Patent Publication Number: US-8535620-B2

Title: Method of filling liquid sample

Description:
CROSS-REFERENCE 
     This application claims priority to Japanese Patent Application No. 2009-282931, filed Dec. 14, 2009, the entirety of which is hereby incorporated by reference. 
     BACKGROUND 
     1. Technical Field 
     The present invention relates to a method of filling a liquid sample. 
     2. Related Art 
     A method of performing chemical analysis and chemical synthesis, biotechnology-related analysis, and the like using a microfluidic chip in which a micro flow channel is provided on a glass substrate or the like attracts attention. The microfluidic chip is called micro total analytical system (micro TAS), lab-on-a-chip, or the like. The microfluidic chip has advantages that, for example, necessary amounts of a sample and a reagent are small, a reaction time is short, and an amount of wastes is small compared with an analyzer in the past. Therefore, the microfluidic chip is expected to be used in wide fields such as medical diagnosis, on-site analysis of environment and foods, production of pharmaceuticals, chemicals, and the like (JP-A-2006-509199). Since the necessary amount of the reagent is small in the microfluidic chip, cost of a test decreases. When the necessary amounts of the sample and the reagent are small, since the reaction time is substantially reduced, the test is efficient. In particular, since a necessary amount of a sample such as blood is small, a burden on a patient can be reduced using the microfluidic chip in medical diagnosis. 
     When an amount of a liquid sample such as a sample or a reagent is small, measurement results tend to fluctuate because dispense accuracy falls and the influence of evaporation of the liquid sample on the amount of the sample is large. In general, dispensing work for the liquid sample is complicated and a work time is long. Since consumables such as a pipette and a chip are consumed in large volume, cost of a test increases. Manual dispensing work for the liquid sample tends to cause mistakes and it is highly likely that undesirable substances are mixed in the liquid sample. According to such a background, there is a demand for a technique for accurately and precisely dispensing a small amount of a liquid sample. 
     SUMMARY 
     An advantage of some aspects of the invention is to provide a method of filling a liquid sample that can precisely and accurately dispense the liquid sample at low cost in a simple and easy way while preventing mixing of foreign matters. 
     According to an aspect of the invention, there is provided a method of filling a liquid sample including:
         supplying the liquid sample to a first well of a biochip,       

     the biochip includes:
         the first well on a first surface of a substrate,   a plurality of second wells that are provided on a first surface of a substrate separated from the first well and include a reagent;       

     adhering a cover and the substrate on a loop-shaped area that surrounds the first well and the second wells in contact surfaces of the cover and the substrate in a state in which the cover is arranged on the substrate to cover the first well and the second wells; 
     moving, using centrifugal force, the liquid sample from the first well to the second wells through a space formed between the cover and the substrate in an area further on an inner side than the loop-shaped area by rotating the biochip around a rotation axis in a state in which the biochip is arranged such that a distance from any one of the second wells to the rotation axis is longer than a distance from the first well to the rotation axis; and 
     sealing the first well and the second wells by adhering the cover and the substrate to. 
     In the aspect of the invention, “the second wells separated from the first well” means that the second wells are provided independently from the first well. For example, this means that the first well and the second wells are not connected by a flow channel. In the invention, “adhere” is a concept including both “welding: adhering contacting portions of plural members by melting” and “bonding: adhering plural members using an adhesive”. 
     In the method of filling a liquid sample, in the moving the liquid sample from the first well to the second wells includes, when the biochip is rotated, the biochip may be arranged such that the first surface is opposed to the rotation axis. The term “opposed” is a concept including not only a case in which the first surface is opposed to the rotation axis in parallel to each other but also a case in which, for example, an angle θ 1  of an acute angle among angles formed by the first surface and the rotation axis is in a range of 0&lt;θ 1 &lt;90. 
     In the moving the liquid sample from the first well to the second wells, when the biochip is rotated, the biochip may be arranged such that a distance from the first surface to the rotation axis in the vertical direction with respect to the rotation axis is shorter than a distance from a second surface opposed to the first surface to the rotation axis in the vertical direction with respect to the rotation axis. The term “opposed” is a concept including not only a case in which the first surface is opposed to the second surface in parallel to each other but also a case in which, for example, an angle θ 2  of an acute angle among angles formed by the first surface and the second surface is in a range of 0&lt;θ 2 &lt;90. 
     In the method of filling a liquid sample, the cover may have a surface on which an adhesive is arranged. In the adhering the cover and the substrate on the loop-shaped area, the loop-shaped area may be pressed to bond the substrate and the cover on the loop-shaped area. 
     In the method of filling a liquid sample, the substrate and the cover may have a heat-melting characteristic. In the adhering the cover and the substrate on the loop-shaped area, ultrasound may be irradiated on the loop-shaped area to weld the substrate and the cover on the loop-shaped area. 
     In the method of filling a liquid sample, ends of the second wells may respectively include projections. In the sealing the first well and the second wells, the projections of the biochip and the cover may be adhered. 
     In the method of filling a liquid sample, the cover may have an elastically deforming characteristic. 
     The method of filling a liquid sample includes: supplying the liquid sample to the first well of the biochip; adhering the cover and the substrate on the loop-shaped area surrounding the first well and the second wells in the contact surfaces of the cover and the substrate in the state in which the cover is arranged on the substrate to cover the first well and the second wells; moving, using centrifugal force, the liquid sample from the first well to the second wells through the space formed between the cover and the substrate in the area further on the inner side than the loop-shaped area by rotating the biochip around the rotation axis in the state in which the biochip is arranged such that the distance from any one of the second wells to the rotation axis is longer than the distance from the first well to the rotation axis; and sealing the first well and the second wells by adhering the cover and the substrate. Therefore, it is possible to fill the liquid sample in the second wells in a simple and easy way. Further, it is possible to precisely and accurately dispense the liquid sample at low cost while preventing mixing of foreign matters. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The invention will be described with reference to the accompanying drawings, wherein like numbers reference like elements. 
         FIG. 1  is a diagram for explaining a step of a method of filling a liquid sample according to a first embodiment of the invention (an upper diagram is a plan view and a lower diagram is a sectional view corresponding to the upper diagram; the same holds true in  FIGS. 2 ,  5 ,  7 , and  9 ). 
         FIG. 2  is a diagram for explaining a step of the method of filling a liquid sample according to the first embodiment of the invention. 
         FIG. 3  is a diagram for explaining a step of the method of filling a liquid sample according to the first embodiment of the invention. 
         FIG. 4  is a perspective view schematically showing a pressing member shown in  FIG. 3 . 
         FIG. 5  is a sectional view for explaining a step of the method of filling a liquid sample according to the first embodiment of the invention. 
         FIG. 6  is a diagram for explaining a step of the method of filling a liquid sample according to the first embodiment of the invention. 
         FIG. 7  is a diagram for explaining a step of the method of filling a liquid sample according to the first embodiment of the invention. 
         FIG. 8  is a sectional view for explaining a step of the method of filling a liquid sample according to the first embodiment of the invention. 
         FIG. 9  is a diagram for explaining a step of the method of filling a liquid sample according to the first embodiment of the invention. 
         FIG. 10  is a diagram for explaining a step of a method of filling a liquid sample according to a second embodiment of the invention (an upper diagram is a plan view and a lower diagram is a sectional view corresponding to the upper diagram; the same holds true in  FIGS. 11 and 13 ). 
         FIG. 11  is a diagram for explaining a step of the method of filling a liquid sample according to the second embodiment of the invention. 
         FIG. 12  is a diagram for explaining a step of welding a biochip and a cover shown in  FIG. 10  using an ultrasonic welding device. 
         FIG. 13  is a diagram for explaining a step of the method of filling a liquid sample according to the second embodiment of the invention. 
         FIG. 14  is a diagram for explaining a step of welding the biochip and the cover shown in  FIG. 11  using the ultrasonic welding device. 
     
    
    
     DESCRIPTION OF EXEMPLARY EMBODIMENTS 
     Methods of filling a liquid sample according to embodiments of the invention are specifically explained below. 
     1. First Embodiment 
     Method of Filling a Liquid Sample 
       FIGS. 1 to 3  and  FIGS. 5 to 9  are diagrams for explaining steps of a method of filling a liquid sample according to a first embodiment of the invention (in  FIGS. 1 ,  2 ,  5 ,  7 , and  9 , upper diagrams are plan views and lower diagrams are sectional views corresponding to the upper diagrams). FIG.  4  is a perspective view schematically showing a pressing member shown in  FIG. 3 . 
     The method of filling a liquid sample according to the embodiment of the invention includes: supplying a liquid sample  20  to a first well  12  of a biochip  100  in which the first well  12  and plural second wells  14  separated from the first well  12  and including a reagent are provided on a first surface  10   a  of a substrate  10  ( FIGS. 1 and 2 ); adhering a cover  16  and the substrate  10  on a loop-shaped area  18  (hereinafter also simply referred to as “area”) surrounding the first well  12  and the second wells  14  in contact surfaces of the cover  16  and the substrate  10  in a state in which the cover  16  is arranged on the substrate  10  to cover the first well  12  and the second wells  14  ( FIGS. 3 to 5 ); moving, using centrifugal force, the liquid sample  20  from the first well  12  to the second wells  14  through a space  17  formed between the cover  16  and the substrate  10  in an area further on an inner side than the loop-shaped area  18  by rotating the biochip  100  around a rotation axis A in a state in which the biochip  100  is arranged such that a distance from any one of the second wells  14  to the rotation axis A is longer than a distance from the first well  12  to the rotation axis A ( FIGS. 6 and 7 ); and sealing the first well  12  and the second wells  14  by adhering the cover  16  and the biochip  100  to ( FIGS. 8 and 9 ). In the explanation in this embodiment, the biochip  100  is used in order to apply a PCR (Polymerase Chain Reaction) to the liquid sample  20 . 
     1.1. Step of Supplying the Liquid Sample  20   
     As shown in  FIG. 1 , the biochip  100  used in the method of filling a liquid sample according to this embodiment includes, on the first surface  10   a  of the substrate  10 , the first well  12  and the plural second wells  14  separated from the first well  12  and including a reagent. As shown in  FIG. 1 , the first well  12  and the second wells  14  are recesses provided in the substrate  10 . These recesses do not pierce through the substrate  10 . In the substrate  10 , the first well  12  is provided independently from the plural second wells  14 . The first well  12  and the second wells  14  are not connected through a flow channel or the like. 
     1.1.1. Substrate 
     In this embodiment, the first surface  10   a  is a surface on which the first well  12  and the second wells  14  are provided in the substrate  10 . 
     The liquid sample  20  is stored in the first well  12  (see  FIG. 2 ). The reagent included in the second wells  14  is, for example, a reagent used for a test for the liquid sample  20 . The reagent included in the second wells  14  can be arranged on inner wall surfaces of the second wells  14 . After liquid including the reagent is injected into the second wells  14 , a solvent in the liquid is dried, whereby the reagent can be arranged on the inner wall surfaces of the second wells  14 . 
     The capacities of the first well  12  and the second wells  14  are respectively determined as appropriate according to conditions such as a test target and a test method. The capacity of the first well  12  is preferably larger than the total capacity of the plural second wells  14  because an amount of the liquid sample  20  sufficient for filling all the plural second wells  14  in a step explained later can be stored. 
     As shown in  FIG. 1 , the plural second wells  14  can be arranged to form plural columns and rows. The plural second wells  14  are provided independently from one another and are not connected to one another through a flow channel or the like. For example, the plural second wells  14  are recesses having the same capacity. 
     When the PCR is performed using the biochip  100 , for example, it is also possible that the first well  12  does not include a reagent and the second wells  14  include a reagent containing a primer for amplifying target DNA included in a sample. In this case, in the plural second wells  14  of the biochip  100 , primers for respectively amplifying different target DNAs are contained in the reagent and the PCR is performed after the reagent in the second wells  14  are dissolved into the liquid sample  20  in the second wells  14 , whereby amplification and analysis of two or more kinds of nucleic acids can be performed at a time using the biochip  100 . 
     A material of the substrate  10  is not specifically limited. However, the substrate  10  is preferably formed of a material that does not damage components included in the liquid sample  20 . The substrate  10  can be formed of, for example, an inorganic material (e.g., single-crystal silicon or pyrex (registered trademark) glass) or an organic material (e.g., resin such as polycarbonate). When the substrate  10  is formed of the inorganic material, the first well  12  and the second wells  14  can be formed in the substrate  10  by dry etching employing a photolithography method. When the substrate  10  is formed of resin, the first well  12  and the second wells  14  can be formed on the substrate  10  by, for example, die molding, injection molding, or hot embossing. In the explanation in this embodiment, the substrate  10  is formed of polycarbonate. 
     1.1.2. Cover  16   
     A material of the cover  16  is not specifically limited. However, the cover  16  is preferably formed of a material that does not damage the components included in the liquid sample  20 . The cover  16  preferably has an elastically deforming characteristic in order to surely generate the space  17  in the step of rotating the biochip  100  explained later (see  FIG. 6 ). Examples of such a cover  16  include resin and rubber. 
     When the biochip  100  is used for measurement of fluorescent intensity, at least the cover  16  is preferably formed of a transparent and low-autofluorescent material. Both of the substrate  10  and the cover  16  are preferably formed of a transparent and low-autofluorescent material. When the biochip  100  is used for the PCR, the substrate  10  and the cover  16  are preferably a material that can withstand heating in the PCR. Examples of such a material include transparent and low-autofluorescent resin (e.g., polycarbonate). 
     The cover  16  may have the surface  16   a  on which an adhesive is arranged. The cover  16  having the surface  16   a  on which the adhesive is arranged can be adhered to an object by strongly pressing the surface  16   a  on which the adhesive is arranged against the object (in this embodiment, the first surface  10   a  of the substrate  10 ). Examples of such a cover  16  include LightCycler 480 Sealing Foil, 04 729 757 001, manufactured by Roche Diagnostics K.K., polyolefin micro plate sealing tape, 9793, manufactured by Sumitomo 3M Limited, and amplification tape 96, 232702, manufactured by Nalge Nunc International Corporation. The surface  16   a  on which the adhesive arranged of the cover  16  may be porous because the surface  16   a  does not show adhesiveness in a non-pressed state and can show adhesiveness when pressed. Alternatively, the adhesive arranged on the surface  16   a  of the cover  16  may be, for example, an adhesive that shows adhesiveness according to application of energy (e.g., an electron beam). 
     1.1.3. Liquid Sample  20   
     As shown in  FIG. 2 , the liquid sample  20  is supplied to the first well  12 . For example, the liquid sample  20  can be stored in the first well  12  manually (using a pipette) or mechanically. When the biochip  100  is used for, for example, the PCR, the liquid sample  20  includes a sample that may contain target DNA, a primer for amplifying the target DNA, a fluorescent reagent (e.g., SYBR GREEN (trademark)) for measuring an amount of an amplified product, and a PCR master mix respectively by proper concentrations. 
     An amount of the liquid sample  20  is appropriately determined according to the capacities of the first well  12  and the second wells  14 . The amount of the liquid sample  20  is preferably the same as the total capacity of the plural second wells  14  or larger than the total capacity. The amount of the liquid sample  20  is preferably larger than the total capacity of the plural second wells  14  because the liquid sample  20  can be more surely filled by the plural second wells  14 . 
     The liquid sample  20  is prepared from a sample. When the liquid sample  20  is a target of the PCR, examples of target DNA as a measurement target include DNA extracted from a sample such as blood, urine, saliva, or cerebrospinal liquid or cDNA reverse-transcribed from RNA extracted from the sample. 
     1.2. Step of Adhering the Cover  16  and the Substrate  10  on the Loop-Shaped Area  18   
     Subsequently, as shown in  FIG. 3 , the cover  16  is arranged on the biochip  100  to cover the first well  12  and the second wells  14 . In this state, with the substrate  10  and the cover  16  set in contact with each other, the loop-shaped area  18  (an area indicated by hatching in  FIG. 5 ) surrounding the first well  12  and the second wells  14  in the biochip  100  is pressed to adhere (bond) the substrate  10  and the cover  16  in the area  18 . 
     As shown in  FIG. 5 , the area  18  surrounds the first well  12  and the second wells  14  in a loop shape. The area  18  is formed by arranging the pressing member  40  on the cover  16  and pressing the cover  16  in an arrow direction in  FIG. 3  to adhere the substrate  10  and the cover  16 . 
     For example, as shown in  FIG. 4 , the pressing member  40  has a hollow section  42  and a loop-shaped end face  44  located at an inlet of the hollow section  42 . In a state in which the end face  44  of the pressing member  40  is in contact with the cover  16 , the pressing member  40  is pressed against the biochip  100 . The cover  16  is pressed in the arrow direction in  FIG. 3  while being in contact with the end face  44 , whereby the cover  16  is bonded to the substrate  10  and the loop-shaped area  18  is formed. 
     Therefore, in this step, although the substrate  10  and the cover  16  are bonded on the area  18 , the substrate  10  and the cover  16  are simply in contact with each other and are not bonded in areas on the inner side and the outer side of the area  18 . Specifically, in an area on the inner side than the area  18 , the substrate  10  and the cover  16  are not bonded and the cover  16  is simply in contact on the substrate  10 . Therefore, the liquid sample  20  enters between the substrate  10  and the cover  16  with centrifugal force, whereby a space is formed in the area further on the inner side than the area  18 . 
     1.3. Step of Moving the Liquid Sample  20  from the First Well  12  to the Second Wells  14   
     Subsequently, as shown in  FIG. 6 , in a state in which the biochip  100  is arranged such that a distance from any one of the second wells  14  to the rotation axis A is longer than a distance from the first well  12  to the rotation axis A, the biochip  100  is rotated around the rotation axis A, whereby, as shown in  FIG. 7 , the liquid sample  20  is moved using centrifugal force from the first well  12  to the second wells  14  through the space  17  formed between the cover  16  and the substrate  10  in the area further on the inner side than the loop-shaped area  18 . Consequently, the liquid sample  20  is filled in the second wells  14 . The distance from the first well  12  (the second wells  14 ) to the rotation axis A means, as shown in  FIG. 6 , a distance from an end d 1  (d 2 ) of the first well  12  (any one of the second wells  14 ) to the rotation axis A in a rotating state of the biochip  100 . As a device for rotating the biochip  100  around the rotation axis A, for example, a commercially available centrifuge may be used. 
     In the area further on the inner side than the area  18 , since the substrate  10  and the cover  16  are simply in contact with each other, when the biochip  100  is rotated as explained above, the centrifugal force is applied to the liquid sample  20  in a direction away from the rotation axis A on a plane perpendicular to the rotation axis A. Therefore, as shown in  FIG. 7 , the liquid sample  20  moves from the first well  12  to the second wells  14  through the space  17 . On the other hand, since the substrate  10  and the cover  16  are adhered in the area  18 , the liquid sample  20  does not leak to the area further on the outer side than the area  18  and remains further on the inner side than the area  18 . 
     More specifically, the cover  16  is elastically deformed by the liquid pressure of the liquid sample  20  and the centrifugal force applied to the cover  16  and distortion occurs in the cover  16 . As a result, the space  17  is formed between the cover  16  and the substrate  10 . The liquid sample  20  moves from the first well  12  to the second wells  14  through the space  17 . 
     When the biochip  100  is rotated, as shown in FIG.  6 , the biochip  100  is arranged such that the first surface  10   a  of the biochip  100  is opposed to the rotation axis A. More specifically, the biochip  100  may be arranged such that a distance in the vertical direction with respect to the rotation axis A from the first surface  10   a  to the rotation axis A is shorter than a distance in the vertical direction with respect to the rotation axis A from a second surface  10   b  opposed to the first surface  10   a  to the rotation axis A. 
     1.4. Step of Sealing the First Well  12  and the Second Wells  14   
     Subsequently, the cover  16  is adhered to the substrate  10  to seal the first well  12  and the second wells  14 . Consequently, the contact surfaces of the substrate  10  and the cover  16  are entirely bonded (see  FIG. 9 ). 
     Examples of a method of sealing the first well  12  and the second wells  14  include, as shown in  FIG. 8 , a method of pressing a roller  30  against the cover  16  while rotating the roller  30  in an arrow direction (a direction from the second wells  14  to the first well  12 ) on the cover  16 . With this method, the liquid sample  20  present in the space  17  between the substrate  10  and the cover  16  moves to the first well  12  and the contact surfaces of the cover  16  and the substrate  10  are bonded. As a result, the first well  12  and the second wells  14  are sealed by the cover  16 . The same pressing operation may be performed using a blade (not shown) instead of the roller  30 . 
     1.5. Application of the Biochip  100   
     With the method of filling a liquid sample according to this embodiment, various kinds of tests can be applied to the biochip  100  in which the liquid sample  20  is filled in the second wells  14 . When the PCR is performed using the biochip  100 , the PCR can be performed by setting the biochip  100  in which the liquid sample  20  is filled in the second wells  14  in a thermal cycler (not shown) including a flat heat block (not shown). 
     Since the second wells  14  of the biochip  100  are sealed by the cover  16 , evaporation of the liquid sample  20  in temperature cycle processing of the PCR is prevented. Since the cover  16  is formed of a transparent and low-autofluorescent material, quantitative determination of the target DNA (realtime-PCR) may be performed by measuring fluorescent luminance simultaneously with amplification. Analysis of various nucleic acids (DNA and RNA) employing the principle of the PCR including variation of genes such as SNP and methylation of DNA can be performed using the biochip  100 . 
     1.6. Characteristics 
     The method of filling a liquid sample according to this embodiment includes: supplying the liquid sample  20  to the first well  12  and plural second wells  14  separated from the first well  12  and including a reagent are provided on the first surface  10   a  of the substrate  10 ; adhering a cover  16  and the substrate  10  on the loop-shaped area  18  surrounding the first well  12  and the second wells  14  in the contact surfaces of the cover  16  and the substrate  10  in the state in which the cover  16  is arranged on the first surface  10   a  to cover the first well  12  and the second wells  14 ; moving, using centrifugal force, the liquid sample  20  from the first well  12  to the second wells  14  through the space  17  formed between the cover  16  and the substrate  10  in the area further on the inner side than the loop-shaped area  18  by rotating the biochip  100  around the rotation axis A in the state in which the biochip  100  is arranged such that the distance from any one of the second wells  14  to the rotation axis A is longer than the distance from the first well  12  to the rotation axis A; and sealing the first well  12  and the second wells  14  by adhering the cover  16  and the biochip  100 . Therefore, with the method of filling a liquid sample according to this embodiment, the liquid sample  20  supplied to the first well  12  can be filled in the second wells  14  in a simple and easy way of a centrifugal action. In filling the liquid sample  20  in the second wells  14 , since the cover  16  and the substrate  10  are adhered to each other on the loop-shaped area  18  surrounding the first well  12  and the second wells  14 , foreign matters are not mixed externally. Since the liquid sample  20  is moved from the first well  12  to the second wells  14  through the space  17  between the cover  16  and the substrate  10 , it is unnecessary to manufacture a flow channel for connecting the first well  12  and the second wells  14  in the substrate  10 . Therefore, it is possible to precisely and surely dispense the liquid sample  20  at low cost in a simple and easy way. 
     With the method of filling a liquid sample according to this embodiment, for example, it is possible to perform dispensing of a very small amount of the liquid sample, which is difficult in manually dispensing the liquid sample using a pipette. 
     With the method of filling a liquid sample according to this embodiment, the first well  12  and the plural second wells  14  of the biochip  100  are sealed in a state in which each of the plural wells  14  is separated from the first well  12 . Therefore, it is possible to prevent backflow of the liquid sample  20  from the second wells  14  to the first well  12 . This enables to perform accurate measurement of the liquid sample  20 . 
     With the method of filling a liquid sample according to this embodiment, it is possible to substantially reduce steps for preparation of a reagent and dispensing of the liquid sample. Since it is unnecessary to use expensive equipment such as an automatic dispensing device, it is possible to dispense the liquid sample at low cost. 
     When both of the substrate  10  and the cover  16  are formed of a transparent and low-autofluorescent material, with the method of filling a liquid sample according to this embodiment, it is possible to perform fluorescent measurement using the biochip  100  in which the liquid sample  20  is filled in the second wells  14 . This enables to perform simple and easy measurement. 
     In the step of moving the liquid sample  20  from the first well  12  to the second wells  14 , when the biochip  100  is rotated, as shown in  FIG. 6 , the biochip  100  may be arranged such that the first surface  10   a  is opposed to the rotation axis A and the distance in the vertical direction with respect to the rotation axis A from any one of the second wells  14  to the rotation axis A is longer than the distance in the vertical direction with respect to the rotation axis A from the first well  12  to the rotation axis A. 
     An adhesive may be arranged on the cover  16 . In the step of adhering the cover  16  and the substrate  10  on the loop-shaped area  18 , the loop-shaped area  18  may be pressed to bond the substrate  10  and the cover  16  on the loop-shaped area  18 . With this method, since the substrate  10  and the cover  16  are bonded with the adhesive arranged on the cover  16  by pressing the loop-shaped area  18 , the substrate  10  and the cover  16  can be adhered simply and easily and at low cost. Since the loop-shaped area  18  is simply pressed in the step of adhering the cover  16  and the substrate  10 , heat is not generated, a rise in the temperature of the biochip  100  can be suppressed, and damage to the liquid sample  20  can be reduced. 
     The cover  16  preferably has an elastically deforming characteristic. With this method, the cover  16  is elastically deformed by the liquid pressure of the liquid sample  20  and the centrifugal force applied to the cover  16  and distortion occurs in the cover  16 . As a result, the space  17  can be easily formed between the cover  16  and the substrate  10 . The liquid sample  20  can move from the first well  12  to the second wells  14  through the space  17 . 
     In the explanation in this embodiment, the biochip  100  is used for the PCR. However, the biochip  100  obtained by the method of filling a liquid sample according to this embodiment may be used for, for example, tests of viruses, bacteria, protein, low-molecular and high-molecular compounds, cells, particles, colloid, allergy substances such as pollens, poison, hazardous substances, and environmental pollution substances. In the explanation in this embodiment, the second wells  14  of the biochip  100  include the reagent. However, depending on a test content, the second wells  14  may not have to include the reagent. 
     2. Second Embodiment 
       FIGS. 10 ,  11 , and  13  are diagrams for explaining steps of a method of filling a liquid sample according to a second embodiment of the invention (in  FIGS. 10 ,  11 , and  13 , upper diagrams are plan views and lower diagrams are sectional views corresponding to the upper diagrams).  FIG. 12  is a diagram for explaining a step of welding the biochip  200  and a cover  26  shown in  FIG. 10  using an ultrasonic welding device  300 .  FIG. 14  is a diagram for explaining a step of adhering (welding) the biochip  200  and the cover  26  shown in  FIG. 11  using the ultrasonic welding device  300 . 
     In the method of filling a liquid sample according to this embodiment, the biochip  200  shown in  FIG. 10  is used. The biochip  200  is different from the biochip  100  in the first embodiment not including projections  11  in that ends  24   a  of plural second wells  24  are respectively formed by the projections  11 . The biochip  200  may be used in the method of filling a liquid sample according to the first embodiment. The biochip  100  used in the method of filling a liquid sample according to the first embodiment may be used in the method of filling a liquid sample according to the second embodiment. A first well  22  and the second wells  24  of the biochip  200  have configurations and functions same as those of the first well  12  and the second wells  14  of the biochip  100  in the first embodiment. 
     In the method of filling a liquid sample according to this embodiment, components same as those used in the method of filling a liquid sample according to the first embodiment are denoted by the same reference numerals and sigs and detailed explanation of the components is omitted. Among components used in the method of filling a liquid sample according to this embodiment, the components denoted by the reference numerals and signs same as those in the method of filling a liquid sample according to the first embodiment have the same configurations and functions. 
     The method of filling a liquid sample according to this embodiment is different from the method of filling a liquid sample according to the first embodiment for adhering the substrate  10  and the cover  16  by welding by ultrasound irradiation. Therefore, in the method of filling a liquid sample according to this embodiment, explanation of steps common to those of the method of filling a liquid sample according to the first embodiment is omitted. Steps different from those of the method of filling a liquid sample according to the first embodiment are mainly explained. 
     Like the cover  16  in the first embodiment, the cover is preferably formed of a transparent and low-autofluorescent material. In the biochip  200 , the substrate  110  and the cover  26  have a heat-melting characteristic. The substrate  110  and the cover  26  are preferably formed of the same material because the substrate  110  and the cover  26  can be surely welded. When the biochip  200  is used for the PCR, the substrate  110  and the cover  26  are preferably formed of a material that can withstand heating in the PCR. Examples of such a material include transparent and low-autofluorescent resin (e.g., polycarbonate). 
     First, the liquid sample  20  is supplied to the first well  22  by a method same as the method of filling a liquid sample according to the first embodiment (see 1.1. above). 
     Subsequently, as shown in  FIG. 10 , in a state in which the cover  26  is arranged on the substrate  110  to cover the first well  22  and the second wells  24 , as shown in  FIG. 11 , ultrasound is irradiated on a loop-shaped area (hereinafter simply also referred to as “area”)  28  surrounding the first well  22  and the second wells  24  in contact surfaces of the cover  26  and the substrate  110  to weld the substrate  110  and the cover  26  on the area  28 . Consequently, the substrate  110  and the cover  26  are adhered in the area  28 , while the substrate  110  and the cover  26  are simply in contact with each other in areas further on the inner side and the outer side than the area  28 . In other words, in the area further on the inner side than the area  28 , there is a space (not shown) in which the liquid sample  20  can move between the substrate  110  and the cover  26 . 
     For the irradiation of the ultrasound, for example, the ultrasonic welding device  300  is used. The ultrasonic welding device  300  converts electric energy into mechanical oscillation energy (ultrasound) with an ultrasonic oscillator  304  and irradiates the ultrasound from a horn  302 . The irradiated ultrasound is, for example, 20 kHz. 
     The ultrasonic welding device  300  includes, as shown in  FIG. 12 , the ultrasonic oscillator  304  and the horn  302  attached to the ultrasonic oscillator  304 . As shown in  FIG. 12 , the horn  302  has a hollow section  305 . The horn  302  has shape same as that of the pressing member  40  shown in  FIG. 4 . Specifically, the horn  302  has the hollow section  305  and an end face  306 . The hollow section  305  and the end face  306  respectively have structures same as those of the hollow section  42  and the end face  44  of the pressing member  40  shown in  FIG. 4 . 
     In a state in which the end face  306  of the horn  302  is pressed against the cover  26  on the biochip  200 , the horn  302  is pressed against the biochip  200  in an arrow direction in  FIG. 12  and ultrasound is intensively emitted from the end face  306 . As a result, the ultrasound is intensively irradiated on the area  28  (see  FIG. 11 ) in contact with the end face  306  and frictional heat is generated. The substrate  110  and the cover  26  are melted and adhered (welded) on the area  28 . 
     Subsequently, the liquid sample  20  is moved using centrifugal force from the first well  22  to the second wells  24  through the space (not shown) formed between the cover  26  and the substrate  110  in the area further on the inner side than the loop-shaped area  28  by a method same as the method of filling a liquid sample according to the first embodiment (see 1.3. above). 
     Subsequently, the ultrasound is irradiated on the entire cover  26 , whereby, as shown in  FIG. 13 , the entire contact surfaces of the substrate  110  and the cover  26  are adhered (welded). Consequently, the first well  22  and the second wells  24  are sealed. 
     For the irradiation of the ultrasound, for example, the ultrasonic welding device  300  shown in  FIG. 14  can be used. The ultrasonic welding device  300  includes, as shown in  FIG. 14 , the ultrasonic oscillator  304  and a horn  303  attached to the ultrasonic oscillator  304 . The horn  303  has a configuration same as that of the horn  302  shown in  FIG. 12  except that the hollow section  305  is not provided on the inside. 
     As shown in  FIG. 14 , the ultrasound is irradiated on the entire cover  26  by the ultrasonic welding device  300  from above the cover  26  laminated on the biochip  200 . In a state in which an end face  307  of the horn  303  is pressed against the cover  26 , the horn  303  is pressed against the biochip  200  in an arrow direction in  FIG. 14 . The ultrasound is irradiated on contact surfaces of the end face  307  of the horn  303  and the cover  26 . Consequently, the contact surfaces of the substrate  110  and the cover  26  are adhered (welded). As a result, the first well  22  and the second wells  24  are sealed. 
     The method of filling a liquid sample according to this embodiment has actions and effects same as those of the method of filling a liquid sample according to the first embodiment. With the method of filling a liquid sample according to this embodiment, since the substrate  110  and the cover  26  are adhered (welded) by the ultrasound irradiation, application of heat on the liquid sample  20  can be suppressed. Therefore, damage to the liquid sample  20  is small. This enables to maintain activity of the reagent included in the second wells  24 . Since bonding power of the substrate  110  and the cover  26  is strong, it is possible to surely prevent liquid leakage from the second wells  24 . 
     In the biochip  200  used in the method of filling a liquid sample according to this embodiment, the ends  24   a  of the second wells  24  are respectively formed of the projections  11 . Therefore, when the projections  11  and the covers  26  are welded by the ultrasound irradiation, the ultrasound tends to be concentrated on contact surfaces of the projections  11  and the cover  26 . This makes it possible to more surely weld the projections  11  and the cover  26 . Since the welding by the ultrasound irradiation can suppress a rise in the temperature of the biochip  200 , damage to the liquid sample  20  is small. 
     The embodiments of the invention have been explained. The invention includes configurations substantially the same as the configurations explained in the embodiments (e.g., configurations having the same function, method, and results or configurations having the same object and results). The invention includes configurations in which unessential parts of the configurations explained in the embodiments are replaced. The invention includes configurations that realize actions and effects same as the configurations explained in the embodiments or can attain an object same as that of the embodiments. The invention includes configurations in which a technique in the past is added to the configurations explained in the embodiments.