JP2832117B2 - Sample measuring device and sample measuring system - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the technical field of, for example, measuring components in a sample using an antigen-antibody reaction or a nucleic acid hybridization reaction, or measuring minute substances such as cells, microorganisms, or chromosomes. .

[0002]

2. Description of the Related Art Recent advances in the technology for detecting trace components include:
In the field of clinical testing, it has played a major role in the early diagnosis and prognosis diagnosis of various diseases. 1958, S.M. A. Bers
on, et al., published a method of quantifying insulin by labeling with radioiodine, since IgE, IgG, C
Plasma proteins such as RP and microglobulin, AFP, CE
A, tumor markers such as CA19-9, TSH, hormones such as T _3, blood drug, HBV, viruses and specimen such as HIV, such as DNA or RNA nucleic acid become measurable, yet many Automated Sample processing is now possible.

For many of these biological trace components, an immunological method utilizing an antigen-antibody reaction or a method utilizing nucleic acid-nucleic acid hybridization is used.
As an example of such an analysis method, an antibody or antigen or a single-stranded nucleic acid that specifically binds to a substance to be detected is immobilized on a solid surface such as a fine particle, a bead, or a wall of a storage portion as a probe, and the substance to be detected is And an antigen-antibody reaction or nucleic acid hybridization is performed. Enzymes, fluorescent substances, labeled substances having a specific interaction carrying a highly sensitive labeling substance such as a luminescent substance, for example, using a labeled antibody or a labeled antigen or a labeled nucleic acid, etc. It detects antibody compounds and double-stranded nucleic acids to quantify test substances.

FIG. 20 is a diagram showing an example of a conventional apparatus for performing the above measurement. An insoluble carrier 202 having a reagent immobilized on its surface is placed in the reaction tank 201, where a sample solution is accumulated and reacted to form a reaction solution 203. Reaction liquid 203
Is temporarily stored in a syringe type pump 206 through a liquid feeding pipe 204 and a valve 205. Next, the valve 205 is switched to send the stored reaction solution to the optical cell 207 at the measurement position. The optical cell 207 is irradiated with light from the light source 208, and the light passing through the reaction solution is detected by the light receiving element 209,
Colorimetric measurement or fluorescence measurement of the color reaction of the reaction solution is performed. The reaction solution after the measurement is discharged as waste liquid.

On the other hand, an apparatus for separating and flowing a large number of fine particles such as cell particles and chromosomes contained in a particle suspension such as a blood sample one by one and measuring each particle by optical or electrical means. Have been put to practical use as flow cytometers, particle counters, and the like.

FIG. 21 is a diagram showing an example of the configuration of a conventional flow cytometer. The test tube 220 is filled with a sample liquid that is a particle suspension, while the sheath bottle 223 is
Contains a sheath solution such as a PH buffer solution and a physiological saline solution. Each liquid is temporarily stored in the syringe type pumps 222 and 225, respectively, and then the valves 221 and 224 are switched to press the stored liquid to send it. The sample liquid is supplied to the sample nozzle 226, and the sheath liquid is supplied to the periphery 227 of the sample nozzle. According to the sheath flow principle, the sample liquid is hydrodynamically converged while being surrounded by the sheath liquid, and the fine particles in the sample liquid are separated and flow through the flow part 228 by one. The light source 22
9 and the light receiving elements 230 and 231 measure the fluorescence and scattered light emitted from the irradiated particles. Measurements are performed on a large number of particles sequentially, and the types and properties of the particles are analyzed using statistical methods based on their outputs. Although not explicitly shown, it is necessary to wash the path by flowing a cleaning liquid through the liquid supply path each time one sample is measured, and a cleaning mechanism for this purpose is provided. A specific example of the cleaning mechanism is described in, for example, Japanese Patent Application Laid-Open No. 1-118748.

[0007]

However, in the above-mentioned conventional apparatus, a dead space in the flow path is unavoidable due to the long liquid feeding path and the pressure pump, so that a wasteful sample liquid is required. The use of a large amount of sample liquid increases the amount of waste liquid generated, but this is not preferable in view of environmental problems such as biohazard.

Further, in the above-mentioned conventional apparatus, each liquid is supplied under pressure in order to supply the sample liquid to the measuring section. However, since a piping and a pressurizing mechanism such as a pump are required, the apparatus is large. Control becomes complicated, and there is a limit to miniaturization of the device.
Furthermore, there is a certain time lag in forming a flow in the measuring unit or in stopping the flow, and control responsiveness is poor.

The present invention has the following objects in view of the above problems. (1) To provide a miniaturized and integrated measurement cartridge and system in which a dead space in a flow path of a liquid sample is reduced as much as possible and a required sample amount is reduced. (2) To provide a measurement cartridge and a system capable of performing measurement without damaging a specimen and addressing environmental issues such as biohazard countermeasures. (3) To provide a measurement cartridge with stable performance at low cost.

[0010]

According to one aspect of the present invention, there is provided a sample measuring device, comprising: an inlet for injecting a sample; and a flow section through which the injected sample is flown and including a measuring position in the middle. And pump means having a heating element provided at a position substantially facing the flow path downstream of the measurement position of the flow section.

One embodiment of the sample measuring system of the present invention has a holding mechanism for holding a cartridge for measuring a sample, and measuring means for measuring a sample of the cartridge, wherein the cartridge stores a sample. An inlet for injecting, a flow section through which the injected sample is flown, including a measurement position in the middle, and a feeding action of the sample in the flow section, and a flow near an outlet downstream of the measurement position of the flow section. Pump means having a heating element provided at a position substantially facing the road.

[0012]

【Example】

<Example 1> As a first example of the present invention, a sample sample was reacted with a reagent, and a reaction solution was obtained by repeating washing and reaction as necessary, and the reaction solution was optically measured. A cartridge for measuring a sample will be described. FIG. 1 is a side view showing the structure of the cartridge of the first embodiment, FIG. 2 is a top view of the first substrate and the second substrate viewed from above, and FIG. 3 is an assembly diagram of the cartridge.

The cartridge of this embodiment has a structure in which a first substrate 1, a second substrate 2, and a third substrate 3 are joined, the first substrate 1 is a silicon substrate, and the second substrate 2 and the third substrate 3 are It is a glass substrate. By joining these substrates, a space is formed inside the cartridge that forms the storage section 4 that is a reaction tank. The third substrate 3 is provided with an injection port 5 which is a hole for injecting a liquid such as a sample specimen liquid, and the sample liquid can be injected into the accumulation section 4 from outside. An insoluble carrier 6 having a spherical shape and a reagent immobilized on the surface is enclosed in the storage section 4. The insoluble carrier 6 is made of a material such as a ceramic such as glass, a plastic made of a polymer compound, a metal such as a magnetic material, or a composite material thereof.
A surface treatment is introduced in which a covalent bonding group or the like is introduced so that the reagent can be easily fixed. The shape of the insoluble carrier 6 is not limited to a spherical shape, but may be other shapes such as a polyhedron, and the number thereof is not limited to one and may be many. Alternatively, the reagent may be directly immobilized on the inner wall surface of the storage section 4 without using an insoluble carrier. The details of the reagent will be described later.

A flow section 7 is connected to the storage section 4, and an outlet at the tip thereof is a nozzle opening 8. The nozzle opening 8 has a pipeline resistance action by having a tapered shape. A micropump 9 is formed on the first substrate 1 near the nozzle opening 8. Specifically, the micropump 9 has a configuration in which a heat generating element is attached to a circulation part by a manufacturing method described later. When a pulse voltage is applied to the heating element, the sample liquid heated by the heat is instantaneously vaporized and bubbles are generated, and the sample liquid is dropped from the nozzle by the pressure generated by the shock when the bubbles expand and contract. And discharge
A small amount of the sample liquid is discharged from the distribution section. Then, the sample liquid is drawn in and supplied from the accumulation section 4 toward the nozzle of the circulation section by the discharged amount. By continuously performing the discharge and discharge at a high frequency, a function of feeding the sample liquid is obtained, and a flow of the fluid can be created in the flow section 6.

FIG. 7 is a view for explaining a specific state in which bubbles are generated and droplets are discharged. In the initial state of FIG. 7A, when the heating element is heated by instantaneously applying a pulse voltage to the heating element, moisture near the heating element is vaporized and bubbles are generated as shown in FIG. Then, the volume expands by the amount of vaporization, and the liquid near the nozzle opening is pushed out from the nozzle opening as shown in (C). The bubbles that have been expanding initially are cooled and start contracting as shown in (D), and the liquid discharged from the opening due to the reduction in volume flies in the air as droplets as shown in (E). The liquid is supplied by the amount of the discharged liquid by capillary action, and returns to the initial state of (A). The basic principle of the droplet discharge by the bubbles is described in detail in US Pat. No. 4,723,129 and US Pat. No. 4,740,796.

[0016]

On the surface of the first substrate 1, a sensitive element for measuring a sample liquid is provided together with the micropump. Specifically, in order to optically detect the state of the sample liquid, a first light detection element 10, a first optical filter 11 having a wavelength selection function, and a second light detection element 1
2. The second optical filter 13 is formed on the substrate by a manufacturing method described later. These members constitute an optical detection unit for selectively receiving the first and second lights that arrive via the sample liquid. In this embodiment, an example in which the sample liquid is optically measured is described. However, the present invention is not limited to this. For example, the sample liquid may be measured using an electric or magnetic method. Furthermore, these may be combined and measured. In this case, as in the case of the optical detection unit in FIG. 1, a sensitive element (electrode, magnetic detection element, etc.) suitable for each measurement is bonded to the substrate.

As shown in FIG. 2, the first substrate 1 has a heating element 9 of a micropump and first and second light detecting elements 1.
0 and 12 are joined, and conductive patterns 18, 19 and 20 are connected to these elements, respectively, and are patterned on the surface of the first substrate 1 as shown in the figure. Then, when the first substrate 1 and the second substrate 2 are joined, the ends of the conductive patterns 18, 19, 20 are exposed to the outside, so that contact with the external terminals can be conducted.

The above members are all integrated into a cartridge. A method for manufacturing the cartridge will be described later. On the other hand, as shown in FIG. 1, in order to examine the degree of coloration of the sample liquid by applying irradiation light, which is the measurement energy, to the sample liquid inside the circulation unit 7 or to generate fluorescence or scattered light from the sample liquid. Light sources 14, 16,
A light irradiating unit including the condenser lenses 15 and 17 is provided separately from the cartridge. As the light sources 14 and 16, for example, semiconductor lasers, LEDs, halogen lamps, tungsten lamps, mercury lamps and the like are suitable. In the case where the measurement is performed by detecting light emitted by the specimen itself, such as chemiluminescence or bioluminescence, light irradiation is not required, so that there is no need to provide a light irradiation unit.

In this embodiment, a micropump is provided near the outlet on the downstream side of the measurement position, so that the following significant advantages unique to sample measurement can be obtained.

If the micropump is provided upstream of the measurement position, the mutagenized pressure and heat generated by the pumping action will adversely affect the sample liquid and cell particles near the pump, such as denaturation and damage, and this will be measured. There is a risk of doing it. On the other hand, by providing the micropump on the downstream side, it is possible to prevent the sample before measurement from being adversely affected. In addition, since pressure and heat are applied to the waste liquid after measurement at the downstream position,
The bactericidal action and sterilization action of the waste liquid are obtained, and measures are taken against biohazard.

Also, a micro pump is provided near the outlet,
Since the flow is created in the flow section by drawing in the fluid by the amount discharged from the nozzle, a stable fluid system is obtained and the control response of the fluid control is high. Further, the dead space of the fluid system is almost eliminated, and the amount of used specimen can be extremely small.

Here, several modified examples of the cartridge will be described. FIG. 4 shows an example in which condenser lenses 21 and 22 are integrally formed on the upper surface of the substrate. As the condenser lens, a spherical lens, a Fresnel lens, a zone plate, or the like can be used. FIG. 5 shows that the irradiation light is introduced into the optical fibers 23 and 24.
This is an example in which the optical axis alignment between the light source and the cartridge is not required. FIG. 6 shows an example of a cartridge obtained by further developing the above-described embodiment, in which measurement modules each including a storage unit, a distribution unit, and each element are arranged in a high-density and parallel manner on a single substrate to form an array. is there.

Next, a method of manufacturing the cartridge will be described in detail. Since the cartridge of this embodiment can be manufactured relatively easily by micromechanics technology including a semiconductor manufacturing process, it is suitable for mass production by batch processing, and an array as shown in FIG. 6 is also easy. The manufacturing process mainly includes the following four steps.

(Step 1) A hole serving as a storage section 4 is provided in a glass substrate serving as a second substrate 2, and a groove serving as a flow section 7 is formed. A groove is formed in the glass substrate by performing photolithography using photosensitive glass or by etching the glass to a desired depth with hydrofluoric acid. As another method, for example, a resist may be used as a groove by applying a resist to a glass substrate or a silicon substrate, developing and solidifying the resist by a photolithography process. Alternatively, the grooves can be formed by anodically bonding a silicon substrate formed by etching the patterns of the accumulation section and the circulation section to a glass substrate.

In this embodiment, the grooves are formed by processing a glass substrate, but the second substrate is manufactured by molding using a translucent resin material by a molding method or the like. Is also good.

(Step 2) A heating element of a micropump is bonded to a silicon substrate serving as the first substrate 1. FIG. 8 shows a detailed configuration of a heating element of a micropump formed on a silicon substrate. This manufacturing process is as follows. After forming a silicon oxide film on the silicon substrate 31, Hf
The B ₂ layer 32 and the Al layer 33 are stacked and formed as a heat generating portion and an electrode portion by using a photolithography process. Further, SiO ₂ is used as the insulating layer 34 and T
a is sequentially laminated on a portion of the electrode portion excluding the wire bonding portion, and thereafter, only Ta is patterned in a belt shape around the heating portion by a photolithography process. And Ta
A resin layer 36 is formed in a pattern on the uncoated SiO ₂ layer in order to enhance the isolation between the electrode and the liquid as a specimen, thereby producing a heating element serving as a pump unit.

(Step 3) Two optical detectors are joined to a silicon substrate serving as the first substrate 1. FIG. 9 shows a detailed configuration of an optical detection unit formed on a silicon substrate. The manufacturing process of this optical detection unit is as follows. Glass substrate 4
A Cr film 42 having a thickness of 0.1 μm is formed on the substrate 1 by a resistance heating method. The substrate is heated to 350 ° C., and SiH ₄ and NH ₃ (10
An SiN: H layer 43 is formed to a thickness of 0.3 μm by a plasma CVD method using a mixed gas of CCM: 20 CCM). In the same vacuum, the substrate is heated to 200 ° C., and a 0.6 μm α-Si: H layer 44 is formed by a plasma CVD method using SiH ₄ gas. N + -α-S by adding PH ₃ gas to SiH ₄ gas
i: An H layer 45 is formed to a thickness of 0.2 μm. An Al layer is formed to a thickness of 0.1 μm by an electron beam evaporation method. The electrode 46 is formed by patterning the Al layer by a photolithography process.
An opening is provided in the n + -α-Si: H layer 5 by RIE (reactive ion etching) using CF ₄ gas. After the optical filter 47 is formed in the opening by spin coating, the resist is peeled off to produce an optical detection unit.
The wavelength selection characteristics of the optical filter are selected according to the emission wavelength or the fluorescence wavelength of the measurement object.

It should be noted that the Cr layer can be used as the back surface light shielding portion. In addition, by using a Cr layer as a gate electrode, it is possible to detect a field-effect sensor with an optimum current value. In this embodiment, the example of the α-Si light receiving element has been described, but other pn photodiodes and pi photodiodes may be used.
n photodiode, Schottky photodiode, CC
D or the like is acceptable.

(Step 4) As shown in FIG. 3, a first substrate 1 which is the silicon substrate, a second substrate 2 which is the glass substrate, and a third substrate 3 provided with a hole of an injection port are prepared. Join. At this time, the insoluble carrier 6 with the reagent fixed on the surface
Is enclosed in the storage unit 4. As a method for bonding the substrates, there are methods such as bonding with an adhesive and anodic bonding. For example, if anodic bonding is performed by CO ₂ laser irradiation, the bonding temperature can be lowered, and the adverse effect of heat on the substrate can be suppressed.

Next, the reagent used in the present embodiment will be described in detail. The reagent is immobilized on the surface of the insoluble carrier enclosed inside the accumulator, or directly on the inner wall surface of the accumulator. The reagent used in the present embodiment contains at least a bio-related substance, and the selection of the bio-related substance depends on the substance to be analyzed or the analyte. That is, specific detection becomes possible by selecting a biologically relevant substance that shows biological specificity to the subject.

Examples of the biologically-related substances include natural or synthetic peptides, proteins, enzymes, saccharides, lectins, viruses, bacteria, nucleic acids such as DNA and RNA, and antibodies. Among them, the following substances are particularly useful clinically. IgG, immunoglobulins, such as IgE, complement, CRP, ferritin, plasma proteins and their antibodies such as alpha ₁ or beta ₂ microglobulin,
α-fetoprotein, carcinoembryonic antigen (CEA), CA
19-9, tumor markers such as CA-125 and their antibodies, luteinizing hormone (LH), follicle-stimulating hormone (FSH), human ciliary gonadotrobin (hCG), hormones such as estrogen, insulin, and their antibodies; Viral hepatitis-related antigen, HIV, ATL
Virus infection related substances and their antibodies, such as diphtheria, botulinum, mycoplasma, bacteria such as syphilis treponema and their antibodies, protozoa such as toxoplasma, trichomonas, leishmania, trypanozoma, malaria parasite and their antibodies,
Phenytoin, antiepileptic drugs such as phenobarbital, quinidine, cardiovascular drugs such as dicoxinin, anti-asthmatic drugs such as theophylline, chloramphenicol, drugs such as antibiotics such as gentamicin and antibodies thereof,
In addition, there are enzymes, bacterial exotoxins (such as streptolysin O) and antibodies thereof, and a substance which causes an antigen-antibody reaction with the substance to be detected in the sample is appropriately selected according to the type of the substance to be detected. When nucleic acid hybridization is used instead of the antigen-antibody reaction, a nucleic acid probe having a base sequence complementary to the base sequence of the nucleic acid to be tested is used.

FIG. 10 is a diagram showing the configuration of an entire system for performing measurement by mounting the cartridge. The cartridge 100 described above is a cartridge holder 1
01 is held. Although only one cartridge is shown in the figure, a plurality of samples can be simultaneously or simultaneously mounted by arranging a plurality of similar cartridges in parallel or using a cartridge in which measurement modules are arrayed as shown in FIG. It can be measured sequentially.

A plurality of sample containers 104 are arranged in the rack 103, and each contains a plurality of sample liquids. The dispenser device 102 uses the pipette 105 to sequentially supply the sample liquid in each sample container 104 to the cartridge 100.

On the other hand, the washing solution container 106 contains a washing solution for B / F separation, and the reagent solution container 107 contains a reaction reagent solution. The flow path from each container is connected to a valve 108 and one of them is selectively switched by the valve 108, and the selected liquid is supplied to the cartridge 100 via the tube 109. Both the pipette 105 and the tube 109 of the dispenser device 102 can be connected to the inlet of the cartridge 100, and a desired liquid is supplied to the cartridge.

The stirrer 1 is placed on the cartridge holder 101.
The cartridge 10 to which the cartridge 10 is attached and attached and held
It has the effect of stirring the sample liquid and the reagent in the zero accumulation part and promotes the reaction. The stirring is performed by, for example, remotely moving a magnetic carrier reagent using a magnet or applying vibration to the sample liquid by ultrasonic waves.

Further, in order to improve the accuracy of the measurement data, it is necessary to control the temperature of the storage section in the cartridge with high accuracy. For this purpose, the entire cartridge is held in a constant temperature box (not shown). In addition, it is preferable to take a constant temperature means so that the washing water, the reaction reagent and the specimen are kept at a constant temperature as needed.

Electrodes are provided on the cartridge holder 101 and are connected to the exposed conductive patterns of the cartridge 100 when the cartridge is mounted. This electrode is electrically connected to the drive / detection circuit 111 and the drive / detection circuit 1
Reference numeral 11 denotes driving of the light sources 14 and 16 for measurement, driving of the stirrer 110, driving of the dispenser device 102, driving of the valve 108, driving of the micropump in the cartridge, and detection of output from the two optical detection elements in the cartridge. Do. The computer 112 controls the entire system and performs sample measurement based on the detection results. Using antigen-antibody reaction or nucleic acid hybridization reaction, etc.
The color reaction or the fluorescence or scattered light is detected and subjected to data processing by a known method such as a rate assay method or an end point method. Further, comparison processing is also performed with the calibration curve data prepared in advance. The result of this analysis is
6. Output to a display, printer, etc. attached to 6.

When the measurement is performed by driving the micropump, waste liquid is discharged from the nozzle of the cartridge 100.
This is received by the waste liquid container 113 and the waste liquid is stored in the container. In this embodiment, since the heating element is used as the micropump, the waste liquid discharged by the heating action and the pressure action of bubble generation is sterilized or sterilized.
It is further preferable that the waste liquid is sterilized using heat, ultraviolet rays, chemicals, or the like in the chamber 13.

As described above, the present system uses the cartridge 10
Since 0 is disposable and is replaced with a new one each time one sample is measured, the system is simplified and a small and low-cost sample measurement system is provided. Further, by making the disposable, the micropump and the sensitive element do not require much durability, and the cartridge can be supplied at a lower cost.

Hereinafter, as an example of measurement by the above measurement system, a step of detecting a specific DNA in a sample specimen will be described.

(Step 1) A cartridge is prepared in which a single-stranded DNA probe that specifically performs a hybridization reaction with a specific target DNA (single-stranded) is immobilized as a reagent in a storage section. When the cartridge is mounted on the cartridge holder of the measurement system, the pipette of the dispenser device automatically injects a sample liquid containing a large number of DNAs that have been previously formed into a single strand by pre-processing into the storage section of the cartridge.

(Step 2) The sample liquid in the storage section of the cartridge is stirred by the stirring means provided in the measurement system to promote the reaction. If the target single-stranded DNA is present in the sample solution, the DN immobilized in the accumulation portion
A hybridization reaction specifically occurs with the A probe to generate a double-stranded DNA.

(Step 3) In order to remove single-stranded DNA that has not undergone a hybridization reaction, B / F separation is performed by injecting and discharging a washing solution.

(Step 4) Next, an enzyme-labeled probe is injected into the accumulation part, and the double-stranded DNA produced by the hybridization reaction is specifically labeled with an enzyme.

(Step 5) B / F separation is again performed by washing to wash away excess enzyme-labeled probe.

(Step 6) A color reaction is performed by reacting with the enzyme label, or a reagent solution containing a substrate exhibiting fluorescence emission or chemiluminescence is injected into the accumulating portion and reacted.

(Step 7) The micropump of the cartridge is operated to flow the reaction solution of (Step 6) into the flow section.
The color reaction or fluorescence or chemiluminescence light is detected by the light receiving element, and the amount of the target DNA can be quantified from the detected light amount. Further, by measuring the change over time of the detected light amount using a rate assay method, the quantification can be performed more accurately.

According to the sample measuring cartridge and system described above, the following effects can be obtained. (1) A micropump is provided downstream of the measurement position, so that the measurement can be performed without denaturing or damaging the sample liquid, and sterilization action is provided by applying a mutation pressure or heating to the waste liquid after the measurement. Alternatively, a sterilization effect is obtained. (2) A stable fluid system is obtained, and the control response of fluid control is also high. Further, since there is almost no dead space in the flow path, a small amount of the sample liquid is required. (3) Since the amount of generated waste liquid is small and sterilized or sterilized, it is preferable from the viewpoint of environmental problems such as measures against biohazard. (4) Batch production can be performed using a semiconductor manufacturing process, and cartridges with stable quality can be mass-produced at low cost. (5) By integrating the light receiving element, alignment adjustment of the optical system becomes unnecessary. (6) Since a cartridge having an integrated sample measurement function is supplied at a low cost and the cartridge is replaced every time one sample is measured, the configuration of the fluid system is simplified, and the entire measurement system is extremely compact and highly reliable. It will be.

<Embodiment 2> Next, as another embodiment of the present invention, a sheath flow is generated in a flow section by a sample solution and a sheath solution to sequentially separate individual fine particles (for example, blood cells and chromosomes) in the sample. An example of a cartridge for measuring particles to be measured will be described. FIG. 11 is a side view showing the structure of the cartridge, and FIG. 12 is a top view seen from above.

The cartridge of this embodiment is also manufactured by a micromechanics technique including a semiconductor manufacturing process similar to that of the above embodiment. The cartridge has a configuration in which a first substrate 51 and a second substrate 52 are joined, and the first substrate 51 is a silicon substrate and a second substrate 52 is a glass substrate. On the upper surface of the second substrate 52, a sample receiving portion 53 for receiving a sample solution in which the particles to be measured are suspended is formed, and a sample tube 54 is connected thereto. The tip of the sample tube 54 projects into the flow section 56. In addition, a sheath liquid supply port 55 is provided for supplying a sheath liquid such as physiological saline or distilled water, and is connected to a circulation unit 56. A nozzle opening 57 is formed at the outlet at the tip of the flow portion 56, and has a tapered shape to have a pipeline resistance action. A micropump 58 is formed on the first substrate 51 near the nozzle opening 57. Specifically, the micropump 58 has a configuration in which a heating element (or a piezoelectric element) is attached to the distribution section by the same manufacturing method as in the previous embodiment. A liquid feeding action is obtained. Furthermore, by using the heating and pressurization of the sample liquid by the heating element of the micropump, a sterilizing action or a sterilizing action of the measured sample liquid is obtained, which is preferable from the viewpoint of biohazard countermeasures. .

A fluid flow is formed in the flow portion 56 by the liquid sending action. At this time, the sample liquid is wrapped like a sheath according to the sheath flow principle to form a thin flow. Of fine particles flow one by one in the flow section.

On the surface of the first substrate 51, a sensitive element for measuring fine particles in the sample liquid is provided together with the micropump 58. Specifically, in order to optically measure fine particles, a first light detecting element 59, a first optical filter 60 having a wavelength selecting function, and a second light detecting element 6 are provided.
The first and second optical filters 62 are formed by the same manufacturing method as in the above embodiment, and detect fluorescence and scattered light from fine particles. It is preferable that the light detecting element is provided with a light stopper for blocking direct light incident from the light source. Further, in order to electrically measure the fine particles, two electrodes 63 and 64 are formed on the substrate, and the electric impedance between them is measured. The measured value of the electrical impedance mainly contains the volume information of the particles. The above-mentioned members constitute a measuring unit for measuring fine particles in the sample liquid. In the present embodiment, an example in which the fine particles are optically and electrically measured is described.

As shown in FIG. 12, a heating element 58 of a micropump, first and second light detecting elements 59 and 61, and electrodes 63 and 64 are joined to the first substrate 51. In the same manner as in the previous embodiment, the conductive patterns are connected to each other, and the end portions 74 of the conductive patterns are exposed to the outside so that contact with the external terminals can be made.

The above members are all integrated into a cartridge to form a cartridge. On the other hand, as shown in FIG. 11, a light source
5, 67 are provided separately from the cartridge. Light sources 14 and 16 that generate light having a wavelength suitable for fluorescence excitation are used. For example, a semiconductor laser or the like is suitable. Lens units 66 and 68 for condensing a light beam from the light source are integrally formed on the upper surface of the second substrate. The form of the light irradiation unit is not limited to this, and various variations are conceivable as in the previous embodiment. Further, it is easy to form an array of measurement modules as shown in FIG.

FIGS. 13 and 14 show a modification of the cartridge, and are a side view and a top view, respectively. In this example, the sample liquid is supplied from the outside together with the sheath liquid, and one end of the sample tube 70 is exposed to the outside of the cartridge, and is connected to an external sample supply mechanism. Optical fibers 70 are provided on both side surfaces of the two optical detection units to receive fluorescence or scattered light diverging to the side due to light irradiation on the fine particles.
71, 72, and 73 are embedded and guided to a detection element (not shown) such as a photomultiplier. Also, the nozzle opening 57
Has a configuration in which holes are formed in the upper substrate so as to discharge droplets upward. Other configurations are the same as in the above embodiment.

FIG. 15 is a diagram showing a configuration of an entire system for performing measurement by mounting the cartridge. The cartridge 100 described above is a cartridge holder 1
01 is held. Although only one cartridge is shown in the figure, a plurality of samples can be measured simultaneously or sequentially by mounting a plurality of similar cartridges in parallel or using an arrayed cartridge as shown in FIG. can do.

A plurality of sample containers 104 are arranged in the rack 103, and each accommodates a plurality of sample liquids which have been subjected to pretreatment (eg, purification processing and reaction with reagents).
The dispenser device 102 uses the pipette 105 to sequentially supply the sample liquid in each sample container 104 to the cartridge 100.

On the other hand, a sheath liquid container 114 containing a sheath liquid such as a PH buffer solution / physiological saline solution or distilled water therein contains:
Tube 1 via regulator 115 that controls the flow rate
09 is connected, and the distal end of the tube 109 is
1 is connected to the sheath liquid supply port of the cartridge 100 attached to the cartridge 1. The state of the sheath flow is controlled by adjusting the regulator 115.

Electrodes are provided on the cartridge holder 101, and are connected to the exposed conductive patterns of the cartridge 100 when the cartridge is mounted. This electrode is electrically connected to the drive / detection circuit 111 and the drive / detection circuit 1
11 is a drive of the light sources 65 and 67, a dispenser device 102
, The regulator 115, the micropump in the cartridge, the output from the two optical detection elements in the cartridge, and the electrical impedance between the two electrodes. The computer 112 controls the entire system and performs particle analysis based on the detection results. Many detection data can be obtained by measuring individual particles, and various analysis methods using statistical methods such as histograms and cytograms are known as methods for analyzing particles using the data. The analysis result is output to a display, a printer, or the like attached to the computer 112.

When measuring by driving the micropump, waste liquid is discharged from the nozzle of the cartridge 100.
This is received by the waste liquid container 113 and the waste liquid is stored in the container. In this embodiment, since the heating element is used as the micropump, the waste liquid discharged by the heating action and the pressure action of bubble generation is sterilized or sterilized.
It is further preferable that the waste liquid is sterilized using heat, ultraviolet rays, chemicals, or the like in the chamber 13.

As described above, the present system uses the cartridge 10
Since 0 is disposable and is replaced with a new one each time one sample is measured, the system is simplified and a small and low-cost sample measurement system is provided. Further, by making the disposable, the micropump and the sensitive element do not require much durability, and the cartridge can be supplied at a lower cost. In the cartridge and the system according to the present embodiment, various effects can be obtained similarly to the previous embodiment.

<Embodiment 3> FIG. 16 shows the configuration of a measurement system according to a further embodiment. In the figure, 150 is a frame, 151 is a holder, and the drawing shows the cartridge 100.
Indicates a state in which the cartridge is mounted on the holder 151, and the cartridge is replaced every time one sample is measured. Cartridge 10
Numeral 0 has substantially the same structure as any of the above-described ones, but a condenser lens 120 is formed on the upper surface. As the condenser lens 120, a spherical lens, a Fresnel lens, a zone plate, or the like can be used. 152, a light source such as a semiconductor laser or a lamp; 153, a lens for converting light from the light source 152 into a parallel light flux; 154, a half mirror;
55 is a condenser lens, and 156 is a light receiving element.

In this configuration, light emitted from the light source 152 is converted into parallel light by the lens 153. This light passes through the half mirror 154 and reaches the condenser lens 120 of the cartridge 100. The parallel light is condensed on the channel 121 by the condensing lens 120, and the sample in the channel is irradiated with the condensed light. Here, of the scattered light and fluorescent light emitted from the sample in all directions by the irradiation, the component condensed again by the condensing lens 120 to become parallel light and reflected by the half mirror 154 is condensed by the lens 155, and is condensed by the lens 155. Photoelectric conversion is performed to obtain measurement data. A wavelength filter (not shown) is provided in front of the light receiving element 156 so as to extract only a target fluorescence or scattered light component. It should be noted that the half mirror 154 may be replaced with a dichroic mirror having a characteristic of transmitting the irradiation light wavelength and reflecting the target fluorescence wavelength.

Here, the features of this embodiment will be described. In putting the system of this embodiment into practical use, the mechanical mounting position accuracy when the cartridge is mounted on the holder 151 is as follows:
Generally, it is about several tens μm to several hundreds μm in the horizontal direction. Then, there is a possibility that the optical axis of the light beam emitted from the light source 152 and the center axis of the flow path 121 of the cartridge mounted on the holder may be shifted relative to each other by the mechanical accuracy described above. At this time, if the irradiation light is not parallel light, the condensing position of the irradiation light changes according to the relative displacement between the cartridge and the irradiation light as shown in FIG. Here, if the width of the channel 121 of the cartridge is, for example, 10 μm, the light-collecting position may deviate from the channel 121 in some cases, and a change in the light-collecting position greatly affects the measurement accuracy.

On the other hand, the present embodiment is characterized in that the light incident on the cartridge is parallel light, and this light is condensed at the center position of the flow path 121 by the condenser lens 120. . Thus, as shown in FIG. 17A, the irradiation light is always focused on a certain position of the flow path 121, and is not affected by the mounting position accuracy of the cartridge.

FIG. 18 shows a configuration of a measurement system according to a modification of FIG. This is similar to the system shown in FIG. 16, but the light receiving system is different, so that more detailed measurement data can be obtained. 16 denote the same members, and a description of the same members will be omitted. In FIG. 18, 16
Reference numeral 0 denotes a spectral element such as a grating or a prism, 161 denotes a lens, and 162 denotes an array-type light receiving element for individually receiving each split wavelength component. When light is condensed and irradiated on the sample of the cartridge similarly to the previous embodiment, fluorescence or scattered light is generated from the sample. Of these, the light is condensed again by the condensing lens 120 to become parallel light,
The components reflected at 4 are split by the spectroscopic element 160 and are individually detected by the light receiving element 162 for each wavelength component via the lens 161. More detailed analysis is possible based on the data thus obtained.

FIG. 19 is a diagram showing the configuration of a measurement system according to a modification of FIG. The present embodiment is characterized in that the cartridge itself has a light collecting function and a spectral function. FIG.
In 9, 170 is a light source for generating irradiation light, and includes a light emitting element, a lens, and the like. 171 is a reflection mirror for directing the irradiation light from the light source 170 to the measurement position of the cartridge 100 mounted on the holder 151.
An irradiation system including the light source 170 and the mirror 171 is built in a lower part of the frame 150. Also, frame 15
Above 0, a light receiving system including a lens 161 and an array type light receiving element 162 is provided. On the other hand, cartridge 1
Reference numeral 00 denotes a gradient index lens 175, and further thereon a spectral element 176 such as a grating or a prism. Note that the set of the lens 175 and the spectral element 176 can be replaced with a zone plate having both a light collecting function and a spectral function.

In this configuration, the mounted cartridge 10
The sample 0 is irradiated with light from below. The generated fluorescence is condensed by the refractive index distribution type lens 175, becomes parallel light, and is separated by the spectroscopic element 176. This light is received by the array type light receiving element 162 above the cartridge 100 for each wavelength component.

[0070]

According to the present invention, the pump means having the heating element is provided at a position substantially facing the flow path on the downstream side of the measurement position, so that the measurement can be performed smoothly without denaturing or damaging the sample liquid. By applying pressure and heating, especially heat to the sample liquid after the measurement, a sufficient bactericidal action or sterilization can be obtained.

Further, a stable fluid system can be obtained, and the control response of fluid control is high. Further, since there is almost no dead space in the flow path, a small amount of the sample liquid is required.

Therefore, the amount of waste liquid generated is small, and it is sufficiently sterilized or sterilized by heating. Therefore, the effect of the present invention is great also from the viewpoint of environmental problems such as measures against biohazard. In particular, since such a biohazard countermeasure can be performed by the pump means for sending the sample itself, the efficiency of measurement and the simplification of the apparatus can be achieved.

[Brief description of the drawings]

FIG. 1 is a side view illustrating a configuration of a cartridge according to an embodiment.

FIG. 2 is a top view of each of a second substrate and a first substrate constituting the cartridge.

FIG. 3 is an assembly view of the cartridge.

FIG. 4 is a diagram showing a first modification of the embodiment.

FIG. 5 is a diagram showing a second modification of the embodiment.

FIG. 6 is a diagram showing a third modification of the embodiment.

FIG. 7 is a diagram for explaining the operation of the micropump.

FIG. 8 is a diagram showing a structure of a heating element of the micropump.

FIG. 9 is a diagram illustrating a structure of an optical detection unit.

FIG. 10 is a diagram illustrating an entire measurement system according to an embodiment.

FIG. 11 is a side view illustrating a configuration of a cartridge according to a second embodiment.

FIG. 12 is a top view of the second embodiment.

FIG. 13 is a side view of a modification of the second embodiment.

FIG. 14 is a top view of a modification of the second embodiment.

FIG. 15 is a diagram illustrating an entire measurement system according to a second embodiment.

FIG. 16 is a diagram illustrating a configuration of a measurement system according to a third embodiment.

FIG. 17 is a diagram for explaining features of the system in FIG. 16;

FIG. 18 is a diagram showing a configuration of a measurement system according to a modification of FIG.

FIG. 19 is a diagram showing a configuration of a measurement system of another modified example.

FIG. 20 is a configuration diagram of a conventional device.

FIG. 21 is a configuration diagram of another conventional apparatus.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 1st substrate (silicon substrate) 2 2nd substrate (glass substrate) 3 3rd substrate (glass substrate) 4 Accumulation part 5 Inlet 6 Insoluble carrier reagent 7 Distribution part 8 Nozzle opening 9 Micropump heating element 10 12 Light receiving element 11 13 Optical filter 14 16 Light source 15 17 Condensing lens 21 22 Condensing lens part 23 24 Optical fiber 51 First substrate (silicon substrate) 52 Second substrate (glass substrate) 53 Sample liquid receiving part 54 Sample tube 55 Sheath liquid supply Port 56 Flowing part 57 Nozzle opening 58 Heating element of micropump 59 61 Light receiving element 60 62 Optical filter 63 64 Electrode 65 67 Light source 66 68 Condensing lens part 70 71 72 73 Optical fiber 74 Conductive pattern

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI G01N 21/01 G01N 21/01 Z 21/75 21/75 Z (72) Inventor Shunichi Onishi 3-30 Shimomaruko, Ota-ku, Tokyo No. 2 Canon Inc. (72) Inventor Kazumi Tanaka 3-30-2 Shimomaruko, Ota-ku, Tokyo No. 2 Canon (72) Inventor Masanori Sakuranaga 3-30-2 Shimomaruko, Ota-ku, Tokyo (72) Inside the Canon Inc. Takayuki Yagi 3-30-2 Shimomaruko, Ota-ku, Tokyo Inside Canon Inc. (72) Yoshito Yoneyama 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Stock In-company (72) Inventor Kazuo Isaka 3-30-2 Shimomaruko, Ota-ku, Tokyo Within Canon Inc. (56) References JP-A-3-178641 (JP, A) JP-A-5-306683 (JP, A) JP-A-5-296914 (JP, A) "Nikkei Electronics August 21" Nikkei BP August 21, 2001 p. 135-139 (58) Fields surveyed (Int. Cl. ⁶ , DB name ) G01N 35/08 G01N 1/10 G01N 1/10 101 C12M 1/34 G01N 21/75 G01N 21/01 JICST file (JOIS)

Claims (19)

(57) [Claims] 1. An inlet for injecting a sample, a flow section through which the injected sample is flown, including a measurement position in the middle, and a position substantially facing a flow path downstream of the measurement position of the flow section. And a pump means having a heating element provided in the sample measuring device. 2. The device according to claim 1, wherein a sensitive element is provided near the measurement position. 3. The device according to claim 2, wherein a plurality of said sensitive elements are provided along a flow direction of the flow part. 4. The device according to claim 3, wherein said sensitive element is a light receiving element, and said light receiving element is provided with an optical filter in front of said light receiving element. 5. The device according to claim 1, wherein an optical function portion having a light condensing function is formed on the measurement position. 6. The device according to claim 5, wherein the optical function unit has a function of converging a parallel light beam supplied from the outside to a measurement position of the circulation unit. 7. The device according to claim 1, further comprising a storage part for storing the injected sample, wherein the storage part is filled with a reagent. 8. The reagent according to claim 7, wherein the reagent includes a biological substance.
Devices. 9. The device according to claim 1, further comprising an introduction port for introducing a sheath liquid together with the sample, wherein the flow section forms a sheath flow. 10. The device according to claim 1, wherein a plurality of said distribution sections are formed in an array in parallel. 11. A method for manufacturing a sample measuring device, wherein the device according to claim 1 is manufactured by a step including a semiconductor manufacturing process. 12. The semiconductor manufacturing process includes a step of forming a pattern on a substrate by a method including lithography, and a step of bonding the substrate to another substrate.
Manufacturing method. 13. A cartridge having a holding mechanism for holding a cartridge for measuring a sample, and measuring means for measuring a sample of the cartridge, wherein the cartridge has an inlet for injecting a sample, and the injected sample. Is flowed, a distribution part including a measurement position in the middle, and has a feeding action of the sample in the distribution part, and is provided at a position substantially facing a flow path near an outlet downstream of the measurement position of the distribution part. And a pump means having a heating element. 14. The system of claim 13, wherein said measurement means comprises means for applying measurement energy to said measurement location. 15. The system of claim 14, wherein said measured energy comprises light energy. 16. The system according to claim 15, wherein said measuring means includes a spectroscopic element and a light receiving element. 17. The system according to claim 15, wherein a light receiving element for receiving light emitted from the measurement position is provided inside the cartridge. 18. The system according to claim 15, wherein a light receiving element for receiving light emitted from the measurement position is provided outside the cartridge. 19. The system according to claim 15, further comprising means for applying a parallel light beam for measurement to the cartridge, wherein the cartridge is provided with an optical function unit for condensing the parallel light beam at the measurement position.