Methods and electroanalytical sensors for detecting and / or quantifying a panel of hematological parameters in a sample of blood or derived biological fluids

Abstract

The present invention relates to a method and sensor for detecting and / or quantifying in a sample of blood or a derived biological fluid a panel of hematological parameters comprising at least one parameter selected from the group consisting of the number of blood cell elements (RC) in the blood or derived biological fluid, the mean corpuscular volume (MCV) of said blood cell elements, the percentage of the sample volume occupied by blood cell elements (HCT), and the amount of at least one substance comprising a protoporphyrin metal complex (Hb). The present invention further relates to a method for manufacturing said electroanalytical sensor.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims priority to Italian Patent Application No. 102023000001014, filed January 24, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to methods and sensors for detecting and / or quantifying in a sample of blood or a derived biological fluid a panel of hematological parameters comprising at least one parameter selected from the group consisting of the number of blood cell elements (RC) in the blood or derived biological fluid, the mean corpuscular volume (MCV) of said blood cell elements, the percentage of the sample volume occupied by blood cell elements (HCT), and the amount of at least one substance comprising a protoporphyrin metal complex (Hb).

[0003] The present invention further relates to a method for manufacturing said electroanalytical sensor. [Background technology]

[0004] Hemoglobin (Hb) is a tetrameric metalloprotein found in red blood cells (RBCs). It contains four iron atoms, essential for transporting oxygen from the lungs to the body's tissues. The molecule also transports a portion of carbon dioxide from the tissues that produce it to the lungs. Traditionally, measuring Hb concentrations in blood has been a specific and sensitive indicator for diagnosing anemia and other diseases associated with impaired Hb levels in the blood. Normal hemoglobin levels depend entirely on the subject's age and gender. The traditional method used for measuring hemoglobin is the Drabkin test, developed in 1930. Detection is based on the formation of cyanohemoglobin through the interaction between cyanide ions and oxyhemoglobin. Dosage is determined by spectrophotometric measurement of absorbance at a wavelength of 540 nm, corresponding to the characteristic absorbance peak of cyanohemoglobin. Over the years, other methods for determining hemoglobin have been developed, primarily colorimetric and chromatographic methods, such as fluorimetry, colorimetry, HPLC, and chemiluminescence. Although these techniques allow for the quantification of low concentrations of hemoglobin, they may be subject to several drawbacks: - Use of bulky equipment (analysis cannot be performed at the point of care or in clinics with insufficient facilities); - Use of expensive equipment; - Long analysis time; - Use of highly qualified personnel; - complex analytical procedures; - Use of expensive materials and reagents.

[0005] All these drawbacks have stimulated research to develop new techniques to overcome or reduce these problems. Electrochemical methods are a particularly promising alternative for analyzing hemoglobin in biological fluids. Depending on the type of modification made to the electrode surface, these methods can be subdivided into four main classes: 1) Immobilization of hemoglobin on the electrode surface; 2) the use of organic and inorganic catalysts; 3) the use of molecularly imprinted polymer (MIP) membranes; 4) Immunological methods.

[0006] The closest prior art study is a study by Brett C.M. et al. (1999), which involved the development of an electrochemical sensor suitable for monitoring hemoglobin in whole blood using methylene blue as an electrochemical mediator. This study utilized a glassy carbon electrode, a classic three-electrode system. Specifically, the electrochemical cell consisted of a graphite working electrode, a calomel reference electrode, and a platinum counter electrode. The cell was filled with 0.1 M phosphate or acetate buffer, which acted as the supporting electrolyte.

[0007] The electrochemical mediator (methylene blue) was electrochemically deposited on the surface of the working electrode by immersing the sensor in a beaker containing a solution consisting of 0.2 mM methylene blue, 0.1 M phosphate buffer (pH = 8.2), and 0.1 M Na2SO4. Electrochemical deposition was performed by applying a potential scan ranging from -0.6 to 1 V and performing 20 scans by cyclic voltammetry. Immediately after electrodeposition, the sensor was left immersed in the electrolyte for several hours or overnight, which allows for the disappearance of unpolymerized monomers and therefore more stable electrochemical measurements. Hemoglobin was determined by a batch injection analysis (BIA) technique, which allows for the measurement of the solution contained in the beaker after immersion of the sensor and subsequent addition of whole blood (approximately 50 μL). Signal transduction was performed by chronoamperometry; a current signal proportional to the concentration of hemoglobin present in the solution was obtained by applying a potential of +550 V. To reduce memory effects caused by potential retention or absorption of metalloproteins within the film deposited on the electrode, the BIA technique waits 20 seconds before subsequent additions, allowing the residual current to stabilize. In this study, eight whole blood samples from eight volunteer donors were analyzed. The matrix was immediately treated with EDTA to prevent coagulation and then diluted 1:5 with phosphate buffer before electrochemical measurements. Six of the analyzed samples yielded results equivalent to the hemoglobin dosage measured using the reference method.

[0008] All of the above methods have drawbacks, among which the common problem is that hemoglobin detection cannot be performed on whole blood without other treatments. Secondly, some of the above methods require the use of expensive and bulky equipment, well-equipped laboratories, qualified personnel, long run times, and limited specificity and selectivity.

[0009] In addition to hemoglobin quantification, there may be a need to measure an entirely different set of hematological parameters using minimal amounts of biological fluids without relying on analytical laboratories. Some of these parameters are cellular components of blood, such as erythrocytes, reticulocytes, erythroblasts, particularly their numbers, mean corpuscular volume, and relative volumes, as well as substances containing (proto)porphyrin metal complexes, such as hemoglobin, myoglobin, and zinc protoporphyrin.

[0010] Therefore, there is a need to develop methods and sensors that allow for the detection and quantification of the number of blood cell elements, the mean corpuscular volume of said blood cell elements, the percentage of the sample volume occupied by blood cell elements, and / or the amount of at least one substance comprising a protoporphyrin metal complex in blood or a derived biological fluid, and that can be applied both outside the laboratory and in the laboratory in emergency situations or in small, less equipped facilities. In particular, from a diagnostic point of view, there is a strong need to provide methods and sensors that are cheap, rapid, simple, specific and selective, and can be adapted to laboratory and non-specialized environments. Summary of the Invention [Problem to be solved by the invention]

[0011] It is therefore an object of the present invention to provide a method for detecting and / or quantifying in a sample of blood or a derived biological fluid a panel of hematological parameters comprising at least one parameter selected from the group consisting of the number of blood cell elements (RC) in the blood or derived biological fluid, the mean corpuscular volume (MCV) of said blood cell elements, the percentage of the volume of the sample occupied by blood cell elements (HCT), and the amount of at least one substance comprising a protoporphyrin metal complex (Hb), which solves the aforementioned problems in a simple and efficient manner. [Means for solving the problem]

[0012] This object is achieved according to the invention in relation to the method defined in claim 1.

[0013] It is a further object of the present invention to provide an electroanalytical sensor as defined in claim 19 and a method for manufacturing the sensor as defined in claim 20. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 depicts a schematic diagram of an example electroanalytical sensor according to an embodiment of the present invention. [Figure 2] FIG. 2 is a graph showing a cyclic voltammetry (CV) obtained by measuring 10 μl of a solution containing 2 mM methylene blue, 0.1 M phosphate buffer (pH 7.4), and 0.1 M Na 2 SO 4 . [Figure 3] FIG. 3 is a graph in which the horizontal axis represents impedance (Z) and the vertical axis represents the surface coefficient (St) multiplied by the corresponding current (i). [Figure 4] FIG. 4 is a graph in which the horizontal axis represents the concentration of hemoglobin ([Hb]) and the vertical axis represents the surface coefficient (St) multiplied by the impedance (Z) minus the current value (i). [Figure 5]FIG. 5 is a graph in which the horizontal axis represents hematocrit value (HCT) and the vertical axis represents the surface coefficient (St) multiplied by impedance (Z) minus the current value (i). [Figure 6] FIG. 6 is a graph in which the horizontal axis represents the number of red blood cells (RC) and the vertical axis represents the value obtained by multiplying the surface coefficient (St) by the impedance (Z) and subtracting the current value (i). [Figure 7] FIG. 7 is a graph showing the concentration of hemoglobin [Hb] as a function of current (i) normalized to the corresponding impedance (Z). [Figure 8] Figure 8 shows a graph showing how the impedance measurements (Z) corrected by the coefficient of electroactive surface (St) correlate highly significantly with the hemoglobin concentration in mol / L (M). The horizontal scale is logarithmic. [Figure 9] FIG. 9 is a diagram depicting a comparison between the new measurement model of blood hemoglobin concentration and hemoglobin concentrations determined using a reference method. [Figure 10] FIG. 10 shows a graph of the resistance measurements (ΔV / I) normalized to the number of red blood cells contained in a capillary blood volume (60 μl) deposited on the surface of the electrochemical cell as a function of the impedance measurements, expressed as a natural logarithm. DETAILED DESCRIPTION OF THE INVENTION

[0015] A method according to the present invention for detecting and / or quantifying in a sample of blood or a derived biological fluid a panel of hematological parameters comprising at least one parameter selected from the group consisting of the number of blood cell elements (RC) in the blood or derived biological fluid, the mean corpuscular volume (MCV) of said blood cell elements, the percentage of the volume of the sample occupied by blood cell elements (HCT), and the amount of at least one substance comprising a protoporphyrin metal complex (Hb), comprises the following steps:

[0016] In a first step, a sample of blood or derived biological fluid is applied to an electroanalytical sensor that includes at least a first working electrode, a reference electrode, and a counter electrode.

[0017] In a second step, a current signal, which is a function of the amount of a substance containing a protoporphyrin metal complex in the blood or derived biological fluid, is detected by chronoamperometry using an electroanalytical sensor in which an insoluble electrochemical mediator is deposited on at least one working electrode. In a third step, an impedance signal, which is a function of the number of at least blood cell elements in the blood or derived biological fluid, is detected by electrochemical impedance spectroscopy. More precisely, the electronic circuitry that enables the implementation of electrochemical impedance spectroscopy allows the measurement of a quantity of resistance, called Rs, which corresponds to the transport of charge in solution and is closely related to impedance. For this reason and for simplicity, this parameter will be referred to herein by the more general term "impedance."

[0018] In a fourth step, at least one parameter of the panel is quantified using at least one specific mathematical formula, the at least one parameter being a function of the current signal and the impedance signal.

[0019] The sample is preferably venous or capillary blood. By the definition of derived biological fluid, it is intended to include different fractions of blood as well as solutions containing blood and anticoagulant substances.

[0020] Blood cell elements are intended to mean cellular elements selected from the group consisting of erythrocytes, reticulocytes, and erythroblasts.

[0021] The protoporphyrin metal complex is preferably an iron porphyrin metal complex, more preferably a metal complex of hemoglobin, myoglobin, or zinc porphyrin, and more preferably zinc protoporphyrin.

[0022] The percentage of the sample volume occupied by blood cell elements (HCT) is preferably the hematocrit. The number of blood cell elements (RC) in blood or a derived biological fluid is preferably the red blood cell count. The amount of at least one substance comprising a protoporphyrin metal complex (Hb) is preferably the hemoglobin concentration.

[0023] The volume of the sample of blood or derived biological fluid is preferably less than or equal to 50 μl, more preferably less than 15 μl.

[0024] Preferably, the panel of parameters comprises two or more of said parameters.

[0025] Preferably, the method comprises a step of detecting by chronoamperometry and a step of detecting by electrochemical impedance spectroscopy. The steps of detecting by chronoamperometry and electrochemical impedance spectroscopy may be sequential or simultaneous, preferably simultaneous.

[0026] Preferably, the step of chronoamperometric detection is carried out at a potential of -1V to +1V, more preferably at a potential of about 0.55V.

[0027] Electrochemical mediator, in the context of electroanalytical detection systems, refers to a substance that is the subject of a redox equilibrium such that it generates a current at the interface with the working electrode over a given range of potentials and spontaneously reduces or oxidizes the analyte.

[0028] Electrochemical mediators are defined as insoluble in that once electrodeposition has occurred and the mediator is then adsorbed on the electrode surface, it remains insoluble in the matrix (e.g., blood) during the measurement, whereas potassium ferrocyanide becomes soluble when a drop cast is performed (as occurs in the prior art) and a hemoglobin measurement is made.

[0029] Preferably, the insoluble electrochemical mediator is electrodeposited onto at least one working electrode, thereby avoiding the use of a ligand to trap the mediator on the working electrode.

[0030] Preferably, the insoluble electrochemical mediator is polymerizable.

[0031] Preferably, the insoluble electrochemical mediator is selected from the group consisting of methylene blue, methyl viologen, o-cresol indophenol, 2-6 dibromophenol indophenol, toluidine blue, hematoxylin / eosin, and periodic acid-Schiff stain. More preferably, the insoluble electrochemical mediator is methylene blue.

[0032] In a preferred embodiment of the method according to the invention, a parameter (Hb) is measured which is the amount of at least one substance comprising a protoporphyrin metal complex, the specific formula being: [Hb]=Z·(α4·i+β4) and where [Hb] is the concentration of the substance containing the protoporphyrin metal complex; In the formula, α4 and β4 are the slope and intercept, respectively, of the least squares fit line obtained by setting the ratio between the concentration of a substance containing a protoporphyrin metal complex and impedance on the vertical axis and the current intensity on the horizontal axis.

[0033] In the specific formula, at least one parameter is the total surface area of ​​all blood cell elements (S t ) is a function of

[0034] Total surface of blood cell elements (S t ) represents the ideal total surface area if all morphologies of red blood cells were assumed to be spherical.

[0035] In a broad sense, assuming that cells and blood cell elements, e.g., erythroblasts, reticulocytes, and red blood cells, are spherical in shape rather than doughnut-shaped, MCV ≒ 4 / 3 πr3 We can move forward by writing where MCV is the mean corpuscular volume in fL (femtoliters) and r is the radius of a cell / blood cell assumed to be spherical in shape.

[0036] Therefore, since r = d / 2 (where r is the radius of the sphere and d is the relative diameter) is known, we can substitute it into the above equation and solve for d,

[0037]

number

[0038] is obtained.

[0039] In practice, red blood cells pile up on each other and on the electrodes, so it is more convenient to consider the diameter rather than the radius.

[0040] The surface of the sphere is S=4πr 2 Considering that the surface of the cells on the sensor surface / the surface of the i-th blood cell is given by

[0041]

number

[0042] It can be easily verified that the surface of the ith sphere is given by

[0043] Considering also that the total surface of cells / blood cells interacting with the electroactive surface depends on the volume of the blood / derived biofluid droplet deposited on the sensor, on the hematocrit (volume percentage of red blood cells in blood), as well as on the number of red blood cells, St (the ideal total surface of interaction) is S t ∝RC·HCT·V can be assumed to be given by where RC is the red blood cell count, HCT is the hematocrit, and V is the volume of blood / derived biofluid deposited on the sensor.

[0044] The unit of measurement commonly used in blood chromatophores is not an SI unit, but is defined as k, which is 4.83598 10 -6 It is necessary to use a conversion factor equal to S t m 2 Note that the expression is

[0045] therefore,

[0046]

number

[0047] It is possible to obtain RC is the number of blood cell elements [n. 10 12 / l], HCT is the relative corpuscular volume [%V / V] of the blood cell element; V is the volume of the blood or derived biological fluid sample applied to the sensor; MCV is the mean corpuscular volume [fl].

[0048] St is the formula S t =(α3·Z+β3) / i is a function of the impedance signal and the current signal according to In the formula, α3 and β3 are experimentally obtained, and Z(Ω) is taken as the horizontal axis, and St·i(m 2 These are the slope and intercept of the calibration curve traced on a graph with the vertical axis being σ (µA).

[0049] In a preferred embodiment of the method according to the invention, a parameter (Hb) is measured which is the amount of at least one substance comprising a protoporphyrin metal complex, the specific formula being: [Hb]=(S t ·Z-β0-i) / α0 and where [Hb] is the concentration of the substance containing the protoporphyrin metal complex; α0 and β0 are experimentally obtained, [Hb] (g / dL) is plotted on the horizontal axis, and (St·Zi) (m 2 *These are the slope and intercept of the calibration curve traced on a graph with Ohm-μA on the vertical axis.

[0050] In another preferred embodiment of the method according to the invention, the percentage parameter of the volume occupied by blood cell elements / volume of the sample is measured, the specific formula being: HCT=(S t Z-β1-i) / α1 and HCT is the relative packed cell volume of said blood cell elements; α1 and β1 are experimentally obtained, and the horizontal axis is HCT (%v / v), and the horizontal axis is (St·Zi)(m 2 *These are the slope and intercept of the calibration curve traced on a graph with Ohm-μA on the vertical axis.

[0051] In another preferred embodiment of the method according to the invention, a parameter is measured which is the number of blood cell elements in blood or a derived biological fluid, and the specific formula is: RC=(S t Z-β2-i) / α2 and where RC is the blood cell element count; α2 and β2 are experimentally obtained and RC(n blood cell elements 10 12 / l) is taken on the horizontal axis, and (S t Zi)(m 2 *These are the slope and intercept of the calibration curve traced on a graph with Ohm-μA on the vertical axis.

[0052] In another preferred embodiment of the method according to the invention, a parameter is measured which is the mean corpuscular volume of blood cell elements in blood or derived biological fluids, the specific formula being: MCV=1 / (6 √π) (S t / (·RC·HCT·V))^(3 / 2) and where MCV is the mean corpuscular volume.

[0053] In a preferred embodiment of the method according to the invention, all parameters of the panel are measured.

[0054] An electroanalytical sensor according to the present invention for detecting and / or quantifying in a sample of blood or a derived biological fluid a panel of hematological parameters comprising at least one parameter selected from the group consisting of the number of blood cell elements (RC) in the blood or derived biological fluid, the mean corpuscular volume (MCV) of said blood cell elements, the percentage of the volume of the sample occupied by blood cell elements (HCT), and the amount of at least one substance comprising a protoporphyrin metal complex (Hb), comprising: a polyester, cellulose or ceramic support, at least one first working electrode, a reference electrode, and a counter electrode, preferably printed by screen printing onto said support; Including, An insoluble electrochemical mediator is deposited on the at least one first working electrode.

[0055] The method for manufacturing the aforementioned electroanalytical sensor comprises: - providing a polyester, cellulose or ceramic support; - printing at least one first working electrode, a reference electrode, and a counter electrode onto the support using screen printing to sequentially deposit conductive inks; - electrodepositing an insoluble electrochemical mediator onto at least one first working electrode, preferably by drip electrodeposition. Includes:

[0056] The sensors can be used individually, together with microfluidic elements, or even in combination with other sensors.

[0057] The method and sensor according to the invention have numerous applications of use: drug monitoring, screening, diagnosis and monitoring of hematological conditions, monitoring in sports subjects. [Example]

[0058] Example 1 Sensor configurations with screen-printed electrodes according to preferred embodiments of the present invention Screen-printed electrodes (SPEs) were fabricated using transparent, flexible polyester (or paper) as the support and 245 DEK (Weymouth, UK) as the screen-printing device. Four-electrode cells were fabricated using graphite-based inks for the two working and counter electrodes and silver or silver / silver chloride (Ag / AgCl)-based inks for the pseudo-reference electrode. This novel configuration can simultaneously detect hemoglobin and related blood parameters, such as hematocrit, MCV, red blood cell count, and iron. A sensor according to this embodiment is shown in Figure 1, where reference numeral 1 denotes the polyester support, reference numeral 2 denotes the Ag / AgCl ink reference electrode, and reference numeral 3 denotes the first working electrode, second working electrode, and counter electrode, all of which are graphite ink.

[0059] Example 2 Modification of screen-printed electrodes The first working electrode was modified with methylene blue (MB) by electrochemical deposition. MB is an electrochemical mediator that can selectively oxidize Fe(II) coordinated to hemoglobin heme. Specifically, electrodeposition was performed by cyclic voltammetry (CV) using the parameters specified in Figure 2. CV cycles were performed by measuring 10 μl of a solution containing 2 mM methylene blue, 0.1 M phosphate buffer (pH 7.4), and 0.1 M NaSO. The results are shown in Figure 2.

[0060] Example 3 Electrochemical measurements In the blood matrix, hemoglobin is bound to the cell membrane of red blood cells via protein clusters involving several specific membrane proteins. Hemoglobin localization is driven by the simultaneous diffusion of red blood cells within the blood volume deposited on the surface of an electrochemical cell. For these reasons, hemoglobin was determined by applying two different techniques: chronoamperometry (CA) and electrochemical impedance spectroscopy (EIS). Through CA, iron(II) can be oxidized by interacting with methylene blue, which is reduced to its semiquinone form. The current values ​​obtained from CA measurements are then correlated with the EIS response, which is determined by the volume and number of red blood cells.

[0061] In detail, CA and EIS were performed by applying the following parameters:

[0062] [Table 1]

[0063] Measurements were performed by depositing a volume of blood between 4 and 10 μl and immersing the electrochemical cell.

[0064] Example 4 Electrochemical detection of hemoglobin Hemoglobin is one of several substances that contain protoporphyrin metal complexes, in particular Fe2-protoporphyrin(IX).

[0065] In the particular case of hemoglobin, the present invention provides that the first working electrode is modified by electrodeposition with at least one redox mediator: preferably methylene blue, methyl viologen, o-cresol indophenol, 2-6 dibromophenol-indophenol, of an Fe(II)-protoporphyrin(IX) metal complex having chemical and physical properties that allow electrodeposition.

[0066] Zinc-protoporphyrin, in this particular case Zn2-protoporphyrin(IX), is one of the substances that comprise the protoporphyrin metal complex.

[0067] In the particular case of hemoglobin, the present invention provides that the first working electrode is modified by electrodeposition with at least one redox mediator of a Zn(II)-protoporphyrin(IX) metal complex, preferably diphenylbenzidine.

[0068] Hemoglobin, anchored in the plasma membrane of blood cells, exists in its optimal oxygen-binding conformation, with each hemoglobin molecule itself in the (+2) oxidation state, coordinated to four protoporphyrin structures in a 1:4 stoichiometry. The iron (+2) allows its own detection by being oxidized at a potential of +0.55 V using a solid-state electrochemical cell. The working electrode is suitably functionalized with an electrochemical mediator that allows for optimization of the kinetics of the oxidation-reduction current at the electrode interface.

[0069] In its optimal state, heme iron is reduced to (II). Apohemoglobin consists of four subunits containing protoporphyrin rings bound to iron(II) in a 1:4 stoichiometry. The sensor is based on the detection of iron(II) by oxidation with the mediator methylene blue as follows: MB+nFe 2+ ←→(S+L)+ nFe 3+ MB+nFe 2+ ←→S+L+nFe 3+ where MB is methylene blue that is reduced to the semiquinone form (S) and leuco-methylene blue (L). Depending on the pH shift, one of the two species will predominate.

[0070] In other words, the above reaction represents a redox reaction between heme iron(II) and methylene blue (MB), in which MB is reduced to two molecular forms: semiquinone (S) and leucoquinone (L). The working electrode (modified with methylene blue) is able to detect iron(II) at an applied potential of +0.55 V.

[0071] Here, iron(II) coordinated to hemoglobin heme is reliably protected from oxidation by antioxidant mechanisms (such as glutathione) and molecular mechanisms involving the conformation of the protein. Therefore, the oxidation process at the electrode of the electrochemical cell cannot occur in a "simple" manner, but must be "guided" by the use of mediators, such as methylene blue or its analogues.

[0072] A further feature already mentioned is that hemoglobin is anchored to the plasma membrane of blood cells via a series of protein clusters involving several specific membrane proteins, e.g., some spectrins, ankyrins, etc.

[0073] These two phenomena pose the experimental challenge of being able to detect hemoglobin within the context of a complex system, such as blood, where the analyte of the present invention is "protected" by the protein conformation itself or by its binding to a structure that is more dynamic in space and time, as befits a cell (red blood cell).

[0074] This type of "interference" does not take long to appear when one is attempting to detect hemoglobin using an electrochemical system consisting of a three-electrode cell printed on a polyester surface. In this state, the working electrode of the cell is functionalized by electrodeposition with methylene blue. There is no possibility of correlation between the current determined by applying a potential of 0.55 V for 60 seconds and measurements made on blood samples whose hemoglobin concentration has already been determined using a clinical reference method: the data appear to be completely random.

[0075] In this context, it is necessary to recall the phenomena already mentioned, and in particular to investigate the mechanism of interaction between the matrix and the mediator. Indeed, hemoglobin is transported by localization phenomena and at the same time by the diffusion of red blood cells within the volume of blood deposited on the surface of the electrochemical cell, in particular on the working electrode. Therefore, if the carrier is a cell, the interaction between the protein and the mediator will depend on the steric factors of the cell itself and on the shape of its distribution on the electrode surface. As is generally known, the surface factor is necessarily linked to the volume of the object being considered, and in such a respect, if there are many objects and they are all the same, the total interaction will depend on the number of objects present.

[0076] Thus, considering the hemochromatometric test, it is possible to visually identify a series of parameters necessary to be able to estimate the mentioned interaction: MCV (mean corpuscular volume), HCT (hematocrit), RBC (red blood cell count).

[0077] Therefore, what has so far been defined as the interaction between the analyte-transporting cells and the functionalized surface of the working electrode can be further defined as an estimation of the electroactive surface (matrix) at the working electrode.

[0078] According to some observations (see above), the total electroactive surface of the red blood cell is

[0079]

number

[0080] It can be analytically inferred as follows: During the ceremony, RC is the number of blood cell elements [n. 10 12 / l], HCT is the relative corpuscular volume [%V / V] of the blood cell element; V is the volume of the blood or derived biological fluid sample applied to the sensor; MCV is the mean corpuscular volume [fl].

[0081] In order to be able to establish whether the hypothesis published and supported so far by this first evidence was correct, it was necessary to investigate the matrix using impedance measurement techniques, thus measuring the impedance via a second working electrode.

[0082] In this regard, having verified that the obtained impedance values ​​could then be normalized by the number of red blood cells contained in the volume deposited on the electrode surface and correlated to MCV, the inventors proceeded to study the correlation between hemoglobin concentration and other parameters considered in hemocytometry tests.

[0083] Measurements of red blood cell count (RC), hematocrit (HCT), hemoglobin, and mean corpuscular volume (MCV) can be derived from measuring the surface coefficient (St) as shown below.

[0084] The surface coefficient St is related to the current (i) and impedance (Z) by the following relationship: S t =(α3Z+β3) / i where α3 and β3 are the slope and intercept of the curve shown in Figure 3, respectively. The least-squares curve interpolated through the points and shown as "Straight line (St(Z))" has a slope of α3 = 1.17 10 -7 and intercept β3 = -9.46·10 -5 It has.

[0085] To be able to measure the red blood cell count (RC), hematocrit (HCT), mean corpuscular volume (MCV), and hemoglobin [Hb] concentration of an unknown blood sample, we proceeded as follows.

[0086] Once a calibration curve is obtained from the impedance and current values ​​measured in unknown blood samples, as shown in Figure 3, the St value can be obtained via the formula shown above.

[0087] Then, [Hb] (g / dL), HCT (%v / v), and RC (10 12 Values ​​for the number of cells in 100 ml / L can be obtained using the respective calibration curves as shown in Figures 4, 5, and 6.

[0088] Interpolate the points and create a "straight line (St * The least-squares curve, denoted as "α0 = 0.111" and intercept β0 = -0.893 (Figure 4).

[0089] Therefore, the unknown measurement of hemoglobin concentration can be obtained by solving the following equation: [Hb]=(S t ·Z-β0-i) / α0 In the formula, α0 and β0 are the slope and intercept, respectively, of the calibration curve shown in FIG.

[0090] Similarly, HCT will result from relationships similar to those above, HCT=(S t Z-β1-i) / α1 In the formula, α1 and β1 are the slope and intercept, respectively, of the calibration curve shown in FIG.

[0091] Interpolate the points and create a "straight line (St * The least-squares curve, denoted as "α0 = 0.045" and intercept β0 = -1.226 (Figure 5).

[0092] Similar to the relationship shown above, the number of red blood cells in an unknown sample is RC=(S t Z-β2-i) / α2 can be obtained from the relationship In the formula, α2 and β2 are the slope and intercept of the calibration curve shown in Figure 6, respectively. *The least-squares curve, denoted as "Zi"), has a slope α2 = 0.224 and an intercept β2 = -0.433 (Figure 6).

[0093] Finally, to obtain the MCV of an unknown sample, it is sufficient to insert the measurements of the parameters obtained so far (St, HCT and RC) into the "inverse" of the equation for the total electroactive surface given above, in which case MCV=1 / (6 √π) (S t / (·RC·HCT·V))^(3 / 2) is obtained.

[0094] Based on these observations, a second mathematical formula for the specific measurement of hemoglobin was also derived that directly correlates to both current and impedance values ​​measured using the same sensor: [Hb]=Z(α4·i+β4) where α4 and β4 are the slope and intercept, respectively, of the least squares interpolation curve shown in FIG.

[0095] To illustrate what has been shown so far, the values ​​obtained by measuring the current and impedance of eight unknown samples are shown in the table below.

[0096] [Table 2]

[0097] The types of matrices analyzed in this context are capillary and venous blood. The signals (i and Z) obtained in capillary blood samples differed in absolute value from those in venous blood samples. The correlation coefficient between the two calibration curves (capillary and venous) was equal to 1.33.

[0098] In particular, the optimal sampling conditions are: i. The test time for electrochemical sensors must be less than 4 hours from the time of collection (for venous blood collection by butterfly and vacutainer systems); ii. Capillary blood collection can be performed using an anticoagulant system (EDTA-K) or within the anticoagulant system It was.

[0099] The measurements shown in the table above were obtained with capillary blood samples. Venous blood has different current and impedance values ​​than capillary blood: the ratio value between the current obtained with the venous blood sample and the current of the capillary blood sample was equal to 1.33.

[0100] Quantification of hemoglobin concentration and other blood chromatocytometry parameters was performed by calibration curves between samples processed by the reference method (using venous blood) and samples processed by the electrochemical method (using capillary blood from the same patient).

[0101] The results obtained using this analytical approach are shown in the table below.

[0102] [Table 3]

[0103] Thus, correlations were reported for 11 blood samples initially analyzed with the reference method for hemochromatometry testing.

[0104] In Figure 8, it can be verified that the impedance measurements (Z) corrected by the electroactive surface coefficient (St) correlate highly significantly with the hemoglobin concentration expressed in mol / L (M). The horizontal scale is logarithmic. The hemoglobin values ​​are expressed in g / dL and range from 10.4 to 16.2.

[0105] Figure 9 shows a comparison between the new measurement model for blood hemoglobin concentration and the hemoglobin concentration determined using the reference method. Both concentrations are shown in [g / dL]. The samples compared are always 11 models. A significant correlation is observed. The maximum retention rate is 6.9%.

[0106] Figure 10 shows the resistance measurements (ΔV / I) normalized to the number of red blood cells contained in a capillary blood volume (60 μl) deposited on the surface of the electrochemical cell as a function of the impedance measurements, expressed as a natural logarithm. Thus, the inverse proportionality between the number of red blood cells and the impedance can be inferred therefrom. Seven samples are considered.

[0107] All this evidence leads to what currently represents a general model on which the analytical method is based: this model is an empirical relationship obtained from correlable quantities, such as resistance, calculated in the ohmic region from the current measured by chronoamperometry (first part of the measurement) and the impedance obtained in the second part of the measurement.

[0108] In this sense, it should be specified that the sensor consists of two working electrodes at which the aforementioned measurements are carried out independently.

[0109] advantage Compared to the methods according to the prior art, the method and device according to the invention have the following advantages: - Devices (sensors) are becoming smaller; - the methods and devices are easier to industrialize; - low costs of the method and device; - Significantly smaller sample volume required; - No pretreatment of the biological matrix is ​​required; - The time required for analysis is reduced; - the method is simple to perform and the device is easy to use; - Measurements can be performed both in the laboratory and at the point of care; - The device is reagent-free; - The device allows for accurate and reliable measurement of hemoglobin; - the method can be applied to whole blood, capillary blood, serum, and plasma without the need for pretreatment of the biological matrix; the method can also be used in the presence of anticoagulants; - the method may also allow the detection of all metalloproteins, in particular iron(II) and iron(III) containing metalloproteins such as myoglobin, and may also allow the amount of iron itself in biological fluids to be dosed; The same reading device can be used to determine other tests for which single-use diagnostic units exist (eg blood glucose).

[0110] Declaration pursuant to Article 170.2(2) of the Italian Intellectual Property Code The biological material of human origin used in the present invention was obtained in accordance with applicable legal regulations.

Claims

1. 1. A method for detecting and / or quantifying in a sample of blood or a derived biological fluid a panel of hematological parameters comprising at least one parameter selected from the group consisting of the number of blood cell elements in the blood or derived biological fluid (RC), the mean corpuscular volume (MCV) of said blood cell elements, the percentage of the volume of the sample occupied by blood cell elements (HCT), and the amount of at least one substance comprising a protoporphyrin metal complex (Hb), comprising: - applying a sample of blood or derived biological fluid to an electroanalytical sensor comprising at least one first working electrode, a reference electrode and a counter electrode; - detecting a current signal by chronoamperometry using an electroanalytical sensor, the current signal being a function of the amount of a substance comprising a protoporphyrin metal complex in the blood or derived biological fluid, wherein an insoluble electrochemical mediator is deposited on the at least one first working electrode; and detecting an impedance signal by electrochemical impedance spectroscopy, the impedance signal being a function of the number of at least blood cell elements in the blood or derived biological fluid; - quantifying at least one parameter of the panel using at least one specific mathematical formula, the at least one parameter being a function of the current signal (i) and the impedance signal (Z); Including, A method in which in a particular mathematical formula at least one parameter is a function of the total surface area of ​​the blood cell element (St).

2. St is of the formula St=(α ３ ・Z+β ３ ) / i is a function of the impedance signal (Z) and the current signal (i) according to In the formula, α ３ and β ３ 2. The method of claim 1, wherein Z and S are the angle coefficient and intercept, respectively, of a calibration curve experimentally obtained and traced on a graph with Z on the horizontal axis and St·i on the vertical axis.

3. A parameter (Hb) is measured, which is the amount of at least one substance containing a protoporphyrin metal complex, and a specific mathematical formula is used to calculate the Hb. [Hb]=Z・(α ４ ・i+β ４ ) and where [Hb] is the concentration of the substance containing the protoporphyrin metal complex; α ４ and β ４ 3. The method according to claim 1 or 2, wherein a slope and an intercept of a least-squares fit line obtained by setting the ratio between the concentration of the substance containing the protoporphyrin metal complex and the impedance on the vertical axis and the current intensity on the horizontal axis, respectively.

4. [Equation 1] and In the formula, RC is the number of blood cell elements [n. 10 １２ / l], HCT is the relative corpuscular volume [%V / V] of the blood cell element; V is the volume of the blood or derived biological fluid sample applied to the sensor; MCV is the mean corpuscular volume [fl]; St is represented by the formula St=(α ３ ・Z+β ３ ) / i is a function of the impedance signal and the current signal according to In the formula, α ３ and β ３ are experimentally obtained, Z(Ω) is taken on the horizontal axis, and St・i(m ２ 4. The method according to claim 1, wherein the slope and intercept of a calibration curve are traced on a graph with the vertical axis being μA.

5. A parameter (Hb) is measured, which is the amount of at least one substance containing a protoporphyrin metal complex, and a specific mathematical formula is used to calculate the Hb. [Hb]=(St・Z-β ０ -i) / α ０ and where [Hb] is the concentration of the substance containing the protoporphyrin metal complex; α ０ and β ０ are experimentally obtained, [Hb] (g / dL) is plotted on the horizontal axis, and (St·Zi) (m ２ * The method according to any one of claims 1 to 4, wherein the slope and intercept of a calibration curve are traced on a graph with Ohm-μA as the vertical axis.

6. The parameter percentage of the volume occupied by blood cell elements / volume of the sample is measured and a specific formula is HCT=(St・Z-β １ -i)・α １ and where HCT is the relative packed cell volume of said blood cell element; α １ and β １ are experimentally obtained, and the horizontal axis is HCT (% v / v), and the horizontal axis is (St·Zi)(m ２ * The method according to any one of claims 1 to 5, wherein the slope and intercept of a calibration curve are traced on a graph with Ohm-μA as the vertical axis.

7. The parameter, which is the number of blood cell elements in blood or derived biological fluids, is measured and a specific mathematical formula is used. RC=(St・Z-β ２ -i) / α ２ and where RC is the blood cell element count; α ２ and β ２ are experimentally obtained, and RC (n blood cell elements × 10 １２ / l) is taken on the horizontal axis, and (St·Zi)(m ２ * The method according to any one of claims 1 to 6, wherein the slope and intercept of a calibration curve are traced on a graph with Ohm-μA as the vertical axis.

8. The parameter measured is the mean packed cell volume in blood or derived biological fluids, and a specific mathematical formula is used to MCV=1 / (6√π)・(St / (RC・HCT・V))^(3 / 2) and 8. The method of claim 1, wherein MCV is the mean corpuscular volume.

9. 9. The method of claim 1, wherein all parameters of the panel are measured.

10. 10. The method of claim 1, wherein the blood cell element is a cellular element selected from the group consisting of red blood cells, reticulocytes, and erythroblasts.

11. 11. The method according to any one of claims 1 to 10, wherein the protoporphyrin metal complex is an iron protoporphyrin complex, preferably a complex of hemoglobin, myoglobin, or zinc protoporphyrin, preferably zinc porphyrin.

12. 12. The method of any one of claims 1 to 11, wherein HCT is hematocrit, RC is red blood cell count, and Hb is hemoglobin.

13. 13. The method of any one of claims 1 to 12, wherein the insoluble electrochemical mediator is selected from the group consisting of methylene blue, methyl viologen, o-cresol indophenol, 2-6 dibromophenol indophenol, toluidine blue, hematoxylin / eosin, and periodic acid-Schiff stain.

14. 14. The method of claim 13, wherein the insoluble electrochemical mediator is methylene blue.

15. 15. The method of any one of claims 1 to 14, wherein the sample volume in blood or derived biological fluid is less than or equal to 50 μl.

16. 16. The method of claim 15, wherein the volume of blood or derived biological fluid is less than 15 μl.

17. 17. The method according to any one of claims 1 to 16, wherein the step of chronoamperometric detection is carried out at a potential of -1 V to +1 V.

18. 18. The method of claim 17, wherein the step of chronoamperometric detection is carried out at a potential of about 0.55 V.

19. 1. An electroanalytical sensor for detecting and / or quantifying in a sample of blood or a derived biological fluid a panel of hematological parameters comprising at least one parameter selected from the group consisting of the number of blood cell elements (RC) in the blood or derived biological fluid, the mean corpuscular volume (MCV) of said blood cell elements, the percentage of the volume of the sample occupied by blood cell elements (HCT), and the amount of at least one substance comprising a protoporphyrin metal complex (Hb), - a polyester, cellulose or ceramic support, at least one first working electrode, a reference electrode and a counter electrode, preferably printed by screen printing onto said support; Including, An electroanalytical sensor, wherein an insoluble electrochemical mediator is deposited on the at least one first working electrode.

20. 20. A method for manufacturing an electroanalytical sensor according to claim 19, comprising: - providing a polyester, cellulose or ceramic support; - printing at least one first working electrode, a reference electrode, and a counter electrode onto said support using screen printing to successively deposit conductive inks; - electrodepositing an insoluble electrochemical mediator onto at least one first working electrode; A method comprising:

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