Non-enzymatic electrochemical glucose sensor and related fabrication process

Abstract

Non-enzymatic electrochemical glucose sensor (100), for the detection of glucose in biological fluids, characterized in comprising: - a support (101), having a first surface and a second surface opposite to the first surface; - a first metallic electrode (102a), a second metallic electrode (102b), and a third metallic electrode (102c) deposited on said support (101); - a sensitive element consisting of a compound, having a nanoporous structure, deposited on a surface of the second metallic electrode (102b). The invention is also related to a fabrication process of the non-enzymatic electrochemical glucose sensor (100).

Description

[0001] DESCRIPTION

[0002] "Non-enzymatic electrochemical glucose sensor and related fabrication process"

[0003] The present invention is related to a non-enzymatic electrochemical glucose sensor .

[0004] In particular, the present invention is related to a non-enzymatic sensor able to determine the concentration of glucose in biological fluids .

[0005] The present invention is also related to a fabrication process of a non-enzymatic electrochemical glucose sensor .

[0006] The development of glucose sensors has driven the evolution of monitoring diabetes mellitus ( DM) and the progression of the same disease . Enzymatic sensors have been the cornerstone of scienti fic research in this technical field for over five decades , despite their intrinsic low sensitivity and thermal / chemical stability . Furthermore , as sel f-monitoring of blood glucose is extremely frequent , the economic cost of this simple operation is becoming unsustainable for national health systems , discouraging the performance of frequent tests .

[0007] Furthermore , the recent clinical need for continuous glucose monitoring for timely treatment and / or inj ection of arti ficially synthesi zed insulin in diabetic patients has led to new technological challenges . In addition to continuous monitoring, the debate on the use of blood as a biological sample , rather than other alternative fluids such as interstitial fluid, or ISF, or even better the use of non-invasive fluids such as saliva, sweat and tears , is still open .

[0008] Non-enzymatic or fourth-generation glucose sensors can meet both requirements , since they have a continuous and non-invasive glucose source available and, in addition, research and clinical practice are working on standardi zing the use of these devices , whose accuracy is influenced by multiple factors , such as the pre-analytical phase , the features of the sample and environmental parameters .

[0009] However, the widespread di f fusion of fourth-generation glucose sensors is currently limited by technology that does not make them competitive , and limits their commercial di f fusion .

[0010] Consequently, in order to achieve a reliability comparable to that of the enzymatic blood glucose sensors , alternative and new technological approaches are currently being developed . The development of sensors based on a direct electron trans fer, compared to traditional enzymatic sensors , has encouraged the development of optimi zed materials and structures . Non-enzymatic glucose sensors , exploiting metallic, metal-oxide and carbon materials , having a conductive porous structure , are currently known . These non-enzymatic sensors are characteri zed by a remarkable sensitivity, allowing the detection of low concentrations of glucose in non-invasive biological fluids .

[0011] In particular, the surface area, the electrocatalytic activity and the ef fective electron trans fer to the conductive substrate are among the factors that influence the performance of non-enzymatic sensors .

[0012] The patent literature discloses several non-enzymatic electrochemical glucose sensors , such as the one described in the Chinese patent application CN109342518A, whose subj ect is a preparation method of a glucose sensor based on screen-printed electrodes , produced starting from first , carbon nanotubes pretreated, and nickel and copper oxide loaded on the surface of carbon nanotubes , fixed by a liquid phase reduction method to prepare a composite catalytic material .

[0013] In addition, the patent application CN110632146A discloses an enzyme- free glucose electrochemical sensor and a related detection method . The sensor comprises a three- electrode system consisting of a reference electrode , a counter electrode and a working electrode , in which the working electrode comprises a metal organic framework coated metal nanoparticle composite material with electrochemical catalytic activity . The metal organic framework and the metal nanoparticles are matched to generate a hydrogen overflow ef fect . The glucose detection method comprises the steps of electrochemically pretreating the working electrode , namely, applying high negative potential , electrochemically oxidi zing glucose , i . e . , applying a potential required by glucose oxidation, and electrochemically cleaning the working electrode , i . e . , applying a positive potential .

[0014] The purpose of the present invention is to provide a non-enzymatic electrochemical sensor for the detection of glucose in biological fluids , which is reliable , sensitive and stable , thanks to the use of optimi zed structure and materials .

[0015] In addition, a purpose of the present invention is to provide a process aimed at the fabrication of a non-enzymatic electrochemical glucose sensor, which is optimi zed and which allows the use of materials and easily scalable and inexpensive production techniques , preferably reusable .

[0016] According to the present invention, a non-enzymatic electrochemical glucose sensor is provided, as defined in claim 1 . According to the present invention, a fabrication process of a non-enzymatic electrochemical glucose sensor is also provided, as defined to claim 8 .

[0017] For a better understanding of the present invention, a preferred embodiment is now described, purely by way of nonlimiting example , with reference to the attached figures , in which :

[0018] - figure 1 shows a schematic representation of a non- enzymatic electrode for detecting glucose modi fied by means of zeolite , according to the invention;

[0019] - figure 2 shows SEM images of the zeolite / oil compound with LTA zeolite deposited on a metal electrode , according to the invention;

[0020] - figure 3 shows a diagram concerning the operation of an electrochemical cell based on the glucose sensor according to the invention .

[0021] In particular, the non-enzymatic electrochemical glucose sensor 100 according to the present invention is based on a particular porous structure , on the ion exchange capacity and on the catalytic activity of the LTA zeolite .

[0022] The non-enzymatic electrochemical glucose sensor 100 according to the invention comprises a support 101 , preferably made of glass , on which three metallic electrodes are deposited and which comprises a sensitive element produced by means a compound based on a material having a nanoporous structure .

[0023] In particular, the non-enzymatic electrochemical glucose sensor 100 comprises a first metallic electrode 102a and a second metallic electrode 102b, comprised within the three electrodes , deposited on a first surface of the support 101 , and a third metallic electrode 102c deposited on the first or on a second surface of the support 101 , with such a second surface opposite to the first surface .

[0024] According to one aspect of the invention, the first metallic electrode 102a and the second metallic electrode 102b have a planar geometry, with the shape of a truncated circular crown and a circumference respectively, as shown schematically in figure 1 .

[0025] According to one aspect of the invention, the third metallic electrode 102c also has the shape of a truncated circular crown .

[0026] According to one aspect of the invention, the non- enzymatic electrochemical glucose sensor 100 therefore comprises a glassy support 101 superficially covered with a metallic element , preferably silver, so as form the metallic electrodes 102a, 102b, 102c .

[0027] According to one aspect of the invention, the second metallic electrode 102b, having a circular shape , is coated on the surface by a compound based on a nanoporous material which constitutes the sensitive element .

[0028] According to one aspect of the invention, the sensitive element consists of a vegetable oil and zeolite powder having a thickness comprised between 50 microns and 500 microns , preferably between 100 microns and 300 microns , even more preferably equal to 200 microns .

[0029] Advantageously, the thickness of the coating layer of the second metallic electrode 102b, i . e . , of the sensitive element , influences the amount of the nanoporous material and therefore the ability to convert the detected glucose into a useful electrical signal .

[0030] According to one aspect of the invention, the non- enzymatic electrochemical glucose sensor 100 , in use , is placed in contact with an alkaline solution which encloses the analyte , as schematically shown in figure 3 . The non- enzymatic electrochemical glucose sensor 100 is then immersed in an alkaline solution which encloses the glucose . Through the application of electric fields , an electrical current between two of the three metallic electrodes placed on the same surface of the support 101 is promoted . Such a flow is proportional to the concentration of the analyte .

[0031] According to one aspect of the invention, in use , electrical fields are applied to the non-enzymatic electrochemical glucose sensor 100 , so as to promote the electro-oxidation of the analyte . The transduction is detected through the electrical current flowing between the electrodes placed on the same surface of the support 101 .

[0032] According to one aspect of the invention, the electrooxidation mechanism that occurs on the non-enzymatic electrochemical glucose sensor 100 involves an isomeri zation process of the target molecule catalyzed by the zeolite .

[0033] The transduction mechanism involves zeolite-catalyzed glucose isomeri zation to D-Fructose and D-Mannose under alkaline conditions . The isomeri zation includes glucose epimeri zation and aldose-ketose interconversion, in which protons are removed from the oxygen at position 0- 1 , resulting in the opening of the sugar ring and trans fer of the charge to the oxygen at position 0-5 . The latter resulted in the removal of a proton from the oxygen in 0-2 , forming an enediol anion .

[0034] According to one aspect of the invention, the non- enzymatic electrochemical glucose sensor 100 comprises an electrode , in the case shown the second metallic electrode 102b, coated with a nanoporous zeolite having high electrocatalytic characteristics , high cost-ef fectiveness and reusability . The non-enzymatic electrochemical glucose sensor 100 according to the present invention is a sensor having zeolite-modi fied electrodes , or ZME . The non-enzymatic electrochemical glucose sensor 100 , in addition to being highly innovative from a technological point of view, represents an advantageous device since the analyte detection and electron trans fer process can be selectively influenced by the nanoporous zeolite layer in correspondence with the electrode-solution interface , exploiting the si ze selectivity, cation exchange and catalytic properties of the zeolite .

[0035] The uni form porosity of the glucose sensor 100 based on the LTA zeolite layer on a silver layer has demonstrated, during experimental tests performed by the Applicant , a high sensitivity and a wide linear range for glucose detection . Such a range for glucose detection is preferably comprised between 0 . 1 mM and 20 mM, more preferably between 10 pM and 20 mM, even more preferably between 5 pM and 20 mM .

[0036] The Applicant performed experimental tests for the development of an optimi zed fabrication process of the sensor 100 , subj ect of the present invention, and relating to an easy deposition of the zeolite / vegetable oil mixture on a silver-coated glassy substrate / support , thus avoiding a hydrothermal synthesis . According to the invention, the fabrication process of the non-enzymatic glucose sensor 100 is characteri zed by a temperature below 150 ° C .

[0037] Such a process comprises the following steps :

[0038] - cleaning a glass support 101 and metalli zing, preferably using silver, the same support 101 using a stencil mask made , for example , by means of 3D printing, to obtain three metallic electrodes 102a, 102b, 102c ;

[0039] - heating of the zeolite powder at 150 ° C for Ih, in order to remove water and other adsorbed molecules ;

[0040] - preparing a mixture by mixing vegetable oil and the zeolite powder, preferably soybean oil and zeolite powder, even more preferably 60% by weight of soybean oil and 40% by weight of zeolite powder ;

[0041] - mixing the mixture for a time comprised between 5 minutes and 1 h, preferably for 30 minutes , by means of a homogeni zer, preferably at 24000 rpm, and placing the compound in an ultrasonic bath, preferably for 20 minutes at 50 ° C, so as to reduce the aggregation of the nanoporous material particles and the viscosity of the compound obtained; - pouring a known volume of the compound onto a metallic electrode, in particular onto the second metallic electrode 102b, preferably using a pipette;

[0042] - annealing the obtained electrodes 102a, 102b, 102c, at a temperature comprised between 140°C and 160°C, for a time comprised between 2 hours and 4 hours, preferably for 2h at 150°C, in order to create a carbon matrix with nanoporous material grains embedded inside, and store the electrodes under vacuum, so as to avoid oxidation of the silver and water saturation of the zeolite.

[0043] According to one aspect of the invention, the metallization to which the glassy support 101 is subject is a two-step process.

[0044] According to the invention, during a first step, the working electrode and the reference electrode, i.e., the first metallic electrode 102a and the second metallic electrode 102b, are deposited on a first surface of the support 101. During a second step, the counter-electrode, i.e., the third metallic electrode 102c, is deposited on the opposite side of the support 101, i.e., on its second surface, opposite to the first surface. All the deposited silver layers have a thickness preferably equal to 250nm. The electrode annealing step decomposes the oil , creating a carbon matrix that acts as a mechanical support , promoting iono-electronic conduction of encapsulated zeolite particles , as shown in figure 2 . Soybean oil , characteri zed by an iodine value of about 143 , allows to reduce the annealing temperature . In this way the zeolite content in the mixture and then in the deposited film can be considered mechanically and electrically stable and repeatable .

[0045] Advantageously, the use of commercial grade zeolite allows to avoid the calcination process at a high temperature , over 450 ° C , necessary for the synthesis of the same zeolite .

[0046] Figure 1 shows a bare electrode , without zeolite coating, immersed in an alkaline NaOH solution, with an appropriate voltage applied between the working electrode 102a and the reference electrode 102b .

[0047] The reduction reaction that occurs at the cathode produces Ag+and OH~ in the solution, according to the reaction :

[0048] 2Ag + 2Na++ 2H2-> 2Ag+ + 2H2+ 2Na++ 2OH

[0049] At the anode, the oxidation of the silver electrode in the presence of OH~ forms silver oxide according to the reaction :

[0050] Silver can further react with OH~ to produce silver oxide , AgO, and electrons in the electrode .

[0051] The deposition of a zeolite compound on the silver electrode allows to exploit its electrocatalytic, ion exchange and si ze exclusion features . The electron trans fer mechanisms can occur when the electroactive species , small enough, which move freely through the pores of the zeolite , are exchanged by the cations of the electrolyte .

[0052] Another trans fer process occurs inside the zeolite when an electroactive species is physically trapped inside the structure . In the case under consideration, the transduction mechanism towards glucose takes into account two aspects . The first aspect concerns the reaction involving glucose immersed in an alkaline solution . D-Glucose in the presence of Lewis acids and BrGnsted bases is characteri zed by its isomeri zation to D-Fructose and D-Mannose through two distinct mechanisms . Therefore , considering that the working temperature of the electrochemical cell is maintained at 20 ° C with a 0 . IM solution (pH 13 ) for a time interval shorter than 3 minutes , it is expected that the glucose isomeri zation can be considered negligible . The second aspect , more interesting, concerns the reaction mechanism of zeolite with glucose .

[0053] The mechanism of the proposed non-enzymatic electrochemical glucose sensor 100 is therefore based on the indirect detection of glucose molecules through appropriate charge trans fer variations . In the speci fic case , the isomeri zation process catalyzed by the zeolite , as discussed above , proceeds with the formation of open and closed saccharide anions . It is therefore expected that the zeolite catalyzes the deprotonation of hexoses which, in general , can be assumed to proceed much faster than the isomeri zation and closed hexose anions .

[0054] Therefore , the consumption of OH~ during deprotonation can be considered faster and closely related to the glucose concentration in the solution . In other words , the use of the non-enzymatic electrochemical glucose sensor 100 allows , during isomeri zation, to indirectly measure an active species directly related to the glucose concentration .

[0055] A method of use of the non-enzymatic electrochemical glucose sensor 100 comprises the steps of :

[0056] - placing the non-enzymatic electrochemical glucose sensor 100 in contact with an alkaline solution in which the analyte is present ; - applying to the non-enzymatic electrochemical glucose sensor 100 electric fields in order to promote the electro-oxidation of the analyte ;

[0057] - detecting the transduction through the electrical current flowing between the metallic electrodes 102a,

[0058] 102b placed on the same surface of the support 101 .

[0059] Advantageously, the non-enzymatic electrochemical glucose sensor and the fabrication process of the sensor itsel f according to the invention are inexpensive , reliable and highly sensitive .

[0060] It is finally clear that the non-enzymatic electrochemical glucose sensor and its fabrication process , described and illustrated herein, may be subj ect to modi fications and variations without departing from the protective scope of the present invention, as defined in the attached claims .

Claims

CLAIMS1. Non-enzymatic electrochemical glucose sensor (100) , for the detection of glucose in biological fluids, characterized in comprising:- a support (101) , having a first surface and a second surface opposite to the first surface;- a first metallic electrode (102a) , a second metallic electrode (102b) , and a third metallic electrode (102c) deposited on said support (101) ;- a sensitive element consisting of a compound, having a nanoporous structure, deposited on a surface of the second metallic electrode (102b) ; characterized in that said first metallic electrode (102a) , said second metallic electrode (102b) and said third metallic electrode (102c) are deposited on at least one surface of the support (101) by means of a metallization process .

2. Non-enzymatic electrochemical glucose sensor (100) according to claim 1, characterized in that said first metallic electrode (102a) , said second metallic electrode (102b) and said third metallic electrode (102c) have planar geometry .

3. Non-enzymatic electrochemical glucose sensor (100) according to claim 1, characterized in that said firstmetallic electrode (102a) has the shape of a truncated circular crown and said second metallic electrode (102b) has a circular shape.

4. Non- enzymatic electrochemical sensor (100) according to claim 1, characterized in that said first (102a) and said second electrode (102b) are deposited on the first surface of the support (101) while said third electrode (102c) is deposited on the second surface, opposite to the first surface.

5. Non-enzymatic electrochemical glucose sensor (100) according to claim 1, characterized in that said third metallic electrode (102c) has the shape of a truncated circular crown.

6. Non-enzymatic electrochemical glucose sensor (100) according to claim 1, characterized in that said sensitive element consists of a mixture of vegetable oil and zeolite powder .

7. Non-enzymatic electrochemical glucose sensor (100) according to claim 6, characterized in that the surface layer of mixture of vegetable oil and zeolite powder, which covers a surface of the second metallic electrode (102b) , has a thickness comprised between 50 microns and 500 microns.

8. Non-enzymatic electrochemical glucose sensor (100) according to claim 1, characterized in that said first metallic electrode (102a) , second metallic electrode (102b)and third metallic electrode (102c) are made of silver and said support (101) is made of a glassy material.

9. Fabrication process of a non-enzymatic electrochemical glucose sensor (100) according to one of the previous claims, characterized in comprising the following steps : cleaning a glass support and metallizing said support using a stencil masks; heating a zeolite powder to 150°C for Ih, so as to remove water and other adsorbed molecules; preparing a mixture by mixing vegetable oil and zeolite powder; mixing the mixture with a homogenizer and subsequently placing said mixture in a heated ultrasonic bath, in order to reduce the aggregation of the particles and the viscosity of the compound obtained; pouring a known volume of the compound onto one electrode ; annealing the obtained sensor at a temperature comprised between 140°C and 160°C, for a time comprised between 2 hours and 4 hours, in order to create a carbon matrix in which the grains of nanoporous material are dispersed.

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