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ANALYTICAL CHEMISTRY, VOL. 50, NO. 2, FEBRUARY 1978
Micro-Size Potentiometric Probes for Gas and Substrate Sensing C. P. Pui, G. A. Rechnitz,"' and R. F. Miller Departments of Chemistry and Physiology, State University of New York, Buffalo, New York 14214
The design, construction and evaluation of micro gas sensing probes for ammonia and carbon dioxide is described in detali. It Is shown that the resulting sensors have promise for biological measurements In terms of tip size (less than 10 hm), sensitivity, and dynamic response. The micro ammonia sensing probe is further utilized to prepare a micro urea sensor by immobilizing urease enzymes at the electrode tip.
Potentiometric gas sensing probes have been used as the basis of many enzyme sensor systems in addition t o their utility for direct gas measurement. A classical gas sensing membrane probe employs a double membrane system in which a thin layer of a n appropriate filling solution separates a n external gas permeable hydrophobic membrane from a n internal sensing electrode of the ion-selective membrane type. T h e solution layer a n d internal sensing electrodes are so chosen that t h e gaseous species to be measured shifts a chemical equilibrium in the filling solution to produce an ion activity change that can be sensed by the internal membrane. Recently, a new type of gas sensor known as the air-gap sensor has been reported ( I ) . T h e sensor is based on the same principle as other gas sensors except t h a t t h e gas permeable membrane is replaced by an air gap which separates the electrolyte layer (adsorbed as a very thin film on the surface of a n ion-selective electrode) from the sample, the entire system being contained in a gas-tight measuring chamber. The advantages of each type of gas sensor have been well established. (1-6). Currently, potentiometric gas sensors for measuring a wide range of gases are commercially available. T h e application of these sensors has been extended to include assays of biological samples. However, one limitation of these commercially available sensors results from their relatively large size which makes in vivo measurements in biological systems difficult, if not impossible. It would clearly be advantageous for many biological applications to reduce the sensing tip size so that its insertion into living tissue is practical. We report here a gas sensing probe design made of glass micropipets, with a tip diameter of less than 10 km; i t seems likely t h a t this tip diameter might be further reduced with the possibility of achieving small enough tips for intracellular applications. Such sensors can be fabricated t o serve as micro gas sensing devices or, with the addition of an enzyme immobilized at the tip, can form t h e basis of a highly selective micro biological substrate sensor. Micro-size ammonia and carbon dioxide sensors are reported here and a micro urea sensing enzyme probe based on t h e ammonia system is described. EXPERIMENTAL Materials. Analytical grade reagents were used to prepare buffers, constant ionic strength solutions, and standard ammonium chloride, sodium bicarbonate, and urea solutions. Distilled and deionized water was used throughout for these preparations. Enzyme urease (Type VI: Highly purified powder from Jack Beans) and Bovine serum albumin (BSA, Fraction V powder) were IPresent address, Department of Chemistry, University of Delaware, Newark, Del. 19711. 0003-2700/78/0350-0330$0 1.OO/O
obtained from Sigma Chemical Company and glutaraldehyde (25% solution in water) from J. T. Baker Chemical Co. Dimethyl dichlorosilane, obtained from Ventron Corp., was made up as a 4 70 solution in analytical reagent grade carbon tetrachloride. Sealing of micropipets and electrodes was carried out using halocarbon wax Series 6-00 (Halocarbon Products Corp., Hackensack, N.J.), glass cover cement (Kroenig) (ChromeGessellschaft Schmid & Co.), high vacuum wax Apiezon WlOO (Apiezon Products Limited, London) and 5 minute epoxy (Devon Corp., Mass.). Corning Code 0150 pH sensitive glass tubings (1 mm o.d., 0.5 mm i.d.) were obtained from Corning Glass Works while all other glass tubing was obtained from Glass Company of America, Inc. Micro Sensor Fabrication. The gas sensing probes reported in this work function via pH changes within an appropriate internal solution. For the fabrication of various micropipets, we found it convenient to use two different vertical pipet pullers, one from David Kopf Instruments (Mode 700C) and the other from Narishige Scientific Instruments Laboratory. A noninverting binocular microscope (Ernst Leitz Wetzlar) was used to monitor much of the sensor fabrication. Micro p H Electrodes. Methods for making various types of micro glass pH electrodes have been well established (7-9). For the purpose of this work, spear-type glass micro pH electrodes were prepared as follows. A 7-cm length of pH tubing was pulled into two very finely tapered micropipets using the DKI vertical puller. A long heating coil, a strong solenoidal pull, and high heat were necessary to achieve the desired pipet profile. The tips of the micropipets were then sealed using a De Fonbrune microforge. An incomplete seal was generally indicated by the taking up of water when the electrodes were soaked in distilled water. The sealed pH electrodes were then filled by boiling in deionized distilled water under vacuum for about 20 min. The water in the electrodes was replaced by 0.01 M HC1 using a fine steel tubing attached to a hypodermic syringe. The dc resistances of the electrodes were then measured using a Keithley electrometer (Model 601B). An electrode with a sealed tip generally had a dc resistance close to io9 Q. Insulating pipets were pulled from ordinary Pyrex glass tubing (1.6-mm o.d., 1.2-mm id.) under moderate heat and a moderate pull, using the Narishige vertical puller equipped with a long heating coil. Under microscopic observation, the tip of the insulating pipet was broken off mechanically until a tip diameter of a few micrometers was obtained. The pipet tip was filled with insulating wax or cement by simply dipping the tip into heatmelted halocarbon wax or glass cover cement and by applying a slight vacuum at the open end of the pipet. The insulating pipet was cut to a length of about 1 cm shorter than the pH electrode to be insulated. The insulating pipet was then mounted on a Plexiglas holder, with plasticine, on the microscope platform and the pipet tip was gently heated with a heating coil. The micro pH electrode, held by a micromanipulator (PRIOR, England), was carefully inserted into the insulating pipet such that only about 50 pm of the sensitive tip protruded out of the insulating pipet. Any excess wax or cement adhering to the tip was removed using a fine electrically heated wire. At the wide end, the space between the pipet and the electrode was filled with glass cover cement so that the air trapped would help to prevent the entry of aqueous solution through the lower seal. The pH response of the insulated pH electrode was measured against a calomel electrode (Corning Catalogue No. 476017) in standard buffers. Electrodes with the slopes of at least 50 mV per unit pH were used. The dc resistance of the pH electrodes after insulation was generally increased by a hundred-fold. 0 1978 American
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 2, FEBRUARY 1978
Because of the high resistances associated with these electrodes, all resistance and potential measurements were carried out in a Faraday cage. The potential measurements were made using a Q ) Orion digital 801 meter and were high input impedance recorded using a Heath/Schlumberger Model EC1200-02 recorder. Micro Air-Gap Gas Sensor. Glass tubings of 5 mm o.d., 3.4 mm i.d. were thoroughly cleaned, rinsed with millipore-filtered distilled water, and dried in an oven. By means of the D.K.I. vertical puller equipped with a short heating coil, the tubing was first extended 1 to 2 cm under high heat. I t was then realigned and subsequently pulled by gravity using minimum heat. The pipet had a very short tapered profile with a tip diameter usually of a few micrometers. The total length of the pipet was cut to about 2.5 cm. The tip of this micropipet was then made hydrophobic by repeatedly dipping into millipore-filtered dimethyl dichlorosilane (4% solution in carbon tetrachloride) and heating with a heating coil. Slight vacuum was also applied at the wide end of the pipet during this operation. This pipet is designated as the outer pipet. A second pipet, designated as the inner pipet, was pulled from a glass tubing of size 3-mm o.d., 2.4-mm i.d. with the Narishige vertical puller. A medium length heating coil, low heat, and a low pull were used. The profile of this pipet should be such that the shank of the pipet is sufficiently large to accommodate an insulated micro pH electrode and itself fit into the outer pipet. The tip of the pipet was then broken off to a diameter of about 10 pm. A small drop of high vacuum wax was then melted onto the shank of the pipet close to the tip. The outer pipet was mounted on a Plexiglas holder on the microscope platform and firmly secured with a Plexiglas clamp. The inner pipet, held by a micromanipulator, was carefully inserted into the outer pipet. Gentle heating was applied to slightly melt the vacuum wax on the inner pipet so that the tip could be inserted to a distance approximately 150 to 200 pm from the tip of the outer pipet. Continuing gentle heating brought the wax seal very close to the tip of the inner pipet, thus forming an air space within the tip of the outer pipet. The space between the outer and the inner pipets at the wide end was sealed with quick setting epoxy. The inner pipet was then filled with an appropriate internal solution (0.1 M NaCl and 0.005 NH4Cl or NaHC03, saturated with silver chloride). Some agitation might be necessary to bring the internal solution right to the tip of the inner pipet, and this could be achieved readily by means of a finely etched tungsten wire (ac current in sodium nitrite) or a very fine micropipet. An insulated micro pH electrode was then very carefully inserted so that the pH tip almost protruded out of the internal solution. A fine silver wire (0.127 m) electrolytically coated with silver chloride was finally inserted to contact the internal solution. The pH and the reference electrodes were firmly sealed to the inner pipet at the wide end using glass cover cement. The complete assembly of a micro air-gap sensor is shown in Figure 1. Measuring Techniques for Ammonia and Carbon Dioxide. To test the micro air-gap ammonia or carbon dioxide sensors, the standard addition technique was used. Gaseous ammonia was generated by adding a standard solution of 1 M ammonium chloride to a 100-mL sample of 0.1 M Na2P04NaOHbuffer (pH 11.0) in a thermostated cell ( T = 25 "C). In the case of carbon dioxide, 1 M sodium bicarbonate solution was added to a 100-mL sample of 0.5 M sodium chloride solution, acidified to pH 1.9 with concentrated sulfuric acid. Potential readings were taken after a steady state was reached as indicated on the digital meter and strip chart recorder. Preliminary Investigation of a Micro Biosensor for Urea. Five milligrams of urease enzyme was dissolved in 25 pL of 15% BSA solution (prepared in Tris buffer, 0.2 M, pH 8.51, and this enzyme solution was stored under refrigeration until use. The inner pipet, after assembly with the outer pipet as described above, was filled with a solution containing 5 X M ammonium chloride and lo-' M sodium chloride saturated with silver chloride. The tip of the outer pipet was then broken off very slightly to a tip diameter of about 10 p m in order to make the very tip hydrophilic. A drop of enzyme solution held a t the tip of a graduated micropipet (Gilmont) was brought in contact with the tip of the outer pipet and a small column of enzyme solution collected within the tip was allowed to dry by evaporation. The
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Figure 1. Construction of a micro air-gap gas sensing probe. (1)
Ag/AgCI wires, (2) insulated micro pH electrode, (3)ordinary Pyrex glass, (4) hydrophobic surface, (5) glass cover cement, (6) 0.01 M HCI, (7) epoxy resin, (8)0.005 M NH,CI or NaHC03 -4- 0.01 M NaCI, (9) air space, (10) high vacuum Apiezon wax, (11) glass cover cement or halocarbon wax, (12) air gap process was repeated until a thin layer of enzyme residue formed at the tip. A drop of 25% glutaraldehyde was then brought in contact with the enzyme layer in the tip for several minutes and polymerization formed a sturdy layer of enzyme at the pipet tip. The top was then washed thoroughly with distilled water or Tris buffer to remove excess glutaraldehyde or unbound enzyme. The micro pH electrode and the silver-silver chloride wire were then inserted to complete the sensing unit. The potential response of the micro urea probe was tested by the standard addition technique. Urea solution, 1 M, in Tris buffer, 0.2 M, pH 8.5, was added to a 100-mL sample of Tris buffer, 0.2 M, pH 8.5, contained in a thermostated cell ( T = 25 "C) and steady-state potentials were recorded. Tris buffer, 0.2 M, pH 8.5, was prepared by dissolving a weighed amount of tris(hydroxymethy1)aminomethane in distilled deionized water. The pH was adjusted with hydrochloric acid.
RESULTS A N D D I S C U S S I O N Sensor Design. The construction of a micro air-gap gas sensor is shown in Figure 1 and a photograph of an actual gas sensing tip is shown in Figure 2. T h e internal ion sensing electrode (the p H electrode) fits snuggly in the inner pipet containing the appropriate filling electrolyte. The electrolyte is held in place within the pipet by capillarity, and it contains a fixed activity level of t h e ion (chloride ion) to which t h e reference electrode (silver-silver chloride) responds. T h e hydrophobic tip of the outer pipet essentially acts as a gas permeable membrane. Thus, when t h e micro probe is immersed into a sample solution, the air trapped in the gap and t h e hydrophobicity of the sensing tip prevents t h e entry of the aqueous solution but allows the gas to pass freely through the tip into the air gap. The process will continue until t h e partial pressure of t h e gas in the internal electrolyte a t t h e tip of the inner pipet equals that of the sample solution. Since the amount of electrolyte surrounding the micro ion-sensitive electrode tip is small, as is the size of the air gap, this equilibrium is quickly established. T h e equilibrium concentration of the gas in the thin layer of electrolyte determines its ionic activity (the p H ) which is measured by t h e micro
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 2, FEBRUARY 1978
20
i
Figure 2. A micro air-gap sensing tip under 400 times magnification (internal electrolyte was not Incorporated in the Inner pipet for clarity) ion-sensitive electrode (micro p H electrode). The ionic activity of the thin layer of electrolyte is proportional to the logarithm of the partial pressure of gas in the sample. In general, the probe potential is related to the determinand concentration by the equation
E = Constant
k
2.3 RT -log,, [XI F
where X is the determinand, R is the gas constant, T is the temperature, F is the Faraday constant and E is the potential; the sign of the last term is positive for an acidic gas and negative for a basic gas. The theoretical significance of this equation has been published previously ( I , IO). Although the micro air-gap gas sensor is in an early stage of development, there are several unique features associated with the basic design which should be emphasized. Apart from the relatively simple construction, the sensor shares the advantage of a conventional air-gap probe in that the lack of a membrane phase facilitates the free diffusion of gaseous molecules into the internal electrolyte. However, the electrolyte layer on the pH-sensitive tip does not need to be removed after every measurement in order to maintain accuracy as required in the conventional air-gap sensors. The inner pipet serves to retain the bulk of the internal electrolyte, which stabilizes the composition of the thin layer of the sensitive tip by slow but continuous exchange. The micro air-gap gas sensor can be used in direct contact with the sample and requires no special reaction vessel. As is evident from Figure 2, it should be possible to further miniaturize the micro air-gap gas sensor without too much difficulty. Micro Air-Gap Gas Sensing Probes. The potential response of a micro air-gap ammonia sensor is shown in Figure 3. The internal electrolyte employed was a solution of 0.005 M ammonium chloride and 0.1 M sodium chloride, and the measurement of ammonia gas was carried out by total conversion of ammonium chloride over the concentration range of to 5 X M. Also included in the figure is the calibration curve obtained under the same experimental conditions using a commercial barrel type Orion ammonia probe (Model 95-10). As can be seen, a linear calibration curve with a slope of -60 mV per decade concentration change of ammonia is obtained over the total range studied, comparable t o that obtained for the commercial barrel type ammonia sensor. The micro ammonia sensor had the following dimensions: outer tip diameter, 8 pm; inner tip diameter, 15 Fm; insulating tip diameter, 8 pm; p H sensitive tip length, 50 pm; distance between inner and outer tips, 165 Fm. For the micro ammonia sensors studied, in addition to the general factors (temperature, osmotic pressure, electrolyte composition, etc.) which affect the response of all gas sensing probes ( I , 5 , 6, I I ) , the sensitivity was found to be largely determined by the sensitivity of the internal micro p H electrode and by the geometry of the electrolyte surrounding the p H sensitive tip. The sensitivity decreased significantly
I
-5
-3
-4
Log
I
I-1 50 -2
hH3:
Figure 3. Potential response of a micro air-gap ammonla sensor. (A) micro air-gap sensor, (0)Orion ammonia sensor (Model 95-10)
Flgure 4. Dynamic response curves of a micro airgap ammonia sensor with increasing thickness of the electrolyte layer a t the p H sensitive tip. Hence, for a good micro ammonia sensor, the tip of the micro p H electrode should almost protrude out of the electrolyte. The wax-sealed micro p H electrodes were satisfactory for the present work and have the advantage of being relatively easy to prepare; however, with prolonged use (several hours), the wax-seal insulation slowly broke down because of the intrusion of electrolyte, leading to decreasing sensitivity of the micro p H electrode which in turn affected the ammonia response. This problem can be circumvented by using glass-to-glass-sealinsulated micro pH electrodes (7-9). The dynamic response at two different concentration levels obtained for the micro ammonia sensor is shown in Figure 4. The response time is short (2-3 min) particularly a t high ammonia concentrations. The response appeared to be predominantly a function of the geometry of the sensing probe itself, since a good micro pH electrode is effectively instantaneous in its response. Thus, with an outer tip size of a few micrometers, the response time is largely determined by the tip size and the shape of the inner pipet, the p H sensitive tip length, and the size of the air gap. The potential reproducibility of the micro ammonia probe is, of course, dependent on the reproducibility of the internal micro pH electrode. For the micro ammonia sensor described above, the potentials between successive measurements were reproducible to about 1mV above the ammonia concentration of M, until the micro p H electrode showed sign of de-
ANALYTICAL CHEMISTRY, VOL. 50, NO. 2. FEBRUARY 1978
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-1
'40
t
15'
t
/
150
-4
-3 LOQ \
-2
CO2 1
Flgure 5. Potential response of a micro air-gap carbon dioxlde sensor. (A) micro air-gap sensor, (0)Corning carbon dioxide sensor (Catalogue No. 476107)
terioration. Equilibration between two successive measurements was slow, and this was almost certainly due to the slow diffusion of the confined layer of electrolyte surrounding the relatively long p H sensitive tip with the bulk electrolyte. Re-equilibration times were tested in alkaline buffer (pH 11.01, air, and dilute sulfuric acid, and were found to be quickest (- 1 h) in acid solution. This would also appear to be consistent with the continuous rapid decrease in ammonia partial pressure in the air gap and internal electrolyte as a result of the continuous decrease of partial pressure of ammonia in acid solutions due to the formation of ammonium ions. T h e use of the micro air-gap sensor is not limited to ammonia; fabrication of other gas sensors also seems feasible. Figure 5 shows the response of a micro air-gap carbon dioxide M sodium probe, made by employing a solution of 5 X bicarbonate and 0.1 M sodium chloride as an internal electrolyte. The measurement of carbon dioxide was tested in determining sodium bicarbonate by means of the total conversion method. As can be seen, the calibration curve has a slope of 50 mV per decade concentration change of carbon dioxide which is comparable to that obtained for the commercial barrel carbon dioxide sensing probe (Corning Catalogue No. 476107) over the concentration range studied. This micro air-gap carbon dioxide sensor appeared to have some advantages over the recently reported micro carbon dioxide membrane probes (12,13)whose potential responses for carbon dioxide were sub-Nernstian and whose micro hydrophobic membranes were difficult to fabricate. The lower detection limit of the micro air-gap gas sensors has not been fully examined but is expected to be largely a function of the sensitivity of the internal micro p H electrode, the geometry of the electrolyte surrounding the pH sensitive tip and the composition of the internal electrolyte. The overall results obtained from this work indicate that it may be feasible to develop additional micro gas sensors by judiciously selecting the inner ion sensing electrodes, for instance, a H2S sensor with a micro sulfide electrode, a H F sensor with a micro fluoride electrode, a HCN sensor with a micro silver electrode, and a C12 sensor with a micro chloride electrode. Micro Biosensor for Urea. The simplicity in design of the micro air-gap sensors also makes them attractive as building blocks for fabricating micro biosensors. This is particularly true because the sensing tip does not require a hydrophobic gas permeable membrane which could make immobilization of enzyme(s) difficult and could itself act as a partial barrier for gas diffusion. The tip may also be bevelled for tissue penetration and thus, if an enzyme can be immobilized within the sensing tip, the sensor may eventually
t
-5
-4
-3
-2
-1
I
L:g(urea:
Flgure 8. Potential response of a micro urea sensor
prove to be useful for extra- and intracellular recording of biological substrates. We have carried out a preliminary study of the feasibility of fabricating such a biosensor using the well established ureaseurea system (14,15) and the results obtained are highly promising as shown in Figure 6. The micro urea probe was fabricated simply by chemically binding a thin layer of urease enzyme within the sensing tip (10-pm tip size) of a micro air-gap ammonia sensor. Hence, when the probe was immersed into a urea substrate sample. the gaseous ammonia produced by the urease-catalyzed reaction at the tip diffused through the air gap and altered the hydrogen ion activity of the internal electrolyte monitored by the internal micro p H electrode. Such a micro sensor is highly selective, if not specific, because the enzyme provides the specificity of the chemical reaction and the micro air-gap sensor provides the high selectivity and sensitivity for measuring the gaseous product produced from the reaction. The response of the micro urea probe was quick, requiring only several minutes to reach a steady-state potential. Although the sensitivity of the probe is low, which may be due to the low sensitivity of the micro p H electrode used (50 mV per unit pH) and insufficient quantities of enzyme, further optimization of the probe is likely to improve urea response.
ACKNOWLEDGMENT We thank M. E. Meyerhoff for his continued interest in this work and R. Dacheux for some technical assistance.
LITERATURE CITED (1) J. Ruzicka and E. H. Hansen, Anal. Chim. Acta, 69, 129 (1974). (2) E. H. Hansen, H. B. Filho, and J. Ruzicka, Anal. Chim. Acta, 71, 225 (1974). (3) J. Ruzicka, E. H. Hansen, P. Bisgarrd, and E. Reymann, Anal. Chim. Acta, 72, 215 (1974). (4) E. H. Hansen and J. Ruzicka, Anal. Chim. Acta, 72, 353 (1974). (5) P. L. Bailey and M. Riley, Analyst(London), 100, 145 (1975). (6) J. W. Ross, J. H. Riseman, and J. A . Krueger. Pure Appi. Chem., 36, 473 (1973). (7) G. Eisenman, "Glass Electrodes for Hydrogen and Other Cations", Marcel Dekker, New York, N.Y., 1967. (E) M. Lavallee, 0. F. Schanne, and N. C. Hebert, "Glass Microe\ectrodes", John Wiley & Sons, New York, N.Y., 1969. (9) L. C. Clark, Jr., D. W. Lubber, I. A. Silver, and W. Simon, "Ion and Enzyme Electrodes in Biology and Medicine", University Park Press, Baltimore, Md., 1976. (IO) D. Midgley and K. Torrance, Ana/yst(London), 97, E26 (1972). (11) P. L. Bailey and M. Riley, Ana/yst(London), 102, 213 (1977). (12) C. R. Caflisch and N. W. Carter, Anal. Blochem., 60, 252 (1974). (13) M. Sohtell and B. Karlmark, Pfiugers Arch., 363, 1979 (1976). (14) D. S. Papastathopoulous and G.A. Rechnitz, Anal. Chim. Acta, 79, 17 (1975). (15) M. Mascini and G. G. Guilbault, Anal. Ghem., 49, 795 (1977).
RECEIVED for review September 16,1977. Accepted November 29, 1977. Financial support by a grant from the National Institutes of Health is gratefully acknowledged.
