PROCESS FOR MODIFYING THE SURFACE OF ELECTRODES FOR THE CONSTRUCTION OF ELECTROCHEMICAL BIOSENSORS

Abstract

Development of a technique that is intended to modify, stabilize, functionalize, and reuse the surface of screen-printed electrodes, by means of the application of Rhodamine 6G as a working area modifying organic compound, enabling the creation of immunosensors that use proteins or their biological or synthetic fragments, antigens, antibodies, peptides, DNA, enzymes, RNA, and aptamers as analytes or as an element of biological recognition.

Claims

1) An electrode surface modification process for construction of electrochemical biosensors characterized in that it comprises the following steps: a) 2 μL to 4 μL of rhodamine 6G were applied to the working electrode surface; b) physical adsorption was carried out between 10 to 20 minutes at room temperature; c) then, the electrode was connected to the receiver of the PalmSens 3 potentiostat (Compact Electrochemical Interfaces) and electrodeposition was performed in three consecutive measurements in cyclic voltammetry (V=10 to 200 mV.s−1) using 50 to 120 μL of potassium ferroferricyanide from 1 mM to 5 mM/0.1M Kcl as supporting electrolyte; d) after this step, the electrode was washed with 100 μL of distilled water and then dried at room temperature.

2) The modification process as in claim 1, wherein it comprises a graphite electrode (screen-printed DRP 110) or other working electrodes preferably of conductive material, presenting electrochemical inertia in the range of −0.4V and +1.4V (versus Ag/AgCl or Ag), such as vitreous carbon, carbon paste, diamond, gold, platinum, and may be a combination of nanotechnological materials such as polymeric films, graphene, carbon nanotubes and nanoparticles on the surface of the electrodes, as well as in the probes used for recognition.

3) The modification process as in claim 1, wherein it encompasses the use of rhodamine 6G in concentrations ranging from 100 μg to 100 mg diluted in an organic solvent, and surfactants, chemical compounds or nanotechnological materials can be added to improve the bond with the surface of the electrodes and with the biomolecules.

4) The modification process as in claim 1, wherein it encompasses the use of specific antibodies and their respective native antigens, mimetics, bacterial cultures and samples of patient scrapes, not being restricted to use only in mycobacteria, but for immunosensors in general, using biomolecules that have proteins or their biological or synthetic fragments, antigens, antibodies, peptides, DNA enzymes, RNA and aptamers, as analytes or as a biological recognition element.

5) The modification process as in claim 1, wherein it makes the biosensor reusable by washing the modified electrode with organic solvent at different concentrations.

6) The modification process as in claim 1, characterized by being the modification of electrodes and the detection of biological materials through electrochemical analysis, following the variation of signals by differential pulse voltammetry, cyclic voltammetry, square wave voltammetry or other appropriate electrochemical technique, of the oxidation peak or reduction of the binding of specific recognition between probe and targets.

7) The modification process as in claim 1, characterized in that the modification of the transducer surface of electrodes with rhodamine 6g is responsible for obtaining an electrochemical sensor that can be used in the detection of biomolecules that have proteins or their biological or synthetic fragments, antigens, antibodies, peptides, DNA enzymes, RNA, and aptamers, as analytes or as a biological recognition element.

Description

DESCRIPTION OF FIGURES

[0047] For a better understanding of the characteristics of the present invention, which uses rhodamine 6G as a modifier of the transducer surface of a commercial screen-printed electrode, exemplifying graphic results are presented which represent a way of producing the immunosensor for the diagnosis of mycobacteria such as leprosy and tuberculosis, by way of example of the invention.

Interaction of Rhodamine 6G with the Electrode Surface

[0048] FIG. 1 illustrates the cyclic voltammetry (VC) scan (FIG. 1A) and the differential pulse voltammetry (VPD) (FIG. 1B) for three different electrodes modified with rhodamine 6G (4.5 and 6), and the same electrodes before adsorption of this component (1, 2 and 3). It is observed that in the electrode voltammetries without modification there was a great variation, over 50 uA, in the oxidation current of VPD and VC, and the potential also varied from 0.2 to 0.4V. While in the modified electrodes with rhodamine 6G, in addition to a significant increase in current peaks in VC and VPD, indicating greater electron passage, the currents and potentials of these modified electrodes remained very homogeneous between the voltammetric analysis between different electrode areas. Rhodamine 6G has efficient adsorption on carbon, possibly due to Van der Waals interactions between the planar benzene ring and the electrode surface. Thus, it was proved that this modification was useful to homogenize, standardize and increase the conductivity of the transducer surface of the electrodes, preparing them for sample application.

Investigation of Rhodamine 6G Concentration

[0049] FIG. 2 shows the adsorption of different concentrations of rhodamine 6G on the surface of the electrodes, in order to study their interaction to choose the best dilution to be used in the next tests. FIGS. 2A and B show cyclic and differential pulse voltammograms for the following rhodamine dilutions: (1) 50 μg, (2) 100 μg, (3) 500 μg, (4) 1 mg, (5) 5 mg, (6) 10 mg, (7) 50 mg, (8) 100 mg. Through the VC analysis we observed that as the rhodamine concentration increases, the oxidation and reduction current also increases, however, the design of the voltammetric curves are distorted and the peaks start to become more spaced from the concentration of 1 mg, probably due to the accumulation and saturation of rhodamine on the electrodes surface. In the VPD in FIG. 2B, the oxidation peak decreases with a concentration above 500 μg, proving once again that this is the limit concentration to efficiently modify the electrodes, and that concentrations above this margin saturate the transducer surface, making it difficult to bind probes and targets. FIG. 2C shows a table with the values of scan speeds of the peaks of the oxidation currents in cyclic voltammetry for different studied concentrations of rhodamine. These values were used to formulate the Randles-Sevcik equation that measures the active area and the passage of electrons on the transducer surface, thus showing that the modification of electrodes with rhodamine increases up to 99% of the active area in relation to unmodified electrodes.

Detection of Antigens with Modified Electrodes

[0050] For the construction of the bioelectrode, the specific antibody for micobacterium leprae Anti-PGL-1 antigens was immobilized on the surface of the electrodes modified with rhodamine 6G, and recognition was tested with some mimetic, synthetic and native antigens that specifically recognize this probe. In FIG. 3A, cyclic voltammetry was performed to determine the difference between the binding of the antibody with the PGL-1 native antigen (curve 1), the antibody with the M3R mimetic antigen (curve 5) and the antibody without recognition antigen (curve 3), curve 2, electrode without modification and curve 4, electrode modified with rhodamine, but without having the targets serving as baselines.

[0051] FIG. 3B presents the bar graph of the same analysis as above, with the values of the currents of the oxidation peaks of the connection in each electrode at the potential of 0.25V. Analyzing the current peaks at this potential, it is possible to verify the difference when there is antibody binding to the antigens and when there is no recognition. In FIG. 3C, differential pulse voltammetry analyzes were performed with the same anti-PGL-1 antibody recognizing other specific antigens for comparison. CURVE 1 represents the binding of the antibody with the native PGL-1 antigen, in CURVE 2 the antibody is without recognition antigen, in CURVE 3 the antibody is recognizing the LAM antigen of M. leprae, in CURVE 4 the binding is with the synthetic PGL-1 antigen, in CURVE 5 with mimetic PGL-1 antigen, the M3R, and in CURVE 6 the antibody recognized another mimetic antigen, MPML14. In FIG. 3C the same connections were analyzed by the bar graph.

[0052] It can be concluded with such analyzes that each type of antigen has a specific recognition, and that all curves differ from the curve without the recognition target, showing the specificity of the constructed bioelectrode.

Investigation of Bioelectrode Cleaning and Reuse

[0053] FIG. 4 shows the cleaning and reuse of the bioelectrode modified with rhodamine 6G, even after adsorption of antibody and antigens In FIG. 4A we have the cyclic voltammograms of the modified electrodes immobilized with the Anti-PGL-1 antibody with native PGL-1 antigen (curve 1), without recognition antigen (curve 2), and with M3R mimetic antigen (curve 3). After these readings and curve differentiation, the electrodes were washed with alcohol and reused for new adsorptions. In FIG. 4B, the electrodes that were washed and returned to their original baseline were modified again with rhodamine 6G, and it is possible to note that curves that were different in the previous figure due to the immobilizations were homogeneous, proving the cleanliness of the electrode with alcohol and homogeneity of the surface with the deposition of rhodamine. The electrodes modified for the second time were then immobilized with the same antibody, but the order of adsorption of the antigens was modified. The electrode that had not been immobilized with antigen was immobilized with native PGL-1 antigen (FIG. 4C curve 1) in the second test, the electrode that had been immobilized with M3R antigen had no added antigen (FIG. 4C curve 2) in the second test, and the electrode that had been adsorbed with native PGL-1 antigen in the first modification, had the M3R antigen added in the second immobilization. Even after being washed and used in immobilizations on different electrodes, the bioelectrode maintained its ability to differentiate antigens with the oxidation and reduction peaks remaining very close to the modified and immobilized electrodes for the first time. This study demonstrated that electrodes modified with rhodamine 6G have the ability to be cleaned and reused even after adsorption of probes and targets, as they bind to the chemical compound previously adsorbed and not directly on the electrode surface, and when the modified bioelectrode is washed with some organic solvent, alcohol in the case of the present test, it manages to undo the bonds of the benzene ring between the rhodamine and the carbon on the electrode surface, washing together all the biological material that had been deposited on the rhodamine.

Prototype for Differentiating Between Clinical Samples

[0054] In FIG. 5, clinical samples of dermal scrapings from patients and contacts suspected of leprosy were used. These were similarly immobilized on carbon electrodes modified with rhodamine 6G and anti-PGL-1 antibody. CURVES 1 refer to immobilization with positive dermal scraping and CURVES 2 with negative dermal scraping. It is observed that the curves of the positive samples have a lower oxidation and reduction current peak than the negative samples, since the antibody-antigen binding hinders the passage of electrons on the electrode surface, and when there is no specific binding, electron transfer is greater. In FIG. 5A, the test was performed in cyclic voltammetry with V=200 mV.s−1 and showed differentiation between the current of the oxidation peaks of the positive and negative sample of 88 μA. In FIG. 5B, however, the scan speed was increased to 500 mV.s−1 and the differentiation between samples also increased to 141 μA at the oxidation peaks. In FIG. 5C, the scanning curves were analyzed in differential pulse voltammetry and the difference between the oxidation currents of the positive and negative samples were even more significant (306 μA).

Study with Other Mycobacteria

[0055] FIG. 6 shows cyclic voltammetry (A and B) and differential pulse voltammetry (C) with the tuberculosis-specific antigen Anti-LAM immobilized on the modified electrode, as was done in the previous tests. In CURVES 1, the electrodes were incubated with TB culture (positive samples), whereas in CURVES 2 there was no target (negative control) being stored. There is a significant difference between the peaks of the oxidation currents of the positive and negative samples, 74 μA in FIG. 6A, 139 μA in FIG. 6B and 262 μA in FIG. 6C. The results were similar to those of FIG. 5, with clinical samples of dermal scraping due to the same form of recognition of the specific antibody, and the antigen with rhodamine 6G immobilized on bioelectrodes, thus confirming that this form of modification is useful in the diagnosis of other diseases and/or recognition of diverse biomolecules.

DESCRIPTION OF THE INVENTION

[0056] The electrode used was screen-printed graphite type, consisting of a working electrode (4 mm in diameter), a counter electrode and a reference electrode (Ref. DRP 110).

[0057] 2 μL to 4 μL of rhodamine 6G were applied on the working electrode surface, spread over the entire area (tested at different concentrations), and physical adsorption was done between 10 to 20 minutes at room temperature. Then, the electrode was connected to the receiver of the PalmSens 3 potentiostat (Compact Electrochemical Interfaces) and electrodeposition was performed in three consecutive measurements in cyclic voltammetry (V=10 to 200 mV.s−1) using 50 to 120 μL of potassium ferroferricyanide from 1 mM to 5 mM/0,1M Kcl as supporting electrolyte After this step, the electrode was washed with 100 μL de of distilled water and dried at room temperature.

[0058] Unmodified electrodes were also measured under the same conditions as above to determine the difference between them.

Rhodamine 6G Concentrations

[0059] To define the best rhodamine concentration to be used in the preparation of the electrode, variations from 100 ug to of dye diluted in ultrapure water were tested. Cyclic voltammetry and differential pulse voltammetry with different scan speeds were performed to determine the active area of the electrode through the Randles-Sevcik equation. Biological antibody recognition tests were also performed at all concentrations.

Detection of Antigens on the Surface of the Modified Electrode

[0060] For the formulation of the baseline, differential pulse voltammetry and cyclic voltammetry measurements at different scanning speeds on the modified and unmodified screen-printed electrode connected to a PalmSens 3 potentiostat (Compact Electrochemical Interfaces) were obtained, using 50 to 120 μL of potassium ferroferricyanide from 1 mM to 5 mM/0.1M Kcl as supporting electrolyte.

[0061] In electrodes modified with rhodamine 6G, 2 to 10 μL of antibody specific for M. leprae (anti-PGL-1) or specific for M. tuberculosis (anti-LAM of TB) or other antibodies specific for mycobacteria and incubated at 25 to 37° for 5 to 50 minutes. Subsequently, they were incubated with 2 to 10 μL of antigen specific to native or synthetic antibodies for another 5 to 50 min from 25 to 37° and washed with distilled water. The readings were taken by differential pulse voltammetry and cyclic voltammetry in a portable potentiostat using potassium ferroferricyanide as supporting electrolyte.

Cleaning and Reusing Electrodes

[0062] A technique for cleaning the used and modified electrodes with rhodamine was investigated, knowing that it is very soluble in organic solvents, ethyl alcohol was used, which can undo the bonds of such dye with the carbon on the surface of the electrodes. The electrodes used after adsorption of probes and targets were immersed in alcohol for 5 to 30 min and then washed with distilled water and dried at room temperature. Rhodamine 6G was again electrodeposited on said electrodes, preparing them for reuse.

Detection in Clinical Samples

[0063] Dermal scrapes from patients and contacts suspected of leprosy were used. The slit skin technique assumes the collection of contaminated samples from the ear lobe, elbows, knees, and the active lesion if present, which are stored in phosphate buffer. The scrapes were previously quantified by real-time PCR and their concentrations were already known as well as samples classified as negative that were used as controls.

[0064] The M. leprae specific antibody (anti-PGL-1) was coupled to magnetic nanoparticles COFe.sub.2O.sub.4 with treatments for bioconjugation. After this process, the positive and negative scrape samples were incubated with the conjugated antibody for 30 min to 2 hours at 37 to 45° C. The antigen-antibody conjugate was washed with PBS1X in a magnetic shelf and the part attached to the magnet was resuspended and applied 2 to 20 μL on the working area surface of the electrode modified with rhodamine 6G and incubated at a temperature between 25 to 37° for 5 to 50 minutes. The analyzes were performed by differential pulse voltammetry and cyclic voltammetry measurements at different scanning speeds on the electrode in a portable potentiostat and 50 to 120 μL of potassium ferroferricyanide from 1 mM to 5 mM/0.1M Kcl was used as support electrolyte.

Detection of Other Mycobacteria

[0065] A test was performed with electrodes modified with rhodamine 6G to detect another type of mycobacterium, M. tuberculosis. For this purpose, the specific antibody of this Anti-LAM tuberculosis pathogen was used, which was immobilized in the same way as in the previous tests, and for specific recognition, a tuberculosis culture sample was subsequently adsorbed in TLN medium (positive sample), and as a negative control only TLN medium without any type of pathogen was adsorbed and incubated at 25 to 37° for 5 to 50 minutes. The analyzes were performed by differential pulse voltammetry and cyclic voltammetry measurements at different scanning speeds on the electrode in a portable potentiostat and 50 to 120 μL of potassium ferroferricyanide from 1 mM to 5 mM/0.1M Kcl were used as supporting electrolyte.

Proposal for Field Use

[0066] Lyophilized kits containing anti-PGL1/anti-LAM or other antibodies specific for mycobacteria coupled or not to magnetic nanoparticles will be made available for subsequent agglutination, with various types of biological samples to be tested in the field that will be applied to electrodes already prepared with rhodamine 6G, which facilitates and speeds up the in-house diagnosis process.

CONCLUSIONS

[0067] The present invention shows that rhodamine 6G worked as a versatile modifier of the transducer surface of commercial screen-printed electrodes. It was able to stabilize and functionalize the working area, as it easily adsorbs onto graphite carbon through the benzene ring and binds to proteins and other biological molecules through hydrogen bonds. Furthermore, its bond is easily undone when washed with ethyl alcohol, making the biosensor reusable The surface-modified immunosensor promoted the detection of antigens when specific antibodies were used as a target and differentiated dermal scraping samples from leprosy patients from contacts without the disease. It presented advantages inherent to the rapid diagnosis of these pathogens, applicability, specificity, sensitivity, stability, selectivity, and low cost. For example, this type of sensor can be used in the detection of biomolecules that have proteins or their biological or synthetic fragments, antigens, antibodies, peptides, DNA, RNA and aptamers, as analytes or as a biological recognition element.

[0068] In the process of modifying the surface of electrodes for the construction of biosensors, graphite working electrode (screen-printed DRP 110) or other working electrodes, preferably made of conductive material, presenting electrochemical inertia in the range, can be used of −0,4V and +1,4V (versus Ag/AgCl or Ag), such as glassy carbon, carbon paste, diamond, gold, platinum, and may be a combination of nanotechnological materials such as polymeric films, graphene, carbon nanotubes and nanoparticles on the surface of electrodes as well as probes used for recognition.

[0069] Rhodamine 6G was used in concentrations ranging from 100 μg to 100 mg, diluted in organic solvent, and surfactants, chemical compounds or nanotechnological materials can be added to improve the bond with the surface of the electrodes and with the biomolecules.

[0070] Specific antibodies and their respective native antigens, mimetics, bacterial cultures, and samples of patient swabs can be used, not restricted to use only for mycobacteria, but for immunosensors in general using biomolecules that have proteins or their biological fragments or synthetics, antigens, antibodies, peptides, DNA, RNA, and aptamers, as analytes or as a biological recognition element.

[0071] The great advantage of the present invention is to make the biosensor reusable by washing the modified electrode with organic solvent at different concentrations.

[0072] The modification of electrodes and detection of biological materials occurs through electrochemical analysis, following the variation of signals by differential pulse voltammetry, cyclic voltammetry, square wave voltammetry or other appropriate electrochemical technique, of the oxidation peak or reduction of the specific recognition binding between probe and targets.

[0073] The modification of the transducer surface of electrodes with rhodamine 6g may be responsible for obtaining an electrochemical sensor that will be used in the detection of biomolecules that have proteins or their biological or synthetic fragments, antigens, antibodies, peptides, DNA, RNA enzymes and aptamers, as analytes or as a biological recognition element.