Abstract:
A measuring apparatus for a biochemical compound has a channel structure having an inlet port and an outlet port. A passageway extends between the inlet and outlet ports for passing a liquid sample therethrough. The passageway has its inside walls lined with a layer of hydrophilic material. A biosensor is provided in the passageway to detect a biochemical compound contained in the liquid sample. Preferably, the hydrophilic material comprises a metal oxide having a photocatalytic characteristic, which is illuminated with ultraviolet rays.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    1. Field of the Invention  
           [0002]    The present invention relates to biosensors for the determination of a biochemical compound contained in a liquid sample, and more specifically to a channel structure in which a biosensor is located for determining a compound contained in a liquid sample flowing through the channel.  
           [0003]    2. Description of the Related Art  
           [0004]    The method generally employed in the measurement of biochemical components of a liquid sample such as blood and body fluid involves passing the liquid sample through a narrow passageway to bring it into contact with a biosensor provided in the passageway. The sensor of this type essentially comprises a permeable membrane, an immobilized enzyme and a working electrode. As the liquid flows in the channel, it diffuses through the permeable membrane to the immobilized enzyme. A reaction between the enzyme and a species being analyzed causes a current to flow through the working electrode on which a measurement is made. The amount of the species that can be detected depends largely on its concentration in the vicinity of the membrane. If the liquid sample is too viscous, a smooth flow is impeded, causing the substrate concentration to fluctuate violently.  
           [0005]    In order to ensure a smooth sample flow in the channel, the prior art utilized the hydrophobic characteristic of fluorocarbon resin by coating the inner walls of the channel with a thin layer of the resin. However, a small amount of the liquid sample still reacts with the fluorocarbon resin and adheres to the channel walls to eventually form a rugged surface. As a result, measurement was severely affected and reading became unstable.  
         SUMMARY OF THE INVENTION  
         [0006]    It is therefore an object of the present invention to provide a measuring apparatus having a channel structure which maintains its passageway under impurity-free condition for an extended period of time to ensure smooth flow of liquid sample being analyzed by a biosensor located inside of the passageway.  
           [0007]    According to the present invention, there is provided a measuring apparatus comprising a channel structure having an inlet port and an outlet port and a passageway between the inlet and outlet ports for passing a liquid sample therethrough, the passageway having inside thereof lined with a layer of hydrophilic material. A biosensor located in the passageway detects a biochemical compound contained in the liquid sample. Preferably, the hydrophilic material comprises a metal oxide having a photocatalytic characteristic, which is illuminated with ultraviolet rays. Specifically, the channel structure is formed by a first member having a channel between the inlet port and the outlet port with the inside of the channel being lined with a layer of the metal oxide, and a second member secured to the first member for enclosing the channel, the second member being coated with a layer of the metal oxide so that the metal oxide layer forms a ceiling portion of the enclosed channel. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0008]    The present invention will be described in detail further with reference to the following drawings, in which:  
         [0009]    [0009]FIG. 1 is a block diagram of a measuring apparatus of a first embodiment of the present invention for measuring a biochemical compound using a biosensor;  
         [0010]    [0010]FIG. 2 is a cross-sectional view taken along the lines  2 - 2  of FIG. 1;  
         [0011]    [0011]FIG. 3 is a cross-sectional view taken along the lines  3 - 3  of FIG. 1;  
         [0012]    [0012]FIG. 4 is a cross-sectional view of a biosensor;  
         [0013]    [0013]FIG. 5 is a graphic representation of the speed of liquid sample flowing in a channel plotted against time for comparison between the present invention and the prior art;  
         [0014]    [0014]FIG. 6 is a cross-sectional view of a modified form of the main unit;  
         [0015]    [0015]FIG. 7 is a plan view of a modified main unit of the present invention; and  
         [0016]    [0016]FIG. 8 is a cross-sectional view taken along the lines  8 - 8  of FIG. 7. 
     
    
     DETAILED DESCRIPTION  
       [0017]    Referring now to FIG. 1, there is shown a measuring apparatus according to a first embodiment of the present invention for measuring a chemical compound by using a biosensor.  
         [0018]    The apparatus is comprised of a main unit  10  which is essentially a channel structure with a liquid passageway or channel  11  that extends between an inlet port  12  and an outlet port  13 . In order to produce a smooth liquid flow in the liquid channel  11 , the inner walls of this channel are lined with hydrophilic material as indicated by numeral  14 . Preferably, the liner  14  is composed of a metal oxide whose photocatalytic function renders it significantly hydrophilic when the liner is subjected to ultraviolet radiation. Because of the, excellent hydrophilic property, the metal oxide liner  14  produces no reaction at all with the liquid sample. Thus, the inner walls of the channel  11  are not contaminated with impurities with a resultant elimination of stagnation and irregular flow pattern.  
         [0019]    A supply tube  21  is connected to the inlet port  12  to introduce a liquid sample such as blood or body fluid from a collecting cell, not shown. To the outlet port  13  is connected a drain tube  22  in which a sucking pump  23  is provided to pull the liquid sample through the channel  11 . A flow detector  24  is provided to measure the flow rate of the liquid sample supplied to the channel  11 . A comparator  25  compares the output of the flow detector  24  with a reference voltage and drives a flow regulator  26  with an offset voltage that indicates an amount by which the detected flow rate deviates from the reference value. This feedback control proceeds such that when the offset voltage reduces substantially to zero the output signal of biosensor  15  proportionally represents the concentration of a biochemical compound being analyzed.  
         [0020]    In the channel  11  is provided a biosensor  15 , which is connected via a lead line  16  to an electrode pad  17 . A reference electrode  30  and a counter electrode  31  are attached to the inner wall of drain tube  22  and connected to a measuring instrument  32  to which the electrode pad  17  is also connected. Measuring instrument  32  applies a reference voltage to the reference electrode  30  to detect an output signal produced across the working electrode  41  and the counter electrode  31 . With amperometric measurement, glucose, lactic acid, uric acid, cholesterol, choline and cholic acid can be detected. With potentiometric measurement, the concentration of ions such as hydrogen, sodium, potassium and chroline can be detected.  
         [0021]    As shown in detail in FIGS. 2 and 3, the main unit  10  is fabricated on a silicon layer  18 . Using the conventional wet-etching technique, a groove portion of the channel  11  is formed on the silicon layer  18 . The width and the depth of the channel  11  are 500 μm, for example. The use of the dry-etching method is preferred for applications where high precision measurement is required. A desired shape of the cross-section may be obtained by selecting a suitable etching solution.  
         [0022]    Biosensor  15  is fabricated on the bottom of the channel  11  by initially covering the inner walls of channel  11  with a resist to expose an area in which the biosensor is to be formed and then successively performing deposition processes on the exposed area. As shown in FIG. 4, one example of the biosensor is comprised of a working electrode  41 , a bonding layer  42  with which the working electrode is brought into intimate contact with an immobilized enzyme layer  43 , on which a diffusion limiting (semi-permeable) membrane  44  is secured. In order to ensure a smooth flow in the channel  11  the biosensor  15  has a thickness of 1 mm or less.  
         [0023]    More specifically, platinum is used as a target material in a sputtering method to form the working electrode  41 , the connecting lead  17  and the electrode pad  17  on the silicon layer  18 . In a typical example, the working electrode  41  has an area of 500 μm×1000 μm and the connecting lead  16  has a width of 1 mm and the electrode pad  17  has a size of 2 mm×2 mm. Bonding layer  42  is formed by spin-coating 1 v/v % γ-aminopropyltriethoxysilane solution. Immobilized enzyme layer  43  is subsequently prepared by spin-coating an enzyme solution containing 56.5 U/μl oxidase and a 2.5 w/v % albumin solution containing 1 v/v % glutaraldehyde. The oxidase that can be used for the layer  43  includes lactic oxidase, lactose, glucose oxidase, uric oxidase, galactose oxidase, lactose oxidase, sucrose oxidase, ethanol oxidase, methanol oxidase, starch oxidase, amino acid oxidase, monoamine oxidase, cholesterol oxidase, choline oxidase and pyrubic acid oxidase. These can be used singly or in combination. Diffusion limiting membrane  44  is prepared by spin-coating a polyfluoroalcoholester solution containing 2 v/v % methacrylic acid resin (such as Fluorad FC-722, a registered trademark of Sumitomo 3M) which may be appropriately diluted by perfluorohexthane.  
         [0024]    Biosensor  15  is then coated with a resist and metal oxide is deposited on the bottom and sidewalls of the channel  11  by using a sputtering method and a spin-coating method. Alternatively, the liner  14  is formed by submerging the silicon layer  18  in an oxide-dispersed inorganic binder solution, or introducing an oxide (such as antifouling material) solution into the channel  11  using a syringe.  
         [0025]    The metal oxide that can be advantageously used in the present invention includes titanium oxide, zinc oxide, strontium titanate, tungstic trioxide, ferrous oxide, bismuth trioxide, and tin oxide. These metal oxides can be used singly or in combination. Preferably, the thickness of metal oxide liner  14  is in the range between 0.1 μm and 10 μm.  
         [0026]    As shown in FIGS. 2 and 3, a UV-transparent layer  19  is secured on the silicon layer  18  to allow penetration of ultraviolet radiation from a UV source  20 . Before the layer  19  is brought into face-to-face contact with the silicon layer  18 , a layer of metal oxide is deposited on a portion of the contact surface of layer  19  that serves as a top wall of the channel  11 . The deposited metal oxide layer thus constitutes a ceiling portion of the liner  14 . With the deposited contact surface facing downwards, the transparent layer  19  is cemented to the silicon layer  18 , using an anodic bonding system. An alternative method of forming the metal oxide liner  14  is to initially secure the UV-transparent layer  19  to the silicon layer  18  so that the channel  11  is enclosed, and to introduce an appropriate solution just mentioned above to simultaneously coat all the inner walls of the channel.  
         [0027]    The ultraviolet rays impinged on the upper surface penetrate the transparent layer  19 . Some of the UV rays illuminates the top liner  14  and others diffract as they penetrate their way into the silicon layer  18  and illuminate the sidewall portions of the liner  14 ; Part of the penetrating rays will bounce off the bottom surface of silicon layer  18  and illuminates the bottom portion of the liner  14 . In this manner, the metal oxide liner  14  exhibits a significant hydrophilic characteristic that allows the channel  11  to remain in an impurity free condition over a long period of time.  
         [0028]    Experiments show that the present invention compares favorably with the prior art in which the channel is lined with a hydrophobic material. In the experiments, body fluid sample was introduced at a rate 10 μl/min into the channel  11  and the variation of the liquid speed was observed for a period of 25 hours. As shown in FIG. 5, the prior art suffers a significant decrease in the liquid speed, whereas no appreciable decrease is observed in the present invention. It is apparent that in the prior art the inner walls of the channel are contaminated with flow-impeding impurities which build up in an increasing number with time.  
         [0029]    [0029]FIG. 6 shows a modified form of the main unit  10  in which a plurality of biosensors  15 A,  15 B and  15 C of different types are arranged within the channel  11  to allow simultaneous analysis of a number of different biochemical components. This eliminates the need to replace the main unit with another to analyze a different species and reduces the total time to determine more than one biochemical component.  
         [0030]    The provision of reference and counter electrodes  30  and  31  in the drain tube  22 , rather than within the channel  11 , serves to advantageously reduce the amount of resistance the liquid sample will encounter in the channel  11 .  
         [0031]    Since the inner walls of channel  11  are free from contaminating impurities, the concentration control system provided at the supply tube  21  operates advantageously for precision measurement of biochemical compounds.  
         [0032]    The present invention can be inexpensively implemented by a modified form of the main unit  10  shown in FIGS. 7 and 8. According to this modification, a channel  51  is formed by lower and upper grooves which are respectively formed on a lower block  58  and an upper block  59  of UV-transparent plastic material, Channel  51  extends between an inlet port  52  and an outlet port  53 , both of which are formed in the upper block  59 . A metal oxide liner  54 A is deposited on the upper groove of the channel  51  and the inner walls of inlet and outlet ports  52 ,  53 , and a metal oxide layer  54 B is deposited on the lower groove of the channel  51 . A biosensor  55  is located in the channel  51 . A reference electrode  60  and a counter electrode  61  are also provided in the channel  51 . Biosensor  55  is identical to that of the previous embodiment. The working electrode of biosensor  55  and the reference and counter electrodes  60  and  61  are connected to a measuring instrument  62 .  
         [0033]    Lower and upper blocks  58 ,  59  are detachably coupled together by means of a pair of claw hooks  70 , each of which is secured to the upper block  59  to detachably engage a groove  71  of the lower block  58 . Blocks  58  and  59  are detached from each other to expose the channel grooves for maintenance purpose.  
         [0034]    The measuring apparatus of the present invention were verified for clinical applications by the following examples.  
       EXAMPLE I  
       [0035]    Using the apparatus of FIGS. 2 and 3, the glucose value of an adult male (34 years old, 68 kilograms) was measured at 10-minute intervals for a period of two hours. For purposes of comparison, a standard full-scale clinical measurement apparatus (Hitachi Jidou Sokutei Souchi 7050) was used to measure the glucose value of the adult male under the same condition. A correlation value of 0.989 was obtained between the data of these apparatus.  
       EXAMPLE II  
       [0036]    Using the apparatus of FIGS. 7 and 8, the glucose, lactic acid and uric acid of the adult male was measured at 10-minute intervals for a period of two hours. The same measurements were made using the standard full-scale clinical measurement apparatus. For glucose, lactic acid and uric acid, correlation values of 0.987, 0.981 and 0.979 were respectively obtained between these apparatus.