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    • 专利摘要:
    Portable microfluidic device to detect nitrite-nitrate. The invention describes an ionogel comprising a hydrogel in an ionic liquid, and a reactive system which changes color by reaction with nitrite. The invention also discloses a microfluidic device comprising: a substrate 1 comprising: calibration reservoirs 2 separated from each other comprising ionogel, a reservoir 3 for calibration of the hydrogel in the ionic liquid, a reservoir 6 for depositing sample to be analyzed, connected to through a microfluidic channel 5 with a reducing agent, to a measurement reservoir 4 comprising ionogel, and a measurement reservoir 7 comprising ionogel of the invention where sample to be analyzed is deposited. The invention also discloses methods for making the ionogel and the device as well as a method for detecting and colorimetrically determining nitrite and nitrate in a contaminated water using the ionogel and the device. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2665790A1
    申请号:ES201631376
    申请日:2016-10-26
    公开日:2018-04-27
    发明作者:Fernando BENITO LÓPEZ；Janire SAEZ CASTAÑO
    申请人:Euskal Herriko Unibertsitatea；
    IPC主号:
    专利说明:

    PORTABLE MICROFluoro IV DISPOSIT TO DETECT NITRITE-NITRATE
    Field of the InventionThe present invention falls within the techniques for detection and determination of contamination ofa mass of water per nitrate. In particular it refers to a portable microfluidic device capable of detecting
    nitrite-nitrate combined in a contaminated water quickly and efficiently, and in the same placepollution.
    Background of the inventionIn recent years, various environmental monitoring devices have appeared in response to the growingpollution of natural, industrial and municipal waters with toxic substances and pollutants fromwater such as harmful chemical agents. Environmental monitoring agencies operategenerally manually, by taking samples in the contaminated place, and transporting thesame to the corresponding facilities where the analysis is carried out by highly qualified personnelusing sophisticated instruments. This strategy has benefits such as obtaining very good results.precise and exact and the adjustment to legal regulatory procedures, but presents disadvantages such asthe high costs associated with the maintenance of these facilities, instrumentation and personnelskilled. In addition, this type of protocols prevents working on site, thus extending the response times incase of unexpected contamination. Other types of methods allow obtaining information in situ, but theequipment used is generally expensive and sophisticated, so you also need qualified personnelfor useThe determination of the concentration of nutrients in a body of water is very important because theNutrient concentration influences and modifies the balance of life in water. The increase inUncontrolled concentration of nutrients in water could lead to an environmental disaster. In particular, theincrease in nitrates causes the detriment of human health and degradation of aquatic life, such aseutrophication of algae that leads to the death of fauna and flora.Current techniques for the detection of nitrate and nitrite are amperometric, electrochemical, biosensor, orbased on spectrophotometric methods that are the most popular due to the excellent limits ofdetection (nanomolar), dynamic range and cost efficiency.Classic colorimetric methods have been traditionally applied as a quick tool fordetermination and detection of the concentration of analytes in several matrices. The calorimetric technique uses theability of reagents to bind to analytes of interest that generate a colored reaction. The intensityThis reaction will depend directly on the analyte concentration. In particular, the reagent ofGriess that is used for the determination and detection of nitrite in an aqueous matrix. the determinationCalorimetric nitrite using Griess reagent is chemically robust, offers excellent performanceanalytical and has been applied in the development of several analytical platforms. However, these systems aregenerally complicated devices consisting of reagent tanks, pumps and control valvesof the flow of liquids in the device and detector modules making them expensive and unsuitable foruse in the place of contamination.The present invention arises from the need in the state of the art to provide systems ofcombined nitrite / nitrate detection, alternatives that overcome at least part of the mentioned disadvantagesof the current methods.
    Description of the FiaurasFigure 1: Scheme of a microfluidic device of the invention consisting of a substrate (1), fiveyou would reserve calibration (2), a reserve (6) to deposit sample to analyze, a reserve (3) calibrationof the "blank", a reservoir (4) of measurement, a reservoir (7) of measurement to deposit sample to be analyzed, and amicrofluidic channel (5) that communicates the reserve (6) and the reserve (4), and which comprises a reducer.Figure 2A: shows a UV / UV-Vis spectrum of an ionogel 1 (10-1) comprising a hydrogel in a liquidionic with Griess reagent (10) and said hydrogel in the ionic liquid with Griess reagent and with nitrite (11).Figure 28: shows the UV / UV-Vis spectrum of another ionogel 2 (10-2) comprising another hydrogel in another liquidionic with Griess reagent (12) and said hydrogel in said ionic liquid with Griess reagent and with nitrite (13). TheMaximum Absorbance for Griess reaction with nitrite was found at A = 532 nm. In this Figures 2A and 28,They observe the absorbance differences.Figure 3: represents the lines of a multivariate calibration model (17) formed by triangles (15) andof validation (16), formed by points (14), generated after image analysis of theconcentrations: (O ppm), (2.5 ppm), (5 ppm), (7.5 ppm) and (10 ppm).
    Description of the inventionIn one aspect the invention relates to an ionogel, hereinafter ionogel of the invention, comprising:
    a hydrogel in an ionic liquid, and
    a reactive system that provides a color change by reaction with nitrite.
    The hydrogel that is a polymerized and crosslinked structure, porous, and with hydrophilic properties capable of retaining a significant fraction of water within it. While hydrogels are generally prepared from hydrophilic monomers, hydrophobic monomers can also be used to regulate properties for specific applications.
    In accordance with the present invention, the hydrogel is obtained in an ionic liquid, by polymerization and cross-linking of two or more precursor monomers selected from acrylic acid, alkali metal acrylates, acrylamide and their acrylic derivatives, well known to a person skilled in the art , such as methyl acrylate, methyl methacrylate etc. In a particular embodiment the hydrogel is a polyacrylamide.
    In a preferred embodiment the hydrogel is obtained by polymerization of N-isopropylacrylamide and N, N'-methylene bis (acrylamide), which in addition to comonomer is crosslinking agent. The proportions of the monomers in the linal hydrogel polymer vary between wide margins and are determined lazily by the lunation expert of the desired hardness and the degree of crosslinking. For example in the case of Nisopropylacrylamide and N, N'-methylene bis (acrylamide), the proportions can vary from 100: 1, to 10: 1, and for example they can be 90: 1, 70: 1, 50: 1 020: 1.
    The reactive system that provides a color change by reaction with the nitrite that can be used in the present invention may in principle be any conventional colorimetric reactive system known to a person skilled in the art. Non-limiting examples of such reactive systems are enzymes such as nitrite and nitrate reductases, which are described for example the "Nitrite Biosensing via Selective Enzymes-A Long but Promising Route; M. Gabriela Almeida, Alexandra Serra, Celia M. Silveira, and Jose JG Moura; Sensors (Basel). 2010; 10 (12): 1153D-11555H diphenylbenzidine, the minoxidine described in "Detection 01 Nitrite in Water Using
    ,
    Minoxidil as a Reagent, Mario Gonzalez-Jimenez, Jorge Arenas-Valganon, Isaac F. Cespedes-Camacho, Juan Carlos Garela-Prieto, Emilio Calle, and Julio Casado, J.Chem.Educ; 2013, 90, 1053-1056 ", or Griess reagent. In a preferred embodiment of the invention, Griess reagent is used.
    Ionic liquids are liquid organic salts at temperatures close to room temperature, and comprise an organic cation and an anion that can be organic or inorganic. They have notable properties such as zero volatility, high ionic conductivity, as well as catalytic properties. They are currently used in numerous fields, in particular as electrolytes. Among the possible organic cations that can be used in the present invention are those derived from imidazolium, pyridinium, pyrrolidinium, ammonium, or loslonium. Notably, among others, 1-alkyl-3-methylimidazolium, 1-alkylpyridinium, N-methyl-Nalkylpinolidinium, ammonium salts, loslonium salts, etc. Non-limiting examples of cations are: trihexyltetradecyl loslonium [P6,6,6,14t; tributyl tetradecyl loslonio (P4,4,4, 14r; tretrabutyl loslonio (p4,4,4r; triisobutyl methyl loslonio [PI, 4,4,41 '; l-butyl-l-methyl pyrrolidine, l-ethyl-3-methylimidazolium (emim) ', l-Butyl-3-methylimidazolium (bmimr, N-propyl N-methyl-pyrrolidinium [C3mpyrf, N-butyl N-methyl-pyrrolidinium [C4mpyrf, trioctyl methyl ammonium [OcbNMef.
    Among the possible anions that can be used in the present invention are sulfonates, borates, phosphates, halides, etc. Non-limiting examples of anions are among others: tosylate [tosldodecylbenzenesulfonate (dbsar. Ethyl sulfate, bis (trifluoromethanesulfonyl) amide (NTfzr. Dicyanoamide (dear, Tetracyanoborate (BCN3J ", hexafluorophosphate [PFsr tetrafluoroborate (fluorofluoroethyl) Trifluoro loslate, chloride, bromide, iodide, Iluoride.
    In a particular embodiment the ionic liquid is eti! l-ethyl-3-methi imidazolium sullate (10 -1). In another particular embodiment the ionic liquid is trihexyltetradecyl loslonio dicianoamide (10-2).
    The reactive system is embedded inside the hydrogel in the ionic liquid, whose structure allows the storage of the reactive system, for long periods of time without deterioration, while acting as a calorimetric sensor as explained below.
    In another aspect the invention relates to a microlluidic device comprising:
    a) a substrate 1 comprising:
    b) two or more calibration reservoirs 2 separated from each other comprising ionogel of the invention,
    c) a calibration reservoir 3 comprising hydrogel in the ionic liquid ("white"),
    d) a reservoir 6 for depositing sample to be analyzed, connected through a microfluidic channel 5
    comprising a reducing agent, to a measuring reservoir 4 comprising ionogel of the
    invention,
    e) a measuring reservoir 7 comprising ionogel of the invention where sample to be deposited is deposited.
    The ionogel of the invention that is used in a particular microlluidic device is the same in all reservoirs; where this ionogel comprises a hydrogel determined in a deionic ionic liquid which is used in the calibration reservoir 3 ("blank").
    The substrate 1 that serves as a support for the device is a plastic plate. In principle, the plate can be of any material as long as it is inert to the materials with which it will come into contact, that is, it does not react, and does not interfere with the chemistry of the sensor microlluidic device and remains unchanged.
    Examples of suitable plastic materials for the substrate are, among others, high density polyethylene, low density polyethylene, ethylene polyterephthalate, vinyl polychloride, polypropylene, polystyrene or polycarbonate, polymer or copolymer of cyclic olefins or acrylic resins. According to a particular embodiment, the substrate is a polymethylmethacrylate plate. These plates can be obtained from commercial law.
    The dimensions of the substrate can be variable and there is no particular limitation in this regard. The device size may vary depending on the analysis requirements, the number of samples that you want to analyze in a single device, the number of repetitions that you want to make of a given sample, among other parameters. Ideally, the size is defined in such a way that it can be handled easily in the hand (portable). In a particular embodiment, the device is 1 mm thick and 22 mm x 10 mm in size. Likewise, the dimensions and shapes of the reservoirs can also vary in the device, without any particular limitation in this regard.
    The accuracy of the device in determining and quantifying the nitrite content in a sample will depend, among other factors, on the number of calibration reservoirs 2 separated from each other. In this sense it is preferred that the device has at least 3, preferably at least 4, and more preferably at least 5 calibration reservoirs 2. These reservoirs contain different concentrations of nitrite (standard solutions) and in them a color change is caused by reaction of the reactive system with nitrite that is proportional in color intensity to the concentration of nitrite in a linear range.
    According to a particular embodiment, the microfluidic channel 5 comprises a microfluidic paper. In said channel, a reducing agent is also provided, which reduces the nitrate present in the sample to be analyzed that is disposed in the reservoir 6 to nitrite, which is the analyte for which the ionogel reactive system of the invention shows sensitivity. In a particular embodiment the reducing agent comprises Zn (O), and is arranged for example in the form of an emulsion of Zn (O) in ultrapure water. Other reducers that can be used according to the present invention are nitrate reductase; reducing agents in acidic medium such as formic acid, Fe (O), and ammonium ions; and reducers in basic medium such as: solutions of Al, Zn and Fe (11) and hydrazine, among many others.
    In a further aspect the invention relates to a process for manufacturing the ionogel of the invention comprising the following steps:
    A) - prepare a mixture of the hydrogel precursor monomers and, where appropriate, a polymerization initiator in an ionic liquid;
    B) -. polymerize the monomers in the ionic liquid to obtain the hydrogel in the ionic liquid, and
    e) -incorporate the reactive system, which provides a color change by reaction with nitrite, to the hydrogel in the ionic liquid.
    Depending on the monomers selected in each particular embodiment for polymerization, the person skilled in the art can select a suitable initiator, as well as the polymerization conditions such as UV or visible light, and / or temperature and the particular ionic liquid. As explained above, at least one monomer also acts as a crosslinking agent in the reaction. In a particular embodiment the monomers are N-isopropylacrylamide and N, N'-methylene bis (acrylamide), and they are light-cured with a photopolymerization initiator, such as 2,2-dimethoxy-2-phenylacetophenone and UV light. The polymerization of step B) is carried out in an ionic liquid as defined above and the result is a hydrogel in said ionic liquid which, as explained below, serves as a "blank" in the reservoir 3 of the device.
    In a preferred embodiment said ionic liquid is selected from among ethyl l-ethyl-3-methylimidazolium sulfate and trihexyltetradecyl phosphonium dicyanoamide. It may sometimes be convenient or even necessary to heat the reaction mixture to facilitate the dissolution of the monomers.
    Once the hydrogel in the ionic liquid is obtained, it is optionally washed to remove the remains of unreacted monomers, and of any other reagent, with ultrapure water and / or a suitable organic solvent for example an alcohol.
    Then in step C) the hydrogel reactive system is incorporated into the ionic liquid, for example by simple arrangement or impregnation thereof. In a particular embodiment the reactive system is the Griess reagent. Then the resulting ionogel is allowed to dry. In a particular embodiment, the same amount in microliters of Griess reagent as of ionogel mixture is added thereto.
    In a further aspect the invention relates to a process for manufacturing the microfluidic device of the invention. The procedure comprises the steps of:
    - provide on a substrate 1:
    - one or more calibration reserves 2 separated from each other;
    - a reservoir 6 to deposit sample to analyze,
    - a calibration reservoir 3 (-white "),
    - a reserve 4 of measure,
    - a reserve 7 of measure to deposit the sample to be analyzed,
    - placing a microfluidic channel 5 comprising a reducer between reservoir 6 and reservoir 4, and - obtaining the ionogel in situ in reservoirs 2, 4, and 7, and the hydrogel in the ionic liquid in reservoir 3, according to the procedure of manufacturing defined above.
    For the fabrication of the microfluidic device a laser can be used in a conventional manner. In addition, a microfluidic channel 5 is provided that connects reservoirs 6 and 4. Said channel consists of a particular embodiment of a microfluidic paper that allows the passage through it by capillarity of the sample that is deposited in reservoir 6 towards reservoir 4 of measure. Said microfluidic channel 5 comprises a reducing agent capable of reducing the nitrate of the sample to nitrite, a species that is the one detected and determined in the present invention. In a particular embodiment said reducing agent is Zn (O), and can be incorporated into channel 5 in the form of an emulsion in ultrapure water. The amount of emulsion that is incorporated may vary in each particular case; typically 1 to 20 IlL are incorporated into channel 5, in particular 5 to 10 IlL of reducing agent suspension.
    The hydrogel in the ionic liquid is obtained directly in situ in each reservoir 2, 3, 4, 6 and 7 of the microfluidic device, following the procedure for manufacturing the ionogel of the invention set forth above (steps A and B). In the reservoirs 2, 4, 6 and 7, the ionogel is subsequently obtained by incorporating the reactive system (according to step C)).
    In this sense, in order to obtain the hydrogel in the ionic liquid, a mixture of the precursor monomers, and if necessary a polymerization initiator, is first obtained in an ionic liquid. The mixture can be obtained in situ in each reservoir or can be obtained in a separate container and from it a quantity of mixture that is deposited in the corresponding reservoirs can be taken. Polymerization is then carried out in situ in the device. Once the polymerization reaction is finished, the resulting product is washed, and then the reactive system is incorporated into reservoirs 2, 4, 6 and 7, (but not in reservoir 3) and allowed to dry. The amount of mixture deposited in the corresponding reservoirs is variable and depends in each case on the design of the microfluidic device. Typically these are volumes between 1 IlL-20 IlL mixture, for example between 5-10 1LL.
    The manufacturing process of the microfluidic device of the invention may further comprise an additional step of incorporating into the ionogel in the calibration reservoirs 2, calibration solutions with different concentrations of nitrite, (standard solutions), in which a color change originates. by reaction of the reactive system with nitrite, proportional in color intensity to the concentration of each solution in a linear range. From the processing of the color changes in the calibration reservoirs 2 as explained below, a calibration line is obtained which is used to colorimetrically determine the nitrite concentration in the analyzed sample. Figure 3 shows a particular embodiment where calibration solutions with concentrations other than nitrite were used, so that in a first reservoir 2 O ppm was placed, in the second reservoir 2 2.5 ppm was disposed, in the third reservoir 2 were arranged 5 ppm, in the fourth reservoir 2, 7.5 ppm and in the fifth 10 ppm of nitrite were placed.
    In another aspect the invention relates to a method for detecting and determining colorimetrically the concentration of nitrite and / or nitrate in a sample, hereinafter method of the present invention. The method of the invention comprises the use of the microfluidic device of the invention, allows to detect and determine colorimetrically the concentration of nitrite in a sample; and / or the concentration of nitrate in the sample.
    The method is defined below in reference to the microfluidic device manufactured as described above.
    The method of the invention comprises the following steps:
    (i) - deposit equal amounts of sample to be analyzed in reservoirs 3, 6 and 7 of the microfluidic device of the invention,
    (ii) -reduction of nitrate present in the sample deposited in reservoir 6 to nitrite by the action of the reducing agent as it passes through the microfluidic channel 5;
    (ni) -determination by colorimetry of nitrite concentrations in calibration reservoirs 2 and establishment of the calibration line;
    (iv) -detection and determination by colorimetry of the concentration of nitrite present in the sample in reservoir 7, and of the concentration of nitrite in the sample in reservoir 4, and
    (v) determination of the nitrate concentration in the sample by difference between the concentration obtained in reservoir 7 and the concentration obtained in reservoir 4.
    In a particular embodiment, part of the microfluidic device to which the calibration solutions of different concentrations of nitrite have already been incorporated into the calibration reservoirs 2, and therefore this stage would not be a stage of the method of the invention. In another particular embodiment, part of the microfluidic device has not yet been incorporated with calibration solutions with different concentrations of nitrite in the calibration reservoirs 2, and therefore this stage is defined and understood as an additional stage of the method of invention.
    The calorimetric detection of nitrite and / or nitrate in the sample can be done with the naked eye if reservoirs 4 and 7 develop color. In a particular embodiment the volume of sample to be analyzed that is available in the
    reservoirs 3, 6 and 7 is the same.
    The determination of concentrations is carried out by analyzing the color of reservoirs 2, 4 and 7 of an imagetaken by means of a camera, or video of the microfluidic device 1, and processing of the differentdevice reservoirs to determine concentrations. In a particular embodiment of the method ofIn determining the invention, the value determined in reservoir 3 ("blank") is taken into account.
    the sample analyzed is liquid, and it can be any taken from any liquid of natural origin,industrial or municipal that may present these nitrite / nitrate contaminants.
    In another aspect the invention relates to the use of the ion ion of the invention and / or the use of the devicemicrofluidic of the invention, for the detection and calorimetric determination of the concentration of nitrite and / ornitrate, in a sample.
    The use of the ionogel and the device allows the arrangement to be carried out in a single unit (for example byinjection) of the sample to be analyzed, the chemical reactions and the detection and determination of the analyte in aonly step. The device is manufactured from a substrate of a flexible material, at low cost and can beEasily modified depending on the desired structure. In addition, its use has other advantages, among whichits easy storage, transport and disposability stand out which is very suitable forcheap and fast on-site diagnosis by untrained personnel, without the need for a power source orelectronic components, and that can be easily interrogated with a camera. Also how do I knowIllustrated below in the Examples its use provides high sensitivity and reliability.
    The device is very small, portable, uses small sample volumes, thus reducingthe amount of reagents and providing a response in a short period of time.
    The calibration points (number to reserve 2) are variable, and are included in the device itself. itshandling is simple and requires minimal manipulation for the in situ characterization of the analyte to be analyzed atfrom real samples (for example, a contaminated water)
    The range of application can be varied and selected in each case. The ranges are usually determinedtypical of water contaminated by nitrates ranging from 1 ppb to several ppm.
    The following are illustrative examples of the invention that in no case should be construed aslimiting the scope of protection of the invention.
    Examples
    For the manufacture of ionogels were used: N-isopropylacrylamide, N, N'-methylene-bis (acrylamide), and asphotoinitiator: 2,2-dimethoxy-2-phenylacetophenone. The ionic liquids used were: l-ethyl-3-methylimidazolium ofethyl sulfate and trihexyltetradecyl phosphonium dicianamide (Sigma-Aldrich, Spain).Griess reagent. Griess reagent is commercial; but it can be prepared conventionally by mixingsulfanilamide, naithylenediamine dihydrochloride, and phosphoric acid.The UV light source used for photo-polymerization was a BONDwand UV-365 nm (Electrolyte Corporation,USA).The UV-Vis spectra were recorded on a Perkin-Elmer Lambda 900 UV-VIS-NIR spectrometer.The photos were taken with a Canon EOS 10000 model camera and calibrated by using a cardX-Rite (X-Rite Inc., USA) with the Color Checker Passport v. 1.0.2 (X-Rite Inc., USA) and followedby the Photoshop CC program (Adobe Photoshop CS5 Extended, Adobe Systems Inc., USA).For the characterization of Griess reagent storage in ionogel, ionogels spectra were taken andWith nitrite The maximum absorbance for the Griess reaction was found to be at A = 532 nm. TheFigures 2A and 2B show the difference between ionogel and ionogel absobance with nitrite.
    Example 1: Manufacture of the device (see Figure 1).
    The sensor was manufactured from a 1 mm poly (methyl) methacrylate (PMMA) plate (Goodlellow, United Kingdom)thick, which was cut with a C02 laser ablation system (Universal Laser Systems, Austria),setting both the size of the device (rectangle) and the different components of that device(reserve them) using different laser energies.Each device was designed with five calibration reservoirs 2, a reservoir 3 for hydrogel calibration inthe ionic liquid in contact with the sample ("blank"), a reservoir 6 to deposit the sample to be analyzed; areserve it 4 of measure, and reserve it 7 of measure and to deposit also the sample to analyze. HeThe device was 1 mm thick and 22 mm x 10 mm in size.A microfluidic channel 5 connecting reservoirs 6 and 4 was made from Whatman Grade filter paperwith 595 and an emulsion of metallic Zn (Sigma-Aldrich, Spain) was placed in ultrapure water.
    Preparation of hydrogel and iooogelTwo hydrogels were synthesized in an ionic liquid by mixing N-isopropylacrylamide, N, N'-methylenebis (acrylamide) and a photoinitiator: (2,2-dimethoxy-2-phenylacetophenone) dissolved in ionic liquids (IL): acetateof l-ethyl-3-methyl-limidazolium sulfate or dicylamide trihexyltetradecyl phosphonium respectively, byheating at 45 e C for 10 min
    5111 of the above mixtures were deposited in the device reserves and were formed ("drop-casting")
    S

    by light-curing in situ (1600 mW cm'2) for 20 and 30 minutes, respectively.
    the hydrogels in Il were washed well with ultrapure water and ethanol, and all but reservoir 3 wereembedded with the Griess reagent and allowed to dry for 12 hours to obtain two ionogels (10-1 and 10).
    To the ionogels of the 2 calibration reservoirs were added: 3 III of nitrite standard solutionsprepared with the following concentrations in the linear range of O-t O ppm: (see Figure 3): (O ppm), (2.5 ppm)(5 ppm), (7.5 ppm) and (10 ppm).After a few minutes they developed different quoting intensities in each calibration reservoir 2,proportional in intensity to each of the nitrite concentrations.The same amount was deposited in microliters of liquid sample to be analyzed in reservoirs 3 and 6 and 7 with aamount of nitrate of 2.5 ppms and nitrite of 2.5 ppms.the sample deposited in 6 moved by capillarity to reservoir 4 passing through microfluidic channel 5where Zn (O) reduced all the nitrate present in the sample as it passed to nitrite.Certain color intensities were developed in reservoirs 4 and 7 of measurement.Photos of the device were then taken with the camera and processed by image analysis.The parameters of luminance (l), chromaticity (e) and tone (H) were taken by PhotoShop cePixelating each reservoir and the nitrate concentration of the sample was calculated using a calibration modelmultivariate (see Figure 3).In particular, the nitrite concentration (determined in reservoir 7), the nitrite concentration was determinedplus nitrate reduced to nitrite (determined in reservoir 4) and the concentration determinednitrate The effect of the sample on the hydrogel on the ionic liquid ("white") in the reservoir was also evaluated
    3.
    The results obtained showed a concentration of 5 ± 0.5 ppm of nitrite (in reservoir 4) which is ofaccording to the concentration of the nitrite sample plus that of nitrate, added to reservoir 6.The nitrite concentration determined in reservoir 7 was 2.5 ± 0.5 ppm.Therefore the difference between the two (reserve 4 less reserve 7) resulted in a nitrate concentration inthe sample of 2.5 ± 0.6 ppm.
    The invention is not limited to the specific reatizaciones that have been described but also covers, byFor example, the variants that can be made by the average expert in the field within what isIt follows from the claims.
    权利要求:
    Claims (21)
    [1]
    1. An ionogel comprising: a hydrogel in an ionic liquid, and a reactive system that provides a color change by reaction with nitrite.
    [2]
    2.-. An ionogel according to claim 1, wherein the hydrogel is a polyacrylamide.
    [3]
    3.-An ionogel in which the hydrogel is obtained by polymerization of the monomers: N-isopropylacrylamide and N, N'-methylene bis (acrylamide).
    [4]
    4. An ionogel according to one of claims 1-3, wherein the reactive system is Griess reagent.
    [5]
    5. An ionogel according to any of claims 1 to 4, wherein the ionic liquid comprises a cation and an anion wherein the at least one cation is selected from: trihexyltetradecyl phosphonium, tributyl tetradecylphonium, tretrabutyl phosphonium, triisobutyl methyl phosphonium, 1-butyl-l-methyl pyrrolidine, l-ethyl-3-methylimidazolium, 1-butyl-3-methylimidazolium, N-propyl N-methyl-pyrrolidinium, N-butyl N-methyl-pyrrolidinium, trioctyl methyl ammonium, and The at least one anion is selected from: tosylate, dodecylbenzenesulfonate, ethyl sulfate, bis (trifluoromethanesulfonyl) amide, dicyanoamide, tetracyanoborate, hexafluorophosphate, tetrafluoroborate, ethyl sulfate, tris (penta fluoride ethyl) trifluoro phosphate, chloride, bromide, bromide .
    [6]
    6.-Lonogel according to claim S, wherein the ionic liquid is selected from 1-elyl-3-methylimidazolium ethyl sulfate and trihexyl aryldecyl phosphonium dicyanoamide.
    [7]
    7.-A microfluidic device comprising
    a) -a substrate (1) comprising:
    b) - two or more calibration reservoirs (2) separated from each other comprising ionogel,
    c) -a calibration reservoir 3 comprising hydrogel in the ionic liquid,
    d) -a reservoir (6) to deposit sample to be analyzed, connected through a microfluidic channel (5)
    comprising a reducer, to a measuring reservoir (4) comprising ionogel,
    e) - a measurement reservoir (7) comprising ionogel, where the sample to be analyzed is deposited, wherein the ionogel and the hydrogel in the ionic liquid is as defined in the preceding claims.
    [8]
    8. A microfluidic device according to claim 7, wherein the calibration reservoirs 2 separated from each other are at least five.
    [9]
    9. A microfluidic device according to claim 7 or 8, wherein the calibration reservoirs (2) contain different concentrations of nitrite and in which a color change is caused by reaction of the reactive system with the nitrite proportional in color intensity at the concentration of nitrite in a linear range.
    [10]
    10. A microfluidic device according to any one of claims 7 to 9, wherein the substrate (1) is a polymethylmethacrylate plate.
    [11 ]
    11. A microfluidic device according to any one of claims 7 to 10, wherein the microfluidic channel (5) comprises a microfluidic paper.
    [12]
    12. A microfluidic device according to any one of claims 7 to 11, wherein a reducing agent is provided in the microfluidic channel (5).
    [13]
    13. A microfluidic device according to claim 12, wherein the reducing agent is Zn (O).
    [14]
    14. A process for manufacturing the ionogel according to any one of claims 1 to 6 comprising the following steps:
    A) - prepare a mixture of the precursor monomers of a hydrogel and, if appropriate, an initiator of the
    polymerization in an ionic liquid;
    B) -. polymerize the monomers in the ionic liquid to obtain the hydrogel in the ionic liquid, and
    e) -incorporate the reactive system, which provides a color change by reaction with nitrite, to the hydrogel in the ionic liquid.
    [15]
    15. A process for manufacturing the ionogel according to the preceding claim wherein the monomers are Nisopropylacrylamide and N, N'-methylene bis (acrylamide), and photopolymerized with 2,2-dimethoxy-2-phenylacetophenone and UV light.
    [16]
    16. A process for manufacturing the ionogel according to claim 14 or 15, wherein the ionic liquid is selected from l-ethyl-3-methylimidazolium ethyl sulfate and trihexyltetradecyl phosphonium dicyanoamide.
    [17]
    17. Process for manufacturing the microfluidic device of any one of claims 7 to 13, comprising:
    S
    the

    -provide on a substrate (1):-one or more calibration reservoirs (2) separated from each other;-a reservoir (6) to deposit sample to analyze,-a reservoir (3) for calibration of the hydrogel in the ionic liquid,-a reservoir (4) of measurement,-a reservoir (7) of measurement, to deposit the sample to be analyzed,-situar a microfluidic channel (5) comprising a reducer between the reservoir (6) and the reservoir (4), and- obtain the hydrogel in the liquid in situ in the reservoirs (2), (3), (4) Y (7) and subsequently the ionogelin reservoirs (2), (4) and (7) by adding the reactive system, according to the procedure defined in theclaims 14 to 16.
    [18]
    18. A method according to claim 17, which comprises the additional step of incorporating to the ionogel in the calibration reservoirs (2) calibration solutions of different concentrations of nitrite in which a color change is caused by reaction of the reactive system with nitrite proportional in color intensity to the concentration in a linear range.
    [19]
    19.-Method to detect and determine colorimetrically the concentration of nitrite and nitrate in a sample
    comprising the steps of: (i) depositing equal amounts of sample to be analyzed in reservoirs 3, 6 and 7 of the microfluidic device of the invention, (ii) -reduction of nitrate present in the sample deposited in reservoir 6 to nitrite by the action of the reducing agent as it passes through the microfluidic channel 5; (i¡¡) -determination by colorimetry of nitrite concentrations in calibration reservoirs 2 and establishment of the calibration line; (iv) -detection and determination by colorimetry of the concentration of nitrite present in the sample in reservoir 7, and of the concentration of nitrite in the sample in reservoir 4, and
    (v) determination of the nitrate concentration in the sample by difference between the concentration obtained in reservoir 7 and the concentration obtained in reservoir 4.
    [20]
    20. Method according to claim 19, wherein the determination of the concentrations is carried out by analyzing the color of the reservoirs (2), (4) and (7) of an image taken by means of a camera, or video of the device microfluidic 1, and processing the different device stores to determine concentrations.
    [21]
    21. Use of the ionogel and the microfluidic device according to any one of claims 1 to 6 and 7 to 13 respectively for the detection and calorimetric determination of the concentration of nitrite and nitrate in a sample.
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    同族专利:
    公开号 | 公开日
    WO2018078208A1|2018-05-03|
    ES2665790B1|2019-02-15|
    EP3534156A4|2020-06-24|
    US20190271675A1|2019-09-05|
    JP2019537006A|2019-12-19|
    EP3534156A1|2019-09-04|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    ES2185462A1|2000-10-31|2003-04-16|Univ Granada|Single use sensor quantifying nitrite content of water consists of a polyester sheet with a circular central activated zone detecting radiation absorption|WO2021005254A1|2019-07-05|2021-01-14|Universidad Del País Vasco / Euskal Herriko Unibertsitatea|Microfluidic sensor for the detection of analytes|
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    ES201631376A|ES2665790B1|2016-10-26|2016-10-26|PORTABLE MICROFLUIDIC DEVICE FOR DETECTING NITRITO-NITRATE|ES201631376A| ES2665790B1|2016-10-26|2016-10-26|PORTABLE MICROFLUIDIC DEVICE FOR DETECTING NITRITO-NITRATE|
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