WO2016097431A1 - Method for producing an amperometric lactate biosensor, biosensor produced by means of said method, and use thereof in toxic complex media - Google Patents

Method for producing an amperometric lactate biosensor, biosensor produced by means of said method, and use thereof in toxic complex media Download PDF

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WO2016097431A1
WO2016097431A1 PCT/ES2015/070791 ES2015070791W WO2016097431A1 WO 2016097431 A1 WO2016097431 A1 WO 2016097431A1 ES 2015070791 W ES2015070791 W ES 2015070791W WO 2016097431 A1 WO2016097431 A1 WO 2016097431A1
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lactate
biosensor
amperometric
enzyme
lod
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PCT/ES2015/070791
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Spanish (es)
French (fr)
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Naiara HERNÁNDEZ IBÁNEZ
Jesús INIESTA VALCÁRCEL
Vicente Montiel Leguey
Craig Edward Banks
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Universidad De Alicante
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means

Definitions

  • the present invention is framed in the field of amperometric sensors for use in assisted reproduction, whose purpose more specifically focuses on the detection and determination of lactate / lactic acid in complex media such as cell culture media, used in development and growth of human embryonic cells.
  • lactate oxidase contains the cofactor Flavin adenine dinucleotide (FAD) in its structure, which catalyzes the conversion of lactate to pyruvate in the presence of O2 while generating H2O2 as a product [VP Zanini, B. López De Mishima, V Sol ⁇ s, Sensors and Actuators, B: Chemical 155 (201 1) 75].
  • FAD Flavin adenine dinucleotide
  • NADH cofactor is present in embryonic culture media, which would interfere in the measure if a biosensor based on lactate dehydrogenase is used, it is necessary to develop electrochemical lactate biosensors in which it is used as Lactate oxidase enzyme.
  • Amperometric lactate biosensors which use lactate oxidase as a biocatalyst for the oxidation reaction of lactate to pyruvate generating hydrogen peroxide, work when subjected to a potential between +0.5 and +0.65 V against the AgCI / Ag reference electrode ( 3M chloride). This is the range of potentials required to oxidize H2O2 on the electrode surface and, therefore, establish a quantifiable response that relates the intensity of current obtained with the amount of lactate present in the study solution [L. Rassaei, W. Olthuis, S. Tsujimura, EJR Sudholter, A. Van Den Berg, Analytical and Bioanalytical Chemistry 406 (2013) 123].
  • amperometric lactate biosensors The problem of these amperometric lactate biosensors is the high potential to which the measurement should be taken when the medium in which the lactate is found can contain other substances that can also contribute to the reading of the recordable current at these potentials (they are the so-called interferents). To avoid this circumstance, we must find a range of potentials where these substances do not contribute to the current reading that should only be due to the presence of lactate. Therefore, it is necessary to develop amperometric lactate biosensors that can operate at low electrode working potentials and, consequently, interferences due to those electroactive species that can oxidize to higher potentials can be avoided.
  • the ferrocene methanol is oxidizes to ferrocinium (1), the ferrocinium subsequently oxidizes the H2O2 (2) formed after the oxidation of lactate by the enzyme lactate oxidase (hereinafter LOD), finally detecting the subsequent electrochemical signal at a formal potential of +0.19 V against a reference electrode AgCI / Ag (3 M KCI) (3).
  • LOD lactate oxidase
  • the electrode area can be increased using nanomaterials such as carbon nanotubes, graphene or metal nanoparticles. Some authors prefer the combination of nanomaterials with conductive polymers.
  • the combination of both gives the biosensor special properties due to the synergistic effect of the individual components, such as the combination of MWCNTs and polysulfone for the manufacture of a biosensor in order to determine lactate in wine and beer samples [S. Pérez, E. Fábregas, Analyst 137 (2012) 3854] using a screen-printed graphite platform.
  • the results showed detection limits of 0.05 mg / L, a linearity range between 0.1 ⁇ 0.5 mg / L, but with a stability of 40% after 2 weeks, the electrode being stored in a phosphate buffer solution at neutral pH.
  • the protein was immobilized by interleaving chitosan sheets (1% w / w, aqueous solution in acetic acid).
  • the enzymatic solution consisted of an aqueous solution of LOD and HRP.
  • the deposit of each layer was made just after drying the previous layer. The measurements were carried out at a potential of -50 mV.
  • This biosensor presented a linearity range of 5-244 ⁇ , a correlation coefficient of 0.994 and a long-term storage stability of 90% after 15 months.
  • the biosensor application focused on the analysis of lactate in solid food and wine samples. However, the biosensor preparation is still too complex for the potential commercialization of the biosensor.
  • Zhenyu Zhou et al [Zhou, Z.; Xu, L; Wu, S.; Su, B. Analyst 139 (2014), 4934] also manufactured a biosensor using chitosan and MWCNT for the manufacture of an electrochemiluminescent biosensor for the determination of glucose, lactate and choline.
  • they dissolved chitosan in acetic acid (1% w / w) by adjusting the pH to 5 with NaOH and left sonicating for 40 minutes.
  • 4 mg of MWCNTs were dispersed in 2 ml_ of the previous solution, and then 200 ⁇ _ of the resulting dispersion was mixed with 100 ⁇ _ of LOD.
  • the Cui group [X. Cui, CM. L., J. Zang, S. Yu, Biosensors and Bioelectronics 22 (2007) 3288] also prepared a solution of chitosan (1% w / w) in HCI by adjusting the pH to 5 with NaOH, subsequently adding: CNTs carbon nanotubes (up to a concentration 5 ⁇ g ml_ "1 ); LOD (up to a concentration 10 mg-mL " 1 ); and an osmium-with polyvinylimidazolium-Os complex PVI-Os (up to a concentration of 4 mg mL "1 ).
  • the metal substrate used was gold modified with a self-assembled layer of MPA mercaptopropionic acid.
  • the metal substrate dried deposit 3 ⁇ of the solution Chitosan / CNTs / LOD / PVI-Os on the surface of the gold electrode modified with MPA and allowed to dry in an incubator at 37 ° C for 3 hours.
  • it was washed under stirring with a phosphate buffer solution at neutral pH for 2 hours and finally stored in air at 4 ° C.
  • the biosensor presented a detection limit of 5 ⁇ , with a good linearity range up to 1 mM, but again the authors did not present data on the stability of the amperometric lactate biosensor or its applications in real samples.
  • Chitosan is a linear polysaccharide composed of randomly distributed chains of ⁇ -D-glucosamine and N-acetyl-D-glucosamine.
  • any biosensor for the detection of lactate that jointly uses the enzyme LOD and MWCNTs together with other polysaccharides or biopolymers other than chitosan.
  • amperometric lactate biosensors The success of the introduction of amperometric lactate biosensors in the market must be based on their simplicity, rapid response, sensitivity, low cost and portability. To achieve some of these aspects, miniaturization of amperometric biosensors is essential. In recent years, the use of screen-printed platforms for the manufacture of lacto biosensors has been increasing. To date, there are no authors in the state of the art who use the combination of biopolymers, together with carbon nanotubes (CNT) exclusively for the manufacture of amperometric lactate biosensors that use lactate oxidase and also necessarily use screen-printed platforms.
  • CNT carbon nanotubes
  • Rawson et al. [FJ Rawson, WM Purcell, J. Xu, RM Pemberton, PR Fielden, N. Biddle, JP Hart, Talanta 77 (2009) 1 149] used screenprinted carbon ink platforms containing CoPC as electrocatalyst, using the amperometric biosensor as a platform to monitor cell metabolism in vitro. Therefore, biosensors immobilized on screen-printed platforms for the analysis of lactate in complex media are described, which necessarily implies the use of external layers to protect the biosensor and avoid interference.
  • the process described in the present invention makes it possible to manufacture biosensors capable of overcoming the drawbacks of current amperometric biosensors, thanks to the materials used and the method of preparation.
  • An object of the present invention thus relates to the process of preparing an amperometric biosensor for the determination of lactate comprising the following steps: one . Dispersion of a biopolymer in organic solvent.
  • a mediator and carbon nanotubes are added to the previous dispersion to give rise to the mediator solution.
  • the biopolymer used is based on glucose or amylose polysaccharides, functionalized with groups, amino, acetyl or carboxylic, which contain glycosidic bonds.
  • These materials are biocompatible polymers of the type chitosan, starch, chitin, hyaluronic acid, glycogen and cellulose, which have glycosidic bonds of glucose, amylose or N-acetyl-D-glucosamine.
  • the organic solvent used for dispersion of the biopolymer is dimethylformamide.
  • the solvent may be a combination of dimethylformamide and ethanol, in a 1: 3 ratio (v: v).
  • the mediator used in step 2 of the process is ferrocene methanol (FcMe).
  • the nanotubes used in step 2 are multi-wall carbon nanotubes functionalized with oxygenated groups (MWCNT).
  • the bienzymatic system formed by the LOD enzyme is used together with the enzyme horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • the electrochemical platform of choice for the preparation of the amperometric lactate biosensor in stage 4 has been a disposable screen-printed graphite platform (SPGE) with a pseudo basal character, which allows an adequate deposition of the conductive carbon matrix and, consequently, a adequate reproducibility in immobilization of the LOD enzyme.
  • SPGE screen-printed graphite platform
  • the screen-printed platform used can be of the conventional type, of the microelectrode type, of the array type, of the microarray type, of the micro-band type or the micro-band type in series. In this way, the biosensor can be applied to the simultaneous determination of several samples, or even perform analyzes with a very small amount of sample, conferring versatility and speed to the system.
  • step 6 of drying the biosensor for solvent removal can be carried out at 2-5 ° C, or at 25 ° C under high vacuum conditions for 1 hour and then stored at 2-5 ° C in air .
  • Another object of the invention relates to a new portable and disposable amperometric lactate biosensor, obtained by the procedure described above.
  • the amperometric lactate biosensor obtained utilizes disposable screen-printed graphite electrochemical platforms, on which a conductive carbonaceous matrix composed of a combination of an enzymatic system containing at least the LOD enzyme, functionalized carbon nanotubes and a biopolymer for the immobilization of the enzyme that gives stability to the biosensor, but that is biocomplatible.
  • Another object of the invention relates to the use of the biosensor obtained by the described procedure, for application in various complex media, including embryo culture media used in human assisted reproduction techniques and other media used in cell development.
  • amperometric lactate biosensor is also extrapolated to other fields of application such as food, where the purpose of the application of the amperometric lactate biosensor would be the detection and determination of lactate / lactic acid in samples from the wine and beer sector; or in the sports field, in which the amperometric lactate biosensor would address the determination of athletes' blood lactate levels.
  • the biosensor is biocompatible and non-toxic. This allows its use for in situ measurement during cell growth of human embryos.
  • the degradation rate of the biosensor is reduced.
  • the biosensor is stable for at least 4 months at a temperature of 4 ° C without the need to protect it with any type of membrane, gel or solution.
  • biosensor allows to measure the concentration of lactate in real time, in embryo culture media used in assisted reproduction techniques, after removal of the embryo. and.
  • the biosensor can be prepared on different types of platforms with different designs such as conventional, micro electrode, array, microarray, microband or serial microband
  • biocompatible biopolymers such as chitosan, starch, chitin, hyaluronic acid, glycogen or cellulose, also contribute to significantly improve the stability of the amperometric lactate biosensor, achieving retention of the biosensor activity after 4 months of storage between 2-5 ° C. This becomes a great advance on the biosensors described in the literature so far.
  • the described amperometric biosensor does not need to be protected with a film of aqueous or organic nature (for example, membrane, gel, or solution) for stable storage of the biosensor, which differentiates it from some of those currently known and gives it a New added value.
  • a film of aqueous or organic nature for example, membrane, gel, or solution
  • FIGURE 1 Amperometric lactate biosensor.
  • Scheme of the disposable screen-printed platform showing: 1) multi-wall carbon nanotubes functionalized with oxygenated groups, 2) ferrocene methanol (FcMe), 3) HRP enzyme, 4) LOD enzyme, 5) biopolymer, 6) lactate, 7) counter electrode, 8) reference electrode and 9) working electrode.
  • the invention describes a new process for the preparation of an amperometric biosensor for the determination of portable and disposable lactate using screen-printed electrochemical graphite platforms, as well as the biosensor obtained by said method.
  • the biosensor described can be used in various applications for the determination of lactate / lactic acid in different media, including embryo culture media and other media used in cell development.
  • the procedure for the preparation of the amperometric lactate biosensor consists of the following stages: 1. Dispersion of the biopolymer in organic solvent.
  • a mediator and carbon nanotubes are added to the previous dispersion to give rise to the mediator solution.
  • MWCNT multi-wall carbon nanotubes functionalized with oxygenated groups
  • This dispersion was subjected to an ultrasonic field (ultrasonic cleaning bath) for 1 hour, which is called mediator dispersion B.
  • mediator dispersion B The final result is a dispersion with a black pasty appearance, due to the MWCNTs.
  • the preparation of solution B is modified as follows: it contains 2.5 mg of MWCNTs, 12.5 mg of FcMe and 5.25 mg of biopolymer dispersed in 500 ⁇ _ of an organic solution 1 : 3 (v: v) DMF-Ethanol. The mixture was dispersed by using an ultrasonic bath for one hour. 3. Preparation of the enzyme solution by dissolving at least the LOD enzyme in a buffer solution at neutral pH.
  • an enzymatic solution consisting of 5 mg / mL of the LOD enzyme, 0.5 Units of activity per biosensor, is prepared, which is stored at -20 ° C, previously dissolved in 0.1 M phosphate buffer; and 19 mg / mL of the enzyme horseradish peroxidase (HRP) in 0.1 M of a sodium phosphate buffer at pH 7.5 (solution C).
  • HRP horseradish peroxidase
  • the film formed on the electrode surface is protected with a plastic Petri dish and allowed to dry at a temperature between 2-5 ° C, or at 25 ° C under high vacuum conditions for 1 hour and then stored in air in a refrigerator between 2-5 ° C.
  • the biosensor obtained by this procedure is formed by a screen-printed graphite substrate supported on a flexible insulating polymer (screen-printed platform SPGE), a conductive carbonaceous matrix formed by the combination of an enzymatic system that contains at least the LOD enzyme, functionalized carbon nanotubes and the biopolymer for immobilization of the enzyme.
  • a scheme of the biosensor obtained in Figure 1 can be observed. It shows the conductive carbonaceous matrix with the multi-wall carbon nanotubes functionalized with oxygenated groups (1), the FcMe mediator (2), the bienzimatic system formed by the HRP (3) and the LOD (4) and the biopolymer (5).
  • the carbonaceous matrix is deposited on the screen-printed platform that contains the counter electrode (7), the reference electrode (8) and the working electrode (9).
  • the sample with lactate (6) to be determined is deposited on this biosensor.
  • a disposable screen-printed electrochemical platform is used to manufacture the biosensor.
  • These platforms consist of a work electrode of graphite of basal character with a diameter of 3.1 mm, a counter electrode of graphite and a pseudo-reference formed by a paste of silver / silver chloride, all electrodes supported on a polystyrene base.
  • the manufacturing of the platforms is carried out by screen printing a carbon ink on a flexible sheet of polystyrene to define the contacts, the counter electrode and the working electrode. This sheet is dried in an oven at 60 degrees for 30 minutes.
  • the reference electrode is then incorporated by screen printing an Ag / AgCI paste on the plastic substrate and dried at 60 degrees for 30 minutes. It is subsequently printed with a dielectric paste to cover the connections and define the graffiti work electrode.
  • the amperometric lactate biosensor is ready for use.
  • a preconditioning of the amperometric biosensor is performed by immersing the biosensor in a 0.1 M sodium phosphate buffered solution at pH 7.5 for 5 minutes under magnetic stirring, for proper cleaning.
  • an electrochemical pretreatment of the biosensor is performed for 2 minutes, applying a potential of -0.2 V in 0.1 M sodium phosphate pH 7.5 to the biosensor.
  • the biosensor is ready to perform the measurement.
  • the measurement can be carried out by two conventional electrochemical techniques. The first of these is cyclic voltammetry.
  • a drop of the standard solution or problem to be measured is placed in order to cover the three electrodes present in the screen-printed platform and then apply a linear sweep of potential versus time from -0.1 V to -0.4 V and the current intensity value is taken at a potential of -0.2 V.
  • the measurement is carried out at different lactate concentrations.
  • the other electrochemical technique is chronoamperometry.
  • the biosensor is immersed in a vessel containing 7 mL of 0.1 M sodium phosphate buffer pH 7.5, with magnetic stirring, a constant potential of -0.2 V is applied and a known volume of a lactate-containing solution is added which It is desired to determine.
  • 25 ⁇ of a 10 mM sodium lactate solution is added during chronoamperometry to subsequently relate the current intensity values obtained with the different lactate concentrations.
  • EXAMPLE 1 Preparation of an amperometric lactate biosensor based on a hybrid MWCNT / LOD / chitosan biopolymer system.
  • a series of amperometric lactate biosensors were prepared by the procedure presented: first a solution containing 84 mg of chitosan in 1 ml_ of dimethylformamide was prepared and an ultrasound field was applied for 30 minutes (solution A). From the previous solution (solution A) 50 ⁇ _ were taken on which 5 mg of FcMe and 1 mg of MWCNT were added. This new solution was maintained for one hour in an ultrasonic bath (solution B).
  • the bienzyme solution was prepared in an eppendorf tube containing 20 ⁇ _ of a 2-unit enzyme solution of the enzyme lactate oxidase LOD in 0.1 M sodium phosphate buffer pH 7.5, previously stored at -20 ° C, and 0.5 mg was incorporated of the enzyme HRP peroxidase (solution C).
  • solution B mediating solution
  • enzyme solution solution C
  • the amperometric lactate biosensor was immersed in a 0.1 M solution of sodium phosphate buffer with a solution pH of 7.5 , under continuous stirring for 5 minutes for proper cleaning. Subsequently, a pretreatment was performed.
  • the calibration lines were carried out by taking the subtracted value of the current intensity at -0.2 V obtained for each of the lactate concentrations added on the 0.1 M sodium phosphate buffer solution at a pH of 7.5, at current intensity at the same potential in the absence of sodium lactate in the solution versus the concentration of sodium lactate in the solution.
  • the calibration lines showed correlation values between 0.995 and 0.999 in a concentration range between 0.01 and 0.20 mM lactate.
  • electrochemical platforms of the array or microarray type can also be useful as an amperometric lactate biosensor, since it allows its use in the simultaneous determination of several biological samples and gives the analytical system versatility and speed.
  • manufacture of the amperometric lactate biosensor can be carried out using screen-printed electrochemical platforms such as microelectrode, micro-band or micro-band in series to determine the concentration of lactate in embryo culture media whose volumes range between 25 and 50 microliters allowing determinations with very little sample.
  • EXAMPLE 2 Use of the amperometric lactate biosensor in embryo culture media.
  • lactate For the determination of lactate by chromatographic method, an HPLC high pressure liquid chromatography device (Agilent 1 100 series, Santa Clara, USA) with a visible ultraviolet detector was used.
  • the mobile phase consisted of a 20 mM aqueous solution NaH 2 P0 4 adjusting the pH of the solution with H 3 P0 4 to pH 2.5.
  • the column used was a C18 hypersil and the flow rate was 0.5 mL min "1 with a wavelength of 210 nm.
  • a calibration line was made using sodium lactate standards prepared with the same mobile phase.
  • lactate for the sample used by the HPLC-UV-Vis chromatographic method was 11.9 ⁇ 0.10 mM.
  • the biosensor used in this case was the biosensor manufactured in accordance with the preferred embodiment of the described procedure, using a mixture of DMF and ethanol as solvent for the chitosan and drying the biosensor at 25 ° C under high vacuum conditions. Eight amperometric lactate biosensors were prepared and 4 of them were used for the realization of the calibration line and the other 4 remaining for the measurement of lactate in the test sample.
  • the lactate measurement in the culture medium problem sample was carried out as follows: first, the biosensor was cleaned in the manner indicated above, then the biosensor was introduced into a vial containing 5 mL of buffer 0.1 M sodium phosphate at pH 7.5 and lactate determination was carried out using the chronoamperometry technique, at a constant potential of - 0.2 V. After two minutes of pretreatment, 60 ⁇ of the G1 embryonic culture medium is added. The intensity value obtained was recorded and the concentration of lactate in the culture medium was calculated using the calibration line obtained above. For each lactate determination in the culture medium, 4 disposable biosensors were used.
  • the stability tests of the amperometric lactate biosensors were performed once prepared following the preferred procedure and after 8 days, 14 days, and 4 months, respectively.
  • the amperometric lactate biosensors used on day 1 of its preparation presented a calibration line with a linear regression of 0.99932, the slope being -0.0034 ⁇ - ⁇ "1 +/- 0.0002 ⁇ ⁇ " 1 .
  • EXAMPLE 3 Preparation of an amperometric lactate biosensor based on a hybrid MWCNT / LOD / biopolymer starch, chitin, hyaluronic acid, glycogen or cellulose system.
  • the test was performed with the biosensor manufactured in accordance with the preferred embodiment of the described procedure, using a mixture of DMF and ethanol 1: 3 v: v as a solvent for the FcMe mediator, the MWCNTs and the biopolymer.
  • the different biopolymers mentioned have been used: starch, chitin, hyaluronic acid, glycogen and cellulose, respectively.
  • the previous dispersion was sonic for 1 hour.
  • the enzyme solution containing the enzyme LOD and HRP was prepared. Once the corresponding mediating dispersion and the enzymatic solution were deposited, the drying process was carried out at 25 ° C under high vacuum conditions for 1 hour, and later Storage at 2-5 ° C in air. Calibration lines were made for each amperometric lactate biosensor corresponding to the MWCNT / LOD / starch, MWCNT / LOD / chitin hybrid systems,
  • MWC NT / LO D / hyaluronic acid MWCNT / LOD / glycogen
  • MWCNT / LOD / cellulose MWCNT / LOD / cellulose
  • the biosensor was cleaned in the manner indicated above in the procedure and then, the biosensor was introduced into a vial containing 5 ml_ of 0.1 M sodium phosphate buffer at pH 7.5. Lactate determination was carried out using the chronoamperometry technique, at a constant potential of -0.2 V with respect to an Ag / AgCI reference electrode. After two minutes of pretreatment, 25 ⁇ _ of sodium lactate in sodium phosphate buffer at pH 7.5 were added. Current intensity values obtained with each aliquot addition of the lactate solution were recorded.
  • the calibration lines obtained with the amperometric lactate biosensors manufactured with the different biopolymers presented the following linearity ranges and linear regression coefficients:
  • Table 1 Linearity obtained with different amperometric lactate biosensors obtained by the procedure described and using different biopolymers.

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Abstract

The invention relates to a method for producing an amperometric biosensor for the determination of lactate based on the lactate oxidase (LOD) enzyme, comprising the following steps: dispersion of a biopolymer in an organic solvent; preparation of a mediating solution by adding a mediator and carbon nanotubes; preparation of an enzymatic solution with at least the LOD enzyme; addition of the mediating and enzymatic solution to a disposable screen-printed platform; and drying of the biosensor. The invention also relates to the biosensor produced by means of said method, where the biopolymer is based on amylose and glucose polysaccharides with functional hydroxyl, amine, acetyl or carboxylic groups and has glycosidic bonds. The invention also relates to the use of same in complex media such as embryonic cell media, food media or blood samples.

Description

PROCEDIMIENTO DE PREPARACIÓN DE BIOSENSOR AM PEROM ÉTRICO DE LACTATO, BIOSENSOR OBTENIDO MEDIANTE ESE PROCEDIMIENTO Y SU USO EN MEDIOS PROCESSING OF PREPARATION OF BIOSTRACTOR AM PEROM ETRIC OF LACTATE, BIOSENSOR OBTAINED THROUGH THAT PROCEDURE AND ITS USE IN MEDIA
COMPLEJOS COMPLEXES
DESCRIPCIÓN DESCRIPTION
Procedimiento de preparación de biosensor amperométrico de lactato, biosensor obtenido mediante ese procedimiento y su uso en medios complejos. Procedure for preparing amperometric lactate biosensor, biosensor obtained by this procedure and its use in complex media.
CAMPO DE LA INVENCIÓN FIELD OF THE INVENTION
La presente invención se enmarca en el campo de los sensores amperométricos de uso en reproducción asistida, cuya finalidad más concretamente se centra en la detección y determinación de lactato/ácido láctico en medios complejos como los medios de cultivos celulares, empleados en el desarrollo y crecimiento de células de embnones humanos. The present invention is framed in the field of amperometric sensors for use in assisted reproduction, whose purpose more specifically focuses on the detection and determination of lactate / lactic acid in complex media such as cell culture media, used in development and growth of human embryonic cells.
ESTADO DE LA TECNICA ANTERIOR STATE OF THE PREVIOUS TECHNIQUE
La mayoría de las investigaciones para el desarrollo de biosensores electroquímicos de lactato se centran en mejorar: Most research for the development of electrochemical lactate biosensors is focused on improving:
(i) la inmovilización de la enzima empleada sobre el material del electrodo,  (i) immobilization of the enzyme used on the electrode material,
(¡i) la mejora de la transferencia electrónica,  (¡) The improvement of electronic transfer,
(iii) el tipo de mediadores y coenzimas,  (iii) the type of mediators and coenzymes,
(iv) la mejora de la estabilidad del sensor, y  (iv) improved sensor stability, and
(v) la miniaturización del biosensor. Teniendo en cuenta estas premisas, durante las últimas tres décadas han ¡do creciendo las investigaciones sobre el desarrollo de biosensores de lactato debido a su importancia en distintas áreas de aplicación. En la mayoría de estudios que se pueden encontrar en la actualidad, se opta por la fabricación de biosensores amperométricos basados en dos tipos de enzimas: la lactato oxidasa y la lactato deshidrogenasa. Esta última requiere de la presencia del cofactor nicotinamida adenina dinucleótido oxidado (NAD+) que es reducido a NADH, lo que conlleva la fabricación de un biosensor sofisticado, caro y con un tiempo de vida limitado [L. Agüí, M. Eguílaz, C. Peña-Farfal, P. Yáñez-Sedeño, J.M. Pingarrón, Electroanalysis 21 (2009) 386; A.C. Pereira, M.R. Aguiar, A. Kisner, D.V. Macedo, L.T. Kubota, Sensors and Actuators, B: Chemical 124 (2007) 269]. Por el contrario la enzima lactato oxidasa contiene el cofactor Flavín adenín dinucleótido (FAD) en su estructura, que cataliza la conversión de lactato a piruvato en presencia de O2 generándose al mismo tiempo H2O2 como producto [V. P. Zanini, B. López De Mishima, V. Solís, Sensors and Actuators, B: Chemical 155 (201 1 ) 75]. Por tanto y, debido fundamentalmente a que el cofactor NADH se encuentra presente en medios de cultivo embrionario, lo que interferiría en la medida si se utilizara un biosensor basado en la lactato deshidrogenasa, es necesario desarrollar biosensores electroquímicos de lactato en los cuales sea empleada como enzima la lactato oxidasa. (v) biosensor miniaturization. Taking these premises into account, during the last three decades, research on the development of lactate biosensors has been growing due to their importance in different areas of application. In the majority of studies that can be found today, the manufacture of amperometric biosensors based on two types of enzymes is chosen: lactate oxidase and lactate dehydrogenase. The latter requires the presence of the cofactor nicotinamide adenine dinucleotide oxidized (NAD + ) which is reduced to NADH, which entails the manufacture of a sophisticated, expensive biosensor with a limited lifetime [L. Agüí, M. Eguílaz, C. Peña-Farfal, P. Yáñez-Sedeño, JM Pingarrón, Electroanalysis 21 (2009) 386; AC Pereira, MR Aguiar, A. Kisner, DV Macedo, LT Kubota, Sensors and Actuators, B: Chemical 124 (2007) 269]. By contrast, the enzyme lactate oxidase contains the cofactor Flavin adenine dinucleotide (FAD) in its structure, which catalyzes the conversion of lactate to pyruvate in the presence of O2 while generating H2O2 as a product [VP Zanini, B. López De Mishima, V Solís, Sensors and Actuators, B: Chemical 155 (201 1) 75]. Therefore and, mainly because the NADH cofactor is present in embryonic culture media, which would interfere in the measure if a biosensor based on lactate dehydrogenase is used, it is necessary to develop electrochemical lactate biosensors in which it is used as Lactate oxidase enzyme.
Los biosensores amperométricos de lactato, que emplean lactato oxidasa como biocatalizador para la reacción de oxidación de lactato a piruvato generando peróxido de hidrógeno, funcionan cuando se les somete a un potencial entre +0.5 y +0.65 V frente al electrodo de referencia AgCI/Ag (cloruro 3 M). Este es el rango de potenciales necesario para oxidar el H2O2 sobre la superficie del electrodo y, por consiguiente, establecer una respuesta cuantificable que relaciona la intensidad de corriente obtenida con la cantidad de lactato presente en disolución de estudio [L. Rassaei, W. Olthuis, S. Tsujimura, E.J. R. Sudhólter, A. Van Den Berg, Analytical and Bioanalytical Chemistry 406 (2013) 123]. El problema de estos biosensores amperométricos de lactato es el elevado potencial al cual se debe realizar la medida cuando el medio en el que se encuentra el lactato puede contener a otras sustancias que también pueden contribuir a la lectura de la corriente registrable a estos potenciales (son los denominados interferentes). Para evitar esta circunstancia, debemos encontrar un rango de potenciales dónde dichas sustancias no contribuyan a la lectura de corriente que sólo debe ser debida a la presencia del lactato. Por tanto, es necesario el desarrollo de biosensores amperométricos de lactato que puedan operar a potenciales de trabajo del electrodo bajos y, consecuentemente, se puedan evitar interferencias debidas a esas especies electroactivas que pueden oxidarse a potenciales más elevados. Amperometric lactate biosensors, which use lactate oxidase as a biocatalyst for the oxidation reaction of lactate to pyruvate generating hydrogen peroxide, work when subjected to a potential between +0.5 and +0.65 V against the AgCI / Ag reference electrode ( 3M chloride). This is the range of potentials required to oxidize H2O2 on the electrode surface and, therefore, establish a quantifiable response that relates the intensity of current obtained with the amount of lactate present in the study solution [L. Rassaei, W. Olthuis, S. Tsujimura, EJR Sudholter, A. Van Den Berg, Analytical and Bioanalytical Chemistry 406 (2013) 123]. The problem of these amperometric lactate biosensors is the high potential to which the measurement should be taken when the medium in which the lactate is found can contain other substances that can also contribute to the reading of the recordable current at these potentials (they are the so-called interferents). To avoid this circumstance, we must find a range of potentials where these substances do not contribute to the current reading that should only be due to the presence of lactate. Therefore, it is necessary to develop amperometric lactate biosensors that can operate at low electrode working potentials and, consequently, interferences due to those electroactive species that can oxidize to higher potentials can be avoided.
La estrategia elegida hasta el momento en la bibliografía relacionada es la búsqueda de mediadores que favorezcan la disminución del potencial de oxidación del H2O2 producido por la especie enzima lactato oxidasa. De esta forma el proceso de oxidación no se lleva a cabo directamente sobre la superficie del sensor sino a través de un mediador anclado a ésta y ello se produce a un potencial sensiblemente inferior al descrito habitualmente [Costa Eric D, Electrochemical biosensor stability. Patent Number: EP546019-A; WO9204466-A1 ; AU9184377-A]. En la literatura se pueden encontrar mediadores redox de distinta naturaleza, entre los más utilizados se encuentran la ftalocianina de cobalto [F.J. Rawson, W.M. Purcell, J. Xu, R.M. Pemberton, P. R. Fielden, N. Biddle, J. P. Hart, Talanta 77 (2009) 1 149], el ferroceno [S. Pérez, E. Fábregas, Analyst 137 (2012) 3854] o derivados de éste como el ferroceno metanol (FcMe) [V. P. Zanini, B. López De Mishima, V. Solís, Sensors and Actuators, B: Chemical 155 (201 1 ) 75, X. L¡, J. Zang, Y. Liu, Z. Lu, Q. L¡, C.M. L¡, Analytica Chimica Acta 771 (2013) 102]. A potenciales de +0.19 V el ferroceno metanol se oxida a ferrocinio (1 ), el ferrocinio oxida posteriormente al H2O2 (2) formado tras la oxidación del lactato por la enzima lactato oxidasa (en adelante LOD), finalmente detectándose la posterior señal electroquímica a un potencial formal de +0.19 V frente a un electrodo de referencia AgCI/Ag (3 M KCI) (3). Como se puede observar, el potencial se reduce notablemente de +0.65 V a +0.19 V, pudiéndose evitar la interferencia de otras sustancias electroactivas presentes en el medio de estudio. The strategy chosen so far in the related literature is the search for mediators that favor the reduction of the oxidation potential of H2O2 produced by the enzyme species lactate oxidase. In this way the oxidation process is not carried out directly on the sensor surface but through a mediator anchored to it and this occurs at a potential significantly lower than the one normally described [Costa Eric D, Electrochemical biosensor stability. Patent Number: EP546019-A; WO9204466-A1; AU9184377-A]. Redox mediators of different nature can be found in the literature, among the most commonly used are cobalt phthalocyanine [FJ Rawson, WM Purcell, J. Xu, RM Pemberton, PR Fielden, N. Biddle, JP Hart, Talanta 77 (2009 ) 1 149], the ferrocene [S. Pérez, E. Fábregas, Analyst 137 (2012) 3854] or derivatives thereof such as ferrocene methanol (FcMe) [VP Zanini, B. López De Mishima, V. Solís, Sensors and Actuators, B: Chemical 155 (201 1) 75, X. L¡, J. Zang, Y. Liu, Z. Lu, Q. L¡, CM L¡, Analytica Chimica Acta 771 (2013) 102]. At potentials of +0.19 V the ferrocene methanol is oxidizes to ferrocinium (1), the ferrocinium subsequently oxidizes the H2O2 (2) formed after the oxidation of lactate by the enzyme lactate oxidase (hereinafter LOD), finally detecting the subsequent electrochemical signal at a formal potential of +0.19 V against a reference electrode AgCI / Ag (3 M KCI) (3). As can be seen, the potential is significantly reduced from +0.65 V to +0.19 V, being able to avoid the interference of other electroactive substances present in the study medium.
FcMe→ FcMe+ + e~ ( 1 ) H202 + 2FcMe+ <→ 02 + 2H+ + 2FcMe ( 2 ) FcMe → FcMe + + e ~ (1) H 2 0 2 + 2FcMe + <→ 0 2 + 2H + + 2FcMe (2)
H202 + FcMe+ <→ 02 + 2H+ + FcMe + e~ ( 3 ) H 2 0 2 + FcMe + <→ 0 2 + 2H + + FcMe + e ~ (3)
El grupo de Fábregas y Pérez [S. Pérez, E. Fábregas, Analyst 137 (2012) 3854] inmovilizan simultáneamente la enzima lactato oxidasa junto con la enzima peroxidasa de rábano silvestre (HRP). En este sistema bienzimático la oxidación de lactato está catalizada por la enzima lactato oxidasa dando como subproducto peróxido de hidrógeno que posteriormente se reduce a H2O por la acción catalítica de la enzima HRP en su forma reducida, dando lugar a la enzima HRP en su forma oxidada. Finalmente, la enzima HRP en su forma oxidada se reduce por el FcMe dando lugar a la especie ferrocinio metanol (FcMe+), que posteriormente se reduce electroquímicamente a un potencial de -200 mV frente a un electrodo pseudoreferencia de pasta de plata / cloruro de plata. The group of Fábregas y Pérez [S. Pérez, E. Fábregas, Analyst 137 (2012) 3854] simultaneously immobilize the lactate oxidase enzyme together with the enzyme horseradish peroxidase (HRP). In this bienzimatic system, the oxidation of lactate is catalyzed by the enzyme lactate oxidase giving hydrogen peroxide as a by-product that is subsequently reduced to H 2 O by the catalytic action of the HRP enzyme in its reduced form, giving rise to the enzyme HRP in its oxidized form Finally, the enzyme HRP in its oxidized form is reduced by the FcMe giving rise to the species ferrocinio methanol (FcMe + ), which is then electrochemically reduced to a potential of -200 mV compared to a silver paste / chloride pseudo-reference electrode silver.
Por otra parte y para que estos sistemas puedan funcionar correctamente, es necesario inmovilizar la enzima LOD sobre la superficie del biosensor. Entre los distintos procedimientos referenciados algunos autores apuestan por el empleo de matrices de sol-gel [J. Huang, J. L¡, Y. Yang, X. Wang, B. Wu, J.i. Anzai, T. Osa, Q. Chen, Materials Science and Engineering C 28 (2008) 1070; J. Weber, A. Kumar, S. Bhansali, Sensors and Actuators, B: Chemical 1 17 (2006) 308] por ser inertes químicamente, por la posibilidad de modificar la porosidad y por permitir trabajar a bajas temperaturas. Un ejemplo de lo anterior es el biosensor fabricado por Huang y colaboradores, quienes emplearon una matriz de sílice sol-gel mediante la mezcla de Tetraetil ortosilicato (TEOS), H20 y HCI que adicionaron sobre un electrodo de carbón vitreo modificado con nanotubos de carbón de pared múltiple (MWCNT) y nanopartículas de platino, obteniendo un biosensor para la medida de muestras de sangre con una sensibilidad de 6.36 μΑ/mM, un rango de linealidad de (0.2~2) x 10"3 M, pero con una estabilidad de 4 semanas [J. Huang, J. L¡, Y. Yang, X. Wang, B. Wu, J. i. Anzai, T. Osa, Q. Chen, Materials Science and Engineering C 28 (2008) 1070]. On the other hand and for these systems to work properly, it is necessary to immobilize the LOD enzyme on the surface of the biosensor. Among the different procedures referenced, some authors are committed to the use of sol-gel matrices [J. Huang, J. L¡, Y. Yang, X. Wang, B. Wu, Ji Anzai, T. Osa, Q. Chen, Materials Science and Engineering C 28 (2008) 1070; J. Weber, A. Kumar, S. Bhansali, Sensors and Actuators, B: Chemical 1 17 (2006) 308] for being chemically inert, for the possibility of modifying porosity and for allowing work at low temperatures An example of the above is the biosensor manufactured by Huang et al., Who used a sol-gel silica matrix by mixing Tetraethyl orthosilicate (TEOS), H 2 0 and HCI, which added on a vitreous carbon electrode modified with nanotubes of multiple wall carbon (MWCNT) and platinum nanoparticles, obtaining a biosensor for measuring blood samples with a sensitivity of 6.36 μΑ / mM, a linearity range of (0.2 ~ 2) x 10 "3 M, but with a 4-week stability [J. Huang, J. L¡, Y. Yang, X. Wang, B. Wu, J. i. Anzai, T. Osa, Q. Chen, Materials Science and Engineering C 28 (2008) 1070 ].
Otros autores han preferido como sustrato la combinación de una matriz de sílice y un biopolímero, de modo que la matriz de sílice aportaba el carácter hidrofílico, el biopolímero mejoraba la inmovilización de la proteína y, con ello, el biosensor amperométrico mejoraba la transferencia iónica, proporcionando estabilidad mecánica y buena adhesión. Un ejemplo sobre la estrategia anterior corresponde al biosensor realizado por Zanini y col. , quienes emplearon un hidrogel de laponita en presencia de quitosano sobre un sustrato masivo de carbón vitreo para la medida de lactato en alimentos presentando una sensibilidad de 0.326 ± 0.003 A cm" 2 M"1 y un límite de detección de (3.8 ± 0.2) x 10"3 M [V. P. Zanini, B. López De Mishima, V. Solís, Sensors and Actuators, B: Chemical 155 (201 1 ) 75; X. L¡, J. Zang, Y. Liu, Z. Lu, Q. L¡, CM. L¡, Analytica Chimica Acta 771 (2013) 102]. Sin embargo el biosensor fue probado únicamente para la determinación de lactato en bebidas alcohólicas y productos lácteos. No obstante, el empleo de un electrodo de carbón vitreo, conlleva el requerimiento de un montaje para la determinación más complejo de lo deseable. Un aspecto fundamental para la aplicabilidad de un sensor es su sensibilidad para la respuesta analítica buscada. Una forma de aumentar la sensibilidad de un biosensor consiste en el aumento de la carga de enzima. Esto se lleva a cabo normalmente aumentando el área superficial del electrodo. El área del electrodo se puede aumentar empleando nanomateriales como nanotubos de carbono, grafeno o nanopartículas metálicas. Algunos autores prefieren la combinación de nanomateriales con polímeros conductores. La combinación de ambos confiere al biosensor propiedades especiales debido al efecto sinérgico de los componentes individuales, como por ejemplo, la combinación de MWCNTs y polisulfona para la fabricación de un biosensor con la finalidad de determinar lactato en muestras de vino y cerveza [S. Pérez, E. Fábregas, Analyst 137 (2012) 3854] utilizando una plataforma serigrafiada de grafito. Los resultados mostraron unos límites de detección de 0.05 mg/L, un rango de linealidad entre 0.1 ~0.5 mg/L, pero con una estabilidad del 40 % tras 2 semanas, almacenándose el electrodo en una disolución tampón fosfato a pH neutro. Other authors have preferred as a substrate the combination of a silica matrix and a biopolymer, so that the silica matrix provided the hydrophilic character, the biopolymer improved the immobilization of the protein and, thus, the amperometric biosensor improved ionic transfer, providing mechanical stability and good adhesion. An example of the above strategy corresponds to the biosensor carried out by Zanini et al. , who used a laponite hydrogel in the presence of chitosan on a massive substrate of vitreous carbon for the measurement of lactate in food presenting a sensitivity of 0.326 ± 0.003 A cm " 2 M " 1 and a detection limit of (3.8 ± 0.2) x 10 "3 M [VP Zanini, B. López De Mishima, V. Solís, Sensors and Actuators, B: Chemical 155 (201 1) 75; X. L¡, J. Zang, Y. Liu, Z. Lu, Q. L¡, CM. L¡, Analytica Chimica Acta 771 (2013) 102] However, the biosensor was only tested for the determination of lactate in alcoholic beverages and dairy products, however, the use of a vitreous carbon electrode , entails the requirement of an assembly for the more complex determination than desirable.A fundamental aspect for the applicability of a sensor is its sensitivity to the analytical response sought. The sensitivity of a biosensor consists in increasing the enzyme load. This is usually done by increasing the surface area of the electrode. The electrode area can be increased using nanomaterials such as carbon nanotubes, graphene or metal nanoparticles. Some authors prefer the combination of nanomaterials with conductive polymers. The combination of both gives the biosensor special properties due to the synergistic effect of the individual components, such as the combination of MWCNTs and polysulfone for the manufacture of a biosensor in order to determine lactate in wine and beer samples [S. Pérez, E. Fábregas, Analyst 137 (2012) 3854] using a screen-printed graphite platform. The results showed detection limits of 0.05 mg / L, a linearity range between 0.1 ~ 0.5 mg / L, but with a stability of 40% after 2 weeks, the electrode being stored in a phosphate buffer solution at neutral pH.
Con la finalidad de conseguir una mayor biocompatibilidad junto con una buena sensibilidad del biosensor amperométrico de lactato, varios autores han empleado materiales híbridos formados por biopolímeros y nanotubos de carbono. Este es el caso del empleo del biopolímero quitosano junto con nanotubos de carbono, aplicado a la fabricación del biosensor amperométrico de lactato realizado por el grupo de Monosík [Monosík, R. ; Stred'ansky, M.; Greif, G. ; Sturdík, E. , Food Control 23, (2012) 238.], cuya preparación se lleva a cabo preparando una mezcla de MWCNT y n- icosane la cual se deposita sobre un electrodo de oro de 1 .6 mm de diámetro que incluía un electrodo de referencia de Ag/AgCI. Una vez seco, la proteína se inmovilizaba intercalando láminas de quitosano (1 % w/w, disolución acuosa en ácido acético). La disolución enzimática consistió en una disolución acuosa de LOD y HRP. El depósito de cada capa se realizaba justo después del secado la capa anterior. Las medidas se llevaron a cabo a un potencial de -50 mV. Este biosensor presentaba un rango de linealidad de 5-244 μΜ, un coeficiente de correlación de 0.994 y una estabilidad de almacenamiento a largo plazo del 90 % tras 15 meses. La aplicación del biosensor se centró en el análisis de lactato en muestras de alimentos sólidos y vino. Sin embargo, la preparación del biosensor resulta todavía demasiado compleja para la potencial comercialización del biosensor. In order to achieve greater biocompatibility together with a good sensitivity of the amperometric lactate biosensor, several authors have used hybrid materials formed by biopolymers and carbon nanotubes. This is the case of the use of the chitosan biopolymer together with carbon nanotubes, applied to the manufacture of the amperometric lactate biosensor carried out by the Monosík group [Monosík, R.; Stred'ansky, M .; Greif, G.; Sturdík, E., Food Control 23, (2012) 238.], whose preparation is carried out by preparing a mixture of MWCNT and nicosane which is deposited on a gold electrode of 1 .6 mm in diameter that included a Ag / AgCI reference electrode. Once dry, the protein was immobilized by interleaving chitosan sheets (1% w / w, aqueous solution in acetic acid). The enzymatic solution consisted of an aqueous solution of LOD and HRP. The deposit of each layer was made just after drying the previous layer. The measurements were carried out at a potential of -50 mV. This biosensor presented a linearity range of 5-244 μΜ, a correlation coefficient of 0.994 and a long-term storage stability of 90% after 15 months. The biosensor application focused on the analysis of lactate in solid food and wine samples. However, the biosensor preparation is still too complex for the potential commercialization of the biosensor.
Zhenyu Zhou y colaboradores [Zhou, Z. ; Xu, L ; Wu, S. ; Su, B. Analyst 139 (2014), 4934] también fabricaron un biosensor empleando quitosano y MWCNT para la fabricación de un biosensor electroquimioluminiscente para la determinación de glucosa, lactato y colina. Para su fabricación disolvieron quitosano en ácido acético (1 % w/w) ajusfando el pH a 5 con NaOH y se dejó sonicando durante 40 minutos. Por otro lado, 4 mg de MWCNTs se dispersaron en 2 ml_ de la disolución anterior, y a continuación 200 μΙ_ de la dispersión resultante se mezclan con 100 μΙ_ de LOD. Finalmente, 20 μΙ_ de la mezcla se depositan sobre un sustrato de óxido de indio y estaño (ITO) (área 1 .2 cm x 2.5 cm) y se extendieron sobre la superficie y se dejó secar. El biosensor presentó un límite de detección para lactato de 97 μΜ. Desafortunadamente no mostraron datos sobre la estabilidad del biosensor amperométrico de lactato y su aplicación en muestras reales. Zhenyu Zhou et al [Zhou, Z.; Xu, L; Wu, S.; Su, B. Analyst 139 (2014), 4934] also manufactured a biosensor using chitosan and MWCNT for the manufacture of an electrochemiluminescent biosensor for the determination of glucose, lactate and choline. For its manufacture, they dissolved chitosan in acetic acid (1% w / w) by adjusting the pH to 5 with NaOH and left sonicating for 40 minutes. On the other hand, 4 mg of MWCNTs were dispersed in 2 ml_ of the previous solution, and then 200 μΙ_ of the resulting dispersion was mixed with 100 μΙ_ of LOD. Finally, 20 μΙ_ of the mixture is deposited on a substrate of indium tin oxide (ITO) (area 1.2 cm x 2.5 cm) and spread on the surface and allowed to dry. The biosensor presented a detection limit for lactate of 97 μΜ. Unfortunately, they did not show data on the stability of the amperometric lactate biosensor and its application in real samples.
Por otro lado, el grupo de Cui [X. Cui, CM. L¡, J. Zang, S. Yu, Biosensors and Bioelectronics 22 (2007) 3288] también prepararon una disolución de quitosano (1 % w/w) en HCI ajusfando el pH a 5 con NaOH, adicionando posteriormente: nanotubos de carbono CNTs (hasta una concentración 5 μg ml_"1); LOD (hasta una concentración 10 mg- mL"1); y un complejo de osmio-con polivinilimidazolio-Os PVI-Os (hasta una concentración 4 mg mL"1). Sin embargo, el sustrato metálico utilizado era oro modificado con una capa autoensamblada de ácido mercaptopropiónico MPA. Una vez seco el sustrato metálico se depositan 3 μί de la disolución quitosano/CNTs/LOD/PVI-Os sobre la superficie del electrodo de oro modificado con MPA y se deja secar en una incubadora a 37 °C durante 3 horas. Una vez preparado se lavaba bajo agitación con una disolución tampón de fosfato a pH neutro durante 2 horas y finalmente se almacena al aire a 4 °C. El biosensor presentó un límite de detección de 5 μΜ, con un buen rango de linealidad hasta 1 mM, pero de nuevo los autores no presentaron datos sobre la estabilidad del biosensor amperométrico de lactato ni sus aplicaciones en muestras reales. El quitosano es un polisacárido lineal compuesto de cadenas distribuidas aleatoriamente de β-D-glucosamina y N-acetil-D-glucosamina. Sin embargo, hasta la fecha no existe la descripción de ningún biosensor para la detección de lactato que emplee conjuntamente la enzima LOD y MWCNTs junto con otros polisacáridos o biopolímeros distintos al quitosano. On the other hand, the Cui group [X. Cui, CM. L., J. Zang, S. Yu, Biosensors and Bioelectronics 22 (2007) 3288] also prepared a solution of chitosan (1% w / w) in HCI by adjusting the pH to 5 with NaOH, subsequently adding: CNTs carbon nanotubes (up to a concentration 5 μg ml_ "1 ); LOD (up to a concentration 10 mg-mL " 1 ); and an osmium-with polyvinylimidazolium-Os complex PVI-Os (up to a concentration of 4 mg mL "1 ). However, the metal substrate used was gold modified with a self-assembled layer of MPA mercaptopropionic acid. Once the metal substrate dried deposit 3 μί of the solution Chitosan / CNTs / LOD / PVI-Os on the surface of the gold electrode modified with MPA and allowed to dry in an incubator at 37 ° C for 3 hours. Once prepared, it was washed under stirring with a phosphate buffer solution at neutral pH for 2 hours and finally stored in air at 4 ° C. The biosensor presented a detection limit of 5 μΜ, with a good linearity range up to 1 mM, but again the authors did not present data on the stability of the amperometric lactate biosensor or its applications in real samples. Chitosan is a linear polysaccharide composed of randomly distributed chains of β-D-glucosamine and N-acetyl-D-glucosamine. However, to date there is no description of any biosensor for the detection of lactate that jointly uses the enzyme LOD and MWCNTs together with other polysaccharides or biopolymers other than chitosan.
El éxito de la introducción de los biosensores amperométricos de lactato en el mercado debe fundamentarse en su simplicidad, respuesta rápida, sensibilidad, bajo coste y portabilidad. Para conseguir algunos de estos aspectos es esencial la miniaturización de los biosensores amperométricos. En los últimos años se ha ¡do incrementando el uso de plataformas serigrafiadas para la fabricación de biosensores de lactado. A día de hoy no existe en el estado del arte autores que empleen la combinación de los biopolímeros, junto con los nanotubos de carbono (CNT) exclusivamente para la fabricación de biosensores amperométricos de lactato que empleen lactato oxidasa y que además necesariamente empleen plataformas serigrafiadas. The success of the introduction of amperometric lactate biosensors in the market must be based on their simplicity, rapid response, sensitivity, low cost and portability. To achieve some of these aspects, miniaturization of amperometric biosensors is essential. In recent years, the use of screen-printed platforms for the manufacture of lacto biosensors has been increasing. To date, there are no authors in the state of the art who use the combination of biopolymers, together with carbon nanotubes (CNT) exclusively for the manufacture of amperometric lactate biosensors that use lactate oxidase and also necessarily use screen-printed platforms.
Uno de los campos donde más impacto ha supuesto el empleo de plataformas serigrafiadas corresponde al análisis clínico, como por ejemplo en el análisis de muestras sanguíneas. Un ejemplo es el biosensor amperométrico fabricado por Shimomura y col [T. Shimomura, T. Sumiya, M. Ono, T. Ito, T.A. Hanaoka, Analytica Chimica Acta 714 (2012) 1 14], que consistía en una plataforma serigrafiada carbonosa modificada con ftalocianina de cobalto (II) (CoPC), que resultaba ser un buen electrocatalizador para la oxidación de H2O2. En este caso es reseñable la adición de una capa de Nafion con la finalidad de proteger al biosensor de posibles interferentes. Por otro lado, Rawson y col. [F.J. Rawson, W.M. Purcell, J. Xu, R.M. Pemberton, P.R. Fielden, N. Biddle, J.P. Hart, Talanta 77 (2009) 1 149] emplearon plataformas serigrafiadas de una tinta de carbono que contenían CoPC como electrocatalizador, empleando el biosensor amperométrico como plataforma para monitorizar el metabolismo celular in vitro. Por tanto, se encuentran descritos biosensores inmovilizados en plataformas serigrafiadas para el análisis de lactato en medios complejos, lo que implica, necesariamente, el uso de capas externas para proteger el biosensor y evitar los interferentes. One of the fields where the use of screen-printed platforms has had the greatest impact corresponds to clinical analysis, such as in the analysis of blood samples. An example is the amperometric biosensor manufactured by Shimomura et al [T. Shimomura, T. Sumiya, M. Ono, T. Ito, TA Hanaoka, Analytica Chimica Acta 714 (2012) 1 14], which consisted of a carbonated screenprint platform modified with cobalt (II) phthalocyanine (CoPC), which turned out to be a good electrocatalyst for the oxidation of H2O2. In this case, it is worth mentioning the addition of a layer of Nafion in order to protect the biosensor from possible interference. On the other hand, Rawson et al. [FJ Rawson, WM Purcell, J. Xu, RM Pemberton, PR Fielden, N. Biddle, JP Hart, Talanta 77 (2009) 1 149] used screenprinted carbon ink platforms containing CoPC as electrocatalyst, using the amperometric biosensor as a platform to monitor cell metabolism in vitro. Therefore, biosensors immobilized on screen-printed platforms for the analysis of lactate in complex media are described, which necessarily implies the use of external layers to protect the biosensor and avoid interference.
Cabe destacar que no existen descritos en la literatura biosensores amperométricos aplicados en la determinación de lactato en medios de cultivo embrionario, como son los utilizados en reproducción asistida, que utilicen plataformas serigrafiadas desechables para realizar las medidas en tiempo real o in situ. Los cambios metabolómicos en medios de cultivo celular después de retirar el embrión humano -o incluso durante su desarrollo- en términos de consumo o formación de nuevos metabolitos, puede reflejar la calidad y, por lo tanto, la viabilidad del embrión para su transferencia al útero materno. El lactato es un componente vital consumido durante los primeros días del desarrollo embrionario, por lo que se considera un metabolito significativamente diferente de un desarrollo celular adecuado. Por lo tanto la monitorización de lactato en tiempo real e in situ permite al embriólogo tener un dato adicional para la selección y valoración del embrión humano. Así pues, según todo lo anterior, los mayores inconvenientes de los biosensores amperométricos de lactato en la actualidad son: It should be noted that there are no described in the literature amperometric biosensors applied in the determination of lactate in embryo culture media, such as those used in assisted reproduction, that use disposable screen-printed platforms to perform measurements in real time or in situ. Metabolomic changes in cell culture media after removing the human embryo - or even during its development - in terms of consumption or formation of new metabolites, may reflect the quality and, therefore, the viability of the embryo for transfer to the uterus maternal. Lactate is a vital component consumed during the first days of embryonic development, so it is considered a significantly different metabolite from adequate cell development. Therefore, real-time and on-site lactate monitoring allows the embryologist to have additional data for the selection and evaluation of the human embryo. Thus, according to all of the above, the major drawbacks of amperometric lactate biosensors today are:
(I) la baja estabilidad que generalmente presentan, bien por el deterioro del electrodo empleado o por la poca estabilidad enzimática,  (I) the low stability that they generally present, either due to the deterioration of the electrode used or due to the low enzyme stability,
(II) la necesidad de emplear un montaje tedioso para la realización de la medida,  (II) the need to use a tedious assembly to carry out the measurement,
(III) la baja reproducibilidad de los biosensores amperométricos de lactato, y  (III) the low reproducibility of amperometric lactate biosensors, and
(IV) que no existe hasta la fecha ningún dispositivo electroquímico desechable para la determinación y cuantificación de lactato en medios complejos como los medios de cultivo embrionario.  (IV) that to date there is no disposable electrochemical device for the determination and quantification of lactate in complex media such as embryo culture media.
EXPLICACIÓN DE LA INVENCIÓN EXPLANATION OF THE INVENTION
Se hace necesario a la luz de lo anteriormente expuesto, buscar un procedimiento de preparación de biosensores de lactato que permitan la detección y cuantificación de lactato en cualquier medio de cultivo, especialmente en medios complejos como los medios de cultivo embrionario, que sean estables, reproducibles, de fácil manejo, con una rápida respuesta y que sean económicos. It is necessary in the light of the above, to look for a procedure for the preparation of lactate biosensors that allow the detection and quantification of lactate in any culture medium, especially in complex media such as embryonic culture media, which are stable, reproducible , easy to use, with a quick response and that are economical.
El procedimiento descrito en la presente invención permite fabricar biosensores capaces de superar los inconvenientes que presentan los biosensores amperométricos actuales, gracias a los materiales empleados y al método de preparación. The process described in the present invention makes it possible to manufacture biosensors capable of overcoming the drawbacks of current amperometric biosensors, thanks to the materials used and the method of preparation.
Un objeto de la presente invención se refiere, pues, al procedimiento de preparación de un biosensor amperométrico para la determinación de lactato que comprende las siguientes etapas: 1 . Dispersión de un biopolímero en disolvente orgánico. An object of the present invention thus relates to the process of preparing an amperometric biosensor for the determination of lactate comprising the following steps: one . Dispersion of a biopolymer in organic solvent.
2. A la dispersión anterior se adicionan un mediador y nanotubos de carbono, para dar lugar a la disolución mediadora.  2. A mediator and carbon nanotubes are added to the previous dispersion to give rise to the mediator solution.
3. Preparación de la disolución enzimática disolviendo menos la enzima LOD en una disolución tampón a neutro.  3. Preparation of the enzyme solution by dissolving less the LOD enzyme in a neutral buffer solution.
4. Adición de la disolución mediadora obtenida en la etapa 2 sobre una plataforma serigrafiada.  4. Addition of the mediating solution obtained in stage 2 on a screen-printed platform.
5. Adición de la disolución enzimática obtenida en la etapa 3 sobre la plataforma serigrafiada y la disolución mediadora. 5. Addition of the enzymatic solution obtained in step 3 on the screen-printed platform and the mediating solution.
6. Secado del biosensor para la eliminación del disolvente. En una realización preferente, el biopolímero utilizado está basado en polisacáridos de glucosa o amilosa, funcionalizados con grupos, amino, acetilo o carboxílico, que contienen enlaces glucosídicos. Estos materiales son polímeros biocompatibles del tipo quitosano, almidón, quitina, ácido hialurónico, glucógeno y celulosa, que presentan enlaces glicosídicos de glucosa, amilosa o N-acetil-D-glucosamina. 6. Drying the biosensor for solvent removal. In a preferred embodiment, the biopolymer used is based on glucose or amylose polysaccharides, functionalized with groups, amino, acetyl or carboxylic, which contain glycosidic bonds. These materials are biocompatible polymers of the type chitosan, starch, chitin, hyaluronic acid, glycogen and cellulose, which have glycosidic bonds of glucose, amylose or N-acetyl-D-glucosamine.
En otra realización preferente, el disolvente orgánico utilizado para la dispersión del biopolímero es la dimetilformamida. En otra realización preferente, el disolvente puede ser una combinación de dimetilformamida y etanol, en proporción 1 :3 (v:v). In another preferred embodiment, the organic solvent used for dispersion of the biopolymer is dimethylformamide. In another preferred embodiment, the solvent may be a combination of dimethylformamide and ethanol, in a 1: 3 ratio (v: v).
En una realización preferente adicional, el mediador utilizado en la etapa 2 del procedimiento es el ferroceno metanol (FcMe). Preferentemente, los nanotubos utilizados en la etapa 2 son nanotubos de carbono de pared múltiple funcionalizados con grupos oxigenados (MWCNT). In a further preferred embodiment, the mediator used in step 2 of the process is ferrocene methanol (FcMe). Preferably, the nanotubes used in step 2 are multi-wall carbon nanotubes functionalized with oxygenated groups (MWCNT).
Preferentemente, en la disolución enzimática de la etapa 3 se emplea el sistema bienzimático formado por la enzima LOD junto a la enzima peroxidasa de rábano silvestre (HRP). Preferably, in the enzymatic solution of step 3 the bienzymatic system formed by the LOD enzyme is used together with the enzyme horseradish peroxidase (HRP).
La plataforma electroquímica de elección para la preparación del biosensor amperométrico de lactato en la etapa 4, ha sido una plataforma serigrafiada de grafito (SPGE) desechable con un carácter pseudo basal, que permite un adecuado depósito de la matriz carbonosa conductora y, consecuentemente, una adecuada reproducibilidad en la inmovilización de la enzima LOD. Preferentemente, la plataforma serigrafiada utilizada puede ser de tipo convencional, de tipo microelectrodo, de tipo array, de tipo microarray, de tipo microbanda o tipo microbanda en serie. De esta forma se puede aplicar el biosensor a la determinación simultánea de varias muestras, o incluso realizar análisis con muy poca cantidad de muestra, confiriendo versatilidad y rapidez al sistema. The electrochemical platform of choice for the preparation of the amperometric lactate biosensor in stage 4, has been a disposable screen-printed graphite platform (SPGE) with a pseudo basal character, which allows an adequate deposition of the conductive carbon matrix and, consequently, a adequate reproducibility in immobilization of the LOD enzyme. Preferably, the screen-printed platform used can be of the conventional type, of the microelectrode type, of the array type, of the microarray type, of the micro-band type or the micro-band type in series. In this way, the biosensor can be applied to the simultaneous determination of several samples, or even perform analyzes with a very small amount of sample, conferring versatility and speed to the system.
Preferentemente, la etapa 6 de secado del biosensor para la eliminación del disolvente se puede llevar a cabo a 2-5 °C, o a 25 °C bajo condiciones de alto vacío durante 1 hora y después se almacena a 2-5 °C al aire. Preferably, step 6 of drying the biosensor for solvent removal can be carried out at 2-5 ° C, or at 25 ° C under high vacuum conditions for 1 hour and then stored at 2-5 ° C in air .
Otro objeto de la invención se refiere a un nuevo biosensor amperométrico de lactato portable y desechable, obtenido por el procedimiento descrito anteriormente. El biosensor amperométrico de lactato obtenido utiliza plataformas electroquímicas serigrafiadas de grafito desechables, sobre las que se deposita una matriz carbonosa conductora compuesta de una combinación de un sistema enzimático que contiene al menos la enzima LOD, nanotubos de carbono funcionalizados y un biopolímero para la inmovilización de la enzima que dé estabilidad al biosensor, pero que sea biocomplatible. Otro objeto de la invención se refiere al uso del biosensor obtenido mediante el procedimiento descrito, para su aplicación en distintos medios complejos, incluidos los medios de cultivo embrionario utilizados en técnicas de reproducción asistida humana y otros medios utilizados en el desarrollo celular. El uso del biosensor amperométrico de lactato es también extrapolable a otros campos de aplicación como son el alimentario, donde el objeto de la aplicación del biosensor amperométrico de lactato sería la detección y determinación de lactato/ácido láctico en muestras provenientes del sector vinícola y cervecero; o en el campo deportivo, en el que el biosensor amperométrico de lactato abordaría la determinación de los niveles de lactato en sangre de los deportistas. Another object of the invention relates to a new portable and disposable amperometric lactate biosensor, obtained by the procedure described above. The amperometric lactate biosensor obtained utilizes disposable screen-printed graphite electrochemical platforms, on which a conductive carbonaceous matrix composed of a combination of an enzymatic system containing at least the LOD enzyme, functionalized carbon nanotubes and a biopolymer for the immobilization of the enzyme that gives stability to the biosensor, but that is biocomplatible. Another object of the invention relates to the use of the biosensor obtained by the described procedure, for application in various complex media, including embryo culture media used in human assisted reproduction techniques and other media used in cell development. The use of the amperometric lactate biosensor is also extrapolated to other fields of application such as food, where the purpose of the application of the amperometric lactate biosensor would be the detection and determination of lactate / lactic acid in samples from the wine and beer sector; or in the sports field, in which the amperometric lactate biosensor would address the determination of athletes' blood lactate levels.
La introducción de las realizaciones preferentes descritas en el procedimiento persigue los siguientes objetivos: The introduction of the preferred embodiments described in the procedure pursues the following objectives:
(i) la mejora de la dispersión del biopolímero gracias a la combinación de los disolventes orgánicos, y así conseguir la mejora de la reproducibilidad a la hora del depósito de la disolución mediadora,  (i) the improvement of the dispersion of the biopolymer thanks to the combination of organic solvents, and thus achieve the improvement of reproducibility at the time of deposit of the mediating solution,
(¡i) la optimización de la cantidad de los productos con el fin de reducir el coste consiguiendo el mismo rendimiento y (iii) mejorar la estabilidad del biosensor amperométrico evitando el posible deterioro del sustrato carbonoso del electrodo de trabajo debido a un alto porcentaje de DMF.  (¡) The optimization of the quantity of the products in order to reduce the cost achieving the same performance and (iii) improve the stability of the amperometric biosensor avoiding the possible deterioration of the carbonaceous substrate of the working electrode due to a high percentage of DMF
El procedimiento de preparación del biosensor amperométrico de lactato descrito y el uso del sistema híbrido compuesto por biopolímero y nanotubos de carbono funcionalizados, proporciona una serie de ventajas frente a los biosensores conocidos hasta el momento: The process for preparing the amperometric lactate biosensor described and the use of the hybrid system composed of biopolymer and functionalized carbon nanotubes, provides a series of advantages over the biosensors known so far:
a. Se consigue una interacción interfacial fuerte que mejora la estabilidad y robustez mecánica del biosensor. b. El biosensor es biocompatible y no tóxico. Esto permite su uso para la medida in situ durante el crecimiento celular de embriones humanos. to. A strong interfacial interaction is achieved that improves the stability and mechanical robustness of the biosensor. b. The biosensor is biocompatible and non-toxic. This allows its use for in situ measurement during cell growth of human embryos.
c. Se reduce la velocidad de degradación del biosensor. El biosensor es estable durante al menos 4 meses a una temperatura de 4 °C sin necesidad de protegerlo con ningún tipo de membrana, gel o disolución.  C. The degradation rate of the biosensor is reduced. The biosensor is stable for at least 4 months at a temperature of 4 ° C without the need to protect it with any type of membrane, gel or solution.
d. Permite medir la concentración de lactato en tiempo real, en medios de cultivo embrionario usados en técnicas de reproducción asistida, después de la retirada del embrión. e. El biosensor se puede preparar sobre distintos tipos de plataformas con diferentes diseños como convencional, micro electrodo, array, microarray, microbanda o microbanda en serie  d. It allows to measure the concentration of lactate in real time, in embryo culture media used in assisted reproduction techniques, after removal of the embryo. and. The biosensor can be prepared on different types of platforms with different designs such as conventional, micro electrode, array, microarray, microband or serial microband
El uso de biopolímeros biocompatibles como quitosano, almidón, quitina, ácido hialurónico, glucógeno o celulosa, también contribuyen a mejorar notablemente la estabilidad del biosensor amperométrico de lactato, consiguiéndose la retención de la actividad del biosensor tras 4 meses de almacenamiento entre 2-5 °C. Esto se convierte en un gran adelanto sobre los biosensores descritos en la bibliografía hasta el momento. The use of biocompatible biopolymers such as chitosan, starch, chitin, hyaluronic acid, glycogen or cellulose, also contribute to significantly improve the stability of the amperometric lactate biosensor, achieving retention of the biosensor activity after 4 months of storage between 2-5 ° C. This becomes a great advance on the biosensors described in the literature so far.
Debido al sistema bienzimático empleado es posible la realización de la medida a un potencial de -0.2 V frente a un electrodo de referencia formado por una pasta de plata y cloruro de plata, con lo que se consigue evitar la interferencia de sustancias electroactivas presentes en los medios de cultivo embrionario. Due to the bienzimatic system used, it is possible to carry out the measurement at a potential of -0.2 V compared to a reference electrode formed by a paste of silver and silver chloride, which avoids the interference of electroactive substances present in the embryonic culture media.
Todas estas ventajas se consiguen gracias al uso del biopolímero, el uso de la plataforma serigrafiada de grafito pseudo basal utilizada para la fabricación del biosensor de lactato, la etapa de secado del sistema híbrido nanotubos de carbono/biopolímero/enzima y un almacenamiento más simple del biosensor de lactato. Además, el biosensor amperométrico descrito no necesita estar protegido con una película de naturaleza acuosa u orgánica (por ejemplo, membrana, gel, o disolución) para un almacenamiento estable del biosensor, lo que lo diferencia de algunos de los conocidos actualmente y le confiere un nuevo valor añadido. All these advantages are achieved thanks to the use of the biopolymer, the use of the silkscreen graphite platform pseudo basal used for the manufacture of the lactate biosensor, the drying stage of the system Hybrid carbon / biopolymer / enzyme nanotubes and simpler storage of the lactate biosensor. In addition, the described amperometric biosensor does not need to be protected with a film of aqueous or organic nature (for example, membrane, gel, or solution) for stable storage of the biosensor, which differentiates it from some of those currently known and gives it a New added value.
BREVE DESCRIPCIÓN DE LAS FIGURAS FIGURA 1 : Biosensor amperométrico de lactato. Esquema de la plataforma serigrafiada desechable mostrando: 1 ) los nanotubos de carbono de pared múltiple funcionalizados con grupos oxigenados, 2) ferroceno metanol (FcMe), 3) la enzima HRP, 4) la enzima LOD, 5) biopolímero, 6) lactato, 7) contraelectrodo, 8) electrodo de referencia y 9) electrodo de trabajo. BRIEF DESCRIPTION OF THE FIGURES FIGURE 1: Amperometric lactate biosensor. Scheme of the disposable screen-printed platform showing: 1) multi-wall carbon nanotubes functionalized with oxygenated groups, 2) ferrocene methanol (FcMe), 3) HRP enzyme, 4) LOD enzyme, 5) biopolymer, 6) lactate, 7) counter electrode, 8) reference electrode and 9) working electrode.
EXPOSICIÓN DETALLADA DE MODOS DE REALIZACIÓN DETAILED EXHIBITION OF REALIZATION MODES
La invención describe un nuevo procedimiento para la preparación de un biosensor amperométrico para la determinación de lactato portable y desechable utilizando plataformas electroquímicas serigrafiadas de grafito, así como el biosensor obtenido por dicho procedimiento. El biosensor descrito se puede utilizar en diversas aplicaciones para la determinación de lactato/ácido láctico en distintos medios, incluidos los medios de cultivo embrionario y otros medios utilizados en el desarrollo celular. The invention describes a new process for the preparation of an amperometric biosensor for the determination of portable and disposable lactate using screen-printed electrochemical graphite platforms, as well as the biosensor obtained by said method. The biosensor described can be used in various applications for the determination of lactate / lactic acid in different media, including embryo culture media and other media used in cell development.
El procedimiento para la preparación del biosensor amperométrico de lactato está formado por las siguientes etapas: 1. Dispersión del biopolímero en disolvente orgánico. The procedure for the preparation of the amperometric lactate biosensor consists of the following stages: 1. Dispersion of the biopolymer in organic solvent.
En primer lugar se dispersaron 84 mg de biopolímero en 1 ml_ de dimetilformamida (DMF) bajo un campo de ultrasonidos (baño ultrasónico de limpieza (ultrasons-H selecta)) durante 30 minutos. A esta dispersión se le denomina A.  First, 84 mg of biopolymer was dispersed in 1 ml_ of dimethylformamide (DMF) under an ultrasonic field (ultrasonic cleaning bath (select H-ultrasonic)) for 30 minutes. This dispersion is called A.
2. A la dispersión anterior se adicionan un mediador y nanotubos de carbono, para dar lugar a la disolución mediadora. 2. A mediator and carbon nanotubes are added to the previous dispersion to give rise to the mediator solution.
A 50 μΙ_ de la dispersión A se le adicionaron 5 mg del mediador ferroceno metanol (FcMe) (Aldrich > 97 %, España) y 1 mg de nanotubos de carbono de pared múltiple funcionalizados con grupos oxigenados (MWCNT) (DropSens, España). Gracias a la realización de un estudio de caracterización se sabe que el porcentaje atómico en carbono y oxígeno para el material de carbono MWCNT es del 91 .1 % y del 8.9 %, respectivamente, con una relación de carbono/oxígeno de aproximadamente 10. Un análisis más completo de los materiales de MWCNT reveló la ausencia de otros elementos asociados con la síntesis, por lo que la pureza de estos materiales es muy alta, ya que está exento de materiales de carbono amorfo y catalizadores metálicos. Esta dispersión se sometió a un campo de ultrasonidos (baño ultrasónico de limpieza) durante 1 hora, a la cual se le denomina dispersión mediadora B. El resultado final es una dispersión con aspecto pastoso de color negro, debido a los MWCNTs. Con la finalidad de mejorar la reproducibilidad del biosensor amperométrico de lactato, se modifica la preparación de la disolución B de la siguiente manera: contiene 2.5 mg de MWCNTs, 12.5 mg de FcMe y 5.25 mg de biopolímero dispersos en 500 μΙ_ de una disolución orgánica 1 :3 (v:v) de DMF-Etanol. La mezcla se dispersó mediante el uso de un baño de ultrasonidos durante una hora. 3. Preparación de la disolución enzimática disolviendo al menos la enzima LOD en una disolución tampón a pH neutro. 50 mg of the dispersion A was added with 5 mg of the ferrocene methanol (FcMe) mediator (Aldrich> 97%, Spain) and 1 mg of multi-wall carbon nanotubes functionalized with oxygenated groups (MWCNT) (DropSens, Spain). Thanks to the conduct of a characterization study, it is known that the atomic percentage in carbon and oxygen for the MWCNT carbon material is 91.1% and 8.9%, respectively, with a carbon / oxygen ratio of approximately 10. A More complete analysis of MWCNT materials revealed the absence of other elements associated with the synthesis, so the purity of these materials is very high, since it is free of amorphous carbon materials and metal catalysts. This dispersion was subjected to an ultrasonic field (ultrasonic cleaning bath) for 1 hour, which is called mediator dispersion B. The final result is a dispersion with a black pasty appearance, due to the MWCNTs. In order to improve the reproducibility of the amperometric lactate biosensor, the preparation of solution B is modified as follows: it contains 2.5 mg of MWCNTs, 12.5 mg of FcMe and 5.25 mg of biopolymer dispersed in 500 μΙ_ of an organic solution 1 : 3 (v: v) DMF-Ethanol. The mixture was dispersed by using an ultrasonic bath for one hour. 3. Preparation of the enzyme solution by dissolving at least the LOD enzyme in a buffer solution at neutral pH.
Por otro lado se prepara una disolución enzimática formada por 5 mg/mL de la enzima LOD, 0.5 Unidades de actividad por biosensor, la cual se encuentra almacenada a -20 °C, previamente disuelta en tampón fosfato 0.1 M; y 19 mg/mL de la enzima peroxidasa de rábano silvestre (HRP) en 0.1 M de un tampón de fosfato sódico a pH 7.5 (disolución C).  On the other hand, an enzymatic solution consisting of 5 mg / mL of the LOD enzyme, 0.5 Units of activity per biosensor, is prepared, which is stored at -20 ° C, previously dissolved in 0.1 M phosphate buffer; and 19 mg / mL of the enzyme horseradish peroxidase (HRP) in 0.1 M of a sodium phosphate buffer at pH 7.5 (solution C).
4. Adición de la disolución mediadora obtenida en la etapa 2 sobre la plataforma serigrafiada. 4. Addition of the mediating solution obtained in stage 2 on the screen-printed platform.
Con una micropipeta se depositan 0.6 μί de la dispersión mediadora B sobre la superficie de trabajo de la plataforma serigrafiada de grafito.  With a micropipette, 0.6 μί of the mediating dispersion B is deposited on the work surface of the graphite silkscreen platform.
5. Adición de la disolución enzimática obtenida en la etapa 3 sobre la plataforma serigrafiada y la disolución mediadora.5. Addition of the enzymatic solution obtained in step 3 on the screen-printed platform and the mediating solution.
Seguidamente se adicionan lentamente 5 μί de la disolución enzimática C que contiene 0.5 unidades de la enzima LOD, sobre la matriz carbonosa anterior, de modo que se observe un cambio de color. Then 5 μ lentamente of the enzymatic solution C containing 0.5 units of the LOD enzyme is slowly added onto the previous carbonaceous matrix, so that a color change is observed.
6. Secado del biosensor para la eliminación del disolvente. 6. Drying the biosensor for solvent removal.
Finalmente, la película formada sobre la superficie del electrodo se protege con una placa Petri de plástico y se deja secar a una temperatura entre 2-5 °C, o a 25 °C bajo condiciones de alto vacío durante 1 hora y después se almacena al aire en una nevera entre 2-5 °C.  Finally, the film formed on the electrode surface is protected with a plastic Petri dish and allowed to dry at a temperature between 2-5 ° C, or at 25 ° C under high vacuum conditions for 1 hour and then stored in air in a refrigerator between 2-5 ° C.
Como ya se ha indicado previamente, el biosensor obtenido por este procedimiento está formado por un sustrato de grafito serigrafiado soportado sobre un polímero aislante flexible (plataforma serigrafiada SPGE), una matriz carbonosa conductora formada por la combinación de un sistema enzimático que contiene al menos la enzima LOD, nanotubos de carbono funcionalizados y el biopolímero para la inmovilización de la enzima. Se puede observar un esquema del biosensor obtenido en la Figura 1 . En ella se muestra la matriz carbonosa conductora con los nanotubos de carbono de pared múltiple funcionalizados con grupos oxigenados (1 ), el mediador FcMe (2), el sistema bienzimático formado por la HRP (3) y la LOD (4) y el biopolímero (5). La matriz carbonosa está depositada sobre la plataforma serigrafiada que contiene el contraelectrodo (7), el electrodo de referencia (8) y el de trabajo (9). Sobre este biosensor se deposita la muestra con lactato (6) a determinar. As previously indicated, the biosensor obtained by this procedure is formed by a screen-printed graphite substrate supported on a flexible insulating polymer (screen-printed platform SPGE), a conductive carbonaceous matrix formed by the combination of an enzymatic system that contains at least the LOD enzyme, functionalized carbon nanotubes and the biopolymer for immobilization of the enzyme. A scheme of the biosensor obtained in Figure 1 can be observed. It shows the conductive carbonaceous matrix with the multi-wall carbon nanotubes functionalized with oxygenated groups (1), the FcMe mediator (2), the bienzimatic system formed by the HRP (3) and the LOD (4) and the biopolymer (5). The carbonaceous matrix is deposited on the screen-printed platform that contains the counter electrode (7), the reference electrode (8) and the working electrode (9). The sample with lactate (6) to be determined is deposited on this biosensor.
Más concretamente, para la fabricación del biosensor se emplea una plataforma electroquímica serigrafiada desechable. Estas plataformas consisten en un electrodo de trabajo de grafito de carácter basal con un diámetro de 3.1 mm, un contraelectrodo de grafito y un pseudo-referencia formado por una pasta de plata/cloruro de plata, todos los electrodos soportados sobre una base de poliestireno. La fabricación de las plataformas se lleva a cabo serigrafiando una tinta de carbono sobre una lámina flexible de poliestireno para definir los contactos, el contraelectrodo y el electrodo de trabajo. Esta lámina se seca en una estufa a 60 grados durante 30 minutos. Después se incorpora el electrodo de referencia serigrafiando una pasta de Ag/AgCI sobre el sustrato de plástico y se seca a 60 grados durante 30 minutos. Posteriormente se imprime con una pasta de dieléctrico para cubrir las conexiones y definir el electrodo de trabajo grafitico. More specifically, a disposable screen-printed electrochemical platform is used to manufacture the biosensor. These platforms consist of a work electrode of graphite of basal character with a diameter of 3.1 mm, a counter electrode of graphite and a pseudo-reference formed by a paste of silver / silver chloride, all electrodes supported on a polystyrene base. The manufacturing of the platforms is carried out by screen printing a carbon ink on a flexible sheet of polystyrene to define the contacts, the counter electrode and the working electrode. This sheet is dried in an oven at 60 degrees for 30 minutes. The reference electrode is then incorporated by screen printing an Ag / AgCI paste on the plastic substrate and dried at 60 degrees for 30 minutes. It is subsequently printed with a dielectric paste to cover the connections and define the graffiti work electrode.
En cualquier caso, transcurrido un mínimo de unas 12 horas de su preparación, el biosensor amperométrico de lactato está listo para su uso. En primer lugar, se realiza un acondicionamiento previo del biosensor amperométrico sumergiendo el biosensor en una disolución tamponada de fosfato de sodio 0.1 M a pH 7.5 durante 5 minutos bajo agitación magnética, para una correcta limpieza. Posteriormente se realiza un pretratamiento electroquímico del biosensor durante 2 minutos aplicando al biosensor un potencial de -0.2 V en fosfato de sodio 0.1 M pH 7.5. Una vez finalizado el pretratamiento el biosensor está listo para realizar la medida. La medida se puede llevar a cabo mediante dos técnicas electroquímicas convencionales. La primera de ellas es la voltamperometría cíclica. Para ello, se coloca una gota de la disolución patrón o problema a medir con el fin de que cubra los tres electrodos presentes en la plataforma serigrafiada y a continuación se aplica un barrido lineal de potencial frente al tiempo desde -0.1 V hasta -0.4 V y se toma el valor de intensidad de corriente a un potencial de -0.2 V. Para la construcción de la recta de calibrado, se realiza la medida a distintas concentraciones de lactato. La otra técnica electroquímica es la cronoamperometría. En esta técnica se sumerge el biosensor en un vaso que contiene 7 mL de tampón fosfato de sodio 0.1 M pH 7.5, con agitación magnética, se aplica un potencial constante de -0.2 V y se adiciona un volumen conocido de una disolución que contiene lactato que se desea determinar. Para la construcción de la recta de calibrado se van adicionando 25 μί de una disolución de lactato sódico 10 mM durante la cronoamperometría para posteriormente relacionar los valores de intensidad de corriente obtenidos con las distintas concentraciones de lactato. In any case, after a minimum of about 12 hours of its preparation, the amperometric lactate biosensor is ready for use. First, a preconditioning of the amperometric biosensor is performed by immersing the biosensor in a 0.1 M sodium phosphate buffered solution at pH 7.5 for 5 minutes under magnetic stirring, for proper cleaning. Subsequently, an electrochemical pretreatment of the biosensor is performed for 2 minutes, applying a potential of -0.2 V in 0.1 M sodium phosphate pH 7.5 to the biosensor. Once the pretreatment is finished, the biosensor is ready to perform the measurement. The measurement can be carried out by two conventional electrochemical techniques. The first of these is cyclic voltammetry. To do this, a drop of the standard solution or problem to be measured is placed in order to cover the three electrodes present in the screen-printed platform and then apply a linear sweep of potential versus time from -0.1 V to -0.4 V and the current intensity value is taken at a potential of -0.2 V. For the construction of the calibration line, the measurement is carried out at different lactate concentrations. The other electrochemical technique is chronoamperometry. In this technique, the biosensor is immersed in a vessel containing 7 mL of 0.1 M sodium phosphate buffer pH 7.5, with magnetic stirring, a constant potential of -0.2 V is applied and a known volume of a lactate-containing solution is added which It is desired to determine. For the construction of the calibration line, 25 μί of a 10 mM sodium lactate solution is added during chronoamperometry to subsequently relate the current intensity values obtained with the different lactate concentrations.
EJEMPLO 1. Preparación de un biosensor amperométrico de lactato basado en un sistema híbrido MWCNT/LOD/biopolímero quitosano. Se prepararon una serie de biosensores amperométricos de lactato mediante el procedimiento presentado: primero se preparó una disolución que contenía 84 mg de quitosano en 1 ml_ de dimetilformamida y se aplicó un campo de ultrasonidos durante 30 minutos (disolución A). De la disolución anterior (disolución A) se tomaron 50 μΙ_ sobre los cuales se adicionaron 5 mg de FcMe y 1 mg de MWCNT. Esta nueva disolución se mantuvo durante una hora en un baño de ultrasonidos (disolución B). Paralelamente se preparó la disolución bienzimática en un tubo eppendorf que contenía 20 μΙ_ de una disolución enzimática de 2 unidades de la enzima lactato oxidasa LOD en tampón 0.1 M fosfato de sodio pH 7.5, previamente almacenado a -20 °C, y se incorporó 0.5 mg de la enzima peroxidasa HRP (disolución C). Estas cantidades corresponden a una disolución para la preparación de 4 biosensores amperométricos de lactato. Una vez preparadas la disolución mediadora (disolución B) y la enzimática (disolución C) se depositaron, sobre una plataforma serigrafiada con un electrodo de trabajo grafitico que presentaba un carácter pseudo basal. Para ello, se depositaron 0.6 μΙ_ de la disolución B con el fin de recubrir completamente la superficie del electrodo de trabajo y seguidamente, sobre este depósito, se incorporaron 5 μΙ_ de la disolución C, lentamente. Finalizado este proceso, los biosensores amperométricos de lactato se sometieron al proceso de secado y se almacenaron entre 2-5 °C al aire dentro de una placa Petri. Los biosensores amperométricos de lactato se testearon con el tiempo una vez preparados. EXAMPLE 1. Preparation of an amperometric lactate biosensor based on a hybrid MWCNT / LOD / chitosan biopolymer system. A series of amperometric lactate biosensors were prepared by the procedure presented: first a solution containing 84 mg of chitosan in 1 ml_ of dimethylformamide was prepared and an ultrasound field was applied for 30 minutes (solution A). From the previous solution (solution A) 50 μΙ_ were taken on which 5 mg of FcMe and 1 mg of MWCNT were added. This new solution was maintained for one hour in an ultrasonic bath (solution B). In parallel, the bienzyme solution was prepared in an eppendorf tube containing 20 μΙ_ of a 2-unit enzyme solution of the enzyme lactate oxidase LOD in 0.1 M sodium phosphate buffer pH 7.5, previously stored at -20 ° C, and 0.5 mg was incorporated of the enzyme HRP peroxidase (solution C). These amounts correspond to a solution for the preparation of 4 amperometric lactate biosensors. Once the mediating solution (solution B) and the enzyme solution (solution C) were prepared, they were deposited on a screen-printed platform with a graffiti work electrode that had a pseudo basal character. For this, 0.6 μΙ_ of solution B was deposited in order to completely cover the surface of the working electrode and then, on this deposit, 5 μΙ_ of solution C was slowly incorporated. After this process, the amperometric lactate biosensors were subjected to the drying process and stored between 2-5 ° C in the air inside a Petri dish. The amperometric lactate biosensors were tested over time once prepared.
Antes de realizar la medida de corriente-tiempo correspondiente al experimento de cronoamperometría en función de las concentraciones de lactato de sodio, el biosensor amperométrico de lactato se sumergió en una disolución de 0.1 M de tampón fosfato de sodio con un pH de la disolución de 7.5, bajo agitación continua durante 5 minutos para su correcta limpieza. Posteriormente se realizó un pretratamiento electroquímico durante dos minutos a -0.2 V frente a un electrodo de pseudo referencia constituido por una pasta de plata y cloruro de plata en una disolución tampón fosfato de sodio 0.1 M a un pH 7.5 y, por último, se llevó a cabo la determinación de lactato mediante la técnica de cronoamperometría a un potencial constante de -0.2 V frente al electrodo de pseudo referencia, adicionando sucesivas alícuotas de una disolución estándar de lactato de sodio con una concentración de 10 mM en un tampón fosfato de sodio 0.1 M a un pH 7.5. Las rectas de calibrado se llevaron a cabo tomando el valor substraído de la intensidad de corriente a -0.2 V obtenido para cada una de las concentraciones de lactato adicionadas sobre la disolución de tampón de fosfato de sodio 0.1 M a un pH de 7.5, a la intensidad de corriente a un mismo potencial en ausencia de lactato de sodio en la disolución frente a la concentración de lactato de sodio en la disolución. Las rectas de calibrado presentaron valores de correlación comprendidos entre 0,995 y 0,999 en un rango de concentración entre 0.01 y 0.20 mM de lactato. Before performing the current-time measurement corresponding to the chronoamperometry experiment based on sodium lactate concentrations, the amperometric lactate biosensor was immersed in a 0.1 M solution of sodium phosphate buffer with a solution pH of 7.5 , under continuous stirring for 5 minutes for proper cleaning. Subsequently, a pretreatment was performed. electrochemical for two minutes at -0.2 V against a pseudo reference electrode consisting of a paste of silver and silver chloride in a 0.1 M sodium phosphate buffer solution at pH 7.5 and, finally, the determination of lactate using the chronoamperometry technique at a constant potential of -0.2 V against the pseudo reference electrode, adding successive aliquots of a standard sodium lactate solution with a concentration of 10 mM in a 0.1 M sodium phosphate buffer at pH 7.5 . The calibration lines were carried out by taking the subtracted value of the current intensity at -0.2 V obtained for each of the lactate concentrations added on the 0.1 M sodium phosphate buffer solution at a pH of 7.5, at current intensity at the same potential in the absence of sodium lactate in the solution versus the concentration of sodium lactate in the solution. The calibration lines showed correlation values between 0.995 and 0.999 in a concentration range between 0.01 and 0.20 mM lactate.
Se realizó un estudio sobre cómo afectan algunos de los posibles interferentes presentes en medios de cultivo embrionario utilizados en técnicas de reproducción asistida. Entre los candidatos examinados se eligieron la glucosa, la proteína de suero bovino BSA y piruvato. Los resultados obtenidos revelaron que la determinación de lactato de sodio mediante el uso del biosensor amperométrico de lactato obtenido es independiente de la presencia de los interferentes descritos anteriormente. A study was conducted on how they affect some of the possible interference present in embryo culture media used in assisted reproduction techniques. Among the candidates examined were glucose, bovine serum protein BSA and pyruvate. The results obtained revealed that the determination of sodium lactate by using the amperometric lactate biosensor obtained is independent of the presence of the aforementioned interferents.
El uso de plataformas electroquímicas de tipo array o microarray también puede ser útil como biosensor amperométrico de lactato, ya que permite su uso en la determinación simultánea de varias muestras biológicas y le confiere versatilidad y rapidez al sistema analítico. Finalmente la fabricación del biosensor amperométrico de lactato se puede realizar utilizando plataformas electroquímicas serigrafiadas tipo microelectrodo, microbanda o microbanda en serie para determinar la concentración de lactato en medios de cultivo embrionario cuyos volúmenes oscilan entre 25 y 50 microlitros lo que permite realizar determinaciones con muy poca cantidad de muestra. The use of electrochemical platforms of the array or microarray type can also be useful as an amperometric lactate biosensor, since it allows its use in the simultaneous determination of several biological samples and gives the analytical system versatility and speed. Finally, the manufacture of the amperometric lactate biosensor can be carried out using screen-printed electrochemical platforms such as microelectrode, micro-band or micro-band in series to determine the concentration of lactate in embryo culture media whose volumes range between 25 and 50 microliters allowing determinations with very little sample.
EJEMPLO 2: Uso del biosensor amperométrico de lactato en medios de cultivo embrionario. EXAMPLE 2: Use of the amperometric lactate biosensor in embryo culture media.
Para la validación del uso del biosensor amperométrico de lactato en medios de cultivo embrionarios, se empleó un medio de cultivo embrionario G-1 (vitrolife) y se comparó la determinación de la concentración de lactato en el medio de cultivo obtenida mediante una técnica cromatográfica HPLC-UV-Vis con la obtenida mediante el uso del biosensor amperométrico descrito en la invención. For the validation of the use of the amperometric lactate biosensor in embryonic culture media, a G-1 embryonic culture medium (vitrolife) was used and the determination of the lactate concentration in the culture medium obtained by an HPLC chromatographic technique was compared. -UV-Vis with that obtained by using the amperometric biosensor described in the invention.
Para la determinación de lactato por método cromatográfico se utilizó un equipo de cromatografía líquida de alta presión HPLC (Agilent 1 100 series, Santa Clara, USA) con un detector ultravioleta visible. La fase móvil consistió en una disolución acuosa 20 mM NaH2P04 ajusfando el pH de la disolución con H3P04 hasta pH 2.5. La columna empleada fue una C18 hypersil y la velocidad de flujo fue de 0.5 mL min"1 con una longitud de onda de 210 nm. Se realizó una recta de calibrado utilizando patrones de lactato de sodio preparados con la misma fase móvil. Todos los patrones de lactato se filtraron utilizando los filtros con un tamaño de poro de 0.5 mieras. Se realizó una dilución 1 : 10 (v/v) de la muestra problema (medio de cultivo) en agua y posteriormente se filtró. El valor de la concentración de lactato para la muestra empleada mediante el método cromatográfico HPLC-UV-Vis fue de 1 1 ,9 ± 0, 10 mM. Una vez determinado el valor de concentración de lactato mediante la técnica cromatográfica utilizando el HPLC-UV-Vis se realizaron las medidas de lactato en la muestra problema (medio de cultivo) mediante el empleo del biosensor amperométrico de lactato. El biosensor empleado en este caso fue el biosensor fabricado de acuerdo con la realización preferente del procedimiento descrito, utilizando una mezcla de DMF y etanol como disolvente para el quitosano y secando el biosensor a 25 °C en condiciones de alto vacío. Se prepararon 8 biosensores amperométricos de lactato y se emplearon 4 de ellos para la realización de la recta de calibrado y los otros 4 restantes para la medida de lactato en la muestra problema. For the determination of lactate by chromatographic method, an HPLC high pressure liquid chromatography device (Agilent 1 100 series, Santa Clara, USA) with a visible ultraviolet detector was used. The mobile phase consisted of a 20 mM aqueous solution NaH 2 P0 4 adjusting the pH of the solution with H 3 P0 4 to pH 2.5. The column used was a C18 hypersil and the flow rate was 0.5 mL min "1 with a wavelength of 210 nm. A calibration line was made using sodium lactate standards prepared with the same mobile phase. All patterns of lactate were filtered using filters with a pore size of 0.5 microns A 1: 10 dilution (v / v) of the test sample (culture medium) in water was made and subsequently filtered. Lactate for the sample used by the HPLC-UV-Vis chromatographic method was 11.9 ± 0.10 mM. Once the lactate concentration value was determined by the chromatographic technique using HPLC-UV-Vis, the lactate measurements were made in the test sample (culture medium) by using the amperometric lactate biosensor. The biosensor used in this case was the biosensor manufactured in accordance with the preferred embodiment of the described procedure, using a mixture of DMF and ethanol as solvent for the chitosan and drying the biosensor at 25 ° C under high vacuum conditions. Eight amperometric lactate biosensors were prepared and 4 of them were used for the realization of the calibration line and the other 4 remaining for the measurement of lactate in the test sample.
La medida de lactato en la muestra problema de medio de cultivo se llevó a cabo de la siguiente manera: en primer lugar, se realizó limpieza del biosensor de la manera indicada anteriormente, posteriormente se introdujo el biosensor en un vial que contenía 5 mL de tampón fosfato de sodio 0.1 M a pH 7.5 y la determinación de lactato se llevó a cabo empleando la técnica de cronoamperometría, a un potencial constante de - 0.2 V. Tras dos minutos de pretratamiento se adicionan 60 μί del medio de cultivo embrionario G1 . Se registró el valor de intensidad obtenido y mediante la recta de calibrado obtenida anteriormente se calculó la concentración de lactato en el medio de cultivo. Para cada determinación de lactato en el medio de cultivo se utilizaron 4 biosensores desechables. Se realizaron tres medidas de la concentración de lactato en el medio de cultivo elegido en tres días diferentes, y las medias de las concentraciones junto con sus respectivas desviaciones estándar fueron 1 1 .5 ± 2.0 mM, 12.2 ± 1 .8 mM y 1 1 .8 ± 1 .7 mM, respectivamente. The lactate measurement in the culture medium problem sample was carried out as follows: first, the biosensor was cleaned in the manner indicated above, then the biosensor was introduced into a vial containing 5 mL of buffer 0.1 M sodium phosphate at pH 7.5 and lactate determination was carried out using the chronoamperometry technique, at a constant potential of - 0.2 V. After two minutes of pretreatment, 60 μί of the G1 embryonic culture medium is added. The intensity value obtained was recorded and the concentration of lactate in the culture medium was calculated using the calibration line obtained above. For each lactate determination in the culture medium, 4 disposable biosensors were used. Three measurements of the lactate concentration were made in the culture medium chosen on three different days, and the mean concentrations together with their respective standard deviations were 1 1 .5 ± 2.0 mM, 12.2 ± 1 .8 mM and 1 1 .8 ± 1 .7 mM, respectively.
Los test de estabilidad de los biosensores amperométricos de lactato se realizaron una vez preparados siguiendo la realización preferente del procedimiento y transcurridos 8 días, 14 días, y 4 meses, respectivamente. Los biosensores amperométricos de lactato empleados a día 1 de su preparación presentaron una recta de calibrado con una regresión lineal de 0.99932 siendo la pendiente de -0.0034 μΑ- μΜ"1 +/- 0.0002 μΑ μΜ"1. Después de los 14 días transcurridos de la preparación de los biosensores, éstos presentaron unas rectas de calibrado con una regresión lineal de 0.99579 y 0.99839, sin ninguna pérdida de la sensibilidad del biosensor amperométrico de lactato. Finalmente, pasados 4 meses tras la preparación de los biosensores se realizó una nueva recta de calibrado, comprobando que los biosensores seguían siendo válidos para su empleo en la determinación de lactato, a pesar de que la sensibilidad del biosensor amperométrico de lactato se redujo en un 10- 20%. The stability tests of the amperometric lactate biosensors were performed once prepared following the preferred procedure and after 8 days, 14 days, and 4 months, respectively. The amperometric lactate biosensors used on day 1 of its preparation presented a calibration line with a linear regression of 0.99932, the slope being -0.0034 μΑ- μΜ "1 +/- 0.0002 μΑ μΜ " 1 . After the 14 days after the preparation of the biosensors, they presented calibration lines with a linear regression of 0.99579 and 0.99839, without any loss of the sensitivity of the amperometric lactate biosensor. Finally, after 4 months after the preparation of the biosensors, a new calibration line was made, verifying that the biosensors were still valid for use in the determination of lactate, despite the fact that the sensitivity of the amperometric lactate biosensor was reduced by a 10-20%.
EJEMPLO 3. Preparación de un biosensor amperométrico de lactato basado en un sistema híbrido MWCNT/LOD/biopolímero almidón, quitina, ácido hialurónico, glucógeno o celulosa. EXAMPLE 3. Preparation of an amperometric lactate biosensor based on a hybrid MWCNT / LOD / biopolymer starch, chitin, hyaluronic acid, glycogen or cellulose system.
La detección y determinación de lactato empleando el uso de diferentes biopolímeros para la fabricación del biosensor amperométrico de lactato se llevó a cabo de la siguiente manera. The detection and determination of lactate using the use of different biopolymers for the manufacture of the amperometric lactate biosensor was carried out as follows.
El ensayo se realizó con el biosensor fabricado de acuerdo con la realización preferente del procedimiento descrito, utilizando una mezcla de DMF y etanol 1 :3 v:v como disolvente para el mediador FcMe, los MWCNTs y el biopolímero. En este ejemplo se han utilizado los distintos biopolímeros mencionados: almidón, quitina, ácido hialurónico, glucógeno y celulosa, respectivamente. La anterior dispersión se sónico durante 1 hora. Paralelamente se preparó la disolución enzimática que contenía la enzima LOD y la HRP. Una vez depositadas la dispersión mediadora correspondiente y la disolución enzimática, se realizó el proceso de secado a 25 °C en condiciones de alto vacío durante 1 hora, y posterior almacenamiento a 2-5 °C al aire. Se realizaron rectas de calibrado para cada biosensor amperométrico de lactato correspondiente a los sistemas híbridos MWCNT/LOD/almidón, MWCNT/LOD/quitina,The test was performed with the biosensor manufactured in accordance with the preferred embodiment of the described procedure, using a mixture of DMF and ethanol 1: 3 v: v as a solvent for the FcMe mediator, the MWCNTs and the biopolymer. In this example, the different biopolymers mentioned have been used: starch, chitin, hyaluronic acid, glycogen and cellulose, respectively. The previous dispersion was sonic for 1 hour. In parallel, the enzyme solution containing the enzyme LOD and HRP was prepared. Once the corresponding mediating dispersion and the enzymatic solution were deposited, the drying process was carried out at 25 ° C under high vacuum conditions for 1 hour, and later Storage at 2-5 ° C in air. Calibration lines were made for each amperometric lactate biosensor corresponding to the MWCNT / LOD / starch, MWCNT / LOD / chitin hybrid systems,
MWC NT/LO D/ácido hialurónico, MWCNT/LOD/glucógeno y MWCNT/LOD/celulosa. MWC NT / LO D / hyaluronic acid, MWCNT / LOD / glycogen and MWCNT / LOD / cellulose.
Una vez fabricados los biosensores amperométricos de lactato, se llevó a cabo la limpieza del biosensor de la manera indicada anteriormente en el procedimiento y seguidamente, se introdujo el biosensor en un vial que contenía 5 ml_ de tampón fosfato de sodio 0.1 M a pH 7.5. La determinación de lactato se llevó a cabo empleando la técnica de cronoamperometría, a un potencial constante de -0.2 V respecto a un electrodo de referencia de Ag/AgCI. Tras dos minutos de pretratamiento se fueron adicionando 25 μΙ_ de lactato sódico en tampón fosfato de sodio a pH 7.5. Se registraron los valores de intensidad de corriente obtenidos con cada adición de alícuota de la disolución de lactato. Las rectas de calibrado obtenidas con los biosensores amperométricos de lactato fabricados con los distintos biopolímeros, presentaron los siguientes rangos de linealidad y coeficientes de regresión lineal: Once the amperometric lactate biosensors were manufactured, the biosensor was cleaned in the manner indicated above in the procedure and then, the biosensor was introduced into a vial containing 5 ml_ of 0.1 M sodium phosphate buffer at pH 7.5. Lactate determination was carried out using the chronoamperometry technique, at a constant potential of -0.2 V with respect to an Ag / AgCI reference electrode. After two minutes of pretreatment, 25 μΙ_ of sodium lactate in sodium phosphate buffer at pH 7.5 were added. Current intensity values obtained with each aliquot addition of the lactate solution were recorded. The calibration lines obtained with the amperometric lactate biosensors manufactured with the different biopolymers, presented the following linearity ranges and linear regression coefficients:
Tabla 1 : Linealidad obtenida con diferentes biosensores amperométricos de lactato obtenidos mediante el procedimiento descrito y utilizando distintos biopolímeros. Table 1: Linearity obtained with different amperometric lactate biosensors obtained by the procedure described and using different biopolymers.
Figure imgf000026_0001
Todos los biosensores amperométricos de lactato desechables presentaron una alta estabilidad mecánica y enzimática.
Figure imgf000026_0001
All disposable amperometric lactate biosensors had high mechanical and enzymatic stability.

Claims

REIVINDICACIONES
1 . Procedimiento de preparación de un biosensor amperométrico de lactato basado en la enzima lactato oxidasa (LOD) que comprende las siguientes etapas: one . Method of preparing an amperometric lactate biosensor based on the enzyme lactate oxidase (LOD) comprising the following steps:
1 . Dispersión del biopolímero en disolvente orgánico.  one . Dispersion of the biopolymer in organic solvent.
2. A la dispersión anterior se adicionan un mediador y nanotubos de carbono, para dar lugar a la disolución mediadora.  2. A mediator and carbon nanotubes are added to the previous dispersion to give rise to the mediator solution.
3. Preparación de la disolución enzimática disolviendo al menos la enzima LOD en una disolución tampón a pH neutro.  3. Preparation of the enzyme solution by dissolving at least the LOD enzyme in a buffer solution at neutral pH.
4. Adición de la disolución mediadora obtenida en la etapa 2 sobre una plataforma serigrafiada.  4. Addition of the mediating solution obtained in stage 2 on a screen-printed platform.
5. Adición de la disolución enzimática obtenida en la etapa 3 sobre la plataforma serigrafiada y la disolución mediadora.  5. Addition of the enzymatic solution obtained in step 3 on the screen-printed platform and the mediating solution.
6. Secado del biosensor para la eliminación del disolvente.  6. Drying the biosensor for solvent removal.
2. Procedimiento de preparación de un biosensor amperométrico de lactato según la reivindicación 1 , donde el biopolímero utilizado es quitosano. 2. Method of preparing an amperometric lactate biosensor according to claim 1, wherein the biopolymer used is chitosan.
3. Procedimiento de preparación de un biosensor amperométrico de lactato según la reivindicación 1 , donde el biopolímero utilizado está basado en polisacáridos de glucosa o amilosa, funcionalizados con grupos hidroxilo, amino, acetilo o carboxílico, que contienen enlaces glucosídicos, como son el almidón, la quitina, el ácido hialurónico, el glucógeno y la celulosa. 3. Method of preparing an amperometric lactate biosensor according to claim 1, wherein the biopolymer used is based on glucose or amylose polysaccharides, functionalized with hydroxyl, amino, acetyl or carboxylic groups, containing glycosidic bonds, such as starch, chitin, hyaluronic acid, glycogen and cellulose.
4. Procedimiento de preparación de un biosensor amperométrico de lactato según la reivindicación 1 , donde el disolvente orgánico es dimetilformamida o una combinación de dimetilformamida y etanol. 4. Method of preparing an amperometric lactate biosensor according to claim 1, wherein the organic solvent is dimethylformamide or a combination of dimethylformamide and ethanol.
5. Procedimiento de preparación de un biosensor amperométrico de lactato según la reivindicación 4, donde la combinación de dimetilformamida y etanol es en proporción 1 :3 (v:v). 5. Method of preparing an amperometric lactate biosensor according to claim 4, wherein the combination of dimethylformamide and ethanol is in a 1: 3 ratio (v: v).
6. Procedimiento de preparación de un biosensor amperométrico de lactato según la reivindicación 1 , donde el mediador utilizado es el ferroceno metanol. 6. Method of preparing an amperometric lactate biosensor according to claim 1, wherein the mediator used is ferrocene methanol.
7. Procedimiento de preparación de un biosensor amperométrico de lactato según la reivindicación 1 , donde los nanotubos de carbono utilizados son de pared múltiple y están funcionalizados con grupos oxigenados. 7. Method of preparing an amperometric lactate biosensor according to claim 1, wherein the carbon nanotubes used are multiple wall and are functionalized with oxygenated groups.
8. Procedimiento de preparación de un biosensor amperométrico de lactato según la reivindicación 1 , donde la disolución enzimática se prepara con la enzima lactato oxidasa (LOD), junto a la enzima peroxidasa de rábano silvestre (HRP). 8. Method of preparing an amperometric lactate biosensor according to claim 1, wherein the enzyme solution is prepared with the enzyme lactate oxidase (LOD), together with the enzyme horseradish peroxidase (HRP).
9. Procedimiento de preparación de un biosensor amperométrico de lactato según la reivindicación 1 , donde la plataforma serigrafiada es de grafito desechable, de tipo convencional, microelectrodo, array, microarray, microbanda o microbanda en serie, para conferir versatilidad y rapidez al sistema. 9. Method for preparing an amperometric lactate biosensor according to claim 1, wherein the screen-printed platform is made of disposable graphite, of the conventional type, microelectrode, array, microarray, microband or microband in series, to confer versatility and speed to the system.
10. Procedimiento de preparación de un biosensor amperométrico de lactato según la reivindicación 1 , donde el secado se realiza a 2-5 °C o a 25 °C bajo condiciones de alto vacío durante 1 hora y después se almacena a 2-5 °C al aire. 10. Method of preparing an amperometric lactate biosensor according to claim 1, wherein drying is carried out at 2-5 ° C or at 25 ° C under high vacuum conditions for 1 hour and then stored at 2-5 ° C at air.
1 1 . Biosensor amperométrico de lactato basado en la enzima lactato oxidasa (LOD) obtenido por el procedimiento descrito en la reivindicación 1 , formado por un sustrato de grafito serigrafiado soportado sobre un polímero aislante flexible, una matriz carbonosa conductora formada por la combinación de un sistema enzimático que contiene al menos la enzima LOD, nanotubos de carbono funcionalizados y el biopolímero para la inmovilización de la enzima. eleven . Amperometric lactate biosensor based on the enzyme lactate oxidase (LOD) obtained by the method described in claim 1, formed by a screen-printed graphite substrate supported on a flexible insulating polymer, a conductive carbonaceous matrix formed by the combination of an enzymatic system that It contains at least the LOD enzyme, functionalized carbon nanotubes and the biopolymer for immobilization of the enzyme.
12. Uso del biosensor amperométrico de lactato basado en la enzima lactato oxidasa (LOD) descrito en la reivindicación 1 1 en medios complejos, incluidos los medios de cultivo embrionario utilizados en técnicas de reproducción asistida humana y otros medios utilizados en el desarrollo celular. 12. Use of the amperometric lactate biosensor based on the enzyme lactate oxidase (LOD) described in claim 1 1 in complex media, including embryo culture media used in human assisted reproduction techniques and other media used in cell development.
13. Uso del biosensor amperométhco de lactato basado en la enzima lactato oxidasa (LOD) descrito en la reivindicación 1 1 en muestras alimentarias provenientes del sector vinícola y cervecero. 13. Use of the amperometric lactate biosensor based on the enzyme lactate oxidase (LOD) described in claim 1 1 in food samples from the wine and beer sector.
14. Uso del biosensor amperométhco de lactato basado en la enzima lactato oxidasa (LOD) descrito en la reivindicación 1 1 en la determinación de los niveles de lactato en sangre. 14. Use of the lactate amperometric biosensor based on the enzyme lactate oxidase (LOD) described in claim 1 1 in the determination of blood lactate levels.
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