Mass Production of Biosensors Manuel Alvarez-lcaza and Ursula Bilitewski Gesellschaft für Biotechnologische Forschung mbH Department of Enzyme Technology Mascheroder Weg 1 D-W-3300 Braunschweig, Germany
Biosensors are analytical devices based on the combination of a biological component with a suitable transducer. The biological component is in an immobilized form, generally in proximity to the transducer. It may catalyze chemical r e a c t i o n s (enzymes, m i c r o o r g a n i s m s , or organelles) or specifically bind the analyte (antibodies or receptors). The transducer monitors the biochemical reaction (i.e., 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 t r a p p i n g a biochemical layer between mem0003 - 2700/93/0365 -525A/$04.00/0 © 1993 American Chemical Society
branes was combined with electrodes and introduced more than 30 years ago ( i ) , it was t h o u g h t to be the beginning of an analytical revolution. Efforts were focused 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
REPORT samples, and very few are commercially available. A prerequisite for commercial production is that the biosensor must be fabricated at least on a m e d i u m batch scale, with appropriate quality assurance (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 t h u s they are not suitable for large-scale production.
Additionally, not all experimental parameters affecting sensor response are completely understood. Therefore, t h e y 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 II (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 to 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 Po t e n s o m e t r i c devices. For analytical purposes, the two basic elec-
ANALYTICAL CHEMISTRY, VOL. 65, NO. 11, JUNE 1, 1993 · 525 A
REPORT Table 1. Main biological components and measuring principles used in biosensor systems Transducer/measuring principle
Biological component
pH, 0 2 , H 2 0 2 , modified electrode ISFETs 0 2 , pH optode, NADH fluorescence, NADH absorption Conductivity Thermistors/calorimetry Surface plasmon resonator Grating coupler, interferometer Piezoelectric device Fluorescence (competitive assay) Enzyme-linked assays: electrodes, absorption, fluorescence, luminescence Q2, C 0 2 electrodes Electrodes
Enzyme
Antibody
Microorganisms Plant and animal tissue
trochemical methods are potentiometry and amperometry. Potentiometric m e a s u r e m e n t s involve d e t e r m i n a tion of t h e p o t e n t i a l b e t w e e n two electrodes when there is no c u r r e n t flowing b e t w e e n t h e m . T h e elec t r o d e s can be s i m p l e m e t a l w i r e s whose surfaces are modified to make them selective for a p a r t i c u l a r ion. T h e y a r e dipped directly i n t o t h e sample or separated from 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 elec trode and the other is the indicator (or working) electrode. The most common potentiometric devices are pH electrodes and other ion-selective electrodes. The poten tial of these electrodes (E) is depen dent on the activity (concentration) of a defined ion (aj, as described by the Nernst equation, if only a single ion is relevant and if electrochemical e q u i l i b r i u m b e t w e e n t h e solution and the electrode is obtained RT £ = £ ° + — lnai zF
(1)
E° is the standard potential, R is the gas constant, F is the F a r a d a y con s t a n t , ζ is t h e n u m b e r of electrons transferred between each molecule of the analyte and the electrode, and Τ is the temperature. The potential difference with r e spect to the reference electrode is de pendent on all potential differences that appear at the various phase b o u n d a r i e s of t h e electrochemical setup, including the potential of the reference electrode itself and differ ences b e t w e e n e l e c t r o l y t e s , s e p a rated by membranes or porous plugs (4). Reproducible fabrication of po tentiometric measuring systems de
pends on the reproducible fabrica tion of reference electrodes and on the junctions between electrolytes. An important variation of the sys tems used to determine ion concen t r a t i o n s are the ion-sensitive field effect t r a n s i s t o r s (ISFETs), which a r e composed of a n i o n - s e l e c t i v e membrane built directly over the in sulation of the t r a n s i s t o r gate (5). The device is s i m i l a r to a conven tional MOSFET (metal oxide semi conductor FET) except t h a t the metal contact at the gate is removed to expose the underlying modified in sulator to the solution. A m p e r o m e t r i c d e v i c e s . Amper ometry is based on the oxidation or reduction of a n electroactive com pound a t an electrode while a con stant potential is applied to this elec t r o d e w i t h r e s p e c t to a s e c o n d electrode. The r e s u l t i n g c u r r e n t is measured by using either these two electrodes (working and counter elec trodes) or a three-electrode arrange ment (working, counter, and refer ence electrodes) to compensate for the potential drop caused by passage of the current through the solution. The m e a s u r e d c u r r e n t / is a direct measurement of the electrochemical reaction rate (oxidation or reduction rate of the analyte at the electrode), as described by Faraday's law (Equa tion 2)
I = zF%
(2)
eu
where dn/di is the oxidation or r e duction rate (in mol s - 1 ). Because of the heterogeneous nature of the pro cess, t h e reaction r a t e depends on the r a t e of electron transfer at the surface of the electrode and on the mass transport of the analyte to the surface. The rate of electron transfer
526 A · ANALYTICAL CHEMISTRY, VOL. 65, NO. 11, JUNE 1, 1993
can be accelerated by increasing the p o t e n t i a l difference b e t w e e n t h e electrodes. When the reaction at the surface of t h e electrode is fast, be cause of the increased potential, a maximum overall rate of reaction is reached. This fast reaction, known as the reversible case, is limited by the m a x i m u m r a t e of m a s s t r a n s p o r t . This maximum occurs when the con centration of analyte at the surface of the electrode is zero. The total rate of m a s s t r a n s p o r t to t h e electrode surface depends on the bulk concen tration of analyte, on the area of the electrode, and on diffusion and con vection conditions (6). For example, in a quiescent solution, after a po t e n t i a l b e t w e e n t h e e l e c t r o d e s is poised, the current (/) decreases with time (f) because of the slow spread of the diffusion layer out into the bulk solution combined with a decrease of the concentration gradient. The cur rent change is described by the Cottrel equation / = zFA
—C
(3)
where C is the concentration of the electroactive compound (in mol cm - 3 ), A is the electrode area (cm 2 ), and D is t h e diffusion coefficient of t h e electroactive compound in the solu tion (cm 2 s _ 1 ). U n d e r these conditions, t h e cur rent should become zero after a rela tively long time. Nevertheless, this process stops because of random con vection in the solution; even after a very long time, low currents can be observed. In most practical s i t u a tions, forced convection is provided by moving the electrode with respect to t h e liquid or vice versa. A s t a g nant layer, which has a thickness (L) t h a t depends on the relative move ment of the electrode and the liquid, is formed on the surface of the elec trode (7). Consequently, m a s s t r a n s p o r t to the electrode surface is controlled by diffusion through this layer. This ap p r o a c h is c o n v e n i e n t b e c a u s e a steady state is reached in a relatively s h o r t t i m e a n d b e c a u s e t h e final value of the current is different from zero and depends on the analyte con centration, described by the follow ing equation I = zFAj
cB
(4)
where cB is the analyte concentration in the bulk solution. Equation 4 can be obtained from Fick's laws (steady state). The
boundary conditions assume uniform analyte distribution in the bulk solution up to the stagnant layer, and at 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 m e m b r a n e 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. In o t h e r practical s i t u a t i o n s , it may be preferable to increase mass t r a n s p o r t . T h i s r e s u l t can be obtained by using microelectrodes (S). 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. Commercially available biosensor systems Company
Application
Model
Analyte
Principle
Genetics International, U.K.
ExacTech
Glucose
Blood
Prufgerâtewerk Medingen GmbH, Freital, Germany Metertech Inc., Nan Kang Taipei, Taiwan Yellow Springs Instrument Co., USA
ESAT 6660-2
Glucose Lactate
Disposable mediated enzyme electrode Enzyme electrode
Model 5000
Glucose
Glucose strip
Blood
2700 Select
Blood, serum, plasma
Glucose Enzyme Lactate electrode Ethanol Lactose Sucrose Galactose Methanol Starch 2300 Stat Glucose Enzyme Lactate electrode 1500 G Glucose Enzyme electrode FGA-1 Glucose Glucose Enzyme TOA Electronics electrode + Ltd., Tokyo, Japan analyzer flow injection analysis Enzyme Glu-11 Glucose electrode Kalger GmbH, Microzym-L Lactate Enzyme Neuberg, Germany electrode Electrolux Fermentation Getinge AB, Getinge, Sweden
Electrolux
Glucose
Sigma, Russia
EXAN
Glucose
Dosivit, Nantes, France
MC2 Multisensor
La Roche, Switzerland Aucoteam GmbH, Berlin, Germany Central Kagaku Corp., Tokyo, Japan Kelma, Niel, Belgium
LA 640
Glucose Sucrose Lactose Lactate Ethanol Lactate
BODyPoint
BOD
BOD-2000
BOD
RODTOX
Pharmacia Biosensor AB, Uppsala, Sweden
BIAcore
Short time Microbial BOD, reactor + toxicity 0 2 electrode Immunologic surface plasmon resonance
Biotechnology, pharmaceuticals, food
Blood, plasma, serum Blood, plasma Biotechnology
Food, medicine
with time and is not affected by convection in the bulk solution. Microelectrodes h a v e opened m a n y new possibilities in electrochemistry and may have a positive effect on the biosensors field. Suitable electrode m a t e r i a l s for working and counter electrodes for amperometry are conductive, i n e r t m a t e r i a l s s u c h a s noble m e t a l s , graphite and other modified forms of carbon, a n d conducting polymers. Ag/AgCl is the most common reference electrode. F u n d a m e n t a l s of e n z y m a t i c r e a c t i o n s . Enzymes, proteins with m o l e c u l a r w e i g h t s r a n g i n g 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 advant a g e of c h a n g e s in t h e i r a c t i v i t y caused by the presence of substrates, inhibitors, or activators. The activity is related to the conversion r a t e of the substrate. Under standard conditions (25 °C, optimal pH) it is reported in units representing the amount of enzyme required for the conversion of 1 umol of substrate in 1 min. 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 compounds using oxygen or NAD, and t h e hydrolases, which catalyze the hydrolysis of compounds.
Food, biotechnology, medicine Biotechnology
Bulk solution
Enzyme reactor + flow injection analysis Enzyme electrode Agriculture, Electrochemical food enzyme sensor Enzyme electrode Microbial electrode Microbial electrode
C=cB
Wastewater Sewage and wastewater Municipal and industrial wastewater Biomolecular interactions
x=-L
x=0
Figure 1. Schematic diagram of the concentration profile of an electrochemically active compound at an amperometric electrode.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 11, JUNE 1, 1993 · 527 A
REPORT The general form of an enzymatic reaction is (9) Ε + S -> ES; £S-> Ε + Ρ
(5)
where Ε is the enzyme, S is the sub strate, ES is the e n z y m e - s u b s t r a t e complex, and Ρ is the product. When S is the only limiting substrate, the reaction r a t e is limited by t h e de c o m p o s i t i o n of t h e e n z y m e - s u b s t r a t e complex, leading to t h e Michaelis-Menten equation
w h e r e v0 i s t h e r e a c t i o n r a t e (mol s - 1 ), 7 m a x is the maximum reac tion rate, [S] is the substrate concen t r a t i o n , a n d KM is t h e MichaelisMenten constant (concentration for which v0 = 0.5 VmaJ. Vmax depends on the amount of enzyme or the en zyme activity. This parameter can be determined by measuring the initial reaction rate at high substrate con centrations ([5] » KM). At low sub strate concentrations ([S] « KM) the reaction r a t e is linearly related to the substrate concentration v0 = a[S] where
Vmax
α = ——
(7) (8)
When immobilized enzymes a r e used, t h e overall reaction rate may also be limited by m a s s t r a n s p o r t conditions. Enzyme e l e c t r o d e s . Enzyme electrodes are based on t h e electro c h e m i c a l d e t e r m i n a t i o n of com pounds involved in an enzymatic r e action such a s t h e o x i d a t i o n of glucose, catalyzed by glucose oxidase
overall sensor response depends on the detector, the enzymatic reaction, and the mass transfer processes. When designing enzyme elec trodes, detector characteristics need not be considered. The electrochemi cal detector can be considered a pas sive witness t h a t observes the reac t i o n t a k i n g p l a c e in t h e e n z y m e region. To analyze t h e s i t u a t i o n w i t h i n the enzyme region, a simplified model is proposed (Figure 2). T h e model consists of t h r e e regions: a convective region, for χ > L, where t h e a n a l y t e c o n c e n t r a t i o n is con s t a n t ; a diffusion-limiting region, between χ = 0 a n d χ = L, w h e r e a p u r e diffusion process occurs; a n d the region where the enzyme is im mobilized a n d a diffusion reaction process takes place (x < 0). To keep the model simple, the fol lowing a p p r o x i m a t i o n s a r e m a d e . T h e p a r t i t i o n coefficients for t h e three regions are considered equal to unity. The supply of cosubstrate for the enzyme is considered to be plen tiful, which implies that for low sub strate concentrations the enzymatic reaction rate depends linearly on the substrate (Equation 7). The enzyme is distributed uniformly for χ < 0. B o u n d a r y conditions a r e e s t a b lished a t t h e two interfaces of t h e d i f f u s i o n - l i m i t i n g layer a n d very
Enzyme layer
Diffusion control
Bulk solution
Glucose + 0 2 -> Gluconic acid + H 2 0 2 (9) This reaction can be monitored by the electrochemical reduction of 0 2 , the oxidation of H 2 0 2 , or changes in pH. The hydrolysis of urea catalyzed by urease Urea + H 2 0 -> N H ; + HCO3 + OH" (io) can be monitored by pH m e a s u r e ments, NH4-selective electrodes, or conductivity m e a s u r e m e n t s . In all enzyme electrodes, the enzyme is im mobilized. The enzymatic reaction t a k e s place only in a region sepa rated from the bulk solution. The en zyme substrate must reach t h e e n zyme 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, cit of the substrate is defined by Equation 13.
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deep within t h e enzyme region, where t h e s u b s t r a t e concentration should reach zero. The interface a t χ = 0 is particularly interesting be cause several characteristics of the sensors depend on the concentration of substrate located here. Although t h i s b o u n d a r y condition is e s t a b lished because of the continuity of the mass flow between the regions, it is convenient to use t h e concentra tion at this interface Cj as a n inter mediate variable to express the solu tions of the differential equations at both sides of the interface. Using cit it is possible to express the concen tration profile within the enzyme r e gion by C(x) = c, e x p ^ H * ; * < 0
(")
where De is t h e diffusion coefficient in the zone with enzyme and α is de fined as in Equation 8. The concentration profile within the enzyme region is described by a simple exponential decay (Equation 11). This simple form r e s u l t s from the boundary conditions chosen, but they are not applicable in every problem. Specifically, t h e condition of zero concentration as χ -» - ° ° is difficult to apply in many cases be cause t h e region with enzyme h a s only a finite thickness. A condition in which no flow is allowed beyond χ < -d, where d is the thickness of the e n z y m e r e g i o n , h a s to be i m p l e mented. This leads to an expression that is complicated in mathematical language b u t describes a physical situation similar to that described by exponential decay (10, 11). The sensor response is controlled by mass t r a n s p o r t if the enzymatic reaction is faster t h a n t h e diffusion process—that is, if the concentration fj is 0 or a t l e a s t n e g l i g i b l e . T h e value of c{ is small if the s u b s t r a t e concentration reaches zero at least at a distance χ = -d inside t h e region with enzyme. From Equation 11, this is possible when t h e dimensionless p a r a m e t e r σ 2 = α d2/DB = VmBXd2/ KMOe is large. The parameter σ rep resents t h e ratio between the maxi mum rate of reaction and the rate of diffusion (maximum Thiele modulus). The concentration profile in t h e p u r e diffusion region c a n also be w r i t t e n by u s i n g t h e i n t e r m e d i a t e variable c{ C(x)= ^ ( c B - q ) + ς ; 0
