TW588159B - Electrical potential type urea sensing device and the manufacturing method thereof - Google Patents

Abstract

The present invention discloses an electrical potential type urea sensing device and the manufacturing method thereof, wherein a conductive layer is first formed on the substrate, then deposit a sensing film as the ion sensing electrode, the sensing film has a sensing region and a non-sensing region; use a conductive wire to extend from the conductive layer as the electrical contact with the exterior; encapsulate the non-sensing region of the sensing film by an encapsulant, define a sensing window in the sensing region; finally, use enzyme immobilization technology to immobilize the urea enzyme on the sensing window in the sensing film, so as to complete the fabrication of an electrical potential type urea sensor device. The present invention is a disposable urea sensing device, and has the functions of mass duplication, low cost, and ease of packaging.

Description

588159 ———__ 92JI8955- ± _L Revision _____ V. Description of the invention (1) 1. [Technical field to which the invention belongs] The present invention relates to a potential-type urea sensing element and a method for manufacturing the same, and particularly to a potential urea sensor Tin oxide serves as an extended ion sensing field for the acid-base sensing film of an EGFET and cooperates with a urea enzyme to produce a urea sensing element. 2. [Previous technology] According to the above, the urea concentration in the blood reflects protein intake and alienation 'and is closely related to kidney, liver function and adrenal endocrine function, so the concentration of urea nitrogen in blood or urine is the human body. An important indicator of health is an important parameter used to judge renal function in clinical diagnosis. However, traditional organic quantitative analysis methods have many shortcomings in practical applications, such as complicated operations, long analysis time, expensive equipment, and cannot be applied to the detection of continuous processes. Therefore, in order to make up for the shortcomings of traditional quantitative analysis, A biosensor was developed, which combined the principles of biochemistry, electronic circuits, materials science, and optics to design a biosensor that fits all fields. Ion-sensing field-effect transistor system appeared in 1970 and ΐ = This element itself has an ion-selective electrode: Element: The characteristics of 微 wei 丨 ΐ are completely different from traditional electrodes; this type of sensing: 2 Miniaturization, easy instrumentation and suitable for the design of automated systems. Fixed in the cabin in 1980. Caras and Janata firstly penicillin

'Between ion-sensing field-effect transistors; C2 A-sensors, & 卽 Yunxi: f-gate making penicillin This is called an enzyme-sensing field-effect transistor.

Page 6 588159 _ Case No. 921189 阽 5. Description of the invention (2) ― Year-finance March 26th, n has been developed so far, and many related patents have been proposed, such as US Patent No. 5,992,183 Metal oxide matrix biosensors It discloses a metal oxide thin film as a substrate for biomolecules, and also provides a current or potential type biosensor, and cooperates with enzymes, antibodies, antigens, and nucleic acid sequences for sensing. U.S. Patent No. 5,858, 1 86, MJrea biosensor for hemodialysis monitoring ', which is an electrochemical sensor that can quantitatively detect the concentration of urea by using the dialysis waste liquid during hemodialysis. This sensor uses enzymes The urea is hydrolyzed, and changes in the pH value are detected. U.S. Patent No. 5,833,824, 'Dorsal substrate guarded ISFET sensor, which uses an ion-sensing field effect transistor to sense the ionic activity of a solution. U.S. Patent No. 4,877,582, Chemical sensor device with field effect transistor, which uses an ion-sensing field-effect transistor as an enabler and uses a suitable biometric substance to capture specific detection The chemical signals are converted into electrical signals by using field effect transistors to quantitatively detect specific substances to be tested in the solution. Due to the wide range of biosensing technologies, the present invention is directed to the urea concentration in blood or urine, and proposes a urea sensing element combined with semiconductor process technology to detect a biosensor based on pH (pH). The sensing element makes it an architect suitable for development into a disposable sensing element. [Summary of the Invention] The main object of the present invention is to provide a potential-type urea sensing element 588159

=, Manufacturing method ’, which uses a non-insulating solid-state ion-sensing thin film as the sensing electrode of a ^ @% efficiency transistor and combines it with semiconductor process technology to make a disposable urea sensing element. = Another object of the invention is to provide a method for manufacturing a potential-type urea sensing element, which can be copied in large quantities, has low cost, and is easy to package *, so it can reduce the cost of conventional ion sensing field effect transistors. And other issues. Another object of the invention is to provide a method for manufacturing a potential-type urea sensing element 3, which has the advantages of simple manufacturing, low cost, dry storage, easy packaging and processing, adjustable sensing area, and convenient transportation. One embodiment of the present invention is a structure of a potential-type urea sensing element, which is provided with a sensing film on a substrate having a conductive layer on the surface as an ion sensing electrode, and the sensing film has a sensing The detection area is a non-sensing area; a conductive line is extended from the conductive layer, and the electrical contacts are externally connected; a non-sensing area of the sensing film is covered with a packaging gel, and the sensing A sensing window is defined in the testing area; and a urease is fixed in the sensing window on the sensing film, and the urea sensing element is used to detect the urea concentration in blood or urine, and it is used as a clinical Judging parameters on the break. Another embodiment of the present invention is a method for manufacturing a potential-type urea sensing element, the steps of which include: first providing a substrate, and then forming a conductive layer on the surface thereof; and forming a sensing film on the surface of the conductive layer of the substrate, It is used as an ion sensing electrode, and the sensing film has a sensing area and a sensing area.

588159 i 92118955

V. Description of the invention (4) The detection area; then a conductive line extending from the conductive layer is formed as an external electrical contact; and then a packaging step is performed to cover the non-sense on the sensing film with a packaging gel. In order to define the sensing area, a sensing window is used; finally, a urea enzyme is fixed in the sensing window on the sensing film by using enzyme fixing technology. In this way, the production of a urea sensing element can be completed Alternatively, the sensing element can be used to detect the pH value, and the urea concentration can be known. f Detailed descriptions are provided below with specific examples and accompanying drawings to make it easier to understand the purpose, technical content, features and effects of the present invention. 0. [Embodiment] The present invention uses tin dioxide As an extended ion sensing field effect transistor (EGFET) acid-base ion sensing film, urea is fabricated using standard silicon chip manufacturing processes or using a separate structure of tin dioxide / indium tin oxide / substrate The structure of the sensing element is an extended gate ion-sensing field effect transistor; this framework is applicable to various types of object sensors based on the detection of Qiu. The present invention is a potential-type urea sensing element. As shown in the first figure, it includes a glass substrate 12 on which an indium tin oxide (ITO) conductive layer 14 is formed on the surface; A non-insulating solid-state ion-sensing film 16, such as tin dioxide, is located on the surface of the indium tin oxide conductive layer 14 and serves as a solid-state ion-sensing electrode to detect the pH value of the solution. The 16 series has a sensing area and a non-sensing area. 588159 Λ Modified case No. 921189 D. Field description (5) of the invention; a conductive line 18 is extended from the indium tin oxide conductive layer 14 as a signal transmission line And external electrical contacts; and an encapsulating gel 20 covering the non-sensing area of the sensing film 16 and defining the sensing area as a sensing window 22 to define sensing using the encapsulating gel 20 The sensing area of the element 10 is about 2 * 2 mm2; and a urease 24 is fixed in the sensing window 22 on the sensing film 16, and the urease 24 is urea embedded in pVA_sbQ colloid. Composed of enzymes. This urea sensing element is easier to fabricate and package than the conventional ion-sensing field-effect transistor, and can reduce costs to meet the requirements of disposable sensing elements. The embodiment of the above-mentioned potential-type urea sensing element uses a glass substrate 2 as an element substrate. In addition, the substrate may be selected from other insulating-based insulating substrates, and the insulating substrate is selected from a dream substrate ceramic substrate. Polymer substrates, etc .; therefore, 'this sensing element has better basicity'. The substrate method can be changed due to different practicability and process conditions. The structure shown in Figure r illustrates the manufacturing method of the present invention. Potential-type urea sensing element: the first manufacturing method shown in the step c is a sectional view; as shown in the first (refer to the second (a)), shake ^ ^ and form a steel tin oxide on it A glass substrate 12 is formed, the thickness of Π is 23 angstroms, and the resistance is ^ ~ = q. This oxide tin conductive layer is then broadcasted on the glass substrate 12 and the money method is used to grow a negative dielectric tin. The surface of the conductive layer 14 is made of warfare | sensing film 16 of tin oxide, as described in the Second Institute, page 10, 588159. 5. Description of the invention (6)

Amendment 11 When the tin oxide is used as the target, argon and oxygen are passed in (4: ,,, milk, and the sensing film 16 of tin dioxide grows, the degree of the glass plate 12 is maintained At 150 ° C, the deposition pressure is maintained at 20 mTorr: the helmet emits a power of 50 watts to form a thin film 16 with a thickness of 2000 angstroms, and the electrode is sensed by F 2 Z 5; wherein the sensing film 16 is It can be divided into sensing & domain and non-sensing area. Then a conductive wire is provided, which is guided by steel tin, and the layer 14 is extended as an external electrical contact. See also the second ( c) As shown in the figure, an encapsulation step is performed, and a non-sensing area and a part of the sensing area of the sensing film 16 are covered with an encapsulation gel 20 such as epoxy resin so as to define a sensing area for the sensing area. Window 2 2. Finally, as shown in the second (d) figure, a urea enzyme 24 is fixed in the sensing window 22 on the sensing film 16 by using an enzyme fixing technology; here, the present invention uses photopolymerization The property of photopolymerization of matter meets the role of fixing urea enzyme 24 on sensing window 22 to complete the production of urea sensing element 10. It can also use other immobilized processes to form potential electrochemical biosensors' in order to reduce the cost of large-scale instruments such as optical biological analysis and the carrying characteristics of sensing elements, and is suitable for real-time detection and disposal of discarded elements. Please refer to the following instructions for detailed enzyme immobilization technology: First, prepare urease (urease, EC 3.5.1.5, 50000 ~ 1 00000 units / g); PVA-SbQ colloid, where PVA (DP = 1 700 , DS = 88), SbQ (1 · 25 mol%), N · ν · = 12 · 69 wt ° /., PH = 5.6, viscosity at 25 ° C is about 5750cp; urea (NH2CONH2 = 60 · 06), with a purity of 99%; and phosphate (kh2P04 = 136.09), which is a general reagent grade, used for the configuration of buffer solutions.

Page 11 588159 93. 3. 26

0 的 ， Then, PVA-SbQ was diluted (100 mg / 100 microliters of 55 mM phosphate solution of 7.0) and then with an enzyme solution (7 mg / ΙΟΟμΙ of millimol pH value of 7.0. Phosphate solution) After mixing in the proportion of]: 丨, take 1 microliter of drop onto the sensing window 22, and then place the sensing element 10: 4 watts, 365 nanometers of ultraviolet light, and perform light irradiation. The polymerization reaction is about "unique. Finally, after the photopolymerization reaction is completed, the sensing element is placed in a dark box at 4 ° C for about 12 hours to complete the enzyme immobilization process." As shown in the third figure, It is a potential type urea sensing element 10 with a separate tin dioxide / indium tin oxide / glass substrate structure as a transducer and it is electrically connected to a high input impedance potentiometer using a conductive wire 18, such as a metal half-% potentiometer The transistor and the op amp's readout circuit in this figure is the LTii instrument amplifier 26 'and is provided with a silver / gasified silver electrode μ to provide a reference stable potential for measuring the urea sensing element 1 in the solution. The reaction voltage is calculated, and the solution concentration is calculated. The fourth figure is a circle of the pH correction curve of the urea sensing element of the present invention. As shown in the figure, it is the "base and acid sensing characteristics of the tin dioxide sensing film" to determine the sensitivity of each batch of elements. The measurements are all within a stable range; the voltage response caused by the above-mentioned instrumentation amplifier readout elements in different pH environments is different. Among the pH values between 2 · 2 ~ 10.2 ·, the tin dioxide sensation The film's sensing degree is about 58 · 85 ± 0.41 millivolts / acid test value. The fifth graph is the state diagram of the response time and recovery time of the urea sensing element of the present invention, as shown in the figure, which shows the urea sensing Required for measuring element response

Fifth, the time of invention reward -----_ § —— ^ and the time of 60 seconds after recording the emperor's record. From the experimental results, it can be seen that about 90% of the maximum reaction voltage has been reached after the A-dry reaction, and right, in the buffer solution of 5 millimoles twice, about 10 minutes after the left, That is, it will slowly return to the voltage before the reaction. Because the purpose of sensing the architecture of the object 4 = is to develop a disposable life: sense =, the length of the response time is not necessary to consider. parameter. The system-12 is a positive curve diagram of the urea sensation in the measurement environment of different pH values according to the present invention, which is the change of the initial acid value of the test solution, the reaction voltage and the calibration curve. The impact. When the initial pH value is higher, the range in which the reaction can be changed is significantly reduced. The maximum changeable limit of the urea hydrolysis reaction is pH 9 · 3; therefore, if the initial acid value of the test solution is too high, , It will make the changeable range of the reaction relatively narrow J. Conversely, if the initial acid test value of the test / valley solution is too low, it will also affect the enzyme activity, and will also make the reaction electric willingness smaller, so how to Find the _ equilibrium point between the two, from this experimental result, we can know that the acid test value 6.0 is the best reaction environment. As shown in the seventh figure, it is a calibration curve diagram of the urea sensing element at different buffer solution concentrations according to the present invention. It shows the effect of different buffer solution concentrations on the reaction, based on acid-base sensing. The sensor is susceptible to the buffering capacity of the buffer solution, and the concentration of the buffer solution is related to the strength of the buffer solution. The higher the buffer solution concentration, the stronger the buffering capacity; otherwise, the lower the delta buffer solution concentration, the buffering The worse the capability is, the more the acid-base value generated near the sensing window will change, and it will quickly respond to the voltage.

5 ^ 8159

Case No. 92118955 V. Description of the invention (9) Changes, so its calibration curve will shift to the direction of low urea concentration. The eighth figure is a schematic diagram of the change of the maximum reactable voltage of the urea sensing element of the present invention with the increase of time. This figure shows that, when discussing the stability of the preservation state, it is prepared in advance. Urea sensor made of tin dioxide / indium tin oxide / glass ion-sensing field-effect transistor, and the completed sensor is stored in a dark box of 4 and measured every 5 to 10 days , Observe how long the sensor can be saved after the production is completed. From the experimental results, it can be seen that the sensor can still work normally after 99 days, and the maximum response voltage of the sensor still has no obvious attenuation phenomenon. Therefore, the present invention makes use of an extended tin dioxide / oxygen = indium tin [isolated field-effect transistor on a glass substrate to produce disposable urine, and measurement elements using a separate structure. The millimolar acid-base value of 6 has the best response curve under the working environment of the acid solution. The range of urea = 31 mg / 100 ml to 120 mg / 100 ml can be measured. The sensitivity of the linear knife is 16 9 · 1 mV / p [urea concentration]. Induction ΐ ΐ: 2 uses a non-insulating solid-state ion-sensing film as the ion detection electrode, and combines semiconductor process technology to produce a low # i; 5 1 A 4+ element sensing element, which has a large number of replicable and It has the characteristics of the electric device, so it can reduce the problems of the conventional ion sensing field effect and packaging. In addition, the present invention also has the advantages of integrity of the product, easy storage, easy packaging, adjustment, sensing area, and convenient transportation. ^ The above-mentioned embodiment is only for explaining the # 术 田 相 芨 锊 点, the objective # mmj of the present invention, the technical thoughts and special features of the moon ... people who are more familiar with this technology can understand the contents of the present invention. U page 588159 9a 3. 26 _Case No. 92118955_ Year and month Amendment _ V. Description of the invention (10) Content and implementation according to it, when the scope of the patent of the present invention cannot be limited, that is, the spirit disclosed by the present invention All equal changes or modifications should still be covered by the patent scope of the present invention. Description of drawing number: 10 Potential-type urea sensing element 12 Glass substrate 14 Conductive layer of indium tin oxide 16 Sensing film 18 Conductive wire 20 Encapsulated gel 22 Sensing window 24 Urease 26 Instrumentation amplifier 28 Silver / Vaporized silver electrode

Page 15 Brief Description of Drawings The cross-section of the structure of the T-type urea sensing element of the figure to the J-month. This is a cross-sectional view of the structure of each step in the production of a potential-type urea sensing element. The third figure is a measurement architecture diagram of the urea sensing element of the present invention. The fourth figure is a graph of the pH correction curve of the urea sensing element of the present invention. The fifth graph is a response time and recovery time state diagram of the urea sensing element of the present invention. The sixth figure is a calibration curve diagram of the urea sensing element according to the present invention when measuring environments with different pH values. The seventh figure is a calibration curve diagram of the urea sensing element of the present invention at different buffer solution concentrations. The eighth figure is a schematic diagram of the change of the maximum reactable voltage of the urea sensing element of the present invention over time.

Claims (1)

J2118955 Amendment No. 588159 6. Potential-type urea sensing element 'applicable to patents' includes: a substrate 'a surface is formed with a conductive layer; a sensing film is located on the surface of the conductive layer of the substrate and serves as an electrode' the sensing film It has a sensing area and a non-sensing conductive line, which is extended from the conductive layer as a sexual contact; an encapsulating gel, which defines the non-sensing and sensing area of the sensing film A sensing window; and a urea enzyme, the sensing fixed on the sensing film 2, the potential type urea sensing as described in the first patent application range, the substrate is selected from an insulating substrate and a non-insulating substrate . 3. According to the potential-type urea sensor described in item 2 of the patent application scope, the insulating substrate is selected from the group consisting of a silicon substrate, a glass substrate, and a ceramic polymer substrate. 4. According to the potential-type urea sensor described in item 1 of the patent application scope, the conductive layer is indium tin oxide. 5. The sensing film in the potential-type urea sensor described in item 1 of the scope of the patent application is a non-insulating solid-state ion sensing film 6. The sensor in the potential-type urea sensor described in item 1 of the scope of the patent application The material of the test film is tin dioxide. 7. According to the potential-type urea sensor described in item 1 of the scope of the patent application, the sensing film is formed by a coin forging method. 8. According to the potential type urea sensor described in the scope of the patent application, the conductive wire system provides a high-impedance electric current as an ion sensing area; it is an external electrical area, and the inside of the window . Test element, its test element, its substrate and south test element, its test element, its test element, its test element, its test element, its count Wei 92〗 9a3.26 Patent application range 9 Potential type urea sensing element as described in item 1 of the V ° urine application H range, its J is fixed in the sensing window with enzyme fixing technology. n: The enzyme system is composed of urease embedded in PVA_SbQ gel. 2 A method for manufacturing a urea-type sensation element, including the following steps. A substrate is provided on which a conductive layer is formed; and a sensing film is formed on the surface of the conductive layer of the substrate to make it a ionic sensor. The sensing film has a sensing area and a non-sensing area; ~ forming a conductive line extending from the conductive layer as an external electrical contact; and performing a packaging step, using a The encapsulating gel covers the non-sensing area of the sensing film, and defines a sensing window for the sensing area; and the enzyme fixing technology is used to fix a urea enzyme in the sensing window on the sensing film. . 12. The manufacturing method according to item n of the scope of patent application, wherein the substrate is selected from an insulating substrate and a non-insulating substrate. 1 3. The manufacturing method as described in item i 2 of the scope of patent application, wherein the insulating substrate is selected from the group consisting of a silicon substrate, a glass substrate, a ceramic substrate, and a polymer substrate. 14. The manufacturing method as described in item n of the scope of patent application, wherein the conductive layer is copper tin oxide. 15. The manufacturing method according to item 丨 丨 in the scope of patent application, wherein the sensing Page 18 ^ 8159 Six plug No. 11 is called M. Patent application scope 1 l1 film non-insulating solid-state ion sensing film. Wherein the sensing wherein the sensing wherein the urea is made of tin dioxide as described in item 11 of the scope of patent application.疋 之 制 -method thin 7 film circumference = the manufacturer mentioned in the item * formed by the Hada sputtering method, the manufacturing method described in item 11 of the scope of patent application of the winter, its 3 elements :, using physical embedding The method is fixed to the sensing opening window :: ... Yeast 2 please refer to the manufacturing method described in item 11 of the patent scope. The urea and hydrazone in the invention are composed of urea enzyme embedded in PVA-SbQ colloid. , Page 19 588159 铖 SS