Patent Application
20250033050
The present disclosure relates to a micro analysis chip in which a micro flow passage is formed in the interior of a porous substrate, an electrolyte concentration measuring system including the micro analysis chip, and an electrolyte concentration measuring method.
In recent years, development of a micro flow passage device capable of efficiently (very small amount, rapid, and simple) performing biochemical analysis in a chip by utilizing a micro-sized, fine flow passage has attracted attention in wide fields of biochemical research, medicine, drug discovery, health care, environment, food, and the like.
In particular, a paper micro analysis chip based on paper uses an inexpensive material such as paper as a substrate, enables a specimen or an inspection liquid to be driven by utilizing a capillary phenomenon of paper itself without using an electric power, and, therefore, enables any one to readily realize inspection and diagnosis based on POC (point of care) at low cost due to a small size, a low cost, ease of carrying, high disposability (disposal is completed by only incineration), and no need for a large apparatus. Consequently, there are global expectations as an inspection device in medical services in developing countries and remote areas where medical equipment is not sufficiently prepared, at disaster sites, and the like, and at air terminals and the like where infections must be prevented from spreading at the water's edge. In addition, attention is attracted as a health care device capable of controlling and monitoring the health state by oneself and various pathological diagnosis devices in usual medical sites.
Electrolyte measurement is one of biochemical inspections of the pathological diagnosis. The electrolyte measurement is an inspection for measuring an ion concentration (Na+, K+, Cl−, or the like) in blood or in urine. That is, the electrolyte measurement is an inspection to measure “ionic concentration balance in a body”. The ionic concentration in a body reflects the amount of water and the pH in a body, and to maintain life by normally functioning transmission through nerves, a heart, and muscles, it is important that the ionic concentration is appropriately controlled.
In this regard, for example, when a change occurs in the electrolyte concentration, there is a high likelihood that an irregularity occurred in kidney functions or hormone effects. Therefore, the electrolyte measurement is an indispensable inspection for screening disease and, further, is a very important inspection for examining the physiological functions (life state) of a patient in a disaster site or the like.
Various universities and companies perform research on the electrolyte measurement by using μPADs.
NPL 1 proposes a filter-paper-based measuring device for the concentration of a Na ion and a K ion. This device includes a dispensation portion to dispense a specimen, and a dispensed specimen permeating from the dispensation portion into the respective regions of a working electrode and a reference electrode electrically couples the two electrodes to each other so as to enable the potentiometry to be performed. In this regard, in the above-described device, to obtain a stable potential at the reference electrode, a KCl ion crystal is accumulated on the reference electrode, and KCl dissolving in the specimen during measurement maintains a high Cl ion concentration in the reference electrode region and obtains a stable potential of the reference electrode. Further, only a measurement target ion being selected by an ion-selective film formed covering the working electrode enables the measurement to be performed without being affected by other ions.
In addition, PTL 1 discloses a method in which a stable potential is obtained at the reference electrode by dispensing a specimen in a working electrode portion and simultaneously dispensing a saturated solution of KCl or NaCl serving as a standard solution in a reference electrode portion.
However, arrangement of an electrolyte layer (KCl layer) is very difficult, and the potential of the reference electrode is not stabilized by only simply stacking and arranging the KCL layer on a support electrolyte or AgCl. Consequently, even when the concentration of the specimen is the same, variations between devices increase, and it is difficult to obtain stable measurement results. This problem was found on the basis of the investigation by the present inventors.
It is an object of the present disclosure to provide a micro analysis chip in which an electrolyte layer is arranged at an appropriate position, which indicates an appropriate potential of a reference electrode, and which is capable of performing stable analysis.
The present disclosure relates to a micro analysis chip including a flow passage region surrounded by a flow passage wall disposed in the interior of a porous substrate,
Further features of the present disclosure will become apparent from the following description of exemplary embodiments with reference to the attached drawings.
The operation of the present disclosure will be described in detail.
The role of the reference electrode is to serve as a reference potential relative to a potential generated at the working electrode. Therefore, the reference electrode has to always indicate the same potential relative to any concentration of specimen. The potential between the reference electrode and the working electrode is incorrect unless the potential of the reference electrode is stable.
In general, Ag/AgCl is frequently used as the base electrode of the reference electrode. Regarding Ag/AgCl, the following equilibrium reaction occurs at an interface to the specimen, and the potential is determined in accordance with a Cl− concentration.
Consequently, the Cl− concentration being constant enables the potential to be stabilized.
Since a Cl− concentration is constant in a saturated sodium chloride (NaCl) solution or a saturated potassium chloride (KCl) solution, the same interfacial potential is always obtained by reacting the saturated NaCl solution or the saturated KCl solution with AgCl.
When the NaCl concentration, that is, the Cl− concentration, is increased, the potential of the Ag/AgCl electrode is decreased. When the NaCl concentration reaches a concentration close to saturation (about 5.2 mol/L when a temperature is 25° C.), since NaCl does not dissolve any more, the Cl− concentration becomes constant, the above-described equilibrium reaction is stopped, and the potential is not changed any more. In this regard, since it is conjectured that the measurement is frequently performed at room temperature (about 25° C.), in the following explanation, use at room temperature (about 25° C.) is assumed, and the design is based on the saturated concentration at 25° C. An amount of an electrolyte arranged in Example described later is an amount on the assumption of 25° C. However, when use in a different environment is assumed, it is sufficient that the design is performed in accordance with the use environment, and the configuration is not limited to the configuration (amount of electrolyte) on the assumption of 25° C.
Based on the above-described principle, in general, regarding an internal-liquid-type reference electrode, saturated KCl or NaCl is used as an internal liquid, and regarding a solid-electrode type, a technique to ensure a stabilized potential of a reference electrode by directly dispensing the saturated liquid serving as a standard liquid in a Ag/AgCl electrode is adopted.
According to the above, however, a reference liquid has to be prepared separately from a specimen, and regarding a micro analysis chip that is a simple, low-cost inspection device, there is a time and effort issue and a cost issue.
To address the above-described issues, NPL 1 describes a configuration in which, as illustrated in
However, even when the electrolyte is stacked on the Ag/AgCl electrode or the support electrolyte film by using various printing techniques, such as ink jet and dispenser, and adopting various electrolyte concentrations and pitch intervals, it is difficult to obtain a stable potential.
A test to ascertain that it is difficult to obtain a stable potential by the above-described configuration was performed as described below.
A sheet including a first flow passage chamber, a second flow passage chamber, and a flow passage to connect the first flow passage chamber to the second flow passage chamber was prepared. The reference electrode (Ag/AgCl electrode) 3 was arranged in the first flow passage chamber of the sheet, the support electrolyte film 9 was stacked on the electrode, further, the electrolyte layer (KCl layer) 3a was stacked on the support electrolyte film 9, and a commercially available electrode 5 was disposed in the second flow passage chamber so as to form a micro analysis chip. Regarding the micro analysis chip, a KCl solution serving as a specimen was dripped to the flow passage portion connected to the Ag/AgCl electrode and the commercially available electrode, and a potential difference between the Ag/AgCl electrode and the reference electrode was measured where the concentration of the KCl solution was changed. The concentration of the KCl solution was changed in the range of 10−4 mol/L to 800 mmol/L. The results of the test are illustrated in
The reason for an occurrence of variations in the potential is considered to be as described below.
A specimen that reaches the electrolyte layer first dissolves the electrolyte and comes into contact with the Ag/AgCl electrode while the Cl− concentration is saturated. But the specimen is swept away by a specimen that flows thereafter (
In addition, as illustrated in
That is, it was ascertained that the measured interfacial potential is not stabilized unless a configuration in which the specimen having a saturated Cl− concentration is stably supplied to the Ag/AgCl electrode is established.
The micro analysis chip according to the present disclosure is characterized in that the electrolyte is arranged upstream of the reference electrode with reference to a forward direction of the specimen in the flow passage region, and the potential of the reference electrode is stabilized by the specimen coming into contact with the reference electrode while the Cl− concentration is saturated.
That is, the micro analysis chip according to the present disclosure desirably has a configuration in which a predetermined amount of the electrolyte is arranged at a position so that an upstream end portion of the reference electrode is located downstream of a position where an electrolyte concentration in the specimen is a concentration close to saturation (88% of the saturated concentration) due to the electrolyte being dissolved in the specimen passing through the flow passage. In this regard, “88% of the saturated concentration” corresponds to about 4.6 mol/L with respect to a NaCl solution in which saturation is 5.2 mol/L.
Further, the micro analysis chip according to the present disclosure may have a configuration in which, for example, a third flow passage chamber provided with a working electrode is included in addition to the first flow passage chamber and the second flow passage chamber, these being connected by a flow passage. In this regard, there is no particular limitation regarding the number of flow passage chambers arranged on the micro analysis chip.
Exemplary embodiments according to the present disclosure will be described below with reference to the drawings. In this regard, the following embodiments are exemplifications, and the present disclosure is not limited to the contents of the embodiments. Regarding the following drawings, constituent elements unnecessary for explanation of the embodiments are omitted from the drawings.
Example 1 will be described with reference to
The passage was formed by a method described in (Japanese Patent Laid-Open No. 2021-37612). Specifically, a flow passage forming particle (toner) characterized by meltability was used, an electrophotographic system was adopted, and a predetermined flow passage pattern in an unfixed state was formed on filter paper. Thereafter, the flow passage pattern was made to permeate the interior of the paper by an oven or a heater so as to form a flow passage pattern. The resulting flow passage pattern is as illustrated in
Subsequently, the reference electrode and the working electrode were formed on the flow passage pattern by screen printing, an ink jet apparatus, a dispenser, or the like. Since the present disclosure relates to a reference electrode, the configuration is described focusing on the reference electrode.
As illustrated in
Regarding the electrolyte, a chloride is favorable, and in particular, sodium chloride (NaCl) or potassium chloride (KCl) which are easy to handle are favorable. Hereafter, explanations will be performed with reference to NaCl having solubility that exhibits more stable temperature dependence.
The electrolyte (NaCl) was arranged in layers on the flow passage before the reference electrode so that the amount was set to be 3.0×102 g/L or more and preferably 3.4×102 g/L or more per liter of the amount of the specimen supplied to the reference electrode. This is a value obtained by dividing the amount of NaCl necessary for saturation by the amount of the specimen supplied to the reference electrode since the saturated concentration of the electrolyte also depends on the amount of the specimen. When 3.0×102 g/L or more per liter of the amount of the specimen supplied to the reference electrode of NaCl is arranged, the concentration in the specimen passed through the NaCl layer is 4.6 mol/L or more and is close to the saturated concentration at 25° C. so as to enable stable measurement to be performed. Preferably, 3.4×102 g/L or more of NaCl is arranged, and in such an instance, the concentration in the specimen passed through the NaCl layer is almost the saturated concentration (5.2 mol/L) and is a concentration sufficient for maintaining the state in which the Cl− concentration is almost saturated. There is no particular limitation regarding the upper limit, and 5.6×102 g/L or less is appropriate since NaCl may overflow during NaCl coating.
In this regard, when the electrolyte is KCl, it is sufficient that the amount of the electrolyte used is 3.0×102 g/L or more and preferably 3.4×102 g/L or more per liter of the amount of the specimen supplied to the reference electrode. In such an instance, the concentration in the specimen is 3.6 mol/L or more and 4.2 mol/L or more, respectively, and is a value of the saturated concentration of a KCl solution or close to this.
In this regard, there is an appropriate amount of specimen supplied to the dispensation portion in accordance with the size and the performance of each device, and in general, the amount is about 10 μL to 50 μL. For example, in the instance in which the size of the reference electrode is 3 mm×3 mm, the size of the working electrode is 3 mm×3 mm, and the thickness of the paper is 200 μm as an example of a small device, the volume of the total flow passage including the flow passage between these is about 3.6×10−9 m3 (3.6 μL) (reference electrode is 1.8 μL and working electrode is 1.8 μL). Consequently, the specimen being about 10 μL enables the specimen to be sufficiently supplied to the reference electrode and the working electrode.
When the size of the device increases, a necessary amount of the specimen increases. However, since the base of the specimen is human blood or urine, a small amount is favorable, and, in general, the amount is 50 μL or less. The present disclosure does not depend on the size of the device and the necessary amount of the specimen. However, for the above-described reason, the explanation will be provided on the assumption that the amount of the specimen is within the range of about 10 μL to 50 μL.
Regarding the micro analysis chip of Example 1, 10 μL of the specimen is dispensed and supplied to a dispensation portion 11 located at the center of the flow passage region, about a half thereof is supplied to the reference electrode 3 side (S portion in
For example, when the amount of the specimen supplied to the reference electrode is 5 μL, 4.6 mol/L or more of NaCl concentration is obtained by arranging 1.5 mg of NaCl. Consequently, when NaCl is arranged having a thickness of 200 μm in a 3 mm×3 mm region, the NaCl concentration in the specimen passed through the NaCl layer becomes 4.6 mol/L or more that is close to saturation by arranging 8.3×105 g/m3 or more of NaCl per unit volume of a NaCl arrangement region. Further, when 9.4×105 g/m3 or more of NaCl per unit volume of the NaCl arrangement region is arranged, the concentration becomes close to the saturated concentration (about 5.2 mol/L) in the specimen passed through the NaCl layer and is a concentration sufficient for making the Ag/AgCl electrode into an equilibrium state. In this regard, to specify a concentration of a solution in which NaCl is dissolved in the specimen, the specific gravity of the solution has to be taken into consideration, and the above-described NaCl concentration is a value in consideration of it.
When the amount of the specimen supplied to the reference electrode is 25 μL, and NaCl is arranged having a thickness of 200 μm in a 3 mm×3 mm region, as described above, the NaCl concentration in the specimen passed through the NaCl layer becomes 4.6 mol/L or more by arranging 4.2×106 g/m3 or more of NaCl per unit volume of the NaCl arrangement region. To set the NaCl concentration in the specimen passed through the NaCl layer to be almost saturated concentration (about 5.2 mol/L), it is sufficient that 4.7×106 g/m3 or more of NaCl per unit volume of the NaCl arrangement region is arranged.
In addition, when the amount of the specimen supplied to the reference electrode is 5 μL, and NaCl is arranged having a thickness of 200 μm in a 6.7 mm×6.7 mm region, the NaCl concentration in the specimen passed through the NaCl layer becomes 4.6 mol/L or more by arranging 1.7×105 g/m3 or more of NaCl per unit volume of the NaCl arrangement region. To set the NaCl concentration in the specimen passed through the NaCl layer to be almost saturated concentration (about 5.2 mol/L), it is sufficient that 1.9×105 g/m3 or more of NaCl per unit volume of the NaCl arrangement region is arranged.
When the specimen reaches the arranged electrolyte (NaCl) layer 3a, the specimen moves in the direction of the reference electrode (Ag/AgCl electrode) 3 while dissolving the arranged electrolyte (NaCl). In such an instance, the Cl− concentration in the specimen passed through the electrolyte (NaCl) layer is always maintained in the state of saturation or close to saturation, and since the saturated specimen is continuously supplied to the reference electrode (Ag/AgCl electrode), a stable potential is obtained.
In the present configuration (
The reference electrode potential was measured using the specimen where the electrolyte (NaCl) concentration was changed from 10−5 mol/L to 1 mol/L. The results are illustrated in
As a result of the measurement, it was ascertained that the potentials obtained by using specimens having different concentrations were constant. Variations when specimens having different concentrations were used were within about 0.3 mV (
Further, as illustrated in
Regarding the working electrode (ion-selective electrode), a solid-contact-type ion-selective electrode in which an ion-selective film having target-ion selectivity was stacked on a base electrode was used. In the present example, the target ion of the ion-selective electrode was set to be Na+.
Regarding the base electrode, efforts to use Ag/AgCl, carbon, and PEDOT/PASS have been proposed, and there is no particular limitation regarding use in the present disclosure. It is sufficient that the base electrode is selected in accordance with characteristics, such as cost and performance, necessary for the device. In the present example, a Ag/AgCl electrode was adopted as the base electrode.
Regarding the ion-selective film, commonly used ion-selective films may be used provided that the film has sensitivity to a target ion and having sufficient selectivity for an interfering ion. Examples of the material used for the ion-selective film includes ionophores, such as 12-crown-4-ether having a crown ether structure, anion removers, such as sodium tetraphenylborate (NaTPB), plasticizers, such as NPOE and DOS, polymer agents, such as simple PVCs and copolymers of vinyl chloride and vinyl acetate.
Subsequently, the ion-selective film is produced by mixing appropriate amounts of components, dissolving or dispersing them in THE or cyclohexanone serving as a solvent, applying the resulting liquid by an ink jet method to a base electrode (Ag/AgCl electrode) in which intermediate layers such as NaCl are stacked. In this regard, the application method is not limited to the ink jet method, and it is sufficient that the ion-selective film is stacked on the base electrode after the viscosity of the solution is adjusted in accordance with the printing method, such as a dispenser or screen printing.
Regarding this test, the potential profile between the electrodes is obtained in accordance with the Na+ concentration in the specimen, and it is clear that the results indicating sufficient sensitivity (gradient of the graph) and selectivity are obtained.
Further, this device was used, a commercially available electrode was set in a dispensation portion between the reference electrode and the working electrode (ion-selective electrode), the potential between the reference electrode and the commercially available electrode was measured (□plot in
It is clear that there was substantially no change in the reference electrode-side potential (D) even when the Na+ concentration in the specimen changed and that the ion-selective electrode-side potential (o) changed in accordance with the change in the Na+ concentration. In this regard, the difference of the two is the potential difference between the reference electrode and the working electrode and is indicated by the • plot in
In this regard, the present disclosure is not limited to the micro analysis chip in which the Ag/AgCl electrode is used alone as the reference electrode. As described in NPL 1, a configuration in which Ag/AgCl is used as the base electrode and a support electrolyte layer to stabilize the interfacial potential to the specimen and to decrease the influence of an interfering ion is disposed thereon may be adopted. For example, as described in NPL 1, the support electrolyte layer may be formed by mixing appropriate amounts of TBA-TBB (tetrabutylammonium tetrabutylborate) or TDMACl (tridecylmethylammonium chloride), a plasticizer, and PVC and applying and drying a solution produced by mixing these into THE or cyclohexane serving as a solvent.
In the present example, as illustrated in
Regarding this configuration, the electrolyte is not limited to a chloride. When the ion-selective film used for the reference electrode is a Na+-selective film, the electrolyte may be a hydroxide such as sodium hydroxide (NaOH), and the electrolyte may be selected in accordance with the target ion.
A configuration to make the operation in Example 1 more effective will be described.
In a configuration in which an electrolyte layer (NaCl or KCl) 3a is arranged upstream of the Ag/AgCl electrode, a laminate layer 14 is disposed on the surface and the back surface of the reference electrode (
In general, the Ag/AgCl electrode is printed on the paper by screen printing. Since a Ag/AgCl paste ink for screen printing has a certain level of viscosity, the ink is not completely printed in the thickness direction of the filter paper and is printed leaving a certain portion of filter paper on the back surface side. In addition, regarding the flow passage, in consideration of deviation of the Ag/AgCl electrode due to printing, the ink is printed leaving at least about 0.2 mm of clearance from the flow passage wall. Therefore, a filter paper portion functioning as a flow passage remains to the right and left of and under the reference electrode. When the surface of the reference electrode is covered with a laminate layer, a region in which the saturated specimen can move is limited, and the saturated specimen preferentially passes through the filter paper portion remaining to the right and left of and under the reference electrode.
When such a configuration is adopted, only the specimen passed through the limited flow passage comes into contact with the reference electrode, and the specimen in contact with the reference electrode is readily limited to the saturated specimen. Consequently, an output potential is further stabilized.
In this regard, the relation between the electrolyte 3a and the reference electrode 3 according to the present disclosure is not limited to the arrangement configuration illustrated in
According to the present disclosure, a micro analysis chip capable of performing stable analysis is provided.
While the present disclosure has been described with reference to exemplary embodiments, it is to be understood that the disclosure is not limited to the disclosed exemplary embodiments. The scope of the following claims is to be accorded the broadest interpretation so as to encompass all such modifications and equivalent structures and functions.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022-065895 | Apr 2022 | JP | national |
This application is a Continuation of International Patent Application No. PCT/JP2023/010501, filed Mar. 17, 2023, which claims the benefit of Japanese Patent Application No. 2022-065895, filed Apr. 12, 2022, both of which are hereby incorporated by reference herein in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2023/010501 | Mar 2023 | WO |
| Child | 18913277 | US |
