Method for preparing a monolithic support immobilized thereon with uranyl cations, and related methods for capture and recovery

Abstract

The present invention relates to a method for preparing a monolithic support on which uranyl cations are immobilized in the internal volume of at least one channel. According to the present invention, such a method comprises: (a) activating the inner surface of the channel; (b) introducing into the internal volume of the channel a polymerization solution comprising: - a monomer comprising a phosphoric acid group, - at least one crosslinking agent, - several solvents, and - a free radical polymerization initiator; (c) polymerizing the polymerization solution; (d) rinsing the monolithic support obtained in step (c); and (e) contacting the previously rinsed monolithic support with a solution comprising uranyl cations. The present invention also relates to a method for capturing proteins that selectively bind uranium by means of a monolithic support prepared by the above method, and to a method for recovering proteins that selectively bind uranium using the capture method.

Description

Technical Field

[0001] The present invention relates to a method for preparing a monolithic support on which uranyl cations are immobilized, which monolithic support is more particularly synthesized and anchored in situ into the channels of a miniaturized analysis system.

[0002] The present invention also relates to a method for capturing proteins that selectively bind uranium, which uranium is immobilized on such a monolithic support.

[0003] Finally, the present invention relates to a method for recovering proteins that selectively bind uranium, which recovery method implements the previous capture method. Background Art

[0004] Low concentrations of uranium are suspected of having neurotoxic effects in humans. Although the biodistribution of uranium in the human body has been well described, the biochemical mechanisms occurring at the cellular and molecular levels and the mechanisms responsible for these neurotoxic effects remain to be elucidated. Identifying target molecules, in particular proteins that selectively bind uranium, in human neuron cell models should make it possible to predict the uranium-protein species that may form and to describe in detail the biochemical mechanisms associated with uranium neurotoxicity.

[0005] To identify such target molecules, the publication by C. Bassett et al. ("Specific capture of uranyl protein targets by metal affinity chromatography", Journal of Chromatography A, 2008, 1185, 233-240), reference [1], cited at the end of the present specification, describes the use of an immobilized metal affinity chromatography (IMAC) separation mode in order to selectively capture proteins that bind uranium in the uranyl form UO2 2+ of uranium.

[0006] In the following of the present specification, the expression "protein that selectively binds uranium" may be used instead of "protein that binds uranium in the uranyl form UO2 2+ of uranium".

[0007] In publication [1], the selective capture experiments based on the IMAC mode were carried out with a support formed by styrene-divinylbenzene copolymer microbeads functionalized with aminophosphonate groups ( C467), through which uranyl ions were immobilized. The presence of the aminophosphonate groups proved satisfactory for immobilizing uranyl ions by complexation and for retaining the free uranyl bonds that bind to proteins (in particular the uranium target proteins contained in human complex serum samples).

[0008] As described in the publication by A. Dedieu et al. ("Identification of uranyl binding proteins from human kidney-2 cell extracts by immobilized uranyl affinity chromatography and mass spectrometry", Journal of Chromatography A, 2009, 1216, 5365-5376), i.e., reference [2], the support described in publication [1] was used to capture and identify the uranium-selective binding proteins contained in human kidney cell HK-2 extracts. These proteins were captured in batch mode and then identified by proteomics.

[0009] Thus, publications [1] and [2] reported the first batch of work that has been carried out for immobilizing UO2 2+ ions for the capture and identification of uranium-selective binding proteins.

[0010] However, capturing and identifying the uranium target proteins present in cell extracts remains challenging because these cell extracts are only available in very limited amounts. Consequently, the target proteins are only present in low abundance in these cell extracts. In addition, the batch capture method requires the use of beads with a minimum volume of 50 microliters in a suspension system, which in turn requires protein samples ranging from 20 micrograms to 50 micrograms. One of the main direct consequences is the difficulty in performing experimental replicates, which is crucial for validating the capture method for these uranium target proteins contained in these cell extracts and the reproducibility of the identification.

[0011] Therefore, given the very low availability of biological samples, the low abundance of these target proteins, and the limitations of the batch mode, there is an urgent need to scale down the uranium target protein capture method.

[0012] However, there are a number of technical obstacles to the miniaturization of the support formed by beads: not only is it laborious and less reproducible on the one hand to fill the channels of a miniaturized analysis system with beads, but it also requires the installation of frits for maintaining these beads. However, the frits can be the cause of bubble formation and / or solute adsorption phenomena during the analysis process. All these factors can cause serious reproducibility problems in the selective capture experiments.

[0013] Therefore, the object of the present invention is to overcome the drawbacks of the prior art and to provide a method for preparing a support immobilized with UO2 2+ cations, which allows for the continuous capture of uranium-selective binding proteins contained in a biological sample, regardless of its volume.

[0014] Another object of the present invention is to provide a method for capturing proteins that selectively bind uranium, which are contained in a biological sample, and which method has improved reproducibility and robustness compared to the capture methods described in publications [1] and [2]. Summary of the Invention

[0015] First, by a method for preparing a support on which UO2 2+ cations are immobilized, and more specifically, by a method for preparing a monolithic support on which UO2 2+ cations are immobilized in the internal volume of at least one channel of a miniaturized analysis system, the foregoing and other objects are achieved.

[0016] According to the present invention, the method comprises the following successive steps (a) to (e):

[0017] (a) Activating the inner surface of the channel;

[0018] (b) Introducing into the internal volume of the channel a polymerization solution for synthesizing a monolithic support, the polymerization solution comprising:

[0019] - a monomer comprising a phosphate group,

[0020] - at least one crosslinking agent,

[0021] - several solvents, and

[0022] - a free radical polymerization initiator;

[0023] (c) Polymerizing the polymerization solution obtained in step (b), thereby obtaining a monolithic support anchored to the wall of the channel;

[0024] (d) Rinsing the monolithic support obtained in step (c); and

[0025] (e) Contacting the rinsed monolithic support obtained in step (d) with a solution comprising UO2 2+ cations, thereby obtaining a monolithic support on which UO2 2+ cations are immobilized.

[0026] The monomer comprising a phosphate group is selected, in combination with the crosslinking agent, the solvents and the free radical polymerization initiator, to allow the preparation in situ in the internal volume of the channel of a miniaturized analysis system of a monolithic support having a mechanically and chemically stable porous three-dimensional polymer structure, on which UO2 2+ cations are immobilized.

[0027] As previously mentioned, for the preparation according to the present invention of a support on which UO2 2+The method for the overall carrier of cations comprises successive steps (a) to (e).

[0028] The activation step (a) has the effect of functionalizing the inner surface of the channel, thereby allowing subsequent anchoring of the monolith carrier to its inner wall.

[0029] In the case where the channel is made of glass, the activation step (a) can be carried out by silanization. In fact, the silanization reaction is capable of modifying the silanol groups on the inner surface of the glass channel into vinyl groups.

[0030] For example, it is possible to consider using γ-methacryloxy-propyltrimethoxysilane (γ-MAPS) as the silanizing agent.

[0031] The polymerization solution introduced into the internal volume of the channel in step (b) of the preparation method according to the invention can be made of a mixture comprising a monomer containing a phosphate group, a crosslinking agent, a solvent and a free radical polymerization initiator.

[0032] The polymerization solution generally comprises a single monomer containing a phosphate group.

[0033] This monomer can in particular be selected from methacrylate monomers containing a phosphate group or any other monomer capable of covalently bonding a phosphate group to the surface of the monolithic solid support.

[0034] In a variant of the preparation method according to the invention, based on the total mass of the polymerization solution, the mass proportion of the monomer containing a phosphate group is between 4% by mass and 10% by mass.

[0035] It is clarified that the expressions "between... and..." and "comprising... to..." used in the present application should be understood as defining not only the values of the range, but also the boundary values of the range.

[0036] More particularly advantageously, based on the total mass of the polymerization solution, the mass proportion of the monomer containing a phosphate group is between 8.7% by mass and 9.7% by mass.

[0037] In a preferred variant of the preparation method according to the invention, the polymerization solution implemented in step (b) comprises a methacrylate monomer containing a phosphate group, which is even more preferably polyethylene glycol methacrylate phosphate (EGMP).

[0038] The polymerization solution implemented in step (b) of the preparation method according to the invention further comprises at least one crosslinking agent.

[0039] In other words, the polymerization solution can comprise only one crosslinking agent, but can also comprise a mixture of two, three or more crosslinking agents.

[0040] The crosslinking agent or these crosslinking agents may in particular be selected from the group consisting of acrylamide (AA) and bisacrylamide (BAA), bisacrylamide (BAA), divinyl dimethacrylate and a mixture of divinyl dimethacrylate derivatives.

[0041] In an advantageous variant, the crosslinking agent consists of a mixture of acrylamide / bisacrylamide (AA / BAA), preferably in a mass ratio of 19 / 1.

[0042] In a variant of the preparation method according to the invention, based on the total mass of the polymerization solution, the mass proportion of the AA / BAA mixture, preferably in a mass ratio of 19 / 1 (AA / BAA), is between 3% by mass and 8% by mass.

[0043] More particularly advantageously, based on the total mass of the polymerization solution, the mass proportion of the AA / BAA mixture, preferably in a mass ratio of 19 / 1 (AA / BAA), is between 6.6% by mass and 7.3% by mass.

[0044] The polymerization solution implemented in step (b) of the preparation method according to the invention also comprises several solvents.

[0045] These solvents may in particular be selected from dodecanol (DOC), dimethyl sulfoxide (DMSO) and dimethylformamide (DMF).

[0046] In an advantageous variant of the preparation method according to the invention, the polymerization solution implemented in step (b) comprises three solvents, which are preferably dodecanol, dimethylformamide and dimethyl sulfoxide.

[0047] In this advantageous variant, based on the total mass of the polymerization solution, the mass proportion of dodecanol is between 28% by mass and 59% by mass, preferably between 52.7% by mass and 58.3% by mass; based on the total mass of the polymerization solution, the mass proportion of dimethyl sulfoxide is between 22% by mass and 53% by mass, preferably between 22.8% by mass and 25.2% by mass; and based on the total mass of the polymerization solution, the mass proportion of dimethylformamide is between 7% by mass and 9% by mass.

[0048] The polymerization solution implemented in step (b) of the preparation method according to the invention also comprises a free radical polymerization initiator.

[0049] The free radical polymerization initiator can be particularly selected from free radical polymerization initiators that can be used to prepare monolithic supports, namely azobisisobutyronitrile (AIBN), 2,2 - diethoxyacetophenone (DEA), α - dialkoxyacetophenone, 2,2 - dimethyl - 2 - hydroxyacetophenone (DARO), α - hydroxyacetophenone, benzoin methyl ether (BME), 2 - methyl - 4'-(methylthio)-2 - morpholinopropiophenone (IRG), α - alkylaminobenzophenone or alternatively 2,2 - dimethoxy - 2 - phenylacetophenone (DMPA).

[0050] In a variant of the preparation method according to the invention, based on the total mass of the polymerization solution, the mass ratio of the free radical polymerization initiator is between 0.05% by mass and 0.2% by mass.

[0051] More particularly advantageously, based on the total mass of the polymerization solution, the mass ratio of the free radical polymerization initiator is between 0.14% by mass and 0.16% by mass.

[0052] In a preferred variant of the preparation method according to the invention, azobisisobutyronitrile (AIBN) is used as the free radical polymerization initiator in the polymerization solution.

[0053] It should be noted that recently in the publication by M. Araya - Farias et al. ("A lab - on - chip formonolith - based preconcentration and electrophoresis separation of phosphopeptides", Analyst, 2017, 142, 485 - 494), i.e., reference [3], the polymerization solution for synthesizing monolithic supports includes polyethylene glycol methacrylate phosphate, acrylamide, bisacrylamide, dodecanol, dimethyl sulfoxide, dimethylformamide and azobisisobutyronitrile to preconcentrate phosphopeptides, which are potential biomarkers for Alzheimer's disease. For this purpose, Zr 4+ cations are immobilized on the surface of the in - situ synthesized phosphated monolithic support and anchored into the microchannels of a commercial cross - shaped chip. The miniaturized analytical system incorporating this monolithic support exhibits excellent performance in terms of the selectivity and enrichment factor of phosphopeptides.

[0054] Now, unexpectedly and surprisingly, the inventors have observed that the support described in publication [3] is fully applicable to applications other than those described in that publication, no longer for the immobilization of Zr 4+ cations and the capture of some phosphopeptides, but for the immobilization of UO2 2+ cations, the capture of uranium - binding proteins and their recovery.

[0055] In a particularly preferred embodiment, based on the total mass of the polymerization solution, the polymerization solution introduced into the internal volume of the channel in step (b) of the preparation method according to the invention comprises:

[0056] - 4% to 10% by mass, advantageously 8.7% to 9.7% by mass of polyethylene glycol methacrylate phosphate,

[0057] - 3% to 8% by mass, advantageously 6.6% to 7.3% by mass of a mixture of acrylamide / bisacrylamide, preferably at a mass ratio of 19 / 1,

[0058] - 28% to 59% by mass, advantageously 52.7% to 58.3% by mass of dodecanol,

[0059] - 22% to 53% by mass, advantageously 22.8% to 25.2% by mass of dimethyl sulfoxide,

[0060] - 7% to 9% by mass of dimethylformamide, and

[0061] - 0.05% to 0.2% by mass, advantageously 0.14% to 0.16% by mass of azobisisobutyronitrile.

[0062] After step (b) of introducing the polymerization solution into the internal volume of the channel of the miniaturized analysis system, step (c) of polymerizing the polymerization solution is carried out to obtain an integral support.

[0063] This polymerization step (c) of the preparation method according to the invention is a free radical chain polymerization initiated by a free radical polymerization initiator.

[0064] This polymerization step (c) is advantageously photopolymerization, i.e., polymerization carried out by irradiating the polymerization solution with ultraviolet light. This irradiation can in particular be carried out on a local area of the channel.

[0065] In an advantageous variant of the preparation method according to the invention, the wavelength of the ultraviolet light is between 320 nanometers and 380 nanometers, preferably between 330 nanometers and 370 nanometers, and even more preferably 347 nanometers, which is the absorption maximum of AIBN.

[0066] In an advantageous variant of the preparation method according to the invention, the duration of the irradiation with ultraviolet light is between 5 minutes and 60 minutes, advantageously between 15 minutes and 45 minutes, and preferably 25 minutes.

[0067] At the end of step (c), an integral support that has been synthesized in situ and immobilized on the wall of the channel of the miniaturized analysis system is obtained.

[0068] According to the preparation method of the present invention, after the polymerization step (c), it includes a step (d) of rinsing the monolithic support. This rinsing step (d) can remove the unreacted excess reagents from the polymerization solution.

[0069] This rinsing step (d) can especially be carried out successively with an alcohol and water, and the alcohol is advantageously methanol.

[0070] Then, the preparation method includes a step (e) of bringing the rinsed monolithic support obtained at the end of step (d) into contact with a solution comprising UO2 2+ cations. In this way, a monolithic support on which UO2 2+ cations are immobilized is obtained in the internal volume of the channels.

[0071] In an advantageous variant of the preparation method according to the present invention, a solution comprising UO2 2+ ions is prepared in an aqueous ammonium acetate solution.

[0072] By coupling with an inductively coupled plasma mass spectrometer (ICP-MS), the binding of UO2 2+ ions to the monolithic support can be advantageously monitored.

[0073] In an advantageous variant of the preparation method according to the present invention, the step (e) of bringing the monolithic support into contact with a solution comprising UO2 2+ cations is carried out by circulating the solution comprising UO2 2+ cations through the monolithic support.

[0074] Therefore, the preparation method according to the present invention can in-situ synthesize a monolithic support on which uranyl UO2 2+ cations are immobilized in a miniaturized analysis system, and the size of this miniaturized analysis system is much smaller than that of an analysis system formed by microbeads (such as those described in publications [1] and [2]), at least 1000 times smaller.

[0075] In particular, the method according to the present invention can prepare a support on which UO2 2+ cations are immobilized in a channel, and the inner diameter of the channel can advantageously be less than or equal to 300 microns, and preferably can be between 50 microns and 90 microns.

[0076] The scale reduction that can be achieved with the preparation method according to the present invention can also reduce the consumption of solvents and reagents as well as the amount of reaction by-products and their treatment, which is an obvious advantage from an industrial perspective. This scale reduction is more beneficial for applications in the nuclear field because it allows reducing the restrictions related to the handling of radioactive samples, but also limits the amount of waste and the costs associated with the specific management of waste.

[0077] Secondly, the present invention relates to a method for capturing proteins that selectively bind uranium, which are contained in a biological sample.

[0078] According to the present invention, the capture method comprises the following steps (i) and (ii):

[0079] (i) By implementing the preparation method defined above, a monolithic support immobilized with UO2 2+ cations is prepared in the internal volume of at least one channel of a miniaturized analysis system, and

[0080] (ii) At least circulating a solution containing the biological sample through the monolithic support immobilized with UO2 2+ cations obtained at the end of step (i), thereby achieving the capture of proteins that selectively bind uranium on the monolithic support.

[0081] In the capture method according to the present invention, during step (i), the monolithic support is prepared in situ by the preparation method defined above. The advantageous features of this preparation method are elucidated, in particular the features related to the implementation details of steps (a) to (e) and the features related to the channels of the miniaturized analysis system, which can be used alone or in combination.

[0082] As described above, the monolithic support is anchored to the inner wall of the channel and includes immobilized uranyl ions.

[0083] During the circulation step (ii), selective capture of the protein occurs, which among the protein or proteins contained in the biological sample, has an affinity for the UO2 2+ ions immobilized on the monolithic support.

[0084] Therefore, according to the IMAC mode and this mode, these proteins that selectively bind uranium are selectively captured from the biological sample, and the amount involved can be greatly reduced compared to the amount required for capture using the supports of publications [1] and [2].

[0085] Thirdly, the present invention relates to a method for recovering proteins that selectively bind uranium, which are contained in a biological sample.

[0086] According to the present invention, the method comprises the following steps (1) to (3):

[0087] (1) Capturing the proteins that selectively bind uranium by implementing the above capture method,

[0088] (2) Removing unbound proteins by circulating a protein-free solution through the monolithic support, and

[0089] (3) At least one elution step, by circulating an elution solution through the monolithic support obtained at the end of step (2), thereby recovering the protein that selectively binds uranium in the elution solution.

[0090] In the recovery method according to the present invention, in step (1), the protein that selectively binds uranium is captured by implementing the capture method defined above, where the advantageous features of the capture method are specified to be used alone or in combination.

[0091] Thus, the proteins that selectively bind uranium, which have been captured in step (1), are recovered from the monolithic support by implementing step (3), which includes at least one elution step with an elution solution.

[0092] The recovery method according to the present invention is particularly simple to implement and enables the selective recovery by elution of the protein that selectively binds uranium, which has been previously captured on a monolithic support on which UO2 2+ cation is immobilized.

[0093] It is specified that the biological sample involved in the method for capturing the protein that selectively binds uranium and thus in the method for recovering the protein that selectively binds uranium can in particular consist of a protein solution, a cell extract or a biological fluid, and can in particular be derived from a human neuronal cell line.

[0094] Other features and advantages of the present invention will become apparent upon reading the following additional description, which relates to an example of preparing an 8-mm monolithic support on which uranyl cations are immobilized, and to an example that can illustrate the performance of such a monolithic support for capturing and subsequently recovering the protein that selectively binds uranium contained in a biological sample.

[0095] Of course, these examples are given solely for the purpose of illustrating the subject matter of the present invention and in no way limit the subject matter. Detailed Description

[0096] The polymerization solution for synthesizing the monolithic support is prepared from the following compounds in the following mass ratios:

[0097] - 2.22 mg (0.15% by mass) of azobisisobutyronitrile (AIBN),

[0098] - 100.05 mg (6.7% by mass) of a mixture of acrylamide (AA) and bisacrylamide (BAA) (the ratio of AA to BAA is 19 / 1),

[0099] - 130.60 mg (8.7% by mass) of polyethylene glycol methacrylate phosphate (EGMP),

[0100] -346.03 mg (23.1 wt%) of dimethyl sulfoxide (DMSO),

[0101] -799.93 mg (53.4 wt%) of dodecanol (DOC), and

[0102] -118.80 mg (7.9 wt%) of dimethylformamide (DMF).

[0103] AIBN, AA-BAA, EGMP, DMSO, DOC and then DMF were successively introduced into a vial, and all these compounds were mixed to obtain a polymerization solution for synthesizing the monolithic support.

[0104] After degassing, the obtained polymerization solution was introduced into one or more of the four straight channels of a glass analytical microsystem sold by ChipShop. The four channels are parallel to each other, 50 μm in width, 50 μm in depth, and 58.5 mm in length.

[0105] It is clarified that before introducing the polymerization solution, the inner surface of the channels has been functionalized with a mixture (50 / 50, v / v) including γ-methacryloxypropyltrimethoxysilane (γ-MAPS) dissolved in acetone.

[0106] After introducing the polymerization solution into one or more of the four channels, the microsystem was placed in an oven under ultraviolet radiation with a maximum wavelength of 365 nm at a distance of 14.5 cm from the source for 25 minutes so as to have a power of 3.1 mW·cm -2 to achieve the polymerization of the polymerization solution.

[0107] The monolithic support thus formed in the channels has been rinsed with a mixture containing methanol and water.

[0108] A solution containing 500 parts per billion (ppb) of uranyl ions in an ammonium acetate buffer has been prepared. The molar concentration of ammonium acetate is between 25 mmol / L and 50 mmol / L, and the pH value is between 4 and 5.

[0109] Then, the solution was circulated through the monolithic support in situ synthesized and anchored to the walls of the channels of the microsystem at a flow rate of 0.24 mL / h (4 μL / min) for 60 minutes, thereby immobilizing the uranyl ions on the surface of the monolithic support.

[0110] The immobilization efficiency of the uranyl ions was determined by inductively coupled plasma mass spectrometry (ICP-MS).

[0111] This step can be carried out offline by recycling the fractions. However, this step can advantageously be carried out online, i.e., continuously, by coupling the microsystem to the mass spectrometer via a microsprayer. In the case of the present example, 50 ng of uranium is bound to an 8-mm monolithic support.

[0112] In the case where the mass spectrometer is a triple quadrupole ICP, when monitoring the capture of the protein, in addition to the signal of uranium, the signals of phosphorus and / or sulfur contained in the protein can also be monitored.

[0113] The ability to monitor uranyl ions and their target molecules is a considerable advantage, which enables the determination of the immobilization efficiency of these ions on the monolithic support and their ability to capture target molecules through the interaction with the immobilized ions. For example, the amount of transferrin desferri that is captured by the monolithic support on which uranyl ions are immobilized and recovered is 0.75 μg.

[0114] Table 1 below shows a comparison of the amount of uranium immobilized on the monolithic support prepared by the method according to the present invention, and the amount of transferrin desferri recovered after being captured by the same support with the amount obtained by the reference support formed by microbeads described in the publication [1].

[0115] Table 1

[0116]

[0117] References

[0118] [1] C.Basset et al., Journal of Chromatography A, 2008, 1185, p.233 - 240

[0119] [2] A.Dedieu et al., Journal of Chromatography A, 2009, 1216, p.5365 - 5376

[0120] [3] M.Araya - Farias et al., Analyst, 2017, 142, p.485 - 494

Claims

1. A method for preparing a monolithic support immobilized with UO2 cations in the internal volume of at least one channel of a miniaturized analysis system, the method comprising the following successive steps (a) to (e): 2+ Cation, the method comprising the following successive steps (a) to (e): (a) Activate the inner surface of the channel; (b) Introduce a polymerization solution for synthesizing a monolithic support into the internal volume of the channel, the polymerization solution comprising: - polyethylene glycol methacrylate phosphate as a monomer, - at least one crosslinking agent selected from bisacrylamide and mixtures of acrylamide and bisacrylamide, - several solvents, and - a radical polymerization initiator; (c) Polymerize the polymerization solution obtained in step (b), thereby obtaining a monolithic support anchored to the wall of the channel; (d) Rinse the monolithic support obtained in step (c); and (e) Contact the rinsed monolithic support obtained in step (d) with a solution comprising UO2 2+ cations, thereby obtaining a monolithic support on which the UO2 2+ cations are immobilized.

2. The method according to claim 1, wherein the crosslinking agent consists of a mixture of acrylamide / bisacrylamide.

3. The method according to claim 1 or 2, wherein the solvent is selected from dodecanol, dimethylformamide, and dimethyl sulfoxide.

4. The method according to claim 1 or 2, wherein the radical polymerization initiator is azobisisobutyronitrile (AIBN).

5. The method according to claim 4, wherein based on the total mass of the polymerization solution, the polymerization solution comprises: - 0.05% to 0.2% by mass of azobisisobutyronitrile, - 3% to 8% by mass of acrylamide and bisacrylamide, - 4% to 10% by mass of polyethylene glycol methacrylate phosphate, - 22% to 53% by mass of dimethyl sulfoxide, - 28% to 59% by mass of dodecanol, and - 7% to 9% by mass of dimethylformamide.

6. The method according to claim 5, wherein based on the total mass of the polymerization solution, the polymerization solution comprises: - 0.14% to 0.16% by mass of azobisisobutyronitrile, - 6.6% to 7.3% by mass of acrylamide and bisacrylamide, - 8.7% to 9.7% by mass of polyethylene glycol methacrylate phosphate, - 22.8% to 25.2% by mass of dimethyl sulfoxide, - 52.7% to 58.3% by mass of dodecanol, and - 7% to 9% by mass of dimethylformamide.

7. The method according to claim 1 or 2, wherein the polymerization step (c) is carried out by irradiation with ultraviolet light.

8. The method according to claim 7, wherein the wavelength of the ultraviolet light is between 320 nanometers and 380 nanometers.

9. The method according to claim 8, wherein the wavelength of the ultraviolet light is between 330 nanometers and 370 nanometers.

10. The method according to claim 7, wherein In step (c), the duration of irradiation with the ultraviolet light is between 5 minutes and 60 minutes.

11. The method according to claim 10, wherein, In step (c), the duration of irradiation with the ultraviolet light is between 15 minutes and 45 minutes.

12. The method according to claim 1 or 2, wherein the rinsing step (d) is carried out successively with alcohol and then with water.

13. The method according to claim 12, wherein the alcohol is methanol.

14. The method according to claim 1 or 2, wherein The channel is made of glass, and the activation step (a) is carried out by silanization.

15. The method according to claim 14, wherein, The silanization is carried out by means of γ-methacryloxypropyltrimethoxysilane (γ-MAPS) as a silanizing agent.

16. The method according to claim 1 or 2, wherein the inner diameter of the channel is less than or equal to 300 micrometers.

17. The method according to claim 16, wherein the inner diameter of the channel is between 50 micrometers and 90 micrometers.

18. The method according to claim 1 or 2, which comprises said UO2 2+ The solution of said cations is prepared in an aqueous ammonium acetate solution.

19. The method according to claim 1 or 2, wherein the step (e) of contacting the monolithic support with the solution comprising the UO2 2+ cations is carried out by circulating the solution comprising the UO2 2+ cations over the monolithic support.

20. A method for capturing proteins that selectively bind uranium, which proteins are contained in a biological sample, the method comprising the following steps (i) and (ii): (i) By implementing the preparation method according to any one of claims 1 to 19, a monolithic support immobilized with UO2 2+ cations is prepared in the internal volume of at least one channel of the miniaturized analysis system, and (ii) At least circulate the solution containing the biological sample through the monolithic support immobilized with UO2 cations obtained at the end of step (i), thereby achieving the capture of proteins selectively binding uranium on the monolithic support. 2+ Cations of the monolithic support, thereby achieving the capture of proteins selectively binding uranium on the monolithic support.

21. A method for recovering proteins that selectively bind uranium, which proteins are contained in a biological sample, the method comprising the following steps (1) to (3): (1) Capturing the proteins that selectively bind uranium by implementing the capture method according to claim 20, (2) Removing unbound proteins by rinsing the monolithic support with a protein-free solution in a loop, and (3) At least one elution step by passing an elution solution in a loop through the monolithic support obtained at the end of step (2), whereby the proteins that selectively bind uranium are recovered in the elution solution.

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