Electrochemical microcell

Abstract

An electrochemical microcell comprising: - a chamber adapted to insert an electrode; - at least one microchannel adapted to load a solution of analysis reagent and an analyte solution; wherein the at least one microchannel is in fluid communication with the chamber of the electrochemical microcell, and wherein the chamber is integral with the at least one microchannel; and a method of analysis by means of said electrochemical microcell.

Description

[0001]Title: Electrochemical microcell DESCRIPTION Field of Application In its most general aspect, the present invention relates to the sector of microfluidic devices, precisely to a reagentless electrochemical microcell. In particular, the present invention relates to a reagentless electrochemical microcell and to a method for the use thereof in the scope of a detection process through an electrochemical technique. Prior art Most of the known microfluidic devices are based on polydimethylsiloxane (PDMS) processed by injection molding and soft lithography. Another relevant type of microfluidic devices is based on paper. Moreover, it is known that said microfluidic devices are devised to operate in continuous flow on microvolumes of sample and to optimize the analytic response. Some of the various analytes detectable by said type of devices are, for example, heavy metals, antigens, enzymes, and pesticides. Despite the advantages provided by the miniaturization of the system, the use of microfluidic devices in continuous has some disadvantages, including the fact that the techniques used for making common microfluidic devices are difficult to transfer to an industrial scale, the choice of the materials used is limited since sometimes they are not suitable for the reagents used and often are characterized by a certain brittleness. The device described in the publication “Amperometric measuring cell for the determination of putrescine oxidase activity”, Talanta 47, 367–376 (1998) by Nagy et al. consists of a PDMS microcell made by photolithography, said microcell being loaded with a disc made of porous material containing the reagent, which is immobilized. The publication “Microfluidic Device Directly Fabricated on Screen-Printed Electrodes for Ultrasensitive Electrochemical Sensing of PSA” Nanoscale Res. Lett., vol. 14, pp. 0–6, 2019 by Chen et al. describes a PDMS microfluidic device made by photolithography and comprising a magnetic foil, on which magnetic particles conjugated with anti-PSA antibodies are loaded. Said device is designed to operate in continuous flow. However, there remains the need to replace the microfluidic devices suitable for a use in continuous, since said procedure is almost impossible to be carried out except by expert operators and in the context of an analytical laboratory, thereby excluding in-situ use. Instead, patent CN102620959 describes an electrochemical- microcell device made to carry out electroluminescence measurements and comprising at least one chamber, into which the analysis reagent is to be placed, and a sample chamber, said chambers being connected to each other. The reagents are immobilized inside the system. However, reagent immobilization, which, in the context of printed electrodes, is normally carried out on the working electrode, generally causes reduced sensitivity and versatility of the system. Moreover, in particular, there remains the need to provide a technology using an electrochemical cell, in which reagents do not necessarily have to be immobilized inside the system. Thus, the technical problem underlying the above-mentioned invention is to provide an electrochemical microcell that is convenient and easy to produce and more practical to use, thereby solving the above- mentioned problems. Summary of the invention Said problem was solved by providing an electrochemical microcell comprising: - a chamber adapted to insert an electrode; - at least one microchannel adapted to load a solution of analysis reagent and an analyte solution, wherein the at least one microchannel is in fluid communication with the chamber of the electrochemical microcell, and wherein said chamber is integral with the above-mentioned at least one microchannel. According to the present invention, the expression “integral with” means that the chamber and the at least one microchannel are made as a single piece. Preferably, the chamber of the microcell comprises a housing pocket adapted to insert an electrode and a reaction zone, said reaction zone being in fluid communication with said housing pocket. According to a specific embodiment of the present invention, the electrode is a printed electrode, in particular a screen-printed electrode. According to the present invention, the expression “printed electrode" means a miniaturized electrode obtained by printing and supported on a solid support, for example printed by screen printing, also called “Screen Printed Electrode” or SPE electrode. In particular, printed electrodes are typically a constituent part of an electrochemical cell, having length and width of a few tens of millimeters, and comprise a working electrode, being the signal transducer, a reference electrode, and an auxiliary electrode or counter electrode; in the cell, said electrodes are printed on a support, in particular a thin film made of polyester or of ceramic material. Advantageously, said electrochemical microcell according to the invention can be defined “reagentless”, i.e., as will be seen below, it has a wide range of applications and its use is not limited to the type of solvent used for dissolving the reagent types. In particular, there is no need to immobilize the reagents in the microcell. Even more advantageously, said electrochemical microcell can also be used by not very experienced employees. In particular, according to one of its modes of use which is described below with reference to the process of the invention, the electrochemical microcell of the invention may be pre-loaded with a reagent and does not need the use in continuous flow, thereby enhancing the worker’s safety, since the contact with said reagent is avoided, and allows to carry out analyses in situ or on field. Indeed, because of the microchannel into which the reagents are pre-loaded through simple techniques, the electrochemical microcell can be used in the context of a method of analysis even outside a chemical laboratory. Preferably, said reaction zone has a volume between 80 µL and 250 µL, preferably between 120 µL and 180 µL, and / or said microchannel has a volume between 10 µL and 22 µL. Preferably, said electrochemical microcell is adapted to the use in discontinuous flow. Preferably, said electrochemical microcell is a monolithic structure formed as one piece. Preferably, said electrochemical microcell is made of a thermoplastic elastomer or a mixture of thermoplastic elastomers. According to the present invention, the term “thermoplastic elastomer” means a copolymer (in particular, a plastic and a rubber) consisting of a material having both thermoplastic and elastomeric properties. Thermoplastic elastomers are characterized by the ability to undergo moderate elongations and to substantially return to their original shape, thus resulting in a longer life and a better capacity compared to other materials. The main difference between thermosetting elastomers and thermoplastic elastomers is the type of cross-linking binding in their structures. In particular, the thermoplastic elastomer is a polymer that is suitable for the use in 3D printing or the above-mentioned mixture of thermoplastic elastomers comprises polymers that are suitable for the use in 3D printing. More preferably, the thermoplastic elastomer is a thermoplastic polyurethane (TPU) or the above-mentioned mixture of thermoplastic elastomers comprises thermoplastic polyurethane. Advantageously, the size of said microcell is adjustable according to analysis needs. In particular, as will be seen below in the examples, said microcell is ready to use and is capable of working even with a small volume of solution, thanks to the increased effectiveness in detecting analytes, even when analytes are in a minimum amount. Moreover, the fact of being able to work with small volumes of sample allows to use a reduced amount of reagents. The above-mentioned technical problem has been solved by providing a method of analysis through an electrochemical technique comprising the following steps: a) providing an electrochemical microcell according to the invention; b) pre-loading the solution of analysis reagent into said electrochemical microcell, thereby obtaining a pre-loaded electrochemical microcell; c) inserting an electrode device into the chamber of said pre- loaded electrochemical microcell, preferably into said housing pocket; d) loading an analyte solution into said pre-loaded electrochemical microcell; e) carrying out a measurement through an electrochemical technique. Preferably, said electrode device is a printed electrode, more preferably a screen-printed electrode. Preferably, said electrode device comprises a reference electrode, a working electrode, and a counter electrode or auxiliary electrode. Preferably, in said step e), the electrochemical technique may be selected from cyclic voltammetry and chronoamperometry. Preferably, in said step a), the electrochemical microcell is 3D- printed. Advantageously, thanks to 3D printing, said method of production of said microcell is simple, reproducible, and easily transferable to an industrial scale. In an equally preferred manner, the electrochemical microcell is made of a thermoplastic elastomer or a mixture of thermoplastic elastomers, more preferably selected of thermoplastic polyurethane or a mixture of thermoplastic elastomers comprising thermoplastic polyurethane. Preferably, steps a) to e) are carried out in discontinuous. Preferably, the pre-loading step b) comprises the following substeps: i) introducing a solution of analysis reagent into the at least one microchannel of said electrochemical microcell; ii) freezing or drying said solution of analysis reagent so introduced into the electrochemical microcell, thereby obtaining a pre- loaded electrochemical microcell containing at least one analysis reagent; iii) optionally, storing said pre-loaded electrochemical microcell so obtained in step ii) under controlled environmental conditions. The expression “solution of analysis reagent” means a solution comprising analysis reagents, and solvents or further solutes, such as, for example, a buffer. More preferably, in step ii), the solution of analysis reagent introduced into the electrochemical microcell is frozen, wherein freezing of the solution of analysis reagent inside the microchannel is carried out by means of a freezing device operating at about -18°C. Alternatively, in step ii), the solution of analysis reagent introduced into the electrochemical microcell is dried, wherein drying is carried out under nitrogen flow, using a specific desiccator placed at inert atmosphere, or by freeze-drying. Preferably, in said step b), the solution of analysis reagent comprises at least one reagent, more preferably said reagent may be selected from enzymes, bioactive molecules or organic or inorganic molecules. Preferably, the loading step b) comprises the following substeps: - introducing an analyte solution into the at least one microchannel of said pre-loaded electrochemical microcell, thereby obtaining an electrolytic solution comprising said analyte solution and said at least one analysis reagent; and - letting said electrolytic solution flow from the microchannel to the chamber of said electrochemical microcell. Preferably, in said step d), said analyte may be selected from enzymes, bioactive molecules or organic or inorganic molecules. Said problem has been solved by a use of the electrochemical microcell according to the invention in a method of analysis through an electrochemical technique, preferably in the analysis of organic and inorganic compounds, bioactive molecules or enzymes. Said method is very advantageous in the environmental, medical-health, and food sectors. The above-mentioned technical problem has also been solved by means of a kit comprising the electrochemical microcell of the above- mentioned invention and an electrode device, said electrode device being a printed electrode, more preferably a screen-printed electrode. Preferably, said electrode device comprises a reference electrode, a working electrode, and a counter electrode or auxiliary electrode. Preferably, the above-mentioned electrode device may adapted to be placed in the housing pocket of the chamber of the above-mentioned electrochemical cell, thereby leading to the advantages explained in relation to the process of the invention in the summary and in the following detailed description. Preferably, the above-mentioned electrode device is placed in the housing pocket of the chamber of the above-mentioned electrochemical cell. The features and the advantages of the present invention will become clearer from the following description, which is provided by way of non-limiting example, with reference to the attached figures. Brief description of the drawings Figure 1 shows a perspective view of the microcell according to the invention. Figure 2 shows a sectional view in the direction of the height of the microcell according to the present invention. Figure 3: Figure 3a illustrates a 3D-printed microcell according to the invention without an electrode device. Figure 3b illustrates a 3D- printed microcell according to the invention, with a screen printed electrode placed inside the housing pocket of the chamber of the microcell. Figure 3c illustrates the screen printed electrode of Figure 3b. Figure 4 illustrates the cyclic voltammograms (CV) recorded using the microcells according to the invention in the presence of sodium ascorbyl phosphate (AAP). Figure 5: Figure 5a illustrates the response straight line obtained using the storing mode by freezing (a) and calculating the average of all the signals of peak current (Ip) at the different AAP concentrations, obtained from three replicated measurements. The linear interpolation on the average signals was made using the method of least squares. The error bars express the standard deviation associated to each average signal. Figure 5b illustrates a response straight line obtained using the storing mode by drying (b). Figure 6: Figure 6a illustrates the cyclic voltammogram relative to the tests carried out after different storage periods. Figure 6b illustrates the trend of the average sensitivity of the microcells according to the invention with varying storage times. Figure 7: Figure 7a shows the cyclic voltammogram (CV) and Figure 7b shows the response straight line obtained by the standard addition method on the analyzed sample of Example 1.2. Figure 8: Figure 8a illustrates the chronoamperograms relative to one of the three replicated tests. Figure 8b shows the response straight line obtained from the average of the signals obtained in the three replicated tests. Detailed description of a preferred embodiment With reference to Figures 1 and 2, the electrochemical microcell according to the present invention, according to a particular embodiment thereof, is referred to as a whole with reference number 100. The microcell 100 comprises a chamber 5, namely, a compartment intended to contain a printed electrode. In particular, the electrochemical microcell, which is substantially shaped as a parallelepiped and made of a thermoplastic elastomer, comprises a base 1 and four side walls, in particular two first walls 3a and 3b and two second walls 3a’ and 3b’, opposite said first walls 3a and 3b, respectively, which extend from the perimeter of said base 1, defining an overall lateral envelope and an upper opening 12, adapted to insert the electrode. In detail, as shown in Figure 2, said chamber 5 further comprises a housing pocket 11, in particular comprising two zones: - an upper zone located at the opening 12 and adapted to facilitate the insertion of a screen printed electrode, and - a lower zone located at the reaction zone 13, which is in fluid communication with said housing pocket 11, the lower zone being thus adapted so that the reaction zone is placed at and in fluid communication with a detection zone of the screen printed electrode comprising a working electrode, being the signal transducer, a reference electrode, and an auxiliary electrode or counter electrode, once the screen printed electrode is placed in the chamber 5 of the electrochemical microcell 100, as shown in Figure 3b. Specifically, the electrodes that constitute the detection zone of the screen printed electrode are printed on a support, in particular made of ceramic material. In the reaction zone 13, the electrochemical reaction takes place when the electrolytic solution contacts the detection zone of the screen printed electrode. The chamber 5 is integral with a microchannel 10, wherein the microchannel 10 extends from the lower zone of the chamber 5 and is in fluid communication therewith. As illustrated, the microchannel 10 substantially extends in a parallel direction with respect to the chamber 5 and along the wall 3a of the electrochemical microcell 100. As is evident and generally speaking, the electrochemical microcell 100 also comprises a duct 7, adapted to put the chamber 5 and the microchannel 10 in fluid communication. The microchannel 10, having a substantially tubular shape, is adapted to store both the analyte solution and the solution of analysis reagent. Said solutions are introduced into said microchannel 10 through an upper opening 15 and said solutions are let flow into said chamber 5 of the electrochemical microcell 100 through the duct 7. As illustrated, the duct 7 extends substantially transversely with respect to the chamber 5 and the microchannel 10. The electrochemical microcell 100 may also be provided with closing means 6 and 6’, which are adapted to simultaneously close both the chamber 5, precisely the opening 12 thereof, and the microchannel 10, precisely the opening 15 thereof, respectively, so as to improve the storage of the reagents, once they are pre-loaded. The closing means 6 and 6’ are formed as one piece and comprise a safety filament 16 and a fastening ring 17. The safety filament 16 and the fastening ring 17 are formed as one piece. The fastening ring 17 is adapted to externally surround the electrochemical microcell 100. Advantageously, thanks to the filament 16 and the fastening ring 17, during the operations of loading the reagents or the analyte, there is no risk of contaminating or losing the closing means 6 and 6’. Said electrochemical microcell 100 has been printed by means of a 3D printer Prusa i3 MK3S+, and the material used is a thermoplastic elastomer (filament “Ninjaflex” of Adafruit Industries LLC). During printing, the nozzle was set at a temperature of 225°C and placed at a distance of 0.1 mm from the build plate, which had a temperature of 40°C. The electrochemical microcell described in the following examples has a reaction zone with a volume of 150 µL, when the SPE electrode is inserted, whereas the volume of the lateral microchannel is 15 µL. 1. Examples 1.1 Production of microcells according to the invention having alkaline phosphatase (ALP) as the element for biological recognition The enzyme alkaline phosphatase (ALP) was used as the element for biological recognition. The enzyme, the solvents, and further solutes required for the measurement (TRIS-HCl buffer at pH 8 and MgCl2 as a cofactor) were pre-loaded into the lateral microchannel, in concentrated solution. In particular, for each microcell, first of all 10 microliters of an aqueous solution of 1.5 M TRIS-HCl + 3.75 mM MgCl^were injected into the microchannel. Then, 5 microliters of an aqueous solution of 0.1 M TRIS-HCl with a concentration of 500 mg / L of ALP were injected into the microchannel 5, to make a total of 15 microliters of reagent solution. The storage of the pre-loaded solution of analysis reagent was tested with two different modes: a) Freezing the pre-loaded solution of analysis reagent in the microchannel inside a freezing device with a temperature set at -18°C; b) Drying the pre-loaded solution of analysis reagent in the microchannel, in a specific desiccator placed at inert atmosphere and under nitrogen flow; thus, the solvent of said solution evaporates and the pre-loaded solution of analysis reagent remains in the solid phase. The time required to completely dry the solution of analysis reagent in the microchannel ranges from 2 to 3 days. At the time of the analysis, the solution of analysis reagent is rehydrated. 1.1.1 Performance of electrochemical microcells Afterward, a calibration curve of five points was prepared using sodium ascorbyl phosphate (AAP) as the enzymatic substrate. In the presence of enzyme ALP, the substrate AAP undergoes the dephosphorylation reaction outlined below (reaction 1). The ascorbic acid (AA) produced in reaction 1 can be oxidized on the surface of the working electrode, thereby generating a current which is proportional to its concentration, which in turn is proportional to the concentration of AAP, initially present in the electrolytic solution. In five electrochemical microcells made according to the invention, aqueous AAP solutions (analyte solutions) having increasing concentrations of 0 to 0.13 mM were respectively loaded. The measurements were carried out at 37°C so as to operate within the range of maximum enzymatic activity of ALP. The electrochemical measurements were carried out using the technique of cyclic voltammetry, applying a controlled potential variation at a screen printed electrode, precisely a carbon-membrane SPE electrode Drop-sens DS150 of the firm Metrohm, and recording current intensity. After each measurement, the SPE electrode is washed with water and inserted into the reaction zone. Specifically, the screen printed electrode used is formed by a reference electrode made of silver, a working electrode made of carbon, and a counter electrode or auxiliary electrode made of platinum, wherein the electrical circuit contacts are made of silver. The overall dimensions of the ceramic substrate are 34 mm (length) x 10 mm (width) x 0,5 mm (thickness). The following parameters were evaluated: sensitivity, R2, LOD (Limit Of Detection) and LOQ (Limit Of Quantification) from the response straight lines obtained. The sensitivity was calculated by dividing the slope of each response straight line by the geometric area of the working electrode. The LOD and LOQ values were obtained from the linear regression parameters of each response straight line. The performances of the electrochemical microcells were tested using the two storing modes a) and b) described above, and the obtained results were compared with each other. The performances obtained with the electrochemical microcells made according to the invention were compared with that of a classic electrochemical cell (below defined “cell”) operating with an SPE electrode having the enzyme ALP immobilized on its surface. The comparison of said performances is illustrated in Table 1. Range Sensitivity m ALP R^st LOD LOQ AAP] (μA mM- raight Device [1(mg) li ) mm- ne (mM) (mM) (mM2) a) Microcell according to the 0.0027 0-0.13 *0.66 ± 0.09 0.998 0.008 0.024 invention -SPE frozen reagents b) Microcell according to the 0.0027 0-0.13 0.64 ± 0.02 0.996 0.004 0.012 invention -SPE dried reagents c) Cell- immob. 0.10 0-5.0 0.119 ± 0.002 0.997 0.2 0.6 ALP on SPE *The sensitivity was obtained from the average of the sensitivities calculated on the three replicates made and the associated error is represented by the standard deviation of the average of the sensitivities (RSD %= 9%). Table 1 5 As can be seen from Table 1, the electrochemical microcells made according to the invention (“a” and “b”) have a sensitivity that is more than five times greater compared to the electrochemical cells in which the enzyme ALP is immobilized on the electrode (“c”). Such result is obtained using an enzyme amount almost 40 times lower in the 10 production of the electrochemical microcells “a” and “b”. The LODs of the electrochemical microcells “a” and “b” are also two orders of magnitude lower than the LOD of the “classic” electrochemical cells “c". The greater sensitivity of the electrochemical microcells “a” and “b” is due to the fact that the free enzyme in the solution of analysis reagent allows to have a kinetics of the enzymatic reaction not affected by diffusion phenomena or alterations of the structure of active sites, which instead occur when the enzyme is immobilized. Moreover, the reduced amount of ALP to be used for the electrochemical microcells “a” and “b” compared to the traditional cell “c” cause them to be more advantageous from the economic standpoint. Moreover, Table 1 also shows that the different storing mode has little influence on the sensitivity of the final device, and therefore it is also possible to use devices having pre-dried reagents without incurring particular performance alterations. 1.1.3 Stability of the reagents pre-loaded into the electrochemical microcells according to the invention The stability of the constructed microcells was evaluated by carrying out a measurement of cyclic voltammetry on an analyte solution at a specific AAP concentration (0.1 mM) after 0, 6, 15 and 30 days of storage of the pre-loaded microcells placed in a refrigerator at 4°C. The modes and the measurement conditions used are similar to those used in paragraph 1.1.2. The blank sample without AAP was prepared. The sensitivity was calculated from the obtained signals, after subtraction of the blank sample. Three replicates were made for each measurement, and the average sensitivities and relative standard deviations were calculated. The results are shown in Table 2. Storage time (days) Average sensitivity (µA mM-1mm-2) 0 (immediate use) 0.60 ± 0.04 6 0.56 ± 0.06 15 0.57 ± 0.06 30 0.47 ± 0.05 Table 2 As can be observed in Table 2, there are no significant sensitivity variations after 15 days of storage, whereas there is a slight reduction (ca. 20%) starting from 30 days of storage. As a consequence, the solution of analysis reagent pre-loaded and stored in the above- described modes can also be used after several days. After six days of storage, the stability of the constructed microcells was compared with the stability of a traditional cell having the enzyme ALP immobilized. The degree of stability (GS) was calculated using equation 1 (eq. 1). wherein: S0 represents the sensitivity of the electrochemical microcell when it is used immediately; S 6 days represents the sensitivity of the electrochemical microcell used after 6 days of storage. Table 3 shows a comparison between the degree of stability relative to the developed microcell and the one relative to a traditional electrochemical cell having the enzyme ALP immobilized. Degree of Device Storage time (days) stability Cell with immobilized ALP 6 0.45 Electrochemical microcell according to the 6 0.93 invention Table 3 Table 3 shows that the degree of stability of the microcell developed is about twice as great as the one of an electrochemical cell having the enzyme ALP immobilized. This is possible because the enzyme ALP stored inside the microcell does not undergo denaturation or deactivation processes, that instead can occur following immobilization, and thus the stability of the pre-loaded electrochemical microcell according to the invention is better. 1.2 Analysis of a real sample and evaluation of the matrix effect A suitably diluted sample of anti-aging serum “NIVEA LUMINOUS630” containing AAP as an antioxidant agent was analyzed. The analysis was carried out by the technique of cyclic voltammetry and the slope of the response straight line obtained with the standard addition method was compared with that of the straight line in Figure 5a (external-calibration curve). The measurements were carried out under the same conditions used in paragraph 1.1.2. The comparison between the slopes of the two straight lines was carried out by means of a t-test, in which the presence of a significant difference between the two values with a confidence level equal to 95% was assumed as the null hypothesis (H0). Before calculating the parameter t, an F-test was carried out to evaluate whether the variances s2(y / x)1 and s2(y / x)2 belonged to the same Gaussian distribution, namely an hypothesis test based on the Fisher- Snedecor F distribution, which is used to verify the hypothesis that two populations, both following normal distribution, have the same variance. The parameter Fcalc was calculated with the formula expressed by equation 2 (eq.2). wherein s2(y / x)2 represents the variance associated to the points of the calibration curve obtained with the standard addition method (Fig.7b) s2(y / x)1 represents the variance associated to the points of the external-calibration curve (Fig.5a) The parameter Fcalc was compared with the F value tabulated at 95% confidence through the degrees of freedom (df) associated to the two series of calibration points. According to equation 3 (eq. 3): ^^^= ^^− 2 (eq.3) wherein nirepresents the number of points used to construct each calibration curve. When the parameters df1and df2are known, the value of Ftab(95%, 2 tails) is equal to 39.17; whereas the value of Fcalcis equal to 4.22. Since Fcalc< Ftab, it can be stated that the two variances belong to the same Gaussian distribution (homoscedastic variances). It is then possible to calculate the parameter tcalc, using wherein s2(y / x)poolrepresents a common variance and is expressed by the equation 5: b1and b2represent the slopes of the two calibration curves compared; ∑(xi1-xaverage1)2and ∑(xi2-xaverage2)2represent the sums of the squares of the residuals of the abscissa values used to construct the two calibration curves (eq.5) The value of tcalc. was compared with the value of ttab at 95% confidence, using the total number of degrees of freedom of the system (df1+df2). When the sum df1+df2 is known, the value of ttab (95%, 2 tails) is equal to 2.571 whereas the value of tcalc is equal to 2.194. Since tcalc < ttab, the initial null hypothesis (H0) according to which the two slope values compared significantly differ with a confidence level of 95% can be rejected. The outcome of the t-test carried out showed the absence of a significant matrix effect during the measurements carried out on the real sample. Table 4 shows the parameters sensitivity and R2of the two compared calibration curves. Device Type of Range [AAP] Sensitivity R2calibration (mM) (μA / mM mm2) straight with respect to line the sample Microcell External 0-0.135 0.66 ± 0.09 0.998 according to the invention- SPE frozen reagents Microcell Standard 0-0.075 0.81 ± 0.07 0.974 according to additions the invention- SPE frozen reagents Table 4 From the negative intercept of the straight line with the x-axis in Fig.7b, the AAP concentration in the diluted sample was determined, and said concentration, multiplied by the dilution factor used and divided by the weighted mass of serum, gave the % m / m of AAP in the analyzed serum. ^ % ^^^ ^^^^^^^^^^^^^ ^^= 1.4 ± 0.1^% ^^.The % m / m of AAP present in the real sample was also determined using the external-calibration curve shown in Fig. 5a. ^ % ^^^ ^^^^^^^^^^= 1.48 ± 0.05^% ^^^^ ^^^.It can be observed that the results obtained through the two methods are comparable with each other, which further confirms the absence of a significant matrix effect during the analysis of a real sample. The result obtained is consistent with the typical values of % m / m of AAP present in commercial sera (0.01-3%). 1.3 Electrochemical microcell produced according to the invention for the determination of the enzyme ALP Another application tested for the microcells made according to the present invention is the determination of enzyme ALP’s activity. The determination of enzyme ALP is widely carried out in both medical and food fields. The normal level of ALP concentration in human serum, expressed as enzyme units (U), is between 20 and 140 U / L. The enzyme substrate AAP (the analysis reagent) and all the solvents and solutes required for the measurement (TRIS-HCl buffer at pH 8 and MgCl2 as a cofactor) were pre-loaded into the lateral microchannel in the form of concentrated solutions, so as not to exceed the maximum volume that can be loaded, i.e. 15 µL. Specifically, for each microcell, first of all 10 microliters of an aqueous solution of 1.5 M TRIS-HCl + 3.75 mM MgCl^were injected into the microchannel. Then, 5 microliters of an aqueous solution of AAP with a concentration of 0.15 M were injected into the microchannel 5, to make a total of 15 microliters of reagent solution. The enzyme ALP used in the tests has a specific activity of 38 U / mg. The tests were carried out starting from a set of five microcells made according to the invention and respectively loaded with aqueous solutions of ALP (analyte solution) at increasing concentration in a concentration range from 0 to 10 mg / L (0-380 U / L). The measurements were carried out at 37°C, so as to operate within the range of maximum enzymatic activity of ALP. The electrochemical microcells with the pre-loaded analyte solution and solution of analysis reagent were tested evaluating, also in this case, the parameters sensitivity, R2and LOD obtained from the response straight lines, recorded in a concentration range from 0 to 10 mg / L (mg / L = ppm) of ALP. A screen printed electrode SPE DS150 placed in the microcells made according to the invention was used, wherein said microcells had been previously stored in a freezing device. The electrochemical measurements were carried out using the technique of classic chronoamperometry and the cyclic voltammogram was calculated by the Cottrell equation, which describes the change of electric current with respect to time in an experiment under controlled potential. The currents (I) were sampled through chronoamperograms up to a time of 194s, and subsequently plotted as a function of ALP concentration. From the slopes of the response straight lines obtained for each replicate, the average sensitivity with the associated RSD% (standard deviation associated with the average of the sensitivities) was obtained, whereas the values of LOD (Limit Of Detection) and LOQ (Limit Of Quantification) were obtained from the standard deviation associated with five repeated measurements. The goodness of linearity (R2) was obtained from the straight line in Fig. 8b. The following results were obtained: Average sensitivity = 0.015 ± 0.001 μA ppm-1mm-2(RSD %= 6.2%) R2= 0.998 LOD = 0.077 mg / L (2.93 U / L) LOQ = 0.231 mg / L (8.79 U / L) Range of ALP concentration in human serum = 20-140 U / L The LOD and LOQ values obtained are respectively approximately 7 and 2 times lower than the lower limit concentration of ALP present in samples of human serum, under normal conditions. Moreover, the electrochemical microcell has a linear response even at concentrations 3 times higher than the upper limit of ALP concentration acceptable in serum (140 U / L). The performances of the electrochemical microcells made according to the invention with ALP as reagent have proved to be better, in terms of sensitivity (higher), LOD (lower), and amount of enzyme used (lower) compared to traditional electrochemical microcells, in which the traditional approach of immobilized enzyme was used. It was also confirmed that said devices can be used as sensors for the determination of the activity of the enzyme ALP in concentration ranges characteristic of biological samples. The 3D-printed microcells produced have proved to be devices suitable for making biosensors and microcells capable of using small amount of reagents and sample. Said devices have proved to be reproducible in terms of manufacturing and response. Thanks to the application flexibility of said devices, it is possible to make different types of electrochemical microcells that also use reagents and biological elements (enzymes, etc.) other than those used herein.

Claims

CLAIMS 1. An electrochemical microcell (100) comprising: - a chamber (5) adapted to insert an electrode; - at least one microchannel (10) adapted to load a solution of analysis reagent and an analyte solution; wherein the at least one microchannel (10) is in fluid communication with the chamber (5) of the electrochemical microcell (100), and wherein the chamber (5) is integral with the at least one microchannel (10).

2. The electrochemical microcell (100) according to claim 1, wherein said chamber (10) comprises a housing pocket (11) adapted to insert an electrode and a reaction zone (13), said reaction zone (13) being in fluid communication with said housing pocket (11).

3. The electrochemical microcell (100) according to claim 2, wherein said reaction zone (13) has a volume between 80 µL and 250 µL, preferably between 120 µL and 180 µL, and / or said microchannel (10) has a volume between 10 µL and 22 µL.

4. The electrochemical microcell (100) according to any one of the preceding claims, wherein said electrochemical microcell (100) is adapted to the use in discontinuous flow.

5. The electrochemical microcell (100) according to any one of the preceding claims, wherein said electrochemical microcell (100) is a monolithic structure formed as one piece.

6. The electrochemical microcell (100) according to any one ofthe preceding claims, wherein said electrochemical microcell (100) is made with a thermoplastic elastomer or a mixture of thermoplastic elastomers, preferably the thermoplastic elastomer being thermoplastic polyurethane or the mixture of thermoplastic elastomers comprising thermoplastic polyurethane.

7. A method of analysis through an electrochemical technique comprising the following steps: a) providing an electrochemical microcell (100) according to any one of claims 1 to 6; b) pre-loading a solution of analysis reagent into said electrochemical microcell (100), thereby obtaining a pre-loaded electrochemical microcell (100); c) inserting an electrode device into the chamber (5) of said pre-loaded electrochemical microcell (100), preferably into said housing pocket (11); d) loading an analyte solution into said pre-loaded electrochemical microcell (100); e) carrying out a measurement through an electrochemical technique.

8. The method of analysis through an electrochemical technique according to claim 7, wherein in said step a) the electrochemical microcell (100) is 3D-printed and / or said electrochemical microcell (100) being made with a thermoplastic elastomer or a mixture of thermoplastic elastomers, preferably the thermoplastic elastomer being thermoplastic polyurethane or the mixture of thermoplasticelastomers comprising thermoplastic polyurethane.

9. The method of analysis through an electrochemical technique according to claim 7 or 8, wherein in said step e) the electrochemical technique is cyclic voltammetry or chronoamperometry.

10. The method of analysis through an electrochemical technique according to any one of claims 7 to 9, wherein steps a) to e) are carried out in discontinuous.

11. The method of analysis through an electrochemical technique according to any one of claims 7 to 10, wherein the pre-loading step b) comprises the following substeps: i) introducing a solution of analysis reagent into at least one microchannel (10) of said electrochemical microcell (100); ii) freezing or drying said solution of analysis reagent so introduced into said electrochemical microcell (100), thereby obtaining a pre-loaded electrochemical microcell (100) containing at least one analysis reagent; iii) optionally, storing said pre-loaded electrochemical microcell (100) so obtained in step ii) under controlled environmental conditions.

12. The method of analysis through an electrochemical technique according to any one of claims 7 to 11, wherein the loading step d) comprises the following substeps: - introducing an analyte solution into at least one microchannel (10) of said pre-loaded electrochemical microcell (100), thereby obtaining an electrolytic solution comprising said analyte solution and said at least one analysis reagent; and- letting said electrolytic solution flow from the microchannel (10) to the chamber (5) of said electrochemical microcell (100).

13. The method of analysis through an electrochemical technique according to any one of claims 7 to 12, wherein the said electrode device is a printed electrode.

14. The method of analysis through an electrochemical technique according to claim 13, wherein said electrode device is a screen-printed electrode.

15. The method of analysis through an electrochemical technique according to any one of claims 7 to 14, wherein the said electrode device comprises a reference electrode, a working electrode, and a counter electrode or auxiliary electrode.

16. A use of the electrochemical microcell (100) according to any one of claims 1 to 6 in a method of analysis through an electrochemical technique, preferably in the analysis of organic compounds, inorganic compounds, bioactive molecules or enzymes.

17. Kit comprising the electrochemical microcell according to any one of claims 1 to 6 and an electrode device, said electrode device being a printed electrode.

18. Kit according to claim 17, wherein said electrode device is a screen-printed electrode.

19. Kit according to claim 17 or 18, wherein said electrode device comprises a reference electrode, a working electrode, and a counter electrode.

20. Kit according to anyone of claims 17 to 19, wherein saidelectrode device is adapted to be placed in the housing pocket of the chamber of said electrochemical cell.

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