 A
REPORT the top of the sensor results in greater stability with respect to variations in the enzyme activity.
Fabrication technoiogles for electrodes As long as the cosubstrate is provided, there is no basic difference between amperometric and potentiometric detection in enzyme sensors. The decision of whether to use a n amperometric or a potentiometric detector should be based on the capabilities of the detector for measuring a particular component of the enzymatic reaction. By contrast, if a mediator has to be used to complete the enzyme reaction, an amprometric detector is necessary to turn over the mediator. But even when a particular detection method must be used, the many manufacturing techniques offer a wide range of materials for obtaining different characteristics of the surface or of the whole electrode. Potentiometric devices. To give electrodes used in potentiometric devices a n ion-selective character, the electrode surface must be modified. Depending on the ion-sensitive material used, one can distinguish between crystalline and noncrystalline electrodes. Crystalline electrodes can be further divided into homogeneous (e.&, single crystal) and heterogeneous (e.&, AgCl in PVC) solid elec-
Figure 4. Influence of a on the concentration profile of an enzyme substrate (when the enzyme layer is not covered with a membrane), neglecting the stagnant layer at the boundary between the enzyme layer and the solution.
trodes. Noncrystalline electrodes can be subdivided into porous-supported (e.&, glass frit), nonporous-supported (e.g., PVC), liquid ion exchanger, and neutral carrier electrodes. Given t h e poor electrical conductivity of these electrcde materials, t h i n electrode membranes (0.1-5 mm, depending on the material) can be used. Many of these devices a r e described in the literature (4). Electrodes selective for many ions and neutral molecules are commercially available. The most important are glass electrodes; the composition of the glass influences the selectivity. The potential-determining step occurs at the membrane-sample interface by exchanging ions between the two phases. The resulting potential is measured by using a reference electrode connected electrically to t h e sample solution a n d a metal electrode COMected to the inner side of the membrane. The former junction can be done by a salt bridge and the latter either by direct contact of the metallic conductor with the active phase (coated-wire electrodes) or by contact through an electrolyte solution with defined composition. Various solutions are described in the literature and explained theoretically by suitable models. Coated-wire electrodes are formed by dipping a metal wire (Cu, Ag/AgCl, Pt) in a polymer solution (e.g., W C in tetrahvdrofuran) dooed with a suitable iinophore and ahowing the solvent to evaporate. Amperometric devices. As described earlier, amperometric measurements require two or three different electrodes (6): a working, a reference, and possibly a counter electrode. Because the reference electrode has to provide a constant potential, conventional reference electrodes such as AglAgCl or Hg/HgCl electrodes are used. In many cases, metal wires such as Ag, F’t, or Au are suitable because the potential is less critical in amperometric than in potentiometric measurements. The working and the counter electrodes should be chemically inert and allow rapid electron transfer from the electrode to the electroactive compound and vice versa. Snitable electrode materials are noble metals; carbon in forms such as carbon paste, amorphous graphite, pyrolytic graphite, or glassy carbon; or other conducting materials such as conducting polymers. They can be used in any size and form, provided the electrode area is defined by isolating surrounding parts. Simple
530 A * ANALYTICAL CHEMISTRY, VOL. 65, NO. ll,JUNE l,1993
metal wires, metal tubes, carbon rods, or carbon fibers can be used. Several commercially available electrochemical detectors (e.g., from Hewlett Packard, Biometra, or Metrohm) and enzyme-based analyzers (e.g., the Yellow Springs Analyzer, ESAT 6660) are designed in this fashion. Because of the interest in miniaturized sensors and the development of inexpensive, disposable sensors, electronic production techniques have been applied to the manufacture of electrodes. These techniques are used to produce amperometric devices or may be used to obtain potentiometric ion-selective devices. Two different approaches, thick- and thin-film technology, result in planar structures, but they differ in the method used for deposition of the various layers on a substrate. Thickfilm technology is based on screen printing pastes on substrates, followed by drying and (if required) firing. Thin films are produced by various vacuum techniques. T h i c k - f i l m technology. The thick-film process is well established for the production of miniaturized hybrid circuits; miniaturization is limited to patterns of lOO-pn remlution (12). The technology is based on screen printing suitable pastes, differing in composition and electrical conductivity, onto substrates. The most common substrate material in hybrid fabrication is fired A1,0, ceramics. This technology, which was established for the production of electronic circuits, is applied to the fabrication of various sensors. It is suitable for both mass and small-batch production. The basic steps are paste preparation, design and screen production, printing of the paste, drying, firing, and encapsulation. A variety of pastes differing in composition and electrical behavior a r e commercially available. The main components of a paste a r e binder glass, such as borosilicates or aluminosilicates; solvents, such as terpineol and ethyl cellulose; and additives, depending on the application. Pastes with high conductivity, used for the production of conducting paths, contain powder of metals such as Au, Pt, Ag, and Pd (13, 14). For special applications, such as the production of r e s i s t o r s a n d c a r b o naceous electrodes, graphite-based pastes are available. In screen printing, a squeegee is used to force the thick-film paste through a screen onto a substrate. The open pattern in the screen de-
-
fines the pattern that will be printed onto the substrate. After printing, the film is dried and usually firedgenerally in air a t a peak temperature of 500-1000 "C. Through this pmcess a hard, adherent, 10-20-pnthick film with the desired electrical and mechanical properties is formed. Because of the high temperatures during the firing, ceramics are the main substrate materials used. In recent years pastes have been developed that can be annealed a t lower temperatures. a s the binder is no longer glass but a polymer. The simplicity of this process makes it suitable for low-cost sensor production, even on a medium production scale, and it has been used to manufacture several sensors (1.5). Thin-film technology.This technology, used for the production of integrated circuits, is based on microlithographic techniques in which patterns of materials on a substrate surface are created (16).The degree of miniaturization is limited by the wavelength of the radiation required to produce patterns on the photoresist fdm. Hence, resolutions down to 1 p or less are possible. The technique involves, for example, the selective removal (lift-off7 of photores i s t from t h e surface in regions where radiation is allowed to pass through a mask to define the shape of the electrodes (27, 28). New material can then be deposited on t h e surface of the unprotected regions of the substrate by using vacuum evaporation, sputtering, or chemical vapor deposition. In vacuum evaporation, metals are deposited on t h e surface of s u b strates by t h e r m a l evaporation within a vacuum chamber (pressure below lo-' mbar). Originally, thermal evaporation was produced by the heat generated from a n electrical current passing through a tungsten filament. This technique, however, produced tungsten contamination in the evaporated material. Recently, heat has been produced by electron beam bombardment. The method has also been used for materials other than metals, such as glass. A prerequisite for this method is a favorable vapor pressure difference. Not all evaporated materials adhere well to the substrate. An intermediate layer of chromium or titanium between the electrode and the substrate is required to provide adequate adhesion when using Au and Pt.However, the use of intermediate materials can result in problems with the electrochemical behavior of the electmdes.
THE IMNOBZZIZATZON
IS ONE PREREQUISITE
FOR M S S PRODUCTION OF BIOSENSORS... Sputtering takes place within a chamber in the presence of an inert gas a t low pressure. Two electrodes are used and a potential difference is established, producing a glow discharge. Material from the cathode is removed, because of the bombardment of gas ions, and projected toward the anode. The substrate on which the new material will be deposited is placed between the two electrodes intercepting the material removed from the cathode. This technique can be used for the deposition of conductors and, in conjunction with radio frequency, for the d e p s i tion of dielectrics. The nature of the deposited film can be modified by the addition of reactive gases, such a s oxygen, to define the final characteristics of the surface. Chemical vapor deposition has the disadvantage of requiring relatively high temperatures (900 "C). New techniques using plasma have lowered the temperature (350 "C). The deposited dielectric material, such as Al,O,, SO,, or Si,N,. can lead to improvements in the mechanical and electrical properties of the substrate.
lmmoblllzatlon technlques A large variety of immobilization techniques, many of which can be used to immobilize enzymes on various carriers, are described in the literature. In biosensor development, techniques based on physical adsorption, entrapment in a gel or a polymer, covalent binding to a carrier, and cross-linking of proteins a r e used. Optimization of the immobilization is one prerequisite for mass produc-
tion of biosensor8 because it has an impact on some of the characteristic parameters of the enzyme sensors. In some cases, cosubstrates such as mediators have to be added. They can also be incorporated into t h e sensors by using the physical and chemical properties of the materials. Optimized immobilization techniques for mediators or other approaches are required for efficient electron transfer from the enzyme to the electrode, especially if oxidases are used. Various approaches, such as physical adsorption or covalent binding of mediators, the use of molecular-weight-enlarged mediators, modifications of enzymes, and oriented immobilization of enzymes. have been examined with respect to effective electron transfer for the development of "reagentless" enzyme electrodes. To date, physical adsorption of mediators is mainly used for the production of mediator-modified electrodes. But these sensors have limited stability when they are in permanent use and show some interference with oxygen, the natural electron acceptor of oxidases. Thus, alternatives are still being investigated, and even from a scientific viewpoint a final discussion is difficult. Physical adsorption. The physical adsorption method, based on van der Waals attractive forces of the enzymes and the surface of solid supports, is t h e oldest a n d simplest method of enzyme immobilization. I t can be performed by dropping an aliquot of the protein solution on the carrier and allowing the solvent to evaporate. The preferred application is adsorption on graphite electrodes (19). The major disadvantage is that the binding forces between the enzyme and the support cannot be controlled easily. They may be too weak, and thus the adsorbed enzymes are liable to be desorbed during use, depending on experimental conditions such a s pH, ionic strength, temperature, and type of solvent. They may also be too strong (a), causing much of the enzyme to be denatured during immobilization. Entrapment in a matrix. Further improvement in stability is a t tained by increasing the difision resistance: the entrapment of enzymes in matrices such as gels, polymers, pastes, or inks. Immobilization is as simple a s physical adsorption because. in general, the enzyme need only be mixed with the paste, ink material. or a prepolymer. This mixture is applied to the electrode or to a support such a s a n additional membrane, where i t is allowed to dry
ANALYTICAL CHEMISTRY. VOL. 65,NO. 11, JUNE 1.1993
531 A
REPORT or where t h e polymerization is started. Suitable matrix materials include gelatin, polyurethanes that can be surface-cross-linked with isocyanates, polyvinyl alcohol (21), carbon paste (22,231,carbon ink, or polypyrrole (24). The enzyme is immobilized in a membrane, which is formed either directly on the electrode (allowing enzyme immobilization even on microstructured electrodes) or on a n additional membrane that has to be fixed on the electrode. Even if the enzyme kinetics are not affected by immobilization, membrane fabrication must be controlled carefully because the diffusion behavior (De) changes with the composition of the membrane and with polymerization conditions. The main advantage of this technique is its compatibility with mass production methodologies such as screen printing or photopolymerization. C o v a l e n t binding. Generally, stable attachment of proteins to a support is best attained by covalent binding. Prerequisites for this immobilization procedure a r e suitable functional groups on the protein and on t h e support (25).Functional groups in the protein are provided by amino acid residues, the most important of which are amino groups from L-lysine and carboxyl groups from Laspartate or L-glutamate. The amino acids essential for the catalytic activity of t h e enzyme should not be involved in the covalent linkage to the support. Enzymes immobilized in this fashion generally lose activity. Additionally, the affinity of the enzyme to the substrate may be affected by conformational changes. Covalent binding of enzymes to electrodes has been attained on metals, graphite, and conducting poly-
mers. These materials have to be activated before covalent binding of an enzyme. The surface of a metal electrode generally is oxidized to some degree; some hydroxylic groups are present on the surface. These groups can react with silanizing reagents, such a s (aminopropy1)triethoxysilane, creating amino groups on the surface. By using glutaraldehyde, a bifunctional aldehyde, as a coupling reagent, proteins a r e covalently bound. All reactions can be performed at room temperature with aqueous solutions of glutaraldehyde and the protein. To automate the process, the solutions can be handled by suitable dispensers or i n k j e t printers (26). The degree of oxidation of graphite electrodes can be increased by electrochemical oxidation, resulting in carboxylic groups on t h e surface. These groups can be activated for protein coupling via peptide binding using dicyclohexylcarbodiimide.If t h e coupling reaction can be controlled, these immobilization methods result in a monolayer of enzyme on the electrode, allowing for fast diffusion of the substrate. At the same time, however, the amount of enzyme that can be covalently immobilized is limited by the electrode size. Both features are changed if the enzyme is coupled covalently to a 3 0 matrix. C r o s s - l i n k i n g of proteins. An immobilized enzyme preparation can be obtained by inter- and intramolecular cross-linking of the enzyme molecules. This method is based on the production of 3D cross-linked enzyme aggregates by means of bior multifunctional reagents. These reagents are completely insoluble in water and can be adsorbed onto a solid surface. Glutaraldehyde has been the most extensively applied
I
Table 111. Optimal values of the important parameters for the design of enzyme electrodes msl valu.
Small
RMl.*.
Parameters Ds and L are constrained by maximum response time. Highly dependent on immobilization method. De and dare constrained to have a large a. In some cases a compromise between dand D is needed.
-2
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Cross-linking agent (271,although many other multifunctional reagents have been tested (28).Thus the immobilization procedure is similar to that described above. Covalent binding of enzymes t o electrodes generally is a combination of attachment to the electrode surface and cross-linking. The resulting 3D network has an impact on the enzyme kinetics and the dimLsion characteristics. They have to be optimized for each enzyme to achieve good insolubility while retaining the enzymatic activity and producing appropriate diffusion properties. Factors such a s proteinlcross-linking reagent ratio, pH, temperature, and reaction time have to be balanced. These conditions are generally found empirically.
Production technologies for mass transfer control For mass transfer control in biosensors (increased linear range and lower sensitivity with variations in enzyme activity), it is necessary to produce diffusion control barriers (membranes)a t the top of the sensor. Furthermore, the use of a membrane on top of a sensor also helps to prevent the loss of weak immobilized components (enzymes and mediators with high molecular weight) and the passage of substances that might interfere with electrochemical detection or damage the enzyme. These membranes also reduce noise attributable to stirring. Possible technologies for membrane deposition are limited by the fact that the membranes have to be built on top of a layer containing enzyme. High temperatures, highenergy radiation, or dipping the sensors i n t o chemically aggressive solutions should be avoided. Polymer deposition, fixation of commercially available membranes, and electropolymerization are used. P o l y m e r deposition. During some part of their preparation, several polymers, gels, and pastes are liquids with different degrees of viscosity, and their fluidity can be exploited to deposit them on the sensor surface. The sensor can simply be dipped into the solution containing t h e polymer. Because t h e l a y e r thickness is of critical importance, the viscosity of this solution has to be controlled carefully. Additionally, the deposition of the solution on the sensor can be controlled by devices similar to those used for the formation of Langmuir-Blodgett films (29) or by being sprayed (30) or spincoated (31). .
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With good viscosity control, screen printing can be used to produce the diffusion control membrane. Therefore, for the production of the whole sensor, t h e same technology a n d equipment may be used (32). Fixation ofcommercially available membranes. Substrate diffusion can also be limited by cutting and affixing commercially available membranes t o the top of the electrode. This approach has been the most popular way to limit diffusion in biosensors. The application is very simple in individual cylindrically shaped electrodes (e.g., using 0-rings). Some commercially available enzyme analyzers use entrapment of the enzyme between two commercial membranes. The problem of contacting the membranes with the electrode is solved by adequate design of t h e electrode receptacle in the measuring cell. The planar characteristics of sensors produced by microelectronic techniques create problems t h a t must be solved. A good and reproducible contact between the membrane and the electrode, as well as a good seal and fixation of the membrane around the electrode perimeter, has to be obtained. A possible solution to this problem is to use screen printing to fix the membrane with a fast-setting glue deposited on t h e electrode substrate (33). Electropolymerization. Membranes can be produced on top of electrodes by electropolymerization (e.g., polypyrrole and phenol derivatives [34,351).The advantage is that the membrane is produced only on top of the electrode and not on the rest of the sensor. Physical characteristics such as thickness can easily b e controlled by m e a s u r i n g t h e amount of electrical charge passed during membrane formation. Disadvantages are poor adherence of the polymerized film to the electrode and incompatibility with some enzyme immobilization methods. However, electropolymerization appears to be one of the most promising technologies for the mass production of biosensors. Summary As a result of the accomplishments in biosensor research over the past 30 years, it is simple to use immobilized enzymes to fabricate electrodes of various materials, sizes, a n d forms. In addition, scientists have a greater theoretical understanding of parameters such as enzyme activity and diffusion properties that influence sensor response. We have
summarized the most important design parameters in Table 111. We know which technologies (including membrane fabrication, screen printing, and photolithographic techniques such a s thin-film methods) are, in principle, suitable for the mass production of enzyme electrodes. This knowledge has led to the commercialization of enzyme analyzers that are used mainly for glucose or lactate determination. But there are still many parameters that must be improved to achieve reproducible fabrication of sensors applicable to other fields. For example, the electron transfer between enzyme and electrode has to be improved before reagentless, implantable sensors can be fabricated. Another active area of research is in the development of easy-to-use small immunosensors. Commercialization of these devices will be achieved only when attention to their manufacturing asp& is accelerated.
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Ursula Bilitewski heads the biosensor project in the Department of Enzyme Technologv of the GesellschaPfiir Biotechnologische Fonchung. She received her M.Sc. degree in 1983andherPh.D. in 1987fiom Westfolische Wilhelms Uniuenity. Her researchfomes on the use of electrochemicol techniques to prepare biosenson for the analysis of complex media. Manuel Aluarez-Icazu receiued his B.Sc. degree in 1977fiom the National University of M m i o , his M A . h m MIT in 1985, and his Ph.D. in 1989fiom the Cranfield Institute of Technology, U.K. His research centen on the study of biophysicol phenomen0 for the development of measurement methods and instruments.
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