WO2017043954A1

WO2017043954A1 - Method and material for producing an electro-assisted crystallisation cell for biological macromolecules

Info

Publication number: WO2017043954A1
Application number: PCT/MX2016/000092
Authority: WO
Inventors: Abel MORENO CÁRCAMO; Nuria Victoria SÁNCHEZ PUIG
Original assignee: Universidad Nacional Autónoma de México
Priority date: 2015-09-09
Filing date: 2016-09-07
Publication date: 2017-03-16
Also published as: MX2015011982A

Abstract

The present invention relates to the field of biomacromolecular crystallography, as well as to methods and devices used for the nucleation and growth of biological macromolecule crystals. Unlike existing devices in the prior art, the electro-assisted crystallisation cell described in this invention has a different conception, since the crystallisation cell is a novel design based on a plastic material (polyethylene, PET).

Description

METHOD AND MANUFACTURING MATERIAL OF ELECTRO-ASSISTED CRYSTALLIZATION CELL

FOR BIOLOGICAL MACROÜOLECULES

FIELD OF THE INVENTION

The present invention relates to the field of biomacrorrsolecuiar chryography, as! as to the methods and devices used for the nucieation and growth of biological macromolecule crystals.

BACKGROUND OF THE INVENTION

Crystallography is a science that is part of the Solid State Chemistry, it deals with the study of the three-dimensional structure of organic, inorganic and even biological macromolecules solids. Crystallization is a complex process that involves multiple balances. The three common stages in the crystallization of any molecule are; nucieation, crystal growth and growth term. During nucieation enough moiecuias are associated to form a thermodynamically stable aggregate that is known as "critical nucleus". This core provides the surfaces on which e! Crystal will grow by subsequent addition of dough. The term of growth of a crystal! It occurs when raw moiecuias have been used up in the solution. Nucieation and the growth of a crystal occur in supersaturated solutions where the concentration of the molecule to be crystallized, in this case a protein, exceeds the value of its solubility balance. However, the supersaturation requirements for nucieation and crystal growth are different. At high supersaturations both nucieation and crystal growth can occur, while growth occurs predominantly at low supersaturation. The probability of obtaining crystals increases in solutions with very high supersaturations. However, the risk is that many nuclei that produce many small crystals are produced. The ideal is to be able to control and separate both phenomena, and thus obtain only a few nuclei that originate crystals of adequate size.

For years the control of the nucieation of macromolecular biological systems has been a bottleneck in biological and biomedical research.

The techniques used to crystallize biological macromolecule (for example, proteins) are based on the use of solutions that vary in their chemical parameters (ionic strength, polymers, ρΗ, temperature) and in which most of the biological macromolecule (for example, proteins or protein families) have crystallized. However, how popular these kits are in the crystallization of proteins, by providing only the initial crystallization conditions, they do not allow to separate the nucieation process from the growth of crystals. This concept had not been explored in detail, except for a commercial product of the company TRIA A Sci. And Tech, of Spain, which offers a device called Granada Crystallization Box (GCB), which does not use electric current but that allows to grow crystals in capillary media inside a plastic box, even, obtaining large crystals for structural studies via X-rays is still a big problem. When it comes to obtaining the structure of biological macromolecules by means of X-ray diffraction techniques, there are two ways to obtain the structure of micro- powders. crystals, which is not yet well established for biological macromoecures (for example, proteins) and the other possibility is to obtain the 3D structure using high-quality mono-crystals. In that sense, biomachromolecular crystallography requires crystals of high optical quality - structure! to have the high resolution 3D structure. There are several techniques that have allowed to obtain crystals by conventional routes in small drops (methods of hanging or sitting drops [1-3]) or by routes that are not the classic [4, 5]. In that sense, non-classical methods using electric or magnetic fields have opened a new path in the study of! crystal growth of biological macromoecures by unconventional routes. Since 2005, a preliminary idea emerged about the conception of crystallizing electro-assisted proteins in situ [6,7]. In 2008, a review appears to put the theoretical basis of this type of crystallization methods assisted by electrochemical aspects [8]. It is from this 2008 publication that presents the perspectives in the crystallization of proteins, when a series of articles related to variants and proposals of growth cells arise [9-15],

Recently, the effect of many physical parameters such as electric and magnetic fields in the control of nucleation and crystal growth has been evaluated. The advances made with moderate biological macromoecures (for example, proteins) in which electric fields have been used in their crystallization and which were the preliminary cell models that can be considered as the precursor ideas of electro crystallization are presented in Figure 1. assisted by biological macromoecuias.

US Patent 5,597,457 proposes a method and device for the growth of crystals in a fluid protein substance of sufficient quality to be subjected to diffraction, using the electro-crystallization method to grow protein crystals. Disseminates a system comprising a glass, carbon, or silicon substrate and a pair of parallel electrodes arranged in a solvent containing the protein solution; and it is preferred that the electrodes be in full contact with the substance. Once crystallization is carried out, an X-ray beam is directed towards the protein solution.

US 8,945,303 discloses a device for crystallization of proteins or other biopoiimers comprising a transparent conducting electrode in a glass substrate, an insulating element and a crystallization solution. Moreover, patent 8,945,303 teaches a plate-shaped crystallization device.

The state of the art, which precedes the present invention, are precursor ideas in which none proposes the present invention. All the cells that predate the present invention (Mirkin et al, 2003 and Sazaki et al; and Moreno and Sazaki, 2004) used platinum wire electrodes, which ran the risk of producing water electrolysis. The first cell that used the concept of electro-assisted crystallization in which electric and magnetic fields are combined, is published by Sazaki et al., 2004, Moreno and Sazaki, 2004. This cell contained two parallel platinum electrodes in which crystallization occurs in the internal section thereof or on any of the electrodes depending on the load of the protein under study. For the first time it was shown in the publication of Sazaki et al., 2004 that by applying to the crystallization process an electric field of 2 micro Amps and a magnetic field of 10 Tesia it was possible to grow crystals of the same size and with the same orientation, all oriented in the C crystallographic axis of the protein under study. It is from Gii-Alvaradejo et ai. (2011), where the use of transparent ITO electrodes is presented for the first time to avoid any problem of water electrolysis. While another publication reports the use of graphite electrodes (Espinosa-Montaro et al, 2013). In both cells it was applied only in the protein crystallization method by the "bat" method, for which the crystallization conditions and the exact amounts of both protein and precipitating agent necessary to produce the nucleation and the consequent must be previously known. crystal growth One of the problems that this cell had was mainly the separation of the crystalline samples from its interior for subsequent structural studies by X-rays. In 2013, a variant of this technique was published to be applied in the crystallization of proteins in the concept of hanging drop by diffusion in vapor phase (Flores-Hernández et al., 2013). The best performed in this cell allow to extend the known crystallization conditions obtained in drops using commercial kíís and apply electro-assisted crystallization. The difference in the cell published in 2013 and the previous ones is that it used ITO electrodes mounted on glass.

The present invention deals with a new device for crystallizing biological macromoecures, which joins the crystallization path by the method of hanging or sitting drops (conventional) and an electric field (unconventional), which allows crystallizing biological macromoecures in an electro-assisted manner. In situ, Until now, none of the cells or crystallization devices that combine the method of seated drop and an electric field is similar or similar to the present invention, much less proposes the use of plastic plates to mount the device , and at the same time as a conductive material to assist in crystallization. In one embodiment of the present invention, electro-assisted crystallization proceeds in the cell, automatically when it has a batch type configuration, configuration also within the scope of the present invention.

Tin and Indian Oxide (ITO) - unlike glass a matter! Hard, fragile, transparent or not inorganic, which may contain silica sand, sodium carbonate and calcium - it is a ternary composition of indium, staph and oxygen in varying proportions. Depending on the oxygen content, the ITO can be described as a ceramic material or alloy. ITO is transparent and colorless in thin films, and is even one of the most commonly used transparent conductive oxides due to its two properties, its electrical conductivity and its optical transparency, as well as the ease with which it can be deposited as a thin film. ITO has been frequently used to make transparent conductive coatings for screens or displays, such as liquid crystal displays, for organic light-emitting diodes, solar cells, among others.

Due to its properties, in the present invention, the electro-assisted crystallization cell is manufactured with ITO electrodes mounted on plastic material (ITO on polyethylene) which gives it great advantages over its predecessors: 1) it allows to configure the crystallization of macromoecuias biological that proceeds in the cell according to! method of hanging or sitting drops and an electric field, electro-assisted in situ; 2) the easy separation of the crystals that have been obtained within it by simply cutting the window opposite to where the biological macromolecule crystallized (for example, protelna); and 3) in cases where biological macromoecuias (for example, proteins} have a high structural water content or are very sensitive for cell removal, ITO electrodes allow to obtain X-ray collection of crystals in situ since they are transparent to this radiation; 4) overcomes the difficulty of separating the phenomena of nucleation and growth of a crystal, and thus obtain only a few nuclei that originate crystals of adequate size, thus solving many of the deficiencies that exist in the existing approaches, especially in the case of biological macromolecules such as proteins; among other.

StMAFUG OF THE INVENTION

Unlike the existing devices, the electro-assisted crystallization cell described in this invention has a different conception, since this new crystallization cell is based on plastic material (polyethylene, PEI), which allows crystallization to be configured which proceeds in the cell according to the seated drop method in diffusion in vapor phase or even in bat.

The electro-assisted crystallization cell of the present invention uses ITO electrodes mounted on plastic material (ITO on polyethylene) which gives it great advantages over its predecessors. The three main disadvantages of the cell in which the glass-mounted ITO electrodes were: the stiffness of the glass, the runoff or dripping and the extraction of crystals. Due to the rigidity of the glass, there were sealing problems and runoff problems on some occasions when the experiment was mounted; Protein crystal extraction was also a problem or required great expertise from the experimenter. This problem has been overcome in the present cell made of ITO plastic material (PET).

In a first aspect, the present invention makes it possible to configure the crystallization of biological macromolecules that proceed in the cell according to the method of hanging or sitting drops and an electric field; that is, electro-assisted in situ.

In a second aspect, the electro-assisted crystallization cell allows the easy separation of the crystals that have been obtained within it by simply cutting the window opposite to where the biological macromolecule crystallized (for example, a protein). The crystals of said proteins obtained can be recovered from the cell by cutting the plastic material easily with a conventional knife making a slit and the crystals of said protein can be recovered for diffraction studies.

In a third aspect, the present invention makes it possible that in those cases where biological macromolecules such as proteins have a high structural water content or are very sensitive to remove them from the cell, ITO electrodes allow the collection of rays to be obtained. X of crystals in situ since the plastic material is transparent to this radiation without producing X-ray absorption, and thus being able to carry out high resolution structural studies, even, without having to take the sample of its growth conditions.

In a fourth aspect of the present invention, the electro-assisted crystallization cell overcomes the difficulty of separating the nucleation and growth phenomena of a crystal in situ, and thus obtain only a few nuclei that originate crystals of suitable size, thus solving many of the deficiencies that exist in the existing approaches, especially in the case of biological macromolecules such as proteins. The nuclei grow by means of a classic process like that of the diffusion in vapor phase (water extraction) and also the cell has the advantage of allowing to observe the experiments in a conventional microscope and even the experiments can be recorded with a camcorder with time lapse recording. In addition the procedure to manufacture the cell in matter! Plastic are also presented in this specification.

In a fifth aspect, the present electro-assisted crystallization cell can be used in the presence of magnetic fields without it undergoing changes or variations in its design.

In a sixth aspect of the present invention, the electro-assisted crystallization cell can be used at different temperatures.

The present invention allows the crystallization of biological macromolecules, such as: proteins, nucleic acids and polysaccharides, and macromolecular complexes having combinations of these biomolecules. A direct current is applied in the range of 2-8 micro Amps in parallel plates of electrodes of ΪΤΟ (Tin and Indian Oxide) which allows the electro-crystallization of the biological macromolecule (for example, a protein) on the cathode or anode. Subsequently, the current stops and the crystalline growth process proceeds through vapor phase diffusion. The precipitating agent placed in the largest compartment of the cell draws water from the solution where the protein and the precipitating agent were mixed. Large crystals are obtained in a period of 2-4 weeks. The design of this cell allows to influence the process of nucieation and control the process of crystal growth.

In one embodiment of the invention, the electro-assisted crystallization cell present allows the possibility of being able to have two variants thereof: 1) employing the crystallization conditions of classical methods called hanging drop or sitting drop and 2) being able to crystallize in baích; for this, the cell that is claimed in the present invention would have 4 windows to do 4 batch experiments at the same time.

BRIEF DISCOVERY OF THE FIGURES

Figure 1. Three types of broth used previously are shown, in which electric fields are applied in different experimental configurations, observing the influence of electric fields in the crystallization of proteins. Figure 1a: experimental design using the acupuncture technique in gels (GA E) [18] where the anode (101), the precipitating solution (102), the solution with protein (103), the gel (104), the cathode (105) and the power source (108). Figure 1b: using two parallel platinum electrodes, where the red figures represent protein crystals [7]. Figure 1c: using tin and indium oxide electrodes (ITO) applied in the batch technique [18]. In the three cases the current applied since the beginning of the experiment was 2 μΑ and a polarization of the electrodes in cathode and anode is generated, since the proteins have an í, if they are prepared below this value they are positively charged and will stick to the cathode (electrode charged in this type of cell negatively).

Figure 2, Shows the advanced experimental design of the electro-assisted crystallization cell for a biological macromolecule, such as: proteins, nucleic acids and polysaccharides, and macromolecular complexes having combinations of these biomolecules, in a configuration of a seated drop in diffusion in Vapor phase with de transparent and matter electrodes! plastic; the The cell allows controlling the nucleation in the small reservoir and in the larger reservoir the precipitating agent is coiocated.

Figure 3, Electro-assisted crystallization cell for several biological macromolecules, such as: proteins, nucleic acids and poüsaccharides, and macromoiecuiar complexes that have combinations of these biomolecules, in configuration with! TO electrodes in plastic. Different macromolecules, different precipitating agents or a combination of both in a single experiment can be evaluated in each compartment of this cell. The case of a cell with four windows is exemplified but the design can be extended to a larger number of compartments. Figure 4. Electro-assisted crystallization cell for two different biological macromolecules in a seated drop configuration by vapor phase diffusion with ITO electrodes in plastic using the same precipitating agent.

Figure 5. Cele of electro-assisted crystallization that summarizes the general idea of the invention and shows preferred sizes and cuts in e! manufacturing process of the electro-assisted crystallization cell. Figure 6. They show an image of the electro-assisted crystallization cell mounted for diffraction taken in situ by the inventors of the patent in a synchrotron.

Figure 7. Figure 7 A and 7B shows diffraction images taken in situ by Sos inventors of the patent in a synchrotron, as an effective test that supports one of the advantages and potential of the cell present in the direct collection of X-ray diffraction data.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is about an electro-assisted crystallization cell for biological macromolecules, such as: proteins, nucleic acids and poüsaccharides, and macromoiecuiar complexes having combinations of these biomolecules. which comprises two ITO electrodes (tin oxide and indium). Said electrodes are separated by a rubber insulating membrane. The cell (Figure 2) has two compartments, one small one, which is a reservoir where the biological macromolecule (for example, a protein) is mixed with the precipitating agent and in the other reservoir the precipitating agent is placed only. In addition, the cell is perfectly sealed with an adherent substance to be able to apply a continuous current on a micro-Amp scale (in the range of 2-8 micro Amps) on parallel ITO plates for 48 hours, who suffer a polarization that allows crystallization through the application of said current and the biological macromolecuia (for example, a protein) crystallizes on any of them - (cathode or anode). Subsequently, the current stops being applied and the nucleation process stops and the cores generated (attached to the electrode: cathode or anode) proceed to grow through diffusion processes in the vapor phase, that is, the short section of the cell that It is shown in Figure 2. The crystals of the biological macromolecuia grow, because at first nuclear, assisted the process of nucleation by a current, the nuclei generated either in e! anode or cathode (this depends on the burden of the macromolecule or protein under study), are enriched by the mass supply of the solution, when the compartment containing the biological macromolecule mixture (for example, protein) with precipitating agent it is concentrated in time due to the extraction of water made by the precipitating agent that is in the reservoir higher. In a period of 2-4 weeks, large crystals are obtained due to the extraction of solvent into the small compartment.

The electro-assisted crystallization release of the present invention uses ITO electrodes mounted on plastic material (ITO on polyethylene), which gives it great advantages over its predecessors. The main disadvantage of Sa cell in which the glass-mounted ITO electrodes were had was that due to the rigidity of the glass, there were sealing problems and runoff problems on some occasions when the experiment was mounted; The extraction of crystals from biogenic macromolecules (for example, proteins) was also a problem or required great expertise from the experimenter. This problem has been overcome in the present cell made of ITO plastic material (PET).

The electro-assisted crystallization cell allows easy separation of the crystals that have been obtained within it by simply cutting the opposite window where the macromolecule or protein crystallized. The crystals obtained can be recovered from the cell by easily cutting the plastic material with a conventional knife by making a slit and the crystals of said protein can be recovered, for example, for diffraction studies.

The present invention makes it possible that in cases where biological macromolecules (for example, a protein) have a high structural water content or are very sensitive to remove them from the cell, STO electrodes allow to obtain the X-ray collection of crystals in place since the plastic material is transparent to this radiation without producing X-ray absorption, and thus being able to perform high resolution structural studies, even, without having to take the sample of its growth conditions.

The electro-assisted crystallization cell also overcomes the difficulty of separating the nucleation and growth phenomena of a crystal in situ, and thus obtain only a few nuclei that originate crystals of adequate size, thus solving many of the deficiencies that are found in Sas existing approaches, especially in the case of biogenic macromolecules such as proteins. The nuclei grow by means of a classic process such as the diffusion in the vapor phase (water extraction) and in addition the cell has the advantage of allowing to observe the experiments in a conventional microscope and the experiments can even be recorded with a camcorder with recording in time lapses. In addition the procedure to manufacture the cell and the design of the same in matter! Plastic are also presented in this specification.

The present electro-assisted crystallization cell can be used even in the presence of magnetic fields without it undergoing changes or variations in its design, or it can be used at different temperatures. In other embodiments, it is possible to produce two variants of the same electro-assisted crystallization cell: 1) employ the crystallization conditions of classical methods called pendant drop or sedentary drop, comprising two to three reservoirs and 2) to be able to crystallize in batch, for this the cell that is patented would have 4 windows or reservoirs to do 4 batch experiments at the same time (Figure 3).

Example 1 MateHaíes and manufacturing process of the one-way round crystallization cell for biological macromolecules based on ITO plastic electrodes. The electro-assisted crystallization cell comprises two transparent electrode plates of plastic material (polyethene) with tin and indium oxide deposited on its surface, this is allowed to have adequate conductivity and have a resistance ranging from 4 to 8 Ohms. Sa cell dimensions can be made of different sizes, even without limitations; the ideal case is to cut the plates to a size or dimensions of approximately 3.0 to 5.0 cm long by approximately 2.5 - 5.0 crn in width, as shown in Figures 2-4 because the plastic material It allows, as well as offers the advantage of the proper adjustment of the clamps (alligators). These dimensions are suitable for the size and number of reservoir (s) (from two, three to 4 reservoirs; the latter in the case of a baích configuration; and if the number of reservoirs is greater, from 1 to 98 reservoirs, The dimensions will increase proportionally to the number of reservoirs included) which will contain the biological macromoecuSa (for example, protein) which can be found in limited quantities resulting from its purification of the natural source !; Reservoirs of different dimensions allow the crystallization of molecules of greater or lesser molecular weight. The plates are joined through a black rubber insulating membrane approximately 0.15 to 0.2 cm thick and with dimensions equal to the transparent electrode plates. The conductive part of the plates is placed inside the cell, this can be done using a muitimeter to detect and corroborate, that the conductive part of Sa itself is inside. The sealing of the plate to avoid permeation of Siquádo, is done by placing an adherent substance, such as nail varnish, vacuum grease or silicone on the surface of the rubber and the plates are glued. In the case of using the seated drop configuration with vapor phase diffusion (Figure 2 and 4). the largest internal section of Sa section that will contain a must be varnished! precipitating agent, this with e! in order to prevent Sas salts that are part of it from separating and electrodeposing on Sas ITO walls, this internal varnish coating procedure is not done if the batch configuration from 4 sections or reservoirs will be used (Figure 3) up to 98 reservoirs , where only the biological macromolecule is mixed (for example, a protein, or nucleic acids, polysaccharides or combinations thereof) and the precipitating agent and the growth process proceeds immediately. In the reservoir that will contain said macromolecule or protein and the precipitating agent (Figures 2 and 4), it is not covered with varnish or any adherent substance, since it is desirable that said protein be numbered in the electrode and subsequently grow by phenomena of vapor phase diffusion. Once the varnish is placed on the black rubber, the electrode plates move approximately 0.5 - 1.0 cm from each other, this with e! object of leaving tabs to connect the clamps (colloquially known as alligators) that will be connected to the cathode or to the anode of the electroplating.

The process for manufacturing this electro-assisted crystallization cell comprises Sas following steps:

to. cut two transparent electrode plates of plastic material (polyethylene) with tin and indium oxide deposited on its surface, to an appropriate size and dimensions;

b. cut a rubber insulating membrane, approximately 0.15 to Q.2 cm thick, with appropriate size and dimensions to the electrode pins of the preceding paragraph; C. cut the rubber insulating membrane of! point b. at one end generating from two to ninety-six reservoirs;

d. join the two transparent electrode plates by means of Sa rubber insulating membrane;

and. place the conductive part of the electrode plates into the cell;

F. move the electrode plates approximately 0.5 - 1.0 cm from each other, leaving tabs to connect clamps or alligators; (This is done before putting the adherent substance, so as not to contaminate the ITO electrodes when the plates are moved);

g. sealing the plates and insulating membrane with an adherent substance that is placed on said insulating membrane, wherein the adherent substance is nail varnish, vacuum grease or silicon; preferably, nail varnish;

h. connect the clamps or alligators to the cathode and anode of a galvanostat.

The size and dimensions of the electrode plates are preferably between 3.0 - SO cm long and approximately 2.5 - 5.0 cm wide, while the size and dimensions of the rubber insulating membrane are preferably approximately 2.5 - 5.0. cm long by approximately 2.2 - 2.7 cm wide.

In one embodiment of the invention, when the insulating membrane of the cell comprises from two to three reservoirs, preferably three reservoirs - wherein the size and dimensions of the electrode plates is preferably approximately between 3.Q - 5.0 cm long by approximately 8.5 - 7.5 cm wide and the size and dimensions of the rubber insulating membrane are preferably approximately 2.5 - 5.0 cm long by approximately 6.0 - 7.0 cm wide - then the electro- crystallization cell assisted corresponds to a seated drop configuration with vapor phase diffusion, where at least one of Sos reservoirs is of greater volume containing precipitating agent, and the rest of the reservoirs is of lower volume Sos which will contain a biological macromolecule and precipitating agent ; The largest volume reservoir is approximately 1.5 cm x 0.8 x 0.15 - 2.0 cm x 1.0 cm x 0.2 cm, while the lowest volume reservoir is approximately 0.7 cm x 0.5 cm x 0.15 cm - 1.0 cm x 0.8 cm x 0.2 cm, being that in the sedentary drop configuration with three reservoirs, the reservoir of greater volume is central to the other two lateral reservoirs of smaller volume (Figure 4).

In another embodiment, when the insulating cell membrane comprises from four to ninety-six reservoirs, preferably comprises 98 reservoirs, the electro-assisted crystallization cell corresponds to a batch configuration, and all reservoirs contain a biological macromolecule and precipitating agent .

In the case of using the seated drop configuration with vapor phase diffusion (Figure 2 and 4), during sealing, it should be coated with varnish or the larger internal section of the section that will contain the precipitating agent, this for the purpose to avoid that the salts that compose you do not dissociate and electrodepositen on the walls of the ITO. This internal coating procedure of Varnish is not done if the configuration in bat from 4 sections or reservoirs (Figure 3) to 96 reservoirs will be used, where only the biological macromolecule and the precipitating agent are mixed. At a later stage, the solutions are prepared and added to the cell, following the procedure mentioned below: Approximately 100 microiiters of precipitating agent are added to the largest reservoir in the cell (see Figures 2 and 4), according to The size of the construction of it. Subsequently, approximately 25 microiiters of the biological macromolecule are mixed (for example, a proelel) with approximately 25 microiiters of precipitating agent, or in 1: 1, 1: 2 or 1: 3 ratios depending on the concentration of the available biological macromolecule, and depending on the size of the cell construction; care must be taken to first put the mixture in an eppendorf tube, taking care to put the most viscous solution first; in general, the biological macromolecule (for example, protein} is put first and then the precipitating agent and they are mixed very carefully.If polyether glycols are used at concentrations greater than 25% by weight, the precipitating agent must be put first and then the biological macromolecule (for example, a protein), to prepare the mixture that it will go in the small reservoir (Figures 2 and 4.) This also applies when 4 identical or different proteins are prepared to crystallize in the configuration ón bateh as shown in Figure 3. In this configuration of bat, to be able to introduce the biological macromolecules (for example, proteins) mixed with the precipitating agent in any of the 4 sections of the cell, two needles must be placed and Insulin-type syringes, one to introduce the liquid and another to expel the air stored in the Sa cell section. Since there are 4 compartments for the batch method (Figure 3), 8 needles and syringes of insulin type must be used to be able to introduce 4 equal experiments or 4 different biological macromolecules (for example, proteins) that are intended to crystallize. In the case of batch configurations that comprise a greater number of reserves, from 1 to 96 reservoirs, the introduction of the biogenic macromolecule-precipitating agent mixture is made through! use of automated systems.

Once the crystallization plate is mounted, ensuring that there is no loss of internal liquid (drip), the crystallization plate is connected to the respective electroplating and a direct current is applied in the range of 1 to 2 micro Amps for 24 hours. Subsequently, the equipment is disconnected from the current applied by the Galvanostat and the crystalline growth process is allowed to proceed through vapor phase diffusion, to allow the solution that is in the smallest reservoir (Figures 2 and 3) to be concentrate and allow the growth of crystals of the biological macromolecule (for example, a protein).

Additionally, in one embodiment of the invention, the crystals of the cell can be separated, opening a slit by cutting with a cutter and plastic (polyethylene) and removing the crystals at the end of the experiment.

In another embodiment of the invention, the precipitating agent can also be mixed with a cryoprotectant. In another embodiment of the invention, since the plastic ITO is transparent, an in-situ X-ray collection can be carried out without removing the crystals. It can also be placed in any optical microscope.

The foregoing are non-obvious advantages of this crystalline growth cell with respect to the cells disclosed in the prior art.

Example 2 Efeetro-Assist C Crystallization Procedure

To carry out the crystallization of biological macromolecules and see the effect of electro-assisted crystallization and the separation of nucieacián from crystalline growth, the following procedure is followed:

1. Mix a solution of Lysozyme 80 mg / mL (high purity) prepared in a 0.01 M acetates buffer pH 4.5 with a NaC solution! 80 mg / mL 1: 1. Introduce it to the growth cell in any of its variants in batch mode or the seated drop method as the device published in 2013 {Flores-Hernández et al). In a sedentary drop experiment by vapor phase diffusion, the concentration of precipitating agent must be twice that in the small reservoir.

2. Apply a current of 1 to 2 micro Amps for 24 hours, as defined in example 1.

3. Suspend the electric current and see the crystals that are generated at 24 hours in the case of the batch method. In the case of the vapor phase diffusion process, the crystals can be seen at 24 hours and their growth can be followed over 30 days.

Example 3. fi elities of the efeetro-assistants crystal cell configuration The cell in the seated drop configuration (Figure 2), can also be configured with four or more windows to apply the batch method (Figure 3). Even this cell in batch configuration could be expanded to lines of 8 to facilitate its filling with the sample of a biological macromolecule {for example, a protein) and precipitating agent through the use of muiticanai pipettes. This would allow it to perform aita density tests as each compartment could contain a different precipitating agent according to how they are accommodated in commercial kits.

An important aspect is that the reservoirs in which the precipitating agent and the biological macromolecule (for example, protein) to be crystallized are mixed can vary in size and be able to use minimum amounts of said biological macromolecule. Even the same concept of crystallization can be varied so that instead of having a single well for crystallization of a biological macromolecule (for example, a protein) at two different concentrations or two different proteins, there are two lateral reservoirs and one central one that will contain ai precipitating agent (Figure 4). The nucleation is electro-assisted by the application of current and the crystal growth proceeds through the concentration of the drop by vapor phase diffusion.

The advantages described above outweigh all the cells that have been published both by other authors / inventors and by other research groups. Example 4. Use of the erytheatics chamber © teotro-asfstida of ITO plastic for X-ray radiation m situ

To carry out the collection of diffraction data in situ, it is necessary to place the cell for electro-assisted crystallization, with the crystals formed, previously in the goniometer head. Subsequently, the goniometer head must be adjusted so that one of the crystals inside the cell is centered in a 360-degree rotation with the X-ray beam. Once centered, it is possible to start the collection by irradiating the crystal (see Figures 8, 7A and 7B).

Industrial Application of Invention

The invention is applied to the crystallization of biological macromolecules (proteins, nucleic acids and polysaccharides and for macromolecures complexes having combinations of these biomoléeuias) through an electro-assisted crystallization cell. The electro-assisted crystallization cell allows easy separation of the crystals that have been obtained within it by simply cutting the window opposite where the macromolecule or protein crystallized.

The present invention makes it possible that in cases where the biological macromolecules have a high structural water content or are very sensitive to remove them from the cell, ITO electrodes allow to obtain the X-ray collection of crystals in situ since the Plastic material is transparent to this radiation without producing X-ray absorption, and thus be able to perform high-resolution structural studies, even without having to take the sample of its growth conditions.

In other aspects, the present electro-assisted crystallization cement can also be used in the presence of magnetic fields without it undergoing changes or variations in its design or can be used at different temperatures.

In other applications, this type of cell allows measuring kinetics of crystal growth in situ, establishing nucleation studies and crystal growth. In addition to being able to measure the time for induction of nucleation, since the cell is transparent and can be placed even in any optical microscope, the only requirement is that a potentiostat / gaivanostat device must be used to apply the constant current or the respective voltage.

Finally, a very important advantage is the transparent electrodes of plastered ITO allow the crystal growth process to be recorded with a time-lapse video camera.

References

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[2] McPherson, A. (1999). Crystallization of Biological! Macromolecules, 1st Ed. Cold Spring Harbor Laboratory Press, New York.

[3] Bergfors, T. M. (1999) Protein crystaiüzation. Biotechnology Series, Int. Univ. Line. La Joiia, USA.

[4] Andrea E. Gutiérrez-Guezada, Roberto Arreguín-Espinosa, Abel Moreno "Protein Crystal Growth ethods" Chapier 47 of the Springer Handbook of Crystal Growth, Edited by D anaraj, Byrappa, Prasad, Dudley. 2010. Pages 1583-1605. [5] Abei Moreno, & Ma. Eugenia Mendoza "Cr staílizaíion in Gels" Chapter of the Handbook I heard Crysíal Growth, Second Edition, ELSEVIER. Edited by Peter Rudolph and T. Nishinaga. I saw í !; 2015 p. 1277-1315. ISBN: 9780444833033.

[6] Abei Moreno and Margarita Rivera "Conceptions and First Resulís on the Electrocrystallization Behavior of Ferritin". Act Crysisllograp ica Section D. Biological! Crystallography D61 (2005) 1678-1681

[7]. Francisco Acosta, Désir Eid, Liliana Marín-García, Bernardo A. Frontana-Uribe, and Abel Moreno rom Cytochrome C Grysíals to a Solid-State Eiecíron-Transfer Deviee ", Crysíal Growth and Design 7 (2007) 2187-2191.

[8] Frontana-Uribe, Bernardo; Moreno, Abe! On Bectrochemically Assisted Protein Crystallization and Related ethods ". Crysta! Growth and Design 8 {2008} 4194-4199.

[9] Yobana Pérez, Désir Eid, Francisco Acosta, Liliana Marín-García, Jean Jakoncic, Vivian Síojanoff, Bernardo A. Frontana-Uribe, Abel Moreno "ESectrochemicaliy Assisted Protein Crystallization of Comrrsercial Cytochrome C witbout Previous Purification". Crysta! Growth and Design 8 (2008) 2493-2496.

[10] Gabriela Gií-Aívaradejo, Rayana, R. Ruiz-Arelfano, Christopher Owen, Adela Rodriguez-Romero, Enrique Rudiño-Piñera, Moriamou K. Antwi, Vivian Stojanoff & Abei Moreno "Novel Protein Crysta! Growth Eiectrochemical Ceií for Applications in X-Ray Diffraction and Atornic Forcé Microscopy "Crystal Growth and Design 1 1 (201 1) 3917-3922.

[1 1] akamaísu,!.; Ohnishi.Y. Transparent CeíS for Protein Crystallization under Low Appüed Volíage. Jpn J. Appl. Phys. 50 (20 1) 048003-1-048003-2.

[12] Hammadi, 2., Veesler, S. New Approaches on Crysíaiüzatíon under Electric Fields. Prog. Biophys, And Mol. Biol. 101 (2009) 38-44.

[13] A! -Haq _: MA., Lebrasseur, e., Tsuchiya, H ,, Toril, T, Protein Crystallization under Electric Fields. Crystallography Reviews 13 (2007} 29-64.

[14] Flores-Hernández, E., Stojanoff, V., Arreguln-Espinosa, R., Moreno, A. and Nuria Sánchez-Puig. "An electricaliy assisted devíce for protein crystallization in a vapor diffusion seí-υρ ^" . Journal of Appüed Crystallography, 46 (2013) 832-834.

[15] Espinoza-Montero, P., Moreno-Narváez, M. E., Frontana-Uribe, B.A., Stojanoff, V., Moreno, A.

Investigations on the use of graphite ectrodes using a Hull-Type Growth Ceil for Electrochemcial

Assisted Protein Crystallization. Crystal Growth and Design 13 (20 3) 590-598.

[18] Nurit Mirkin, Bernardo Frontana-Uribe, Adeia Rodríguez-Romero, Alejandra Hernández-Santoyo and Abei Moreno. The influersce of the etecírie field upon protein crystallization using the ge! acupuncíure rnethod ". Acia Crysíallographiea D59, No. 9 (2003) 1533-1538.

[7] Abei Moreno & Gen Sazaki "The use of a new ad oc growth ceü with paraliel eiectrodes for nucleation control of iysozyme". Journal of Crystaí Growth, 264 (2004) 438-444.

[18] Gabriela Gil-Alvaradejo, Rayana, R. Ruiz-Areüano, Christopher Owen, Adeia Rodriguez-Romero,

Enrique Rudiño-Piñera, Moriamou. Antwi, Vivian Stojanoff & Abei Moreno "Novel Protein Crysta!

Growth Eiectrochemical Ceü for Applications in X-Ray Diffraction and Atomic Forcé Microscopy "

Crysíal Growth and Design 11 (2011) 3917-3922.

Claims

1. - An electro-assisted crystallization crystallization for biological macromoecuias, characterized in that it comprises:

to. two transparent electrode plates of plastic material (polyethylene) with tin and indium oxide deposited on its surface, of an appropriate size and dimensions;

b. a rubber insulating membrane, approximately 0.15 cm ~ 0.2 cm thick, with appropriate size and dimensions to those of the electrode plates of the preceding paragraph; C. from two to ninety-six reservoirs, where the reservoir of smaller volume contains a biological macromolecuia and precipitating agent, and the larger precipitating agent;

wherein the plates are joined through said sealed rubber insulating membrane, both with an adherent substance and wherein the conductive part of the plates is placed into the cell.

2. - The electro-assisted crystallization ceid for biological macromoecures according to claim 1, characterized in that the size and dimensions of the electrode plates are preferably between 3.0-5.0 cm long and approximately 2.5-5.0 cm. .width.

3. - The electro-assisted crystallization ceid for biological macromoecures according to claim 1, characterized in that the size and dimensions of the rubber insulating membrane are preferably between about 2.5 - 5.0 cm long by about between 2.2 - 2.7 cm Wide

4. - The electro-assisted crystallization ceid for biological macromoecures according to claim 1, characterized in that when it comprises from two to two reservoirs, the electro-assisted crystallization cell corresponds to a seated drop configuration with vapor diffusion .

5. - The electro-assisted crystallization ceid for biological macromoecures according to claim 1, characterized in that when it comprises from four to ninety-six reservoirs, the electro-assisted crystallization cell corresponds to a baich configuration,

8. The electro-assisted crystallization cell for biological macromoecures according to claim 1-5, characterized in that the electrode plates are displaced approximately between 0.5-1.0 cm with respect to each other.

7. The electro-assisted crystallization cell for biological macromoecures according to claim 1-5, characterized in that it has a resistance ranging from 4 to 8 Ohms.

8. - The electro-assisted crystallization cell for biological maeromolecules according to claim 1-5, characterized in that the adherent substance is nail varnish, vacuum grease or silica.

9. - The electro-assisted crystallization cell for biological maomolecules according to claim 8, characterized in that the adherent substance is preferably nail varnish.

10. - The electro-assisted crystallization cell for biological maomolecules according to claim 1 and 4, characterized in that at least one of the reservoirs is of greater volume, and the rest of smaller volume.

11. - The electro-assisted crystallization cell for biological rrsacromolecules according to claim 10, characterized in that the reservoir of greater volume is approximately 1.5 crn x 0.8 x 0.15 - 2.0 cm x 1.0 cm x 0.2 cm .

12. - The electro-assisted crystallization cell for biological maomolecules according to claim 10, characterized in that the reservoir of smaller volume is approximately 0.7 cm x 0.5 cm x 0.15 cm - 1.0 cm x 0.8 cm x 0.2 cm,

13. - The electro-assisted crystallization cell for biological maomolecules according to claim 10, characterized in that the reservoir of greater volume contains precipitating agent and the reservoir of smaller volume contains a biological macromolecule and precipitating agent.

14 - The electro-assisted crystallization cell for biological macromolecules according to claim 13, wherein the biological maeromolecules are proteins, nucleic acids, posaccharides and combinations thereof.

15 - The electro-assisted crystallization cell for biological maomolecules according to claim 13, characterized in that the sealing with the adherent substance is in the largest internal section of the section that will contain the precipitating agent.

18. The ephedro-assisted crystallization ceid for biological maeromolecules according to claim 4, characterized in that three reservoirs preferably comprise in their configuration of a seated drop with vapor phase diffusion.

17 - The electro-assisted crystallization cell for biological maeromolecules according to claim 16, characterized in that the size and dimensions of the electrode plates is preferably between approximately 3.0 - 5.0 cm long and approximately between 8.5 ··· 7.5 cm wide

18. - The electro-assisted crystallization cell for biological macromoecures according to claim 16, characterized in that the size and dimensions of the rubber insulating membrane are preferably between approximately 2.5 · - 5.0 cm long and approximately between 6.0 - 7.0 cm wide.

19. - The electro-assisted crystallization cell for biological macromoecures according to claim 18, characterized in that one of the reservoirs is of greater volume, and the rest of smaller volume.

20. - The electro-assisted crystallization cell for biographical macromoecures according to claim 19, characterized in that reserving it of greater volume is central to the other two lateral reservoirs of smaller volume.

21. - The electro-assisted crystallization cell for biological macromoecures according to claim 19, characterized in that the reserve of greater volume is approximately between 1.5 cm x 0.8 x 0.15 - 2.0 cm x 1.0 cm x 0.2 cm.

22. - The electro-assisted crystallization cell for biological macromoecures according to claim 19, characterized in that the reservoir of smaller volume is approximately between Q.7 cm x 0.5 cm x 0.15 cm - 1.0 cm x 0.8 cm x 0.2 cm .

23. - The electro-assisted crystallization cell for biological macromoecures according to claim 20, characterized in that the reservoir of greater volume contains precipitating agent and reservoirs of smaller volume contain a biological maeromolecule and precipitating agent.

24. The electro-assisted crystallization cell for biological macromoecures according to claim 23, wherein the biological macromoecures are proteins, nucleic acids, polysaccharides and combinations thereof.

25. - The electro-assisted crystallization cell for biological macromoecures according to claim 1 and 5, characterized in that the reservoirs contain a biological maeromolecule and precipitating agent.

28. The electro-assisted crystallization cell for biological macromoecures according to claim 25, wherein the biological macromoecuias are proteins, nucleic acids, polysaccharides and combinations thereof.

27, - Electro-assisted crystallization cell for biological macromoecules in batch configuration according to claim 28, wherein the introduction of the biological agent-molecular mixture requires the use of insulin-like needles and syringes or automated systems.

28. - The electro-assisted crystallization cell for biological macromolecules according to claim 1 and 5 »characterized in that in the batch configuration, there is no internal coating with the adherent substance.

29. - The electro-assisted crystallization yield for biological macromolecules according to claim 1 and 5, characterized in that preferably the rubber insulating membrane comprises 98 reservoirs,

30. - A process for manufacturing the electro-assisted crystallization cell for biological macromolecules of claim 1, characterized in that it comprises the following steps:

to. cut two transparent electrode plates of plastic material (polyethelene) with tin and indium oxide deposited on its surface, to an appropriate size and dimensions;

b. cut a rubber insulating membrane, approximately 0.15 - 0.2 em thick, with appropriate size and dimensions to those of the electrode plates of the preceding paragraph; C. cut the rubber insulating membrane from point b. at one end generating from two to ninety-six reservoirs;

d. join the two transparent electrode plates by means of the rubber insulating membrane;

and. place the conductive part of the electrode plates into the cell;

F. move the electrode plates approximately 0.5 - 1.0 cm from each other, leaving tabs to connect clamps or alligators;

g. sealing the plates and insulating membrane with an adherent substance that is placed on said insulating membrane;

h. connect the clamps or alligators to the cathode and anode of a galvanostat.

31. - The process for manufacturing an electro-assisted crystallization cell for biological macromolecules according to claim 30, characterized in that the size and dimensions of the electrode pins are preferably between about 3.0 - 5.0 cm long by about between 2.5 · - 5.0 cm wide,

32. - The process for the manufacture of an electro-assisted crystallization cell for biological macromolecules according to claim 30, characterized in that the size and dimensions of the rubber insulating membrane are preferably approximately 2.5-5.0 cm long. for approximately 2.2 - 2.7 cm wide

33. - The process for manufacturing an electro-assisted crystallization cell for biological macromolecules according to claim 30, characterized in that when the insulating membrane comprises from two to three reservoirs, the electro-assisted crystallization cell corresponds to a configuration seated drop with vapor phase diffusion.

34. - E3 process for the manufacture of an electro-assisted crystallization cell for biological macromolecules according to claim 30, characterized in that when the insulating membrane comprises from four to ninety-six reservoirs, the electro-assisted crystallization cell corresponds to a bat setting.

35. - The process for the manufacture of an electro-assisted crystallization cell for biological macromolecules according to claim 30-34, characterized in that the electrode pins of the cell have a resistance ranging from 4 to 8 Ohms.

36. - The process for the manufacture of an electro-assisted crystallization cement for biological macromolecules according to claim 30-34, characterized in that the adherent substance is nail varnish, vacuum grease or silicone.

37. - The process for the manufacture of an electro-assisted crystallization cell for biological macromolecules according to claim 36, characterized in that the adherent substance is preferably nail varnish.

38. - The process for manufacturing an electro-assisted crystallization cell for biological macromolecules according to claims 30 and 33, characterized in that at least one of the reservoirs is of greater volume, and the rest of smaller volume.

39. - The process for the manufacture of an electro-assisted crystallization cement for biological macromolecules according to claim 38, characterized in that the reservoir of greater volume is approximately between 1.5 cm x 0.8 x 0.15 - 2.0 em x 1.0 cm x 0.2 cm

40. - The process for the manufacture of an electro-assisted crystallization cell for biological macromolecules according to claim 38, characterized in that the reservoir of the highest volume is approximately between 0.7 cm x 0.5 cm x 0.15 cm - 1.0 cm x 0.8 cm x 0.2 cm

41. - The process for the manufacture of an electro-assisted crystallization cement for biological macromolecules according to claim 38, characterized in that the reservoir of greater volume contains precipitating agent and reserving it of smaller volume contains a biological macromoecuia and precipitating agent.

42. - The process for manufacturing a cell for electro-assisted crystallization biological macromolécuias according to claim 41, ^'wherein the biological macromolécuias are proteins, nucleic acids, poíisacáridos and combinations thereof.

43. - The process for the manufacture of an electro-assisted crystallization cell for biological macromolecules according to claim 41, characterized in that in the sealing stage, It performs an internal varnish coating of the largest internal section of the section that will contain the precipitating agent.

44. - The process for the manufacture of an electro-assisted crystallization cell for biological macromoecures according to claim 33, characterized in that preferably the eiecrode plates and the rubber insulating membrane comprise in their configuration a seated drop with phase diffusion steam, three reservoirs,

45. - The process for the manufacture of an electro-assisted crystallization cell for biological macromolecules according to claim 44, characterized in that the size and dimensions of the electrode plates is preferably approximately 3.0-5.0 cm long by approximately between 8.5 - 7.5 cm wide.

48.- The process for the manufacture of an electro-assisted crystallization crystallization cell for biological macromoecures according to claim 44, characterized in that the size and dimensions of the rubber insulating membrane are preferably approximately 2.5 ~ 5.0 cm. long by approximately 6.0 - 7.0 cm wide.

47. - The process for manufacturing an electro-assisted crystallization cell for biological macromoecures according to claim 44, characterized in that one of the reservoirs is of greater volume, and the rest of smaller volume.

48. - A process for the manufacture of an electro-assisted crystallization cell for biological macromoecures according to claim 47, characterized in that the reservoir of greater volume is central to the other two lateral reservoirs of smaller volume.

49. - The process for the manufacture of an electro-assisted crystallization cell for biological macromoecures according to claim 47, characterized in that the reservoir of greater volume is approximately between 1.5 cm x 0.8 x 0.15 - 2.0 cm x .0 cm x 0.2 cm

50. - The process for manufacturing an electro-assisted crystallization cell for biological macromoecures according to claim 47, characterized in that the reservoir of smaller volume is approximately between 0.7 cm x 0.5 cm x 0.15 cm - 1.0 cm x 0.8 cm x 0.2 cm.

51. The process for manufacturing an electro-assisted crystallization cell for biological macromoecures according to claim 48, characterized in that the reservoir of greater volume contains precipitating agent and reservoirs of smaller volume contain a biological maeromolecule and precipitating agent.

52. - The process for manufacturing an electro-assisted crystallization cell for biological macromolecules according to claim 51, wherein the biological macromolecules are proteins, nucleic acids, polysaccharides and combinations thereof.

53. - The process for manufacturing an electro-assisted crystallization cell for biological macromolecules according to claim 30 and 34, characterized in that the reservoirs contain a biographical macromolecule and precipitating agent.

54. - The process for the manufacture of an electro-assisted crystallization cell for biological macromolecules according to claim 53, wherein the biological macromolecules are proteins, nucleic acids, polysaccharides and combinations thereof.

55. - The process for manufacturing an electro-assisted crystallization cell for biological macromolecules according to claim 30 and 34, characterized in that in the batch configuration, an internal coating procedure is not performed,

56. - The process for the manufacture of an electro-assisted crystallization cement for biological macromolecules according to claims 30 and 34, characterized in that preferably the rubber insulating membrane comprises 96 reservoirs.

57. - Use of the electro-assisted crystallization cell for biological macromolecules of the claim, useful for applications in crystallography of biological macromolecules.

58. - The use of the electro-assisted crystallization ceid for biological macromolecules according to claim 57, wherein the biological macromolecules are proteins, nucleic acids, polysaccharides and combinations thereof.