Bio-Thermo Tip Sensor

The present invention relates to the detection of organic compounds in solutions and is widely applicable for chemical and clinical analysis and for monitoring of fermentation processes and waste water treatment. The method of measuring optical absorbance has been, in general, used for detecting organic compounds, but this method requires complicated technical manipulation and long assaying times. Therefore, bio-sensors, which could specifically and rapidly detect organic compounds have recently been developed by using immobilized enzymes and an electrochemical detector. For bio-sensors, the particular electrochemical device that is used for detection is determined by the type of enzyme reaction. This is one of the drawbacks of electrochemical bio-sensors, because they can not use the common electrochemical sensor for various enzyme reactions.

The enzyme thermistor system, which is constructed by using an immobilized enzyme column a, thermistor a, heat-exchanger and a high sensitivity thermostatic bath, has been invented as a multifunction bio-sensor system, for detecting enthalpy change that occurs in all enzyme reactions. Several enzyme thermistor systems have been developed and reported, one of them being reported in the following document: Klaus Mosbach, Bengt Danielsson. Anal. Chem. Vol. 53 No. 1 (1981), Page 83A to 94A.

The thermistor used in the enzyme thermistor system has excellent sensitivity to temperature change, but it has several inherent problems; the characteristics curve between temperature and resistance exhibits poor lineality; selfheating of the thermistors creastes unavoidable experimental error and; for, these reasons, the sensor system must be large.

It is therefore an object of the present invention to provide an improved bio-sensor having a very small size.

Another object of the invention is to provide an improved bio-sensor having a very short response time.

A further object of the invention is to provide an improved bio-sensor having a good sensitivity.

In accordance with the present invention, the measuring system utilizing a bio-sensor is made simple.

Other objects and advantages of the invention will appear more fully from the following description.

FIG. 1 is a cross sectional diagram of the bio-thermo tip sensor,

FIG. 2 is the wiring diagram of the bio-thermo tip sensor,

FIG. 3 is a schematic diagram of the measuring system employed for the bio-thermo tip sensor,

FIG. 4 is a schematic diagram of the flow type measuring system employed for the bio-thermo tip sensor,

FIG. 5 is the characteristics curve between glucose concentration and thermal change.

A cross sectional diagram of the bio-thermo tip sensor is shown in FIG. 1. The reference numerals 1 and 2 indicate a high sensitive IC thermal sensor or detector configured as a discrete chip and a membrane on which a biocatalyst or enzyme is immobilized, respectively. As shown in FIG. 1, the bio-thermo tip sensor is constructed by using two IC thermal sensors, which are surface coated with an insulating Si.sub.3 N.sub.4 layer. One of the IC thermal sensor acts as a reference sensor, and the other, which has the enzyme containing membrane 2 fixed onto the surface, of the other IC thermal sensor acts as a detecting sensor. The reference number 11 indicates an electrical connecting wire.

FIG. 2 is the wiring diagram of the bio-thermo tip sensor.

The reference numerals 4, 5, 6 and 7 indicate an output terminal of the detecting sensor, an output terminal of the reference sensor, a GND terminal and a -3 V power supply terminal, respectively.

FIG. 3 is a schematic diagram of the measuring system employed for the bio-thermo tip sensor.

The measuring system utilizing the bio-thermo tip sensor is provided with a bio-thermo tip sensor 3, a water bath 12, a power supply 8, voltmeters 9 and a computer 12.

The flow type measuring system employed for the bio-thermo tip sensor as shown in FIG. 4 is constructed with a persistent pump, a flow tube, a pool of buffer solution, a sample injection port and the measuring system as shown in FIG. 3.

After the temperature of a thermostatic bath and the output voltage of the IC thermal sensors becomes constant, an aliquot of sample solution is injected into the sensor compartment. The enthalpy change or heat generation due to the bio-catalyst or enzymic reaction is detected by the IC thermal sensor and the level of enthalpy change causes a change in the output voltage of the detecting IC sensor. The difference between the output voltages of the two IC thermal sensors is a measure of the enthalpy change. For batch system analysis, the stationary output voltage change during the reaction is used as a measure of enthalpy change, but for flow system analysis, a peak output voltage change is measured. The output voltage change is easily converted to thermal change, since the IC thermal sensors used in this invention have a characteristic voltage/temperature change of 10 mV/.degree. C.

An explanation referring to a typical example follows;

The glucose-thermo tip sensor was constructed by using glucose oxidase as the biocatalyst or enzyme. Two IC thermal sensors were mounted inside a plastic perspex case and encapsulated with insulating epoxy resin except the surface thereof after the electrical connecting wire was attached to the case. The thin membrane used for enzyme immobilization was made by casting a solution of cellose-triacetate (50 mg/ml) dissolved in methylene chloride, 1-8 diamino-4-amino methyl octane (20%) and glutalaldehyde (2%) onto the exposed surfaces of the IC thermal sensors. After 24 hr, this membrane was further treated with glutalaldehyde(2%) for 1 hr and then dipped into glucose oxidase solution (1 mg/ml, pH 7 phoshate buffer, 0.05 M).

The IC thermal sensors used in the present example is as follows: IC thermal sensor includes an integrated circuit constructed by a plurality of transistors connected to form a Darlington connection, wherein a collector of each transistor is connected to a substrate acting as a common collector, the base of a first stage transistor is connected to the substrate and the current source is connected between the common collector and an emitter of final stage transistor.

The glucose-thermo tip sensor specifically responded to the glucose in a solution. In the batch system, the response of the glucose-thermo tip sensor to glucose concentration was linear in the range of 3-100 mM (FIG. 5). In the flow measuring system, the response of the sensor was reduced, but the measuring time was shorter.

As described above, it is possible to make the bio-thermo tip sensor utilizing high sensitivity IC thermal sensors.

The measuring system is very simple, and only a power supply and a voltmeter is added for measurement, because the high sensitivity IC thermal sensors used in this invention have an internal constant current circuit and a operation amplifier for producing an amplified output voltage. The sensors also have a characteristic of 10 mV/.degree. C. response over a wide temperature range so that a super efficient thermostat bath is not required. The thermal detection circuit of the sensors is situated 3 .mu.m below the thermally sensitive surface so that the thermal detection circuit has a very short response time and good sensitivity better than thermistors. Also, the very small structure of the sensor circuits endears their application to in-vivo analysis.

While the invention has been described with respect to its preferred embodiment, it is to be understood that the words which have been used are words of description rather than limitation and that changes within the purview of the appended claims may be made without departing from the true scope and spirit of the invention in its broader aspects.