Now, referring to
The motor driver 107 and the control substrate 108 are used for operations of the transfer stage 103, the valve 105 and pump 113. The power supply 106 supplies electricity to each of various components. The information access panel 109 is used for inputting measuring conditions and outputting measurement results.
The biological material inspection system according to the present invention can detect living body related materials such as DNA, RNA, protein, peptide or the like, while the following description deals with a case for detecting DNA.
First, an inspection chip is inserted from the chip introducing window 101. Beads with immobilized probes are being charged in the inspection chip, and further, sample containing fluorescently-labeled DNA, pre-hybridization solution, washing solution or the like are accommodated. Details of the structure of the inspection chip will be explained later. Next, the inspection chip is transferred to the reaction stage 104 by the transfer stage 103. In the reaction stage 104, the pre-hybridization solution is passed through the beads with probes immobilized thereon in the inspection chip, for initiating pre-hybridization.
Next, a sample solution containing DNA is passed through the beads with probes immobilized thereon to initiate hybridization. By hybridization, DNA fragments in the sample combine with DNA of the probe in a complementary strand fashion. Upon completion of hybridization, the beads are washed with more than one types of washing solutions to remove unreacted DNAs. The pump 113 and valve 105 are used for feeding of the sample solution and washing solution. Details of such solution feeding will be explained later.
Upon completion of the washing, the inspection chip is moved up to the optical stage 102 by the transfer stage 103. In the optical stage 102, a laser from the laser light source 110 is concentrated by a lens and is then irradiated to the probe. Since DNA in the sample captured by the probe is fluorescently-labeled, it emits fluorescence when a laser is irradiated. This fluorescence is wavelength selected by a filter and is detected by an optical detector. As the optical detector, CCD camera and photomultiplier are used. An image obtained by the optical detector is displayed on the information access panel 109.
Beads are arranged along with the flow path in the inspection chip. Probes immobilized on beads are different each other. Therefore, type of the probe is identified according to the position of the bead in the flow path. For the sake of detection of the position of the bead, bead itself may be fluorescently-labeled. APD (avalanche photodiode) which is a light receiving element is used for measurement of fluorescence from the bead. APD separates fluorescence from the bead and fluorescence from the DNA by their wavelength. CCD camera may be used in lieu of APD. CCD camera can detect position of a bead although it does not perform separation by wavelength. PMT (Photomultiplier) which is a light receiving element having sensitivity higher than that of APD may be used. Separation by wavelength is made possible with the use of dichroic mirror.
Referring to
Referring to
Beads are held in the reaction flow path 2 one-dimensionally, two-dimensionally, or three-dimensionally.
The reaction flow path 2 may be a flow path having circular form as represented by a capillary, preferably, may be a flow path composed of PDMS (Polydimethylsiloxane, (C2H6SiO)n), which is one sort of silicon resin, formed on a glass substrate. As advantages of using PDMS as material of the flow path, the following three points are mentioned. First, once a die has been produced, formation of a flow path is very simple and at the same time, less expensive. Second, different from the capillary, it is possible to form a flow path with diversified configurations. Namely, a flow path with sophisticated profiles and sections can be formed with ease. Third, optical characteristics are excellent. In other words, since self-fluorescence thereof is very low, error or noise involved in measuring fluorescence intensity of DNA becomes less significant. In the following explanation, it is assumed that the reaction flow path 2 is formed by PDMS. As for materials of the flow path, glass, hard resin and silicon can be used in addition to PDMS.
Referring to
The solution holding flow paths 3, 4 accommodate pre-hybridization solution, sample solution and washing solution. The inspection chip of the present embodiment is designed to carry out three washing steps as shown in
Solution detection units 3a, 4a are provided respectively at the inner ends of the solution holding flow paths 3, 4. An optical sensor (not shown) comprising a light emitting unit and a light receiving unit is provided at each of the solution detection units 3a, 4a. Whether or not a solution has passed through each of the solution detection units 3a, 4a is detected by the optical sensor. The solution detection units 3a, 4a are made of transparent material to allow for observation of solution in the flow path.
The air holding flow paths 3b, 4b, are provided for ensuring stabilization of solution feeding between the solution holding flow paths 3, 4 and the reaction flow path 2. Function of the air holding flow paths 3b, 4b will be explained later.
The transport ports 3c, 4c are used when transferring the solution. Solution feeding is performed by applying a high-pressure to either of transport ports 3c, 4c and the other is made open to the atmosphere. Since solution feeding is carried out by utilizing pressure difference, a high-pressure may be applied to one of transport ports 3c, 4c and a low-pressure may be connected to the other. When the inspection chip is mounted on the biological material inspection system shown in
According to the present embodiment, a plurality of different solutions are held at the same time in each of solution holding flow paths 3, 4. An air gap is provided between solutions. By providing an air gap, mixing of adjoining two solutions is prevented. Each solution is held being sandwiched by air gaps at both sides, i.e., fore and aft, moves from the solution holding flow paths 3, 4 to the air holding flow paths 3b, 4b in its entirety, and further moves in the reaction flow path 2. When the air gap moves from the solution holding flow path 3 to the air holding flow path 3b, length of the air gap becomes longer since its inside diameter becomes smaller. When the air gap moves from the reaction flow path 2 to the solution holding flow path 4 via the air holding flow path 4b, length of the air gap becomes shorter.
In the present embodiment, dimensions and diameters of the air holding flow paths 3b, 4b are set so that cubic content of each of air holding flow paths 3b, 4b may become greater than cubic volume of one air gap. Therefore, there is no opportunity that one air gap occupies the air holding flow path 3b, reaction flow path 2, and air holding flow path 4b at one time. Further, dimensions and diameters of the air holding flow paths 3b, 4b are set so that pressure losses of the air holding flow paths 3b, 4b may be substantially identical with pressure loss of the reaction flow path 2.
In general, pressure loss across the reaction flow path 2 is large since beads are charged therein. If pressure losses across the air holding flow paths 3b, 4b are small, a sudden change in pressure loss occurs between the air holding flow paths 3b, 4b and the reaction flow path 2. For example, when a solution enters from the air holding flow path 3b to the reaction flow path 2, an air gap moving together with the solution is compressed. Contrary, when a solution moves from the reaction flow path 2 to the air holding flow path 4b, an air gap moving together with the solution is swollen. Such compression and expansion hinder stable solution feeding. In the present embodiment, sharp change in pressure loss does not take place by the fact that pressure losses across the air holding flow paths 3b, 4b are set to be nearly same as that of across the reaction flow path 2. Thus, stable solution feeding can be realized.
Next, referring to
Although the user may charge the first solution holding flow path 3 with the solution as mentioned above, an inspection chip to which such solutions are charged in advance may be used.
Next, as shown in
A high-pressure is applied to a first transfer port 3c and a second transfer port 4c is made open to the atmosphere. As a result, as shown in
First, the pre-hybridization step is executed. The high-pressure is applied to the first transfer port 3c and the second transfer port 4c is made open to the atmosphere. As shown in
Next, the reaction step is executed. The high-pressure is applied to the first transfer port 3c and the second transfer port 4c is made open to the atmosphere. The sample solution 601 in the first solution holding flow path 3 passes through the reaction flow path 2. Thus, DNA of the sample solution and DNA of the probe are hybridized. The sample solution 601 passed through the reaction flow path 2 moves into the second solution holding flow path 4.
Next, as shown in
As shown in
Next, the washing step is executed. The high-pressure is applied to the second transfer port 4c and the first transfer port 3c is made open to the atmosphere. As shown in
In
When a solution moves, air gaps before and after the solution also move. According to the present embodiment, cubic contents of the air holding flow paths 3, 4 are greater than volume of the air gap. Therefore, there is no opportunity that one air gap occupies the air holding flow paths 3, 4 and reaction flow path 2 at one time.
Further, pressure losses of the air holding flow paths 3b, 4b are set to be nearly identical with pressure loss of the reaction flow path 2. According to the present embodiment, since no sudden change in pressure loss occurs, compression of air gap moving from the air holding flow paths 3b, 4b into the reaction flow path 2, and expansion of air gap moving from the reaction flow path 2 to the air holding flow paths 3b, 4b can be prevented. Therefore, solution feeding can be performed in stable fashion.
Further, by inserting an air gap between adjoining solutions, detection of solution feeding at the solution detection units 3a, 4a can be made with ease. For example, it is possible to detect solution feeding by utilizing a difference of optical transmissivity or reflectance between liquid such as sample solution and gas such as air gap.
According to the present embodiment, upon completion of the inspection step, the inspection chip is discarded without modification. In other words, waste solutions of sample solution, hybridization solution and washing solution are discarded being maintained in the inspection chip. Therefore, disposal of these waste solutions can be made safely and simply.
According to the present embodiment, by simply applying the high pressure to one of a pair of transfer ports and by opening the other to the atmosphere, all solutions in the inspection chip can be fed one after another. Accordingly, there is no need for a movement mechanism for moving the inspection chip, which allows for reduced size biological material inspection chip system.
According to the embodiment shown in
Now, explanation will be given referring to
During solution feeding, the valve 41 connects the pressure source 40 to the pipe 45. The valve 42 connects the pipe 45 to either of pipes 46L, 46R. The valve 43L connects two pipes 46L, 47L each other or connects the both to the atmosphere. The valve 43R connects two pipes 46R, 47R each other or connects the both to the atmosphere. The pipe 47L is connected to the first transport port 3c and the pipe 47R is connected to the second transport port 4c.
First, solution feeding in outward direction is performed. As shown in
In step S2, solution feeding is started. A pressure from the pressure source 40 is applied to the first transport port 3c via the pipes 45, 46L, 47L. As a result, as shown in
In step S3, a solution sensor determines whether or not all solutions have passed through the solution detection unit 3a, 4a. When all solutions have not passed through, it returns to step S2, and remaining solutions, i.e., pre-hybridization solution 602 and sample solution 601 are fed. After all solutions have passed through, it proceeds to step S4 and solution feeding is stopped. The pipes 46L, 47L are made open to the atmosphere by change-over of the valve 43L. As a result, the first transport port 3c is made open to the atmosphere, and solution feeding in outward direction is performed.
In step S5, it is determined whether or not a number of times of reciprocation designated is being set. When the number of times of reciprocation designated is not set, the processing is terminated. When the number of times of reciprocation designated is set, it returns to step S1.
In step S1, change-over of the valve is performed. The pipe 45 is connected to the pipe 46R by the valve 42, and the pipe 46R is connected to the pipe 47R by the valve 43R. Thus, the pressure source 40 is connected to the second transport port 4c. The pipes 46L, 47L are made open to the atmosphere by the valve 43L. As a result, the first transport port 3c is connected to the atmosphere. In step S2, solution feeding is started. A pressure from the pressure source 40 is applied to the second transport port 4c via the pipes 45, 46R, 47R. As a result, as shown in
In step S3, the solution sensor determines whether or not the sample solution has passed the solution detection unit 4a. When the sample solution has not passed through, it returns to step S2 and continues solution feeding. When the sample solution has passed through, it proceeds to step S4 and stops solution feeding. The pipes 46R, 47R are made open to the atmosphere by change-over of the valve 43R. As a result, solution feeding in inward direction is performed. When, in step S5, reciprocation is carried out as many as the number of times of reciprocation designated, the processing is terminated.
In the present embodiment, solution feeding is controlled while determination is made by the solution sensor whether or not the solution has passed the solution detection unit. Therefore, it is possible to carry out hybridization accurately without observing a solution in the inspection chip by human.
As mentioned above, according to the present invention, in the inspection chip using beads with immobilized probes, the solution detection unit is provided in the flow path, and fluid control is carried out by detecting passing of solutions such as sample, washing solution, or the like. Therefore, fluid control of solutions can be carried out accurately in the inspection chip, thereby improving stability of amount of sample reaction and amount of washing in the chip.
Although embodiments of the present invention are explained above, the present invention is not limited thereto, and it will readily be understood by those skilled in the art that various modifications may be made within the scope of the invention according to the claims.
Number | Date | Country | Kind |
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2006-111305 | Apr 2006 | JP | national |