JP7148136B2 - Water hardness measurement - Google Patents

Description

The present disclosure relates to measuring water hardness. In one aspect, the present disclosure relates to measuring water hardness using a photoprotein indicator.

Water hardness is an index calculated from the concentrations of calcium and magnesium contained in water. As a method for measuring water hardness, for example, a colorimetric method using a low-molecular-weight compound such as o-cresolphthalein complexone, eriochrome black T, and calmagite as a coloring reagent is known (Patent Documents 1 and 2. ).

Luminescent proteins, such as luciferases, are proteins that can catalyze the reaction of a luminescent substrate, such as luciferin, to generate a radiative signal and emit light completely independently of an external light source. Recently, the brightest luciferase, NanoLuc (Nluc), was developed by genetic engineering using deep-sea shrimp Oplophorus luciferase (Oluc). A substrate of choice, furimazine, has also been developed.
Furthermore, a luminescent protein has also been developed in which a luminescent protein and a fluorescent protein are fused. In this fusion photoprotein, the energy of the donor photoprotein is transferred to the acceptor fluorescent protein by Förster resonance energy transfer (FRET) to generate luminescence.
One example of this fusion photoprotein is the nanolantern. Renilla luciferase and the yellow fluorescent protein Venus were combined to create yellow nanolanterns (YNL). Renilla luciferase was also combined with the orange fluorescent protein KusabiraOrange2 to create orange nanolanterns (ONL).
Additionally, the enhanced nanolanthanum (eNL) series has been developed. Nluc is conjugated with cyan fluorescent protein mTuroquoise2, green fluorescent protein mNeonGreen, yellow fluorescent protein Venus, orange fluorescent protein mKOκ, and red fluorescent protein tdTomato to give cyan eNL (CeNL), green eNL (GeNL), yellow eNL (YeNL), Orange-yellow (OeNL) and red eNL (ReNL) were produced, respectively [1].

JP 2005-283392 A Japanese Patent Publication No. 2004-518113

Suzuki, K et al. Nat Commun. 2016, DOI: 10.1038/ncomms13718

Biological proteins include proteins that bind calcium and proteins that bind magnesium. However, no attempts have been made to use these proteins to measure water hardness.
Accordingly, in one aspect, the present disclosure provides a method of measuring water hardness using a fusion photoprotein.

The present disclosure provides, in one aspect, a method of measuring water hardness using a photoprotein indicator, comprising:
contacting the photoprotein indicator with water and a luminescent substrate; and
determining or calculating water hardness from the luminescence signal of the photoprotein indicator;
The luminescent protein indicator has a chemiluminescent protein portion, a metal ion binding protein portion, and a fluorescent protein portion, wherein the metal ion binding protein portion is linked between the chemiluminescent protein portion and the fluorescent protein portion. is a protein that contains
The metal ion binding protein is a protein having a divalent metal cation binding domain,
The luminescent substrate is a substrate of the chemiluminescent protein.

In one aspect, the present disclosure is a device for measuring water hardness, which includes a reagent part in which a luminescent protein indicator and a luminescent substrate of the indicator are arranged, and a substrate on which the reagent part is formed, wherein the luminescent protein The indicator and the luminescent substrate relate to a device for measuring water hardness, independently arranged in the reagent part.

In one aspect of the present disclosure, a mobile information terminal including an imaging unit captures an image of a luminescence signal emitted by supplying sample water to a device for measuring water hardness, and information processing provided by the mobile information terminal. The present invention relates to a method for measuring water hardness, including determining or calculating the hardness of water from imaging data of a luminescence signal obtained by the imaging unit.

In one aspect, the present disclosure provides a device for measuring water hardness, a mobile information terminal including an imaging unit capable of imaging a luminescence signal emitted by supplying water to the device, and a luminescence signal obtained by the imaging unit. The present invention relates to a water hardness measurement system including application software capable of determining or calculating water hardness from imaging data.

According to the present disclosure, in one aspect, a method for measuring water hardness using a fusion photoprotein can be provided.
According to the present disclosure, in another aspect, a method for measuring water hardness using one type of fusion photoprotein as an indicator can be provided.

FIGS. 1A and B show the emission spectra of photoprotein indicator 1 in the presence (black line) or absence (gray line) of 10 mM CaCl ₂ and 10 mM MgCl ₂ , respectively. Figures 1C and D show titration curves (Hill plot curves) of calcium and magnesium ions, respectively, against photoprotein indicator 1. FIG. 2A shows an example of the result of examining the effect of pH on the photoprotein indicator 1, and FIG. 2B shows an example of the result of examining the effect of metal ions on the photoprotein indicator 1. FIG. FIGS. 3A and 3B show hardness values (Scale) and classification (Classification) according to a method for calculating water hardness (total hardness) prescribed by the USGS (United States Geological Survey). FIG. 3C is the result of the relative chemiluminescence intensity ratio for different _CaCO3 concentrations, FIG. 3D is the result of the relative chemiluminescence intensity ratio for different _aCl2 or MgCl2 concentrations, and _FIG . It is a measurement result of commercially available mineral water. As shown in FIG. 3, photoprotein indicator 1 exhibited a water hardness-dependent chemiluminescence intensity ratio. 4A, C, and E are images captured by a camera of a smart phone, respectively, showing how the sample water used in Experiment 3 was caused to emit light from the protein luminescence indicator. 4B, D, and F respectively show the results of image analysis of the image data of FIGS. 4A, C, and E to calculate the RGB image ratio (Blue/Green). Figures 5A-D illustrate the patterning geometry of the photoprotein indicator 13 and the luminescent substrate 14 in the reagent portion 12 formed on the substrate 11 of the device.

The present disclosure is based on the finding that proteins that function as calcium-binding proteins or magnesium-binding proteins in vivo can be used to measure water hardness.
The present disclosure also provides that a protein that functions as a calcium-binding protein or a magnesium-binding protein in vivo can bind both calcium and magnesium in a range of concentrations in water hardness measurements, providing a single photoprotein indicator. It is based on the knowledge that it can be used to measure the total hardness.
In the present disclosure, "binding calcium" and "binding magnesium" can mean "binding calcium ions" and "binding magnesium ions" unless otherwise specified. .

In the present disclosure, water hardness may mean total hardness calculated from concentrations of calcium ions and magnesium ions in water, unless otherwise specified.

[Photoprotein indicator/fusion photoprotein]
In the present disclosure, a photoprotein indicator, in one or more embodiments, refers to an indicator that emits a luminescence signal depending on the concentration of calcium ions and magnesium ions for measuring water hardness.
In the present disclosure, a luminescent protein indicator, in one or more embodiments, has a chemiluminescent protein portion, a metal ion binding protein portion, and a fluorescent protein portion, wherein the chemiluminescent protein portion and the fluorescent protein portion It is a fusion photoprotein in which a metal ion binding protein moiety is linked between.
In the present disclosure, a photoprotein indicator can mean the fusion photoprotein itself in one or more embodiments, unless otherwise specified. The present disclosure, in one aspect, has a chemiluminescent protein portion, a metal ion binding protein portion, and a fluorescent protein portion, wherein the metal ion binding protein portion is linked between the chemiluminescent protein portion and the fluorescent protein portion. It relates to a fusion photoprotein. The fusion photoprotein can be used as a photoprotein indicator in the method of measuring water hardness according to the present disclosure.

In the present disclosure, in one or more embodiments, the fusion photoprotein is obtained from the chemiluminescent protein moiety by binding calcium ions or magnesium ions to the metal ion-binding protein moiety from the viewpoint of measuring water hardness. It is preferably linked to allow resonance energy transfer to the fluorescent protein moiety.
In the present disclosure, the resonance energy transfer, in one aspect, is Förster resonance energy transfer (FRET).
In one or more embodiments, the portion of the chemiluminescent protein is a FRET donor and the portion of the fluorescent protein is a FRET acceptor.

[Part of chemiluminescent protein]
A "chemiluminescent protein portion" in the present disclosure is, in one or more embodiments, derived from a chemiluminescent protein. In the present disclosure, a chemiluminescent protein is, in one or more embodiments, a protein that catalyzes a luminescence-producing reaction of a specific chemical substrate. The chemiluminescent proteins, in one or more embodiments, include luciferase, aequorin, oberin, and combinations thereof.

Chemiluminescent proteins may be those isolated or cloned from naturally occurring luciferase, aequorin, and obelin, or modified forms thereof. Variants of chemiluminescent proteins, in one or more embodiments, have mutations (e.g., deletions and / or substitutions) in portions that do not affect chemiluminescent functional properties (e.g., luminescence intensity, luminescence color), and A chemiluminescent protein that has substantially the same or similar chemiluminescent functional properties as compared to the state without said mutation. Further, the variant of the chemiluminescent protein, in one or more embodiments, has a mutation (e.g., deletion and / or substitution) in the portion that affects the chemiluminescence functional properties, and compared to the state without the mutation are chemiluminescent proteins having substantially different chemiluminescent functional properties.

Luciferases include, in one or more non-limiting embodiments, Renilla luciferase (Renilla luciferase), deep sea shrimp luciferase (Oplophorus luciferase), Cypridina luciferase (Vargula/Cypridina luciferase), Chia luciferase (Gaussia luciferase, Metridia luciferase), Eddy Flagella luciferase, luminescent mushroom (Mycena chlorophos, Omphalotus japinicus) luciferase, cherry shrimp luciferase, firefly luciferase (Photinus luciferase), bacterial luciferase, Akaluc, Turboluc and the like.

In the present disclosure, "a portion of a chemiluminescent protein" refers to a chemiluminescent protein or a portion thereof incorporated (fused) into a fusion luminescent protein. A "part of a chemiluminescent protein" may be the entire chemiluminescent protein or a portion thereof. The part of the chemiluminescent protein is, in one or more embodiments, a part of the N-terminus and / or C-terminus is deleted, and substantially the same as compared to the state without deletion Alternatively, it is a portion of said chemiluminescent protein with similar chemiluminescent functional properties (eg, luminescence intensity, luminescence color).
Chemiluminescent proteins in the present disclosure can be commercially available.

[Luminescent substrate]
In the present disclosure, a luminescent substrate refers to a luminescent substrate of a chemiluminescent protein in the present disclosure. For example, a luminescent substrate for luciferase is called luciferin.
In general, a chemiluminescent protein and a luminescent substrate are a specific combination, and those skilled in the art can understand the luminescent substrate corresponding to the chemiluminescent protein (luciferin for luciferase), or understand the chemiluminescent protein (luciferase, which corresponds to luciferin).
In one or more non-limiting embodiments, the luminescent substrate includes firefly luciferin, bacterial luciferin, dinoflagellate luciferin, vargrin, coelenterazine, AkaLumine-HCl, variants thereof, and combinations thereof. mentioned.

Firefly luciferin, bacterial luciferin, dinoflagellate luciferin, and vargulin can be luciferins for firefly luciferase, bacterial luciferase, dinoflagellate luciferase, and Cypridina luciferase (Vargula/Cypridina luciferase), respectively.
Coelenterazine can also be a substrate for Renilla luciferase, Oplophorus luciferase, Gaussia luciferase, Aequorin, and Oberin.
The luminescent substrate may be an isolated or synthesized substrate existing in nature, or a modification thereof.

[Fluorescent protein portion]
A "portion of a fluorescent protein" in the present disclosure is, in one or more embodiments, derived from a fluorescent protein. In the present disclosure, the fluorescent protein, in one or more embodiments, can function as an acceptor of the energy of the chemiluminescent protein that is the donor described above, and can absorb the energy of the donor and emit fluorescence. In one or more embodiments, the fluorescent protein can enhance the intensity of the luminescence of the chemiluminescent protein or change the color of the luminescence.
Fluorescent proteins may be those isolated or cloned from those existing in nature, commercially available ones, or modifications thereof. Fluorescent proteins of various colors have been developed, and by using these, the emission colors of the fusion photoproteins according to the present disclosure can be designed in various ways.
A variant of a fluorescent protein, in one or more embodiments, has a mutation (e.g., deletion and / or substitution) in a portion that does not affect fluorescence properties (e.g., luminescence intensity, luminescence color), and the mutation is A fluorescent protein that has substantially the same or similar fluorescence properties as compared to the free state. In one or more embodiments, the variant of the fluorescent protein has a mutation (e.g., deletion and/or substitution) in a portion that affects fluorescence properties (e.g., luminescence intensity, luminescence color), and the mutation Fluorescent proteins that have substantially different fluorescence properties compared to the absence.

.
Fluorescent proteins include, in one or more non-limiting embodiments, green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow-green fluorescent protein, yellow fluorescent protein, orange fluorescent protein, and red fluorescent protein, and more specifically In one or more non-limiting embodiments, Azurite, bsDronpa, Cerulean, Citrine, Clover, CyOFP1, Dendra2, Dreiklang, Dronpa2, Dronpa3, DsRed, EBFP, EBFP2, ECFP, EGFP, Emerald, eqFP650, eqFP670, EYFP, Fast-FT, FusionRed, Gamillus, hmKeima8.5, IFP1.4, IFP2.0, iRFP670, iRFP682, iRFP702, iRFP713, iRFP720, Kaeda, KikGR, Kohinoor, LSSmCherry, LSSmKate2, LSSmOrange, mAG1, mAmetrine, mAmetrine1.2, mApple, mBlueberry2, mCardinal, mCerulean3, mCherry, mCherry2, mClover3, mCyRFP1, Medium-FT, mEOS2, mEOS3.1, mEOS3.2, mGarnet2, mIFP2, miniSOG, miRFP670, miRFP720, mKalama1, mKate2, mK-GO, mKOkappa, mKO1, mKO2, mlris, mMaroon1, mNeonGreen, mNeptune, mNeptune2, mNeptune2.5, mNeptune681, mNeptune684, mOrange, mOrange2, mPlum, mRFP1, mRuby2, mRuby3, mScarlet, mScarlet-H, mScarlet-I, mStable, mStrawberry, mTagBFP2, mTFP1, mTurquoise, mTurquoise2, Neptune, NowGFP, oxFP series, Padron, PA-GFP, PAmCherry1, PAmChe rry2, PAmCherry3, Phanta, PS-CFP, PS-CFP2, PSLSSmKate, PSmOrange, RDSmCherry1, rsCherry, rsEGFP, rsEGFP2, rsTagRFP, SBFP2, shyRFP, Sirius, skylan NS, skylan S, Slow-FT, SPOON, TagBFP, TagCFP, TagFP635, TagGFP, TagGFP2, TagRFP, TagRFP657, TagRFP675, TagRFP-T, TagYFP, T-Sapphire, TurboFP602, TurboGFP, TurboRFP, TurboYFP, UnaG, Venus, and circular permutations thereof. Details of these fluorescent proteins can be referred to, for example, https://sites.google.com/site/ilovegfp/Home/fps.

In the present disclosure, the term “portion of a fluorescent protein” refers to a fluorescent protein incorporated (fused) into a fusion luminescent protein or a portion thereof. A "portion of a fluorescent protein" may be the entire fluorescent protein or a portion thereof. The part of the fluorescent protein is, in one or more embodiments, a part of the N-terminus and/or C-terminus is deleted, and is substantially the same or It is the part of a fluorescent protein that has similar fluorescence properties.
Fluorescent proteins in the present disclosure may be commercially available.

[Metal ion binding protein portion]
A metal ion binding protein portion in the present disclosure, in one or more embodiments, is derived from a metal ion binding protein. In the present disclosure, a metal ion-binding protein is, in one or more embodiments, a protein that binds to calcium and magnesium in water when measuring water hardness. In one or more embodiments, the metal ion-binding protein is a protein that undergoes a structural change by binding with calcium or magnesium in water when water hardness is measured.

Metal ion-binding proteins include proteins known in living organisms or cells as calcium-binding proteins or magnesium-binding proteins, or modifications thereof.
A protein having a function capable of binding calcium in a living body or a cell and a protein having a function capable of binding magnesium bind both calcium and magnesium at the calcium concentration and magnesium concentration when measuring water hardness. The present disclosure is based, in one aspect, on the finding that it functions as a soluble protein.
By having such a metal ion-binding protein portion, the fusion photoprotein according to the present disclosure, in one or more embodiments, enables water hardness to be measured using only one type of fusion photoprotein.

Metal ion binding proteins include proteins with divalent metal cation binding domains.
Divalent metal ions include calcium ions and/or magnesium ions. A divalent metal cation binding domain includes, in one or more embodiments, a calcium binding domain.
Calcium-binding domains, in one or more embodiments, include EF-hand motifs and dockerin motifs.
In one or a plurality of more specific non-limiting embodiments, metal ion binding proteins include Cen3, calmodulin, Twitch-3 and the like, preferably Cen3, from the viewpoint of measuring water hardness.

In one or more embodiments, the metal ion-binding protein preferably has a binding constant Ka with calcium of 0.25 mM to 2.5 mM from the viewpoint of measuring water hardness, and a binding constant Ka with magnesium of 0.41 mM to 1.25 mM is preferred.

In one or more embodiments, the metal ion-binding protein may be an isolated or cloned protein known as a calcium-binding protein or a magnesium-binding protein in a living organism or cell, or a variant thereof. may In one or more embodiments, the variant of the protein has a mutation (e.g., deletion and/or substitution) in a portion that does not affect the ability to bind calcium and magnesium, and compared with a state without the mutation A protein having substantially the same or similar ability to bind calcium and magnesium as a protein. Further, in one or more embodiments, the variant of the protein has a mutation (e.g., deletion and/or substitution) in a portion that affects the ability to bind calcium and magnesium, and a state without the mutation. proteins that have substantially different binding capacities for calcium and magnesium in comparison.

In the present disclosure, "a portion of a metal ion binding protein" refers to a metal ion binding protein or a portion thereof that is incorporated (fused) into a fusion photoprotein. A "portion of a metal ion binding protein" may be the entire metal ion binding protein or a portion thereof. In one or more embodiments, the part of the metal ion-binding protein means that part of the N-terminus and/or C-terminus is deleted, and substantially Portions of said metal ion binding proteins having the same or similar calcium and magnesium binding capacity.

In one or more embodiments, the order of linkage of the three moieties in the fusion photoprotein according to the present disclosure is, from the N-terminal side, the order of the chemiluminescent protein moiety, the metal ion-binding protein moiety, and the fluorescent protein moiety. Alternatively, the fluorescent protein portion, the metal ion-binding protein portion, and the chemiluminescent protein portion may be ordered from the N-terminal side.

In one or more embodiments of the fusion photoprotein according to the present disclosure, the chemiluminescent protein portion, the metal ion-binding protein portion, and the fluorescent protein portion satisfy the following conditions from the viewpoint of measuring water hardness. are preferably fused (linked) as follows.
(1) When calcium ions or magnesium ions are not bound to the metal ion-binding protein portion, resonance energy transfer does not occur from the chemiluminescent protein portion to the fluorescent protein portion, and only the chemiluminescent protein emits light even when a luminescent substrate is added. do not have.
(2) When calcium ions or magnesium ions bind to the metal ion-binding protein portion, the structure of the metal ion-binding protein portion changes, allowing resonance energy transfer from the chemiluminescent protein portion to the fluorescent protein portion, and the luminescent substrate becomes Fluorescence occurs when added.
In one or more embodiments, (1) and the The condition (2) can be easily satisfied.

In one or more embodiments, the fusion photoprotein according to the present disclosure may be a recombinant protein produced by genetic recombination technology or a protein synthesized by chemical synthesis. In one or more embodiments, the production of a recombinant protein by genetic recombination technology includes a method of producing using a host transformed with an expression vector containing a gene encoding a fusion photoprotein according to the present disclosure, or no Methods of making in cell lines are included. A fusion photoprotein according to the present disclosure may be purified, such as by using a tag protein.

[Method for measuring water hardness]
The method for measuring the hardness of water according to the present disclosure includes contacting the photoprotein indicator (fusion photoprotein) according to the present disclosure with water, which is a measurement sample, and a luminescent substrate; including determining or calculating the hardness of
Examples of the water whose hardness is to be measured include water whose hardness has been conventionally measured and whose fluctuation range can be expected. The water whose hardness is to be measured is not particularly limited, but drinking water, tap water, spring water, well water, running water, ground water, industrial water and the like can be mentioned.
When a photoprotein indicator according to the present disclosure contacts water and a luminescent substrate, it produces luminescence dependent on calcium and magnesium concentrations. The water hardness is measured based on this luminescence signal.
Water hardness measurements based on luminescence signals may, in one or more embodiments, be based on calculations based on luminescence signal data. For example, luminescence signal data is taken into a portable information terminal, and the data is measured using an application or the like on the terminal of the portable information terminal or via the Internet.
In one or more embodiments, the measurement of water hardness based on luminescence signals includes measuring the luminescence intensity of a chemiluminescent protein and the luminescence intensity of a fluorescent protein and using their ratio as an index. A calibration curve based on the ratio of emission intensities may be prepared in advance.
In one or more embodiments, water hardness measurement based on a luminescence signal includes capturing an image of luminescence, converting it into imaging data, and using an analysis value of the data as an index. For example, it includes obtaining the brightness of the color derived from the chemiluminescent protein and the brightness of the color derived from the fluorescent protein from imaging data and using their ratio as an index. A calibration curve based on the luminance ratio may be prepared in advance. In one or more embodiments, water hardness measurement using imaging data is performed by an information terminal equipped with a camera for obtaining imaging data and application software for analyzing the imaging data to measure water hardness. (e.g., smart phone).
In one or a plurality of embodiments, the measurement of water hardness based on the luminescence signal may be performed visually by comparing the state of luminescence with a table or the like classified in advance by luminescence color.
In the present disclosure, measurement of water hardness can mean determination of water hardness and/or calculation of water hardness in one or more embodiments. Different countries use different calculation methods for water hardness, but they all share the common point of using the calcium concentration and magnesium concentration as the basis for calculation. It can be determined and/or calculated. One or more embodiments of the water hardness calculation method include a method of calculating calcium and magnesium in water as CaCO ₃ converted values (mg/L).
In the present disclosure, the determination of water hardness is, in one or more embodiments, determining whether it belongs to either soft water or hard water, or determining whether it belongs to soft water, medium hard water, hard water, or ultra-hard water. or determining whether it belongs to a predetermined hardness range.

[device]
In the method for measuring water hardness according to the present disclosure, a device can be used to bring the photoprotein indicator according to the present disclosure into contact with water, which is a measurement sample, and a luminescent substrate.
In one or more embodiments, a device according to the present disclosure includes a reagent portion in which a photoprotein indicator according to the present disclosure and a luminescent substrate of the indicator are arranged, and a substrate in which the reagent portion is formed.
At least a photoprotein indicator and a luminescent substrate according to the present disclosure are arranged in the reagent part.

In the device according to the present disclosure, the photoprotein indicator and the luminescent substrate are arranged independently. In one or more embodiments, the luminescent protein indicator and the luminescent substrate are arranged such that when water is supplied to the reagent part in which they are arranged, the luminescent protein indicator and the luminescent substrate react to generate a luminescent signal. It is Accordingly, when water is supplied to the reagent part, the photoprotein indicator and the luminescent substrate come into contact (mix) to generate a luminescent signal. According to the device of the present disclosure having such a configuration, in one or more embodiments, it has a higher signal-to-noise ratio and does not require excitation light, so no special measurement equipment or equipment is required. Preferably, the effect of being can be exhibited. In addition, according to the device of the present disclosure, in one or more embodiments, the user can detect the emitted luminescence signal by capturing an image with an imaging means such as a personal digital assistant possessed by the user, and the Data obtained by imaging by the user can be used for quick measurement, determination, diagnosis, or the like.

In the present disclosure, "independently arranged" means that, in one or more embodiments, the photoprotein indicator and the luminescent substrate are arranged in a state in which no reaction occurs without contact with a liquid such as a sample. is mentioned. Arranged independently includes, in one or more embodiments, that the adjacent photoprotein indicator and luminescent substrate are physically (or spatially) separated. In addition, in one or more embodiments, the independent arrangement can include a form in which the photoprotein indicator and the luminescent substrate are arranged in contact with each other as long as the photoprotein indicator and the luminescent substrate do not react (for example, in a dry state).

The photoprotein indicator and luminescent substrate may be arranged in a pattern in one or more embodiments. In one or a plurality of embodiments, the shape of the patterning may be dot-shaped, line-shaped, circle-shaped, or the like. In one or more embodiments, the photoprotein indicator and the luminescent substrate are alternately and mosaically patterned in order to increase the contact rate between the luminescent protein indicator and the luminescent substrate and generate a higher luminescent signal. is preferred. Fine patterning is preferable from the point of view of improving reproducibility.

In the device of the present disclosure, the reagent portion is formed on the substrate. In one or a plurality of embodiments, the reagent part may be formed on the same plane of one substrate, or may be formed so as to be sandwiched between two substrates.

FIG. 5 illustrates non-limiting patterning shapes. In FIG. 5A, the reagent part 12 is formed by alternately patterning dot-like luminescent protein indicators 13 and luminescent substrates 14 on one substrate 11 in a mosaic pattern. In FIG. 5B, the photoprotein indicator 13 and the luminescent substrate 14 are patterned in dots on one surface of the two substrates 11, and the patterned surfaces of the luminescent protein indicator 3 and the luminescent substrate 14 face each other. A reagent portion 12 is formed by arranging two substrates 11 . In FIGS. 5C and 5D, the reagent portion 12 is formed by patterning the luminescent protein indicator 13 and the luminescent substrate 14 in lines or circles on one substrate 11, respectively.

In one or a plurality of embodiments, the photoprotein indicator and the luminescent substrate are preferably in a dry state in order to reduce the reaction between the photoprotein indicator and the luminescent substrate before water is supplied.

The material of the substrate is not particularly limited, and in one or more embodiments, paraffin, fluorine-based materials such as Teflon (registered trademark), glass, polypropylene, woven fabric, non-woven fabric, paper, and the like. . In one or a plurality of embodiments, the material of the substrate is preferably hydrophobic in order to detect a high luminescence signal.

[Device manufacturing method]
A manufacturing method according to the present disclosure, in one or more embodiments, is a method for manufacturing the device of the present disclosure. The manufacturing method of the present disclosure involves patterning a substrate such that the photoprotein indicator and the luminescent substrate are independently positioned.

As the patterning of the luminescent protein indicator and the luminescent substrate, in one or more embodiments, when water is supplied to the place where the luminescent protein indicator and the luminescent substrate are arranged, the luminescent protein indicator and the luminescent substrate come into contact (mix). These can be arranged independently so that they can be used together to generate a luminescence signal.

As a patterning method, in one or a plurality of embodiments, a solution of a luminescent protein indicator or a luminescent substrate can be arranged in a predetermined pattern shape and dried. Patterning, in one or more embodiments, can be performed using known techniques such as an inkjet printer.

[Method for measuring water hardness using a device]
A detection method according to the present disclosure, in one or more embodiments, is a method for measuring water hardness using a device according to the present disclosure.

Methods of this aspect include, in one or more embodiments, providing water to the reagent portion of a device of the present disclosure and utilizing a luminescence signal produced by providing a sample. Since the device of the present disclosure does not require excitation light, in one or more embodiments, the luminescence signal can be converted into data using a mobile information terminal (smartphone, etc.) and imaging means such as a digital camera.

The detection methods of the present disclosure, in one or more embodiments, can be performed at room or ambient temperature. In one or a plurality of embodiments, the time from water supply to observation may be several seconds to several minutes, or several seconds to one minute.

[Water hardness measurement system]
In one aspect, the present disclosure provides a device according to the present disclosure, a mobile information terminal including an imaging unit capable of imaging a luminescence signal emitted by supplying water to the device, and a luminescence signal obtained by the imaging unit. The present invention relates to a water hardness measurement system including application software capable of determining or calculating water hardness from imaging data.
In one or a plurality of embodiments, the mobile information terminal including the imaging unit includes a smartphone including a camera, a tablet including a camera, and the like.
In one or a plurality of embodiments, the application software measures water hardness based on imaging data of the obtained luminescence signal and information stored in a mobile information terminal or information obtained via the Internet. and displaying it on the mobile information terminal. In one or a plurality of other embodiments, the application software is, in one or more embodiments, the obtained imaging data of the luminescence signal to the server via the network , receives the results measured by the server, and causes the portable information terminal to perform processing for display on the terminal.

[Nucleic acid]
The disclosure relates, in one aspect, to a nucleic acid encoding a fusion photoprotein of the disclosure. In the present disclosure, nucleic acids include single- or double-stranded DNAs selected from synthetic DNAs, cDNAs, genomic DNAs and plasmid DNAs, and transcription products of these DNAs.

[Expression cassette]
The disclosure relates, in one aspect, to an expression cassette comprising a nucleic acid encoding a fusion photoprotein of the disclosure. In the expression cassette, the nucleic acid is operatively linked with an expression control sequence suitable for the host cell into which it is introduced. Expression control sequences include promoters, enhancers, transcription terminators, etc. Others include initiation codons, intronic splicing signals, and stop codons.

[vector]
The disclosure relates, in one aspect, to a vector capable of expressing a fusion photoprotein of the disclosure. In another aspect of the present disclosure, the vector of the present disclosure is, in one or more embodiments, an expression vector comprising the nucleic acid or expression cassette of the present disclosure. The vector according to the present disclosure can be used by appropriately selecting an expression vector system according to the cell (host) in which expression is desired. Vectors that can be used in the vectors of the present disclosure include, in one or more non-limiting embodiments, plasmids, cosmids, YACS, viral (e.g., adenoviral, retroviral, episomal EBV, etc.) vectors and phage vectors, and agroviral vectors. Binary vectors for bacterium methods are included.

[Transformant]
The present disclosure, in one aspect, relates to a transformant that expresses a fusion photoprotein of the present disclosure. The present disclosure, in one or more embodiments, relates to a transformant having the nucleic acid or vector of the present disclosure. A transformant of the present disclosure, in one or more embodiments, can be produced by introducing a nucleic acid, expression cassette or vector of the present disclosure into a host. Examples of hosts include animal cells, plant cells, insect cells, microorganisms, and the like.

The present disclosure further relates to one or more of the following non-limiting embodiments.
[1] A method for measuring the hardness of water using a photoprotein indicator, comprising:
contacting the photoprotein indicator with water and a luminescent substrate; and
determining or calculating water hardness from the luminescence signal of the photoprotein indicator;
The luminescent protein indicator has a chemiluminescent protein portion, a metal ion binding protein portion, and a fluorescent protein portion, wherein the metal ion binding protein portion is linked between the chemiluminescent protein portion and the fluorescent protein portion. is a protein that contains
The metal ion binding protein is a protein having a divalent metal cation binding domain,
The method, wherein the luminescent substrate is a substrate of the chemiluminescent protein.
[2] The photoprotein indicator is linked to the metal ion-binding protein portion so that resonance energy transfer from the chemiluminescent protein portion to the fluorescent protein portion is possible by binding calcium ions or magnesium ions, [1 ] method described.
[3] The method of [1] or [2], wherein the chemiluminescent protein is a photoprotein selected from the group consisting of luciferase, aequorin, oberin, and combinations thereof.
[4] any one of [1] to [3], wherein the substrate is a substance selected from the group consisting of firefly luciferin, bacterial luciferin, dinoflagellate luciferin, vargulin, coelenterazine, and combinations thereof; described method.
[5] The method according to any one of [1] to [4], wherein the metal ion-binding protein is a protein having an EF-hand motif or a dockerin motif.
[6] having a reagent part in which a luminescent protein indicator and a luminescent substrate of the indicator are arranged, and a base material on which the reagent part is formed,
The luminescent protein indicator has a chemiluminescent protein portion, a metal ion binding protein portion, and a fluorescent protein portion, wherein the metal ion binding protein portion is linked between the chemiluminescent protein portion and the fluorescent protein portion. is a protein that contains
The metal ion binding protein is a protein having a divalent metal cation binding domain,
A device for measuring water hardness, wherein the luminescent protein indicator and the luminescent substrate are independently arranged in the reagent section.
[7] capturing an image of a luminescence signal emitted by supplying water to the device according to [6] with a mobile information terminal having an imaging unit;
A method for measuring water hardness, comprising determining or calculating the hardness of water from imaging data of a luminescence signal obtained by the imaging unit by means of information processing means provided in the mobile information terminal.
[8] The device according to [6], a portable information terminal equipped with an imaging unit capable of imaging a luminescence signal emitted by supplying water to the device, and imaging data of the luminescence signal obtained by the imaging unit A water hardness measurement system including application software capable of determining or calculating water hardness.

EXAMPLES The present disclosure will be described in more detail below with reference to examples, but these are examples and the present disclosure is not limited to these examples.

[Preparation of luminescent protein indicator 1]
A fusion photoprotein that serves as a photoprotein indicator was prepared as follows.
Using NanoLuc as a chemiluminescent protein, calcium/magnesium ion-binding protein Cen3 (human-derived HsCen3) as a metal ion-binding protein, and Venus as a fluorescent protein, Venus, HsCen3, and NanoLuc were linked in this order from the N-terminal side. A fusion photoprotein was prepared and designated as photoprotein indicator 1.
HsCen3 was ligated after deleting 7 amino acid residues from the C-terminal side.

[Experimental example 1]
The luminescence properties of the photoprotein indicator 1 for calcium ions and magnesium ions were confirmed. The results are shown in FIG.
FIGS. 1A and B show the emission spectra of photoprotein indicator 1 in the presence (black line) or absence (gray line) of 10 mM CaCl ₂ and 10 mM MgCl ₂ , respectively. In FIG. 1, the emission spectrum was normalized at the isoluminous point (506 nm).
1A and B show that photoprotein reagent 1 binds to calcium ions to increase the luminescence intensity, and binds to magnesium ions to increase the luminescence intensity.
Figures 1C and D show titration curves (Hill plot curves) of calcium and magnesium ions, respectively, against photoprotein indicator 1. The Kd for calcium ions was 355 μM (Hill coefficient 1.04) and the Kd for magnesium ions was 4.7 mM (Hill coefficient 1.6).
All FIGS. 1A-D were performed on reactions with a photoprotein concentration of 4 nM, a luminescent substrate concentration of 10 nM, and a total volume of 100 μL.

[Experimental example 2]
The effects of pH and other metal ions on Luminescent Protein Indicator 1 were confirmed. The results are shown in FIG.
FIG. 2A compares the emission intensity ratio of photoprotein indicator 1 in the presence (black line) or absence (gray line) of 10 mM MgCl ₂ at various pHs. All measurements were performed in triplicate and the average values are shown. The emission intensity ratio was calculated by dividing the Venus peak (525 nM) by the NanoLuc peak (455 nM).
FIG. 2B shows the presence of 3 μM potassium, arsenic, zinc, copper, cobalt, cadmium, lead, manganese, nickel, or iron ions (left bar) with the same metal ions and 10 mM MgCl ₂ . (middle bar) and in the presence of the same metal ion and 10 mM CaCl ₂ (right bar). All measurements were performed in triplicate and SEM is shown with error bars. The emission intensity ratio was calculated by dividing the Venus peak (525 nM) by the NanoLuc peak (455 nM).
2A and B show that the photoprotein indicator 1 is stable against changes in pH and has high specificity to calcium and magnesium ions even in the presence of different metal ions.

[Experimental example 3]
CaCO ₃ aqueous solution with the concentration shown in FIG. 3A, mixed aqueous solution of CaCl ₂ and MgCl ₂ with the concentration shown in FIG. Emission intensity ratio was measured. The results are shown in Figures 3C-E.
The hardness values (Scale) and classifications (Classification) in FIGS. 3A and 3B are values and classifications according to the water hardness (total hardness) calculation method specified by the USGS (United States Geological Survey).
FIG. 3C is the result of the _CaCO3 aqueous solution with the concentration shown in FIG. 3A, _FIG . 3D is the result of the mixed aqueous solution of CaCl2 and MgCl2 with the concentration shown in _FIG . 3B, and FIG. These are the results of commercially available mineral water (CMW1-6).
As shown in FIG. 3, photoprotein indicator 1 exhibited a luminescence intensity ratio dependent on water hardness.

[Experimental example 4]
The luminescence of the luminescent protein indicator 1 using the sample water used in Experimental Example 3 was imaged with a smartphone camera, and the luminescence intensity ratio was calculated from the imaging data. The results are shown in FIG.
Figures 4A, _C , and E _are sample water used in Experiment ₃ , respectively; 1 is an image captured by a smartphone camera of a commercially available mineral water (CMW1-6) of No. 1, which was illuminated with a protein luminescence indicator.
FIGS. 4B, D, and F respectively show the results of image analysis of the image data of FIGS. 4A, C, and E to calculate the RGB image ratio (Blue/Green). 10 mM MOPS (pH 7.2) and 100 mM KCl were used as controls.
As shown in FIG. 4, the luminescence intensity ratio obtained by image analysis of the image data captured by the smartphone also depended on the hardness of the sample water. As a result, it was shown that water hardness can be determined and calculated based on data captured by a mobile information terminal such as a smartphone.

Claims (8)

A method for measuring water hardness using a luminescent protein indicator, comprising:
contacting the photoprotein indicator with water and a luminescent substrate; and
determining or calculating water hardness from the luminescence signal of the photoprotein indicator;
The luminescent protein indicator has a chemiluminescent protein portion, a metal ion binding protein portion, and a fluorescent protein portion, wherein the metal ion binding protein portion is linked between the chemiluminescent protein portion and the fluorescent protein portion. is a protein that contains
The metal ion binding protein is a protein having a divalent metal cation binding domain,
The method, wherein the luminescent substrate is a substrate of the chemiluminescent protein. 2. The luminescent protein indicator of claim 1, wherein the luminescent protein indicator is linked to allow resonance energy transfer from the chemiluminescent protein moiety to the fluorescent protein moiety upon binding of calcium or magnesium ions to the metal ion binding protein moiety. Method. 3. The method of claim 1 or 2, wherein the chemiluminescent protein is a photoprotein selected from the group consisting of luciferase, aequorin, oberin, and combinations thereof. 4. The method of any of claims 1-3, wherein the substrate is a substance selected from the group consisting of firefly luciferin, bacterial luciferin, dinoflagellate luciferin, vargrin, coelenterazine, and combinations thereof. 5. The method according to any one of claims 1 to 4, wherein said metal ion binding protein is a protein with an EF-hand motif or a dockerin motif. a reagent part in which a luminescent protein indicator and a luminescent substrate of the indicator are arranged; and a base material on which the reagent part is formed,
The luminescent protein indicator has a chemiluminescent protein portion, a metal ion binding protein portion, and a fluorescent protein portion, wherein the metal ion binding protein portion is linked between the chemiluminescent protein portion and the fluorescent protein portion. is a protein that contains
The metal ion binding protein is a protein having a divalent metal cation binding domain,
A device for measuring water hardness, wherein the luminescent protein indicator and the luminescent substrate are independently arranged in the reagent section. capturing an image of a luminescence signal emitted by supplying water to the device according to claim 6 with a mobile information terminal comprising an imaging unit;
A method for measuring water hardness, comprising determining or calculating the hardness of water from imaging data of a luminescence signal obtained by the imaging unit by means of information processing means provided in the mobile information terminal. The device according to claim 6, a portable information terminal comprising an imaging unit capable of imaging a luminescence signal emitted by supplying water to the device, and the imaging data of the luminescence signal obtained by the imaging unit Water hardness water hardness measurement system, including application software capable of determining or calculating

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