|Número de publicación||US20040256685 A1|
|Tipo de publicación||Solicitud|
|Número de solicitud||US 10/887,902|
|Fecha de publicación||23 Dic 2004|
|Fecha de presentación||12 Jul 2004|
|Fecha de prioridad||20 Feb 2001|
|También publicado como||DE10207028A1, DE10207028B4, US6808008, US7210517, US20020112840, US20040256074|
|Número de publicación||10887902, 887902, US 2004/0256685 A1, US 2004/256685 A1, US 20040256685 A1, US 20040256685A1, US 2004256685 A1, US 2004256685A1, US-A1-20040256685, US-A1-2004256685, US2004/0256685A1, US2004/256685A1, US20040256685 A1, US20040256685A1, US2004256685 A1, US2004256685A1|
|Inventores||Jung-Chuan Chou, Yen Sheng Wang|
|Cesionario original||Jung-Chuan Chou, Yen Sheng Wang|
|Exportar cita||BiBTeX, EndNote, RefMan|
|Citada por (44), Clasificaciones (11), Eventos legales (1)|
|Enlaces externos: USPTO, Cesión de USPTO, Espacenet|
 The invention relates to a biosensor, and in particular to a biosensor having an extended gate field effect transistor (EGFET) structure with a urea layer immobilized on a tin oxide layer.
 The first ion sensitive field effect transistor (ISFET) was fabricated by P. Bergveld in 1970. The ISFET unlike the MOSFET has no metal gate electrode. Silicon dioxide (SiO2) was first used as a pH sensitive membrane for the ISFET. Subsequently, Al2O3, Si3N4, Ta2O5, and SnO2 were used as a pH sensitive membrane due to their higher pH response.
 The earliest suggestion of the concept of an enzyme modified FET (EnFET) sensor device appears to be that of Janata and Moss. They described a penicillin responsive sensor which comprises a matched pH-responsive ISFET pair, one device having an overlying, active gate film of the cross-linked albumin-penicillinase, and the other having a reference gate membrane of only cross-linked albumin. When penicillin was present in the analysis, the penicillinase present in the active gate material catalyzed the hydrolysis of penicillin to penicilloic acid, which released protons and created a local decrease in pH, whereas the reference gate remained unaffected. Several applications for the EnFET, such as the glucose, urea, acetylcholine, and alcohol exist.
 A number of patents relating to ISFETs are listed hereinafter.
 U.S. Pat. No. 6,218,208 discloses a sensitive material tin oxide (SnO2) obtained by thermal evaporation or by R.F. reactive sputtering, used as a high pH-sensitive material for a multi-structure ISFET.
 U.S. Pat. No. 5,602,467 discloses a circuit layout for measuring ion concentrations in solutions using ISFET. The circuit layout makes it possible to represent the threshold voltage difference of the two ISFETs directly and independently of technological tolerances, operationally caused parameter fluctuations, and ambient influences.
 U.S. Pat. No. 5,387,328 discloses a biosensor employing an ISFET comprising a source and a drain formed in a substrate, an ion sensitive gate placed between the source and drain, an ion sensitive film formed on the ion sensing gate, an immobilized enzyme membrane defined on the ion sensitive film and, a Pt electrode formed on the ion sensitive film. The sensor has a Pt electrode capable of sensing all biological substances that generate H2O2 in the enzyme reaction, whereby are achieved the high sensitivity and rapid reaction time.
 U.S. Pat. No. 5,350,701 discloses a process for producing a surface gate comprising a selective membrane for an integrated chemical sensor comprising a field effect transistor, and the integrated chemical sensor thus produced, wherein the surface gate is particularly sensitive to alkaline-earth species, and more particularly, sensitive to the calcium ion. The process comprises forming grafts on the surface gate, and making the grafts operative utilizing phosphonate based, ion-sensitive molecules.
 U.S. Pat. No. 5,309,085 discloses a measuring circuit with a biosensor utilizing ion sensitive field effect transistors integrated into one chip. The measuring circuit comprises two ion sensitive FET input devices composed of an enzyme FET having an enzyme sensitive membrane on the gate and a reference FET, and a differential amplifier for amplifying the outputs of the enzyme FET and the reference FET.
 A variety of materials are known to be capable of serving as the sensing film of an ISFETs, such as, Al2O3, Si3N4, a-WO3, a-C:H, and a-Si:H, etc. The manufacture of sensing films is typically accomplished by deposition, such as, sputtering or plasma enhanced chemical vapor deposition (PECVD), therefore, the cost is relatively high and the time required for thin film fabrication is long.
 Thus, an easily fabricated, low cost ISFET and the sensing film thereof, eliminating packing problems, are desirable.
 Accordingly, the biosensor according to the invention, having an extended gate field effect transistor structure, comprises a metal oxide semiconductor field effect transistor, a sensing unit, and a conductive wire. The metal oxide semiconductor field effect transistor is formed on a semiconductor substrate. The sensing unit comprises a substrate, a silicon dioxide layer on the substrate, a tin oxide layer on the silicon dioxide layer, and a urease layer immobilized on the tin oxide layer. The conductive wire connects the MOSFET and the sensing unit.
 The method of manufacturing a sensing unit according to an embodiment of the invention comprises the steps of providing a conductive substrate; forming a silicon dioxide layer on the conductive substrate; forming a tin oxide layer on the silicon dioxide layer; electrically connecting the conductive substrate with a conductive wire; forming an insulating layer on the surface of the sensing unit and exposing part of the tin oxide layer and part of the conductive wire; and immobilizing a urease layer on the exposed part of tin oxide layer by gel entrapment.
 The measuring system according to an embodiment of the invention comprises a biosensor as described above; a reference electrode for supplying a stable voltage; an instrumentation amplifier having two inputs and one output, wherein the two inputs are connected to the biosensor and the reference electrode, respectively; a high-resistance multimeter connected with the output of the instrumentation amplifier; and a computer connected with the high-resistance multimeter through a communication interface card to store, process, or analyze data.
 The biosensor according to an embodiment of the invention has the advantages of short response time, simple packing, easy manufacture, and low cost. When compared with conventional FET, this biosensor is more stable, provides better current-leakage protection, and higher sensitivity. In one embodiment of the invention, the biosensor may have a sensitivity of 58 mV/pH and better linearity in a solution having a pH value in the range of 1 to 9, and may be used as a disposable sensing structure.
 In the method according to embodiments of the invention, a gel entrapment is performed by immobilization of an enzyme with a photosensitive polymer on an ion sensitive film, thus the ISFET is favorably used in biosensors. In addition, a commercialized SnO2/SiO2/glass can be directly used to manufacture the enzyme sensing film, thus manufacturing is simplified and cost is reduced.
 In embodiments of the invention, the sensing structure is disposable, thus eliminating problems of enzyme loss due to long-term use and cross contamination.
 A detailed description is given in the following embodiments with reference to the accompanying drawings.
 Embodiments of the invention can be more fully understood by reading the subsequent detailed description and examples with references made to the accompanying drawings, wherein:
FIG. 1a shows a cross-section of the biosensor of an embodiment of the invention.
FIG. 1b shows a cross-section of the sensing structure before the deposition of urease, of an embodiment of the invention.
FIG. 1c shows a package of the sensing structure of an embodiment of the invention.
FIG. 2 shows the structure of the urea biosensor measurement system of an embodiment of the invention.
FIG. 3a shows the IDS versus VG curves of the sensitive film at the different pH values in an embodiment of the invention.
FIG. 3b shows the VG versus pH curve of the sensitive film at the different pH values in an embodiment of the invention.
FIGS. 4a-4 e shows the voltage variation with respect to urea solutions having concentrations of 1.25, 10, 40, 80, and 120 mg/dl, respectively, of the urea biosensor of an embodiment of the invention.
FIG. 5 shows the correcting curve between 1.25 mg/dl and 120 mg/dl urea solutions of urea biosensor.
 The biosensor according to the invention has an extended gate field effect transistor structure. The sensing film extended from the gate of the ISFET such that the metal oxide semiconductor field effect transistor components can be separated from a tested solution to avoid the instability of the semiconductor and the signal interference from the solution. In an embodiment of the invention, the urease is immobilized by means of gel entrapment and forms a component of a sensing film. The sensing film can be used to determine the concentration of hydrogen ion or urea in a solution.
 The biosensor according to an embodiment of the invention is illustrated in FIGS. 1a-1 c. Referring to FIG. 1 a, the biosensor 110 having an extended gate field effect transistor structure comprises a metal oxide semiconductor field effect transistor, a sensing unit, and a conductive wire.
 The metal oxide semiconductor field effect transistor 112 (shown as an electric circuit) is on a semiconductor substrate (not shown) which may be N- or P-type.
 The sensing unit comprises a substrate 101, a silicon dioxide layer 102, a tin oxide layer 103, and a urease layer 109. The substrate can be a conductive glass, such as an indium tin oxide (ITO) glass. The silicon dioxide is formed on the substrate with a thickness of 1000 Å to 2000 Å and may function as a buffer allowing deposition of tin oxide on the glass. Tin oxide layer has a thickness of 2000 Å to 3000 Å and is formed on the silicon dioxide layer. The substrate, the silicon dioxide layer, and the tin oxide layer may available from a commercial product, such as SnO2/SiO2/glass, Kuanghua Development technical corporation, Taiwan, under the trademark TO-3030, for facilitating manufacture. The urease layer is formed by photopolymerization of a mixture of a photosensitive polymer and urease in a phosphate buffer solution. The photosensitive polymer and the urease are in a ratio ranging from 30:1 to 5:1, preferably 25:1 to 15:1, and more preferably about 20:1 by weight. Furthermore, an insulating layer, for example, epoxy resin, may be formed on the surface of the sensing unit to seal the sensing device but expose part of the urease layer for the contact with the tested solution (not shown) and part of the conductive wire for electrical connect.
 The conductive wire 105 connects the metal oxide semiconductor field effect transistor 112 and the sensing unit 110. The wire material is the metal, such as, aluminum.
 Briefly, the measurement of ion concentration of an acid or base solution is attained by transforming the charge of hydrogen ions adsorbed on the sensing film of the extended gate field effect transistor into electrical signals, using the signals to control the width of the channel of MOSFET, and then determining the concentration of the tested hydrogen ions by the magnitude of the electric current.
 In the determination of the urea concentration of a solution, when urea reacts with the urease on the sensing film, OH− or H+ ions are produced through hydrolysis, the EGFET will change voltage corresponding to the pH value of the solution, and thus the urea concentration can be known from the electric signals produced by the ISFET. The hydrolysis of urea is as follows:
 In the method of manufacturing a sensing unit according to an embodiment of the invention, the steps of providing a conductive substrate, forming a silicon dioxide layer on the conductive substrate, and forming a tin oxide layer on the silicon dioxide layer can be accomplished by simply providing a commercially available SnO2/SiO2/glass 106 product to simplify manufacturing and save time and reduce cost, or accomplished step by step as desired. The tin oxide layer can be deposited on the silicon dioxide layer by chemical vapor deposition (CVD). The deposition temperature may be 250 to 600° C., preferably 580 to 600° C. The layer has a thickness of preferably 0.2 to 0.3 μm and a resistance of 20 to 30 Ω/□. For example, tin tetrachloride and water are used to produce tin oxide layer. The reaction is shown as follows:
 The silicon dioxide layer can be deposited on the substrate by CVD at a temperature of preferably 580 to 600° C. The thickness is preferably 100 to 300Å.
 Referring to FIG. 1b, after the SnO2/SiO2/substrate is prepared or obtained, the tin oxide layer is connected to the conductive wire 105. The surface of the resultant is preferably sealed with an insulating layer 104, while part of the tin oxide layer is exposed for coating urease as a detecting window and part of the conductive wire is exposed for connecting the gate of the metal oxide semiconductor field effect transistor.
 Next, a urease layer is immobilized on the exposed part of the tin oxide layer by gel entrapment. The gel entrapment is performed by mixing the photosensitive polymer and the urease in a phosphate buffer solution, photopolymerizing the resulting mixture, and then placing the resultant in the dark at a low temperature for a proper time, thereby the urease is immobilized on the tin oxide layer. The photosensitive polymer may be, for example, a polyvinyl alcohol, which may have a styrylpyridinium group, such as PVA-SbQ. The photosensitive polymer and the urease are used in a ratio ranging from preferably 30:1 to 5:1, more preferably 25:1 to 15:1, and most preferably about 20:1 by weight. For example, the ratio may be in the range of 300 mg/100 μl PBS:10 mg/100 μl PBS to 50 mg/100 μl PBS:10 mg/100 μl PBS, preferably 250 mg/100 μl PBS:10 mg/100 μl PBS to 150 mg/100 μl PBS:10 mg/100 μl PBS. In the photopolymerization, urease (in 5 mM phosphate buffer solution, pH 7) and photosensitive polymer (in 5 mM phosphate buffer solution) are radiated to react. The radiation may be UV light, such as, a UV light with a wavelength of 365 nm. After the photopolymerization, the result may be placed in the dark, for example, a dark box, at a low temperature of, for example, 4° C. to −10° C., for a proper time, and thus the immobilization of urease on the tin oxide layer, as well as the sensing unit, is accomplished, as shown in FIG. 1c. Note that, in the gel entrapment, white light should be avoided to prevent self photopolymerization of the enzyme.
 The sensing unit can be placed directly in the tested solution for the determination of pH value or urea concentration.
 Referring to FIG. 2, the biosensor 110 described above is used to construct the measuring system according to an embodiment of the invention. The measuring system further comprises a reference electrode 204, an instrumentation amplifier 202, a high-resistance multimeter 203, and a computer 205.
 The reference electrode 204, for example, Ag/AgCl reference electrode, is immersed in the tested solution to help to maintain a stable voltage and provide the function of calibration.
 The instrumentation amplifier 202 amplifies the electric signals and comprises two inputs and one output. The two inputs are connected to the biosensor 110 and the reference electrode 204 through the conductive wires 108 and 206. The instrumentation amplifier may be, for example, a commercially available IC, LT1167. The connection of the biosensor 110 and the instrumentation amplifier 202 can be accomplished by, for example, pin connection, and therefore, after measurement, the biosensor 110 and the instrumentation amplifier 202 are detachable to advantageously renew the biosensor 110.
 The high-resistance multimeter 203 is connected to the output of the instrumentation amplifier 202 to read out the output voltage from the biosensor 110.
 The computer 205 is connected with the high-resistance multimeter 203 through a communication interface card to store, process, or analyze data. The computer 205 may be, for example, a personal computer. The communication interface card may be, for example, HP82350. The parameter measurement and the data storage may be controlled using HP VEE program installed in the computer. Microsoft Origin 6.0 is further used to analyze output signals or plot graphs.
 In the measurement, the urea sensing film 109 of the biosensor 110 and the reference electrode are immersed in the tested solution 201 containing acid, base, or urea.
 A commercial SnO2/SiO2/glass sold by Kuanghua Development technical corporation, Taiwan under the trade mark TO-3030 was cut into squares of 1 cm×1 cm and washed in deionized water of an ultrasonic oscillator. An aluminum conductive wire was bonded to the SnO2/SiO2/glass 106 by silver glue and dried at 120° C. in an oven for 10 minutes, and then cooled to room temperature. The aluminum conductive wire 108 was installed through a capillary 107 and the SnO2/SiO2/glass and the capillary were fixed by epoxy resin forming an insulating layer 104 and dried at 120° C. in an oven for 20 minutes. The SnO2/SiO2/glass was then packaged with epoxy resin but an area of 1.5 mm×1.5 mm was kept to be a sensing window. Then, one end of the aluminum conductive wire was connected to a MOFET.
 Subsequently, the sensing window was cleaned in deionized water of the ultrasonic oscillator. A mixture of 200 mg PVA-SbQ (under trade mark Toyo Gose sold by Kogyo Company, Japan) and 10 mg urease (EC 18.104.22.168, powder, Type IV, from Jack bean, 50000 to 100000 units/g, sold by Sigma Chemical Company) in 200 μl phosphate buffer solution (5 mM, pH 7.0) was prepared. 1 μl of the urease mixture was deposited on the sensing window. The resulting device was exposed under UV light (4 W/365 nm) for photopolymerization for 20 minutes, then placed in a dark box at 4° C. for about 12 hours, obtaining a EGFET with urea sensing film, as shown in FIG. 1c.
 Referring to FIG. 2, the biosensor having a urease film as a sensing film as obtained from Example 1 was used. The sensing film of the biosensor was connected to the input of LT1167, and the output of LT1167 was connected to a digital multimeter. An Ag/AgCl reference electrode was connected to another input of LT1167. A computer was connected with the digital multimeter through a communication interface card HP82350. The parameter measurement and the data storage were performed using HP VEE program installed in the computer. Microsoft Origin 6.0 was used to process and analyze output signals.
 A series of solutions having pH value of 1, 3, 5, 7, and 9 were measured using the measuring system as described above. By changing gate voltage, output electric currents were obtained. The data were processed and analyzed using Microsoft Origin 6.0 to plot a curve of electric current versus gate voltage, as shown in FIG. 3a. As the proper current value, IDS, was about 300 μA, a high linear sensitivity for measuring a solution having a pH value in the range of 1 to 9 was obtained to be 58 mV/pH. A curve of gate voltage versus pH value was also plotted, as shown in FIG. 3b.
 A series of urea solutions having concentrations of 1.25 mg/dl, 10 mg/dl, 40 mg/dl, 80 mg/dl, and 120 mg/dl were measured using the measuring system as described above. Curves of output voltage versus measuring time were plotted as shown in FIGS. 4a to 4 e.
 After linear calibration of the data obtained above, the linearity of the sensor was obtained in the range of 5 mg/dl to 50 mg/dl, as shown in FIG. 5 showing the plot of voltage difference versus urea concentration.
 The results indicate that the sensor and the manufacturing method of the sensing unit of an embodiment of the invention have advantages of low cost, easy attainment, and a simple package due to the utilization of commercially available SnO2/SiO2/glass. The method according to the invention is novel.
 Furthermore, a precise response for the biosensor to solutions having different pH values or urea concentrations can be obtained by the measuring system according to an embodiment of the invention, and in turn the pH value or the urea concentration can be obtained precisely. Furthermore, the sensor and the readout circuit are detachable and the sensing unit is disposable, thus, can be prevented contamination or damage to the sensing film.
 While the invention has been described by way of example and in terms of the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments. To the contrary, it is intended to cover various modifications and similar arrangements (as would be apparent to those skilled in the art). Therefore, the scope of the appended claims should be accorded the broadest interpretation so as to encompass all such modifications and similar arrangements.
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|Clasificación de EE.UU.||257/414|
|Clasificación internacional||B22D17/14, C12Q1/58, G01N33/53, C12Q1/00|
|Clasificación cooperativa||C12Q1/58, C12Q1/005, B22D17/14|
|Clasificación europea||B22D17/14, C12Q1/58, C12Q1/00B6|
|12 Jul 2004||AS||Assignment|
Owner name: NATIONAL YUNLIN UNIVERSITY OF SCIENCE AND TECHNOLO
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:CHOU, JUNG-CHUAN;WANG, YEN SHENG;REEL/FRAME:015568/0742
Effective date: 20040615