G. A. Rechnitz Department of Chemistry University of Delaware Newark, Del. 19711
Bioanalysiswith Potentiometric Membrane Electrodes During the past few years, an almost bewildering array of potentiometric membrane electrodes has been developed for biochemical analysis. Some of the resulting measurement systems have reached a degree of refinement a t which they are useful for routine analysis, and commercial competition has become quite intense. The excellent new review by Meyerhoff and Fraticelli (I ) provides a comprehensive technical assessment of the state of the art and details many of the contemporary application areas. Precisely because of the very large nnmher of possible applications and numerous variations in electrode design, the potential user of potentiometric membrane electrodes for hiochemical analysis who is not a specialist in the field is likely to he overwhelmed when faced with a literature growth approaching 1000 publications per year. The purpose of this article is to provide a brief introduction to the field by showing some of the major themes and interrelationships common to hioselective potentiometric membrane electrodes. It is possible to view the development of potentiometric membrane electrodes in terms of a “family tree” (Figure 1). Since the early work on glass electrodes, further progress has been indebted to the infusion of ideas from other scientific fields (2). The 1194A
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development of crystal membrane electrodes, for example, owes much to solid-state chemistry, while efforts to devise carrier-based membrane electrodes were undoubtedly stimulated hy modern research on hiological membrane transport. In keeping with the precept that “necessity is the mother of invention,” the experimentally challenging development of microelectrodes would probably not have been undertaken without the demand hy physiologists for electrodes that could make measurements within single cells and other very small structures. Once a number of ion-selective membrane electrodes were established as useful analytical devices, impetus for further research and development was provided by the desire to extend the range of species that could be measured, to improve selectivity and sensitivity, and to lower costs and maintenance needs, as well as to lengthen the practical lifetime of such sensors. At the same time, much effort was made toward the integration of the resulting sensors into analytical systems, especially those intended for routine clinical analysis involving the rapid and, often, automated determination of electrolytes and dissolved gases in body fluid samples (I,3). A possible source of confusion with regard to selective potentiometric membrane electrodes is a trend toward unconventional electrode com-
ANALYTICAL CHEMISTRY, VOL. 54. NO. 11, SEPTEMBER 1982
ponents and assemblies. For simplicity, it is possible to categorize ion-selective membrane electrodes into “traditional” (Table I) and “nontraditional” (Table 11) types. The term traditional here refers to those electrodes whose configuration is similar to that of the classical pH glass electrode. Within this category (Table I) we find many of the commerciallyavailable ion- and gas-sensing potentiometric membrane electrodes that have become so popular for general analytical purposes. Those electrodes that have I called nontraditional represent variations where either the construction principle or the electrochemical elements (or both) have been changed. Undoubtedly, some of these electrode types will supplant their traditional counterparts in the future, hut it is difficult to predict a t this time which particular design will ultimately win out. For the present it is important to keep in mind that, although there may be important structural and mechanistic differences between the traditional and nontraditional designs, both types of electrodes frequently have comparable selectivity and sensitivity properties. Moreover, the associated instrumentation and data presentation displays are generally similar for all potentiometric electrodes. Some of these nontraditional electrodes are hecoming commercially 0003.2700/82/A351-1194$0l.W/0 @ 1982 American Chemical Socieh,
Figure 1. Family tree of selective potentiometric membrane electrodes
ANALYTICAL CHEMISTRY, VOL. 54. NO. 11. SEPTEMBER 1982
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Table 1.
POteIltiOmeMC
Traditional Ion-Selective Membrane Electrodes
E-hP.
Membrane Electrode
TY$4.X1-.Pclr
I
H+, Na+,Ag+, Li+. Cs+, Rb+. NH,+. K+. TI+
Glass membrane Liquid membrane ion exchanger Neutral CBnier
Cu2+. CI-. Mg2+. Caw. NOS2-, CIO,-. U0z2+, organic cations and anions. S a m dnms K+. Li+. H+, Ca2+.Na+. N b + , Sr“, Ea2+.cd2+
Gystal membrane
Singlecrystal Polycrystalline Mixed crystal
Gas sensors using
F-
S2-, Ag+. I+$+, CI-, Bc CI-. &-, I-, CN-, SCN-.
Wz. NHs. HzS. SO2. KX
Figure 2. Schematic of bioselective membrane electrodes. B, substance to be measured; 0 , electroactive product;
ion electrodes as elements
0 ,other products
Table II.
Nontraditional Electrodes
-hP.
Flp*arnundo.s*.
ISFETS (loll9electivefield effect
H*, H S ,
w+.K+. H+, Na+, F-.
Ca2+,CI-.
tranSWS)
(2).
I-, CN-
Coated wires
CI-. Br-, I-. SCN-, SO,z-. NQ-, CIO,-. Fe3+,organic I-. amino acids, drugs
Polymer mabix
Table 111.
I
Y~IGUI Biocatalytic Potentiometric Electrode.
slmslmw
d.1.mUlln.d
spc*. rr*ll
moul.h*
-E
-m
urea
urease enzyme
NH,+
Glasoelectmde
Gluconate Cdutemine Cysteine
Enzyme sequence Porcine mitochondria whole bacterial cells Rabbit muscle tissue slices
co, N& HzS N k
Gassensor Gassensor Gassensor Gas 8 ~ n s o ~
Squash plant tissue sections
COZ
Gas sen%
AdenOSlne mophosphat-
Glutamate
available on a limited basis, especially as components of proprietary analysis instrumentation systems. Some tecbnical information on these developments can he found in patents and meeting abstracts, but publication in the open scientific literature bas been sparse (I). 1196,.
electrodes can be extended to many classes of biological compounds. The general schematic is illustrated in Figure 2, where the mediator is some type of biocatalyst. These types of electrodes have recently been reviewed
Bioselectlve Electrodes When ion- or gas-sensing membrane electrodes are combined with appropriate mediators, e.g., a substance or phase that converts the compound to he measured into a species sensed by the electrode, the measurement capabilities of potentiometric membrane
ANALYTICAL CHEMISTRY, VOL. 54. NO. 11. SEPTEMBER 1982
A variety of materials may be employed as bibcatalysts in this connec; tion. In recent years, efforts have been made to extend the range of biocatalysts from isolated enzymes to whole cells and plant or animal tissue sections. Some examples of such electrodes are shown ih Table 111, where they are arranged in order of increasing complexity of the biocatalytic system employed. Much attention has also been given to the methodology of fixing the biocatalyst at the electrode surface. This is particularly important in the case of enzyme-based systems. Some of the common immobilization methods are shown ( 4 ) in Figure 3. The method of choice depends largely upon the properties of the enzyme and the particular requirements of the sensing electrode. The resulting “enzyme” electrodes have been widely accepted and are becoming available commercially. In some cases, the use of bacterial cells or tigsue sections may offer advantages over enzyme electrodes in terms of higher biocatalytic activity, improved lifetime, elimination of added cofactora or activators, and reduced cost. While it is possible that such electrodes may substantially increase the range of biocatalytic membrane electrodes to biochemical constituents not now readily measured with conventional electrodes, anumher of questions need to be answered before bacterial- and tissue-based electrodes will be accepted for routine analytical use. In the case of bacterial electrodes, for example, is it really
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necessary that the cells be “alive,” or will the hiocatalytic activity be retained in dormant or even dead cells? Can selectivity be improved by ha&rial induction or the contemporary genetic manipulation techniques? Similarly, for tissue-based electrodes, could the regulatory as well as metaholic functions of animal and plant tissues he employed to devise improved bioselective electrodes? Current research on such electrodes is being pursued quite vigorously, and a clearer picture regarding analytical poasibilities should emerge in the near future. It has been observed ( 4 ) that approximately 80%of the bioselective potentiometric membrane electrodes (gas-sensor-based) described in the literature to date show a less than theoretical, i.e., sub-Nernstian, response. In practice, this means that frequent
calibration and recalibration will be necessary. The dynamic response times of such electrodes typically fall in the 2- to 7-min range. This would appear to be adequate for most analytical situations, but actually represents a distorted’view hecause the time required to recondition these electrodes betueen analytical measurements, e.g., the recovery time, may he as long as 20 min. A major limiting factor in biocbemical analysis is the sensitiuity of potentiometric membrane electrodes. As long as major body fluid constituents are being measured this limitation is not severe, but the levels of some of the more esoteric substances of interest in biological systems are simply too low to be directly measured with potentiometric electrodes. In such cases, it may be possible to utilize cycling or other amplification processes
Cross-Linking
Covalent Linkage
Gel Entrapment
-Membrane
Encapsulation
Flgure 3. Representation of enzyme immobilization techniques. E is the enzyme 11SOA
ANALYTICAL CHEMISTRY. VOL. 54, NO. 11. SEPTEMBER 1982
to produce a useful analytical response. The selectivity of potentiometric hiosensors varies from exquisite to abysmal. In some cases, “fine-tuning” the system to improve selectivity is possible, but the potential user should aluays take care to investigate the selectivity properties of such electrodes under actual analytical conditions to avoid misleading or even erroneous results.
Applications If one could keep a record of each occasion that a potentiometric membrane electrode is used for biochemical analysis, it would undoubtedly be found that the pH-type glass electrode still leads all others in frequency of use. However, the continuing development of automated analysis systems using potentiometric rather than flame methods has resulted in a very sharp increase in the use of ion electrodes for the measurement of alkali metal ions, alkaline earths, and halide ions in biomedical and environmental samples. Frequently, these measurements are also coupled with potentiometric measurement of dissolved gases. Commercial development of potentiometric measurement systems for major body fluid constituents, such as urea, creatinine, or glucose, is perfectly feasible from a technical standpoint, but has proceeded more slowly. There seems to be more momentum in the direction of very low cost (even disposable) electrode-based systems for inorganic species than for the biological constituents. As the attractions of potentiometric sensors (e.g., no turbidity problems, etc.) become more widely recognized and any residual reluctance toward electrochemical techniques on the part of users is overcome, instrument manufacturers will probably fmd it profitable to extend and broaden their offerings in this area. I offer the latter assertion with some hesitancy, because I recall making a similar prediction a t least 10 years ago!
Future ProQp&cts Aside from the further development of the electrodes mentioned above, there are now several novel research directions that may result in other electrode types suited to hioanalysis. These include: An intensive effort to develop antibody- and antigen-sensing poten: tiometric membrane electrodes. Several technical approaches are being taken, including the use of ISFETS (5) and neutral carriers (6). The development of microbial
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iensors for the screening of mutagens. Uthough an amperometric approach ias been used for this purpose (71, po.entinmetric measurements should ilso be feasible. The possibility of using an entyme competition (8)instead of an entyme sequence approach for the po;entiometric measurement of biologi:al substrates. The development of membrane 4ectrode techniques for the determination of hormone factors using enzyme immunoassays (9). The use of hioselective electrodes to distinguish and elucidate metabolic pathways in microbial cells or tissue portions by virtue of differential potentiometric response characteristics
(IO). These and other current efforts attest to the lively nature of the field and suggest that the development and use of potentiometric membrane electrodes for hiocatalytical purposes will continue in the future.
References (1) Meyerhoff,M. E.;Fraticelli, Y.M.
Anal. Chem. 1982,54,2144. (2) Rechnitz, G. A. Science 1981,214, w - a ~
(3) Kissel, T. R.; Sandifer, J. R.; Zumbulyadis, N. Clin. Chem. 1982,28,449-52. (4) Arnold, M.A. PhD thesis, University of n.i..u.m ll."..".l, i a w ( 5 ) Janata, J.; Huber, R. J. In "Ion-Selective Electrodes in Analytical Chemistry, Vol. 2": Freiser. H..Ed.: Plenum Press: New Ybrk, 1980;p.'156.' (6) Solsky, R. L.; Rechnitz, G. A. Anal. Chim. Acto 1981,123,135-41. ( I ) Karube, I.; Matsunaga, T.; Nakahara, T.: Suzuki. S.: Kada. T. Anal. Chem. l981,53,1024-26. (8) Renneberg, R.; Pfeiffer, D.; Scheller, F.; Janchen, M. Anal. Chim. Acto 1982. 134,359-64. (9) Mascini, M.; Zolesi, F.; Pallesehi, G. Anal. Lett. 1982,15(82),101-13. (10) Swiek, N. Honors thesis. University of Delaware, 1982.
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G. A. Rechnitz is Unidel Professor of
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ANALYTICAL CHEMISTRY, VOL. 54,
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Chemistry at the University of Delaware; during 1982-83 he also holds an appointment in the Center for Aduanced Study. His research interests focus on membrane electrodes.
