RS20201065A1 - Planar electrode for biosensors realized using repetitive fractal geometry - Google Patents

Abstract

The planar electrode for biosensors realized by applying repetitive fractal geometry has for a novelty the way of constructing a system of three biosensor electrodes using the repeating fractal geometry until the level of third order. The realization of the invention includes repetitive geometry of the 1st order of the Sierpine fractal, but the invention also relates to the repetitive geometry of the Koch curve, Pean curve and Hilbert curve, and of the geometric shapes includes the three most common shapes square, circle and triangle. Repetitive geometry is the basis of the innovation of the invention, because the change in the design of the planar electrode with its parameters, which will be discussed in the detailed description, as well as the order of fractals, significantly contributes to the increased sensitivity of biosensors. The biosensor of the invention is realized by LTCC technology and screen printing, i.e. for the substrate from which the biosensor electrode was made, ceramic is used on which gold is applied by screen printing, although the invention includes silver, platinum and carbon pastes, but also other manufacturing technologies as well as substrate materials and further functionalization of electrode surfaces. On non-sintered ceramic strips cut by laser using the screen printing process, through specifically designed masks using a photo procedure, a conductive paste is applied in an innovative fractal shape. The non-sintered strips are then laminated for mechanical strength and baked for the sintering process of the paste and the substrate itself. The electrodes can then be functionalized with appropriate bioreceptors, which do not affect the sensitivity itself, but the specificity of analyte recognition.

Description

Planar electrode for biosensors realized by applying repeating fractal geometry

Field of technology

The invention generally encompasses the field of electrochemistry with a special emphasis on the specific implementation of the biosensor electrode. The biosensor itself, implemented as such, has applications in the detection of nucleic acids and proteins in a sample, primarily from pathogenic microorganisms in contaminated food and water.

The scope of the classification is broad and covers the following classes: C12Q1, G01N27, A61B5/0408, H05K1/0283, A61N1/05 and H01L23.

Technical problem

The invention concerns a new construction of an electrochemical planar biosensor whose electrodes are realized through a repeating fractal geometry. The invention solves the problem of insufficient sensitivity of electrochemical sensors, which is particularly important in the realization of biosensors intended for the detection of biological markers or pathogens present in traces in a contaminated sample, in such a way that the new construction of a repeating geometry (specifically fractal) increases the sensitivity of the sensor due to the increase in the effective area between the electrodes.

The increased sensitivity is demonstrated in the examples of detection of DNA of the pathogenic bacterium Campylobacter jejuni and detection of a model protein (glutathione-s-transferase, GST) in milk, although the invention generally relates to the detection of biological molecules, such as nucleic acids or proteins, present in the analyzed sample in trace amounts. The electrode within the scope of the invention functions in the same way in both examples, i.e. regardless of whether it is a nucleic acid or protein (analyte) detection, the only difference is the type of bioreceptor (molecule that recognizes the analyte) used to functionalize the electrode. This information is provided for illustrative purposes and is not limiting of the invention.

Previous electrode designs have mainly involved the implementation of electrodes using circular geometry, which resulted in poor sensor sensitivity and required additional functionalization (or modification) of electrode surfaces in order to increase selectivity and sensitivity.

The invention contributes to increasing sensitivity, as it allows for changing the design of the biosensor electrode, rather than additional modification of the material from which the electrodes are made.

The existing state of the art includes the following works and protected solutions:

Patent application US20060068381 entitled “Methods for identifying a peptide that binds a geometrical shape" published on March 30, 2006, presents a new technique for immobilizing antibodies, enzymes, or aptamers to the surface of sensor electrodes, but differs substantially from the features of the invention in terms of the method of electrode construction.

Patent application KR20190104041A entitled "Electrophoresis chip for electrophoretic applications" published on September 5, 2019, generally discusses a set of printed electrodes with symmetrical geometry that uses an electrochemical detection method for use in electrophoresis, but, like the previous solution, differs in the method of electrode construction.

Also included in the existing state of the art is patent EP1931248V1 entitled "Biologically integrated electrode devices" published on June 18, 2008, as well as application US20190030318A entitled "Fractal geometry microelectrodes and uses thereof" published on January 31, 2019. The aforementioned patent focuses on the use of electroactive, i.e. conductive polymers within the sensor itself, and does not consider specific geometries in the electrode construction. The aforementioned application relates to implantable pulse generators for stimulation of the nervous system and does not involve the use of bioreceptors.

In addition to these solutions, the existing state of the art also includes patent application US20150380355A1 entitled "Self-similar and fractal design for stretchable electronics" published on December 31, 2015, which presents the application of fractals in the implementation of electronic connections in flexible and bendable electronics, but does not mention the application in the implementation of electrochemical sensors or biosensors.

Also, the paper titled "Study of fractal electrode designs for buckypaper-based microsupercapacitors" mentions fractals, but for application in supercapacitors, while the paper titled "Resistance-Capacitance Gas Sensor Based on Fractal Geometry" explains the application of fractals for the realization of a resistive-capacitive gas sensor, without the use of bioreceptors.

Patent US8009053B2 entitled “Rain sensor with fractal capacitor(s)" published on September 10, 2009 explains the implementation of a capacitive sensor using fractal geometry, but not in accordance with the description of the invention.

Also, patent application JR2016520986A entitled "Self-similar fractal design for stretchable electronics" published on July 14, 2016, belongs to the state of the art, but does not mention that fractal geometry was applied to a planar biosensor electrode.

Patent ES2517919T3 entitled "Nanostructure microelectrodes and detection devices that incorporate them" published on November 4, 2014, talks about the realization of nanostructured electrodes in the form of wires for biosensor applications that can also be made in the form of three-dimensional fractal structures, but the structure realized in the manner envisaged by the invention is not mentioned.

A similar description is given in the application EP3369828 entitled "Bioprobes and methods of use thereof", published on September 5, 2018, which states the general application of fractals, but the implementation of the structure of the invention differs.

Exposition of the essence of the invention

Biosensors today represent one of the most commonly used technological solutions in various areas of rapid diagnostics, such as medicine, agriculture, the food industry, environmental protection, etc. Unlike classical methods of microbiological analysis, which are slow (several hours to one week to produce a result), expensive (based on multiple stages, using a large amount of chemicals and utensils) and require highly trained researchers to apply them and interpret the results, biosensors enable fast and simple, yet specific analysis at a low cost. However, the main condition for the commercialization of biosensors is sufficiently sensitive detection so that they can be directly applied to the analysis of contaminated samples (food, blood and blood plasma, water, plant material).

There are several principles of biosensor operation based on, among others, optical, acoustic and fluorescent methods, but electrochemical detection with all its parameters has proven to be the most reliable, fast, economically viable and, generally speaking, the most applicable technique. The physical size, geometry, number of electrodes and construction of an electrochemical sensor usually depend on the intended purpose. Therefore, different characteristics of these sensors can be expected, in terms of sensitivity, selectivity, response time and operating life. Functionalization of sensor electrodes with different materials (nanomaterials, graphene, dendrimers, etc.) or the use of selective membranes further increases the sensitivity of the sensor. Typical planar electrochemical sensors consist of a working electrode, an auxiliary electrode and a reference electrode, interconnected by a thin layer of electrolyte containing the analyte (detection target). Due to the specific nature of the application, various types of implementation of electrochemical biosensor electrodes are known today in terms of the materials they are made of, but the circular geometry of the working electrode is generally used, which entails a number of limitations in terms of detection and sensitivity. The sensitivity of a biosensor largely depends on the material from which the electrodes are made, the substrate, and the geometric parameters of the electrode. Noble metals (gold, silver, platinum), ceramics, and porcelain are most often used for the manufacture of electrodes because these materials are relatively easily functionalized with biological molecules (DNA probes, antibodies, enzymes, aptamers, liposomes, etc.).

The invention is based on the idea that the sensing characteristics of a biosensor, in terms of sensitivity, can be significantly increased by changing the geometry of the electrodes, without changing the material from which the electrodes are made or their additional functionalization.

The invention relates to the implementation of the aforementioned biosensor electrode, or rather its new geometric construction. It is a three-electrode system consisting of a working, reference and auxiliary electrode, which are structurally implemented using the first-order repeating fractal geometry, although the invention envisages a range up to the third order of repeating geometry. The implementation of the invention itself includes the repeating geometry of the 1st order of the Sierpinski fractal, but the invention also relates to the repeating geometry of the Koch curve, the Pean curve and the Hilbert curve, and of the geometric shapes it includes the three most common shapes - a square, a circle and a triangle. The application of the repeating geometry of fractal curves in the implementation of the biosensor is the basic innovation of the invention, as it directly increases the sensitivity of electrochemical biosensors.

The planar electrode is realized using the Low Temperature Cofired Ceramic (LTCC) technology, where the substrate on which the electrode is realized is ceramic. A gold paste in the geometric shape of a fractal curve is applied to the unsintered ceramic substrate by screen printing, through a previously prepared mask. The invention also includes platinum paste, carbon paste and silver paste. Ceramic unsintered strips with a thickness of 300μm are initially cut and shaped by a laser, and after printing they are laminated and baked at 865°C, for the purpose of sintering the ceramic substrate itself and for the purpose of sintering the conductive paste into electrodes with high electrical conductivity. The process of laminating multiple ceramic strips with a thickness of 1-3 mm takes place in an isothermal press at a temperature of 75°C.

The electrode of the invention itself is not limited to the electrode manufacturing technology and includes the manufacturing of electrodes using inkjet printing, or using other thin-film or thick-film fabrication technologies, as well as manufacturing on other substrates such as paper, glass, organic and inorganic polymers.

The planar electrode can be functionalized with various bioreceptors, such as single-stranded fragments, i.e. oligonucleotides of nucleic acids (DNA and RNA probes), antibodies, aptamers, lectins and enzymes. Bioreceptors are chosen to be specific and selective for the desired analyte. If functionalization is performed with antibodies, i.e. immunoglobulins, the resulting biosensor is a so-called immunosensor. Functionalization with DNA probes results in a genosensor.

Brief description of the draft images

Figure 1 shows the process of making an innovative biosensor electrode

Figure 2 shows an innovative geometric implementation of a biosensor electrode according to the description of the invention

Figure 3 shows the innovative electrode - after fabrication: a) appearance of the fabricated electrode, b) measured dimensions of the electrode, c) SEM (scanning electron microscopy) view of the gold electrode surface magnified 2000 times.

Figure 4 shows the current-voltage characteristic of the implemented biosensor, where Figure 4a and 4b show standard tests with a solution of 0.1M ferrocene-methyl alcohol in 0.9M phosphate buffer and 1mM ferrocene-methyl alcohol in 0.05M sulfuric acid, respectively, at a scan rate of 50mV/s. Figure c) shows the current-voltage characteristic for 1mM ferrocene-methyl alcohol in 0.05M sulfuric acid at different scan rate values, while Figure d) shows the repeatability of measurement results on different electrodes.

Figure 5 shows a genosensor, or biosensor functionalized with specific DNA probes, and its testing for the detection of different concentrations of DNA molecules isolated from Campylobacter jejuni, where a signal is generated when DNA sequences isolated from the bacterium bind, i.e. hybridize, to a specific DNA probe on the surface of the electrode of the invention.

Figure b shows an immunosensor, or rather, testing an immunosensor for different protein concentrations. Namely, it is a procedure for realizing a biosensor electrode, whereby the electrode surface is functionalized by applying immunoglobulins, i.e. antibodies specific for the protein (antigen) to be detected - in this case, the antigen is the GST protein, and an anti-GST antibody is applied. A signal is generated when the GST protein binds to the anti-GST antibody applied to the electrode, thereby achieving electrochemical detection of the presence of the GST protein in the sample, using ferrocene - a redox indicator, which is present in the solution.

Figure 7 shows a comparison of the sensitivity of the implemented genosensors and immunosensors for the detection of Campylobacter jejuni and GST proteins in milk, respectively, as an example of the work of the invention and a comparison with commercial sensors using circular geometry functionalized using the same electrode immobilization procedure.

The figure represents an example of repeating geometry for a given invention when it comes to successive repetition of circular geometry.

Detailed description of the invention

Before presenting the details of the invention, it is important to understand and emphasize that the present invention is not limited to the details of construction, such as the dimensions, shape and order of the fractals, nor to the materials for making the electrodes, the substrate, the manufacturing technology, nor to the type of bioreceptor used for functionalizing the electrode, which are illustrated and described below. The terms used in the description of the invention serve to understand the invention, and not to limit it.

The innovative biosensor electrode consists of three electrodes, the main feature of which is reflected in the new geometric construction of the working electrode in the form of a fractal curve. Namely, it is a construction of a repeating geometry of a fractal shape in order to improve the sensitivity of the sensor itself, while the other electrodes follow the geometry of the working electrode. In this way, the effective area between the working and auxiliary electrodes is increased. The invention is based on the hypothesis that the sensitivity of the biosensor can be significantly increased by changing the geometry of the electrodes without changing the material from which the electrodes are made. The implementation of the invention itself includes the repeating geometry of the 1st order Sierpinski fractal, with the dimensions shown in Figure 2, but the invention also relates to the repeating geometry of the Koch curve, the Pean curve and the Hilbert curve, and of the geometric shapes it includes the three most common shapes - a square, a circle and a triangle. The application of the repeating geometry of fractal curves in the realization of biosensors represents the fundamental innovation of the invention, as it directly increases the sensitivity of electrochemical biosensors.

Figure 1 shows the sensor manufacturing process, which begins with phase 101, where the fractal geometry, fractal order, and electrode dimensions are selected, followed by phase 102, which involves the manufacture of screen printing masks, which are implemented using a photosensitive film. The planar electrode is implemented using Low Temperature Cofired Ceramic LTCC technology, with the substrate on which the electrode is implemented being ceramic. In phase 100, first, 300μm thick ceramic unsintered strips are cut and shaped with a laser. In phase 103, a gold paste manufactured by Heraeus TS7102 in the geometric shape of a fractal curve is applied to the cut unsintered ceramic substrate by screen printing, over a previously prepared mask. The invention also includes platinum paste, carbon paste, and silver paste. To ensure the mechanical strength of the electrode, in phase 104, a process of laminating several unsintered ceramic strips of 3-7 layers is performed in order to achieve a final electrode thickness of 1-2 mm. Lamination is performed in an isothermal press at a temperature of 75°C for 3 minutes. After lamination, phase 105 follows, firing at a temperature of 865°C for 10 hours, for sintering the ceramic substrate itself and sintering the conductive paste into high-conductivity electrodes.

The repeating geometry is implemented through fractal curves, the main characteristic of which is that they fill the allocated space in a highly efficient manner, i.e. as the order of the fractal curve increases, its dimension increases on a limited surface. Therefore, by choosing this construction, the effective surface area between the working and auxiliary electrodes increases by 2.4 times.

The three-electrode system, which is the basis for the invention of the biosensor, is implemented in such a way that the working electrode provides a variation in potential, the auxiliary (counter) electrode balances the current of the working electrode, and the reference electrode has a known potential, so that the principle of operation of the system is such that the potential, the voltage in contact with the analytical substance, is controlled, while the change in current is measured. The reference electrode is a half-cell with a known reduction potential. Its only role is to act as a reference in measuring and controlling the potential of the working electrode.

The fractal curves included in the invention are: Koch curve, Sierpinski fractal triangle and carpet, Peano and Hilbert curves. The invention also uses a square, triangle and circle as a starting point for forming the fractal geometry of the biosensor electrode, the scaled copies of which are periodically repeated, where a set of parameters is defined such as the initial dimensions, scaling factor, fractal order, i.e. the number of repetitions of scaled copies, etc. The importance of repeating geometry in biosensor applications is best explained using the example of a circle.

Figure 8 shows the repeating geometry of the circle, where a series of parameters are suggested already in the second iteration: the number of circle repetitions, the distance between small and large circles, and the scaling factor, i.e. the ratio of the diameters of the small and large circles. Finally, with a large number of iterations, we see that the line describing the entire structure increases, which is significant for the biosensor in terms of increasing sensitivity.

Figure 2 shows a biosensor consisting of an innovative electrode design consisting of a working, auxiliary and reference electrode with appropriate dimensions. The invention does not limit the dimensions of the electrode, but primarily emphasizes the dimensions and range of dimensions listed below. Figure 2 shows the layout of the electrodes, as well as the initial dimensions of the sensor.

Figure 3 shows the implemented innovative electrode, an enlarged view of the surface of the implemented electrode, as well as the measured dimensions of individual parts of the implemented electrode.

Cyclic voltammetry. Figures 4a and 46 show standard tests with a solution of 0.1M ferrocene-methyl alcohol in 0.9M phosphate buffer and 1 mM ferrocene-methyl alcohol in 0.05M sulfuric acid, respectively, at a scan rate of 50 mV/s. Figure 4c shows the current-voltage characteristic for 1 mM ferrocene-methyl alcohol in 0.05M sulfuric acid at different scan ratio values, while Figure 4d shows the repeatability of measurement results on different electrodes.

As stated in the description of the essence of the invention, the planar fractal electrode can be functionalized with various bioreceptors, such as antibodies or single-stranded DNA probes, i.e. deoxyribonucleic acid oligonucleotides. The bioreceptors are chosen to be specific and selective for the desired analyte. If the functionalization is performed with antibodies, i.e. immunoglobulins, the resulting biosensor is the so-called immunosensor. Functionalization with DNA probes results in a genosensor. The potential of the developed planar electrodes for practical applications has been tested in two types of biosensor applications, genosensors and immunosensors.

For the genosensor, the clean surface of the planar electrode is incubated for 1 h at 37°C with a solution containing, at a concentration of 10 ng/μl, molecules of a specific single-stranded DNA probe, i.e. oligonucleotides with a sequence complementary to the nucleotide sequence to be detected in the sample. The DNA bioreceptor is manufactured with a thiol group at the 5'-end because gold was used as the material for making the electrodes. In the specific example, these are DNA molecules specific for Campylobacter jejuni. The electrode is then washed three times with phosphate buffer to remove all DNA nucleotides that have not covalently bound to the electrode surface. A covalent bond is formed between the gold electrode and the -SH group (thiol) added to the DNA bioreceptor at the 5'-end. The electrode is then incubated with a solution of methylene blue in phosphate buffer for 1 h, which is a preparation for testing the efficiency of immobilization of the DNA probe on the electrode surface, as well as the efficiency of hybridization of the immobilized DNA probe with complementary nucleotides in the sample. Methylene blue is a frequently used redox indicator for electrochemical measurements with genosensors, for the detection of DNA hybridization, because it has a different electrochemical response to single-stranded and double-stranded DNA, i.e. the DNA structure obtained by hybridization of a single-stranded DNA probe and nucleotides from the sample. This difference arises as a consequence of the interaction of methylene blue and the guanidine base in single-stranded DNA. The efficiency test itself is performed by cyclic voltammetry (CV), measuring in the range from -0.3 V to 0.6 V, using a silver/silver chloride (Ag/AgCI) reference electrode at 100 mV/s, in phosphate buffer, pH 7.2, at room temperature. To evaluate hybridization, the CV values obtained with the addition of complementary or non-complementary DNA proportionally complementary nucleotides from the sample, i.e. analyte, are compared.

Figure 5a shows the procedure for binding the DNA sequence of Campylobacter jejuni to the electrode of the invention. Figures 5b and 5c show the results of testing at different concentrations of the positive and negative test sample, while Figure 5r shows the electrode microstructure obtained by SEM imaging of the immobilized electrode with DNA.

For the immunosensor, the clean surface of the planar electrode is first treated with a solution of mercaptoundecanoic acid (MUK) in ethanol, then washed and exposed to an activation reagent that activates the applied MUK layer. The activation reagent consists of a mixture of N-hydroxysuccinimide (i.e. NHS) and N-(3-dimethylaminopropyl)-N-ethylecarbodiimidehydrochloride (i.e. EDC) in sodium acetate buffer, pH5. This treatment allows the immobilization of proteins that form amino bonds with the activated MUK surface. Then, a solution of monoclonal antibody in phosphate buffer is applied to the surface of the electrode with the MUK layer and incubated for 1 h at room temperature to allow the formation of an amine bond between the applied antibody and the MUK layer. The functionalized immunosensor electrode is then washed with phosphate buffer, pH 7.2, and stored at 4°C in a humidified state (using phosphate buffer) until use, to avoid denaturation of the applied antibodies. The electrode is then incubated with ethanolamine in phosphate buffer for 30 minutes, and then washed with phosphate buffer. In this way, the remaining binding sites to which antibodies have not previously bound on the functionalized electrode are neutralized and non-specific recognition of the analyte is prevented. The electrochemical measurement itself is performed in a solution of 0.1M ferrocene methyl alcohol in 0.9M phosphate buffer, since ferrocene is a redox indicator, i.e. it generates an electrochemical signal. When antigen binds to the antibody on the electrode, the electrochemical signal of ferrocene changes, which is detected by cyclic voltammetry.

Figure 6 shows the procedure for implementing a biosensor electrode as an immunosensor, where the electrode is functionalized with a specific antibody, in this case a monoclonal anti-GST antibody. Figure 6a schematically shows the process of immobilizing the antibody on the electrode surface, while Figure 6b shows testing for different concentrations of GST proteins, and Figure 6c shows testing on real samples in milk. Figure 6g shows an enlarged SEM image of the electrode surface with GST proteins from milk bound to antibodies on the electrode surface. GST was used as a model protein to check the efficiency of the sensor. Measurements were performed in an aqueous solution (phosphate buffer) and in milk to demonstrate that the electrode can be directly used for the analysis of contaminated food (milk). In both cases, ferrocene is added to the solution, i.e. the sample, as a redox indicator.

Figure 7 shows the sensitivity of the implemented innovative sensors and a comparison of the characteristics with the test results on a circular electrode implemented on the same surface. The comparison was performed for both types of sensors, i.e. for both the genosensor and the immunosensor. It can be seen that the sensitivity is increased by more than 5 times compared to the circular geometry.

Method of industrial or other application of the invention

Biosensors are applied in various fields where rapid diagnostics are required, and field applications, in food production and storage processes and other aspects of food technology, as well as in biomedicine and in monitoring environmental parameters, etc. Commercialization depends on sensitivity, since samples for analysis most often contain low concentrations of analytes (biomarkers).

Claims (7)

Patent claims: 1. A planar electrode for biosensors implemented using a repeating fractal geometry consisting of a working electrode 201, an auxiliary electrode 202 and a reference electrode 200 which are interconnected by a thin layer of electrolyte containing an analytical substance, wherein said electrodes 200, 201, 202 are implemented by a process consisting of a phase 100 where the substrate for the electrodes 200, 201, 202 is cut by laser into unsintered strips with a thickness of 300μm, then in phase 101 the geometry of the electrodes is selected, and in phase 102 the masks are made, after which in phase 103 a gold paste is applied by screen printing over said mask, after which in phase 104 the strips are laminated at a temperature of 75°C and finally in phase 105 baking is performed at a temperature of 865°C to achieve sintering of said substrate and paste into electrodes 200, 201, 202, characterized in that said electrodes (200, 201, 202) are structurally implemented with a repeating fractal geometry that is selected in phase (101) in order to increase the effective surface area between the working electrode (201) on the one hand and the auxiliary (202) and reference electrodes (200) on the other hand, after which in phase (102) masks are made for said fractal geometry for screen printing using a photosensitive film. 2. A planar electrode for biosensors according to claim 1, characterized in that said substrate is in the (100) phase of ceramic, gold, silver, platinum, carbon and porcelain and that it depends on the functionalization of the biosensor. 3. A planar electrode for biosensors according to claim 1, characterized in that in step (104) 3-7 ceramic strips with a thickness of 1-3 mm are laminated. 4. A planar electrode for biosensors according to claim 1, characterized in that said biosensor is a genosensor if it detects DNA. 5. A planar electrode for biosensors according to claim 1, characterized in that said biosensor is an immunosensor if it detects antibodies. 6. A planar electrode for biosensors according to claim 1, characterized in that said repeating fractal geometry of the electrode structure (200, 201, 202) consists of: Koch curve, Peano curve, Sierpinski fractal triangle and carpet and Hilbert curve, and of the geometric shapes the repeating geometry consists of: square, circle and triangle, whereby a set of parameters is defined in advance: initial dimensions, scaling factor and fractal order. 7. A planar electrode for biosensors according to claim 1, characterized in that the repeating geometry is a first-order Sierpinski fractal. repeating geometry is realized up to the third order.