Patent Application
20070263046
This application is a U.S. Continuation Application of International Application PCT/JP2006/300136 filed Jan. 10, 2006.
The present invention relates to a detection apparatus for use in detecting a detection-target substance target substance contained in a liquid sample. In particular, the present invention relates to a detection apparatus comprising a cartridge and a processing unit adapted to be combined with the cartridge. More specifically, the present invention relates to a cartridge-type detection apparatus for generating information on existence, concentration, and/or composition, or the like in a liquid sample containing a detection-target substance, which comprises a detection cartridge formed with a passage or passages for passing a liquid sample, and a processing unit adapted to be loaded with the cartridge so as to produce information about the detection-target substance contained in the liquid sample being passed through the cartridge.
Japanese Patent Laid-Open Publication No. 10-311829 (JP 10-311829A) discloses a disposable card-type portable analysis system. This analysis system comprises a testing tool which includes a sensor for detecting at least one test parameter value about a human or animal body fluid and generating an output signal indicative of the detected value, and a portable analysis unit which includes a processing section for receiving the signal from the testing tool and processing the received signal, and a display section.
The card-type disposable testing tool comprises two base plates designed to be liquid-tightly superimposed on each other while interposing a thin partition plate therebetween. A first one of the base plates has an inner surface formed with a passage for passing a human or animal body fluid serving as a test sample, and a first body-fluid reservoir portion in liquid communication with one end of the passage. The first base plate is formed with a body-fluid inlet port penetrating therethrough in a thickness direction thereof. The sensor is mounted on an inner surface of a second one of the base plates. The inner surface of the second base plate is formed with a concave portion for receiving therein a reagent container which contains a reagent for calibrating the sensor, and a second body-fluid reservoir portion in liquid communication with the first body-fluid reservoir portion of the first base plate via an opening of the partition plate.
The portable analysis unit has an insertion slot for inserting the testing tool therethrough. When the testing tool is inserted into the portable analysis unit, the reagent container placed in the reagent-container-receiving concave portion is broken, and the reagent is led to the sensor to calibrate the sensor in advance of an actual analysis. Then, a human or animal body fluid is injected from the inlet port. The body fluid is supplied to the sensor through the passage formed in the inner surface of the first base plate, and subjected to a measurement. An electrical signal generated during the measurement is sent to the analysis unit, and processed by the processing section. An obtained analysis result will be indicated on the display section.
This analysis system has advantages of being able to perform an on-site test on its portability, and to allow a body-fluid sample to be directly injected therein using an injector, such as a syringe, so as to prevent the body-fluid sample from contacting ambient atmosphere. However, the analysis system is intended to analyze a high concentration of liquid, such as a human or animal body fluid, and is therefore unusable for a concentration measurement or chromatography analysis of an extremely small amount of detection-target substance, such as a hazardous heavy metal contained in soil.
U.S. Pat. No. 6,110,354 discloses an analyzer equipped with a microband electrode arrangement and adapted for use in an analysis of drinking water, waste water, a biological fluid such as blood or urine, etc. An analytical principle of this analyzer is to detect a faradaic component generated when an electrolyte liquid as a test sample comes into contact with the electrodes. In one illustrated embodiment, the analyzer has a plate-shaped sensor comprising a flat substrate. It would be understood that the microband electrode arrangement makes it possible to suppress the generation of a non-faradaic component so as to provide enhanced sensitivity allowing a detection of a hazardous metal contained in an aqueous solution in a small amount. However, even with such detection sensitivity, this analyzer is hardly used for the concentration measurement of an extremely small amount of target substance, such as a hazardous heavy metal contained in soil.
It is a major object of the present to provide a detection apparatus comprising a detection cartridge a processing unit adapted to be used in combination with the cartridge, usable in a simplified manner with less restriction on the type of targets to be detected and the site where it is used.
It is another object of the present invention to provide a detection apparatus capable of being used in concentration detecting devices, liquid chromatography analysis, and immunoassay processes, and other various detection methodologies for a detection-target substance contained in a liquid sample.
In a specific aspect, it is yet another object of the present invention to provide a cartridge-type simplified detection apparatus which is conveniently used as a portable device.
It is still another object of the present invention to provide a cartridge-type conveniently carried detection apparatus having a structure which allows a passage line for liquid communication essential to detections, to be arranged in a significantly compact manner.
It is yet still another object of the present invention to provide a concentration detection apparatus capable of detecting a concentration of a detection-target substance contained in a sample even if the concentration is extremely low.
It is another further object of the present invention to provide a cartridge-type portable simplified detection apparatus capable of performing an analysis, such as chromatography analysis, of a liquid sample, in a simplified manner even at a site where a sampling is carried out.
It is still a further object of the present invention to provide a readily-portable analysis apparatus capable of readily performing concentration detection, liquid chromatography analysis, immunoassay analysis, or other detection, at any location without restriction on test locations.
It is an additional object of the present invention to provide a detection cartridge and/or a processing unit for use in the above cartridge-type detection apparatus and/or analysis apparatus.
In order to achieve the above objects, according to the broadest aspect of the present invention, there is provided a cartridge-type detection apparatus which comprises a detection cartridge having a passage for passing a sample liquid containing an detection-target substance, and a processing unit adapted to be loaded with the detection cartridge so as to produce information about the target substance contained in the sample liquid passed through the detection cartridge. The detection cartridge includes an storing section for temporarily storing the target substance, at least a part of a detection mechanism, a liquid passage adapted to be selectively routed through either one or both of the storing section and the at least a part of the detection mechanism, and a plurality of ports in liquid communicate with the liquid passage. As used in connection with the present invention, the term “detection” is intended to mean “generating information about whether an target substance is present or absent, or a property of an target substance, such as a concentration or composition of the target substance”, and includes assay or analysis, such as quantitative analysis and/or qualitative analysis.
The processing unit includes a reagent tank and a liquid feed pump. In this aspect of the present invention, the detection cartridge is associated with the processing unit in a manner that the liquid passage is switched between a first route for allowing the sample liquid supplied into the detection cartridge to be passed through the storing section and then discharged out of the detection cartridge, and a second route for allowing a reagent to be supplied from one of the plurality of ports to the storing section of the detection cartridge by means of the liquid feed pump, and successively passed through the storing section and the at least a part of the detection mechanism.
In one embodiment of the present invention, the sample liquid can be supplied to the detection cartridge independently from the processing unit. When the sample liquid supplied into the detection cartridge reaches the storing section, the target substance contained in the sample liquid is temporarily stored in the storing section. The storing section may be made of a material having a relatively large surface area, such as a porous material including a porous ceramic material, a fibrous material or a fine particle material, and may have a surface with a structure modified by a functional group which exhibits a chemical reaction with or an adsorption action on the target substance. In this case, the target substance will be absorbed and held to/by the material of the storing section. The remaining sample liquid passed across the storing section is discharged out of the detection cartridge from one of the ports of the detection cartridge. Alternatively, a waste liquid reservoir may be formed inside the detection cartridge, and the sample liquid from the storing section may be led to the waste liquid reservoir.
Then, a reagent is supplied from the liquid feed pump of the processing unit into the detection cartridge, and passed through the storing section. This reagent has a function of eluting the target substance which is held by the storing section through a chemical reaction or an absorption action. By having the reagent passed through the storing section, the target substance held by the storing section is eluted from the storing section, and passed through the detection cartridge in a downstream direction together with the reagent in a manner dissolved therein. The reagent containing the target substance is temporarily sent to a passage outside of the detection cartridge and then returned to the detection cartridge, or sent toward the detection mechanism disposed on the downstream side through the internal passing the detection cartridge without being sent out of the detection cartridge.
The reagent tank disposed in the processing unit may be connected to the liquid feed pump. In this case, the processing unit may be provided with a plurality of the reagent tanks, and a tank switching valve mechanism may be provided for alternately provide liquid communication between a desired one of the plurality of reagent tanks and the liquid feed pump. The processing unit may also be provided with a waste liquid tank, and the sample liquid discharged from the detection cartridge may be led to the waste liquid tank.
In another embodiment of the present invention, the cartridge-type detection apparatus is designed, when the detection cartridge is unloaded from the processing unit, to define the first route of liquid passage for allowing the sample liquid supplied into the detection cartridge to be passed through the storing section and then discharged out of the detection cartridge, and, when the detection cartridge is loaded into the processing unit, to define the second route of liquid passage for allowing a reagent to be supplied from one of the plurality of ports to the storing section of the detection cartridge by the action of the liquid feed pump, and successively passed through the storing section and at least a part of the detection mechanism. In this case, the cartridge-type detection apparatus may have a valve mechanism for allowing the liquid passage to be switched between these routes, and this valve mechanism may be disposed in the processing unit.
The detection apparatus of the present invention can be applied to detections of various different substances. In one aspect of the present invention, the detection apparatus is designed as a concentration detection apparatus for providing information about a concentration of an target substance contained in a sample liquid. In this case, the detection cartridge is designed to generate an electric signal indicative of a concentration of the target substance. As one example, the sample liquid is prepared by dissolving a sample, such as soil or mud containing a small amount of target substance, in liquid, such as water.
In one embodiment where the present invention is applied to a concentration detection apparatus, the detection cartridge includes a sample-liquid inlet portion for feeding therethrough a sample liquid having a sample dissolved therein, and a liquid passage extending from the sample-liquid inlet portion. The storing section is disposed in the liquid passage. In this embodiment, the storing section is formed as an enrichment section for enriching an target substance contained in the sample liquid. This enrichment section may be provided in the form of a filter having an ability to absorb the target substance. The detection mechanism provided in the detection cartridge is formed as a detection electrode arrangement. The target substance absorbed to the filter is eluted into an eluent liquid supplied as a reagent, and sent to the detection electrode arrangement serving as the detection mechanism. When the eluent liquid containing the eluted target substance reaches the detection electrode arrangement, a detection electric signal is generated by the electrode.
In the detection apparatus according to this embodiment of the present invention, the processing unit includes a read section for reading the electric signal from the detection cartridge, and produce information about a concentration of the target substance. The detection cartridge in this embodiment may include a waste liquid reservoir. In this case, the liquid passage is formed to extend from the sample-liquid inlet portion to the waste liquid reservoir.
The read section includes processing means which is operable, in response to receiving the electric signal from the detection cartridge, to process the received electric signal so as to produce information about a concentration of the target substance in the sample. The processing unit may be optionally provided with a display section for indicating a detection result.
The cartridge-type concentration detection apparatus according to this embodiment of the present invention may be used for detecting a heavy metal contained in soil or mud. In this case, the electrode arrangement in the detection cartridge is configured to generate an electric signal indicative of a concentration of a heavy metal contained in a sample liquid, and the read section is designed to read the electric signal from the detection cartridge so as to produce information about a concentration of the heavy metal.
In the cartridge-type concentration detection apparatus for detecting a concentration of a heavy metal, the detection cartridge includes a sample-liquid inlet portion for feeding the sample liquid therethrough, a liquid passage in liquid communication with the sample-liquid inlet portion, a storing section serving as an enrichment section disposed in the liquid passage extending from the sample-liquid inlet portion and adapted to enrich the sample liquid, and a detection electrode arrangement. The storing section or the enrichment section includes an absorptive element disposed in the liquid passage and adapted to absorb the heavy metal. The storing section includes an eluent-liquid supply section adapted to be associated with the enrichment element and supply an eluent liquid for eluting the heavy metal absorbed to the absorptive element, through the absorptive element and toward the detection electrode arrangement. Thus, the heavy metal absorbed by the absorptive element is eluted by a predetermined volume of the eluent liquid from the eluent-liquid supply section, and the eluted heavy metal is brought into contact with the detection electrode arrangement to allow the detection electrode arrangement to generate an electric signal indicative of a concentration of the heavy metal.
In a cartridge-type concentration detection apparatus according to another embodiment of the present invention, the storing section or enrichment section includes an absorptive element adapted to receive the sample liquid from the sample-liquid inlet portion and absorb the target substance contained in the sample liquid, and the enrichment section is associated with an eluent-liquid supply section whereby an eluent liquid which functions to elute the target substance absorbed by the absorptive element is directed through the absorptive element and toward the detection electrode arrangement, so that the target substance absorbed by the absorptive element is eluted by a predetermined volume of the eluent liquid from the eluent-liquid supply section, and the eluted target substance is brought into contact with the detection electrode arrangement to allow the detection electrode arrangement to generate an electric signal indicative of a concentration of the target substance
In a cartridge-type concentration detection apparatus according to yet another embodiment of the present invention, the detection cartridge includes a sample-liquid inlet portion for feeding therethrough a sample liquid having the sample dissolved therein, an enrichment section for enriching the sample liquid fed in the sample-liquid inlet portion, a detection electrode arrangement, and a passage for providing liquid communication between respective ones of the sample-liquid inlet portion, the enrichment section and the detection electrode arrangement. The enrichment section includes an absorptive element adapted to receive the sample liquid from the sample-liquid inlet portion and absorb the target substance contained in the sample liquid. The concentration detection apparatus further includes an eluent-liquid supply section adapted to be associated with the enrichment section and supply an eluent liquid for eluting the target substance absorbed to the absorptive element, through the absorptive element and toward the detection electrode arrangement, and a passage switching valve mechanism. This valve mechanism is adapted to be selectively shifted between a sample-liquid feed position for opening a sample-liquid feed passage extending from the sample-liquid inlet portion to the enrichment section and closing an eluent-liquid supply passage extending from the eluent-liquid supply section to the enrichment section, and an eluent-liquid supply position for closing the sample-liquid feed passage extending from the sample-liquid inlet portion to the enrichment section and opening the eluent-liquid supply passage extending from the eluent-liquid supply section to the enrichment section. Further, pumping means is provided for supplying an eluent liquid from the eluent-liquid supply section to the enrichment section when the passage switching valve mechanism is at the eluent-liquid supply position.
In this embodiment, the target substance absorbed to the absorptive element is eluted by a predetermined volume of the eluent liquid from the eluent-liquid supply section, and the eluted target substance is brought into contact with the detection electrode arrangement to allow the detection electrode arrangement to generate an electric signal indicative of a concentration of the target substance. Each of the eluent-liquid supply section, the valve mechanism and the pumping means may be housed in a casing of the processing unit.
The detection cartridge may be formed with a discharge passage for discharging the sample liquid after being passed across the absorptive element, outside the detection cartridge, and a waste liquid reservoir for storing the eluent liquid after being passed across the electrode arrangement. In this case, the discharge passage and a passage between the electrode arrangement and the waste liquid reservoir may be opened and closed, respectively, in a process of feeding the sample liquid, and closed and opened, respectively, in a process of supplying the eluent liquid. The absorptive element may be formed as any one of a membrane (or film), a fine particle and a porous body.
The absorptive element may be a cationic substance-absorptive element. In this case, the absorptive element may be formed of a material having a surface modified with a sulfonic acid group. Alternatively, the absorptive element may be an anionic substance-absorptive element. In this case, the absorptive element may be formed of a material having a surface modified with a quaternary amine group. Alternatively, the absorptive element may be formed of a material having a surface treated by a heavy-metal receptor. This heavy-metal receptor may be any one of a chelating substance, a clathrate, and a heavy-metal absorptive substance. The chelating substance may be either one of iminodiacetic acid and ethylene diamine group. The clathrate may be either one of porphyrin and calixarene. The heavy-metal absorptive substance may be either one of apoenzyme and heavy-metal absorptive antibody.
The detection cartridge may be formed in a card shape. In this case, the processing unit preferably has a casing formed with an insertion portion for allowing insertion of the card-shaped cartridge.
Preferably, the electrode arrangement includes at least one microelectrode element of a size not larger than 10 μm. In this case, the microelectrode element is preferably prepared by attaching an insulation sheet on an upper surface of an electrode member and forming in the insulation sheet a hole of a size not larger than 10 μm.
The electrode arrangement may include a plurality of working electrode elements, at least one counter electrode element, and at least one reference electrode element. Each of the plurality of working electrode elements may be formed to have a different area so as to work for a measurement of a different concentration range.
Alternatively, the electrode arrangement may include at least one working electrode element, at least one counter electrode element, and at least one reference electrode element.
The enrichment section serving as the storing section may have a structure where a cation-absorptive element adapted to absorb a cationic substance and an anion-absorptive element adapted to absorb an anionic substance are arranged in parallel relation to each other. Further, the electrode arrangement serving as the detection mechanism may include two electrode pairs each associated with a corresponding one of the cation-absorptive element and the anion-absorptive element.
In a cartridge-type concentration detection apparatus according to still another embodiment of the present invention, the detection cartridge includes therewithin a sample-liquid inlet portion for feeding therethrough a sample liquid as a concentration-detection target, a liquid passage in liquid communication with the sample-liquid inlet portion, and a concentration-detection electrode arrangement disposed in the liquid passage and adapted to generate an electric signal indicative of a concentration of a specific substance contained in the sample liquid which is fed in the liquid passage and passed through the concentration-detection electrode arrangement. The electrode arrangement includes a plurality of working electrode elements, at least one counter electrode element, and at least one reference electrode element. Each of the plurality of working electrode elements is formed to have a different area so as to work for a measurement of a different concentration range.
In a cartridge-type concentration detection apparatus according to yet still another embodiment of the present invention, the detection cartridge has a flat card shape, and the processing unit has a casing formed with an insertion portion for allowing insertion of the card-shaped cartridge. This detection cartridge includes therewithin a sample-liquid inlet portion for feeding therethrough a sample liquid as a concentration-detection target, a liquid passage in liquid communication with the sample-liquid inlet portion, and a concentration-detection electrode arrangement disposed in the liquid passage and adapted to generate an electric signal indicative of a concentration of a specific substance contained in the sample liquid which is fed in the liquid passage and passed through the concentration-detection electrode arrangement.
This detection cartridge includes a first sheet made of a resin material and formed to have one surface with a concave portion defining at least a part of the liquid passage, a second sheet made of a resin material and formed with a through-hole constituting the sample-liquid inlet portion and a concave portion receiving therein the concentration-detection electrode arrangement, and a third sheet made of a resin material and formed with a through-hole constituting the sample-liquid inlet portion. The first, second and third sheets are laminated in turn while interposing an insulation sheet between adjacent ones thereof. The concentration-detection electrode arrangement includes an electrode element disposed in the electrode-receiving concave portion of the second sheet, and the second sheet and the third sheet are laminated to allow the electrode element to face the third sheet. Further, the insulation sheet interposed between the second sheet and the third sheet has a hole formed at a position corresponding to the electrode element to expose a predetermined area of the electrode element, so that a liquid passage for leading the sample liquid to the electrode element is defined on a side of a surface of the third sheet facing the second sheet.
In this case, the first sheet of the cartridge may have a surface located to face the second sheet and formed with a concave portion constituting a waste liquid reservoir for receiving therein a waste liquid from the electrode element.
In the above embodiments of the present invention, the enrichment section for storing the target substance in an enriched manner is provided in the liquid passage. This makes it possible to perform the concentration detection without any problem even if the target substance is contained in the sample liquid in an extremely low concentration. If soil is contaminated by a hazardous substance, such as a heavy metal, it shall be controlled by environmental regulations even if a concentration of the substance is extremely low. While it has been considered that an on-site detection of a substance contained in such an extremely low concentration is impossible, the cartridge-type concentration detection apparatus according to each of the above embodiments of the present invention makes it possible to perform detection of a pollutant in a simplified manner at a location where a soil sample is collected. In this case, a sample liquid may be prepared by dissolving a soil sample. The enrichment section for enriching an target substance is preferably designed to include an absorptive element adapted to absorb the target substance. Alternatively, any other suitable enrichment means may be used. For example, an enrichment technique of heating a sample liquid to evaporate a liquid component, or a technique-type on a reverse osmosis membrane, may be used.
The cartridge-type concentration detection apparatus according to each of the above embodiments of the present invention can be applied to concentration detection of any substance, i.e., target substance, which allows the detection electrode arrangement to generate electric information about a concentration thereof when a liquid containing the target substance comes into contact with the detection electrode arrangement. Typically, the detection electrode arrangement comprises a working electrode, a counter electrode and a reference electrode. The working electrode is operable to absorb an target substance and then release the absorbed target substance into an eluent liquid when it comes into contact with the eluent liquid. A requirement for the working electrode preferably includes a capability to allow a potential to be applied thereto in a relatively wide range, i.e., a relatively wide potential window, and high resistances to corrosion and oxidation. The potential window means a potential range causing no generation/formation of electrochemically undesirable hydrogen ion and oxide film, and this range is varied depending on a material of the electrode and a pH value of a sample liquid.
A material of the working electrode preferably includes platinum, gold, mercury, silver, bismuth and carbon. While the working electrode may be made of one appropriately selected from these materials, it is preferable to select a material having high ability to adsorb an target substance, i.e., a measurement target. When the target substance is cadmium, lead and mercury, an electrode having a carbon surface may be reasonably used as a working electrode. As a working electrode for a measurement of arsenic and mercury, it is reasonable to use an electrode having a gold surface. For detecting hexavalent chrome, an electrode having a carbon surface may be used. The reason is that the electrode with a carbon surface has a property of excellently absorbing an aggregate of hexavalent chrome and diphenylcarbazide.
As the electrode with a carbon surface, a carbon electrode comprising a sintered body of a graphite/carbon mixture having a graphite/glassy carbon ratio of 70/30 is preferable. Generally, in contrast with an advantage of relatively high ability to absorb lead, cadmium and mercury, graphite has a problem about variation in substance-adsorptive capacity due to difficulty in uniforming its crystalline orientation, and swelling due to contact with liquid. As measures against this problem, graphite may be sintered after mixing glassy carbon therein to obtain a densified sintered body capable of suppressing the penetration of liquid. Further, the grassy carbon can randomly orient graphite crystals to minimize the variation in adsorptive capacity. The above sintered body with a graphite/glassy carbon ratio of 70/30 is advantageous in forming a working electrode having high sensitivity and excellent reproducibility.
The electrode with a gold surface is not limited to a specific material. In the present invention, a glass substrate may be coated with gold through a chromium layer to obtain a working electrode having an adequate function. In this case, a chromium film and a gold film may be formed through a sputtering process. A film thickness may be set, but not limited to, at about 40 nm for the chromium layer, and about 400 nm for the gold layer.
The counter electrode is provided as a means to form a current flow in cooperation with the working electrode, and any electrically conductive material may be used for the counter electrode.
The reference electrode is designed to exhibit a known and stable potential usable as a reference potential. A typical reference electrode may include a hydrogen electrode, a saturated calomel electrode (mercury/mercury chloride electrode), and a silver/silver-halide electrode. The silver/silver-halide electrode includes a silver/silver-chloride electrode having a silver surface which forms silver chloride through an equilibrium reaction with a chlorine-containing solution. In this electrode, even when a voltage is being applied thereto, silver and silver chloride are kept in an equilibrium state to allow a resulting potential to be maintained at a constant value so as to serve as a reference electrode. Although a silver/silver-bromide electrode and a silver/silver-iodide electrode may also be used, the silver/silver-chloride electrode is preferable in view of material availability and production costs.
Each of the electrodes is not limited to a specific size. As one example, a sheet, such as a double-faced adhesive tape, formed with a small hole may be attached on a thin plate-shaped electrode having a rectangular planar size of 3×8.4 mm and a thickness of 0.5 mm, to expose a predetermined area of a surface of the electrode. In view of facilitating a forming process and an attaching operation, a pre-formed thin plate-shaped electrode material is preferably attached onto a base plate of the detection cartridge. Alternatively, the electrode may be directly formed in the base plate of the detection cartridge.
The most typical detection is-type on an electrochemical measurement. In an electro-chemical reaction in an aqueous solution, a reaction rate on the electrode is greater than a mass transfer rate in the solution, and therefore a response delay due to the mass transfer rate, so-called “solution resistance”, occurs to cause difficulty in clarifying respective peaks. When a micron-size microelectrode is used, the controversial mass transfer will be changed from planar diffusion to point diffusion to reduce a response delay per unit area. Thus, peaks can be discriminated from each other in a smaller scale to provide enhanced sensitivity. In view of expecting this advantage, the electrode is preferably formed to have a minor axis dimension of 10 μm or less. A large number of such electrodes may be arranged in an array configuration to obtain a large total amount of current. The microelectrode having such a size can be prepared using semiconductor microfabrication techniques. For example, a circular-shaped microelectrode can be obtained by forming an insulation layer on an electrode substrate and then forming in the insulation layer a small hole having a diameter not larger than 10 μm. A comb-shaped electrode having a configuration where a plurality of working electrodes and counter electrodes each having a diameter not larger than 10 μm are alternately arranged may also be used to provide the same advantage.
In case of the detection of a heave metal based on the electrochemical measurement, it is desirable to minimize a charge current for increasing a potential of the working electrode up to a predetermined value. In this respect, it is necessary to facilitate electron transfer in a solution containing a heavy metal, and an electrolyte may be added to the solution for this purpose. The electrolyte may be any material capable of forming a salt in the solution. Preferably, the electrolyte includes potassium chloride, sulfuric acid, nitric acid, potassium nitrate and sodium hydroxide, in view of costs. The potential window of the working electrode is characteristically shifted toward a positive or negative side depending on a pH value. Thus, the electrolyte can also be used for adjusting the pH value to prevent the occurrence of a problem about generation of hydrogen from the working electrode and formation of an oxygen film, in a potential range for an intended measurement.
The eluent liquid is typically a solution containing an electrolyte, as described in detail later. In this case, the electrolyte-containing eluent liquid can be sent at a constant flow volume to allow a series of processes from the elution to the detection to be successively performed. This makes it possible to simplify a passage line and an operation and eliminate the need for managing a mixing ratio and a mixing speed between eluent and electrolyte liquids. Specifically, if an eluent liquid and an electrolyte liquid are prepared separately, it is essential to manage respective absolute volumes of the two liquids. Particularly, when a space for arranging the passage and the electrode is extremely small as in the concentration detection apparatus of the present invention, it is difficult to manage of the absolute volumes of the solutions. Thus, it is absolutely critical to allow both the elution process and the electrochemical measurement process to be performed using only a single solution.
As mentioned above, referring to the reference electrode, silver chloride is produced on silver formed by a printing process by having a chlorine-containing solution brought into contact with the printed silver as a reference-electrode activating liquid, and a weak electric current is delivered to the reference electrode. Thus, chlorine may be contained in the eluent liquid to eliminate the need for using the reference-electrode activating liquid separately, and provide a simplified structure. However, in a measurement of selenium, chlorine cannot be contained in the eluent liquid, because chlorine acts as an interfering substance during the electrochemical measurement process. Therefore, it is necessary to use the following reference-electrode activating liquid.
Under the condition that an extremely-weak current is supplied to the reference electrode, a reference-electrode activating liquid is brought into contact with the printed silver to create silver chloride on the printed silver so as to allow the reference electrode to maintain its original function. The reference-electrode activating liquid typically contains an appropriate amount of chlorine. A preferable content of chlorine is in the range of 0.05 to 3 M. An excessively small content of chlorine causes instability in formation of silver chloride, and an excessively large content of chlorine causes poor handling capability due to precipitation of solids. The activating liquid may be prepared by dissolving a predetermined amount of potassium chloride or sodium chloride in water.
The reference electrode is required to have an electrical interaction with the working electrode and the counter electrode, and therefore the reference-electrode activating liquid has to be in contact with the eluent liquid. One purpose of using the reference-electrode activating liquid and the eluent liquid in a separated manner is to prevent chlorine from acting as an interfering substance during the electrochemical measurement process. That is, it is necessary to prevent the reference-electrode activating liquid from flowing into a working electrode section, so that a separate reference electrode chamber is provided to define a liquid passage for providing liquid communication between the reference electrode chamber and the eluent liquid. Preferably, this liquid passage is formed as a micro-passage to prevent the occurrence of undesirable molecular diffusion. Alternatively, the detection cartridge may be designed to separate the reference electrode chamber and the eluent liquid by a porous film. In this case, the reference-electrode activating liquid is obliged to penetrate through the porous film by taking a relatively long time. Thus, in a concentration detection apparatus intended to detect an target substance in a short time of period, as in the present invention, it is more preferable to use the micro-passage. Further, it is desirable to install the reference electrode in the reference electrode chamber, and pre-contain the reference-electrode activating liquid in the reference electrode chamber or supply pre-contain reference-electrode activating liquid into the reference electrode chamber according to need. In case of pre-loading the reference-electrode activating liquid in the detection cartridge, the reference-electrode activating liquid may be advantageously contained in an aluminum pack to prevent precipitation of solids due to vaporization of a liquid component.
Depending on a type of the absorptive element, it is necessary to pass an absorptive element-activating liquid through the absorptive element before passing the sample liquid therethrough. For example, quaternary amine for use in absorbing arsenic, selenium and/or hexavalent chrome, can exhibit its absorption ability only after it comes into contact with an OH ion, because this absorption ability comes from a reaction where an OH ion is substituted with a target anion. In this case, the absorptive element-activating liquid is used. This absorptive element-activating liquid may include sodium hydroxide and potassium hydroxide.
The absorptive element is disposed in the passage on an upstream side relative to the electrode arrangement. As mentioned above, the absorptive element may be formed as any one of a membrane (or film), a fine particle and a porous body, or any combination thereof. The present invention may be applied to chromatography analysis, immunoassay or other detection, and one of these configurations of the absorptive element may be appropriately selected depending on the intended purposes. Each of the configurations will be specifically described below.
[Membrane]
The absorptive element is formed in a filter shape using fibers. Alternatively, a polymer or metal membrane formed with appropriate holes may also be used. For example, the membrane may be designed to have a surface which is formed in a specific configuration or modified with a functional group so as to exhibit an target substance absorption ability, or to have fibers which carry particles having a specific absorption function.
[Fine Particle]
The fine particle may be designed to have a surface which is formed in a specific configuration or modified with a functional group so as to exhibit an target substance absorption ability. The passage may be partially filled with such fine particles, for example, over a longitudinal length of about 10 mm or more in a column shape to perform chromatography. The particle-filled portion may have a rectangular parallelepiped shape or a cylindrical shape.
[Porous Body]
This absorptive element consists of a porous body having a large number of continuous pores. For example, the porous body includes a monolithic porous inorganic material such as porous ceramics or porous glass, and a porous polymer material such as porous polyacrylamide gel or porous styrene/divinylbenzene copolymer. The porous body may be designed to have a surface of each continuous pore which is formed in a specific configuration or modified with a functional group so as to exhibit a target substance absorption capability. The substrate or element which has continuous pores and an integral or single-piece structure may hereinafter be referred to as “monolithic substrate or element”. Further, a monolithic substrate having a length less than that allowing chromatography will hereinafter be referred to as “monolithic disc”, and a monolithic substrate having a length allowing chromatography will hereinafter be referred to as “monolithic column”. Each of these terms will be used depending on intended purposes. The monolithic column allows a liquid to be passed therethrough at a lower pressure than that in a resin-filled column. Thus, a low-pressure liquid feed pump may be used to facilitate reductions in size and power consumption while maintaining an analytical performance at the same level. For the same reason, the monolithic disc allows a liquid to be passed therethrough at a relatively low pressure, to facilitate a reduction in size of the apparatus. Each of the monolithic column and the monolithic disc having an integral structure can facilitate an operation of installing the absorptive substrate in a cartridge-type microreactor.
The absorptive element may be formed by any of variety of materials including: styrene/divinylbenzene copolymer; polymethacrylate resin; polyhydroxy methacrylate resin; polyvinyl alcohol; polyolefin typified by polyethylene, polypropylene and ethylene/propylene copolymer; olefin/halogenated olefin copolymer typified by ethylene/tetrafluoroethane copolymer and ethylene/chlorotrifluoro-ethylene copolymer; halogenated polyolefin typified by polytetrafluoroethylene, polyvinylidene-fluoride and polychlorotrifluoroethylene; polysulphone; silica; and alumina. The fibrous absorptive element may be made of a fibrous material, such as: various types of natural or regenerated fibers typified by a cellulosic material, a plant fiber including cotton and hemp, and an animal fiber including silk and wool; and various types of synthetic fibers including polyester fiber and polyamide fiber.
As long as a wall surface defining a passage has a function of absorbing an target substance, the absorptive element having any other configuration can also fulfill the same enriching function.
In order to provide the surface of the absorptive element with a property of absorbing a specific target substance, the surface may have a structure complementary to the specific target substance, or by immobilizing onto the surface a functional molecule which exhibits at least one of ion binding, coordinate bonding, chelate bonding, hydrophobic interaction, and interaction due to polarities in molecules.
The functional molecule exhibiting the interaction includes sulfo group, quaternary ammonium group, octadecyl group, octyl group, butyl group, amino group, trimethyl group, cyanopropyl group, aminopropyl group, nitrophenylethyl group, pyrenylethyl group, diethylaminoethyl group, sulfopropyl group, carboxyl group, carboxymethyl group, sulfoxyethyl group, orthophosphate group, diethyl(2-hydroxypropyl)aminoethyl group, phenyl group, iminodiacetate group, and chelate-forming group including ethylenediamine and sulfur atom, for example, functional groups, such as mercapto group, dithiocarbamate group and thiourea group, and atomic groups, such as avidin, biotin, gelatin, heparin, lysine, nicotinamide adenine dinucleotide, protein A, protein G, phenylalanine, castor bean lectin, dextran sulfate, adenosine 5′-phosphate, glutathione, ethylenediamine diacetate, procion red, aminophenylborate, cattle serum albumin, polynucleotide (e.g., DNA), and protein (e.g., antibody). These substances may be used independently or two or more of them may be used in combination.
The eluent liquid has a function of eluting an target substance absorbed to the absorptive element, from the absorptive element. The effectiveness of the eluent liquid depends on absorption mechanisms. Thus, a specific type of eluent liquid is selected in consideration of chemical characteristics of absorption. For example, in case of an eluent liquid containing an ion capable of being easily absorbed to a surface of the absorptive element, when the eluent liquid is passed through the absorptive element, the ion in the eluent liquid is exchanged with an target substance absorbed to the absorptive element in the form of an ion to allow the target substance to be eluted from the absorptive element. In the present invention, an eluent liquid having the following composition may be used depending on target substances.
In the measurement of cadmium, lead and/or mercury, an Empore™ disc cartridge (product name: Cation-SR, available from 3M) is used as the absorptive element, and a liquid (pH=about 4) containing 0.4 M of potassium chloride, 10 mM of citric acid and 3.5 mM of ethylenediamine is used as the elute liquid. This absorptive element is prepared by immobilizing fine particles having a particle size of 50 to 100 μm to Teflon® fibers, and forming the fibers into a membrane having a thickness of 0.5 to 0.75 mm. The fine particles and the Teflon® fibers are mixed at 10% and 90%, respectively. The absorptive element has a surface modified with a sulfonate group.
In the measurement of arsenic, selenium and/or hexavalent chromium, an Empore™ disc cartridge (product name: Anion-SR, available from 3M) is used as the absorptive element, and 1 M of sulfuric acid (pH=about 2) is used as the elute liquid. This absorptive element is prepared by immobilizing fine particles having a particle size of 50 to 100 μm to Teflon® fibers, and forming the fibers into a membrane having a thickness of 0.5 to 0.75 mm. The fine particles and the Teflon® fibers are mixed at 10% and 90%, respectively. The absorptive element has a surface modified with quaternary amine.
It should be noted that, depending on the type of the absorptive element, it is necessary to pass an absorptive element-activating liquid through the absorptive element before passing the sample liquid therethrough. For example, quaternary amine for use in absorbing arsenic, selenium and/or hexavalent chrome, can exhibit its absorption ability only after it comes into contact with an OH ion, because this absorption ability comes from a reaction where an OH ion is substituted with a target anion. In this case, the absorptive element-activating liquid is used. This absorptive element-activating liquid may include sodium hydroxide and potassium hydroxide.
A size of the absorptive element may be freely determined to an extent allowing an absorption capacity of the absorptive element to be not saturated during a course of absorbing a target target substance. For example, it may be calculated how much a substance capable of being absorbed to the absorptive element is contained in a liquid, so as to determine the size of the absorptive element. While an absorptive element having a relatively small absorption capacity makes it easy to increase an enrichment rate, saturation adsorption is likely to occur. Thus, it is necessary to select an absorptive element having a size with a desired absorption capacity. If the absorptive element is expected to have a chromatography function, it should be designed to have a column shape with a length of at least 10 mm or more in a direction of the passage.
In the absorptive element, a porosity and a size of a continuous pore are determined to an extent allowing the continuous pores to reliably contact a sample liquid and causing no problem about clogging. Preferably, as used for the absorptive element, a membrane has a fiber mesh size of about 0.3 μm or more, and a fine particle has a particle size of about 2 to 50 μm. Further, a monolithic column preferably has a continuous pore with a pore size of about 1 to 50 μm.
In the aforementioned embodiments of the present invention, an adequate measurement result can be obtained using the above Empore disc cartridge available from 3M. This disc cartridge has a significantly small thickness. Thus, a volume of the eluent liquid required for eluting a heavy metal absorbed to the absorptive element can be reduced, for example, to an extremely low value of 9 to 15 μl, and therefore a concentration of the heavy metal in the eluent liquid becomes higher to achieve analysis with high sensitivity. In an analysis of a small amount of target substance, it is critical to reduce a required volume of the eluent liquid to an extremely low value, irrespective of the configuration of the absorptive element.
In addition, the absorptive element formed as such a thin membrane allows a liquid feed pressure required for passing a sample liquid therethrough to become almost zero. A reduction in size of a pump is essential to downsizing of the apparatus, and a reduction in the liquid feed pressure is effective in this regard. From this point of view, it is preferable to use the absorptive element formed as a thin membrane.
The detection cartridge is formed with a micro-passage for transferring and storing various liquids therewithin. The liquid transfer passage is defined by a groove having a width of about several hundred μm to several mm, and a depth of several hundred μm. Preferably, the passage has a sectional area of about 100 μm2 to 1 mm2. An excessively large sectional area of the passage is likely to cause a problem about clogging of the passage with fine particles residing therein, and/or difficulty in releasing gas bubbles. An inner wall surface of the groove defining the passage may be subjected to a hydrophilic treatment to ensure the liquid transfer. The hydrophilic treatment additionally provides a function of preventing gas bubbles from staying in the passage.
As to a mechanism for loading the detection cartridge into the read section of the processing unit, the detection cartridge is preferably loaded into a casing of the read section in a mechanical manner. In view of allowing the electrodes and the ports of the detection cartridge to be reliably connected to terminals, the valve mechanism and supply ports of various liquids, in their proper positions, the casing of the processing unit preferably has a holder portion for holding the detection cartridge at a predetermined position. The detection cartridge and the casing are formed, respectively, with a depression and a protrusion to be conformably fitted to each other. Thus, the depression and the protrusion will be engaged with each other to allow the detection cartridge to be reliably held by the casing of the processing unit.
The electrochemical detection is performed through a plurality of pin-shaped terminals fixed to the holder portion. Preferably, each of the terminals is arranged at a position of a corresponding one of the electrodes of the detection cartridge after being loaded into the processing unit, and designed to be moved inwardly and outwardly by an action of a spring located on an inward side thereof, so as to ensure a reliable contact therebetween. Thus, when the detection cartridge is loaded into the processing unit, each of the terminals fixed to the holder portion at the predetermined position immediately above a corresponding one of the electrodes of the detection cartridge will be reliably brought into contact with the electrode by a biasing force of the spring. Then, according to a predetermined measurement profile, a voltage is applied to these electrodes to detect a current flowing through the electrodes, and the detection signal is sent to a storage section and/or the display section.
A liquid feed operation section may comprise the liquid feed pump, the valve mechanism for alternately opening/closing the ports during a liquid feed operation, and an electronic board for controlling the pump and the valve mechanism. The valve mechanism is connected to respective containers respectively containing the eluent liquid, the electrolyte liquid, the absorptive element pretreatment liquid, a cleaning liquid, and others.
Preferably, the liquid feed pump is designed to stably feed a small volume of liquid at a constant flow rate without pulsation. More specifically, the liquid feed pump is designed to stably achieve a flow rate of about 5 to 100 μl/min, and have a liquid feed pressure of 0.01 to 10 MPa. Further, the liquid feed pump preferably has a small and lightweight pump body, and low power consumption. A liquid feed pump meeting these requirements includes a syringe pump. A preferable syringe pump is “Pencil Pump” available from Uniflows Corp of Japan.
In one embodiment of the present invention, it is preferable to perform a measurement while supplying a sample liquid containing an target substance, at a constant flow rate. For this purpose, flow-volume detection means is preferably employed. By having a sample containing a target substance, such as a heavy metals supplied in liquid form, a number of heavy metal ions passed around a surface of the electrode arrangement per unit time can be increased, and thereby an amount of the target substance to be deposited on the electrode is increased to allow the measurement to be performed with higher sensitivity. In addition, a fresh sample liquid can be continuously supplied at a constant flow rate to eliminate the need for taking account of influences of a remaining volume of the absorptive element-activating liquid, the cleaning liquid or the like, and managing a total volume of the sample liquid. This makes it possible to perform a highly accurate analysis only by managing the flow rate. Further, the flow rate can be changed depending on types or concentrations of target substances, so as to perform a measurement under adequate conditions, and measure various types of target substances using a common chip.
In the above embodiment, both analyses of high and low concentration ranges can be simultaneously performed with high measurement accuracy by controlling the flow rate (i.e., linear velocity) of the sample liquid. The control of the flow rate providing this advantage can be achieved only if the flow-volume detection means is employed.
In the above embodiment of the present invention employing the flow rate control, a plurality of working electrodes may be provided, and each of the working electrodes may be formed to have a different surface area, or a portion of the passage receiving therein each of the working electrodes may be formed to have a different dimension, such as width and/or depth, so that an target substance will be deposited onto a surface of each of the working electrodes in a different amount to allow the analyses of high and low concentration ranges to be simultaneously performed. For example, when a first working electrode having a diameter of 1 mm and a second working electrode having a diameter of 2.5 mm are used in combination, two sensitivities having about 19 times disparity therebetween can be utilized. In this case, preferably, the second electrode for the detection of a low concentration range is disposed on a relatively upstream side of the passage, and the first electrode for the detection of a high concentration range is disposed on a relatively downstream side of the passage.
The present invention can be applied to an analysis apparatus for liquid chromatography analysis. That is, in another embodiment of the present invention, the cartridge-type detection apparatus may use a detection cartridge for liquid chromatography analysis. In this case, the detection cartridge may include a sample-liquid adjustment column and an absorbance measurement cell, and the processing unit may include a light source, an incident optical system for directing light from the light source toward the absorbance measurement cell of the detection cartridge, and a spectrometer operable, in response to receiving light transmitted through the absorbance measurement cell, to produce information about an target substance. In this embodiment, it is preferable to perform a measurement while supplying a sample liquid containing an target substance, at a constant flow rate, as with the aforementioned embodiment.
An target substance to be subjected to the liquid chromatography analysis may include protein, nucleic-acid oligomer, DNA, RNA, peptide, agrichemical, synthetic organic molecule oligomer, polymer, additive, monosaccharide, disaccharide, oligosaccharide, polysaccharide, saturated fatty acid, unsaturated fatty acid, glyceride, phospholipid, steroid, anion and cation. A principle of the measurement is commonly known, and a measurement technique-type on the commonly-known principle may be used in the present invention. In the liquid chromatography analysis, the storing section may be designed in the same manner as that of the aforementioned concentration detection apparatus.
In the present invention, the processing unit may have a cartridge loading portion for detachably loading the detection cartridge thereto, and a reagent-tank mounting portion for detachably mounting the reagent tank thereto. Further, the plurality of ports of the detection cartridge may include a waste liquid port, and the processing unit may include therewithin a waste liquid tank for receiving therein a waste liquid from the detection cartridge. In this case, the line switching valve mechanism is designed to selectively provide liquid communication between the waste liquid port of the detection cartridge and the waste liquid tank.
The line switching valve mechanism and the tank switching valve mechanism may be disposed on a line-switching-valve plate and a tank-switching-valve plate, respectively. At least one of the line-switching-valve plate and the tank-switching-valve plate may have a structure prepared by fixedly laminating a plurality of plate elements each formed through an injection molding process using a plastic material or a cutting process using a plate material. In this case, a hole or passage groove necessary for liquid communication is pre-formed in at least one of the plate elements in a desired pattern. Each of the remaining plate members may have a hole or passage groove, or may have no hole or passage groove.
The above structure of the valve plate makes it possible to arrange a required passage line in a compact manner. In addition, this structure is advantageous in view of both production and maintenance, because it can prevent the occurrence of erroneous passage arrangement while reducing the number of components, and allows an operator or user to conveniently find clogging of the passage and liquid leakage, while facilitating a replacement operation. Further, the plate elements may be made of a transparent plastic material to provide an advantage of providing enhanced visibility of an inside of the valve plate. In an actual design, the valve plate can be formed to have a small volume, for example, of several cubic centimeters, and most of the required passage line can be advantageously arranged within this small space. The plate element may be fixedly laminated by a process using an adhesive or sticker, a thermal bonding (joining) process, an ultrasonic bonding process or a diffusion bonding process. The diffusion bonding process comprises exposing to a high-temperature/high-pressure atmosphere a plurality of target members, so as to induce atomic diffusion in the members to allow respective contact surfaces of the adjacent members to be integrally fused with each other. This technique is primarily used for metals, and can also be applied to plastic materials. A plastic material suitable for the diffusion bonding process includes acrylic resin, PEEK (polyether ether ketone) resin and PTFE (polytetrafluoroethylene).
In the present invention, the processing unit may have a housing which houses electronic processing means including a power supply and information processing means. The plurality of reagent tanks may include a cleaning liquid tank, an activating liquid tank and an eluent liquid tank. The eluent liquid being passed through the passage within the detection cartridge allows an target substance temporarily stored in the storing section of the detection cartridge to be eluted therefrom and sent to the passage within the detection cartridge so as to be subjected to a desired analysis.
As mentioned above, the storing section may be comprised, for example, of the absorptive element having a property of absorbing an target substance. Further, at least one of the plurality of reagent tanks may be an eluent liquid tank. In this case, an eluent liquid stored in the eluent liquid tank has a function of eluting an target substance stored in the storing section which may be comprised, for example, of the absorptive element having a property of absorbing the target substance. The effectiveness of the eluent liquid depends on absorption mechanisms. Thus, a specific type of eluent liquid is selected in consideration of chemical characteristics of absorption. For example, in case of an eluent liquid containing an ion capable of being easily absorbed to a surface of the absorptive element, when the eluent liquid is passed through the absorptive element, the ion in the eluent liquid is exchanged with an target substance absorbed to the absorptive element in the form of an ion to allow the target substance to be eluted from the absorptive element. In the present invention, an eluent liquid having various composition may be used depending on target substances.
The present invention can also be implemented as an apparatus for detections-type on immunoassay. In this case, an target substance may include various allergens, such as egg yolk, egg white, bovine milk, peanut, shrimp, crab, fish, shellfish, soybean, mango, other food item known as an allergen, dust mite, feather, pollen, fungus, bacillus, cockroach and dog's or cat's fur. The target substance may further include endocrine disrupting chemical, agrichemical, immunoglobulin including IgE and IgG, histamine, gene (RNA), stress marker, antigen or antibody included in various proteins, human or animal blood, blood components, urine and saliva, and component indicative of a specific disease.
The immunoassay is classified into: labeling assay and nonlabeling assay (sandwich assay); or homogeneous assay and inhomogeneous assay, by types of labels to be used or methods for separating an antigen and an antibody after a reaction therebetween. A specific analysis is established by a combination thereof. While various combinations are conceivable, a preferable combination for use in the present invention will be described later.
Variety of detection techniques have been known and include: the labeling assay, such as immunonephelometry, latex nephelometry, and an assay using an immunosensor comprising an antigen electrode and an antibody electrode; and the nonlabeling assay, such as enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay, spin immunoassay, metallo immunoassay, particle immunoassay and viroimmunoassay, and the present invention can be applied to any one of these processes.
In one embodiment, the storing section of the detection cartridge may be formed as a filter, and an target substance, such as an antigen or antibody, is immobilized onto a surface of the filter. The target substance may be directly immobilized onto the surface of the filter, or may be immobilized through an appropriate ligand. For example, this immobilization may be achieved by immersing a plastic material or a carbon fiber in an antigen and/or antibody solution. Depending on antigens or antibodies to be used, it is desirable to perform the immobilization through a metal as in case of immobilizing mercapto to gold. In this case, a metal coating can be readily formed by subjecting a carbon fiber to a plating process, a sputtering process or a plasma treatment.
An antigen and/or an antibody are appropriately selected or combined depending on an target substance. For example, a conceivable combination may include: avidin for biotin; protein A for immunoglobulin; hormone receptor for hormone; DNA receptor for DNA; RNA receptor for RNA; and drug receptor for drug.
A process of the immunoassay is roughly divided into a first stage of inducing a antigen-antibody reaction, and a second stage of detecting a label reacted with the antigen or the antibody. An target substance and other substance are separated from each other in the storing section,-type on the antigen-antibody reaction in the first stage, and the separated target substance is analyzed qualitatively and/or quantitatively in the detection mechanism disposed on a downstream side relative to the storing section. The detection mechanism used in the second stage may be-type on electrochemical analysis or optical analysis. In the electrochemical analysis, the same electrode arrangement as that in the aforementioned concentration detection apparatus may be employed. In the optical analysis, the same optical cell as that in the liquid chromatography analysis may be used.
A labeled substance for the immunoassay may include protein label, chemiluminescent substance label, and metal ion. In the immunoassay using a protein label, after the label is immobilized to the storing section, a substrate is passed through to the storing section, and a resulting reaction product is detected by the downstream detection mechanism. For example, a combination of the label and the substrate may include: glucose for glucose oxidase; xanthine for xanthine oxidase; amino acid for amino acid oxidase; ascorbate for ascorbate oxidase; acyl-CoA for acyl-CoA oxidase; cholesterol for cholesterol oxidase; galactose for galactose oxidase; oxalate for oxalate oxidase; and sarcosine for sarcosine oxidase.
The optical analysis may be performed using an enzyme label, such as peroxidase, β-galactosidase or alkaline phosphatase. A colorimetric method or a fluorescent method may be used in the optical analysis.
The detection apparatus of the present invention can also be applied to an analysis-type on other principle, such as an analysis-type on a specific binding reaction. In this case, the storing section may be made of a substance which exhibits a specific binding reaction, so as to be applicable to IMAC (immobilized metal affinity chromatography), hybridization of complementary DNAs, and other analysis of various proteins.
In any of the above embodiments, the cartridge-type detection apparatus of the present invention allows an element or component necessary for complicated replacement, cleaning and/or refresh operations for each measurement to be mounted to the detection cartridge. For example, the section corresponds to the electrodes for the electrochemical analysis, the chromatography column for the liquid chromatography, or the solid-phase antigen or antibody for the immunoassay, and this component is mounted to the detection cartridge.
The optical cell can be readily incorporated in the detection cartridge, and this arrangement is desirable In view of reducing a load on a measurer or operator. Alternatively, considering that the optical cell can be simply cleaned with water, the optical cell may be mounted to the processing unit.
FIGS. 6(a) and 6(b) are schematic diagrams showing the processing unit connected with external devices.
FIGS. 14(b)-(i) to (b)-(iii) show an assembled state of the detection cartridge, wherein FIGS. 4(b)-(i), (b)-(ii) and (b)-(iii) are, respectively, a perspective view, a sectional view taken along the line A-A in
FIGS. 33(a) and 33(b) are exploded diagrams showing the structure of the detection cartridge in
FIGS. 34(a) and 34(b) are, respectively, a sectional view taken along the line “a”-“a” in
FIG. is a top plan view showing an internal structure of an upper housing in an analysis unit of the analysis apparatus in
FIGS. 41(a) to 41(c) are process flow diagrams schematically showing several examples where the present invention is applied to immunoassay, wherein:
The present invention will now be described-type on an embodiment thereof illustrated in the drawings.
[Measurement of Various Heavy Metals Using Detection Cartridge]
An absorptive element 22a adapted to absorb an anionic substance, an anion electrode arrangement for detecting an anionic substance, a cation-absorptive element 22b adapted to absorb a cationic substance, a cation electrode arrangement for detecting a cationic substance, are disposed between the base plates 12, 13. The anion electrode array includes two working electrodes 31, 32, a counter electrode 33 corresponding to the working electrodes 31, 32 and a reference electrode 34, and the cation electrode array includes two working electrodes 35, 36, a reference electrode 37 and a counter electrode 38. The base plate 12 is formed with a plurality of concave portions for receiving therein these electrodes at respective predetermined positions. Specifically, as shown in
Generally, a gold electrode (e.g., size: 3.5 mm×8.4 mm×0.5 mm) having a gold layer formed on a glass substrate is used as a working electrode for a measurement of arsenic and selenium (arsenic/selenium measurement), and a plate-shaped carbon electrode (e.g., size: 3.5 mm×8.4 mm×0.5 mm) is used as a working electrode for a measurement of cadmium, lead, mercury and hexavalent chromium (cadmium/lead/mercury/hexavalent-chromium measurement). In this embodiment, the working electrode 31 may be composed of a gold electrode for an arsenic/selenium measurement, and each of the working electrodes 32, 35, 36 may be composed of a plate-shaped carbon electrode for a cadmium/lead/mercury/hexavalent-chromium measurement). Each of the counter electrodes 33, 38 may be composed of the same plate-shaped carbon electrode (e.g., size: 3.5 mm×8.4 mm×0.5 mm) as that used as the working electrode 32, and each of the reference electrodes 34, 35 may be composed of an electrode (e.g., size: 3.5 mm×8.4 mm×0.5 mm) having an alumina substrate coated with a silver paste (6022 available from Acheson (Japan) Ltd.). It is understood that each of the electrodes may be formed in any other suitable configuration.
Each of the electrodes 31, 32, 33, 34, 35, 36, 37, 38 is formed to have a common size, and received in a corresponding one of the concave portions 31a, 32a, 33a, 34a, 35a, 36a, 37a, 38a in such a manner that an upper surface of the electrode becomes flush with an upper surface of the base plate 12. Three adhesive tapes are interposed, respectively, between the base plates 11, 12, between the base plates 12, 13 and between the base plates 13, 14, so as to allow the adjacent base plates to be liquid-tightly fixed together. In
The respective upper surfaces of all the electrodes including the working electrodes 31, 32, 35, 36 are masked by the adhesive tape 24. As shown in
As shown in
The base plate 13 is formed with a pair of through-holes 301 and a pair of concave portions 302 in such a manner that, when the base plate 13 is superimposed on the base plate 12, each of the through-holes 301 overlaps a corresponding one of the through-holes 201 of the base plate 12, and each of the concave portions 302 receives therein an upper portion of a corresponding one of the absorptive elements 22a, 22b. The base plate 13 further has a passage groove 303 formed at a position overlapping the anion electrode array (31, 32, 33, 34), and a passage groove 304 formed at a position overlapping the cation electrode array (35, 36, 37, 38). Furthermore, the base plate 13 is formed with an electrolyte-liquid chamber 305 for receiving therein an electrolyte-liquid pack containing an electrolyte liquid as a reference-electrode activating liquid, and a communication-hole 306 in liquid communication with each of the electrolyte-liquid chamber 305 and the communication-hole 202 of the base plate 12. The base plate 12 is provided with a needle-shaped member 203 protruding inside the electrolyte-liquid chamber 305 of the base plate 13. The base plate 14 is formed with a pair of through-holes 401 in liquid communication with the respective through-holes 301 of the base plate 13, and a communication-hole 402 in liquid communication with the electrolyte-liquid chamber 305 of the base plate 13. The base plate 14 is integrally formed with a flexible thin plate portion 402a on the side of an outer surface thereof in such a manner as to close the communication-hole 402 (see FIGS. 2(a) and 2(b)). An electrolyte liquid serving as a reference-electrode activating liquid is supplied in a state after being contained in an aluminum pack, and this electrolyte-liquid pack is set in the electrolyte-liquid chamber 305. The electrolyte-liquid chamber 305 is in liquid communication with the reference electrode chambers.
The base plate 14 is formed with a pair of passage grooves 403, and each of the passage grooves 403 has one end (i.e., first end) in liquid communication with a corresponding one of the second ends of the passage grooves 111 of the base plate 111 via the through-holes of the base plates 12, 13. As see in
In an operation of detecting an target substance using the detection cartridge according to this embodiment, the external-connection ports 113,114, 116, 117 are closed, and the port 115 is opened. Then, with assistance of a syringe holder, a sample liquid containing an target substance is injected into the sample-liquid inlet portion 1a of the detection cartridge consisting of the through-holes 401, 301, 201, together with an anion-absorptive element activating liquid according to need.
An electrolyte liquid for activating the reference electrode 34 is supplied to the electrolyte-liquid chamber 305 from an external-connection port 117. Then, the valve mechanism is shifted to close the external-connection port 115, and open the external-connection port 113 to provide liquid communication between an after-mentioned eluent-liquid supply section and the external-connection port 113.
The 4-way valve 53b is provided as a means to switch liquid communication between each of the eluent liquid tanks 54, 55, 56 and the pump 52. The eluent liquid tanks 54, 55, 56 contain an eluent liquid for the anion-absorptive element, an eluent liquid for the cation-absorptive element (this eluent liquid is also used as an electrolyte liquid), and a cleaning liquid, such as water, respectively, and the 4-way valve 53b is operable to selectively sent one of these liquid to the pump 52.
The eluent-liquid-tank cassette 50 storing the eluent liquid tanks is designed to be detachably attached to the casing 20 of the processing unit 2. Thus, if a remaining volume of the liquid contained in one of the liquid tanks runs low, the liquid tank can be detached to supply the liquid thereto. As seen in the eluent liquid tank 56 in
The processing device 21 housed in the casing 20 of the processing unit 2 includes an electronic board 66 mounting a microprocessor and various drivers, and has a top surface 67 provided with a display section and a user interface, such as a plurality of manual operation buttons. Further, the processing unit has an outer surface partially formed as a detection insertion case 62. This insertion case 62 is formed as a hinged lid, and designed to be selectively opened and closed relative to a cartridge holder 61 which allows the detection cartridge to be fitted thereinto. The cartridge holder 61 is designed to be selectively opened and closed.
The bottom surface of the presser plate 614 is provided with a pair of resilient sealing members 411 adapted to be engaged with the respective through-holes 401 of the sample-liquid inlet portion 1a of the detection cartridge so as to prevent back-flow of the sample liquid, a protrusion 141 adapted to be inserted into the electrolyte-liquid chamber 305 so as to allow the reference-electrode activating liquid to be directed toward a desired one of the reference electrodes, and a plurality of resilient protrusions 412 adapted to press the detection cartridge 1 against the upper plate 613 so as to prevent leakage of the various liquids. The detection cartridge 1 is inserted into the cartridge holder 61 in a posture as shown in
In the example illustrated in
As shown in
The processing unit 2 may be designed to be connected to a personal digital assistant (PDA) as shown in
FIGS. 8(a) to 8(c) show flowcharts of a process of detecting an target substance using the detection cartridge 1 and the processing unit 2 illustrated in FIGS. 1 to 7. Referring to FIGS. 8(a) to 8(c), a measurement process will be described below while taking some examples.
In a first stage of the measurement process, the detection cartridge 1 is held by the clamp members 15, 16 of the syringe holder without being inserted into the processing unit 2. The syringe holder is designed to close the through-holes 401 of the sample-liquid inlet portion 1a when it holds the detection cartridge 1. In place of the syringe holder illustrated in
Firstly, in order to activate an absorption ability of the anion-absorptive element 22a, an anion-absorptive element activating liquid is injected from the through-hole 401 of the sample-liquid inlet portion 1a. A volume of the activating liquid may be set at a value allowing the anion-absorptive element 22a to be just wetted. Specifically, about 50 μl of the activating liquid may be all that is needed. Then, 10 ml of the sample liquid is injected from the through-hole 401 of the sample-liquid inlet portion 1a, and discharged from the external-connection port 115 after being passed through the anion-absorptive element 22a. During this process, arsenic and selenium as target substances are absorbed and captured by the anion-absorptive element 22a. Thus, no target substance is contained in the sample liquid when it is discharged. Then, in order to fill the reference electrode chamber with a potassium chloride solution as a reference-electrode activating liquid for creating silver chloride in the reference electrode 34, a pack containing the activating liquid is set in the electrolyte-liquid chamber 305 of the base plate 13, and a pressing portion, i.e., the flexible thin plate portion 402a (see
In a second stage of the measurement process, the detection cartridge 1 is insertingly loaded into the processing unit 2. In response to inserting the detection cartridge 1 into the processing unit 2, the valve mechanism 51 is operated to supply potassium chloride from the port 117 to the reference electrode chamber in the anion electrode array. Then, the eluent-liquid supply port 113 is opened to supply an eluent liquid therethrough. Simultaneously, the through-holes 401 of the sample-liquid inlet portion 1a are closed, and each of the electrode-contact pins 615 is brought into contact with a corresponding one of the working electrodes 31, 32, the counter electrode 33 and the reference electrode 34.
Then, when a measurement start button on the processing unit 2 is pressed down to initiate the measurement, an arsenic/selenium measurement eluent liquid (1 M of sulfuric acid; pH=about 2) is supplied from the port 113 located adjacent to the sample-liquid inlet portion. The eluent liquid flows across the absorptive element 22a, and then flows along the electrodes 31, 32, 33, 34. During this process, the through-holes 401 are closed, and therefore there is no risk of back-flow of the eluent liquid toward the sample-liquid inlet portion. Even though the liquid passage zone facing the electrodes 31, 32, 33 is continuous with the liquid passage zone facing to the reference electrode 34 via the liquid junction 133, the extremely-narrowed liquid junction 133 can prevent the potassium chloride solution in the reference electrode chamber to flow back toward the array of the electrodes 31, 32, 33. A downstreammost end of this liquid passage is connected to a waste liquid reservoir of the processing unit via the port 116.
Although the liquid passage zone facing the electrodes 31, 32, 33, the liquid junction 133, and the liquid passage zone facing the reference electrode 34 are illustrated in
A state of electrical connection is checked up after the liquid passage zone facing the electrodes 31, 32, 33 is filled with the eluent liquid. Specifically, a voltage or current in each of the working electrodes 31, 32 is checked up while applying a certain current or voltage between the working electrode and the counter electrode 33, to determine whether the eluent liquid is sufficiently supplied to ensure adequate electrical connection, and whether the contact pins are adequately in contact with the respective electrodes. A volume of the eluent liquid required for filling the electrode-facing liquid passage zone therewith is about 40 μl.
Then, a voltage (−0.4 V) is applied to each of the working electrodes 31, 32 while supplying the arsenic/selenium measurement eluent liquid from the eluent-liquid supply port 113 at a constant flow rate, so as to deposit arsenic and selenium on the working electrodes 31, 32. The arsenic and selenium captured by the absorptive element 22a is eluted by the eluent liquid passed across the absorptive element, and mixed into the eluent liquid. Then, the arsenic and selenium reach around the electrodes together with the eluent liquid. The arsenic and selenium are deposited on the working electrodes 31, 32 through a reduction reaction occurring around the electrodes. The supply of the eluent liquid and the application of the deposition voltage will be continued until the absorbed arsenic and selenium are entirely desorbed. For example, when 300 μl of arsenic and selenium is deposited at a flow rate of 50 μl/min, the absorbed arsenic and selenium is almost fully eluted. Thus, a deposition time may be set at about 6 minutes. In a specific operation, the supply of the eluent liquid is continued for 5 minutes 50 seconds, and the last 10 seconds are used for stabilizing the eluent liquid in the liquid passage. Then, after an elapse of 6 minutes, a potential sweep operation is initiated. The potential sweep may be performed under the following conditions:
Conditions of Anodic Stripping Voltammetry (ASV)
Sweep System: LSV (Linear Sweep Voltammetry: potential sweep without applying a constant frequency)
Deposition potential: −0.4 V
Deposition time: 6 minutes
Sweep rate: 0.2 V/s
Onset sweep potential: −0.4 V
Termination sweep end potential: 1.2 V
A potential-current curve obtained by the above operation is recorded. For example, a graph as shown in
The measurement process for arsenic and selenium is completed through the above operation. Successively, a measurement of cadmium, lead and mercury is initiated. Except for the following points, the cadmium/lead/mercury measurement is performed in the same manner as that in the selenium measurement. In the cation measurement, the mixing of potassium chloride ions into an eluent liquid is not a factor disturbing the measurement. Thus, a common liquid can be used as both the reference-electrode activating liquid and the eluent liquid. That is, there is no need for arranging the reference electrode-facing liquid passage zone independently of the liquid passage zone facing other electrodes, and the four electrodes 35, 36, 37, 38 may be arranged in series. In
Cation Measurement Eluent Liquid
0.4 M of potassium chloride+10 mM of citric acid+3.5 mM of ethyl diamine (pH=about 4)
The cation-absorptive element 22b has no need for an activating liquid. Thus, an operation corresponding to the above first stage of measurement process may be completed only by injecting a sample liquid.
As shown in the flowchart in
Conditions of ASV
Sweep System: SWV (Square Wave Voltammetry: potential sweep while applying a constant frequency)
Deposition potential: −0.9 V
Deposition time: 6 minutes
Sweep rate: 0.225 V/s
Onset sweep potential: −0.9 V
Termination sweep end potential: 0.6 V
Frequency: 100 Hz
Step potential: 2.55 mV
In case of successively performing the arsenic/selenium measurement and the cadmium/ lead/mercury measurement, after setting the detection cartridge in the syringe holder, an absorptive element activating liquid is injected from the through-hole 401 of the arsenic/selenium sample-liquid inlet portion 1a. Then, 10 ml of sample liquid is injected into each of the through-hole 401 of the arsenic/selenium sample-liquid inlet portion 1a, and the through-hole 401 of the cadmium/lead/mercury sample-liquid inlet portion 1a. Then, the detection cartridge 1 is insertingly loaded into the processing unit 2. In response to pressing down the start button, respective operations of supplying an arsenic/selenium-measurement eluent liquid, checking up electrical connection, re-supplying the eluent liquid and carrying out an electrochemical measurement are sequentially performed. Subsequently, respective operations of supplying a cadmium/lead/mercury-measurement eluent liquid, checking up electrical connection, re-supplying the eluent liquid and carrying out an electrochemical measurement are sequentially performed. After completion of the cadmium/lead/mercury measurement, a cleaning liquid is supplied from the cleaning liquid tank 503 to clean the valve mechanism 51, the pump 52 and a plurality of lines connecting therebetween.
Except that the electrochemical measurement is carried out-type on cathodic stripping voltammetry (CSV), a hexavalent-chromium measurement is performed in the same manner as that in the arsenic/selenium measurement. Due to the difference in electrochemical measurement scheme between the hexavalent-chromium measurement and the arsenic/selenium measurement, the hexavalent-chromium measurement and the arsenic/selenium measurement are performed using individual detection cartridges instead of the simultaneous measurement. In contrast, the cadmium/lead/mercury measurement may be performed just after completion of the hexavalent-chromium measurement, using the same detection cartridge.
In the above modification, a sample liquid is fed from the inlet valve 601 after closing the outlet valve 602, and an appropriate pressure is applied to the enrichment chamber 600a so as to enrich the sample liquid in the enrichment chamber 600a. After the enrichment, the outlet port 602 is opened to supply the enriched sample liquid to an electrode arrangement. This modification has an advantage of being able to reduce an enrichment time by freely increasing an area of the porous membrane 603.
FIGS. 13(a), 13(b) and 13(c) are perspective views showing a portable analysis unit usable with the aforementioned detection cartridge, according to one embodiment of the present invention. As shown in
A chamber 701c for housing various functional components is provided on an opposite side of the waste-liquid-tank chamber 701b relative to the partition wall 701a, and a plurality of reagent tanks 706 is disposed in the chamber 701c over a range of from one longitudinal end wall to an approximately central region thereof and arranged in parallel relation to each other. In this embodiment, five reagent tanks are disposed therein. In
The chamber 701c houses a switching valve mechanism 707 comprising a plurality of switching valves, at a position below the reagent tanks 706, and a tank-switching line plate 708 at a position below the switching valve mechanism 707. The chamber 701c further housed a cartridge holder 709 serving as a cartridge loading portion, and a liquid feed pump 710 at a position on a lateral side of the cartridge holder 709. Furthermore, an after-mentioned destination switching valve mechanism 711 and destination-switching line plate are disposed below the cartridge holder 709.
Returning to
FIGS. 14(a), 14(b)-(i), 14(b)-(ii), 14(b)-(iii) and 14(c) show a detection cartridge for use in electrochemical analysis, according to another embodiment of the present invention, which correspond to FIGS. 1, 2(a), 2(b), 2(c) and 3, wherein a corresponding element or component therebetween is defined by the same reference numeral or code. In this embodiment, the detection cartridge has a bottom surface formed with a port 116 adapted to be connected to a waste liquid tank 705, and a port 117 adapted to be connected to a reagent tank for the reference-electrode activating liquid, in addition to three ports 113, 114, 115. A liquid passage between the ports 14, 15 is formed within the detection cartridge instead of forming outside the detection cartridge.
Referring to
The reagent tank 706 has a lower end formed with a convex portion 706a protruding downwardly. The convex portion 706a has a bottom surface formed with an annular-shaped groove 706b corresponding to the annular-shaped protrusion 715a of the plate 708. The reagent tank 706 also has a reagent outlet 706c formed at a position corresponding to the slits 715c of the plate 708. The outlet 706c is provided with a valve 706e biased toward its close position by a spring 706d. The reagent tank 706 has an upper surface formed with a vent hole 706f sealed by a gas permeable and liquid impermeable material.
The above reagent tank 706 is attached at a predetermined position by fitting the annular-shaped groove 706b onto the annular-shaped protrusion 715a. During this operation, the pin 715b of the plate 708 pushes the valve 706e of the reagent tank 706 upwardly to open the reagent outlet 706c so as to provide liquid communication between an internal space of the tank 706 and the slits 715c. The slits 715c are in liquid communication with a passage formed in the plate 708. The O-ring 715e functions to prevent liquid leakage between the reagent tank 706 and the plate 708.
It is desirable that the liquid feed pump 710 is small in size and capable of stably feeding a small volume of liquid in a level of micro liter without pulsation. Further, when the liquid feed pump is used in portable analysis unit, it is required to have low power consumption. The liquid feed pump may have a flow rate of 5 to 100 micro liter/min and a discharge pressure of 0.01 to 10 MPa. A liquid feed pump meeting these requirements includes a syringe pump, such as “Pencil Pump” available from Uniflows Corp., and “Confluent” available from Scivex Inc.
Referring to
Referring to
The concave portion 709c formed in the lower plate of the cartridge holder 709 has a bottom surface formed with a plurality of liquid ports at positions corresponding to respective liquid ports formed in a bottom surface of a detection cartridge. As one example of the ports,
Returning to
The destination-switching line plate 716 has a lower surface having respective openings of ports G, F, H, I, J, K, L, P2, and these ports are selectively connected to the pump 10 by the switching valve mechanism 711 partly shown in
In the above embodiment, the tank-switching line plate 708 and the destination-switching line plate 716 are formed as separate members. Alternatively, these members may be integrally molded in a single piece.
Although not illustrated in
The analysis unit has five switching valves 711-1, 711-2, 711-3, 711-4, 711-5 for switchably providing liquid communication between an outlet of a liquid feed pump 710 and each of the ports. In
A process of this chromatography analysis is as follows:
(1) Before setting to the analysis unit, a sample is injected into the detection cartridge, and passed through the filter serving as the accumulation (i.e., enrichment) portion (a target substance, i.e., target substance, is trapped by the filter);
(2) The detection cartridge is loaded into the analysis unit;
(3) The activating liquid is supplied to the accumulation filter (for pre-removing gas bubbles to prevent gas bubbles from mixing in the column during a substantial measurement);
(4) The eluent liquid is supplied to the column;
(5) The substantial measurement is carried out. The eluent liquid is passed through the storing section→the passage in the analysis unit→the column→an optical detection section of the analysis unit→the waste liquid tank;
(6) An operation of identification and quantification of the target substance is performed-type on a signal detected by the optical detection section; and
(7) The passage is cleaned.
FIGS. 28(a), 28(b), 29(a) and 29(b) are tables showing a time-series operation in case of using the analysis unit in combination with the detection cartridge for concentration measurement. In these figures, the term “first line” means one of the electrode arrays in
FIGS. 30(a) and 30 are tables showing a time-series operation in case of using the analysis unit in combination with the detection cartridge for chromatography analysis. In these figures, the alphabet codes correspond to the respective reference codes of the valves in
The lowermost plastic plate 814 has four ports 814a, 814b, 814c, 814d. The port 814a serves as a reagent supply port, and the port 814b serves as a sample-liquid injection port. The port 814c serves as a sample-liquid circulation port, and the port 814d serves as a waste-liquid port. The intermediate plastic plates 812, 831 are formed to define a filter concave portion 816 between their contact surfaces to receive therein a filter 815 which serves as an storing section for temporarily storing an target substance. Further, the intermediate plastic plates 812, 831 are formed with a passage segment 817 which extends from the port 814b and penetrates therethrough in a thickness direction of the cartridge 810 while crossing the filter concave portion 816, and a liquid circulation passage segment 818 which extends from the port 814a and penetrates therethrough in the thickness direction. The uppermost plastic plate 811 has an inner surface formed with a groove defining a passage segment 819 which provides liquid communication between the passage segments 817, 818.
The plastic plate 813 is formed with a liquid passage segment 820 which has one end extending from the port 814c and penetrates therethrough in the thickness direction. The contact surface of the plastic plate 812 with the plastic plate 813 is formed with a groove defining a liquid-chromatography column 821, and the other end of the liquid passage segment 820 is connected to one end of the liquid-chromatography column 821. The other end of the liquid-chromatography column 821 is connected to one end of a liquid passage segment 823 via a liquid passage segment 822 which penetrates the plastic plate 813 in the thickness direction. The liquid passage segment 823 is defined by a groove formed in a contact surface of the plastic plate 813 with the plastic plate 814.
The other end of the liquid passage segment 823 is connected to one end of an absorbance measurement cell 824 which comprises a liquid passage segment formed to penetrate the plastic plates 812, 813 in the thickness direction. The other end of the absorbance measurement cell 824 is connected to the waste liquid port 814d via a liquid passage segment 825 defined by a groove formed in a contact surface of the plastic plate 812 with the plastic plate 811 and a liquid passage segment 826 formed to penetrate the plastic plates 812, 813 in the thickness direction.
FIGS. 33(a) and 33(b) show an arrangement of ports, groove and concave portions of upper and lower surfaces of each of the plastic plates 811, 812, 813, 814 of the cartridge 810, wherein
Referring to
In the second plastic plate 812, the filter-defining concave portion 816 and the passages 818, 817, 826 are arranged as shown in
FIGS. 34(a) and 34(b) are sectional views taken along the line “a”-“a” and the line “b”-“b” in
In this embodiment, the light source 831 is provided for liquid chromatography analysis. The light source is not limited to a specific type, but may be any suitable type capable of emitting light having a wavelength of 200 to 1100 nm, and being received in the inner space of the upper body 801a. The wavelength is adjusted depending on types of target substances. A light source suitable for the light source 831 includes “FiberLight” (combination of a deuterium lamp and a tungsten lump) available from Sentronic GmbH. A collimating lens 833 is disposed at an exit of the light source 831 to collimate an emitted beam. A slit plate 834 having a slit for reducing a diameter of the emitted beam at a predetermined value is fixed on an exit side of the collimating lens 833, and a cartridge presser plate 835 is disposed outside the slit plate 834.
A destination-switching valve plate 837 associated with five switching valves 836 indicated by codes A, B, C, D, E is disposed in the upper body 801a in fixed relation thereto, in the same manner as that in the switching valve mechanism 707 in the aforementioned embodiment. This destination-switching valve plate 837 is disposed to extend vertically. The cartridge 810 is inserted between the cartridge presser plate 835 and the destination-switching valve plate 837 vertically from above.
In order to facilitate an operation of inserting the cartridge 810, the cartridge presser plate 835 is designed to be moved in a direction away from the destination-switching valve plate 837, i.e., upwardly from the illustrated position. Specifically, each of the light source 831, the collimating lens 833, the slit plate 834 and the cartridge presser plate 835, are mounted on a base plate 838 in an integrally movable manner, and the base plate is supported by a rail (not shown) movably in the direction indicated by the arrow in
As shown in
Referring to
As shown in
Returning to
An operation of the analysis apparatus according to this embodiment will be described below. Firstly, the cartridge 810 is prepared, and a given volume of sample liquid is injected from the port 814b into the cartridge 810. Through this operation, an target substance contained in the sample is stored in the filter 815 of the cartridge 810. The remaining liquid other than the target substance is discharged from the port 814a. Then, the apparatus is turned on to start a measurement program. This measurement program performs a operational process as shown in
Then, after opening the valve G, a predetermined volume of eluent liquid is sucked to the liquid feed pump 830, and then the valve G is closed. Then, after opening the valve D, the pump 830 is operated to feed the eluent liquid to the column 821 via the port 814c. This operation is performed as a pre-treatment of the column. Subsequently, after closing the valve D and opening the valve G, a predetermined volume of eluent liquid is sucked to the liquid feed pump 830. Then, after opening the valves B, E, the liquid pump 830 is operated. The eluent liquid enters the filter 815 via the valve B, the port 814a, and the passage segments 818, 819, 817 to elute the target substance stored in the filter 815, and then reaches the column 821 via the port 814b, the valve E, the port 814c and the passage segment 821. Then, the eluent liquid passed across the column 821 is discharged to the waste liquid tank 832 via the passage segments 822, 823, the absorbance measurement cell 824, the passage segments 825, 826, and the port 814d. During this process, the light source 831 is turned on (see
Each of the filter 815 and the column 821 contains a functional group having a chemical interaction with the target substance. The functional group contained in the filter 815 functions to trap the target substance. The eluent liquid serves as a means to elute the trapped target substance and carry the eluted target substance to the column 821. The functional group contained in the column has a fine particle size, and the column has a relatively large passage length. Thus, the target substance contained in the eluent liquid is passed through the column while chemically interacting with the functional group contained in the column, and a level of the interaction is varied depending of types of target substances. That is, respective rates of absorption and desorption of the target substance by the column 821 during passing the eluent liquid through the column are varied depending on types of target substances. Thus, a timing when the target substance is detected as a change in absorbance by the spectrometer is varied depending on types of target substances. This makes it possible to distinctly detect the target substance contained in the eluent liquid.
The detection result may be indicated on a display window appropriately provided in a top surface of the analysis unit or on a display unit of a computer connected to the analysis unit.
Subsequently, an operation of cleaning the analysis unit is performed. This cleaning operation is performed, for example, in the following process. Firstly, the valve H of the switching valves 846 is opened, and the pump 830 is operated to suck a predetermined volume of cleaning liquid. Then, after closing the valve H and opening the valves A, C, the pump 830 is operated to clean the filter 815. Then, after closing the valves A, C and opening the valve D, the pump 830 is operated to pass the cleaning liquid through the column 821. Further, after closing the valve D and opening the valves B, E, the pump 830 is operated to pass the cleaning liquid through both the filter 815 and the column 821.
A detection apparatus applied to immunoassay, according to another embodiment of the present invention, will be described below. FIGS. 41(a) to 41(c) show a principle of immunoassay analysis.
In the example illustrated in
In the example illustrated in
In the example illustrated in
The present invention can be applied to the above immunoassays and any other conventional immunoassays. There are many publications about immunoassay, for example, JP 2000-155122 A and JP 2003-987171. While the examples in FIGS. 41(a) to 41(c) employ an enzyme label, any other label may be used.
When the present invention is applied to immunoassay, the detection of the reaction product may be performed by an electrochemical analysis or an optical analysis. As mentioned above, in case of detecting hydrogen peroxide produced by an oxidation/reduction enzyme, an electrochemical analysis may be used. Further, depending on types of target substances, an optical analysis may be used.
The present invention will be more specifically described-type on several Examples.
(Production Method for Substance Concentration Detection Cartridge)
Step 1: Injection Molding
1) Preparation of Molded Base Plates
Four base plates 11 to 14 were molded using an injection molding machine (produced by MEIKI Co., Ltd.). The injection molding was performed under the following conditions: cylinder temperature=280° C.; metering-section temperature=290° C.; and a mold temperature=60° C. A runner was separated from each molded product to obtain desired molded base plates.
2) Mounting of Elements to Molded Base Plates
A carbon electrode and silver paste serving as a working electrode and a counter electrode were attached to the molded base plate 12. Specifically, after applying an adhesive on intended attachment positions of the base plate 12, and then each electrode was attached on the adhesive and fixed. A container containing potassium chloride for wetting a reference electrode and creating a silver/silver-chloride electrode during an arsenic/selenium measurement was mounted to the molded base plate 13. This container was arranged immediately above a needle portion formed on the molded base plate 12 and immediately below a pressing portion formed in the base plate 14.
Step 2: Attachment of Adhesive Tape
1) Preparation of Adhesive Tape
A double-faced adhesive tape was set in a punching machine, and subjected to punching in conformity to a shape of the molded base plate to prepare a punched adhesive tape.
2) Fixing of Molded Base Plates
A first one of the molded base plates having the adhesive tape attached on an upper surface thereof, and a second one of the molded base plates to be superimposed on the first base plate, were set in a positioning apparatus with a vacuum chamber. The position apparatus has an image-recognition-type position adjustment mechanism, and a vacuuming mechanism for an adjustment area. This apparatus makes it possible to fix the molded base plates at a proper position without intrusion of gas bubbles therebetween.
The chromatography analysis unit and cartridge according to the aforementioned embodiment illustrated in FIGS. 31 to 37 were used for a separation test of an organic-acid mixed liquid consisting of oxalic acid and succinic acid.
Specifically, the following analysis apparatus was used in the test:
Light source: FiberLight (200 to 1100 nm) produced by Sentronic GmbH
Spectrometer: SAS-series OEM module (equipped with 1024 element CMOS; 200 to 700 nm) produced by Ocean Optics Inc.
Cartridge: the structure illustrated in FIGS. 32 to 37
Column filler: Wakosil-II 5C18-100 (particle size: 5 μm) produced by Wako Pure Chemical Industries, Ltd.
Filter filler: Wakosil-II 25C18 (particle size: 25 to 30 μm; 70% up) produced by Wako Pure Chemical Industries, Ltd.
Flow rate/temperature: 10 μl/min. room temperatures
Measurement wavelength: 210 nm
0.1 g of oxalic acid and 0.1 g of succinic acid were mixed with 100 ml of purified water (produced by Wako Pure Chemical Industries, Ltd.) to prepare the following sample.
type on the process in
The present invention was applied to a measurement of BNP (Brain Natriuretic Peptide) which is a hormone useful in estimating cardiac diseases. A method used in Example 3 is classified into enzyme immunoassay, homogeneous immunoassay, and competitive immunoassay,-type on classification of immunoassay.
A BNP antigen was used as an storing section, and a method of electrochemically detecting an action of an enzyme label was used as the detection mechanism. Example 3 is one application of a technique by Matsuura et al., described in “Analytical Chemistry, Vol. 77, No. 13, 2005, pp. 4235-4240”, to the cartridge and processing unit of the present invention. Related substances were as follows:
AChE: acetylcholinesterase
ACh: acetylcholin
sulfo-SMCC: sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
PBS: phosphate buffer solution (phosphate buffer)
EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
[Preparation]
(1) A carbon fiber filter (Product No. P-1611H produced by Toyobo Co., Ltd., thickness: 0.32 mm) was immersed in 100 mg/l of gold solution (IM-sulfate), and a voltage of −4V was applied for a holding time of 20 minutes while stirring the solution, to deposit gold on a surface of the filter. Then, the filter was subjected to cleaning at a voltage of +0.75 V for 2 minutes to prepare a carbon fiber filter having a surface plated with gold. This gold-plated filter was immersed in 0.1 mM of cysteamine hydrochloride solution for 2 hours to bond cysteamine onto the Au surface. 0.1 g/l of EDC and 30 mg/l of BNP-32 were added to the filter, and cultured for 1 hour. Then, the filter was rinsed with PBS to prepare a gold-plated filter with BNP (antigen) immobilized on the surface thereof.
(2) The BNP immobilized gold-plated filter was cut into a piece having a diameter of 6.5 mm and a thickness of 0.4 mm, and mounted as the storing section as shown in
(3) 0.4 g/l of BNP antigen (rabbit BNP-32 IgG) and 0.4 g/l of sulfo-SMCC were added to 0.1 M of PBS (pH=8), and the mixture was cultured for 1 hour. Then, the cultured mixture was filtered to remove excess sulfo-SMCC. In the same manner, 1 g/l of choline acetylase and 0.3 g/l of s-acetyl mercaptosuccinic anhydride were added to 0.1 M of BNP. Then, the mixture was cultured for 10 minutes, and filtered. These were mixed together at a ratio of antigen:AVh=1:0.7 (mol ratio), and then cultured for 1 hour to obtain an AChE-labeled BNP antigen.
(4) 2 ml of AChE-labeled BNP antigen (0.4 mg/l in PBS solution) was stirringly mixed with a BNP-containing sample (human blood) at a ratio of 1:1, to produce a reaction therebetween for 30 minutes. An obtained reaction product was injected from an inlet port of the cartridge using a commercially available syringe. The injected liquid was passed through the BNP immobilized gold-plated filter, and discharged from an intermediate port 115 (Step (1)). Then, 4 ml of PBS solution was supplied from the same inlet port to clean the BNP immobilized gold-plated filter, and the cleaned filter was set in the analysis unit. Through this operation, a BNP(in the sample)-BNP antigen (AChF-labeled) reaction product was discharged from the intermediate port, and this reaction product was loaded into the analysis unit in such a manner that an unreacted component of the AChF-labeled BNP antigen was captured by the BNA on the gold-plated filter as the storing section.
(5) 1 mM of acetylthiocholine chloride (PBS solution) was supplied as a substrate from the port 1007 at 200 μl/min and in a volume of 1.0 ml, and successively passed through the storing section and the electrode chamber (Step (2)). When the substrate reaches the storing section, thiocholine was produced by the action of the AChE label in an amount proportional to an amount of AChE label, and sent to the electrode chamber. Then, a LSV measurement was performed under the following conditions, and an electrode activity of thiocholine was measured from a voltage-current curve obtained by the LSV measurement. The amount of thiocholine corresponds to an amount of the unreacted BNP antigen, and has a given relationship with an amount of BNP in the sample. Thus, the measurement was performed while changing a concentration of BNP in the sample, so as to obtain an analytical curve.
LSV conditions: −0.7 V×2 min→1.4 V (sweep rate: 50 mV/sec)
The present invention was applied to a separation/quantitative analysis of a specific protein-type on immobilized metal affinity chromatography (IMAC).
(1) Preparation of Cartridge
A detection cartridge having the same outer shape and external-connection ports as those illustrated in
(2) Preparation of Sample
A roughly extracted protein liquid as a sample was prepared as follows.
500 ml of Escherichia coli carrying a poly (histidine)-tag-LacZ protein-expression vector were cultured. An obtained culture was centrifugalized at 4° C. for 15 minutes at 300×g to collect Escherichia coli. The collected Escherichia coli were suspended in 40 ml of extraction buffer using a Vortex mixer. Then, a cleaning buffer was added to the extraction buffer at a concentration of 0.50 mg/ml, and the mixture was left at room temperatures for 30 minutes. Then, Escherichia coli in the mixture were fragmented for 10 seconds using an ultrasonic disintegrator, and the fragmented Escherichia coli liquid was cooled on ice. Then, the fragmented Escherichia coli liquid was centrifugalized at 4° C. for 20 minutes at 10000×g to precipitate insoluble fractions, and supernatant fluid was collected separately. The collected rough extraction liquid was passed through a disposable filter to obtain a sample.
(3) Preparation/Pretreatment of Cartridge
2 ml of 0.1 M sodium chloride solution, 2 ml of 0.5 M nickel sulfate solution and 4 ml of 0.1 M sodium chloride solution were supplied to the cartridge in turn in this other from an inlet port thereof. Then, 2 ml of membrane equilibrating buffer solution (solution of 50 mM NaH2PO4, 300 mM NaCl and 10 mM imidazole; pH=8.0) was passed through the cartridge. In this operation, the solution was supplied from the inlet port 814b, and discharged from the intermediate port 814a via the storing section 816.
(4) Injection of Sample to Cartridge
5 ml of the sample prepared in the Step (2) was injected from the inlet port of the cartridge using a commercially available syringe. The sample liquid was supplied from the inlet port 814b, and discharged from the intermediate port 814a via the storing section 816.
(5) Analysis Operation
The cartridge was set in the analysis unit, and 2 ml of cleaning buffer solution (solution of 50 mM NaH2PO4, 300 mM NaCl and 20 mM imidazole; pH=8.0) was supplied from the port 814b at 1 ml/min, and discharged from the intermediate port 814a. Then, the valve 836 is switched to provide liquid communication between the port 814b and the port 814c, and 0.2 ml of eluent buffer solution (solution of 50 mM NaH2PO4, 300 mM NaCl and 250 mM imidazole; pH=8.0) was supplied from the port 814a at 50 μl/min. This eluent buffer was sent to the cell 814 via the storing section 816. Then, the valve 836 was re-switched, and 0.2 ml of protein assay CBB solution (produced by Nacalai Tesque Inc.) was supplied from the port 814c at 500 μl/min. Through this operation, liquid in the cell 824 was approximately entirely replaced with the eluent buffer and the protein assay CBB solution. Then, the built-in spectrometer and light source (in common with those for high-performance liquid chromatography (HPLC)) of the analysis unit were activated to observe an absorbance at 595 nm, and an amount of collected protein was quantitatively calculated from a peak intensity of the absorbance.
(6) Result
The present invention may be applied to various measurement methods as well as the aforementioned methods. Some examples of other measurement methods which can effectively utilize the present invention are shown in
As described above, the present invention provides a cartridge-type detection apparatus comprising a detection cartridge having a passage for passing a sample liquid containing an target substance, and a processing unit adapted to be loaded with the detection cartridge so as to produce information about the target substance contained in the sample liquid passed through the detection cartridge. The detection cartridge includes an storing section for temporarily storing the target substance, a liquid passage routed through said storing section, and a plurality of ports in liquid communicate with said liquid passage. The detection cartridge is provided with a part or entirety of a detection mechanism on a downstream side relative to the storing section. The processing unit includes a liquid feed pump and a line switching valve mechanism adapted to switchingly provide liquid communication between the liquid feed pump and a selected one of the plurality of ports of the detection cartridge. The valve mechanism is operable to switch between a first passage connection mode for allowing the sample liquid supplied into the detection cartridge to be passed through the storing section and then discharged out of the detection cartridge, and a second passage connection mode for allowing a reagent to be supplied from one of the plurality of ports to the storing section of the detection cartridge by an action of the liquid feed pump, and allowing the reagent passed across the storing section to be discharged out of the detection cartridge from one of the remaining ports.
In a specific embodiment of the present invention, a detection cartridge includes an storing section for temporarily storing the target substance, a liquid passage routed through said storing section, and a plurality of ports in liquid communicate with said liquid passage. A processing unit for performing analysis and/or processing using the detection cartridge includes a plurality of reagent tanks, a liquid feed pump, a tank-switching-valve plate having a tank switching valve mechanism adapted to switchingly provide liquid communication between the liquid feed pump and a selected one of the plurality of reagent tanks, and a line-switching-valve plate having a line switching valve mechanism adapted to switchingly provide liquid communication between the liquid feed pump and a desired one of the plurality of ports of the detection cartridge. The processing unit is operable to selectively shift of the valve mechanism of the tank-switching-valve plate and the valve mechanism of the line switching valve mechanism to desired valve positions while activating the liquid feed pump, so as to perform an analysis of an target substance.
Thus, the cartridge-type analysis apparatus of the present invention can arrange a functional element or component essential to analysis, within a housing of the processing unit in a significantly compact manner to facilitating providing a simplified portable analysis apparatus. Thus, the analysis apparatus of the present invention makes it possible to perform a speedy analysis at a sampling location of an target substance so as to provide a highly useful apparatus.
Although the invention has been specifically shown and described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. The scope of the invention should be determined by the appended claims and their legal equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2005-002540 | Jan 2005 | JP | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP06/00136 | Jan 2006 | US |
| Child | 11827080 | Jul 2007 | US |
