JP3621927B2 - Biological sample measuring method and apparatus - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method and apparatus for measuring a biological sample such as DNA.
[0002]
[Prior art]
Conventionally, when detecting DNA samples or RNA samples, a DNA array to which an appropriate number of ligand DNAs that selectively adsorb or react with these samples is prepared in advance, and a DNA sample or RNA sample as a subject is prepared. Is labeled with a fluorescent substance in advance, and the labeled DNA sample is reacted with the ligand DNA on the DNA array, and then the presence or absence of binding of the DNA sample or RNA sample to each ligand DNA is determined by the fluorescent substance in each ligand. Was measured by observing the amount of the fluorescent light with a fluorescence detection device under excitation light.
[0003]
[Problems to be solved by the invention]
However, in order to observe the amount of fluorescent light, as described above, it is necessary to label a DNA sample or RNA sample in advance with a fluorescent dye. Therefore, there is a problem in that a reaction reagent, oxygen, and the like are required, which increases the processing time for inspection and increases the cost. In addition, the reaction in the pretreatment may vary, and there is a possibility that measurement reproducibility may be reduced.
[0004]
Furthermore, in order to inspect a plurality of ligands provided in this DNA array in order to observe the DNA array itself, the relative position between the fluorescence detection means and each ligand is moved, so that it can be arranged in rows or planes. It was necessary to make point observations. That is, it is necessary to fix the fluorescence detection means and move the position of the DNA array, or to fix the DNA array itself and move the fluorescence detection means, which complicates the whole measuring apparatus. At the same time, measurement errors may occur due to variations in relative position and the like, resulting in a decrease in measurement reliability.
[0005]
In addition, there is a method in which excitation light is irradiated on the entire ligand-attached surface of the DNA array, fluorescence of the entire ligand-attached surface is acquired as image data, and the data is subjected to image analysis to determine fluorescence in each ligand. However, in this case, since the entire observation surface is irradiated, fluorescence (background) is generated even from a portion where the ligand does not exist. On the other hand, the excitation light intensity in the vicinity of the individual ligand attachment point is lowered, and the fluorescence to be captured is reduced. Since it becomes weak, it is considered that the reliability of measurement is lowered.
[0006]
Accordingly, the present invention has been made to solve the above-described problems of the conventional technology, and eliminates the need for pre-processing of a biological sample such as a DNA sample and allows measurement at a fixed point (one point). The main object of the present invention is to provide a method and apparatus for measuring a biological sample that can simplify the overall structure.
[0007]
[Means for Solving the Problems]
In order to achieve the above-mentioned object, the biological sample measurement method according to claim 1 provides (i) an array site in which a plurality of ligand samples are aligned and fixed in a flow direction in a predetermined flow path. (Ii) flowing a biological sample into the flow path, and (iii) immobilizing the biological sample adsorbed or reacted on the ligand sample upstream from the ligand sample immobilized on the downstream side which is the detection means side to reduction by said ligand samples were dissociated from sequential locally the ligand sample, by the detecting means disposed downstream of (iv) the array sites, have the biological sample is attached to the ligand sample It is characterized by detecting whether or not.
[0008]
With such a configuration, the biological sample adsorbed or reacted to the ligand sample immobilized on the array site can be dissociated from the ligand sample by the dissociation means and detected by the downstream detection means. It is possible to observe the presence or absence of a biological sample at a fixed point (one point) without requiring a special pretreatment for a biological sample.
[0009]
Also, by dissociating from the downstream ligand sample, if the biological sample attached to the upstream ligand sample is dissociated and flowed downstream, it will reattach to the downstream ligand sample. Can be prevented. Moreover, it becomes possible to measure continuously by dissociating sequentially from the downstream side.
[0010]
According to another aspect of the present invention, the biological sample measuring device is (i) arranged in a predetermined flow path and flowing in a plurality of ligand samples to which the biological sample as a subject is adsorbed or reacted. (Ii) a detection means disposed on the downstream side of the array site and capable of detecting the biological sample; and (iii) the biological sample adsorbed or reacted to the ligand sample. Dissociating means capable of locally dissociating from the ligand sample sequentially from the ligand sample immobilized on the downstream side, which is the detection means side, to the ligand sample immobilized on the upstream side, It is characterized by providing. With such a configuration, the same effect as the above method can be produced.
[0011]
The dissociation means may have a heating mechanism capable of locally heating a portion where the ligand sample is immobilized at the array site, or alternatively, to the portion where the ligand sample is immobilized at the array site. It is also preferable to have a liquid ejection part capable of locally ejecting a liquid, or to have a magnetic application mechanism capable of applying magnetism to a part where the ligand sample is immobilized in the array part. .
[0012]
DETAILED DESCRIPTION OF THE INVENTION
Next, a preferred embodiment of the present invention will be described with reference to FIG. FIG. 1 is a schematic diagram showing an entire biological sample measuring apparatus 1 according to the present invention. The biological sample measuring apparatus 1 generally includes a detection device 10 that is an inspection means capable of detecting a biological sample, a flow channel 20 for flowing a biological sample as a subject, and a ligand sample 24 immobilized on an array site 22. Dissociation means 30 for dissociating the biological sample 26 attached to the surface. These are described in detail below.
[0013]
In the flow path 20, there is provided an array portion 22 in which a plurality of ligand samples 24 that adsorb a biological sample contained in the solution F or react with the biological sample are fixed. The plurality of ligand samples 24 are aligned along the flow direction of the solution F. And the detection apparatus 10 which can detect a biological sample is arrange | positioned in the downstream of this flow direction. The detection device 10 is located on an extension line in the flow direction along the alignment direction of the ligand sample 24 (see FIGS. 2 and 3).
[0014]
The flow path 20 may have any shape that allows a solution F such as a DNA sample or an RNA sample to flow therein. For example, as shown in FIG. 2, the ligand samples 24 are aligned in a line. The flow path 20a has an elongated or narrow tubular shape, and the flow paths 20b and 20c have a two-dimensional or planar expansion that can align the ligand sample 24 in a plurality of rows as shown in FIG. be able to. The flow method of the solution F in the flow path 20 includes transient flow, circulation, reciprocation, or retention depending on conditions such as the amount and concentration of the biological sample contained in the solution.
[0015]
If a plurality of rows of ligand samples 24 are provided, as shown in FIG. 3A, a plurality of detection devices 10 may be provided corresponding to each row, or an array sensor. May be arranged in the width direction. Alternatively, dissociation from the ligand is performed in the order as shown in the example of FIG. 3 (b) (see arithmetic numbers), and further, the flow path is narrowed between the array region and the sensor region. It is also possible to perform detection with this sensor. This order is merely an example and is not limited thereto. For example, dissociation can be performed for each column, but it is preferable to dissociate from the downstream ligand. In addition, as a detection device, an ultraviolet absorptiometer, a thermal lens microscope, a surface plasmon resonance analyzer, an electrochemical detector, a fluorescence detector, and the like can be considered. The device can be used.
[0016]
In addition, referring to FIG. 1, a plurality of dissociation means 30 are provided at positions corresponding to the ligand sample 24 in the array portion 22. The dissociation means 30 has a function of allowing the biological sample 26 hybridized to the ligand sample 24 to be released, that is, dissociated from the ligand sample 24. In this embodiment, a plurality of heaters 32 capable of locally heating a plurality of ligand samples 24 immobilized on the array portion 22 are provided. By providing a plurality of heaters 32 in this manner, for example, the dissociation process can be sequentially performed from the heater 32 on the ligand sample 24 on the downstream side in the flow direction of the solution F. In addition, without disposing a plurality of heaters, for example, it is possible to provide one dissociation means corresponding to the array part 22 and control the dissociation action locally from the ligand sample 24 on the downstream side. is there.
[0017]
Next, a method for detecting a biological sample using the above-described biological sample measuring apparatus 1 will be described. First, an array portion 22 is prepared in which a plurality (four in the present embodiment) of ligand samples 24 are fixed in a flow direction 20 in a predetermined shape. Next, a biological sample solution F containing a predetermined sample is flowed into the flow path 20. At this stage, the biological sample 26 in the solution adheres to the predetermined ligand sample 24 or reacts to adhere to the ligand sample 24.
[0018]
Next, the heat treatment is started from the heater 32a on the downstream side, that is, the detection device 10 side, and the biological sample 26a is dissociated from the ligand sample 24 as shown in FIG. Thereafter, the biological sample 26b flows downstream and is detected by the detection device 10 (see the biological sample 26c). According to such a measurement method, it is possible to confirm whether or not a specific biological sample capable of reacting with the ligand sample has adhered to the ligand sample 24 subjected to the dissociation process. In addition, it is not necessary to perform a special pretreatment on the biological sample in the solution in order to detect a specific biological sample, and the presence or absence of the biological sample can be observed at a fixed point.
[0019]
Further, the heating process is sequentially started sequentially with the upstream heater 32b, the heater 32c, and the heater 32d, and the above-described measurement process is repeated. Thereby, continuous detection becomes possible. In addition, by sequentially performing the dissociation process from the downstream side, the biological sample dissociated on the upstream side flows down to the detection apparatus 10 without being attached to the ligand sample on the downstream side again.
[0020]
As another embodiment of the heating mechanism constituting the dissociation means, for example, as shown in FIG. 4, there is a method of heating by irradiating a laser beam 24 from above each individual ligand sample 24. The biological sample 26 hybridized to the ligand sample 24 can be released by such heating.
[0021]
Another form of dissociation means is a liquid ejection mechanism. For example, as shown in FIG. 5, a small hole is locally provided in the main body constituting the flow path 20, and the liquid 36 is locally ejected from above the individual ligand sample 24 toward the ligand sample 24. Thus, the biological sample 26 hybridized with the ligand sample 24 is released. As the liquid to be ejected, for example, a heated solution such as hot water that can be locally heated, or a chemical reaction having an effect of dissociating DNA-DNA or DNA-RNA hybrid such as a low ionic strength solution is generated. There are liquids.
[0022]
Furthermore, as another embodiment of the dissociation means, there is a magnetic application mechanism. For example, as shown in FIG. 6, a magnet 38 is disposed in the vicinity of each ligand sample 24, and a magnetic field is generated by applying magnetism to the magnet, so that a biological sample 26 hybridized to the ligand sample 24 is obtained. It is something to be liberated.
[0023]
In addition, as a substance to be immobilized in advance on the array site as a ligand sample, a variety of substances that selectively adsorb or react with other substances such as antibodies, enzymes and other proteins, biological substances such as DNA, and other substances Can be considered. Then, identification of specific components of DNA, hybridization of proteins and nucleic acids, antibody assays, and DNA chips (or DNA arrays) can be detected from biological samples attached to these coupling materials.
[0024]
Moreover, according to the biological sample measuring apparatus according to the present invention, measurement using a fluorescently labeled sample used in a conventional DNA array is also possible. Furthermore, in this biological sample measuring apparatus, DNA or RNA bound to each ligand is released again and then detected. Therefore, it is possible to use the released DNA or RNA as a sample for biochemical and molecular biological manipulations such as mass spectrometry and base sequence analysis, which enables more advanced analysis and research than conventional DNA arrays. It becomes possible.
[0025]
【The invention's effect】
In the biological sample measurement method according to the present invention, a dissociation treatment is performed on a ligand sample immobilized on an array site in a predetermined flow path so as to release the biological sample hybridized to the ligand sample, Since the dissociated biological sample is detected by a detection device on the downstream side, no special pretreatment is required for the biological sample as the subject, and the presence or absence of the biological sample is determined at a fixed point (one point). It can be observed.
[0026]
Further, the biological sample measuring apparatus according to the present invention includes an array part in which a plurality of ligand samples are aligned and fixed in a flow direction in a predetermined flow path, and a detection means disposed on the downstream side of the array part. And a dissociation means that can dissociate the biological sample adsorbed or reacted with the ligand sample, so that the same effect as the above method can be produced.
[Brief description of the drawings]
FIG. 1 is a schematic view showing an entire biological sample measuring apparatus according to the present invention.
FIG. 2 is a schematic view showing a thin tubular channel for flowing a biological sample.
FIG. 3 is a schematic view showing a flow path having a planar spread through which a biological sample flows.
FIG. 4 is a schematic diagram showing an embodiment of dissociation means.
FIG. 5 is a schematic view showing another embodiment of the dissociation means.
FIG. 6 is a schematic view showing still another embodiment of the dissociation means.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Biological sample measuring device, 10 ... Detection apparatus, 20, 20a, 20b, 20c ... Flow path, 22 ... Array part, 24 ... Ligand sample, 26 ... Biological sample, 30 ... Dissociation means, 32, 32a-32d ... Heater 34 ... Laser beam, 36 ... Liquid, 38 ... Magnet, F ... Solution.

Claims (6)

Prepare an array site in which a plurality of ligand samples are aligned and fixed in the flow direction in a predetermined flow path,
Flowing a biological sample into the flow path,
The biological sample adsorbed or reacted with the ligand sample is sequentially and locally from the ligand sample to the ligand sample immobilized on the upstream side from the ligand sample immobilized on the downstream side which is the detection means side. dissociated,
Wherein by said detecting means disposed on the downstream side of the array sites, to detect whether the biological sample to said ligand sample was adhered, biological sample measurement method. The biological sample measurement according to claim 1 , wherein the step of dissociating the biological sample sequentially and locally from the ligand sample is locally performed on each portion of the array site where the ligand sample is immobilized. Method. An array site that is arranged in a predetermined flow path, and in which a plurality of ligand samples to which a biological sample as a subject is adsorbed or reacted are aligned and fixed in the flow direction;
A detection means disposed on the downstream side of the array site and capable of detecting the biological sample;
The biological sample adsorbed or reacted with the ligand sample is locally and sequentially from the ligand sample immobilized on the downstream side which is the detection means side to the ligand sample immobilized on the upstream side. Dissociation means that can be dissociated from,
A biological sample measurement device comprising: The biological sample measuring apparatus according to claim 3, wherein the dissociating means has a heating mechanism capable of locally heating a portion where the ligand sample is immobilized in the array site. The biological sample measuring apparatus according to claim 3, wherein the dissociating means has a liquid ejection portion capable of locally ejecting liquid to a portion where the ligand sample is immobilized in the array portion. The biological sample measuring apparatus according to claim 3, wherein the dissociating means has a magnetic application mechanism capable of applying magnetism to a portion where the ligand sample is immobilized in the array part.

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