JP2005345178A - Biochemical reaction cartridge - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a use method of a DNA chip, which is manufactured by an ink jet method, capable of obtaining an accurate detection result even if a DNA probe dot is adsorbed at a slightly shifted position in consideration of such a point that the DNA probe is shifted from a desired position according to circumstances. <P>SOLUTION: The shift quantity of the DNA probe is acquired at the time of manufacture of the DNA chip to be stored in the memory part on a DNA inspection cartridge. The data of the accurate position of the DNA probe can be acquired by performing image processing based on the data read from the memory part at the time of detection. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to individual information management of a biochemical reaction cartridge having a biochip manufactured by an ink jet method used in an automatic biochemical reaction inspection apparatus.

  When analyzing the base sequence of gene DNA, or simultaneously performing genetic diagnosis with high reliability for multiple items, it is necessary to select a DNA having the target base sequence using a plurality of types of probes. As a means for providing a plurality of types of probes used for this sorting operation, a DNA chip has attracted attention. In high-throughput screening and combinatorial chemistry for drugs, etc., a large number of target proteins and drug solutions (for example, 96 types, 384 types, 1536 types) can be arranged to perform orderly screening. Necessary. Developed a method for arranging many kinds of drugs for that purpose, automated screening technology in that state, a dedicated device, software for controlling a series of screening operations and statistically processing the results Has been.

  These parallel screening operations basically use a so-called probe array in which a large number of known probes are arranged for the substance to be evaluated, and are used for the probes under the same conditions. It detects the presence or absence of action or reaction. In general, what kind of probe action and reaction is used is determined in advance, and therefore, the probe types mounted on one probe array are, for example, a group of DNA probes having different base sequences, etc. It is a kind of substance when roughly classified. That is, a substance used for a group of probes is, for example, DNA, protein, synthesized chemical substance (drug), and the like. In many cases, a probe array consisting of a plurality of probe groups is often used. However, depending on the nature of the screening work, DNA having the same base sequence, protein having the same amino acid sequence, and the same chemistry may be used as the probe. It is possible to use a form in which a large number of substances are arranged in an array. These are mainly used for drug screening and the like.

  In a probe array consisting of a plurality of types of probes forming a group, specifically, a group of DNAs having different base sequences, a group of proteins having different amino acid sequences, or a group of different chemical substances are included in the group. In many cases, the seeds are arranged on a substrate or the like in an array according to a predetermined arrangement order. Among these, the DNA probe array is used for analyzing the base sequence of gene DNA and simultaneously performing highly reliable genetic diagnosis for many items.

  One of the problems in a probe array composed of a plurality of types of probes forming a group is to mount as many kinds of probes as possible, for example, DNA probes having many kinds of base sequences, on one substrate. In other words, how densely the probes can be arranged in an array.

As one method for fixing a plurality of types of probes in an array on a substrate, it is described in US Pat. No. 5,424,186, on a carrier using a photodegradable protective group and photolithography. An example is a method of preparing DNA probes having different base sequences in an array by sequential DNA extension reaction. When this technique is used, for example, it is possible to prepare a DNA probe array on which DNAs having 10,000 or more different sequences per 1 cm ² are mounted. In this method, when DNA is synthesized by a sequential extension reaction, a photolithography process is performed for each of the four types of bases (A, T, C, G) using a dedicated photomask. By selectively extending any of the bases, a plurality of types of DNA having a desired base sequence are synthesized on the substrate with a predetermined sequence. Therefore, as the DNA chain length increases, the cost required for preparation increases and a long time is required. In addition, since the efficiency of nucleotide synthesis at each extension step is not 100%, the proportion of DNA in which the designed base sequence is defective is not small. Furthermore, when using a photodegradable protecting group during synthesis, the synthesis efficiency is lower than when using a normal acid-decomposable protecting group. There is also a problem that the proportion of DNA is reduced.

  Further, since the product directly synthesized on the carrier is used as it is, it is of course impossible to remove DNA having a defective base sequence from the DNA according to the designed base sequence by purification fractionation. In addition, in the finally obtained array, there is a problem that the base sequence of the DNA synthesized on the substrate cannot be confirmed. This is because if there is almost no extension of a given base at a certain extension stage due to a mistake in the process, and it is a completely defective product, screening using this defective product probe array results in an erroneous result. This means that there is no way to prevent it. The inability to confirm this base sequence is the biggest and essential problem in this method.

As a method different from the above method, probe DNA is synthesized and purified in advance. In some cases, after confirming the base length, each DNA is supplied onto a substrate by a device such as a microdispenser,・ Methods for preparing arrays have also been proposed. PCT publication WO 95/35505 describes a method for supplying DNA onto a membrane using a capillary. When this method is applied, in principle, about 1000 DNA arrays can be prepared per 1 cm ² . Basically, a probe array is prepared by supplying a probe solution to a predetermined position on a substrate with one capillary-like dispensing device for each probe and repeating the operation. If you prepare a dedicated capillary for each probe, there is no problem, but if you try to do the same work using a small number of capillaries, in order to prevent cross-contamination, the capillary should be sufficient when replacing the probe type. Need to be cleaned. Moreover, it is necessary to control the supply position each time. Therefore, it cannot be said that the method is suitable for preparing an array in which many types of probes are arranged at high density. In addition, since the probe solution is supplied to the substrate by tapping the tip of the capillary on the substrate, reproducibility and reliability are not perfect.

  In addition, a micro dispenser device for supplying different drug solutions to individual wells, particularly for 96-well or 384-well microplates used for high-throughput screening of drugs, for example, Robbins It is commercially available from Scientific as HYDRATM. This is basically a two-dimensional arrangement of microsyringes with a minimum discharge amount of 100 nl. If this is applied to array formation, the density is limited by the minimum discharge amount, and there is a limit to increasing the density.

  As another technique, when performing solid-phase synthesis of DNA on a substrate, a method of supplying a solution of a substance necessary for synthesis onto the substrate by an ink jet method at each extension stage has been proposed. For example, in European Patent Publication No. EP 0 703 825B1, a nucleotide monomer and an activator used in solid phase synthesis of DNA are supplied from separate piezo jet nozzles, respectively. A method for solid-phase synthesis of multiple types of DNA having a base sequence is described. The supply (application) by the ink jet method is more reliable than the supply (application) of the solution using the capillary, such as the reproducibility of the supply amount, and the nozzle structure can be miniaturized. It has characteristics suitable for increasing the density of the probe array. However, this technique basically also applies a sequential extension reaction of DNA on a substrate, and is therefore the biggest problem in the technique described in US Pat. No. 5,424,186 described above. There still remain problems such as inability to confirm the base sequence of DNA synthesized on the substrate. Although the complexity of performing a photolithography process using a dedicated mask at each extension stage is eliminated, a predetermined probe is fixed to each point, which is an essential requirement for the probe array. , Including some problems. In addition, the above-mentioned EP 0,703,825B1 only describes a method of using a plurality of independently formed piezo jet nozzles, and when using this small number of nozzles, the aforementioned capillary Similar to the technique using, it is not necessarily suitable for preparing high-density probe arrays.

  Japanese Patent Laid-Open No. 11-187900 discloses a method of forming a spot including a probe on a solid phase by attaching a liquid including a probe as a droplet to a solid phase by a thermal liquid discharge unit.

  By the way, when detecting fluorescence-labeled sample DNA by hybridizing with a probe on a DNA chip, conventionally, the excitation light spot is scanned over the entire surface of the probe array, and the light emission information is photoelectrically read to obtain a digital image. The area of the fixed position of the probe is automatically or manually set, and the presence or amount of each probe is detected by integrating the image signal of the set area. However, it takes time to scan the entire surface of the probe array, and because the target DNA is adsorbed non-specifically on the DNA chip due to the presence of the solid-phase agent, the luminescence is also read and noise increases. Cause the S / N ratio to decrease.

  Therefore, a method of irradiating excitation light only to the position of the probe has been proposed. Japanese Patent Application Laid-Open No. 2000-131237 describes a method of relatively intermittently moving the DNA chip and the excitation light so that only the position of the probe is irradiated. Japanese Patent Laid-Open No. 2001-108684 describes a method using a plurality of multi-spot excitation lights having a spot diameter equal to or smaller than the cell size.

  In probe array preparation using the inkjet method, as many probes as possible are fixed within a certain range in order to improve detection efficiency for diagnosis, etc. from the viewpoint of increasing the density, and not to prepare a large amount of specimen. It is desirable to do. In addition, from the viewpoint of high reliability, a specific probe needs to be applied to a desired position. In order to prevent errors during diagnosis and the like, it is desirable that only a desired probe is fixed at a predetermined location on the probe carrier.

  On the other hand, in various sample analysis methods, we also propose disposable biochemical reaction cartridges that carry out necessary reactions internally for the purpose of reducing contamination by samples, improving reaction efficiency, miniaturizing equipment, and simplifying operations. Has been. For example, in Japanese Patent Application Laid-Open No. 11-509094, a plurality of chambers are arranged in a biochemical reaction cartridge including a DNA array, a solution is moved by differential pressure, and DNA in a sample is extracted or amplified inside the cartridge. Alternatively, a biochemical reaction cartridge that enables a reaction such as hybridization is disclosed.

  However, in the ink jet method, depending on the recovery state of the ink jet head nozzle or the like, the discharge direction of the DNA ink droplet may change and land at a position shifted from a desired position. If the DNA probe deviates from a predetermined position, the image information read at the time of detection becomes incorrect information at a position different from the position where the DNA probe is actually located, and a correct detection result cannot be obtained. Further, if the allowable range of the probe droplet landing position at the time of manufacturing the DNA chip is narrowed in order to avoid the problem at the time of detection, the yield is lowered and the cost is increased as a result.

  The present invention has been made in view of the above-described problems, and relates to a method of using a DNA chip that can obtain a correct detection result even when probe dots are adsorbed at slightly shifted positions.

  As a method for solving the above problems, the biochemical reaction cartridge of the first invention has the following configuration.

  It has a biochip for biochemical reaction in which a plurality of types of polymers are arranged at predetermined positions, and means for storing information on these polymers. Thereby, an amount of data that cannot be recorded on the biochip can be stored and used for data processing or the like.

  The biochemical reaction cartridge of the second invention has the following configuration.

  The polymer information is information that associates the type and position of the polymer. Thereby, even biochips in which different types of polymers are arranged can be used without being confused, and the position information can be given to the cartridge as correction data at the time of detection.

  The biochemical reaction cartridge of the third invention has the following configuration.

  The information of the polymer is information at the time of manufacturing the biochip. Thereby, accurate information immediately after manufacture can be stored.

  The biochemical reaction cartridge of the fourth invention has the following configuration.

  The position information of the polymer at the time of manufacture is acquired using a polymer position acquisition unit at the time of manufacturing the biochip. This eliminates the need to detect the polymer position on the automatic inspection device.

  The biochemical reaction automatic inspection device of the fifth invention has the following configuration.

  In the biochemical reaction automatic inspection apparatus using the biochemical reaction cartridge, the detection of the polymer uses the position information of the polymer to acquire data while correcting the detection position. Thereby, even when the polymer is not spotted at a predetermined position on the biochip during manufacturing, accurate detection can be performed.

  The biochemical reaction cartridge of the sixth invention has the following configuration.

  The polymer position acquisition means uses an optical detection method. Thereby, position information can be easily acquired in a short time.

  The biochemical reaction cartridge of the seventh invention has the following configuration.

  The polymer position acquisition means uses a fluorescence detection method using a fluorescently labeled polymer. As a result, the probe is instantly adsorbed to the solidifying agent after the droplet has landed, but the liquid may deviate from the landing position, and only the droplet position is measured in normal optical detection. It is possible to avoid the problem that there is a possibility of acquiring position information different from the probe position.

  The biochemical reaction cartridge of the eighth invention has the following configuration.

  The position information of the polymer at the time of manufacture is information on a deviation between the arranged position and a predetermined position. Thereby, each probe position information can be expressed by a small value of several microns, so that the amount of data to be stored can be reduced.

  The biochemical reaction cartridge of the ninth invention has the following configuration.

  The predetermined position is defined by a positional relationship from a reference mark on the biochip. Thereby, even if there is a position shift due to rotation of the biochip in the biochemical reaction cartridge, the probe position can be grasped by recognizing the reference mark.

  The biochemical reaction cartridge of the tenth invention has the following configuration.

  The predetermined position is defined by a positional relationship from a reference mark on the biochemical reaction cartridge. As a result, the reference position of the probe position can be adjusted simultaneously by positioning the biochemical reaction cartridge for the solution moving means and the reaction means.

  The biochemical reaction cartridge of the eleventh invention has the following configuration.

  The means for storing polymer information has a storage capacity of 2 kBytes or more. If the displacement of the probe position is recorded in the XY direction in ± 7 steps with the required accuracy, 1 byte is required for each probe, 1 kbyte of storage capacity is required for the position information of 1024 probes, and if there is a storage capacity of 2 kbytes or more, 1024 It is possible to store position information, detection process, lot number, and the like beyond the probe.

  According to the present invention, even when the probe on the DNA chip is deviated from a desired position, the probe position information can be stored in the DNA test cartridge, and further, detection can be performed accurately using the probe position information. Data can be acquired and the reliability of the data can be improved.

  In addition, since the tolerance for the positional deviation of the probe is widened, the yield at the time of manufacturing the DNA chip is improved.

(Example 1)
In this embodiment, probe position information acquisition means on a DNA chip, storage means of a DNA cartridge, and a method for using probe position information at the time of detection will be described with reference to the drawings.

  FIG. 1 shows a conceptual diagram of a DNA chip. A plurality of types of DNA solutions 104 are dropped by an inkjet method onto a glass substrate 101 on which a DNA solution obtained by mixing DNA in a solvent is coated with a DNA solidifying agent for each DNA species. The dropped DNA solution 104 is arranged in an array to form a probe. At that time, fluorescent mark dots 103 for array recognition at the time of detection can be simultaneously arranged in or around the array, and are used for determining the array position when the sample amount is small and the array is difficult to recognize at the time of detection. be able to.

  FIG. 2 shows DNA probe position information acquisition means. The DNA chip 202 immediately after dropping of the DNA solution has a solvent 203 mixed with the DNA dropped on the glass substrate and can be detected optically. Therefore, a detection means combining the optical microscope system 201 and the CCD camera is disposed above the chip, and the solvent droplet 203 is detected by irradiating the coaxial incident light. At this time, the irradiated light may be transmitted light from the back surface of the glass substrate. For each of the detected droplets, the barycentric coordinates and the dot diameter are acquired by image processing. The barycentric coordinates are acquired as relative positions with respect to two or more marks provided on the DNA chip. Here, a DNA chip in which an abnormality is found in the barycentric coordinates or dot diameter is excluded as a defective product. As position information with respect to the mark on the DNA chip, the desired probe coordinates 204 are compared with the detected center-of-gravity coordinates, and a deviation amount in each of the XY directions is acquired for each probe.

  The acquired amount of deviation is converted into simple data with the accuracy required for detection. For example, when converting to simple data with 5um accuracy, if the amount of deviation is + 10um in the X direction, it is converted to +2, and if it is -15um in the Y direction, it is converted to -3. If each is expressed as 1 byte data with 4 bits and ± as the upper 1 bit, the X direction as the upper 4 bits, and the Y direction as the lower 4 bits, 0x27 (00101011 binary number) is obtained. Simple data is generated for each probe position deviation amount, and this is used as probe position deviation information.

  FIG. 3 shows a conceptual diagram of the DNA test cartridge. The DNA test cartridge includes a specimen inlet, a treatment solution holding unit 304, each reaction chamber 303 that performs heating and cooling and stirring according to the purpose, a flow path 305 for flowing each liquid, and an air pressure for liquid movement. It has an inflow / outflow port 306, a hybrid chamber 302 capable of holding a DNA chip and observing the chip after the hybridization reaction, and a storage unit 307 for storing information related to inspection. The storage unit stores the displacement information and accuracy of the probe position of the DNA chip as simple data. According to the data format described above, a 1-byte (8-bit) data area is required for one probe, so in order to store 1024 probe information, lot management numbers, inspection procedures, etc. The storage unit requires a storage capacity of 2 kBytes or more.

  FIG. 4 shows a conceptual diagram of the DNA testing apparatus. The DNA testing apparatus includes a table 402 for setting a DNA testing cartridge 401, an electromagnet unit 403 that is operated when DNA or the like from a sample is extracted in the cartridge, and a method for amplifying DNA from a sample by a method such as PCR. A temperature control unit 404 for controlling the reaction temperature at the time of hybridization, a pump unit 405 comprising a pump, a valve and a joint for discharging or sucking air for moving the fluid in the cartridge; A detection unit 406 for observing and detecting the DNA chip after the hybridization reaction, a moving unit for moving each unit relative to the cartridge, and a control unit 407 for controlling the operation and moving unit of each unit . The table 402 for setting the cartridge 401 has a terminal 409 for reading data from the storage unit of the cartridge and is connected to the control unit 407. The means for reading data from the storage unit of the cartridge is not limited to the terminal 409, and may be a non-contact type such as wireless.

  The DNA test cartridge is set in a DNA test apparatus, the sample is injected, DNA is extracted and amplified, hybridized on the DNA chip, and then detected by the detection unit. The detection is performed by exciting the fluorescent agent labeled on the target DNA amplified from the specimen with laser light and measuring the excitation light quantity two-dimensionally. First, a fluorescent mark in the DNA probe array is detected as a reference position. Since the fluorescent mark is also dropped by the inkjet method, it is necessary to compare it with positional deviation information. The detected fluorescence mark position is compared with the displacement information of the probe position read from the storage unit of the cartridge, and the reference position of each probe is determined. At this time, a mark on the DNA chip may be used to determine the reference position. After that, the position of the probe to be read is determined from the reference position and the deviation amount data, the excitation light amount data at the corresponding position is extracted by image processing, the integral value of the appropriate area is calculated, and the data processing is performed as each probe amount data. The test result is output.

  The above process makes it possible to obtain accurate probe position data regardless of whether the probe position is misaligned when manufacturing the DNA chip by the inkjet method, improving the yield of the DNA chip, and further increasing the background data. Noise can be reduced by accurately eliminating the noise.

(Example 2)
In this embodiment, probe position information acquisition means on a DNA chip will be described with reference to the drawings.

  FIG. 6 shows a conceptual diagram of glass probe adsorption of the DNA probe. At the time of DNA chip manufacture, when DNA ink lands on the glass substrate 606, the probe 601 is normally adsorbed on the glass substrate and the ink droplet 602 remains on it. However, although the probe 605 is attracted to the landing position depending on the state of the glass substrate processing or the like, the ink droplet 603 may move to a position 604 shifted from the probe after landing. That is, even if the position of the droplet is acquired as the position information of the probe by normal optical detection, there is a possibility that it is different from the actual probe position.

  Therefore, a DNA probe 607 combined with a fluorescent agent different from the fluorescent label used at the time of detection is dropped on the glass substrate 606, and after preparing the DNA chip, the probe position is obtained by fluorescence detection. Thereby, even if the positional deviation 604 of the liquid droplet occurs, the positional deviation information of the DNA probe 607 can be accurately acquired regardless of this.

(Example 3)
In this embodiment, a probe position information acquisition unit on a DNA chip will be described.

  As a probe position acquisition procedure, after inserting and fixing a DNA chip in a DNA test cartridge, DNA probe position information is acquired as a relative position from a reference mark on the DNA test cartridge. Thereafter, as described above, data is stored in the storage means as the amount of deviation from the reference position.

  As a result, by attaching the cartridge to the table of the inspection device, or by performing alignment based on the reference mark of the cartridge, the deviation amount data of the DNA probe position stored as the deviation amount from the absolute reference position on the device Can save time and calculation error in detecting the fluorescent mark.

Conceptual diagram of DNA chip. (A) A conceptual diagram of probe detection on a DNA chip. (B) A method for simplifying probe position data. DNA test cartridge conceptual diagram. DNA testing device conceptual diagram. The image processing conceptual diagram at the time of detection. Image of droplet position deviation when the DNA probe lands.

Explanation of symbols

101 glass substrate
102 fiducial mark
103 Fluorescent mark
104 DNA probe
201 Optical microscope system
202 DNA chip
203 DNA probe droplet
204 Standard position of DNA probe
301 DNA test cartridge
302 DNA chip
303 reaction chamber
304 Solution holder
305 flow path
306 Air inlet / outlet
307 storage unit
308 Memory connection terminal
401 DNA test cartridge
402 Cartridge loading table
403 Electromagnet
404 Temperature controller
405 Pump part
406 Detector
407 Control unit
408 display
409 Memory data read terminal
501 DNA probe position
502 Read data integration area
601 and 605 probes
602,603 Normal droplet position
604 Moved droplet position
606 glass substrate
607 Probe with fluorescent agent

Claims (11)

  A biochemical reaction cartridge comprising a biochip for biochemical reaction in which a plurality of types of polymers are arranged at predetermined positions, and means for storing information on these polymers.   2. The biochemical reaction cartridge according to claim 1, wherein the information on the polymer is information that associates the type and position of the polymer.   The biochemical reaction cartridge according to claim 1, wherein the information on the polymer is information at the time of manufacturing the biochip.   4. The biochemical reaction cartridge according to claim 3, wherein the position information of the polymer at the time of manufacture is acquired using a polymer position acquisition unit at the time of manufacturing the biochip.   The biochemical reaction automatic inspection apparatus using the biochemical reaction cartridge according to claim 1, wherein the detection of the polymer uses the position information of the polymer to acquire data while correcting the detection position. Biochemical reaction automatic inspection equipment.   The biochemical reaction cartridge according to claim 4, wherein the polymer position acquisition means uses an optical detection method.   The biochemical reaction cartridge according to claim 4, wherein the polymer position acquisition means uses a fluorescence detection method using a fluorescently labeled polymer.   4. The biochemical reaction cartridge according to claim 3, wherein the position information of the polymer at the time of manufacture is information on a deviation between the arranged position and a predetermined position.   The biochemical reaction cartridge according to claim 8, wherein the predetermined position is defined by a positional relationship from a reference mark on the biochip.   9. The biochemical reaction cartridge according to claim 8, wherein the predetermined position is defined by a positional relationship from a reference mark on the biochemical reaction cartridge.   2. The biochemical reaction cartridge according to claim 1, wherein the means for storing the polymer information has a storage capacity of 2 kBytes or more.

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