Principles of Enzyme Thermistor Systems: Applications to Biomedical and Other Measurements Bin Xie · Kumaran Ramanathan · Bengt Danielsson Department of Pure and Applied Biochemistry, Lund University, Box 124, Center for Chemistry and Chemical Engineering, S-221 00 Lund, Sweden E-mail: Bengt.Danielsson@tbiokem.lth.se This chapter presents an overview of thermistor-based calorimetric measurements Bioanalytical applications are emphasized from both the chemical and biomedical points of view The introductory section elucidates the principles involved in the thermometric measurements The following section describes in detail the evolution of the various versions of enzyme-thermistor devices Special emphasis is laid on the description of modern “mini” and “miniaturized” versions of enzyme thermistors Hybrid devices are also introduced in this section In the sections on applications, the clinical/biomedical areas are dealt with separately, followed by other applications Mention is also made of miscellaneous applications A special section is devoted to future developments, wherein novel concepts of telemedicine and home diagnostics are highlighted The role of communication and information technology in telemedicine is also mentioned In the concluding sections, an attempt is made to incorporate the most recent references on specific topics based on enzyme-thermistor systems Keywords: Enzyme thermistor, Calorimetry, Clinical, Telemedicine, Home diagnostics Introduction 1.1 1.2 1.3 Fundamentals of Calorimetric Devices Principle of Calorimetric Measurement The Transducer Description of Instrumentation and Procedures 2.1 2.2 2.2.1 2.2.2 2.3 2.3.1 2.3.2 2.4 2.5 Conventional System Minisystem Plastic Chip Sensor Microcolumn Sensor Microsystems Thermopile-Based Microbiosensor Thermistor-Based Microbiosensor Multisensing Devices Hybrid Biosensors Applications 17 3.1 3.2 3.3 3.3.1 General Applications Industrial and Process Monitoring Clinical Applications In-Vitro Monitoring 10 10 11 12 14 16 17 18 18 18 Advances in Biochemical Engineering / Biotechnology, Vol 64 Managing Editor: Th Scheper © Springer-Verlag Berlin Heidelberg 1999 B Xie et al 3.3.2 3.3.3 3.3.4 3.3.5 3.4 3.4.1 3.4.2 3.4.3 3.4.4 3.4.5 3.5 In-Vivo Monitoring Bedside Monitoring Multianalyte Determination Hybrid Sensors; Enzyme Substrate Recycling Other Applications Enzyme-Activity Measurement Food Environmental Fluoride Sensing Cellular Metabolism Miscellaneous Applications 21 23 23 23 24 24 24 25 26 26 27 Future Developments 28 4.1 4.2 4.3 Telemedicine 28 Home Diagnostics 29 Other Developments 29 Conclusions 31 References 31 Introduction 1.1 Fundamentals of Calorimetric Devices Life is made up of lifeless molecules, but when the molecules react with each other there is an exchange of several forms of energy One well-known form of energy is heat The merits of measuring heat (calorimetry) were identified several decades ago Almost all effects, either physical, chemical or biological involve exchange of heat Specifically, biological reactions involving enzyme catalysis are associated with rather large enthalpy changes Based on the nature of the catalytic reaction, either a single enzyme or a combination of enzymes can be employed for generating a detectable thermal signal In earlier investigations a wider application of calorimetry, especially in routine analysis, was limited due to the need for sophisticated instrumentation, the relatively slow response and the high costs [1] Several simple calorimetric devices based on immobilized enzymes were introduced in the early 1970s that combined the general detection principle of calorimetry with enzyme catalysis [2] The advantages of these instruments were reusability of the biocatalyst, the possibility of continuous flow operation, inertness to optical and electrochemical interference, and simple operating procedures Several of these concepts, developed in the following years, culminated in the development of the enzyme thermistor (ET) designed in our laboratory The technique drew immediate Principles of Enzyme Thermistor Systems: Applications to Biomedical and Other Measurements attention in the area of biosensors and has been successfully exploited in the last two decades The initial biosensing applications focused on the determination of glucose and urea, and has subsequently been applied in the determination of a wide variety of molecules [3] Development of simple, low-cost calorimeters for routine analysis, called thermal enzyme probes (TEP), has been attempted by several groups These are fabricated by attaching the enzyme directly to a thermistor [4, 5] However, in this configuration, most of the heat evolved in the enzymic reaction is lost to the surrounding, resulting in lower sensitivity The concept of TEP was essentially designed for batch operation, in which the enzyme is attached to a thin aluminum foil placed on the surface of the Peltier element that acts as a temperature sensor [6] Although in later designs [7, 8], the sensitivity of TEP was improved, considerable enhancement in detection efficiency was achieved by employing a small column, with the enzyme immobilized on a suitable support In this case, the heat is transported by the circulating liquid passing through the column to a temperature sensor mounted at the top of the column Several models of this configuration were developed in the mid-1970s, including the enzyme thermistor and the immobilized enzyme flow-enthalpimetric analyzer [9, 10] Furthermore, a commercial flow-enthalpimeter combined with an immobilized enzyme column has also been described [11] Recently, several miniaturized prototypes have been fabricated, e.g., a thermal probe for glucose, designed as an integrated circuit, called a biocalorimetric sensor, with total dimensions of only ¥ ¥ 0.3 mm [12] In a different model, a small thermoelectric glucose sensor employing a thin-film thermopile to measure the evolved heat was described These devices were reported to be less affected by external thermal effects compared to thermistor based calorimetric sensors and could be operated without environmental temperature control [13] Active work is in progress in the authors’ laboratory to construct a miniaturized portable biothermal flow-injection system suitable for on-line monitoring An instrument with 0.1–0.2 mm (ID) flow channels and a flow rate of 25–30 ml/min with sample volumes of 1–10 ml is being evaluated at present A ¥ mm enzyme column allows determination of glucose concentrations down to 0.1 mM Recently a device equipped with thin-film temperature sensors of thermistor type (0.1 ¥ 0.1 mm or smaller) for glucose measurements has also been developed [14] 1.2 Principle of Calorimetric Measurement The total heat evolution is proportional to the molar enthalpy and to the total number of product molecules created in the reaction Q = –np (DH) where Q = total heat, np = moles of product, and DH = molar enthalpy change It is also dependent on the heat capacity Cp of the system, including the solvent: Q = Cp (DT) B Xie et al The change in temperature (DT) recorded by the ET is thus directly proportional to the enthalpy change and inversely proportional to the heat capacity of the reaction DT = –DH np/Cp As the heat capacity of most organic solvents is two or three times lower than that of water, enhanced sensitivity is expected in organic solvents This is the case, provided that DH remains unaltered This area of investigation is dealt with in detail in a later section Table presents a list of the molar enthalpy changes in a few enzyme-catalyzed reactions A thermometric measurement is based on the sum of all enthalpy changes in the reaction mixture Thus, it is favorable to co-immobilize oxidases with catalase, which results in doubling the sensitivity, nullifying the deleterious effects of hydrogen peroxide, and simultaneously reducing the oxygen consumption As indicated in Table 1, the high protonation enthalpy of buffer ions like Tris can be utilized to enhance the total enthalpy of proton-producing reactions A notable increase in the sensitivity can also be obtained in substrate- or coenzyme-recycling enzyme systems, in which the net enthalpy change of each Table Linear concentration ranges of substances measured with calorimetric sensors using immobilized enzymes Analyte Enzyme(s) used Linear range (mM) Ascorbic acid ATP (or ADP) Cellobiose Ascorbate oxidase Pyruvate kinase + hexokinase b-Glucosidase + glucose oxidase/catalase Cholesterol oxidase/catalase Creatinine iminohydrolase Alcohol oxidase Hexokinase Glucose oxidase/catalase 0.01–0.6 10 nM a 0.05–5 Cholesterol Creatinine Ethanol Glucose Glucose L-Lactate L-Lactate L-Lactate (or pyruvate) Oxalate Penicillin G Pyruvate Sucrose Urea a Lactate-2-mono-oxygenase Lactate oxidase/catalase Lactate oxidase/catalase + lactate dehydrogenase Oxalate oxidase b-Lactamase Lactate dehydrogenase Invertase Urease 0.01–3 0.01–10 0.0005–1 0.01–25 0.0002–1 –75 c 0.005–2 0.0002–1 10 nM a 0.005–0.5 0.002–200 0.01–10 0.05–100 0.005–200 Enthalpy change (–kJ/mol) 53 + 100 28 (75 b) 80 + 100 ca 225 67 (115 b) 47 (15 b) 61 With substrate recycling b In Tris buffer, c With benzoquinone as electron acceptor Principles of Enzyme Thermistor Systems: Applications to Biomedical and Other Measurements turn in the cycle adds to the overall enthalpy change [15] A later section provides further details on chemical and enzymatic amplification An inherent disadvantage of calorimetry is the lack of specificity: All enthalpy changes in the reaction mixture contribute to the final measurement It is therefore essential to avoid nonspecific enthalpy changes due to dilution or solvation effects In most cases this is not a serious problem An efficient way of coping with nonspecific effects in a differential determination is the incorporation of a reference column with an inactive filling [16] The flow injection technique is usually employed for an ET assay The sample volumes employed are too small to produce a thermal steady state, but generate a temperature peak This is traced by a recorder The peak height of the thermometric recording is proportional to the enthalpy change corresponding to a specific substrate concentration In most instances, the area under the peak and the ascending slope of the peak have also been found to vary linearly with the substrate concentration [17] A sample introduction of sufficient duration (several minutes) leads to a thermal steady state resulting in a temperature change, proportional to the enthalpy change up to a certain substrate concentration [62] 1.3 The Transducer The instrumentation for fabrication of the ET normally employs a thermistor as a temperature transducer Thermistors are resistors with a very high negative temperature coefficient of resistance These resistors are ceramic semiconductors, made by sintering mixtures of metal oxides from manganese, nickel, cobalt, copper, iron and uranium They can be obtained from the manufacturers in many different configurations, sizes (down to 0.1–0.3 mm beads) and with varying resistance values The best empirical expression to date describing the resistance-temperature relationship is the Steinhart-Hart equation: 1/T = A + B (ln R) + C (ln R) where T = temperature (K); ln R = the natural logarithm of the resitance, and A, B and C are derived coefficients For narrow temperature ranges the above relationship can be approximated by the equation: RT = RTo e b (1/T–1/To) where RT and RTo are the zero-power resistances at the absolute temperatures T and T0 , respectively, and b is a material constant that ranges between 4000 and 5000 K for most thermistor materials This yields a temperature coefficient of resistance between –3 and –5.7% per °C In our ET devices resistances of 2–100 kW have been used Other temperature transducers employed in enzyme calorimetric analyzers include Peltier elements, Darlington transistors, and thermopiles Of these, the thermistor is the most sensitive of the common temperature transducers B Xie et al Description of Instrumentation and Procedures 2.1 Conventional System The earlier investigations employed several different types of plexiglas constructions containing the immobilized enzyme column These devices were thermostated in a water bath, and the temperature at the point of exit from the column was monitored with a thermistor connected to a commercial Wheatstone bridge The latter was constructed for general temperature measurements and osmometry Later, we developed more sensitive instruments for temperature monitoring indigenously: the water bath was replaced by a carefully temperature-controlled metal block, which contained the enzyme column The enzyme thermistor concept has been patented in several major countries Such simple plexiglas devices were extremely useful and could be employed for determinations down to 0.01 mM An example of such a simple device will therefore be described here in some detail (Fig 1) The plastic column, which can hold up to ml of the immobilized enzyme preparation, was mounted into a plexiglas holder, leaving an insulating airspace around the column The heat exchanger consisted of acid-proof steel tubing (ID 0.8–1 mm and about 50 cm long) coiled and placed in a water-filled cup The whole device was placed in a water bath with a temperature stability of at least 0.01°C The cap surrounding the heat exchanger reduced the temperature fluctuations considerably and improved the baseline Fig Cross section of a conventional ET system (see text for detailed description) Principles of Enzyme Thermistor Systems: Applications to Biomedical and Other Measurements The temperature was measured at the top of the column with a thermistor (10 kW at 25 °C, 1.5 ¥ mm, or equivalent) epoxied at the tip of a mm (OD) acid-proof steel tube The temperature was measured as an unbalance signal of a sensitive Wheatstone bridge At the most sensitive setting, the recorder output produced 100 mV at a temperature change of 0.01°C Placing the temperature probe at the very top of the column, rather than in the effluent outside the column, reduced the turbulence around the thermistor and gave a more stable temperature recording The solution was pumped through the system at a flow rate of ml/min with a peristaltic pump The sample (0.1–1 ml) was introduced with a three-way valve or a chromatographic sample loop valve The height of the resulting temperature peak was used as a measure of the substrate concentration and was found to be linear with substrate concentration over a wide range Typically it was 0.01–100 mM, if not limited by the amount of enzyme or deficiency in any of the reactants For example, this type of instrument was adequate for the determination of urea in clinical samples The sensitivity was high enough to permit a 10-fold dilution of the samples, which eliminates problems of nonspecific heat The resolution was consequently about 0.1 mM, and up to 30 samples could be measured per hour In order to achieve more sensitive determinations, we developed a two-channel instrument, in which the water bath was replaced by a carefully thermostated metal block A specially designed Wheatstone bridge permits temperature determinations with a sensitivity resolution of 100 mV/0.001°C The calorimeter was placed in a container insulated by polyurethane foam It consists of an outer aluminum cylinder which can be thermostated at 25, 30 or 37 °C with a stability of at least ± 0.01°C Inside is a second aluminum cylinder with channels for two columns and a pocket for a reference thermistor The solution passes through a thin-walled acid-proof steel tube (ID 0.8 mm) before entering the column Two-thirds of this tubing acts as a coarse heat exchanger in contact with the outer cylinder, while the remaining third is in contact with the inner cylinder This has a higher heat capacity and is separated from the thermostated jacket by an airspace Consequently, the column is held at a very constant temperature, and the temperature fluctuations of the solution become exceedingly small The columns were attached to the end of the plastic tubes by which they are inserted into the calorimeter Columns could therefore be readily changed with a minimum disturbance of the temperature equilibrium Inside the plastic tube were the effluent tubing and the leads to the thermistor were fastened to a short piece of gold capillary with heat-conducting, electrically insulating epoxy resin Veco Type A 395 thermistors (16 kW at 25°C, temperature coefficient 3.9%/°C) were used These are very small, dual-bead isotherm thermistors with 1% accuracy; as such, they were interconvertible, comparatively well matched, and follow the same temperature response curve (within 1%) An identical thermistor was also mounted in the reference probe The Wheatstone bridge was built with precision resistors that have a low temperature coefficient (0.1%; temperature coefficient ppm) and was equipped B Xie et al with a chopper-stabilized operational amplifier This bridge produced a maximum change of 100 mV in the recorder signal for a temperature change of 0.001 °C However, the lowest practically useful temperature range was, limited mainly by temperature fluctuations caused by friction and turbulence in the column: typically 0.005–0.01°C Differential temperature recordings were made against a reference thermistor that was inserted in a pocket in the inner aluminum block or against an identical thermistor probe with an inactive reference column The latter arrangement was useful when nonspecific heat effects (e.g., due to heat of solvation or dilution) were encountered The sample was then split equally between the enzyme column and the reference column [18] Alternatively, the second channel could be reserved for another enzyme preparation, permitting a quick change of enzymatic analysis Some instruments were even equipped with a dual Wheatstone bridge, enabling two different, independent analyses to be carried out simultaneously In such conventional systems it is an advantage to replace used-up enzyme columns quickly, although the fresh column requires about 20–30 to achieve thermal equilibrium with the circulating buffer In total, over 30 instruments of the newer type have been assembled at the work-shop of our centre for use in industry and in research institutes A major drawback has been the unwillingness of the public to accept an uncommon technique, resulting in fewer developments The common mode rejection (detection is non specific), non discriminative, compared to spectroscopic (wherein specific wavelength is used) and electrochemical (wherein specific potential is used) for detection In addition the technique is not very sensitive The theoretical sensitivity is 0.1 mM using the GOX/catalase system There are other more sensitive techniques, such as amperometry chemiluminescence and fiber fluorimetry, that have recently been developed in our laboratory which are not as robust as the ET The high concentration of enzyme employed in the column may be treated both as an advantage and disadvantage This increases the operational stability of the column to over a couple of years, compared to a few months with lower enzyme loads The transducer (thermistor) has no fouling or drift, thus making the calibration extremely simple Such an approach is useful for bioprocess monitoring and scaling up or down is simple The general approach can be used for a multiple enzyme system with thermistors at the inlet and outlet of each enzyme block, and the carry-over heat – if any – can be subtracted for each enzyme/substrate combination Oxygen (for oxidase reactions) can be regenerated by electrolysis of water, with the use of platinum electrodes, directly in the flow stream in the vicinity of the enzyme Similar approaches had already been demonstrated by us The pH changes – if any – are negligible and well within the buffering capacity of the circulating buffer, and not pose a major problem while using multiple enzymes 2.2 Minisystem The design of mini and micro systems calls for a multidisciplinary approach involving engineering, materials science, electronics and chemistry With the advances in integrated circuit technology and micromachining of liquid filters, Principles of Enzyme Thermistor Systems: Applications to Biomedical and Other Measurements Fig Schematic cross section of a compact mini ET system (see text for detailed description) transducers, microvalves and micropumps on chips, practically useful microsystem technology has become a reality [19] A compact sensor of greatly reduced dimensions (outer diameter ¥ length: 36 ¥ 46 mm) has been constructed and is shown in Fig In order to conveniently accommodate enzyme columns and to ensure isolation from ambient temperature fluctuations, a cylindrical copper heat sink was included An outer Delrin jacket further improved the insulation The enzyme column (inner diameter ¥ length: ¥ mm), constructed of Delrin, was held tightly against the inner terminals of the copper core Short pieces of well-insulated gold capillaries (outer diameter/inner diameter: 0.3/0.2 mm) were placed next to the enzyme column as temperature-sensitive elements Microbead thermistors were mounted on the capillaries with a heat-conducting epoxy Two types of mini system has been constructed as discussed below 2.2.1 Plastic Chip Sensor The chip sensor (27 ¥ ¥ mm) was constructed of Plexiglas (see Fig 3) The rectangular enzyme cell (5 ¥ ¥ 0.5 mm) and the inlet and outlet parts were A B Fig Schematic diagram of a microcolumn ET sensor (see text for detailed description) 10 B Xie et al milled into the plastic base to a depth of 0.5 mm Electrically-insulated thermistors in direct contact with flow stream were placed outside the enzyme cell after the porous polyethylene filters The enzyme cell was charged with the enzyme preparation prior to compaction Ready access to the enzyme compartment makes replacement of enzymes possible 2.2.2 Microcolumn Sensor The microcolumn sensor (inner diameter x length: 0.6 ¥ 15 mm) was constructed of stainless-steel tubing and is free of auxiliary components (see Fig 4) Microbead thermistors (thermistors shaped like a bead) were directly mounted in the reference and measurement probes on the outer surface of the inlet and outlet gold tubings, using heat-conducting epoxy Both the length (about 200 mm) and inner diameter (0.15 mm) of the inlet tubing between the sample valve and the column were minimized, in order to reduce sample dispersion during transport in the flow system Similarly, in order to reduce heat loss, simple and short connections between the column and the inlet or outlet tubings were required 2.3 Microsystems A planar substrate, such as silicon wafer, could be micromachined by a sequence of deposition and etching processes This results in three-dimensional microstructures which can be implemented in cavities, grooves, holes, diaphragms, cantilever beams etc The process referred to as silicon micromachining often employs anisotropic etchants such as potassium hydroxide and ethylene diamine pyrocatechol The crystallographic orientation is important as the above-mentioned etchants show an etch-rate anisotropy The ratio for the (100)-, (110)- and (111)- planes is typically 100 : 16 : The technique of electrochemical etch stop could be applied for control of the microstructural dimensions An alternative Fig Illustration of a miniaturized ET system (refer to text for details) ... enzyme activities For instance, arginase cleaves buffer-containing arginine to urea and ornithine.Therefore, the ET determines arginases activity 43 Thermal Biosensors in Biotechnology Table TELISA... feasibility of miniaturizing thermal biosensors in different constructions, sizes and materials, by employing conventional machining and micromachining technologies could be further exploited in several... trends in hardware developments are pointed out Thermal biosensors will probably have a promising future in biotechnology Thermal Biosensors: Principles and State of the Art Nearly all biochemical