JP2006126011A - Microchip for specimen sample - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microchip for specimen samples which can allow a liquid-like inspection sample, such as a PCR reaction solution and blood, to flow into a plurality of chambers accurately by using a conventional rotary drive, and dispenses with a conventional, large and expensive spotter apparatus. <P>SOLUTION: A channel pattern comprises a circumferential or arc-like channel Cr that continues from an input port S1 for injecting specimen samples, a channel Ce for dispensing formed wider than the channel Cr on the circumferential or arc-like channel Cr, and chambers T1, T2 for detection that become parts for inspecting the specimen samples continuously via the channel Ce for dispensing and channels p1, p2 for connection that are narrower than the channel Ce for dispensing. The chambers T1, T2 for detection are formed radially via the channel p1 toward the outer periphery from the channel Ce for dispensing, and are formed wider than the channel p1 toward the outer periphery. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a microchip for a specimen sample for quantitatively and simply arranging and examining a small amount of a reaction solution and a specimen sample such as a chemical reaction / biochemical reaction and a blood test.

For example, a specimen sample microchip used for detecting and diagnosing a target gene (DNA) or the like from a very small amount of sample has a microscopic cross-sectional area channel (in a specific shape corresponding to the inspection purpose). Channel pattern), for example, a sample is fed into the channel pattern from a flow-shaped input port, and a predetermined medical evaluation is performed by a gene amplification reaction or the like, or a sample sample is filled in the channel pattern. This is used when a predetermined evaluation is performed by the reaction of the specimen sample that occurs when a voltage is applied. The microchip for specimen sample forms a channel pattern by hot embossing or injection molding using a fine mold on the polymer substrate, and the polymer substrate on which the channel pattern is formed is coated with a sheet-like or plate-like polymer. There are a structure in which a channel pattern is enclosed inside by attaching a substrate, a structure in which a polymer sheet on which the channel pattern is formed is sandwiched between a pair of polymer-coated substrates, and the like.
JP-A-8-62225 JP 2002-340911 A Special table 2003-185627 gazette JP 2003-166975 A

  Patent Document 1 discloses a sample sample microchip (test unit) having a sample chamber, a waste chamber, and the like for receiving a sample, and having a predetermined flow path, and a direction from the sample chamber to the waste chamber. A method for performing an assay by rotating about an axis so that a centrifugal force is applied to a specimen sample toward the object and a device for applying the centrifugal force are disclosed (see FIGS. 1 and 2). Patent Document 2 discloses a microchip for a specimen sample having first to third gaps such as putting a specimen inside a laminated body in which a plurality of flexible sheets are laminated. There are disclosed a rotation driving means for applying a centrifugal force to the specimen of the sample microchip, a moving means for moving the centrifuged specimen, and the like. Patent Document 3 discloses an electrophoresis sample sample microchip having a separation channel and an introduction channel, a multiple pipettor mechanism having a plurality of pipettor chips 70 attached to the tip thereof, and the like in the XY direction. An electrophoretic device that can be operated is disclosed (see FIGS. 2 and 4). In Patent Document 4, in a rectangular chip, channel patterns serving as a plurality of flow paths are formed from one side to the other side facing the chip.

  However, the sample sample microchip has a predetermined channel pattern formed as a plurality of flow paths. However, in the rotating apparatus as described above, the sample sample microchip is accurately arranged at the mounting position of the centrifuge. If the position is slightly deviated, the specimen sample cannot be poured up to a predetermined position (such as a waste chamber), and there is a problem that bias and sometimes back flow occur. In particular, when the width of the flow path is narrowed by increasing the number of channel patterns, it becomes more difficult to flow the specimen sample to a predetermined position (such as a waste chamber). The sample sample microchip (test unit) of Patent Document 1 needs to inject a sample sample such as a PCR reaction solution into a plurality of channel patterns one by one, as well as the size and flow path of the sample chamber and the waste chamber, etc. In addition, there is a case where the specimen sample cannot be flowed to a predetermined position (such as a waste chamber) even if it is rotated by a rotating device. That is, when the plurality of radial channel patterns are formed slightly shifted from the center, the specimen sample microchip is not accurately placed at the mounting position of the rotation drive device as described above, or the rotation drive There is a case where the specimen sample cannot be poured uniformly due to the shake of the shaft of the motor as a means. In addition, in order to test using a conventional specimen sample microchip, a large and expensive spotter device (the above-mentioned multiple pipetter mechanism) is required, and a mechanism for precisely moving the spotter device in the XY directions or the like is required. It was necessary, and the place and equipment became large, and there was a problem that it was expensive.

  Accordingly, an object of the present invention is to use a conventional rotary drive device to accurately flow a liquid sample sample such as a PCR reaction solution or blood into a plurality of chambers, and requires a conventional large and expensive spotter device. An object of the present invention is to provide a microchip for a specimen sample that will not be obtained.

  The sample sample microchip according to claim 1 of the present invention is a sample sample microchip in which a channel pattern serving as a flow path for a liquid sample sample is formed. The channel pattern has a sample sample at the center of the chip. A circumferential or arc-shaped flow path continuous from the input port to be injected, a dispensing channel formed on the circumferential or arc-shaped flow path and wider than the flow path, and the dispensing And a detection chamber that is a part for continuously inspecting the specimen sample through a connecting flow path narrower than the above, and the dispensing channel is configured to separate the specimen sample from the detection chamber. It is characterized by quantifying the amount dispensed when injecting separately.

  According to the present invention, if a specimen sample is injected from an input port and mounted on a rotating disk of a predetermined rotating device and driven to rotate, a dispensing channel for quantifying the dispensing amount of the channel pattern is circular. Since it is formed on a circumferential or arc-shaped flow path, it spreads in a circumferential flow path, and by this spread, dispensing is performed so as to separate and inject the specimen sample into the detection chamber. Become.

  The microchip for specimen sample according to claim 2 of the present invention is arranged so that the detection chamber is positioned on a straight line on the outer peripheral side of the dispensing channel, and the dispensing channel is a detection chamber. It is characterized by being almost the same size.

  According to the present invention, the specimen sample injected from the input port is arranged in the dispensing channel, and then, when given a predetermined rotation, shows a radial spread and is formed wider than the flow path toward the outer periphery. In the detection chamber, a certain amount of specimen sample can be uniformly dispensed into the detection chamber so as to be separated from the channel pattern.

  The specimen sample microchip according to claim 3 of the present invention is provided with two or more channel patterns of the arc-shaped flow path, each of which has an input port for injecting the sample sample and an arc-shaped flow path. A detection chamber which is a portion for inspecting a specimen sample continuously through a dispensing channel formed wider than the flow channel, and the dispensing channel and a connecting flow channel narrower than the dispensing channel. In which different specimen samples are injected into each input port, and at least two kinds of specimens are simultaneously inspected by dispensing that separates and injects the specimen sample into the detection chamber.

  According to the present invention, a liquid sample sample is injected into one channel pattern, and a different liquid sample sample is injected into the other channel pattern. The specimen sample is dispensed simultaneously and uniformly into the detection chamber.

  The sample sample microchip according to the present invention can be used for quantifying the dispensing amount of a channel pattern by injecting a sample sample from an input port and mounting the sample sample on a rotating plate of a predetermined rotating device and rotating the sample. Since the injection channel is formed on the circumference, it spreads in the circumferential flow path, and this spread allows the specimen sample to be uniformly sent to the outer detection chamber. It is not necessary to form a solution dripping portion for each channel pattern, and when a plurality of radial channel patterns are formed slightly shifted from the center, the specimen sample microchip is accurately arranged on the rotating disk. Even when they are slightly deviated from each other, or even when the shaft of the motor that is the rotation driving means is shaken, the arrangement is even. And it is realizable with a comparatively simple structure, and there exists an advantage which does not require the conventional large-sized expensive spotter apparatus.

  DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, exemplary embodiments to which the invention is applied will be described in detail with reference to the drawings.

(First embodiment)
The sample sample microchip of the present embodiment has a circular or square outer shape, and has a circumferential flow channel Cr and a dispensing channel Ce continuous from the input port S1 for injecting the sample sample at the center thereof. The channel pattern is formed so as to be continuous, and detection chambers T1 and T2 that are portions for inspecting the specimen sample are formed continuously on the outer periphery of the circumferential dispensing channel Ce (FIG. 1). ). The detection chambers T1 and T2 are formed radially through the flow channel p1 from the circumferential dispensing channel Ce to the outer periphery, and are formed wider than the flow channel p1 toward the outer periphery.

  The sample sample microchip 1F shown in FIG. 1 has 19 dispensing channels Ce, and the same number of first detection chambers T1 are formed on the outer peripheral side of each dispensing channel Ce. The same number of second detection chambers T2 are formed outside one detection chamber T1. The above-described channels are formed at equal intervals except for the interval between the channels at both ends, and an input port S1 and an air hole S2 are formed at both ends. The input port S1 is for injecting a liquid specimen sample into a circumferentially continuous channel pattern, and the other air hole S2 is for exhausting air pushed out when the specimen sample is injected.

  The circumferential channel Cr is formed with a narrow width, and the dispensing channel Ce formed on the channel Cr is formed in a substantially circular shape, and the width of the circumferential channel Cr It is formed with a wider width. The dispensing channel Ce is formed to feed a liquid specimen sample into the detection chambers T1 and T2 only in a predetermined amount, and quantitatively dispenses into the detection chamber T1 when a rotational drive is applied. The specimen sample poured into the narrow circumferential channel Cr is formed so as to be wide with respect to the narrow circumferential channel Cr. Is ignored, and only the specimen sample injected into the dispensing channel Ce is fed into the detection chambers T1 and T2 by a predetermined amount. That is, the amount of the solution in which the volume of the specimen sample in the dispensing channel Ce is sent to the detection chambers T1 and T2 is determined. A first flow path p1 between the dispensing channel Ce and the first detection chamber T1, and a second flow path between the first detection chamber T1 and the second detection chamber T2. p2 is formed, and the second flow path p2 is narrower than the first flow path p1. Further, the first detection chamber T1 is formed radially through the flow path p1 from the circumferential dispensing channel Ce to the outer periphery, and is formed wider than the flow path p1 toward the outer periphery. The second detection chamber T2 is formed wider than the flow path p1 toward the outer periphery and the flow path p2 toward the outer periphery that is continuous through the first detection chamber T1. The dispensing channel Ce plays a role of feeding only a predetermined amount to the detection chambers T1 and T2, and is therefore almost the same size as the detection chambers T1 and T2, and an extra amount is sent. I'm trying not to get it. As shown in FIG. 2, the detection chamber T1 may be a sample sample microchip 1G arranged so as to be positioned linearly on the outer peripheral side of the dispensing channel Ce.

The specimen sample microchips 1F and 1G of the present embodiment use a hot embossing device, and are processed into an inexpensive polymer substrate or metal or glass into a fine channel shape on the surface thereof. Can be produced by thermal transfer. In addition, a microchip for a specimen sample having a fine channel shape can be manufactured by PDMS (Polydimethylsiloxane) manufactured using a semiconductor lithography technique using a mask or the like. Furthermore, as another method, for example, it can be produced using RIE (Reactive ion etching), laser, NC processing machine or the like. Here, using Rapid Prototyping, a template made of a pressure film photoresist (SU-8) is created on a Si substrate, transferred to PDMS, and a channel pattern Cp or the like serving as a flow path is formed on one side. A polymer base material (upper chip) was prepared, and on the other hand, the Si substrate was used as the lower part and bonded by O ₂ plasma to prepare a microchip 1F for specimen sample. The prepared sample sample microchip 1F has a square shape with an outer shape of about 40 mm × about 40 mm and a thickness of about 3 mm. In addition, the circular shape has a diameter of about 40 mm and a thickness of about 3 mm. The channel depth was designed to be 120 μm, and the amount of sample necessary for the chip (19 channels) was about 8 μl. As the polymer substrate, in addition to PDMS, any of general-purpose resin materials such as acrylic, polypropylene, polyethylene, polystyrene, cycloolefin polymer, and polycarbonate can be used in the present invention.

  As a sample sample rotation driving device using the sample sample microchips 1F and 1G, a conventional rotation driving device, a spin coater, or the like that rotates in a rotating manner is sufficient. That is, it may have a rotating disk or the like on which the specimen sample microchips 1F and 1G are mounted on the shaft of a motor as a driving means. In addition, the rotating plate is formed with radial holes, and pins, fixing jigs, binders, etc. are inserted into the holes at the predetermined positions to adjust the position. In order to make it easier to mount the specimen sample microchips 1F and 1G, it is possible to form positioning marks, grooves, and the like according to the implementation. It is also possible to mount the specimen sample microchips 1F and 1G on the rotating disk using a double-sided tape or the like without using them.

  Next, the sample sample microchips 1F and 1G in the above-described embodiment are mounted on the rotating disk of the sample sample centrifuge, and the sample sample microchip 1F is slightly shifted from the position of each axis. Even in the case where they are arranged, it has been examined whether they can be separated and injected equally into the first detection chamber T1. Here, after injecting mineral oil (oil) into the circumferential flow path Cr and the dispensing channel Ce of the sample sample microchip 1F, it is mounted on a rotating disk of a rotary drive device such as a spin coater and 3000. The specimen sample microchip 1F was rotated at ˜9000 rpm, and oil was poured into the detection chamber T2 located on the outer periphery. Then, when the dye solution R is injected from the input port S1 instead of the specimen sample and the rotating disk is rotated, only the dye solution R corresponding to the volume of the dispensing channel Ce is sent to the detection chamber T2. It was possible to arrange them uniformly (FIG. 1 (b), FIG. 2 (b)). Note that the dye solution R stops at the position of the second detection chamber T2. Even when the sample sample microchip 1F is arranged slightly shifted from the center position of the turntable, the center position of the turntable is a circumferential channel pattern in which the flow path Cr and the dispensing channel Ce are continuous. If it is located inside, the dye solution can be arranged evenly. Furthermore, it was confirmed that the two liquids were replaced due to the difference in specific gravity between the oil and the dye solution R, and that the air could be arranged (dispensed) in the order of Ce for air, T1 for oil, and T2 for dye solution R. Further, it was found that even when the sample sample microchip was heated at a high temperature in this state, the oil could replace the lid, prevent the sample sample from evaporating, and be stable. Therefore, it can be applied to genetic testing using a gene amplification method (such as PCR).

  Next, an example in which a PCR reaction solution is used will be described.

Example 1
First, the PCR (Polymerase Chain Reaction) reaction is also called a polymerase chain reaction, and is a technique for replicating a gene 1 million times in a short time based on the sequence of a specific gene. In addition to its use in the biotechnological field, it has recently been used to diagnose and detect genetic diseases, viruses and pathogens from blood and cerebrospinal fluid. By analyzing this production process, it is possible to quantify the DNA content itself, and the PCR reaction is performed according to the following procedures (1) to (6). (1) Extraction of DNA (2) DNA in the form of a double helix is heated to form a single strand. (3) A gene composed of four bases binds to a specific base. (4) The base sequence is a gene sequence, prepared based on the target gene sequence, and a gene fragment (primer) paired therewith is added. (5) The primer binds to the target gene sequence. (6) The thermotolerant enzyme (Taq enzyme) regenerates the original double-stranded DNA from the binding site when the primer is bound to the single-stranded gene.

  In this process, two identical double helix DNAs are generated from one double helix DNA. This is heated again to unwind the double helix and then bind to the primer and regenerate DNA in a series of steps. As a result, two double helix DNAs become four double helix DNAs. By this repetition, the DNA produced increases exponentially. These series of processes are repeated in succession with accurate temperature control in an apparatus called a thermal cycler.

  Based on the above techniques, conventional reaction detection using PCR reaction is performed by placing several types of primers in a test chamber using a spotter device and then using the spotter device. After dropping the solution, the substrates are bonded together, and the temperature is accurately controlled and amplified by a heater to detect the reaction result. The chambers T1 and T2 are dispensed in nanoliters and picoliters by a spotter device, and a small amount is desired. When different primers are placed in the chambers, the PCR reaction solution is not contacted between the chambers. Must be (separate injection required). In the reaction between the primer and the PCR reaction solution, the temperature may be raised to a predetermined temperature in the PCR reaction.

  In Example 1, first, mineral oil (oil) is injected from the input port S1 into the circumferential channel Cr and the dispensing channel Ce of the sample sample microchip 1F, and then this is applied to a spin coater or the like. The specimen sample microchip 1F was rotated at 3000 to 9000 rpm by being mounted on a rotary disk of a rotary drive device, and oil was poured into the detection chamber T2 located on the outer periphery. Next, a PCR solution was injected from the input port S1, and the rotating disk was rotated to feed only the PCR solution corresponding to the volume of the dispensing channel Ce into the detection chamber T2. At this time, the mineral oil and the PCR solution are replaced due to the difference in specific gravity, and the oil is arranged in T1 and the PCR solution is arranged in T2. Furthermore, an oil film is uniformly formed inside the PCR solution, and the PCR reaction solution does not come into contact with air, and the lid is covered. Next, the specimen sample microchip 1 is removed from the rotating disk of the centrifuge and placed on a heater table (thermal cycler) that performs accurate temperature control, and temperature control is performed by PCR amplification. After completion of the PCR reaction or by real-time detection, the detection chambers T1 and T2 on the circumference were detected with fluorescence, and the reaction result was diagnosed.

In Example 1, the SRY gene (human sex determination gene) was detected from human genomic DNA. First, PCR solution (dNTP mixture: 5 μl, 10 × Buffer: 5 μl, 25 mM MgCl ₂ : 8 μl, Amplitaq Gold DNA polymerase: 1 μl, TaqMan primer: 5 μl, Forward primer: 5 μl, Reverse primer: 5 μl, H ₂ O: 10 μl, 2.5% PVP: 5 μl, Human Male DNA: 1 μl) were prepared, and placed in the sample sample microchip chamber by applying rotation. Next, the microchip was placed in a thermal cycler, and after heating for 95 minutes at a temperature condition of 95 ° C for 45 cycles of heating at 95 ° C for 15 seconds to 60 ° C for 60 seconds, DNA was amplified. Thereafter, the fluorescence intensity change (ex460-490, em510-550) of each chamber was measured using a fluorescence microscope. It was rising twice. From this result, it was found that a target gene can be detected using a sample sample microchip.

Example 2
Next, the sample sample microchip 1F was set as shown in FIG. 3 and rotated by the rotation driving device. In the sample sample microchip 1F, the width H1 of the first detection chamber T1, the width H2 of the second detection chamber T2, and the width H3 of the dispensing chamber Ce are the same width and are 2000 μm. The width p1h of the first channel p1 is set to 500 μm, the width p2h of the second channel p2 is set to 750 μm, and the width Crh of the circumferential channel Cr is set to 100 μm. The mineral oil O is disposed in the second detection chamber T2 and the second flow path p2 (FIG. 4A), and the dye R is injected from the input port S1 into the circular flow path Cr. (FIG. 4 (b)), and when driven to rotate, two liquids are replaced by a predetermined number of rotations due to the difference in specific gravity between the oil O and the dye solution R, and the dispensing chamber Ce has air (air) A. It was confirmed that oil O could be arranged (dispensed) in the first detection chamber T1 and dye R could be arranged (dispensed) in the second detection chamber T2 (FIG. 4C). Here, assuming that the width Crh of the circumferential channel Cr is about 100 μm and the width H3 of the dispensing chamber Ce is about 2000 μm, the specimen sample which is a PCR reaction solution in the dispensing chamber Ce is Ignoring the specimen sample poured into the narrow circumferential channel Cr, only the specimen sample injected into the dispensing channel Ce is fed a predetermined amount into the detection chamber T1. Become.

(Second Embodiment)
As shown in FIG. 5, in the microchip for specimen sample of the present embodiment, the flow path Cr at the center of the chip is formed as an arc-shaped flow path, and two of these are combined to form a circumferential shape. is doing. That is, two channel patterns of the arc-shaped channel Cr are provided, and each of them is formed on the input port S1 for injecting the specimen sample to each of them, and on the arc-shaped channel Cr and wider than the channel Cr. The dispensing channel Ce, the dispensing channel Ce, and detection chambers T1 and T2 that are continuous through the connecting flow paths p1 and p2 narrower than the dispensing channel Ce are provided. Then, different specimen samples are injected into each of the two sets of input ports S1 and S1, and the two specimens are simultaneously examined by separately injecting the specimen samples into the detection chambers T1 and T2. Also in the present embodiment, the outer shape is circular or square, and the two detection chambers T1 and T2 are arranged in the same manner as in the first embodiment. In the present embodiment, there are two channel patterns of the arc-shaped flow path Cr, but two or more channel patterns may be formed in the same manner.

It is a top view which shows the microchip for specimen samples of the 1st Embodiment of this invention. It is a top view which shows the other example of the said 1st Embodiment. It is a figure explaining an example of the magnitude | size of the microchip for specimen samples of this invention. It is a figure explaining the separation injection process of the sample sample of this invention. It is a top view which shows the microchip for specimen samples of the 2nd Embodiment of this invention.

Explanation of symbols

1F, 1G specimen sample microchip,
Cr circumferential or arc-shaped flow path,
Ce dispensing channel,
p1, p2 flow path toward the outer periphery, p1h, p2h flow path width,
R specimen sample (dye solution),
S1 input port,
S2 air hole,
T1, T2, detection chambers,
H1 width of the first detection chamber;
H2 width of the second detection chamber,
The width of the H3 dispensing chamber,

Claims (3)

In the sample sample microchip on which the channel pattern that becomes the flow path of the liquid sample sample is formed,
The channel pattern consists of a circumferential or arc-shaped flow path continuous from the input port for injecting the specimen sample into the center of the chip, and a width wider than the flow path on the circumferential or arc-shaped flow path. And a detection chamber that is a part for inspecting a specimen sample continuously through the dispensing channel and a flow path for connection narrower than the dispensing channel. A sample sample microchip characterized by quantifying a dispensing amount when a channel is separately injected into a detection chamber so as to separate the sample sample.   The said detection chamber is arrange | positioned so that it may be located in a straight line in the outer peripheral side of the said dispensing channel, This dispensing channel is a substantially the same magnitude | size as the detection chamber. The microchip for specimen samples as described.   Two or more channel patterns of the arc-shaped flow path are provided, each of which has an input port for injecting a specimen sample and a dispensing pipe formed on the arc-shaped flow path and wider than the flow path. A channel, a detection chamber that is a part that continuously inspects the sample through the dispensing channel and a narrower connecting flow path, and injects a different sample into each input port. 3. The specimen sample microchip according to claim 1, wherein at least two kinds of specimens are simultaneously examined by dispensing the specimen samples separately into the detection chamber.

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