WO2020250190A1

WO2020250190A1 - Device and method for the rapid dosage of copper ion and other heavy metals on micro-volumes of human capillary blood and other biological fluids

Info

Publication number: WO2020250190A1
Application number: PCT/IB2020/055524
Authority: WO
Inventors: Ilaria SERNESI; Riccardo Ciuti; Gianfranco Liguri; Serena LASCHI
Original assignee: Igea Pharma Nv
Priority date: 2019-06-14
Filing date: 2020-06-12
Publication date: 2020-12-17
Also published as: IT201900009060A1

Abstract

The present invention relates to a device and a method for the electrochemical determination of copper and other heavy metals in human capillary blood and in other biological fluids.

Description

"Device and method for the rapid dosage of copper ion and other heavy metals on micro-volumes of human ca pillary blood and other biological fluids"

DESCRIPTION

Background art

The determination of the levels of metals pre sent in a person's biological fluids has a wide rele vance in diagnosis and prevention, where said levels are known to impact a wide range of pathological con ditions. By way of example, serum copper levels are relevant in pathologies of the cognitive process such as Wilson's disease, Menkes disease and Alzheimer's disease .

In both the oxidation state I and II, the copper ion is present in blood plasma and other biological fluids rich in proteins distributed in different frac tions distinct from each other for the state of asso ciation with different ligands. The copper in serum and plasma is mostly strongly linked to apoceruloplas- min, a transporter plasma protein that can bind 6 cop per ions and which in physiological conditions carries about 70% of the circulating copper.

The remainder, called non-ceruplasmin copper (NCC) , is mainly represented by exchangeable copper

(CuEXC) , also known as a labile fraction, in which the ion can be weakly bound to albumin, transcuprein, small peptides, amino acids and other ligands (Journal of Trace Elements in Medicine and Biology 45 (2018) 176- 180) . The relative exchangeable copper fraction (REC) corresponds to the percentage ratio between ex changeable copper and total copper (CuEXC/total cop per, %) . Non-protein-bound plasma copper is normally less than 5% of total copper. The sum of the different fractions in which serum copper is distributed consti tutes cupremia, corresponding to the total copper con centration in blood serum.

Wilson's disease, also called hepatolenticular degeneration, is a genetic autosomal recessive disor der and is the most common hereditary cause of copper overload in tissues. The genetic alteration that caus es the disease is the mutation of ATP7B, a gene mapped on chromosome 13 which codes for an ATPase which has the function of controlling the hepatocyte's release of copper into bile and regulating its binding to apoceruloplasmin . This protein is highly expressed in the liver, being indispensable for the excretion of excess copper introduced with food. To date, there is no fully reliable rapid test for the diagnosis of Wil son's disease. The reference for diagnosis is the lev els of ceruloplasmin and copper in plasma and urine. The gold standard for diagnosis is liver biopsy. CuEXC and REC have recently been described as sensitive and specific biomarkers for the diagnosis of Wilson dis ease (Liver Int. 2018 Feb;38(2) :350-357, PLoS ONE 2013, 8(12) : e82323.

Menkes disease (MD) is a degenerative genetic disease with recessive heterosomal inheritance charac terized by low copper concentrations in some tissues and high concentrations in others, with consequent progressive neurological deterioration and important connective tissue anomalies. Diagnosis is based on clinical observation and the measurement of serum cop per and ceruloplasmin levels, which are reduced. This can be confirmed by genetic analysis, with the search for mutations in the ATP7A gene.

Alzheimer's disease (AD) represents 50-80% of cases of dementia, is a progressive pathology with life expectancy from diagnosis ranging from 4 to 20 years, in which symptoms gradually worsen up to a to tal loss of motor and cognitive capacity. The etiology of Alzheimer's disease is complex: familiarity and age are the most important risk factors. The central event of the disease is the formation of plaques containing b-amyloid and neurofibrillary tangles consisting of Tau protein filaments. There is extensive evidence re- garding the role of metals in the oxidation-reduction reactions that can cause the formation and precipita tion of Ab peptides. Exposure to copper is one of the environmental factors involved in the onset and pro gression of AD. Singh I et al . 2013 (Low levels of copper disrupt brain amyloid-b homeostasis by altering its production and clearance, PNAS, 110(36) : 14771- 14776), show that copper stimulates the production of amyloid b and prevents its disposal by altering the function of LRP1, the receptor of the protein involved in the elimination of amyloid b. Recent experimental and clinical evidence shows that the dismetabolism of this ion is a characteristic trait in the pathogenesis of Alzheimer's, which is accompanied by high levels of non-protein-bound copper in plasma and total copper deficiency in the brain (Front Aging Neurosci, 23 Jan uary 2018 ) .

Both indirect and direct methods are available for the determination of non-protein bound copper to day. In the indirect methods for the evaluation of non-ceruplasmin copper to total copper values, ob tained using atomic absorption spectroscopy (Evenson MA and Brenda LW 1975 Clin Chem 21(4) : 619) or with the colorimetric technique (Clin Chem 1989, 35 ( 4 ) : 552-4 ) , or with ICP-OES or ICP-MS Spectrometry analysis (Biol Trace Elem Res. 2014, 160(1) : 132-142), the values of bound copper are subtracted, obtained by measuring ceruloplasmin with immunoturbidimetric dosage (Crit Rev Clin Lab Sci Sci 1982, 17 (3) :229-45) .

Techniques are also available for the direct measurement of plasma or serum copper not bound to proteins. Hyo Sung Jung et al., 2009, (J Am. Chem. Soc. 131(5) :2008-2012) and Colabufo NA and Squitti R (EP 2917739 Bl) describe the use of fluorescent probes. G. A. McMillin et al . , 2009, (Am J Clin Pathol 131:160-165) describe a technique based on ultrafil tration .

Extensive experimental and clinical research has recently highlighted a significant correlation between plasma copper levels and the risk of developing degen erative diseases such as Alzheimer's disease, Parkin son's disease, and type II diabetes mellitus (J Alz- heimers Dis 2011; 23(2) :239-48; J Alzheimers Dis 2017 ; 56 ( 3 ) : 1055-1064 : Clinical Science Mar 08, 2016, 130 (8) 565-574; J Alzheimers Dis 38 (2014) 809- 822; Mol Neurobiol 2017; 54 ( 1 ) : 671 -681 , Biol Trace El em Res 2017; 177 (1) : 53-63) .

The techniques currently available for measuring cupremia require expensive laboratory instruments, as well as expert personnel for their execution. Further methodologies have recently been proposed for the direct measurement of the non-protein bound plasma copper fraction (J Trace Elements in Medicine and Biology 2018, 45:176-180); however, also in this case, these are laboratory procedures that require specialist equipment and specialized personnel.

The above for copper also applies to other metals present in traces in the body.

Like copper, the zinc ion is also an essential trace element of diagnostic interest which is distrib uted in human plasma in different fractions in which it is bound with different affinities to albumin, oth er proteins and some amino acids (Hand Book of Trace Metal Analyses for Health, Canterbury Health Laborato ries, 1998; Nutrients 2017, 9(2), 175) .

The determination of zinc in serum or the cop per/zinc ratio (Cu/Zn ratio) is of clinical relevance in conditions of deficit or excess of this metal (re lated to states of chronic inflammation, immune defi ciencies or chronic-degenerative diseases related to age) (J Am Coll Nutr. 1985; 4 ( 6) : 591-8. , Mech. Ag. Dev. 09/2015, 151: 93-100) . Commonly, the concentration of zinc in the blood (zincemia) and urine is determined in clinical chemistry laboratories by means of colori metric, fluorimetric tests or by atomic absorption spectroscopy (Giorn It Chim Clin (1987) 12,2,91-100; Clinica Chemistry Acta 282, 1-2, 1999, 65-76; J Env Prot Ecol (2014) 15, No 1, 309-316) . Test strips are available for the rapid determination of zinc in wa ter, which can be purchased from various manufacturers (Hach Company Loveland, CO - USA; Hanna Instruments Italia S.r.l.), but these methods are not applicable to biological samples.

Lead and cadmium are two heavy metals of diagnos tic relevance in toxicology. In the laboratory, their concentration in blood and other biological samples is carried out by atomic absorption spectroscopy, induc tively coupled plasma mass spectrometry (ICP-MS) and other techniques (Braz. Chem. Soc. (2006) vol.17 no .2 ; Sensors 2010, 10, 11144-11155) . For screening lead on whole blood, instrumentation (LeadCare II, Blood Lead Analyzer, ESA Biosciences, MA, USA) is commercially available which, however, requires at least 50 mΐ of blood volume and preliminary manipulations of the sam ple to be analyzed.

Also for these metals, the need to have a relia ble, inexpensive and fast measurement method has not been met to date.

There is a strong need to have a low-cost, easy- to-use, sensitive and precise method for the direct detection of heavy metals, in particular copper, cad mium, lead and zinc, in whole blood and/or other human biological fluids or of different animal species, which can be used for diagnosis in decentralized situ ations such as a medical clinic or POCT .

In particular, the availability of a technology, a method and devices that allow the rapid and economic measurement of these metal biomarkers to screen popu lations so as to implement primary and secondary pre vention and therapeutic measures against degenerative diseases such as Alzheimer's disease, Parkinson's dis ease and type II diabetes mellitus is desired.

The background art document US 5.284.567 de scribes a sensor for the determination of an analyte in a biological sample; in particular, it reports the use of a reagent that allows the exchange of lead bound to the erythrocyte and, therefore, the possible alteration of the size of the analyte in the compart ment of interest.

Object of the invention

In a first object the present invention de scribes a sensor device for the qualitative and/or quantitative determination of an analyte in biological samples . According to one aspect, these samples are rep resented by blood or human serum.

A second object of the invention is represented by a process for the preparation of the sensor device.

In a third object, the invention describes a method for the qualitative and/or quantitative deter mination of an analyte in a biological sample.

In a fourth object a method is described for the prognosis of a pathology comprising the qualitative and/or quantitative determination of an analyte in a biological sample.

According to one aspect, this pathology is rep resented by a degenerative pathology.

Brief description of the figures

Figure 1: diagrammatic representation of an em bodiment of a sensor device according to the present invention;

Figure 2: voltammogram of Zn, Cd, Pb and Cu ions in a human blood serum sample + DIDA;

Figure 3: correlation between copper levels and voltammometric response in a human serum sample;

Detailed description of the invention

Defini tions

Where not defined differently, the terminology used in the present description has the current mean- ing for the person skilled in the art. For greater clarity, some of the background art techniques, which find application in the present invention are reported below .

Voltammetry is the measurement of the intensity of the electric current flowing in an electrochemical cell as the potential difference imposed across the cell changes.

Voltammetric redissolution is an electrochemical technique, which allows to carry out quantitative analyses of trace substances present in liquid sam ples. A variant of voltammetric redissolution useful for the purposes of the present invention is voltam metric anodic redissolution, which is based on metal analytes' formation of amalgams with the material with which the working electrode, typically mercury, is composed .

Said technique comprises a first step consisting in the application of a constant and negative poten tial to the working electrode, such as to cause a re duction of the element under examination and a second step (redissolution step, or stripping) , carried out by means of an anodic scan of the potential, in which the value of the electrode's potential is increased, so as to cause the reoxidation of the previously re- duced type, starting from those with lower reduction potential, which return to solution.

When the discharge potential of a metal ion is reached during the scan, a peak-shaped current trend is recorded on the voltammogram.

The peak position and height or area are relat ed, respectively, to the type and concentration of the analyte .

The potential scan, during which the current is measured during the redissolution step, can be con ducted in linear, pulse differential, square wave mode. In the case of square wave scanning, the tech nique is called "Square Wave Anodic Stripping Voltam metry" ( SWASV) .

During the redissolution step, the current in the cell is measured.

The technique allows to obtain qualitative in formation on the sample under examination, as each electroactive type provides a distinctive peak poten tial value, and quantitative, since the value of the peak current provides a quantitative evaluation of said type.

In the present description, the term "analyte" means a metal, or rather a metal ion, present in the biological sample, preferably represented by an essen tial trace element.

For the purposes of the present invention, an analyte is preferably selected from the group compris ing: copper, cadmium, lead, mercury and zinc, and is even more preferably represented by copper.

According to the preferred aspect of the inven tion, in which the analyte of interest is copper, this can be present in one of the following forms:

i) bound copper, i.e., bound to the protein apoceruplasmin,

ii) weakly bound copper (labile fraction), i.e., weakly bound to albumin, transcuprein, small peptides, amino acids and other ligand types;

iii) free copper, i.e., not bound to proteins.

The set of all these copper fractions represents cupremia, i.e., the total concentration of total cop per in human serum.

More generally, for all the above-mentioned ana lytes, there may be: a bound fraction, a weakly bound fraction and a free fraction.

"Qualitative determination" means the possibil ity of determining the nature of a certain analyte, while "quantitative determination" means the possibil ity of determining the quantity, understood as concen- tration, of a certain analyte in the biological sam ple .

For the purposes of the present invention, the biological sample is an isolated biological sample represented by: blood, serum, plasma, saliva, sperm, urine, sweat, liquor, and is preferably represented by venous or capillary whole blood.

The biological sample analyzed according to the present invention has a volume between 1 and 50 pL, preferably between 3 and 10 pL .

According to a first object, the invention de scribes a device for the qualitative and/or quantita tive determination of an analyte in a biological sam ple .

Such device is therefore a sensor device.

In particular, such device comprises at least two electrodes, one of which is a working electrode and one is a reference electrode.

For the purposes of the present invention, the working electrode and the reference electrode have a size (area) very similar to one another.

As far as the reference electrode is concerned, this is preferably represented by an Ag/AgCl based electrode . According to an aspect of the present invention, the device can comprise a third electrode, called pseudo-reference electrode, which can be represented by an electrode made of graphite or other electrically conductive materials.

This third electrode, if present, reveals the presence of the biological sample by conductivity, so as to allow the automatic start of the measurement when the sample enters the capillary chamber, or to normalize the sensor response to changes in tempera ture or other parameters which can interfere with the measurement .

The measurements of the signals generated by the sensor device of the invention are carried out by a commercially available potentiostat or by a potenti- ostat with dedicated HW and SW, as a person skilled in the art can easily understand.

In a preferred aspect of the present invention, the sensor device described comprises a working elec trode and a counter-electrode configured for the SWASV technique .

Figure 1 shows the diagram of an embodiment of a sensor device (1) according to the present invention.

In particular, said sensor device (1) comprises a support (or strip) (1) on which a working electrode (2), a reference electrode (3) and possibly a pseudo reference electrode (4) are layered.

The exposed surface of said electrodes (2), (3) and, where present (4), faces inside a capillary cham ber (5) which consists of the measurement cell and re ceives the sample introduced from the outside through the capillary channel (6) .

The capillary chamber (5) comprises an inner surface (5a) intended to come into contact with the biological sample.

Said capillary chamber (5) can have, at the side opposite that of the capillary channel, a small ori fice (7) which allows the outflow of air when the ca pillary chamber (5) is filled by the sample through the capillary channel (6) .

For the purposes of the present invention, the capillary chamber (5) can have a square, triangular, rectangular, polyhedral or irregular shape or other suitable shape, and the orifice (7) for the air out flow can be formed by means of a second capillary channel (not shown) which connects the capillary cham ber (5) with the outside of the sensor device (1) .

In particular, the electrodes (2, 3 and 4) con sist of an electrically conductive material, selected for example from: gold, silver, platinum and graphite. In a preferred embodiment, the working electrode (2) is made of graphite; in fact, the graphite has ad sorbent properties which allow it to retain mercury salt better than any other inert material and, moreo ver, it does not form an amalgam with the mercury once reduced .

Advantageously, said capillary channel (6) is made, with reference to the materials, in particular the inner surface thereof and dimensions, in order to carry out at least the following functions:

i. allow the rapid transport, by capillary ef fect, of the fluid biological sample inside the capil lary chamber (5);

ii. avoid or provoke lysis of the cellular com ponent of the sample under examination;

iii. host substances provided with specific activities aimed at modifying the binding affinities between the proteins and other ligands with the metals of interest in the sample.

For the three purposes listed above, the inner surface of the capillary canal (6a) can be treated or contain in a suitable form:

- acidifying agents or

- protein denaturating agents or

- competing agents or, chelating agents.

In particular, the acidifying agents are strong acidifying agents selected from the group comprising: trichloroacetic acid, metaphosphoric acid, perchloric acid, etc.

In particular, the protein denaturating agents are chaotropic agents selected from the group com prising: urea, guanidine, sodium dodecyl sulfate, etc .

In particular, the competing agents are compet ing agents for the binding sites for the analyte of interest .

If the analyte of interest is represented by copper, such agents compete for the binding of apoceruloplasmin, transcuprein and other plasma pro teins .

For this purpose, divalent cation salts can be used selected from the group comprising: zinc (II) and iron (II) .

In particular, the chelating agents can be rep resented by EDTA ( ethylenediaminotetracetate ) , EGTA, butandioic acid or similar compounds, and preferably by EDTA.

According to an aspect of the present invention, in addition to or as an alternative to the inner sur- face of the capillary channel (6), such agents are contained in the capillary chamber (5) of the device.

In an embodiment of the invention, the dimen sions of the capillary channel (6) can vary between 0.2-5 mm wide (or larger or long side of the channel section), and preferably 0.5-2 mm wide, and 3-30 mm long, and preferably 10-20 mm long, and 0.2-0.5 mm thick (or smaller or short side of the channel sec tion) .

The materials in contact with the capillary channel and with the capillary chamber can instead be represented by metals, alloys, organic polymers, glassy or ceramic materials; in particular, their sur face must have hydrophilic properties.

In a preferred aspect, the polymer is selected from the group comprising: cellulose and derivatives thereof, starch, polyvinylpyrrolidone, polyvinyl alco hol, and the like, preferably hydroxymethylcellulose .

Hydrophilic properties can be obtained either directly by virtue of the intrinsic properties of the material surface or because such surface has been treated with surfactants capable of reducing the wa ter/solid surface tension; for this purpose, for exam ple, the following can be used: Tween, SDS, TRITON, sodium cholate, etc. According to an embodiment of the device of the present invention, the capillary channel (6) can be filled with a filtering material.

In particular, this material can be represented by glass wool, possibly having fibers with a diameter between 0.5 and 10 pm.

It is thereby possible to separate the corpuscu lar fraction present in the sample from the plasma and allow only the plasma to reach the chamber (5) .

Advantageously, this allows to cancel or limit as much as possible the interference on the measure ment caused by abnormal hematocrit values.

Alternatively to the use of glass wool or other filtering material, it is possible to correct the measurement during the calculation phase, once the hematocrit value is known.

For the preparation of the sensor device of the present invention in accordance with the second object of the invention, this is subjected to a coating step, in which a coating solution is applied to the inner surface of the capillary chamber (5a), or to a portion thereof .

In particular, the coating is obtained on the surface of the capillary chamber. More in detail, the coating solution is prepared by mixing a mercury (II) salt with a supporting gelling polymer .

In particular, the mercury salt is preferably represented by mercury chloride.

For the purposes of the present invention, the supporting polymer can be represented by cellulose or by derivatives thereof such as, for example, methyl- cellulose (for example, commercially available as Methocel®) .

The polymer concentration influences the rate of solubilization, viscosity and reproducibility in the formation of the mercury film.

For example, for methylcellulose, preferred con centration values are about 4-8.5 mg/mL; advantageous ly, within this range the analytical performance meets both the precision and accuracy requirements.

A preparation of the coating can preferably be obtained by mixing 0.3 mg/mL Hg2Cl 2 and methylcellu lose 6.25 mg/mL.

If necessary, an acid can be added to the prepa ration so as to obtain an optimal pH.

Such acid is preferably selected from the group comprising: aliphatic or aromatic carboxylic acids such as citric acid, lactic acid, malic acid, benzoic acid, and the like; in a preferred aspect, the acid is represented by citric acid.

The acid concentration is preferably between 100-1000 mM and more preferably between 300-600 mM.

In particular, the addition of acid to the sup port is necessary if no acid is added to the biologi cal sample.

The sensor is subsequently exposed to a pre treatment step, which can be carried out at the begin ning of the analysis or during preparation.

In particular, in the first case, the pretreat ment is carried out directly by the potentiostat , fol lowing the introduction of the sample into the capil lary chamber (5), which applies the electric poten tial.

By applying an adequate negative potential to the working electrode, the Hg²⁺ ions are reduced to el emental mercury, the membrane is dispersed in the liq uid phase of the sample and the graphite adsorbs the metallic mercury on the surface of the electrode (2) in contact with the sample.

If the pretreatment step is instead carried out during the manufacturing process, the working elec trode (2) is already coated with a thin layer of mer cury, possibly protected by a water-soluble polymer film intended to dissolve in the sample at the time of measurement .

The chemical composition of the coating solution is particularly relevant for the purposes of the pre sent invention, as it is essential in order to perform the following functions:

- ensure the optimal pH of the sample for elec trochemical measurement purposes;

- allow the detection of only the fraction (s) of the metal ion whose concentration is to be measured in the sample;

- avoid cytolysis, thereby avoiding altering the plasma analyte concentration, especially useful in cases where the measurement concerns the non- corpuscular fraction in biological fluids containing cells (as described below) ;

- render the sample viscosity uniform.

In a preferred aspect of the invention, the de scribed device is disposable.

In accordance with a third object, the invention describes a method for the qualitative and/or quanti tative determination of an analyte in a biological sample .

As described above, such analyte is preferably selected from the group comprising: copper, cadmium, lead, mercury and zinc, and is even more preferably represented by copper.

i) bound copper, i.e., bound to the protein apoceruplasmin,

iii) plasma copper, i.e., not bound to proteins.

As regards the biological sample, as described above this is represented by blood, plasma, serum, sa liva, sperm, urine, sweat, liquor, and is preferably represented by venous or capillary whole blood.

In a preferred aspect, the method of the inven tion is carried out by using the device described above .

In particular, the method of the invention com prises the qualitative and/or quantitative determina tion of an analyte of interest in a biological sample by square-wave anodic stripping voltammetry. In detail, this process comprises the steps of:

I) putting the biological sample in contact with the capillary chamber (5) of the sensor device (1) of the invention;

II) taking a measurement by voltammetric anodic redissolution of an analyte of interest in said bio logical sample.

According to the present invention, before step I) inside the capillary chamber (5), or alternatively inside the capillary channel (6), the following may be present :

- acidifying agents or

- protein denaturating agents or

- competing agents or

- chelating agents,

which carry out a pretreatment of the biological sample .

In particular, the protein denaturating agents are chaotropic agents selected from the group com prising: urea, guanidine, sodium dodecyl sulfate, etc . In particular, the competing agents are compet ing agents for the binding sites for the analyte of interest .

In a preferred aspect of the method of the in vention, before step II), a step is carried out to check the complete filling of the capillary chamber (5) (saturation condition) .

This check is carried out by the potentiostat , which measures the electrical conductivity between the electrodes of the device (1) and, for example, exceeding a predetermined or constant value of elec trical conductivity or both conditions; alternative ly, other methods are known to the person skilled in the art . As regards step II), this comprises in particu lar the step of:

Ila) applying a negative electric potential higher than the discharge potential of the Hg²⁺ ion and all the metal ions of interest (analytes) to the working electrode (2), which settle on the surface of the electrode in the form of an amalgam.

After step Ila) there is a step lib) of redisso lution (or stripping) in which a potential scan is applied to the working electrode (2) by applying a square wave pulse, starting from the accumulation po tential up to a value lower than the discharge poten tial of the metal of interest (analyte) , such as to cause oxidation of the metal, which is released from the amalgam and returns to solution as a metal ion.

Since the volume of the capillary chamber (5) is constant and the amount of electric charge necessary to bring the metal of interest (analyte) to solution is known, it is possible to determine the concentra tion of the metal of interest in the sample with methods known to those skilled in the art.

In particular, starting from the amount of charge necessary to cause the complete redissolution of the analyte of interest, the concentration of the same analyte can be derived through a calculation al gorithm based on one of the following principles: a. equivalence between electron moles and the product between the ion moles and the electric charge thereof;

b. standard curve interpolation calibrated on the electrochemical sensor during production;

c. quantitative ratio between the signal pro duced by the sample under examination and a calibra tor with known analyte capacity, measured by the same sensor or a different sensor of the same production batch .

For the purposes of the present invention, the addition of acidifying agents is preferably possible until a pH between 3.5-6.0 and preferably 4.5-5.5 is obtained .

Preferably, the pH is modified by adding a weak acid selected from the group comprising: aliphatic or aromatic carboxylic acids such as citric acid, lactic acid, malic acid, benzoic acid, and the like; in a preferred aspect, the acid is represented by citric acid .

The acid concentration is preferably between 10 mM and 1000 mM and more preferably between 100 mM and

600 mM. According to an aspect of the present invention, the biological sample is represented by venous or ca pillary whole blood.

In such circumstances it is necessary to avoid hemolysis, as the hemolysate could interact with the correct measurement.

For this purpose, the conditions inside the ca pillary cell (5) should preferably be the following:

- Osmolarity: about 230-350 mOsm/L

- Surface tension: about 35-80 X 10-3 N/m

- pH: about 4.2-6.2.

According to an aspect of the method of the in vention, if the biological sample is represented by venous or capillary whole blood, step I) is preceded by a filtering step.

In particular, such step has the purpose of sep arating the corpuscular fraction and allowing only the plasma to reach the capillary chamber (5) for the qualitative and/or quantitative determination of the analyte of interest.

For filtration, a material represented by glass wool can be used, possibly comprising fibers having a diameter of about 0.5-10 pm.

According to another aspect of the present in vention, the described method allows determining the total concentration of the analyte of interest when this is present both in whole blood and in intracellu lar fluids.

In this case, the method may comprise a step of pretreating the biological sample with a lysing agent.

Such lysing agent can be selected from the group comprising: sodium dodecyl sulfate, polyethylene gly col dodecyl ether, or other cytolysing agents.

The lysing agent can be present inside the ca pillary canal (6) and thus produce lysis before the biological sample reaches the capillary chamber (5) .

In a preferred aspect of the present invention, the steps of the qualitative and/or quantitative de termination method are carried out in a time of less than 5 minutes and at a temperature between about 10- 37 °C .

In accordance with a fourth object, the present invention describes a method for the prognosis of a pathology comprising the qualitative and/or quantita tive determination of an analyte in a biological sam ple .

In fact, once the nature of the analyte and pos sibly its concentration have been determined by virtue of the comparison with values of healthy patients or for whom a pathology has been diagnosed or who have subsequently developed a pathology, it is possible to reach a prognosis.

According to a preferred aspect of the inven tion, such pathology is represented by a degenerative pathology, possibly linked to age.

For example, such pathology can be represented by Wilson's disease, Menkes disease, Alzheimer's dis ease, type II diabetes mellitus or Parkinson's dis ease .

In addition to degenerative diseases, the method allows to diagnose or prognosticate states of chronic inflammation or immune deficiencies.

In a preferred aspect of the invention, this method may comprise determining the concentration ra tio between two analytes of interest.

For example, the two analytes can be represented by copper and zinc, allowing the calculation of the copper/zinc ratio (Cu/Zn) .

By virtue of the diagnosis or prognosis made possible by the present invention, measures can be put in place to prevent or delay the onset of such pathol ogies, for example by restoring physiological homeo stasis, or to evaluate the effectiveness of such measures . The present invention will be further described by the following examples, which are to be considered repre sentative but not limiting.

Example 1

A pool of heparin-treated human plasma samples, having cupremia 1040 ppb, was aliquoted and increasing amounts of standard copper (II) chloride were added to the fractions. Figure 2 shows the voltammograms ob tained using sensor devices treated with the following coating solution: 0.3 mg/mL Hg2Cl2, citric acid-IhO 0.6 M, Methocel® 6.25 mg/mL.

The voltammograms were obtained by setting the follow ing measurement algorithm in the potentiostat :

- Deposition potential: 1.1 V.

- Deposition time: 30".

- Potential stripping range: 1.1

0.15 V;

- stripping time: 20

- pulse amplitude: 28 mV;

- frequency: 15 Hz

- step potential 3 mV.

Key :

1 : Sample

2: Sample added with 20 ppb of Hg²⁺

3: Sample added with 40 ppb of Hg²⁺

4: Sample added with 80 ppb of Hg²⁺ In order to estimate the measurement inaccuracy, the fraction corresponding to the unfortified sample and that with 80 ppb of copper added were each measured 20 times, under the conditions described, obtaining re spectively the following CVs : 9.7% and 6.5%.

Example 2

A physiological solution containing 6 g/dL of human albumin was aliquoted and scalar quantities of copper ion between 5 and 1200 ppb were added to the frac tions, using a standard solution of copper (II) chlo ride. Figure 3 shows the correlation between the nomi nal copper ion concentrations and the relative voltam- mometric values obtained using the algorithm and sen sors treated with the coating solution and described in Example 1.

The correlation coefficient (Pearson correlation in dex) (p) was 0.983.

Example 3

A pool of human plasma/heparin samples with 1040 ppm cupremia, was aliquoted and increasing amounts of 2 M citric acid were added to the fractions to correct their pH in a range from 7.0 to 3.0. Figure 4 shows the relationship between the pH values of the differ ent plasma fractions and the relative voltammometric values, obtained using sensors treated with the fol- lowing coating solution: 0.3 mg/mL Hg₂<3l₂, Methocel® 6.25 mg/mL.

Total cupremia was determined on the same samples and the concentration of non-protein bound copper was de termined by the technique described in: J Alz Dis 61 (2018), 907-912. Surprisingly, the useful pH value range for the measurements was between 4 and 5.5. At more acidic or more alkaline pH values, the sensitivi ty of the method was less than 50% of the optimal val ue. Furthermore, the correlation between the values found with the method of the present invention and the reference values expressed as R² was found to be less than 0.75 in all samples.

Example 4

In order to verify the effect of hematocrit (HCT) on the results of the measurements made with the device object of the present invention, a venous/heparin blood sample was aliquoted and the hematocrit of five fractions was corrected by subtraction and addition of plasma obtained by centrifugation of an aliquot of the same sample. The fractions thus obtained were analyzed using sensor devices and the voltammetric analysis al gorithm described in Example 1. The hematocrit was al so determined on the same fractions of venous blood, using the standard method. Table 1 shows the averages (from ten measurements) of the results obtained and the corresponding standard deviations of the correlations between the Walshe val ues (copper fraction not linked to ceruloplasmin) and the electrochemical measurement on 20 blood samples. These vary according to the hematocrit, although with in the physiological values (40-48%) the interference from this hematological parameter is not significant from the diagnostic point of view.

Table 1

Example 5

Nineteen capillary blood samples obtained by finger capillary sampling from adult human subjects of both sexes were analyzed using the sensor devices and the algorithm described in example 1. For reference, the concentration of total copper and non-protein bound copper was determined in the plasma samples obtained by centrifugation from venous blood/heparin from the same donors by the technique described in: J Alz Dis 61 (2018), 907-912. The averages (in mg/L) and standard deviations of the results obtained are shown in Table 2. The correlation coefficient R between Cu_f and Cuf_v was 0.938.

Key: Cu_t = Total copper concentration, Cu_t = Concentra tion of non-protein bound copper, CU_fV = Copper con centration obtained by voltammetry, with the sensor devices object of the present invention.

Table 2

Example 6

A physiological solution containing 6 g/dL of human albumin in physiological solution containing 50 pg/L of Pb²⁺, Cu²⁺, Cd²⁺ ions and 200 pg/L Zn²⁺ ions was sub jected to voltammetric examination using the algorithm described in Example 1.

Figure 5 shows the relative voltammogram, in which the four peaks are related to the four metals present (A = Zn, B = Cd, C = Pb, D = Cu) .

From the above description, the advantages offered by the present invention will be immediately apparent to the person skilled in the art. The method according to the present invention is of fast execution, where the entire analysis process takes less than 5 minutes; by virtue of the particular shape of the sensor cell of the invention, an opti mized analysis time of even less than 2 minutes can be obtained, which becomes less than 1 minute if the analysis concerns only one analyte (for example, cop per) .

The possibility of conducting the measurement directly in a whole blood sample makes the method very versa tile and easy, as blood is an easily accessible tis sue .

The portable and disposable device of the present in vention can work even in the absence of an electrical grid connection and does not require an equipped envi ronment .

The device is intuitive to use and does not require additional equipment or staff training.

The advantage associated with electroanalytical tech niques is that these are simple to obtain, sensitive, quick and low-cost.

The use of the SWASV technique, i.e., a pulse tech nique with respect to classic linear-signal anodic re dissolution, also has an additional advantage since it allows to eliminate the effect of dissolved oxygen, an interferer in the classic redissolution techniques, so that it is not necessary to deaerate the sample or use chemical additives to remove the oxygen from the reac tion environment.

The size of the device allows it to be used with re duced sample volumes, typically less than 20 pL; therefore, the necessary blood sample can be taken through capillary finger sampling, being deposited di rectly on the sensor, without requiring any pretreat ment .

Said sensor is also extremely sensitive; by way of ex ample, where applied to the quantitative analysis of copper in blood plasma, the lower detection limit is below 80 nM/L.

By virtue of the device and the method of the inven tion, it is also possible to measure each fraction of the analyte of interest present in different compart ments or bound differently within the biological sam ple .

Furthermore, the ratio of two analytes of interest can be calculated.

k k k

Claims

1. A sensor device (1) for the qualitative and/or quantitative determination of one or more ana lytes in an isolated biological sample comprising a capillary chamber (5) for determining said analytes, a capillary channel (6) comprising an inner surface (6a) for introducing said isolated biological sample in the capillary chamber (5), a working electrode (2) and a reference electrode (3), wherein said capillary cham ber comprises an inner surface (5a) coated with a coating layer comprising Hg²⁺ ions.

2 . A sensor device (1) according to the preced ing claim, wherein said analyte is represented by a metallic ion selected from the group comprising cop per, cadmium, lead, mercury and zinc, and is even more preferably represented by copper.

3 . A sensor device (1) according to claim 1 or 2, wherein said isolated biological sample is repre sented by blood, serum, plasma, saliva, sperm, urine, sweat, liquor, and is preferably represented by venous or capillary whole blood.

4 . A sensor device (1) according to any one of the preceding claims, further comprising a pseudo reference electrode (4) .

5 . A sensor device (1) according to any one of the preceding claims, wherein said capillary channel (6) or said capillary chamber (5) comprise an inner surface (5a, 6a) which can be treated or contain in an appropriate form:

- acidifying agents or

- protein denaturating agents or

- competing agents or

- chelating agents.

6. A sensor device according to the preceding claim, wherein:

- said acidifying agents are strong acidifying agents selected from the group comprising: trichloroacetic acid, metaphosphoric acid, perchloric acid;

- said protein denaturating agents are chaotropic agents selected from the group comprising: urea, guanidine, sodium dodecyl sulfate;

- said competing agents are competing agents for the binding sites for the analyte of interest;

- said chelating agents are selected from the group comprising: ethylenediaminotetracetate, EGTA, butanedioic acid, or similar compounds.

7 . A sensor device (1) according to any one of the preceding claims, wherein the capillary channel (6) is filled with a filtering material to separate the corpuscular fraction from the plasma.

8. A sensor device (1) according to any one of the preceding claims, wherein the dimensions of the capillary channel (6) are between 0.2-5 mm wide, and preferably 0.5-2 mm wide, and 3-30 mm long, and pref erably 10-20 mm long, and 0.2-0.5 mm thick.

9 . A sensor device (1) according to any one of the preced ing claims, wherein said isolated biological sample is represented by venous or capillary whole blood and has the following characteristics:

- Osmolarity: about 230-350 mOsm/L

- Surface tension: about 35-80 X 10-3 N/m

- pH: about 4.2-6.2.

10 . A process for preparing the sensor device (1) according to any one of the preceding claims, wherein said coating layer applied to the inner surface of the capillary chamber (5a) or to a portion thereof is pre pared mixing a mercury (II) salt with a supporting gel ling polymer selected from the group comprising: cel lulose or a derivative thereof.

11 . A process according to the preceding claim, wherein said cellulose derivative is represented by methylcellulose .

12. A process according to the preceding claim, wherein said methylcellulose is present in a concen tration between 4-8.5 mg/mL.

13. A process according to any one of claims 10 to 12, wherein said mercury(II) salt is preferably represented by mercury chloride.

14. A process according to any one of claims 10 to 13, wherein an acid selected from the group com prising: aliphatic or aromatic carboxylic acids such as citric acid, lactic acid, malic acid, benzoic acid, and the like is added; in a preferred aspect, the acid is represented by citric acid.

15. A process according to the preceding claim, wherein the acid concentration is preferably between 100-1000 mM and more preferably between 300-600 mM.

16. A method for the qualitative and/or quanti tative determination of one or more analytes in an isolated biological sample by the voltammetric anodic redissolution technique in a sensor device comprising a capillary chamber (5) for determining said analytes, a capillary channel (6) comprising an inner surface (6a) for introducing said isolated biological sample in the capillary chamber (5), a working electrode (2) and a reference electrode (3), said capillary chamber comprising an inner surface (5a) coated with a coating layer comprising Hg²⁺ ions, said method comprising the steps of:

I) contacting said isolated biological sample with said capillary chamber (5);

II) taking a measurement by voltammetric anodic redissolution of one or more analytes of interest in said isolated biological sample.

17 . A method according to the preceding claim, wherein before step I) said isolated biological sample is pre-treated with one or more of the following agents :

- acidifying agents,

- protein denaturating agents,

- competing agents,

- chelating agents,

- lysing agents.

18 . A method according to the preceding claim, wherein :

- said protein denaturating agents are chaotropic agents selected from the group comprising: urea, guanidine, sodium dodecyl sulfate; - said competing agents are competing agents for the binding sites for the analyte of interest;

- said chelating agents are selected from the group comprising: ethylenediaminotetracetate, EGTA, butanedioic acid or similar compounds;

- said lysing agents are selected from the group comprising: sodium dodecyl sulfate, polyethylene glycol dodecyl ether, or other cytolysing agents.

19. A method according to any one of the preced ing claims 16 to 18, wherein said step II) comprises the steps:

Ila) application to the working electrode (2) of a negative electrical potential higher than the dis charge potential of the Hg²⁺ ion and of all the ana lytes;

lib) redissolution (or stripping) in which a po tential is applied such to oxidize said analyte.

20. A method according to any one of the preced ing claims 16 to 19, wherein before step I) the iso lated biological sample is subjected to filtration in the capillary channel (6) .

21. A method according to any one of the preced ing claims 16 to 20, wherein said steps are carried out in a time less than 5 minutes.

22. A method according to any one of the preced ing claims 16 to 21, wherein said steps are carried out at a temperature between 10°C and 37°C.

23. A method for the prognosis of a pathology comprising the qualitative and/or quantitative deter mination of one or more analytes in an isolated bio logical sample according to the method of any one of claims 16 to 22.

24. A method for the prognosis of a pathology according to the previous claim, wherein said patholo gy is represented by a degenerative pathology selected from the group comprising: Wilson's disease, Menkes disease, Alzheimer's disease, type II diabetes melli- tus, Parkinson's disease.