ES2525769B2 - Method of obtaining a molecular imprint polymer (MIP) structure - Google Patents

Abstract

The present invention relates to a new method for the realization of MIP patterns at micrometric and sub-micrometric scales by direct writing by electron beam. The precursor mixture of MIP contains compounds sensitive to electronic irradiation and is deposited as a film on a substrate. The areas of the film irradiated with an electron beam are removed with a chemical solvent or developer. The resulting film contains the desired patterns and is polymerized using light and / or heat. The method of synthesis of MIP patterns of the invention allows the realization of arrays of numerous sensors based on MIPs on a substrate or chip of small dimensions for the simultaneous detection of multiple (bio) chemical compounds, in addition to other various applications.

Description

METHOD OF OBTAINING A PRINTED POLYMER STRUCTURE

MOLECULAR (MIP)

Field of the Invention The present invention pertains to the field of manufacturing or synthesis of structures for molecular detection. Specifically, in the production of chemical micro sensors based on patterns of molecular imprint polymers (MIPs) on substrates, in the manufacture of chips for analyte detection with application in analytical chemistry and medicine.

Background of the invention The tendency of the technique in the development of chemical sensing devices is their miniaturization on a micrometric or sub-micrometric scale. The use of miniturized components has numerous advantages, among others that the volume of analyte can be reduced, the energy consumption for its operation is also lower, production costs are reduced and applications are diversified.

The technique has developed optical, electrical and mechanical transducers based on the traditional materials of the microelectronic industry, which are inorganic semiconductors and dielectrics, metals and polymeric resins. However, providing these transducers with a sensor function such as molecular recognition to identify analytes of chemical or biological origin requires the incorporation of "soft" sensory or functional materials. Soft materials are high viscosity amorphous organic materials that are not normally compatible with the manufacturing techniques used in the semiconductor industry.

Biosensors integrate biomolecules as recognition elements such as enzymes, antibodies or DNA, often unstable when removed from their natural environment. They usually require conservation at low temperatures and may not withstand certain operating and storage conditions, which limits their usefulness especially in portable systems.

An alternative strategy is the integration into the sensor of biomimetic synthetic receptors, such as molecular imprint polymers or MIPs ("Molecularly Imprinted Polymers") (K. Haupt, "Creating a good impression", Nat. Biotech. 2002, 20,

884). 5

MIPs are synthetic materials that are used as molecular recognition elements. EP 1237936 B1 describes a general procedure for obtaining MIPs based on the formation of a cross-linked macromolecular structure around a molecule that acts as a molecular template or template and

10 which is extracted after polymerization. When the MIPs are structured on a submicron scale, their sensitivity in the recognition of molecules increases because they have a greater detection surface.

Among the advantages of the use of IPMs compared to biomolecules, for

For example, antibodies, nucleic acids, enzymes, etc., consist of their ability to work in extreme conditions of pH, temperature and in the presence of organic solvents, without losing their structure or affinity for the analyte.

Most MIPs are prepared in the art from precursor mixtures of

20 monomers Precursor mixtures that use oligomers or polymers are developed for very specific uses and analytes. Li describes the preparation of a precursor mixture of MIPs for theophylline comprising polymers of a composition very similar to those of the present invention, also with the presence of double bonds in side chains of said polymers (Li et al. $ Ynthesis and Characterization of

25 Functional Methacrylate Copolymers and their Application in Molecular Imprinting ", Macromolecules 2005, 38, 2620-2625). The publication speculates on the possibility of using these MIPs as biosensors, although it does not say or suggest any procedure to perform it, and therefore the expert I would not find it obvious in the art to arrive at the present invention from the teachings of this publication.

30 The development of biochips based on MIPs is done by stamping on a substrate, usually a wafer. In the current technique, this has been achieved on a micrometric scale using different methods, which can be classified as "contactless" and "contactless" techniques.

Among the contact techniques are the deposit with a micropunta (F. Vandevelde et al. "Oirect patterning of molecularly imprinted microdot arrays for sensors and biochips", Langmuir 2007, 23, 6490), "soft" lithography (M. Yan et al. "Fabrication of molecularly imprinted polymer microstructures", Anal. Chim. Acta 2001, 5 435, 163) and, recently, UV radiation contact photolithography (A = 365 nm)

(S. Guillón et al. "Single step patterning 01 molecularly imprinted polymers lor large scale manufacturing of microbiochips", Lab Chip 2009, 9, 2987). The methods of contact present the inconvenience of the chemical non-compatibility of the interacting surfaces. For example, in soft lithography a seal of polydimethyl siloxane (PDMS) 10 contacts the precursor solution of the MIP to form the pattern defined by the seal; however, POMS is incompatible with the most common solvents used in the synthesis of IPMs, and degrades the quality of the material. Guillón's recent demonstration of the use of photolithography to stamp MIPs on a wafer scale with micrometric resolution is a significant step towards

15 parallel manufacturing of biochips based on MIPs; However, these authors used the photolithographic method of contact, that is, that the mask contacts the film of polymeric material with the consequent problem of the final quality of the material.

20 Non-contact methods avoid this inconvenience. However, the number of contactless polymerization strategies applied to MIP and proven in the art is reduced to two.

The first requires the use of a beam of raised electromagnetic radiation

25 to polymerize the material by a photochemical or thermal mechanism. Two embodiments of this approach have been reported: Laser beam micro-stereo lithography of A = 364 nm managed to perform MIP structures of the order of tens of microns

(P.G. Conrad 11, et al. "Functional molecularly imprinted polymer microstructures labricated using microstereolithography", Advanced Materials 2003, 15, 1541); The

The second embodiment employs a 750 mW CO2 laser beam that allows the local temperature to be increased up to 70 ° C using 35 consecutive pulses of 5 ms, to obtain microarray points of 300 Jlm in diameter (OYF Henry, et al. "Fabrication of molecularly imprinted polymer microarray on a chip by mid-infrared laser pulse initiated polymerization ", Biosensors and Bioelectronics, 2008, 23, 1769).

The second strategy uses photo-masks for the polymerization of the areas of interest. This approach has been applied for the manufacture of selective MIP arrays of 70-90 11m diameter F1uorescein (AV Linares and Col. "Patterning Nanostructured, Synthetic, Polymeric Receptors by Simultaneous Projection Photolithography, Nanomolding, and Molecular Imprinting" Small, 2011, 7, 2318). The

The mask is projected through a 10x objective on the precursor solution of the MIP deposited on a microscope slide for polymerization with a mercury lamp. Alternatively, using nanoporous alumina molds functionalized with fluorescein or myoglobin, points of approx. 90 11m in diameter formed by nanofilaments superficially imprinted with the template molecule of interest, arranged in parallel with an individual diameter of 150 nm and 4 11m in length.

In a similar approach, Byrne has prepared microstructured imprinted gels, imprinted with D-glucose, with a thickness of 13 11m and different geometric configurations (Byrne et al. "Microfabrication of intelligent biomimetic networks lor recognition 01 O-glucose" Chern. Mater. 2006, 18, 5869).

Both contact and non-contact techniques of MIP-based biochips have spatial resolutions greater than micron. However, the sensor structures need to be reduced below the micron to significantly increase their sensitivity. This has been achieved thanks to the possibility of creating phenomena of optical, electrical or magnetic confinement in submicron regions, which achieves greater interaction between the analyte and the signal used for its detection. In addition, the use of sub-micrometric structures allows to reduce considerably both the sample volume and the energy required by the sensor.

The most significant advantages of electron beam writing systems for micro-and nano-manufacturing are first that no mask is required, thus eliminating the amortization costs of traditional lithography masks and accelerating the development cycles of chips; and also its capacity of sub-100 nm resolution, which meets all the lithographic requirements programmed by the ITRS (International Technology Roadmap for Semiconductors) for future generations

of integrated circuits. In addition, the vast majority of photolithography masks used in the microelectronic industry today are manufactured by LHE.

The problem of the technique is thus to obtain biochips on a submicron scale for the detection of analytes. The solution proposed by the present invention is a process comprising the partial irradiation of an electron beam on a precursor film of MIPs, to use as a biosensor a motif drawn on said film that would not have been irradiated by the electron beam.

Description of the invention The invention relates to a process of micro-nano-structuring of MIP films deposited on substrates for the manufacture of microbiochips.

So the present invention is a method of obtaining a molecular imprint polymer structure, comprising the following steps:

(to)

partial electron beam irradiation of a film formed by a precursor mixture of molecular imprint polymers on a substrate, preferably a silicon wafer or a glass, wherein said precursor mixture comprises branched polymer chains;

(b)

removal by revelation with a chemical solvent of the molecules affected by said electron beam irradiation,

(and)

polymerization of the film resulting from step b); Y

(d)

Extraction of said analyte from the polymerized film from step e) by chemical washing.

In a preferred embodiment, the method comprises the previous step of preparing the film on the substrate by spinning or spin-coating. This technique consists in depositing a drop or drops of the precursor mixture of the polymer to be formed on a substrate, then rotating it at a certain speed, typically with a rotor capable of adhering the substrate with a vacuum system. The end result is the formation of a highly uniform film on the substrate.

"Precursor mixture" or prepolymerization of MIPs is defined as that mixture of monomers, oligomers and / or polypeptides from which an MIP capable of recognizing an organic molecule is obtained after a polymerization process. Typically the mixture also comprises a chemical solvent, and the organic molecule or analyte.

5 serves as a molecular template. It may also contain a reaction initiator of polymerization.

"Polymer" is defined as a sequence of monomers with molecular weight equal to or greater than 1.5 kD. Below that molecular weight would be chains of 10 oligomers, which are not the subject of the present invention.

In a preferable embodiment of the invention, said irradiation of step a) is carried out with an electron beam lithography machine. The non-contact method for the realization of direct write MIP structures by electron beam results in a

15 tool suitable for nanofabrication thanks to its sub-micrometric resolution, and can be used for the manufacture of biochips of multiple chemical or biochemical nanosensors. So a very preferable embodiment is that the non-irradiated area of the film resulting from step a) is less than 1 ~ m in any of its dimensions.

The precursor mixtures of MIPs whatever their composition have functional groups and double bonds that determine the reaction with the analyte and its own polymerization. The proportion of double bonds in the side chains of the polymers of the precursor mixture of the present invention is 10% with respect to the

25 total functional groups.

In another embodiment of the invention, said analyte is an organic molecule selected from the group consisting of peptides, antibiotics, pesticides, mycotoxins, drugs, dyes, biomolecules and synthetic molecules of biomolecules,

30 preferably a dye. In another very preferable embodiment said organic molecule is a fluorophore molecule.

In yet another preferred embodiment, said chemical solvent of step b) with which the film affected by the electron beam is revealed is tetrahydrofuran, methanol or

35 isopropanol, or mixtures thereof.

In another preferable embodiment, the polymerization of step c) of the precursor mixture is carried out by heat and / or electromagnetic radiation.

5 In a further preferable embodiment, said final chemical wash of step (d) is performed in presence of methanol, ethanol, acetonitrile, water, formic acid, acetic acid, acid trifluoroacetic or tetra-n-butylammonium sulfate, or mixtures thereof.

The most preferable embodiment of the invention is a MIP structure that has a partial functional polymerized area.

The possibility of using functionalization procedures compatible with conventional microelectronic processes increases the scope and utility of micro and nano-biosensors, in addition to simplifying their manufacturing process.

15 From the point of view of the lithographic process, this requires methods to lithograph sub-micrometric motifs in functional materials for the recognition of organic molecules or biomolecules.

The method of the invention comprises the following steps developed (Figure 1):

20 A) Writing (4) of the desired patterns by irradiation with an electron beam (2) on a thin film (1) of the precursor or prepolymerization mixture of MIP. Once polymerized, the IPM film will act as a sensor in those areas that have not been irradiated.

Typically, the constituent functional monomers in an IPM precursor mixture are selected from vinyl monomers, allylic monomers, acetylenes, acrylates, methacrylates, amino acids, nucleosides, nucleotides, carbohydrates and heterocycles; cross-linking monomers that stabilize the polymer structure in the presence of the

Molecular template selected from diacrylates or dimethacrylates of polyols, such as 1,2-bis (methacryloxy) ethane or 2,2-bis (2-methyl-2-propanoyloxymethyl) butyl-2-methyl-2-propanoate; bisacrylamides such as N, N'methylenebisacrylamide; and divinyl compounds such as divinylbenzene and its analogues. In the precursor mixture of the present invention, these

35 monomers are present forming polymers. The precursor mix

Typical of a MIP also comprises a porogen, preferably an organic solvent, and a molecular template or analyte.

The film is deposited on a substrate (5) by any substrate coating technique.

The writing process with the electron beam can be carried out with a lithography machine or any instrumentation that has an electron gun (2) and a translation platform in a plane (6), on which the substrate is deposited ( 5). The translation platform is an electromechanical system that can be moved manually and / or automatically in a plane (X-Y). Thus, once the substrate system (5) -film (1) is fixed on the translation platform, it is possible to write with the electron beam (2), which usually remains in a fixed position, thanks to the displacement of the platform in the XY plane.

B) Elimination by dissolution of the irradiated regions (4) of the film (1) with a chemical solution that acts as a developer. The film (1) is immersed in the developer, which acts on the material of the mixture affected by electronic irradiation (4) with great selectivity on the non-irradiated material (1,7). The development time will depend on the dissolution rate of the irradiated material.

C) Polymerization of the nanostructured film (8, 9) that remains adhered to the substrate and has not been irradiated with the electron beam. The polymerization process begins in the presence of free radicals generated in the homolytic rupture of the carbon-carbon double bond of the side chains of the linear polymer after irradiation with UV light or heat treatment. Alternatively, the polymerization can be initiated in the presence of peroxide and azocomposite free radical initiators, which are compounds containing covalent bonds whose homolytic cleavage causes free radicals (8, 9). The result of polymerization is the formation of a three-dimensional cross-linked polymer structure.

D) Extraction of the molecular template of the nanostructured crosslinked film (10.11). This is done by immersing the film (8.9) in an organic solvent of the alcohol or nitrile, or aqueous type, capable of dissociating the molecular template of the crosslinked polymeric material.

Thus, the method of the invention creates sub-micrometric patterns in the film by the selective removal of the material around them. The IPM would behave like a positive resin because the irradiated part is removed with development. Remaining patterns on the substrate, ie the structured film, do not receive

10 electronic bombardment, which represents a great advantage of the invention since electronic irradiation could damage the molecular templates contained in the mixture, and therefore degrade the performance of the MIP.

In this respect, the electrons incident on the molecules produce a series of

15 chemical bond breakdown reactions that favor the subsequent dissolution in a chemical developer of the irradiated areas. For this effect to take place, the IPM precursor mixture must contain in its composition molecules sufficiently reactive and sensitive to electronic irradiation such as cross-linkable oligomers, polymers or copolymers. The sensitivity to electronic irradiation of these

20 molecules are due in the invention to the presence of double bonds C = C-in the side groups of said chains. Increasing the number of double bonds increases the sensitivity to electronic irradiation. The double bonds are highly reactive when they interact with energetic electrons, and can be broken to generate free radicals and substances that can be dissolved in the developer. The

25 electron beam energy and electronic dose (number of electrons per unit of time and area unit) optimal of the process will depend on the degree of reactivity of the mixture. As illustrative values, the energy of the electronic beam is usually of the order of several KeVs, while the typical electronic doses are of the order of mC / cm2 •

Another key aspect of the method of the invention is that the cross-linking of the prepolymerization mixture takes place after the drawing of the nanopatterns. The cross-linking process is essential to create a MIP. However, this cross-linking usually hardens the material mechanically and chemically, making it difficult to work afterwards in conditions suitable for use as a sensor

35 molecules

The method of the invention can be applied to any precursor mixture of MIPs containing chemical compounds sensitive to irradiation with electrons, so that the result of this irradiation is the rupture of chemical bonds and the consequent

5 formation of molecules capable of being dissolved in an organic solvent.

Electron beam lithography belongs to the nanofabrication sector of electronic, optical and / or magnetic components with resins, while MIPs belong to the area of functional materials for sensor purposes. combining

These technologies in the present invention belong to different technical areas, it is not suggested and is not obvious to the person skilled in the art and therefore must confer inventiveness to the present invention.

The comparison between the IPM of the invention and a PIN shows that the process of

Electron beam irradiation maintains the ability to act as a molecular imprint of the part of the non-irradiated film, compared to a PIN that does not recognize the molecular template (Figure 3). MIP is highly selective for rhodamine 123 since it does not show cross reactivity against structurally similar template molecules such as rhodamine B and rhodamine 6G that do not contain the amino group

20 primary characteristic of rhodamine 123, to which the selective recognition by the IPM is attributed (Figure 4).

The invention enables the manufacture of MIP structures with a size smaller than microns without the need to use any mask or any other contact device.

25 with the MIP, thus avoiding contaminating it. The non-contact method of the invention can be carried out industrially using commercially available equipment to perform Electron Beam Lithography (LHE).

From the economic point of view, the possibility of manufacturing multiple biochips

30 nanosensors based on MIPs using techniques of the semiconductor industry would reduce production costs considerably. This reduction would not only be motivated by the possibility of mass manufacturing; the use of biologically robust materials such as IPMs, which would act as "artificial antibodies" would eliminate the conservation costs required by the

35 delicate biological macromolecules of recognition. On the other hand, sub-micrometric sensor structures, in addition to offering greater sensitivity, require

lower sample volumes and operating power. Both factors would contribute in turn to reduce the costs of using biochips.

5 As mentioned above, the chemical modifications introduced in the MIPs to be able to selectively recognize rhodamine and be able to configure the Sensor of the invention is specific. If the organic molecule that acts as a template is another, the corresponding MIPs should also be developed in a way specific. This is why only one embodiment is provided and yet

The invention shows an inventive line common to all processes within the scope of the main claim.

Brief description of the díbujos

Figure 1: Scheme of the process of the invention. A) Writing by electron beam on the film:

one.

MIP precursor mixing film.

2.

Electron beam

3.

Electron cannon

20 4. Reason written with the electron beam in the film (1).

5. Substrate.

6. X-Y translation platform. B) Movie development:

7.

Desired pattern in the movie. 25 C) Polymerization of IPM:

8. Polymerized or crosslinked film.

9. Desired pattern in the movie. D) Ana lito extraction:

10.

Polymerized film without the template molecule. 30 11. Desired pattern in the movie.

Figure 2: 1 H-NMR spectrum of the polymer synthesized according to the embodiment example (300 MHz. D6-DMSO) O (ppm): 12.39 (br. HOOC-). 5.99 and 5.63 (s. CH2 = C). 4.10 (br s. CO-NH-). 1.85 (s. = C (CH3) -). 1.23. 1.04 and 0.95 (brs. CH3-C).

Figure 3: Graph of the fluorescence intensity of Rhodamine 123 recognized or captured by the MIP synthesized in Example 2 as a function of immersion time in a 20 nM solution of Rhodamine 123 in acetonitrile. It represents the recognition curve of the molecular template Rodamina 123 by the MIP. The fluorescence intensity from a Non-Imprinted Polymer (NIP) with the molecular template under the same incubation conditions is also shown.

Figure 4: Graph of the intensity of fluorescence of Rhodamine 123, Rhodamina By Rhodamine 6G, recognized or captured by the MIP synthesized in Example 2 after immersion in a 5 flM solution of the indicator in acetonitrile. Incubation time 3

h.

DETAILED DESCRIPTION OF THE INVENTION With the intention of showing the present invention in an illustrative way, although in no way limited, the following examples are provided.

Example 1: Preparation of the polymerization mixture The linear polymer subsequently used to obtain the MIP was prepared by mixing 4.5 mL of trimethylsilyl methacrylate (SIMA, ABCR, 97%) with 525 mg of 2-aminoethyl methacrylate (AEM, Aldrich, 90%) in 20 mL of methanol and thermally polymerizing with 50 mg of 2,2-azobis- (2,4-dimethylvaleronitrile) initiator (ABOV, Wako Pure Chemical Industries). The polymer was precipitated in acetone and redissolved in 30 mL of dimethylformamide (DMF, Cario Ebra), adding 543 mg of N (methacryloxy) succinimide (MAOS; ABCR) and 1 mL of triethylamine (TEA, Fischer Scientific, peptide synthesis). Then, it was precipitated on a solution of trifluoroacetic acid (TFA, Fluorochem, peptide synthesis) in 10% volume water and washed with acetone to obtain a powdery white solid. This polymer had a 10% double bond quantity with respect to the total number of functional groups, measured in the ratio of 1 H-NMR spectrum signals (Figure 2). The polymerization precursor mixture was obtained by dissolving 1 g of the polymer synthesized in 50 mL of methanol and adding 1.5 mg of Rhodamine 123, which acted as a molecular template.

Example 2: Manufacture of a MIP nanopattern on a capable Si substrate of recognizing the molecule Rhodamine 123. A film (1) of the pre mix was deposited on a silicon substrate (5) polymerization obtained in Example 1 by the spinning technique (D.W. Schubert,

5 T. Dunkel, 8pin coating from a molecular point of view: its concentration regimes, influence of molar mass and distribution; Materials Research Innovations Vol. 7, p. 314 (2003 »The thickness of this film was approximately 100 nm. The motif (4) of the pattern according to Figure 3 was drawn with an electron beam (2) generated by a cannon (3) (CRESTEC CABL-9500C) and accelerated towards the movie (1), being the

10 electron energy of 50 KeV and the dose of 2 mC / cm, producing chemical bond breakage. The writing was done thanks to the movement of the platform (6) on which the substrate (5) is located. The movie was then submerged

(1) adhered to the substrate (5) in a tetrahydrofuran (THF) developer for one minute that removed the irradiated area (4) giving rise to the desired pattern (7). Then heat (170 oC, 30 min) was applied to the structured film (1.7) making it a polymerized or crosslinked structured film (8.9). Finally, rhodamine molecules were extracted from the crosslinked structured film (8, 9) by immersion of the crosslinked film in a solution of ethanol with 10% (v / v) trifluoroacetic acid (TFA), obtaining a MIP film

20 with sub-micrometric structures (10,11) according to the written pattern (4) with the electron beam (2) (Figure 3). The pattern obtained is capable of detecting Rhodamine 123 molecules, that is, it is a Rhodamine 123 sensor based on MIPs.

Claims (12)

 Claims 1. Method of obtaining a molecular imprint polymer structure, characterized in that it comprises the following steps:

(to)

partial electron beam irradiation of a film formed by a precursor mixture of molecular imprint polymers on a substrate, wherein said precursor mixture comprises branched polymer chains;

(b)

removal by revelation with a chemical solvent of the molecules affected by said electron beam irradiation,

(and)

polymerization of the film resulting from step b); Y

(d)

Extraction of said analyte from the polymerized film from step e) by chemical washing.

2.

Method according to claim 1, characterized in that it comprises the previous step of preparing said film on the substrate by spinning technique.

3.

Method according to one of claims 1 or 2, characterized in that said substrate is a silicon wafer.

Four.

Method according to one of claims 1 or 2, characterized in that said substrate is a glass.

5.

Method according to any of claims 1 to 4, characterized in that said irradiation of step a) is performed with an electron beam lithography machine.

6.

Method according to any one of claims 1 to 5, characterized in that the non-irradiated area of the film resulting from step a) is less than 1 iJm in any of its dimensions.

7.

Method according to any of claims 1 to 6, characterized in that said ana lyte is an organic molecule selected from the group consisting of peptides, antibiotics, pesticides, mycotoxins, drugs, dyes, biomolecules and synthetic molecules of biomolecules.

8.

Method according to claim 7, characterized in that said organic molecule is a dye.

9.

Method according to one of claims 7 or 8, characterized in that said organic molecule is a fluorophore molecule.

10.

Method according to any one of claims 1 to 9, characterized in that

saying

solvent chemical from the stage b) is tetrahydrofuran, methanol or

Isopropanol, or mixtures thereof.

eleven .

Method according to any of claims 1 to 10, characterized in that

5

bliss polymerization from the stage C) be makes by hot I radiation

electromagnetic

12.

Method according to any of claims 1 to 11, characterized in that

said chemical washing of step (d) is performed in the presence of methan01, ethanol,

acetonitrile, water, formic acid, acetic acid, trifluoroacetic acid or sulfate

10

tetra-n-butylammonium, or mixtures thereof.