WO2010055183A1

WO2010055183A1 - Method for cell identification and quantification with gold nanoparticles through hydrogen ion reduction

Info

Publication number: WO2010055183A1
Application number: PCT/ES2009/070489
Authority: WO
Inventors: Arben MERKOÇI HYKA; Alfredo DE LA ESCOSURA MUÑIZ; Africa GONZÁLEZ FERNÁNDEZ; Belén DÍAZ FREITAS; Christian SÁNCHEZ ESPINEL
Original assignee: Fundació Privada Institut Català De Nanotecnologia; Universidade De Vigo
Priority date: 2008-11-12
Filing date: 2009-11-10
Publication date: 2010-05-20
Also published as: ES2338977A1; ES2338977B1

Abstract

The method for cell identification and quantification comprises bonding specific surface proteins of cells immobilised on the surface of the electrochemical transducer with specific antibodies conjugated with gold nanoparticles, and subsequent nanoparticle detection and quantification through hydrogen generation catalysed by said nanoparticles at an appropriate potential. It furthermore comprises the application thereof in a method of diagnosis and/or prognosis of a disease involving the expression of cell-surface proteins and the corresponding kits.

Description

METHOD OF IDENTIFICATION AND QUANTIFICATION OF CELLS WITH GOLD NANOPARTICLES BY REDUCTION OF HYDROGEN IONS

The present invention relates to a method of cell identification, based on the use of gold nanoparticles conjugated to specific antibodies of the cell surface proteins and on the use of an electrocatalytic method for the detection of these nanoparticles, as well as its application in the diagnosis and / or prognosis of a disease.

STATE OF THE TECHNIQUE

The detection of cells, particularly tumor cells, has been a field of great interest in recent years. This interest is due to the fact that the presence of these cells in peripheral blood is indicative of a disease, as well as a low effectiveness in therapeutic treatments.

The current methods used for diagnosis, such as flow cytometry and histochemistry are not very accurate, expensive, with long analysis times and require very advanced instrumentation. In recent years, new methods based on the detection of cells using specific antibodies have been developed.

On the other hand, the use of biosensors for the identification of cells has been developed to monitor the morphological and photochemical changes of cells adhered to these sensors, using an electrical impedance detection system and the study of the cellular response to chemical substances. , such as the monitoring of the acidification of the medium in live cell cultures.

In some of the methods mentioned above, the immobilization of the cells on a surface, such as the surface of an electrochemical transducer, is required. This immobilization is carried out by adsorption, sandwich, trapping or covalent bonding techniques, among others. These techniques have the disadvantage that hinder the fixation of the cells on the surface of the transducer and its subsequent analysis. Likewise, the basic limitation of adsorption and passive entrapment techniques is the instability of the cells during their continued use. It has been shown that an increase in surface porosity favors this cell immobilization. The nanoparticles can be used to induce this increase in porosity, forming non-toxic biomimetic interfaces or matrices between the cell and the surface of the electrochemical transducer. The weak interaction between the nanoparticles and the surface of the cells provides an environment similar to a native system and allows greater freedom of orientation of the biomolecules. These polymeric matrices are formed by nanoparticles and products with a good biocompatibility and high spicy gel capacity such as chitosan. In these specific cases that there is a coating of the transducer surface, the detection of the presence of the cells is carried out by means of the correlation shown between the increase in the resistance of electron transfer and the concentration of the cells on the transducer surface.

In the case of gold nanoparticles (AuNPs), these have electroactive properties that make electrochemical detection especially indicated, thus taking advantage of the intrinsic advantages of electrochemical techniques (such as differential pulse polarography, differential pulse voltammetry, voltammetry of square wave and potentiometry) such as its speed, simplicity and low cost. Usually, this detection is carried out indirectly, dissolving them in a mixture of hydrobromic acid / bromine and subsequently detecting the resulting Au3 + solution. Lately, direct detection methods have been developed, based on an electrochemical oxidation of AuNPs to Au 3+ (cf. Pumera et al., "Direct voltammetric determination of gold nanoparticles using graphite-epoxy composite electrode", Electrochimica Acta 2005, vol. 50, pp 3702-3707) on the surface of the electrochemical transducer and subsequent in-situ detection. There are also other indirect methods based on the catalytic effect that AuNPs exert on the reduction of certain ions. In most of these cases, the sensitivity of the detection is increased by chemical or electrochemical deposition of silver on the surface of the nanoparticle.

Although these catalytic methods have a greater sensitivity than direct methods, there are still certain drawbacks, such as prolonged analysis times due to the complexity of the processes involved in the detection, or the strict control of reaction conditions, as is the case of the deposition conditions of the silver on The surface of the AuNPs.

Thus, from what is known in the state of the art, it follows that there is still a need to provide methods that provide greater sensitivity, accuracy and speed, and at the same time, require simpler and more manageable instrumentation, to improve identification. of cells and establish more quickly the diagnosis and / or prognosis of a disease.

EXPLANATION OF THE INVENTION

The researchers have found a method of identification and quantification of cells, based on the use of gold nanoparticles conjugated to specific antibodies of the surface proteins of the cells to be identified and on the use of an electrocatalytic method that measures the cathodic current generated in The evolution of hydrogen by reduction of the hydrogen ions of the medium, the reduction being catalyzed by the gold nanoparticles.

This method is advantageous because it allows the identification and quantification of cells by a rapid and sensitive method, as well as carrying out the diagnosis and / or prognosis of a disease in isolated samples of a patient susceptible to presenting the disease and its corresponding kits identification, diagnosis and / or prognosis quickly and accurately.

Thus, one aspect of the present invention is to provide a method of cell identification comprising: (a) Contacting an electrochemical transducer and the cells to be identified in an appropriate culture medium at a certain temperature and for the necessary time, to immobilize and grow the cells on the surface of the electrochemical transducer; (b) Contacting a suspension of gold nanoparticles with an antibody or combination of antibodies specific to the surface proteins of the cells to be identified; (c) Contact the immobilized cells on the surface of the electrochemical transducer with the gold nanoparticles 8 resulting from stage (b), at an appropriate temperature; (d) Apply a potential reducer for an appropriate time in an acidic medium, with which the reduction catalyzed by the gold nanoparticles 8 occurs, of the hydrogen ions of the medium to hydrogen; (e) Measure the cathodic current generated in the reduction of hydrogen ions; (f) Subtract the net value of the current generated by the electrochemical transducer without immobilized cells, from the value obtained in section (e) and correlate the difference in current intensity observed with the presence or absence of gold nanoparticles 8 attached to the surface proteins of the cells to be identified.

This method of cell identification is based on the catalytic properties of gold nanoparticles for the generation of hydrogen from hydrogen ions. Gold nanoparticles in an acidic medium at a suitable potential, catalytically reduce hydrogen ions, generating hydrogen and an associated catalytic stream. This current is measured chronoamperometrically. Chronoamperometry consists in subjecting a fixed potential to the working electrode and recording the current generated over time. For this, you can use any potentiostat-galvanostat that integrates a measurement and current recording system. Subsequently, the current intensity difference generated is correlated with the presence or absence of the cells of interest.

A potentiostat-galvanostat system allows electrochemical tests to be performed, providing a controlled potential difference and recording the current flowing through an electrochemical cell (Potentiostat mode) or providing a controlled current recording the potential difference in the cell terminals (mode Galvanostat).

The catalytic method of hydrogen generation catalyzed by metallic particles has been described above for the quantification of metal ions such as platinum (II) and gold (I) complexes. Thus, for example, gold complexes have been used in the detection of specific DNA sequences (cf. M. Díaz-González et al., "DNA hybridization biosensors using polylysine modified SPCEs", Biosensors and bioelectronics 2008, vol. 23 , pp. 1340-1346). On the other hand, gold nanoparticles have also been used as part of immunocomplexes of magnetic particles with specific conjugated antibodies and have been quantified by direct electrochemical detection, applying the differential voltammetry technique pulse (cf. A. Ambrosi et al., "double-codified gold nanolabels for enhanced immunoanalysis", Analvtical Chemistrv 2007, vol. 79, pp. 5232-5240). However, although the catalytic method of hydrogen generation for the identification of gold complexes is known, it has never been suggested for the identification and quantification of AuNPs or their application in the identification of cells.

Another advantage of the method of identification of cells of the invention is that it allows to reduce the analysis time considerably, even reaching values of 30 seconds. In this way, the diagnostic and / or prognostic methods of the invention can be carried out more quickly and efficiently. It has been observed that the net current (difference between the signal and the target) of catalysis is much more stable at short times when using the gold nanoparticles conjugated to specific antibodies of the invention with respect to the gold complexes described in the state of The technique for the purpose of generating current.

Another advantage of the cell identification method of the invention is that it allows discriminating cells that express certain surface proteins, such as HMy2 cells, in the presence of different percentages of cells that express other different surface proteins, such as PC-3 cells. 7. The presence of PC-3 7 cells does not affect the analytical signal from the recognition of HMy2 cells 6. This advantage could be used for the discrimination of tumor cells in biopsies where at least 4000 cells express a specific surface protein ( cf. FIG. 7 section B).

The terms HMY and HMy-2 (6) have been used interchangeably and refer to the same cell. Similarly, the terms PC3 and PC-3 (7) have also been used interchangeably and refer to the same cell.

A preferred embodiment is a cell identification and quantification method comprising: (a) carrying out the cell identification method of the present invention, and (b) quantifying the amount of cells by means of the following equation:

Number of cells = 1000 [(generated current (μA) - 0.497) 70.0641] The value of the recorded signal increases proportionally as the number of immobilized cells on the surface of the SPCE increases with specific antibodies conjugated with the gold nanoparticles 8. In a preferred embodiment, the immobilized cells are tumor cells and inflammatory cells.

The correlation between the number of cells and the value of the registered signal has a linear relationship in the range of 10,000 to 200,000 cells, with a correlation coefficient of 0.9955 and a detection limit of 4000 cells (cf. FIG. 7 section TO).

On the other hand, this method allows the immobilization and growth of cells on the surface of the electrochemical transducer, which also increases the sensitivity and favors the reduction of the analysis time. The detection in the same medium where the cells to be identified are immobilized and / or proliferate, facilitates the reduction of stages and as a consequence the simplification of the method.

As mentioned above, the method of the invention uses an electrochemical transducer. A transducer is a device capable of transforming or converting a certain type of energy, into a different one of output. Specifically, an electrochemical transducer is the device that measures chemical properties of substances such as pH and oxidation potentials by electrochemical means. In a preferred embodiment, the electrochemical transducer used for cell identification is a screen-printed carbon electrode (SPCE) (cf. FIG. 1 and FIG. 8 A).

In a particular embodiment, the immobilization of cells on the surface of this transducer is carried out at a suitable temperature, preferably at 37 ⁰ C.

In another particular embodiment, the step (a) of the cell identification method of the present invention is carried out in an atmosphere comprising a carbon dioxide content of 5%.

Generally, the incubation of the gold nanoparticle with the antibody specific antibodies or combination of surface proteins corresponding to step (b) of the identification method of the invention is performed at 25 ⁰ C, preferably for about 20 minutes.

In a particular embodiment, one way of increasing the sensitivity of the method consists in the amplification of the analytical signal recorded by carrying out an indirect immunoassay, using secondary antibodies. These secondary antibodies are conjugated to the gold nanoparticles and subsequently contacted with a solution of cells to be identified conjugated with specific primary antibodies against membrane proteins. The amplification in the analytical signal is due to the fact that to each primary antibody that binds to the cell surface proteins, a large number of secondary antibodies labeled with nanoparticle will be bound.

Another way to increase the sensitivity and selectivity of the method is by blocking or saturating the surface of the nanoparticle that is free of antibody. To achieve this saturation, different techniques can be used, such as increasing the concentration of the same specific antibodies or fragments thereof in step (b) or adding proteins capable of binding unspecifically to the free surface of the nanoparticles.

In a preferred embodiment, the gold nanoparticles 8 resulting from step (b), are contacted with a substance capable of saturating the surface of the antibody-free nanoparticle, such as bovine serum albumin (BSA) or casein. In a particular embodiment, BSA is used at a temperature of 25 ⁰ C for 20 minutes.

In another particular embodiment, the incubation of the cells immobilized on the surface of the electrochemical transducer with specific antibody or combination of antibodies conjugated with gold nanoparticle Ia 8 and with BSA is performed at 37 ⁰ C for 30 minutes.

Generally, the pH of the acidic medium of step (d) of the cell identification method is equal to or less than 5. Preferably, the pH is equal to or less than 3. In a preferred embodiment, the acidic medium is reached by adding an acid selected from hydrochloric acid, sulfuric acid, phosphoric acid and acetic acid. In a more preferred embodiment, the acid is hydrochloric acid. In another preferred embodiment, the sample to be identified is subjected to a reducing potential between -0.6 v and -1.4 v. Preferably, a pretreatment potential of +1.35 v is applied for 1 minute after stage (c). In a particular embodiment, the reduction potential applied is -1 v and the application time 30 seconds.

In another particular embodiment, the cathodic current generated in stage (d) is measured chronoamperometrically.

The method comprises at a later stage comparing this current generated by cells presenting the surface proteins conjugated with the nanoparticle, such as, for example, HMy2 (HLA-DR +) 6 cells as illustrated in example 7, with the current generated by The addition of a solution of 1 M hydrochloric acid to a blank without immobilized cells or to a blank of immobilized cells that do not express the surface protein conjugated with the gold nanoparticle such as PC-3 cells 7. The latter are cells of the line Tumor of prostate cancer that do not express the surface protein DR. Thus, in this specific case, the difference in intensity observed can be quickly correlated with the presence or absence of gold nanoparticles 8 conjugated with anti-DR mAb and BSA linked with the surface protein of the cells to be identified HMy2 (HLA - DR +) 6 (cf. FIG. 2).

In another preferred embodiment, the cells are selected from tumor cells and inflammatory cells that express specific proteins on the surface of the cell. Preferably tumor cells. And in another particular embodiment, the tumor cells are of the HMy2 6 tumor line that expresses HLA-DR molecules on the surface.

Another aspect of the present invention is the use of the method of identification of cells for diagnosis and / or prognosis of a disease in isolated samples of a patient susceptible to presenting the disease, where the antibody or combination of antibodies specific to stage (b) recognize specific markers of the disease to be identified. A preferred embodiment is the use of the method of identification and quantification of cells for diagnosis and / or prognosis of a disease in isolated samples of a patient susceptible to presenting the disease, where the antibody or combination of antibodies specific to stage (b) recognize specific markers of the disease to be identified.

The specific markers are substances produced by the cells to be identified or induced by the host in the presence of a disease that allow to establish the diagnosis or prognosis of a disease, monitor or verify the effectiveness of a treatment. In this specific case, the markers used must be expressed on the surface of the cell.

In a preferred embodiment, the cells to be identified are selected from tumor cells and inflammatory cells that express the specific markers of the disease on the surface of the cell. Preferably, they are tumor cells.

Another aspect of the present invention is a kit for identification of cells, diagnosis and / or prognosis of a disease in isolated samples of a patient, where the means necessary for the identification of cells comprise gold nanoparticles conjugated with the antibody or combination of antibodies 8.

In a preferred embodiment, the kit further comprises an electrochemical transducer. In a particular embodiment, the electrochemical transducer is an SPCE.

As mentioned during the present invention, gold nanoparticles are conjugated with an antibody or combination of antibodies specific to the surface protein of the cell to be identified.

By the term "antibody" is meant an entire antibody, including without limitation a chimeric, humanized, recombinant, transgenic, grafted and single chain antibody, and the like, or any fusion protein, conjugate, fragment, or derivatives thereof containing one or more domains that selectively bind to specific surface proteins. He The term "antibody" also includes an entire immunoglobulin molecule, a monoclonal antibody, or an immunologically effective fragment of any of these.

By an antibody fragment is meant an Fv, a disulfide attached to fragments of Fv, scFv, Fab, F (ab '), or F (ab') 2, which are well known in the state of the art. A fragment of an antibody represents any part thereof with a suitable size and a conformation to bind to surface proteins.

By the term "Fv" is meant a variable fragment of an antibody. The term "scFv" means a variable single chain variable fragment that corresponds to half of a Fab where only the part that provides the specificity is present. The scFv is obtained by the union of the variable part of the heavy and light chains of an antibody.

The term "Fab" means a fragment of an antibody and antigen binding. The term "F (ab ')" means the antibody fragment obtained by the digestion of an entire antibody with the enzyme pepsin and by the term "F (ab') 2" means the antibody fragment obtained by the digestion of an entire antibody with the enzyme papaϊna.

The method of identification and quantification of gold nanoparticles is also considered part of the invention, comprising: (a) Contacting a mixture of gold nanoparticles in an acid medium, with the surface of an electrochemical transducer; (b) Apply a reducing potential for an appropriate time, whereby the reduction catalyzed by the gold nanoparticles of the hydrogen ions of the medium to hydrogen occurs; (c) Measure the cathodic current generated in the reduction of hydrogen ions; (d) Subtract the net value of the current generated by the electrochemical transducer in an acidic medium, from the value obtained in section (c) and correlate the difference in current intensity observed with the presence or absence of gold nanoparticles present in the medium and / or with the concentration of gold nanoparticles present in the medium.

An advantage of this gold nanoparticle identification and quantification procedure is that the catalytic effect of gold on the nanoparticles is observed at less negative potentials and is presented in a more constant and reproducible, that when gold complexes are used. This entails a shorter analysis time to achieve a stabilization of the signal and, on the other hand, prevents damage to the electrode. In addition, due to the catalytic effect of the nanoparticles, the generated anodic current is of greater measure depending on the concentration of gold nanoparticles (cf. FIG. 6). The intensity of the current recorded by chronoamperometry during the electroreduction stage of hydrogen ions at a fixed potential, that is -1.0 v, can be correlated with the presence (cf. FIG 6A, right, curves b'-g ') or absence (cf. FIG 6A, right, curve a ') of gold nanoparticles on the surface of the SPCE. The increase in the recorded catalytic current is proportional to the increase in gold nanoparticle concentrations. In the case of gold nanoparticles conjugated with antibodies, a similar catalytic response is observed, (cf. FIG 6B, right).

In a preferred embodiment, the cathodic current generated in step (c) of the gold nanoparticle identification method is measured at 200 seconds. The absolute value of the current generated at 200 seconds is selected as an analytical signal for the quantification of the gold nanoparticles 8.

In another preferred embodiment, the number of gold nanoparticles present in the medium is quantified by the following equation: Number of nanoparticles = ant Ln [(generated current (μA) - 7.4554) / 4.7399] x1.51 -10 ⁷ .

In a particular embodiment, the quantity of sample to be quantified that is deposited on the surface of the electrochemical transducer is 25 μl.

In a preferred embodiment, the electrochemical transducer used in step (a) of the gold nanoparticle identification and quantification method is a screen-printed carbon electrode.

In a preferred embodiment, the applied reduction potential is between -0.6 v and -1.4 v and in a particular embodiment, this potential is -1 v and the application time 30 seconds. Generally, the pH of the acidic medium of stage (d) of the cell identification method is equal to or less than 5. Preferably, the pH is equal to or less than 3. In a preferred embodiment, the acidic medium is reached by the addition of an acid selected from hydrochloric acid, sulfuric acid, phosphoric acid and acetic acid. In a more preferred embodiment, the acid is hydrochloric acid.

In another preferred embodiment, the method of identification and quantification of gold nanoparticles also comprises applying a pretreatment potential after carrying out step (a). In a particular embodiment, this potential is +1.35 v and is applied for 1 minute.

In another preferred embodiment, the cathodic current generated in stage (c) is measured chronoamperometrically.

Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 represents an SPCE, used as an electrochemical transducer comprising three electrodes: 1 the silver reference electrode, 2 the 4 mm diameter carbon working electrode for a maximum sample volume of 50 μl and 3 the auxiliary electrode . The insulating layer 4 and the electrical contacts 5 are also represented.

Figure 2 represents in section A an example of the method of identification of HMY 6 cells using PC3 7 cells as blank and nanoparticles 8 conjugated to the mouse anti-human monoclonal antibody (anti-DR mAb) and BSA. Section B shows the chronoamperograms obtained by applying a potential of -1.40 v for 5 minutes by adding 50 μl of a 1 M HCI solution, where 9 represents a SPCE target with hydrochloric acid. The chronoamperogram units are represented below: t is time, s is seconds, i is current intensity and μA are microampehos.

Figure 3 represents an example of a cyclic voltamperogram recorded from +1.35 v to -1.40 a scanning speed of 50 mv / s, after adding 50 μl of a 1 M HCI solution directly on the SPCE surface (white curve IQ) or on the nanoparticle sample 8 conjugated with anti-DR mAb (curve H) and BSA. The voltamperogram units are represented below: E is potential, v is volts, i is current intensity and μA is microamps.

Figure 4 depicts microscope images of human tumor cell lines B HMY 6 and human prostate tumor cell lines PC3 7 growing in chamber (top) or in petri dish (bottom).

Figure 5 depicts photographs of the incubation system using 8-well chamber plate (A, B and C) or Petri dish (D).

Figure 6 shows in section A left examples of cyclic voltamperograms recorded from +1.35 v to -1.40 a scanning speed of 50 mv / s, after the addition of a solution of 1 M HCI on the surface of the SPCE (curve) blank a) or on increasing concentrations of nanoparticles 8 on the surface of the SPCE (concentration of nanoparticles of 0.96pM in curve b; 4.8 pM in curve c; 24 pM in curve d; 12OpM in curve e; 60OpM in curve f and 3nM in curve g).

Examples of cyclic voltamperograms recorded in the conditions mentioned in section A are shown in section B, after the addition of a 1 M HCI solution on the surface of the

SPCE (white curve a) or on a solution of nanoparticles 8 conjugated with specific anti-DR mAb antibodies (curve b).

In sections A and B of the right, the recorded chronoamperograms are applied applying a potential of -1.0Ov for 5 minutes on the surface of the SPCE (section A right curve a '), increasing concentrations of a nanoparticle solution (section A right curves b'-g ') and in a nanoparticle solution conjugated with specific anti-DR mAb antibodies (section B right curve b ').

The units are represented below: E is potential, v are volts, i is current intensity, μA are microamps, t is time, s are seconds, pM are picomoles and nM are nanomoles.

Figure 7 section A represents the effect on electrocatalytic signals of the number of cells expressing the HLA DR surface protein, after incubation with nanoparticles conjugated with specific anti-DR mAb antibodies (αDR). Section B represents electrocatalytic signals obtained with HMy2 cells 6 after incubation with gold nanoparticles conjugated with specific anti-DR mAb antibodies (αDR) in the presence of PC-3 cells 7 at different percentages, where the percentage of 100% corresponds to 200,000 cells The units are represented below: c is current, μA is microamps, BL is the target and in number of cells.

Figure 8 represents in section A the sheet with 45 SPCEs obtained after manufacturing with the screen printing machine (left) and detail of one of the SPCEs (right). Section B shows the experimental setup for performing electrochemical measurements with the camera system: eight SPCEs are mounted in the camera system and connected to the potentiostat sequentially. Section C shows a detail of the eight chamber system where the eight SPCEs are introduced for subsequent cell culture.

Figure 9 depicts scanning electron microscopy (SEM) images of the electrochemical transducer (SPCE) (left) and details of the HMy2 6 (section A) and PC-3 7 (section B) cells that have grown on their surface. The images that appear in the upper right corners correspond to cells that have grown on the plastic surface of the SPCEs. It is verified that the cells have grown on carbon and that their morphology is not affected on this material. The units are represented below: mm is millimeters and μm is micrometers.

Figure 10 section A represents the comparative study of the response Electrochemical obtained for the HMy2 6 and PC-3 7 cell lines against the different antibodies tested. The αlgM and αDR antibodies are those that have been conjugated with gold nanoparticle. The first three columns correspond to the target and direct tests and the last two columns correspond to the indirect tests. Section B represents the response obtained by flow cytometry for the two cell lines against the antibodies used. The specificity that had been obtained with the electrocatalytic method is corroborated. The units are represented below: c is current, μA are microamps, BL is the target, Ab are the antibodies tested and fi /% is a percentage of fluorescence intensity.

EXAMPLES

General considerations

Hydrogen Tetrachloroaurate (III) Trihydrate (HAuCI ₄ -3H ₂ O, 99.9%) and trisodium citrate were purchased from Sigma-Aldrich. All buffer reagents and other inorganic chemicals were supplied by Sigma, Aldrich or Fluka, unless otherwise indicated. All chemicals were used as received and all aqueous solutions were prepared with double distilled water. The phosphate solution buffer (PBS) consists of 0.01 M buffered saline phosphate, 0.137 M NaCI and 0.003 M KCI (pH 7.4). Hydrochloric acid (HCI) is quality analysis. Their solutions were prepared with ultra-pure water. The tumor cell lines used in this study were HMy2 6 (HLA-DR +) and PC-3 7 (HLA-DR-). All cells were cultured in RPMI 1640 medium (Gibco, Life technologies, Grand Island, Scotland) supplemented with 10% fetal bovine serum (FCS) (PAA, Linz, Austria), penicillin (100 U / mi) and glutamine ( 2mM) (Gibco) at 37 ⁰ C in a humidified atmosphere containing 5% CO _2. The measurements of the expression of HLA-DR were carried out using a mouse anti-human HLA-DR monoclonal antibody (mAb) (Immunotech, Marseille, France) and BH1, a human IgM mAb antibody that recognizes human HLA class II molecules in The surface of human monocytes, B lymphocytes, tumor cell lines B (HMy2, Raji and Dausi) and tumor cells of patients suffering from hematological malignancies. As a positive recognition control of the cells tested, human mAbs 32.4 are used. In the case of BH1 and 32.4 mAbs, rabbit anti-human IgM polyclonal antibodies conjugated to fluorescein isothiocyanate (FITC) (DakoCytomation, Spain) are used as secondary antibodies, allowing the electrochemical method to be checked with flow cytometric measurements.

The cells were visualized with inverted and direct microscopes (Olympus 1X50 and BX51 respectively, Olympus Optical. Tokyo, Japan) and the photographs were taken with an Olympus DP71 camera.

The electrochemical transducer used for in-situ cell growth was constructed by the researchers and was an SPCE. The substrate used is a transparent polyester sheet (Austostat HT5 of the McDermid Autotype company) and the total sensor size was 29 mm x 6.7 mm (cf. FIG. 1).

The generated current was recorded by chronoamperometry, using a Ivium Compactstat potentiostat-electroplating (Ivium Technologies, The Netherlands).

Example 1: Cyclic voltamperograms in 1 M HCI.

50 μl of 1 M HCI was added to the SPCE surface and a cyclic voltamperogram was recorded from +1.35 v to -1.40 v, at a scanning speed of 50 mv / s, obtaining a blank curve (curve 10, FIG. 3 and curve a, FIG. 6A). On the other hand, 25 µl of a 2M HCI solution was mixed with 25 µl of an AuNPs solution at increasing concentrations. The resulting mixtures were deposited on the surfaces of other SPCEs. Again, cyclic voltamperograms were recorded from +1.35 v to -1.40 v, at a scanning speed of 50 mv / s, obtaining the IJ curves., FIG. 3 and curves b-g, FIG. 6A, where a potential shift is observed with respect to the curve of the target corresponding to the catalytic effect of the nanoparticles and which increases with the concentration of these.

The same experiments were performed for AuNPs 8 conjugated with anti-DR mAb and BSA, again observing a potential shift between the blank of 1 M HCI (IQ curve, FIG. 3 and curve a, FIG. 6B) and the dissolution of AuNPs 8_conjugated with antibody (curve V \, FIG. 3 and curve b, FIG. 6B). Example 2: incubation of cells in flask.

Cell lines HMy2 6 and PC-3 7 cultured in a culture medium at 37 ⁰ C in a humidified atmosphere containing 5% CO2 for 48 h in a flask tissue culture (Falcon, Belcton Dickinson and Company Franklin Lakes, NJ, USA).

Example 3: Incubation of cells on the surface of the SPCE in chamber.

Working electrode 2 was introduced into a Lab-Tek ™ with 8-well plates (Nunc, Thermo Fisher scientific), after removing the joint, it was fixed with the mounting culture medium. Then each well was added 700 .mu.l 200,000 cells in culture medium and incubated at 37 ⁰ C in a humidified atmosphere containing 5% CO2 for 48 h. (cf. FIG. 4 (top) and 5 (A, B and C) and FIG. 8 (B ₁ C). Figure 9 shows the two cell lines grown on the surface of the SPCE.

Example 4: Incubation of cells on the surface of the SPCE in Petri dish.

SPCEs were introduced into the Corning® petri dishes, DxH35 mm x 10 mm, untreated (SIGMA-ALDRICH). Then 200,000 cells were added 3 ml of culture medium and incubated at 37 ⁰ C in a humidified atmosphere containing 5% CO2 for 48 h (cf. FIG. 4 (bottom) and 5 D).

Example 5: Synthesis of gold nanoparticles and their conjugation with specific antibodies.

The nanoparticles were prepared by reducing hydrogen tetrachloroaurate (III) with trisodium citrate, according to the method described by Turkevich (cf. J. Turkevich et al., Faradav Discussion of the Chemical Society, 1951, vol. 11, pp. 55-75) . To a solution of 200 ml of 0.01% boiling HAuCI ₄ with vigorous stirring, 5 ml of a trisodium citrate solution was quickly added. When the reaction mixture turned deep red, indicating the formation of the gold nanoparticles, the solution was cooled while maintaining the stirring. 2 ml of the mixed suspension of gold nanoparticles with 100 .mu.l (100 .mu.g / ml) of anti-DR mAb and incubated at 25 ⁰ C for 20 minutes. Then a step 150 .mu.l lock (1 mg / ml) of bovine serum albumin (BSA) , maintaining the temperature at 25 ⁰ C for 20 minutes was performed. Finally, the resulting mixture was centrifuged at 14,000 rpm for 20 minutes and nanoparticles 8 conjugated with anti-DR mAb and BSA were reconstituted with PBS.

For indirect tests, nanoparticles were conjugated in a similar manner with anti-IgM antibodies (αlgM) and with BSA.

Example 6: A. Incubation of cells with nanoparticles 8 conjugated with anti-DR mAb v BSA.

Once the cells had grown on the surface of the SPCE, they were washed with PBS. Then 50 μl of a solution of nanoparticles 8 conjugated with anti-DR mAb and BSA were added on the working electrode 2 and kept for 30 minutes at 37 ^° C.

B. Incubation of cells with nanoparticles 8 conjugated to αlgM and BSA.

Once the cells had been incubated with the primary antibodies (BH1 or 32.4 mAbs) for 30 minutes, they were washed with PBS. It was then incubated with a solution of nanoparticles 8 conjugated with αlgM and BSA for 30 minutes.

Example 7: Analytical signal based on the generation of hydrogen catalyzed by nanoparticle 8 conjugated with anti-DR mAb and BSA and, recording of the generated current.

The cells were washed with PBS and then 50 μl of a 1 M HCI solution was added on the surface of the 3 electrodes I ₁ 2 and 3. Then, the SPCE was connected to the potentiostat (cf. FIG. 8 B ₁ C) and the working electrode 2 was subjected to a first potential of +1.35 v for 1 minute and then a second potential of -1 v was applied for 300 seconds, registering the generated cathodic current. This generated current is recorded by chronoamperometry. For this, you can use any potentiostat-galvanostat that integrates a measurement and current recording system. In this case, an Ivium Compactstat is used.

Although in this example the reduction potential is applied for 300 seconds, the application time could be reduced up to 30 seconds, since from 30 seconds the cathodic current generated by the reduction of hydrogen ions is stabilized over time, such and as seen in Figure 2B.

Example 8. Analytical signal based on the generation of hydrogen catalyzed by the nanoparticle conjugated with anti-IgM and BSA and recording of the generated current (indirect test).

A first incubation of the cells with primary antibodies (BH1 or 32.4) was performed in a similar manner to that detailed in Example 7. Subsequently it was washed with PBS and a second incubation was performed under identical conditions with nanoparticles conjugated with anti-IgM and BSA. . The recording of the analytical signal was carried out following the same procedure detailed in example 7. The electrocatalytic responses are shown in Figure 10 A.

REFERENCES MENTIONED IN THE APPLICATION

1. Pumera et al., "Direct voltammetric determination of gold nanoparticles using graphite-epoxy composite electrode", Electrochimica Acta, 2005, vol. 50, p. 3702-3707. 2. M. Díaz-González et al., "DNA hybridization biosensors using polylysine modified SPCEs", Biosensors and bioelectronics, 2008, vol. 23, p. 1340-1346.

3. A. Ambrosi et al., "Double-codified gold nanolabels for enhanced immunoanalysis", Analvtical Chemistrv, 2007, vol. 79, p. 5232-5240.

4. J. Turkevich et al., Faradav Discussion of the Chemical Society, 1951, vol. 11, p. 55-75.

Claims

1. Method of identification of cells comprising:

(a) Contact an electrochemical transducer and the cells to be identified in an appropriate culture medium at a certain temperature and for the time necessary, to immobilize and grow the cells on the surface of the electrochemical transducer;

(b) Contacting a suspension of gold nanoparticles with an antibody or combination of antibodies specific to the surface proteins of the cells to be identified;

(c) Contact the immobilized cells on the surface of the electrochemical transducer with the gold nanoparticles (8) resulting from stage (b), at an appropriate temperature;

(d) Apply a reducing potential for an appropriate time in an acidic medium, whereby the reduction catalyzed by the gold nanoparticles (8) of the hydrogen ions of the medium to hydrogen occurs;

(e) Measure the cathodic current generated in the reduction of hydrogen ions;

(f) Subtract the net value of the current generated by the electrochemical transducer without immobilized cells, from the value obtained in section (e) and correlate the difference in current intensity observed with the presence or absence of gold nanoparticles (8) attached to the surface proteins of the cells to be identified.

2. Identification method according to claim 1, wherein the electrochemical transducer is a screen-printed carbon electrode.

3. Identification method according to any of claims 1-2, wherein the reduction potential is between -0.6 v and -1.4 v.

4. Identification method according to any of claims 1-3, wherein the acidic medium is reached by the addition of an acid selected from the group consisting of hydrochloric acid, sulfuric acid, phosphoric acid and acetic acid.

5. Identification method according to claim 4, wherein the acid is hydrochloric acid.

6. Identification method according to any of claims 1-5, which further comprises contacting the nanoparticles resulting from step (b) with a substance capable of saturating the surface of the nanoparticle.

7. Identification method according to any of claims 1-6, which further comprises applying a pretreatment potential after carrying out step (c).

8. Identification method according to any of claims 1-7, wherein the cathodic current of stage (d) is measured chronoamperometrically.

9. Identification method according to any of claims 1-8, wherein the cells are selected from tumor cells and inflammatory cells.

10. Identification method according to any of claims 1-9, wherein the cells are tumor cells.

11. Method of identification and quantification of cells comprising: (a) carrying out the method of identification of cells of any of claims 1-10, and (b) quantifying the amount of cells by means of the following equation: Number of cells = 1000 [(generated current (μA) - 0.497) /0.0641]

12. Use of the cell identification method according to any of claims 1-10, for diagnosis and / or prognosis of a disease in isolated samples of a patient susceptible to presenting the disease, where the antibody or combination of specific antibodies of the stage (b) recognize specific markers of the disease to be identified.

13. Use of the cell identification method according to claim 12, for diagnosis and / or prognosis of a disease, wherein the cells to be identified are selected from tumor cells and inflammatory cells.

14. Use of the method of identification and quantification of cells according to claim 11, for diagnosis and / or prognosis of a disease, in isolated samples of a patient capable of presenting the disease, where the antibody or combination of antibodies specific to the stage ( b) recognize specific markers of the disease to be identified.

15. Use of the method of identification and quantification of cells according to claim 14, for diagnosis and / or prognosis of a disease, wherein the cells to be identified are selected from tumor cells and inflammatory cells.

16. Kit for identification of cells, diagnosis and / or prognosis of a disease in isolated samples of a patient, where the necessary means for the identification of cells comprise gold nanoparticles conjugated with the antibody or combination of antibodies (8).

17. Kit according to claim 16, which further comprises an electrochemical transducer.

18. Method of identification and quantification of gold nanoparticles comprising:

(a) Contact a mixture of gold nanoparticles in an acid medium, with the surface of an electrochemical transducer;

(b) Apply a reducing potential for an appropriate time, whereby the reduction catalyzed by the gold nanoparticles of the hydrogen ions of the medium to hydrogen occurs;

(c) Measure the cathodic current generated in the reduction of hydrogen ions;

(d) Subtract the net value of the current generated by the electrochemical transducer in an acidic medium, from the value obtained in section (c) and correlate the difference in current intensity observed with the presence or absence of gold nanoparticles present in the medium and / or with the concentration of gold nanoparticles present in the medium.

19. Identification and quantification method according to claim 18, wherein the electrochemical transducer is a screen-printed carbon electrode.

20. Method of identification and quantification according to any of claims 18-19, wherein the reduction potential is between -0.6 v and -1.4 v.

21. Identification and quantification method according to any of claims 18-20, wherein the acidic medium is reached by the addition of an acid selected from the group consisting of hydrochloric acid, sulfuric acid, phosphoric acid and acetic acid.

22. Identification and quantification method according to claim 21, wherein the acid is hydrochloric acid.

23. Identification and quantification method according to any of claims 18-22, which further comprises applying a pretreatment potential after carrying out step (a).

24. Method of identification and quantification according to any of claims 18-23, wherein the cathodic current of stage (c) is measured chronoamperometrically.

25. Method of identification and quantification according to any of claims 18-24, wherein the cathodic current of stage (c) is measured at 200 seconds.

26. Identification and quantification method according to claim 25, wherein the number of gold nanoparticles present in the medium is quantified by means of the following equation:

Number of nanoparticles = ant Ln [(generated current (μA) - 7.4554) /4.7399] x1.51 -10 ⁷