Patent Publication Number: US-2005133367-A1

Title: Electrical urea biosensors and its manufacturing method

Description:
BACKGROUND OF THE INVENTION  
      1. Field of the Invention  
      The present invention generally relates to a electrical urea biosensor and its manufacturing method, and more particularly relates to a technology for forming a urea biosensor by using a pH sensitive film with a tin oxide used as the separative gate ion-sensitive field effect transistor (EGFET) and cooperating the use of the urea enzyme.  
      2. Description of the Prior Art  
      Accordingly, the urea concentration in the blood responses the assimilation and the dissimilation catabolism of the protein and simultaneously has a closely relation of the kidney function, the liver function, and the secretion of the adrenalin. Hence, the urea nitrogen concentration of the blood or the urine is an important health index of the human body and is also an important data of the kidney function in the clinical diagnosis.  
      However, the conventional quantitative analysis of the organic matter has many disadvantages in the practical use, such as the complicated operation, the long analysis time, and expensive equipments, and it cannot be used in the detection of the continuous process. Hence, in order to overcome the prior disadvantage of the prior quantitative analysis, a biosensor is developed and combined with the biochemistry technology, the electronic circuits, the materials science, and the optical theory so as to design the biosensor to conform to the requirement in each fields.  
      The ion-sensitive field effect transistor was presented at 1970 and rapidly developed to the microminiaturized sensor. The sensor provides with the ion-sensitive electrode function and also has the character of the field effect transistor and it is completely different from the conventional electrode. The sensor has the advantages of the microminiaturization, the easy instrumentation ability, and suitable for the automation design. Following, in 1980, Caras and Janata further disclosed the gate provided with an ion-sensitive field effect transistor immobilized the aspirin within for using as the aspirin biosensor, which was called the enzyme field effect transistor.  
      Currently, there are many patents proposed. For example, the U.S. Pat. No. 5,922,183 in titled of “Metal oxide matrix biosensor” disclosed a substrate provided with a thin film matrix for biomolecules belonging to a general class of materials known as hydrous metal oxides and provided an amperometric biosensor or a potentiometric biosensor to perform the sensing test by the enzymes, cofactors, antibodies, antigens and the series of the nucleic acids. The U.S. Pat. No. 5,858,186 in titled of “Urea biosensor for hemodialysis monitoring” disclosed an electrochemical sensor capable of detecting and quantifying urea in fluids resulting from hemodialysis procedures. The sensor is based upon measurement of the pH change produced in an aqueous environment by the products of the enzyme-catalyzed hydrolysis of urea. The U.S. Pat. No. 5,833,824 in titled of “Dorsal substrate guarded ISFET sensor” disclosed an Ion-sensitive Field Effect Transistor (ISFET) sensor for sensing ion activity of a solution. The U.S. Pat. No. 4,877,582 in titled of “Chemical sensor device with field effect transistor” disclosed a chemical sensor having a field-effect transistor as an electronic transducer and used for the analysis of specific constituents in a liquid, the chemical sensor comprising means which permits an externally supplied sample solution to reach a chemical receptor of said chemical sensor.  
      Owing to the biological technology is quiet extensive, the present invention is to provide a urea biosensor belong to the formulation of the semiconductor process technology in accordance with the urea concentration of the blood or the urine and the biosensor is to detect the pH value so as to develop a structure of a disposable sensor.  
     SUMMARY OF THE INVENTION  
      The primary object of the present invention is to provide an electrical urea biosensor and its manufacturing method. The present invention utilizes a non-isolation solid-state ion-sensitive film to use as a sensitive electrode of an ion-sensitive gate field effect transistor and also utilizes the semiconductor process technology to manufacture a disposable urea biosensor.  
      Another object of the present invention is to provide an electrical urea biosensor and its manufacturing method. The present invention can be mass production and provides with the advantage of the low cost and the easy package so as can reduce the cost of the prior ion-sensitive gate field effect transistor simplify the package.  
      A further object of the present invention is to provide an electrical urea biosensor and its manufacturing method. The present invention provides with advantages of the simple production, the low cost, easily dry storage, the adjustable sensitive area, and the easy conveyance.  
      In order to achieve previous objects, one of the embodiments of the present invention is to provide a structure of an electrical urea biosensor. A sensitive film is positioned on a surface of a substrate, wherein a conductive layer is formed on the surface of the substrate. The sensitive film is used as an ion-sensitive electrode. The sensitive film provides with a sensitive region and a non-sensitive region. A conductive line is extended from the conductive layer for using as an external electrical contact point. The present invention utilizes a package encapsulant covering the non-sensitive region of the sensitive film to define a sensitive window at the sensitive region and a urea enzyme is immobilized within the sensitive window of the sensitive film. Then, the present invention can utilize the urea biosensor to detect of the urea concentration of the blood sample or the urine sample.  
      Another embodiment of the present invention is to provide a manufacturing method of an electrical urea biosensor. The manufacturing method comprises the following steps. First, a substrate is provided and a conductive layer is formed on a surface of the substrate. Then, a sensitive film is formed on a surface of the conductive layer of the substrate for using as an ion-sensitive electrode. Wherein, the sensitive film provides with a sensitive region and a non-sensitive region. Next, a conductive line is formed and extended from the conductive layer for using as an external electrical contact point. Following, a package step is performed by utilizing a package encapsulant to cover the non-sensitive region of the sensitive film so as to define a sensitive window at the sensitive region. Last, the present invention utilizes an enzyme-immobilized technology to immobilize a urea enzyme within the sensitive window of the sensitive film. Hence, the present invention completes a urea biosensor and utilizes the urea biosensor to perform the detection of the pH value so as to measure the urea concentration.  
      Other advantages of the present invention will become apparent from the following description taken in conjunction with the accompanying drawings wherein are set forth, by way of illustration and example, certain embodiments of the present invention. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
      The foregoing aspects and many of the accompanying advantages of this invention will become more readily appreciated as the same becomes better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:  
       FIG. 1  is a schematic representation of the structure of the cutaway view of the electrical urea biosensor, in accordance with the present invention;  
       FIG. 2   a  and  FIG. 2   d  are schematic representations structures of the cutaway view at various stages during the formulation the electrical urea biosensor, in accordance with the present invention;  
       FIG. 3  is a schematic representation of the framework view of the measurement of the electrical urea biosensor, in accordance with the present invention;  
       FIG. 4  is a schematic representation of the correction curve view of the pH value of the electrical urea biosensor, in accordance with the present invention;  
       FIG. 5  is a schematic representation of the status view of the response time and the return time of the electrical urea biosensor, in accordance with the present invention;  
       FIG. 6  is a schematic representation of the correction curve view of the pH value of the electrical urea biosensor at the measurement environment with various pH values, in accordance with the present invention;  
       FIG. 7  is a schematic representation of the correction curve view of the pH value of the electrical urea biosensor as buffer solutions with various concentrations, in accordance with the present invention; and  
       FIG. 8  is a schematic representation of the maximum variation of the responsible voltage of the electrical urea biosensor as the time increasing, in accordance with the present invention. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT  
      The present invention utilizes the tin dioxide as the pH ion-sensitive film of the extended ion-sensitive gate field effect transistor (EGFET) and utilizes the separation structure of tin dioxide/indium tin oxide/substrate to form the urea biosensor. All the structure is the separative gate ion-sensitive field effect transistor and the suitable range of the structure is all biosensor based on the pH value detecting.  
      Such as shown in the  FIG. 1 , the present invention is an electrical urea biosensor comprising a glass substrate  12 , wherein an indium tin oxide (ITO) conductive layer  14  on a surface of the glass substrate  12 . Besides, there is a non-isolation solid state ion-sensitive film  16 , such as the solid material of the tin dioxide, positioned on a surface of the indium tin oxide conductive layer  14  to use as the solid state ion-sensitive electrode to detect the pH value of the solution. The ion-sensitive film  16  provides with a sensitive region and a non-sensitive region. Then, a conductive line  18  is utilized to extend from the indium tin oxide conductive layer  14  for using as an external electrical contact point. A package encapsulant is used to cover the non-sensitive region of the ion-sensitive film  16  and to define a sensitive window  22 . The present invention utilizes the package encapsulant  20  to define the sensitive area of the biosensor, wherein the sensitive area is about 2 2  mm 2 . A urea enzyme  24  is immobilized in the sensitive window  22  of the ion-sensitive film  16 , wherein the urea enzyme  24  is composed of urease, which is embedded by a PVA-SbQ encapsulant. The present urea biosensor  10  is more easily than the prior ion-sensitive field effect transistor on the formulation and the package can reduce the cost to conform to the requirement of the disposable biosensor.  
      Wherein, the embodiment of the electrical urea biosensor mentioned above is using the glass substrate as its biosensor substrate. Besides, the substrate can be also selected from the group of an isolation substrate and a non-isolation substrate. Furthermore, the isolation substrate can be selected from the group of a silicon substrate, a glass substrate, a ceramics substrate, and a polymer substrate. Hence, the present biosensor has a better variation of the substrate and can change the substrate material depending on the different practical use and process condition.  
      Now, in order to illustrate the manufacturing method of the present invention in accordance with the structure of the  FIG. 1  shown mentioned above, referring to the  FIG. 2   a  to the  FIG. 2   d,  there are schematic representations structures at various stages to illustrate the formulation of the electrical urea biosensor in accordance with the embodiment of the present invention. The manufacturing method of the present invention comprises following steps:  
      First, referring to the  FIG. 2   a,  a glass substrate  12  is provided and an indium tin oxide conductive layer  14  is formed thereon. The thickness of the indium tin oxide conductive layer  14  is about  230  angstroms and its electric resistance is about 50 ohm to 100 ohm.  
      Following, such as shown in the  FIG. 2   c,  a sensitive film grown a tin dioxide sensitive film  16  on the surface of the indium tin oxide conductive layer  14  of the glass substrate  12  by using the sputtering method. The step uses the tin dioxide as the sputtering target and fills in the mixture gas of the argon (Ar) and the oxygen (O 2 ) at the ratio of 4:1. As the growing of the tin dioxide sensitive film  16 , the temperature of the glass substrate  12  is maintained at 150° C., the depositing pressure is maintained at 20 milli-torr, the radio frequency (RF) power is maintained at 50 watt so as to form the film  16  with a thickness of 2000 angstroms and to use as the solid state ion-sensitive electrode. Wherein, the sensitive film  16  can be dividing into a sensitive region and a non-sensitive region. Then, a conductive line  18  is positioned to extend from the indium tin oxide conductive layer  14  for using as an external electrical contact point.  
      Referring to the  FIG. 2   c  again, performing a package step, a package encapsulant, such as the epoxy, is used to cover the non-sensitive region of the ion-sensitive film  16  and a portion of the package encapsulant so as to define a sensitive window  22  of the sensitive region.  
      A package encapsulant is used to cover the non-sensitive region of the ion-sensitive film  16  and to define a sensitive window  22 . The present invention utilizes the package encapsulant  20  to define the sensitive area of the biosensor, wherein the sensitive area is about 2 2  mm 2 . A urea enzyme  24  is immobilized in the sensitive window  22  of the ion-sensitive film  16 , wherein the urea enzyme  24  is composed of urease, which is embedded by a PVA-SbQ encapsulant.  
      Last, such as shown in the  FIG. 2   d,  the present invention utilizes the enzyme immobilized technology to immobilize a urea enzyme  24  on the sensitive film  16  within the sensitive window  22 . Herein, the present invention utilizes the characteristic of photo-polymerization of the photopolymer to immobilize the urea enzyme  24  on the sensitive window  22  and acts to complete the formulation of the urea biosensor  10 . Besides, the present invention can also use other immobilized technology to form the electrical type electrochemistry biosensor. The present invention can reduce the instrument cost, such as the large-size optics biology analysis instrument, and can improve the portable characteristic of the biosensor. The present invention can use for the formulation of the promptly detecting sensor or the disposable sensor.  
      The detail illustration of the enzyme-immobilized technology is referenced to the following description:  
      First, a urease (urease, EC 3.5.1.5, 50000˜100000 units/g); a PVA-SbQ encapsulant (PVA, D.P.=1700, D.S.=88; SbQ, 1.52 mol %; N.V.=12.69 wt %; and the viscosity is about 5750 cp at 25° C.); the urea (NH 2 CONH 2 =60.06), wherein its degree of purity is 99%; and the phosphate (KH 2 PO 4 =136.09), which is the normal ACS grade and used for preparing the buffer solution, are prepared.  
      Following, the diluted PVA-SbQ (100 mg PVA-SbQ/100 ml-55 millimole phosphate solution of the pH value 7.0) and the urea solution (7 mg urea/100 ml-millimole phosphate solution of the pH value 7.0) are mixed at the ratio of 1:1. Next, 1 ml mixture solution is taken to drop on the sensitive window  22  and then the biosensor  10  is put under the illumination of the ultraviolet of 4 watt and 365 nm to perform the photo-polymerization with about 20 minutes. Last, after finishing the photo-polymerization, the biosensor  10  is put in a dark box of 4° C. with about 12 hours to complete the enzyme immobilized process.  
      Such as shown in the  FIG. 3 , the present invention utilizes an electrical urea biosensor  10  of the separation structure of tin dioxide/indium tin oxide/glass substrate as a transducer. The biosensor utilizes the conductive line  18  to electrically connect to the potentiometer of the high input impedance, such as the metal-oxide-semiconductor field effect transistor and the operational amplifier. As shown in the Figure, the read out circuit is the LT1167 instrumentation amplifier  26  and co-operate with the silver/silver chloride electrode  28  to provide a reference stable potential so as to measure the response potential of the urea biosensor  10  in the solution to calculate the concentration of the solution.  
      Referring to the  FIG. 4 , it is the correction curve view of the pH value of the electrical urea biosensor of the present invention. Such as shown in the figure, it is the pH sensitive characteristics of the tin dioxide sensitive film to make sure that the sensitivity of each batch sensor are in a stable range so as to use the mentioned instrumentation amplifier  26  to read out the different response potential of the sensor under the different pH environment. Where, as the pH value is between 2.2 to 10.2, the sensitivity of the tin dioxide sensitive film is about 58.85±0.41 millivoltage/pH value.  
      Referring to the  FIG. 5 , it is the status view of the response time and the return time of the electrical urea biosensor of the present invention. As shown in the figure, it presents the required response time and the required return time of the urea biosensor. According to the experiment result, after about 60 seconds, the response of the sensor is achieving the 90% degree of the maximum response potential and then the sensor is put into the buffer solution of 5 milli-mole. After 10 minutes, the response potential will slowly return to the potential before the reaction. Owing to the purpose of the extend biosensor structure is to develop the disposable biosensor, so the long or short response time is important to the disposable sensor, but not the necessary considering parameter.  
      Referring to the  FIG. 6 , it is the correction curve view of the pH value of the electrical urea biosensor at the measurement environment with various pH values in accordance with the present invention and it presents the influence on the response potential and correction curve when the initial pH value of the pre-measured solution is changing. When the initial pH value is higher, the response changeable range is obviously decreasing and the maximum chance limitation of the urea hydrolysis reaction is the pH value 9.3. Hence, if the initial pH value of the pre-measured solution is too high, it will decrease the response changeable range. On the other hand, if the initial pH value of the pre-measured solution is lower, the response potential will become very small. According to the experiment result, how to find the balance point between both is that the pH value 6.0 is the best reaction environment.  
      Such as shown in the  FIG. 7 , it is the correction curve view of the pH value of the electrical urea biosensor as buffer solutions with various concentrations in accordance with the present invention and it presents the influence causing from the different concentration of the buffer solution. Hence, the sensor based on the pH-sensitive is easily influenced from the buffer ability of the buffer solution and the concentration relation of the buffer solution is related with the strong or weak buffer solution ability. If the buffer concentration is higher, the buffer ability is more excellent. On the other hand, if the buffer concentration is lower, the buffer ability is more poor so as the pH difference generated near the sensitive window will immediately response on the potential difference and the correction curve will slant move to the direction of the low urea concentration.  
      Referring to the  FIG. 8 , it is the maximum variation of the responsible voltage of the electrical urea biosensor as the time increasing in accordance with the present invention. As shown in the figure, it discusses the stability of the storage status. In the present invention, one hundred disposable urea biosensors formed by the extended tin dioxide/indium tin oxide/glass substrate are prepared to store into the dark box of 4° C. to perform the measurement every 5 to 10 days and to observe the storage time of the formed sensor. According to the experiment, after 99 days, the sensor also can normally work and has no obviously decrement of the maximum response potential.  
      Hence, the present invention utilizes the ion-sensitive field effect transistor of the separation structure of the extended tin dioxide/indium tin oxide/glass substrate to form the disposable urea biosensor. The structure of the urea biosensor has a best response curve under the work environment of the phosphate solution with 5 mmol and the pH value 6.0. So as the present invention can detect the urea concentration of 0.31 mg/100 ml˜120 mg/100 ml and the sensitivity of the linear portion is 169.1 mvol/p(urea concentration).  
      The present invention utilizes a non-isolation solid-state ion-sensitive film to use as a sensitive electrode of an ion-sensitive gate field effect transistor by integrating the semiconductor process technology to manufacture a disposable urea biosensor so as the present invention can be mass production and provides with the advantage of the low cost and the easy package so as to reduce the cost of the prior ion-sensitive gate field effect transistor simplify the package. Furthermore, the present invention simultaneously provides with advantages of the simple production, the low cost, easily dry storage, the adjustable sensitive area, and the easy conveyance.  
      The forgoing description of the embodiments of the invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or to limit the invention to he precise from disclosed. The description was selected to best explain the principles of the invention and practical application of these principles to enable others skilled in the art to best utilize the invention in various embodiments and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention not to be limited by the specification, but be defined by the claim set forth below.