Thermistor, enzyme

In addition to the usual evaluation parameters for analytical methods (Chapter 16), the sensitivity, detection limit, dynamic range, and precision profile, biosensors are also characterized with respect to the rapidity of their response and recovery. This 

Interferences are of particular importance for devices destined for continuous use in very complex matrices. Biosensors are tested for interferences not just from species that are expected to bind to or react with the particular chemical recognition agent employed the end use of the biosensor is considered, and components of that sample matrix are examined for potential interference. Test assays are conducted in the sample matrix, and compared with results obtained in simple buffers in order to determine analyte recovery. 

To date, a significant variety of enzyme-based biosensors have been commercialized for clinical testing laboratories and even for use by lay consumers. The most 

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Figure 3.22 — (A) Scheme of the Mach-Zehnder interferometer/enthalpimeter. (Reproduced from [152] with permission of Elsevier Science Publishers). (B) Cross-section of polyvinylidene fluoride film-based enthalpimeter. (Reproduced from [156] with permission of the American Chemical Society). (C) Schematic diagram of an enzyme thermistor.

Thermistor basedflow-through calorimetric sensors. Enzyme thermistors make the most widely developed type of heat measurement-based sensors. The thermistors are normally used as temperature transducers in these devices. Thermistors are resistors with a very high negative temperature coefficient of resistance. They are ceramic semiconductors made by sintering mixtures of metal (manganese, nickel, cobalt, copper, iron) oxides. Like the two previous groups, thermistor sensors do not comply strictly with the definition of "sensor" as they do not consist of transducers surrounded by an immobilized enzyme rather, they use a thermistor at the end of a small... 

Even though most enzyme thermistors have been used for determining substrates, a few applications to the determination of inhibitors and activators, as well as enzyme activities, have also been reported. The enzymes employed for this purpose are usually isolated previously, though some are used in their original tissues and microorganisms [157]. 

Many of the fairly large number of enzyme thermistor biosensors reported so far have been used for the determination of biological substrates or, to a much lesser extent, inorganic substrates. Experimental set-ups similar to that depicted in Fig. 3.22.C were used to determine the substrates listed in Table 3.3, which also gives the primary enzymes and any auxiliary enzymes or reagents employed to improve the determination [158]. 

Table 3.3. Determinations of substrates by use of enzyme thermistor biosensors...

Methods based on the inhibitory effect of the analyte and the use of an enzyme thermistor have primarily been applied to environmental samples and typically involve measuring the inhibitory effect of a pollutant on an enzyme or on the metabolism of appropriate cells [162]. The inhibiting effect of urease was used to develop methods for the determination of heavy metals such as Hg(II), Cu(II) and Ag(I) by use of the enzyme immobilized on CPG. For this purpose, the response obtained for a 0.5-mL standard pulse of urea in phosphate buffer at a flow-rate of 1 mL/min was recorded, after which 0.5 mL of sample was injected. A new 0.5-mL pulse of urea was injected 30 s after the sample pulse (accurate timing was essential) and the response compared with that of the non-inhibited peak. After a sample was run, the initial response could be restored by washing the column with 0.1-0.3 M Nal plus 50 mM EDTA for 3 min. Under these conditions, 50% inhibition (half the initial response) was obtained for a 0.5-mL pulse of 0.04-0.05 mM Hg(II) or Ag(I), or 0.3 mM Cu(II). In some cases, the enzyme was inhibited irreversibly. In this situation, a reversible enzyme immobilization technique... 

Figure 3.23 — Schematic diagram of a four-channel enzyme thermistor sensor. (Reproduced from [164] with permission of Elsevier Science Publishers).

Enzyme thermistors can be altered for measuring the activity of soluble enzymes. For this purpose, an inactive or empty Teflon column can be used as a reaction chamber. The sample and a buffer containing a suitable substrate in excess are passed through heat exchangers and thoroughly... 

One alternative method for the determination of enzyme activities which is particularly effective at low enzyme concentrations involves enrichment with the enzyme by affinity binding (preferably of the reversible type) to an affinity column in the enzyme thermistor unit. The enzyme activity is determined by introducing a pulse of excess substrate. 

The enzyme thermistors suffer from a notoriously high detection limit when the generated heat is detected as the change of thermistor resistance. Suggest ways of mitigating this problem. 

Using the equivalency relationships in Table 3.1, propose an equivalent electrical circuit diagram for an enzyme thermistor operated (a) in direct detection mode and (b) in the feedback, push-pull mode. 

Enzymatic reactions coupled to optical detection of the product of the enzymatic reaction have been developed and successfully used as reversible optical biosensors. By definition, these are again steady-state sensors in which the information about the concentration of the analyte is derived from the measurement of the steady-state value of a product or a substrate involved in highly selective enzymatic reaction. Unlike the amperometric counterpart, the sensor itself does not consume or produce any of the species involved in the enzymatic reaction it is a zero-flux boundary sensor. In other words, it operates as, and suffers from, the same problems as the potentiometric enzyme sensor (Section 6.2.1) or the enzyme thermistor (Section 3.1). It is governed by the same diffusion-reaction mechanism (Chapter 2) and suffers from similar limitations. 

Conventional calorimetric biosensors with thermistors as the transducer were invented early by proposing a thermal biosensor in a flow stream [10]. So far the design of enzyme thermistors does not entirely match the market demand well, but it seems well suited for special applications [10,11]. A number of devices have utilized discrete pairs of thermistors for differential measurements with immobilized enzymes or with separate enzyme columns [9,10,11],... 

Figure 7.10. Typical configuration of a split-flow enzyme thermistor with an aluminum constant-temperature jacket.

By using devices similar to that shown in Figure 7.10, many immobilized enzymes have been tested. Table 7.1 summarizes the analytes, the enzymes, and the dynamic ranges over which the enzyme thermistors provide substrate quantitation. 

Mosbach, K. Danielsson, B., Enzyme thermistor, Biochim. Biophys. Acta 1974, 364, 140-145... 

Principles of Enzyme Thermistor Systems Applications to Biomedical and Other Measurements... 

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. 

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