ES2303495B1 - SELECTIVE BIOSENSOR TO METALOTIONEINS. - Google Patents

Abstract

Selective biosensor to metallothioneins.

The present invention is encompassed within the field of electrochemistry and generally refers to ion selective electrodes and sensors, more specifically, to selective electrodes to metallothioneins, to the construction method of said electrodes and the method that employs said electrodes to the quantification of different isoforms of metallothioneins in samples of biological origin.

Description

Selective biosensor to metallothioneins.

The present invention is encompassed within the field of electrochemistry and generally refers to ion selective electrodes and sensors, more specifically, to selective electrodes to metallothioneins, to the construction method of said electrodes and the method that employs said electrodes to the quantification of different isoforms of metallothioneins in samples of biological origin.

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State of the art

Metallothioneins (MTs) are low proteins molecular mass (6 to 7 kDa), present in all organisms and capable of joining heavy metals. They show a high content of thiol groups from the amino acid cysteine, which constitutes the 33% of the molecule. Currently more than 200 MTs have been described belonging to a hundred organisms. Specifically in mammal, four isoforms of MTs (numbered from 1 to 4) and from MT1 to 13 subisoforms. MT-1 and MT-2 is synthesized in almost all tissues of the organism, its presence being particularly important in parenchymal organs such as liver, kidney, intestine, testicles, lung, heart and brain. The MT-3 is predominantly located in the brain and in smaller quantities in pancreas and intestine, male and female reproductive tract, stomach, heart and kidney. MT-4 is detected in stratified squamous epithelia.

Gene expression studies have shown that these proteins are synthesized in response to a wide variety of physiological and chemical stimuli, such as: hormones, cytokines, reactive oxygen species, stress and, importantly, metals They have recently been linked to stress processes inflammatory, especially during an acute phase response, although its function in these processes is still to be defined.

The quantification of metallothioneins has been a challenge that has led to the development of a large number of Methods for determination. The identification and characterization Structural structures of the MTs are essential processes to clarify the specific functions that these proteins develop in the organism and the role they play in relation to external factors, as the presence of heavy metals.

So far, techniques for the total quantification of MTs have been developed ( M. Sato, KT Suzuki, Biomed. Res. Trace Elements 6 (1995): 13 ). Thus, electroanalytical techniques, UV-Vis spectrophotometers, metal saturation assays and immunological methods have been used to determine these proteins (Table 1).

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TABLE 1 Techniques for determining metallothioneins GFC-FPLC-ETAAS: gel filtration chromatography; liquid chromatography for rapid protein separation; atomic absorption spectrometry electrothermal; IEC-ETAAS: chromatography of ion exchange; atomic absorption spectrometry electrothermal; IE- HPLC-FAAS: chromatography high resolution liquid ion exchange; spectrometry of atomic absorption of flame; SPE-CAC-TDI gels-AAS: solid phase extraction for covalent affinity chromatography with exchange thiolate disulfide; absorption spectrometry atomic; HPLC: high performance liquid chromatography; DPP: Differential pulse polarography; ELISA: enzyme immunoassay

one

There are two types of approaches: the methods indirect and direct. The use of electroanalytical methods to the quantification of the MTs is based on the redox properties of the complexes formed by the thiol groups of the proteins with the metal ions The saturation capacity of the molecule with metal ions have been exploited by the methods of indirect quantification of MTs, such as tests of metallic affinity.

The colorimetric properties of different Reagents in combination with protein thiolate groups are the basis of spectrophotometric determinations. Of the same way, immunoreactivity with specific antibodies has been used by immunological methods.

A step forward in the analysis of MTs it consists of the detection of the different isoforms present in Same sample. With this objective, techniques of separation such as capillary electrophoresis (EC) and chromatography High resolution liquid (HPLC), typically linked to detectors of specific elements, for the indirect quantification of MTs

The heterogeneity of the samples coming of tissues, biological fluids or cell cultures, requires strategies that imply different approaches, adaptation of an existing methodology or the development of new protocols.

The methods discussed (Table 1) for determination of the MTs present difficulties that make the Quantification of these is an awkward task. Also, in the protocols of most methods, two problems appear implicit in the nature of the protein: the oxidation of MTs and the polymorphism they present.

The metal-MT complexes contents in biological samples can be obtained after isolation and purification processes. Whether the protein is fully saturated with metal as if it is not, the high presence of thiol groups can lead, in aerobic conditions, to the oxidation of these and, consequently, of the protein by formation of intra disulfide bonds or inter-molecular during the isolation process or during sample handling.

In relation to the problems derived from MT polymorphism, the large number of isoforms and subisoforms of these proteins makes the application of the techniques Immunological in biological samples is difficult. To increase the sensitivity and selectivity of these methods, the specificity of the antibodies is very important. The absence of samples MT pattern, makes it difficult to compare results between different laboratories, because each one has its own method of isolation.

Description of the invention

In order to overcome difficulties associated with the methods currently employed, the authors of the present invention have directed their research to development of an ion selective electrode (ISE) for the quantification of MTs, since at present there is no selective electrode to These metalloproteins.

Ion selective electrodes are sensors electrochemicals that, thanks to the electrostatic potential that acquires the ion selective membrane when it is placed in contact with a solution, allow quantitatively analyze the analyte to which the membrane is selective. This selectivity comes conferred by a chemical species (ionophore) that interacts with Selectively with the analyte to be determined.

Until now, the most important polymeric material of the employees as matrix in the development of ISEs, was PVC. Other alternative materials such as silicone rubber, epoxy polyurethanes and modified PVC have been tested ( Moody, GJ et al . Analyst, 112 (1987), p.1143 and Cha, SG et al . Anal. Chem., 63 (1991), p.1666 ) to improve the adhesion on the surface of solid state devices or to reduce the adsorption of macromolecules from analytical samples. Despite this, PVC is still the most widely used polymer in the preparation of membranes for ISEs.

However, the incorporation of biological material in PVC membranes is not easy due, basically, to the use of organic solvents (usually tetrahydrofuran), in which proteins (enzymes, antibodies, etc.) dissolve poorly, which usually alters their activity or properties ( Cha, SG et al . Anal. Chem., 63 (1991), p.1666 ).

Currently, the use of polysulfone membranes, PS, (I) as a polymer matrix has opened up great possibilities in solid contact electrodes ( G. Harsanyi, Polymers Films in Sensor Applications, TECHNOMIC publication, Lancaster 1995 ). The main advantage over PVC is that the polysulfone allows incorporating, in addition to the ionophores that give the potentiometric selectivity to the membrane, biological materials (enzymes, antibodies, etc.) from an aqueous phase.

2

In the present invention, the incorporation of the biological material to the membrane takes place by means of the phase inversion technique, that is, the water penetrates the polysulfone inducing its precipitation by displacement of the dimethylformamide (DMF) where it dissolves ( Prieto-Simón et al . Biosens. Bioelectron. 2006, 22, 131-137; Prieto-Simón et al . Biosens. Bioelectron. 2007, 22, 2663-2668; Prieto-Simón et al . Talanta, 2007, 71, 2102-2107; Sánchez , S. et al . Biosens. Bioelectron., 2007, 22, 965-972; Sánchez, S. et al . Analyst., 2007, 132, 142-147 .; Sánchez, S. et al . Biosens. Bioelectron. 2007 ; Nunes, SP et al . Membrane Technology in the Chemical Industry; WILEY-VCH Verlag GMBH, D-69469, Weinheim, Germany, 2001 ).

This immobilization technique maintains the virtually intact proteins throughout the process, since they always remain in aqueous medium at a controlled pH. Further, By this procedure, the amount of protein required is reduces significantly as it stays together mainly to the outer layers of the membrane.

In recent years and based on their coordinating characteristics, some MTs and other polypeptides that interact with metals have been used for the construction of metal ion biosensors, but always being limited to capacitance sensors ( Bontidean, I. et al . Biosens. Bioelectron. 2003, 18, 547-553; Bontidean, I. et al . Anal. Chem. 1998, 70, 4162-4169; Corbisier, P. et al . Anal. Chim. Acta 1999, 387, 235-244 ). These show low selectivity and require regeneration with EDTA after each use. On the other hand, in WO2006045103, a device based on the use of MTs on a solid support is described in order to selectively remove heavy metals from contaminated liquid samples. In this application, the MTs are incorporated into a commercial membrane by adsorption (Pall biodyne).

Now, the authors of the present invention have developed selective potentiometric electrodes to MTs based in a polysulfone membrane that incorporates different metallothioneins that act as ionophores. These electrodes are selective to specific isothorms of metallothionein, depending on the molecule chosen as ionophore. Also, these electrodes allow the quantification of different metallothionein isoforms and their employment in a wide variety of samples that do not require a special pretreatment

These devices, besides being measured Direct for MTs, they have very significant advantages in as to time of analysis with respect to the methods described in The state of the art. They have a good selectivity and not require neither incubation nor regeneration processes.

They use very simple instrumentation, which allows field measurements, as well as the construction of microelectrodes for the quantification of MTs in cells individual. In addition, the electrode of the present invention can Stored dry and shows a long life time.

Finally, the developed electrode presents the following advantages, typical of chemical sensors, compared to the laboratory equipment currently employed in the MT determination:

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small devices and robust,

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fast answer,

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mass production at low cost,

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procedure integration analytical,

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allows monitoring of parameters and applications in situ , and

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portability. This is an advantage especially remarkable since it allows field measurements no need to transfer the sample to a laboratory (avoiding possible alteration of the samples).

First, the present invention contemplates a selective metallothionein electrode (MTs) comprising a metallothionein, which acts as an ionophore, incorporated inside of a polysulfone (PS) membrane.

Also a subject of the invention is a method for the construction of said selective electrode to metallothioneins.

Finally, a method of the invention is for the quantification of different metallothionein isoforms in samples of biological origin.

In one aspect of the present invention, contemplates a selective electrode to metallothioneins (MTs) that it comprises a metallothionein, which acts as an ionophore, incorporated inside a polysulfone (PS) membrane, which allows incorporate biological material inside.

Metallothioneins are characterized by their low molecular weight and high metal ion binding capacity heavy through the high cysteine content (33%) of its primary structure The electrodes contemplated herein invention use metallothioneins as ionophores, resulting selective themselves, even with the ability to differentiate between the different MT isoforms of the same organism.

In a particular embodiment, the ionophore used comprises a metal complex of said metallothionein (P_ {n} -MT), where P is a heavy metal, preferably zinc.

In a preferred embodiment, the electrode presents selectivity to the mammalian MT1 isoform. In this case The ionophore used is the metal complex Zn_ {7} -MT1. Said electrode allows quantification the MT1 of a given sample although other ones are present mammalian isoforms (such as MT2, MT3 and MT4) that could interfere as far.

In another aspect of the invention the method for the construction of selective electrodes a metallothioneins object of the invention comprising the following stages:

to)

preparation of a solution of PS and N, N'-dimethylformamide (DMF);

b)

preparation of a solution of the species that will act as an ionophore;

C)

PS / DMF solution deposition prepared in a) on the surface of the electrode body; Y

d)

incorporation of the ionophore in the PS membrane by immersing the electrode in the solution prepared in b) so that through the phase inversion technique of place to the final biomembrane.

Thanks to the phase inversion technique, the ionophores are incorporated into the PS membrane without manipulating them, by directly immersing the electrode in the protein solution of the MT considered. The electrodes so constructed do not require any conditioning stage with no solution. The dissolution of protein can be scarce and / or expensive so the absence of a conditioning stage involves economic advantages.

In a particular embodiment, the concentration of the solution obtained in a) is between 50 and 150 mg PS per mL DMF, preferably between 75 and 125 mg PS / mL DMF

In another particular embodiment, the species used as an ionophore in the solution of stage b) is the Zn_ {7} -MT1 molecule, at a concentration between 10-7 and 10-3 mol / L, preferably between 5 · 10-5 and 5 · 10-4 mol / L.

The electrodes described can quantify different metallothionein isoforms in samples of origin biological, operation that includes the following stages:

i.

Calibrate the electrode with solutions Pattern;

ii.

Contact the sample at analyze with the electrode, and,

iii.

Quantify metallothionein present in the sample problem through measurements potentiometric

In a particular embodiment, the calibration of the electrode can be carried out by a procedure that understands:

(one)

immerse the working electrode and the reference in a known volume of H2O;

(2)

make microadditions of a solution of known concentration of the complex P_ {n} -MT (P being a heavy metal) of the metallothionein to quantify (depending on the electrode we are calibrating) that allows to obtain readings in a range of concentrations that include the test sample; Y

(3)

represent the potential (in mV) supplied by the electrochemical cell by varying the concentration of P_n -MT of the solution using a linear regression (based on the Nernst equation (E = A_ {1} + B_ {1} \ cdot log C) to adjust the values of potential obtained with respect to the logarithm of the concentration of P_ {n} -MT.

Figure 1 shows a calibration curve Typical for anions.

Once the electrode is calibrated and the calibration curve, by putting the electrode in contact with the problem sample you get an analytical signal (potential, mV) that interpolates into the calibration line to obtain the Metallothionein concentration to be quantified.

Since metallothioneins are found in the cells of all organisms of the animal and plant kingdoms, any type of sample of biological origin (that comes from these organisms, living or dead) is potentially valid to be quantified by the proposed method. Of special interest They are:

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Organisms (aquatic or terrestrial) that can act as biomarkers of metal contamination by the accumulation of these metals in their tissues (mollusks, some fish, etc.)

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Tissue of the central nervous system involved in pathologies such as Alzheimer's and some types of sclerosis.

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Tissue of liver and kidney related poisoning by metals

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Fabrics of a wide variety of organs as tumor markers.

Brief description of the figures

Figure 1. Calibration curve and key parameters of the characterization of an anion selective electrode . LD: Limit of detection; LIRL: Lower limit of linear response. Log C: Logarithm of the analyte concentration. (1) Null response zone; (2) Nonlinear or subnerntian zone; (3) Linear or Nerntsian zone.

Figure 2. Construction of the ion selective electrode body of conventional configuration : (A) Copper connector and plate; (B) Welding perpendicular from the copper plate to the connector; (C) Insertion of the connector into the PVC pipe; (D) Filling with conductive resin. (E) Final scheme of the constructed electrode.

Figure 3. Procedure of deposition of the polysulfone membranes in the selective electrodes to MTs . (A) Electrode body with the conductive surface. (B) Deposition of the PS / DMF solution (C) Immersion of the electrode in the ionophore solution (D) Polysulfone membrane deposited on the surface of the electrode; (1) Drop of PS / DMF (2) Ionophore.

Figure 4. Calibration of a selective electrode to the mammalian MT1 isoform in water.

Figure 5. Calibration of a selective electrode to the mammalian MT1 isoform in water and in an aqueous solution of MT4 of mammal of constant concentration. (A) Water calibration (B) Calibration in the presence of MT4.

Detailed description of particular embodiments materials

Polysulfone (PS) BASF 3010 natur was used (Frankfurt, Germany) as a polymer matrix and was chosen N, N-dimethylformamide (DMF) from Panreac (Barcelona, Spain). All other reagents used were grade analytical and were obtained from Merck (Darmstadt Germany). It was used Milli-Q (Bedford, MA, USA) throughout the trial. For the preparation of the electrode, graphite powder was obtained from Merck, epoxy resin ARALDIT M from Uneco (Badalona, Spain) and the hardener HY 5162 by VANTICO (Barcelona, Spain).

Biosynthesis of metallothioneins

Recombinant mammalian MT1 synthesized from a DNA construct that allows its production in Escherichia coli bacteria was used. Cultures of bacteria transformed with this construct produced a fusion protein MT-GST (MT-Glutathione-S-Transferase), which was recovered and digested with thrombin, a procedure that allowed the subsequent purification of metallothioneins as independent proteins. The supplement of a certain metal in the bacterial culture medium allowed the direct recovery of the MT in the form of the corresponding metal complex.

Methods Construction of the ion selective electrode body of conventional configuration

In a cylindrical PVC body, 20 mm long and 6 mm internal diameter (Figure 2.C), a female connection (2 mm) to which he had welded perpendicularly a copper plate (2.5 mm radius) (Figures 2.A and 2.B). The metal screw that screwed the connection It allowed it to be fixed inside the PVC pipe. The cavity that was exposed between the plate and the body of the electrode, filled with conductive paste epoxy graphite (1: 1), (epoxy: Araldit M- hardener HR (1: 0.4)) (Figure 2.D), avoiding the formation of bubbles of air. It was allowed to dry in the oven at 40 ° C for 24 hours. Subsequently, the surface was polished producing a depression of 0.3 mm where the membrane would be deposited.

Construction of metalotionein selective electrodes

A selective electrode was constructed at mammalian MT1 isoform. The selective ISE to MT1 incorporated the Zn_ {7} -MT1 species inside the membrane of PS acting as an ionophore.

For the preparation of the selective membrane a MT1, 100 mg of PS per mL of DMF was dissolved in a vial. In other vial, the protein solution (ionophore solution) was diluted to a concentration of 5 · 10-5 M (Table 2). A deposit was deposited drop of PS / DMF solution prepared on the body surface of the constructed electrode that precipitated immediately upon immersion in the protein solution (Figure 3).

TABLE 2 Composition of the selective biosensor membrane a MT1

3

Measurements The potentiometric measurements were carried out using a digital potentiometer, Crison pH 2002, with ± 0.1 mV resolution (Alella, Spain) The readings were controlled by computer, using a serial communication line (RS232C) connected to a Pentium 4 with A specially developed software. The software was developed by TMI (Alella, Spain). As a reference electrode, an Orion Termo Ag / AgCl double junction electrode was used.

The calibration parameters were obtained from the construction of the calibration curve in the range 5.0 • 10-8 - 5.7 • 10-6 of MT1. (Figure 4). The data obtained were represented as potential versus logarithm of the concentration of MT1.

Once the electrode is calibrated, by putting it in contact with the problem sample, an analytical signal was obtained (potential, in mV) that was interpolated in a calibration curve to obtain the concentration of the MT1 isoform (3.1 · 10-7 mol / L of MT1).

In the study of interference, that is, at calibrate the electrode in the presence of another metallothionein (in this case of the mammalian MT4 isoform) it was observed that it affected little significant way to the behavior of the selective ISE to MT1. This can be seen in Figure 5, where the two curves are shown of calibration (with and without the presence of interference).

The analytical response characteristics of the selective electrode to MT1 observed were:

one)

Limit of detection: can be detected MT1 above 1.0 · 10-7 M,

2)

Limit of quantification: you can quantify MT1 above 1.0 · 10-6 M,

3)

The Electrode sensitivity is -52 mV / dec,

4)

He electrode response time is few seconds,

5)

He electrode life is at least 1 month,

6)

He pH range in which the electrode is able to detect and quantify MT1 is broad (2-7),

7)

The Interference study results indicate that the electrode is able to detect MT1 in the presence of MT4 (which acts as interfering) even if the concentration of MT4 is twice that of of MT1, and

8)

He electrode is stored dry and at 4 ° C.

Claims (11)

1. Metalotionein selective electrode (MTs) which comprises a metallothionein, which acts as an ionophore, incorporated into a polysulfone (PS) membrane. 2. Electrode according to claim 1, wherein the ionophore used comprises a metal complex of said metallothionein (P_ -MT), where P is a metal heavy. 3. Electrode according to claim 2, wherein The heavy metal is Zn. 4. Electrode according to claim 2, wherein MT metallothionein is mammalian MT1. 5. Electrode according to claim 2, which is selective to mammalian MT1 metallothionein, and where the ionophore used comprises the zinc complex of said metallothionein (Zn_ {7} -MT1). 6. Method for electrode construction defined in any one of claims 1 to 5 comprising the following stages:

to)

preparation of a solution of PS and DMF;

b)

preparation of a solution of the P_ {n} -MT species that will act as ionophore;

C)

PS / DMF solution deposition prepared in a) on the conductive surface of the body of the electrode; Y

d)

incorporation of the ionophore in the PS membrane by introducing the electrode into the solution prepared in b).

7. Method according to claim 6, wherein the concentration of the PS / DMF solution obtained in a) is between 50 and 150 mg / mL. 8. Method according to claim 7, wherein the concentration of the PS / DMF solution is between 75 and 125 mg / mL. 9. Method according to claim 6, wherein the concentration of the P n -MT solution obtained in b) is between 10 -7 and 10 -3 mol / L. 10. Method according to claim 9, wherein the concentration of the solution of P n -MT is between 5 · 10-5 and 5 · 10-4 mol / L. 11. Method for quantifying different metallothionein isoforms in samples of biological origin that It comprises the following stages:

i.

calibrate the electrode of any of claims 1-5, by solutions Pattern,

ii.

contact the sample at analyze with the electrode, and

iii.

quantify metallothionein present in the sample by measurements potentiometric