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US-12623215-B2 - Method for preparing a monolithic support on which uranyl cations are immobilised, and associated methods for capture and recovery

US12623215B2US 12623215 B2US12623215 B2US 12623215B2US-12623215-B2

Abstract

A method for preparing, in the internal volume of at least one channel, a monolithic support on which uranyl cations are immobilised. The method comprises: (a) activating the inner surface of the channel(s); (b) introducing, into the internal volume of the channel(s), a polymerisation solution comprising: a monomer comprising a phosphate group, at least one crosslinking agent, several solvents, and a radical polymerisation initiator; (c) polymerising the polymerisation solution; (d) rinsing the monolithic support obtained in step (c); and (e) contacting the monolithic support previously rinsed, with a solution comprising uranyl cations. A method for capturing proteins that selectively bind uranium by means of a monolithic support prepared by the above-mentioned method, as well as to a method for recovering proteins that selectively bind uranium with the capture method.

Inventors

  • Carole BRESSON
  • Marta GARCIA-CORTES
  • Claude VIDAUD
  • Thuy Tran

Assignees

  • COMMISSARIAT À L'ENERGIE ATOMIQUE ET AUX ÉNERGIES ALTERNATIVES
  • CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE
  • Universite Paris-Saclay

Dates

Publication Date
20260512
Application Date
20200907
Priority Date
20190912

Claims (20)

  1. 1 . A method for preparing, in an internal volume of at least one channel of a miniaturised analytical system, a monolithic support on which UO 2 2+ cations are immobilised, which method comprises the following successive steps (a) to (e) of: (a) activating the inner surface of the channel(s); (b) introducing a polymerisation solution for synthesising a monolithic support into the internal volume of the channel(s), the polymerisation solution comprising: a monomer comprising a phosphate group, at least one crosslinking agent several solvents, and a radical polymerisation initiator; (c) polymerising the polymerisation solution obtained in step (b), whereby a monolithic support anchored onto the walls of the channel(s) is obtained; (d) rinsing the monolithic support obtained in step (c); and (e) contacting the rinsed monolithic support obtained in step (d) with a solution comprising UO 2 2+ cations, whereby a monolithic support on which the UO 2 2+ cations are immobilized is obtained.
  2. 2 . The method according to claim 1 , wherein the monomer comprising a phosphate group is a methacrylate monomer.
  3. 3 . The method according to claim 1 , wherein the crosslinking agent is consisting of a mixture of acrylamide/bisacrylamide.
  4. 4 . The method according to claim 1 , wherein the solvents are selected from dodecanol, dimethylformamide and dimethylsulphoxide.
  5. 5 . The method according to claim 1 , wherein the radical polymerisation initiator is azobisisobutyronitrile (AIBN).
  6. 6 . The method according to claim 5 , wherein the polymerisation solution comprises, based on the total mass of the polymerisation solution: from 0.05 mass % to 0.2 mass % of azobisisobutyronitrile, from 3 mass % to 8 mass % of acrylamide and bisacrylamide, from 4 mass % to 10 mass % of polyethylene glycol methacrylate phosphate, from 22 mass % to 53 mass % of dimethylsulphoxide, from 28 mass % to 59 mass % of dodecanol, and from 7 mass % to 9 mass % dimethylformamide.
  7. 7 . The method according to claim 1 , wherein the polymerisation step (c) is carried out by irradiation by means of ultraviolet rays.
  8. 8 . The method according to claim 7 , wherein, in step (c), the duration of irradiation by means of the ultraviolet rays is between 5 min and 60 min.
  9. 9 . The method according to claim 1 , wherein the rinsing step (d) is carried out successively with an alcohol and then with water.
  10. 10 . The method according to claim 1 , wherein, the channel(s) being made of glass, the activation step (a) is carried out by silanisation.
  11. 11 . The method according to claim 1 , wherein the internal diameter of the channel(s) is less than or equal to 300 μm.
  12. 12 . The method according to claim 1 , wherein the solution comprising the UO 2 2+ cations is prepared in an aqueous solution of ammonium acetate.
  13. 13 . The method according to claim 1 , wherein step (e) of contacting the monolithic support with the solution comprising the UO 2 2+ cations is carried out by circulating this solution comprising the UO 2 2+ cations on the monolithic support.
  14. 14 . A method for capturing proteins that selectively bind uranium, these proteins being contained in a biological sample, this method comprising the following steps (i) and (ii) of: (i) preparing, in an internal volume of at least one channel of a miniaturised analytical system, a monolithic support on which UO22+cations are immobilised, by implementing the preparation method according to claim 1 , and (ii) at least circulating a solution containing the biological sample through the monolithic support on which UO 2 2+ cations are immobilised, obtained at the end of step (i), whereby the capture of the proteins that selectively bind uranium on the monolithic support is achieved.
  15. 15 . A method for recovering proteins that selectively bind uranium, these proteins being contained in a biological sample, this method comprising the following steps (1) to (3) of: (1) capturing the proteins that selectively bind uranium by implementing the capture method of claim 14 , (2) removing the unbound proteins by rinsing the monolithic support by circulating a solution containing no proteins, and (3) at least one elution step, by circulating an eluting solution through the monolithic support obtained at the end of step (2), whereby the proteins that selectively bind uranium are recovered in the eluting solution.
  16. 16 . The method according to claim 2 , wherein the monomer comprising a phosphate group is polyethylene glycol methacrylate phosphate.
  17. 17 . The method according to claim 7 , wherein the wavelength of these ultraviolet rays is between 320 nm and 380 nm.
  18. 18 . The method according to claim 9 , wherein the alcohol is methanol.
  19. 19 . The method according to claim 10 , wherein the activation step (a) is carried out by means of gamma-methacryloxypropyltrimethoxysilane (γ-MAPS) as silanising agent.
  20. 20 . The method according to claim 11 , wherein the internal diameter of the channel(s) is between 50 μm and 90 μm.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This is a National Stage application of PCT international application PCT/FR2020/051537, filed on Sep. 7, 2020, which claims the priority of French Patent Application No. 1910077, filed Sep. 12, 2019, both of which are incorporated herein by reference in their entirety. TECHNICAL FIELD The present invention is concerned with a method for preparing a monolithic support on which uranyl cations are immobilised, this monolithic support being more particularly synthesised and anchored in situ into the channel(s) of a miniaturised analytical system. The present invention is also concerned with a method for capturing proteins that selectively bind uranium which is immobilised on such a monolithic support. Finally, the invention is concerned with a method for recovering proteins that selectively bind uranium, this recovery method implementing the previous capture method. STATE OF PRIOR ART Neurotoxic effects induced by low concentrations of uranium are suspected in humans. Although the biodistribution of uranium in the human body is well described, biochemical mechanisms that occur at the cellular and molecular level and that would be responsible for these neurotoxic effects have yet to be elucidated. The identification of target molecules, in particular proteins that selectively bind uranium, in a human neuronal cell model, should make it possible to predict the uranium-protein species that are likely to be formed and to describe in detail the biochemical mechanisms associated with uranium neurotoxicity. To identify such target molecules, the publication by C. Basset et al. (“Specific capture of uranyl protein targets by metal affinity chromatography”, Journal of Chromatography A, 2008, 1185, 233-240), referenced [1] at the end of the present description, described the exploitation of the immobilised metal affinity chromatography (IMAC) separation mode in order to selectively capture the proteins binding uranium in its uranyl form UO22+. In the following of the present description, the expression “uranium-selectively binding proteins” may be used instead of “proteins that selectively bind uranium in its uranyl form UO22+”. In publication [1], the selective capture experiments, based on the IMAC mode, were conducted with a support formed by styrene-divinylbenzene copolymer microbeads functionalised with aminophosphonate groups (Duolite® C467) through which the uranyl ions were immobilised. The presence of the aminophosphonate groups proved satisfactory for immobilising, by complexation, the uranyl ions and retaining free uranyl bonds that bind with proteins, and in particular the uranium target proteins contained in samples of human complex serum. As reported 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), referenced [2], the support described in publication [1] was used for the capture and identification of uranium-selectively binding proteins contained in extracts of human kidney cells HK-2. These proteins were captured in batch mode and then identified by proteomics. Publications [1] and [2] therefore report the first works that have been carried out to immobilise UO22+ ions for the capture of uranium-selectively binding proteins and their identification. However, the capture as well as the identification of uranium target proteins present in cell extracts are still challenging, insofar as these cell extracts are only available in very limited amounts. Consequently, the target proteins are only present in these cell extracts in low abundance. Furthermore, the batch capture method requires the use of a minimum volume of 50 μL of microbeads in a suspension system, which in turn requires the involvement of 20 μg to 50 μg of protein samples. One of the major direct consequences is the difficulty in carrying out experimental replicates, which are essential to validate repeatability of the capture method, but also of the identification, of these uranium target proteins contained in these cell extracts. There is therefore an imperative need to reduce the scale of the uranium target protein capture method given the very low availability of biological samples, the low abundance of these target proteins and the limitations of the batch mode. However, the miniaturisation of a support formed by microbeads presents a certain number of technical obstacles: not only filling the channels of miniaturised analytical systems with microbeads is, on the one hand, laborious and, on the other hand, not very reproducible, but it furthermore requires the installation of frits for maintaining these microbeads. However, the frits may be the cause of the formation of air bubbles and/or of solute adsorption phenomena during analyses. All these elements generate significant reproducibility problems for selective c