BR102016019574A2 - INTEGRATED SYSTEM, ANTIGEN OR TUMOR DETECTION METHOD AND DISEASE DIAGNOSTIC METHOD - Google Patents

Abstract

integrated system, antigen or tumor marker detection method and disease diagnosis method. The present invention describes a solution for the detection of antigens and / or tumor markers and / or the rapid diagnosis of diseases comprising the use of an integrated system. Specifically, the present invention comprises a biosensor system utilizing saw technology and necessary microelectronics integrated aptamer molecules, a tumor antigen or marker detection method and a rapid disease diagnosis method. The present invention is in the fields of nanoelectronics, microelectronics, microfluidics, nanofluids and biomedicine.

Description

(54) Title: INTEGRATED SYSTEM, METHOD OF DETECTION OF ANTIGEN OR TUMOR MARKER AND METHOD OF DISEASE DIAGNOSIS (51) Int. Cl .: G01N 29/02; G01N 33/53; G01N 33/543 (52) CPC: G01N 29/022, G01N 33/5302, G01N 33/54373 (73) Holder (s): TAQUION

DEVELOPMENT OF INNOVATIVE PRODUCTS AND SERVICES LTDA (72) Inventor (s): LUIZ EDUARDO DOS SANTOS TAVARES (74) Attorney (s): REMER VILLAÇA & NOGUEIRA ASSESSORIA E CONSULTORIA DE PROP. INTELECTUAL S / S LTDA.

(57) Summary: INTEGRATED SYSTEM,

METHOD OF DETECTION OF ANTIGEN OR TUMOR MARKER AND METHOD OF DIAGNOSIS OF DISEASES. The present invention describes a solution for the detection of tumor antigens and / or markers and / or the rapid diagnosis of diseases comprising the use of an integrated system. Specifically, the present invention comprises a system of biosensors using SAW technology and aptamer molecules integrated with the required microelectronics, a method of antigen or tumor marker detection and a method of rapid disease diagnosis. The present invention is located in the fields of nanoelectronics, microelectronics, microfluidics, nanofluids and Biomedicine.

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Invention Patent Descriptive Report

Integrated System, Antigen detection method or Tumor Marker and Disease diagnosis method

Field of the Invention [0001] The present invention describes a system of biosensors using sAW technology and aptamers integrated with the required microelectronics, a method of detecting antigen or tumor marker and a method of rapid diagnosis of diseases. The present invention is located in the field of nanoelectronics, microelectronics, microfluidics, nanofluids and Biomedicine.

Background of the Invention [0002] Today the vast spread of infectious diseases, tropical diseases like Dengue and Zika, and various types of cancer, have become an international problem; and one of the major flaws is the country's capacity and autonomy to produce rapid tests that allow low-cost, accurate and rapid diagnostics. Today, health plans are already obliged to cover rapid tests for dengue, but even so these are scarce in the market, either due to the cost, delay in the analysis time, or even the low effectiveness.

[0003] Point Of Care (POC) tests, tests done initially at the patient's bedside and which provide rapid qualitative or semi-quantitative results, have gained new space in the laboratory world, allowing answers to be given minutes after the performance of exams, wherever the patient is, that is, far from the limits of traditional laboratories, however there are still no technological solutions widespread in the day-to-day consultations carried out by health professionals that are not expensive, or highly proprietary as those offered by GE, SIEMENS, ROCHE and other major players in the international market.

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2/22 [0004] Within the literature related to disease diagnosis systems using point of care devices, the fastest and most reliable method to obtain test results for contagious infectious diseases using biosensors is through SAW (Surface Acoustic Wave) technology . Like experiments carried out using this technique, are those described in Baca et al. (2015), Yang et al. (2015), who report the accuracy of more than 90% of correct answers in diagnoses of Ebola, with tests ranging from 5 to 10 minutes in duration only.

[0005] SAW sensors are a class of microelectronic systems based on modulation of acoustic waves to detect any physical phenomenon. These sensors are widely used in electronic equipment, such as cell phones, and can assume the functions of a filter, direct current to direct current converter (DC / DC) and delay lines.

[0006] There are thousands of patents in the world in relation to SAWs aimed mainly at applications in telecommunications, being a very promising technology and with applications in different segments.

[0007] Because it is a piezoelectric material, the sensor works through resonant waves that travel across its surface. For use in biosensors, it is necessary to change the pattern of resonance on the sensor surface by any element linked to the disease that is to be diagnosed, this element can be an antibody or antigen. This identification can be highly sensitive, at the molecular level, and in small concentrations of the antigen in the biological sample.

[0008] After being functionalized, the sensor element needs an electronic apparatus for its operation, and the most critical circuit for the construction of the biosensor is the oscillator circuit, since it will dictate how often the SAW will oscillate.

[0009] Aptamers are small, single-stranded nucleic acid molecules that adopt a vast number of complex three-dimensional structures virtually capable of binding strongly and specifically to any molecule

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3/22 target, from ions to large proteins (Li et al, 2013). Aptamers can be considered analogous to monoclonal antibodies, however they do not have immunogenic activity, they are obtained using conventional methods of nucleic acid synthesis and have high stability.

[0010] Although they have a mechanism of action similar to that of antibodies, they have certain advantages over conventional methods of developing molecules with pharmacological potential (Tan et al, 2011). Mainly related to cost and development time.

[0011] About aptamers, due to their high specificity and relative simplicity of the molecule (nucleotide sequence), they can be associated with more complex molecules, such as chemotherapeutic drugs, and direct treatment specifically to cells that overexpress the chosen target (Li et al., 2013). In this sense, recent literature describes various forms of conjugation of molecules to aptamers such as non-covalent conjugation, chemical covalent conjugation as well as different nanoparticles (NP) including gold, liposomes, carbon nanotubes, chitosans and polyethylene glycol. The generation of functionalized aptamers to nanoparticles as drug carriers, interfering RNAs, radioisotopes, among others, are of great pharmacological interest and have been explored in a wide variety of biomedical applications (Dhar, et al, 2008; Maureen et al 2012, Li et al, 2013).

[0012] In addition, the high specificity and binding strength of these molecules can structurally alter the target protein by modifying the conformation of their functional sites and, consequently, inactivate their molecular function, thereby interfering with metabolic pathways essential for development and progression of diseases. Thus, the development of aptamers represents a powerful molecular tool for the treatment of diseases and has been widely studied in recent years. Many aptamers have superior affinity to monoclonal antibodies and can distinguish between chiral molecules and are able to recognize

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4/22 an epitope distinct from a target molecule (Vant-Hull et al 1998; Michaud et al, 2003). Such affinity, whose dissociation constant varies from a few picomoles / L to a few nanomoles / L, provides aptamers with the ability to differentiate closely related molecular targets such as, for example, theophylline and caffeine (Michaud et al, 2003). Therefore, the affinity and specificity of aptamers qualify them as potential substitutes for monoclonal antibodies, since the cost / benefit ratio of producing these molecules is much higher than that of monoclonal antibodies. It is worth mentioning that, because they are nucleic acids, they can resume their original conformation after denaturation, the same does not happen with antibodies that need specific ideal conditions for their production and storage, which makes the process of obtaining and the useful life of biosensors more expensive.

[0013] As described in Almeida et al. (2007), Tumor markers (or biological markers) are macromolecules present in the tumor, blood or other biological fluids, whose appearance and / or changes in their concentrations are related to the genesis and growth of neoplastic cells. Such substances act as indicators of the presence of cancer, and can be produced directly by the tumor or by the body, in response to the presence of the tumor. Most tumor markers are proteins or pieces of protein, including cell surface antigens, cytoplasmic proteins, enzymes and hormones.

[0014] These markers can be useful in the clinical management of cancer patients, assisting in the processes of diagnosis, staging, assessment of therapeutic response, detection of relapses and prognosis, in addition to assisting in the development of new treatment modalities. They can be characterized or quantified by biochemical or immunohistochemical means in tissues or blood, and by genetic tests to search for oncogenes, tumor suppressor genes and genetic alterations. Each tumor marker has a specific reference value;

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5/22 rates above the reference value, presented by patients, should be investigated.

[0015] The ideal marker combines the characteristics of early diagnosis of neoplasms and their origin, establishment of the extent of the disease, monitoring of the therapeutic response and early detection of recurrence, in addition to being specific organ-site and having a short half-life, allowing temporarily monitor changes in the tumor.

[0016] In the search for the state of the art in scientific and patent literature, the following documents dealing with the topic were found:

[0017] The document PI1106312 reveals aptamers that have considerable affinity and specificity of binding to the purinergic receptor, being relevant in the diagnostic process. However, this document does not reveal a diagnosis made quickly and accessible and does not use Surface Acoustic Wave technology [0018] The document BR102013024319 reveals a micro device for detecting the dengue virus in a biological sample different from the one proposed by the invention, it does not use the principle of SAW. This document does not use a quick diagnostic method and does not solve the technical problem of miniaturizing this solution and making it commercially functional by regulating the sensitivity x specificity ratio required for a good biosensor.

[0019] WO02 / 095398A1 discloses a method of analytical detection by biosensors, sensitized by aptamers, using Surface Acoustic Wave technology. However, this document does not reveal a diagnostic method for said biosensor, only for detecting cigarette components present in atmospheric air.

[0020] The document WO2015088446 reveals a sensor with Surface Acoustic Wave technology to detect the Influenza A virus, being of low cost, easy to use in Point of Care. However, this biosensor is sensitized by antibodies, which differs from the proposed invention, since antibodies when

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6/22 applied to biosensors have specificity problems generating major problems of cross-reactions in diagnosis, mainly with other diseases producing monoclonal antibodies, as described in (Arantes, 2008).

[0021] Thus, from what can be inferred from the researched literature, no documents were found anticipating or suggesting the teachings of the present invention, so that the solution proposed here has novelty and inventive activity in view of the state of the art.

[0022] Thus, it is clear the need for new technologies that enable a diagnosis of antigens or tumor markers in a faster, more accurate and accessible way.

Summary of the Invention [0023] Thus, the present invention aims to solve the constant problems in the state of the art from the use of an integrated SAW biosensor system, highly sensitive, functionalized with aptamers, and the oscillator circuit can be embedded in the reader or even on the biosensor device itself, for the purpose of rapid diagnosis of antigen or tumor marker detection or more specifically diagnosis of infectious diseases through a system that effectively solves the sensitivity x specificity problem required.

[0024] The main advantage of this invention is the use of aptamers in biosensors, which are endowed with characteristics that surpass the use of antibodies, mainly its specificity characteristic, and are being used both for therapy and for the diagnosis and development of drugs .

[0025] Another advantage in the use of this technology is the question of cost, because, due to its reduced encapsulation, it needs less raw material to be manufactured. This helps a lot in the fact that the biosensor is disposable, as it maintains the low cost characteristics of the device,

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7/22 thereby reducing expenses for medical examinations. And, even if all the electronics are embedded in the biosensor system, it has as an additional advantage the reliability of the results due to the fact that each SAW is connected to its own oscillator circuit.

[0026] The way in which this integration of the oscillator circuit and the SAW circuit will take place within a single encapsulation, in this case the biosensor, will be through the use of components with SMD technology (Surface-Mount Devices).

[0027] In a first object, the present invention reveals an integrated system comprising:

- at least one Surface Acoustic Wave biosensor comprising:

- at least one metal film;

- at least one substrate of piezoelectric material; and

- at least one binding element;

- at least one oscillator circuit, and

- at least one volumetric or functional microfluidic cell plate; wherein said linking element comprises at least one aptamer.

[0028] In a second object, the present invention discloses a method for detecting the presence of antigen or tumor marker comprising the following steps:

- contact of a sample containing the antigen or tumor marker with the Surface Acoustic Wave biosensor of the integrated system;

- interaction of said antigen or tumor marker with aptamer of the integrated system;

- collection of data from said sample by an integrated system incorporated into the reading circuit with or without a wireless data transmission circuit;

- data transmission to an interface by means of specific firmware and embedded in the reader device.

[0029] In a third object, the present invention presents a method of diagnosing diseases comprising the following steps:

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- contact of a biological fluid sample with the Surface Acoustic Wave biosensor of said integrated system;

- collection of data from said sample by the integrated system incorporated into the reading circuit;

- data transmission to an interface by means of specific firmware and embedded in the reader device; and

- data analysis to determine the presence of components related to the disease and, thus, determining the disease.

- recording of data in a local or remote database.

[0030] Still, the inventive concept common to all the protection contexts claimed is the association of SAW biosensor with aptamers for detecting antigen or tumor marker solving problems related to sensitivity x specificity widely described in the prior art.

[0031] These and other objects of the invention will be immediately valued by those skilled in the art and by companies with interests in the segment, and will be described in sufficient detail for their reproduction in the description below.

Brief Description of the Figures [0032] In order to better define and clarify the content of the present patent application, the following figures are presented:

[0033] Figure 1 shows the SAW sensor with reference channel where 1 is the SAW delay line; 2 is the sensitive film deposited on the surface of the measurement channel; A is the broadband amplifier; M is the mixer; V is the power supply.

[0034] Figure 2 shows the photolithographic mask used in the manufacture of microdevices.

[0035] Figure 3 shows the sensor test plate with the sensor mounted with microfluidic cavities.

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9/22 [0036] Figure 4 shows the phase shift by loading the sensor surface by the aptamer.

[0037] Figure 5 shows the phase shift of the sensor caused by the capture of the anti-NS1 protein.

[0038] Figure 6 shows the functional prototype of the current development stage of the solution.

[0039] Figure 7 shows the biosensor with embedded electronics.

[0040] Figure 8 shows the circuit for reading and transmitting original sensor data.

[0041] Figure 9 shows the integrated solution.

[0042] Figure 10 shows the NS-1 protein binding tests by the experimental ELONA methodology (Enzime-Linked oligonucleotide Assay). [0043] Figure 11 shows the immobilization of aptamer D106 in the biosensor through all experimental procedures: a- glass slide for fluorescent microscopy with double-sided tape for immobilizing the chip; b-fixation of the biosensor to the blade; c and d- adding the aptamer solution to the biosensor; e- incubation of the biosensor in the dark.

[0044] Figure 12 shows the results obtained by observing the slides under a Leica BS 400 inverted fluorescence microscope. A- Bright field image; B- Image acquired with the FAM fluorescence filter on the control chip. C- Image acquired with the FAM filter on the chip with the aptamers. [0045] Figure 13 shows the images of the immobilization of the high-resolution aptamer D106 FAM obtained in Multiphoton Microscopy. A- Control, BAptamer D106 FAM immobilized on the biosensor (green dots in detail).

Detailed Description of the Invention [0046] The present invention describes an integrated disposable system of the biosensor with embedded or not microelectronics, on the sensor side, a method of antigen or tumor marker detection and a method of rapid diagnosis of diseases. The present invention uses biosensors

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10/22 SAW (Surface Acoustic Wave) with aptamers.

[0047] The present invention describes an integrated SAW biosensor system sensitized with aptamers, and the oscillator circuit may be embedded in the reader or even in the biosensor device itself, for purposes of rapid diagnosis of antigen detection or more specifically diagnosis of infectious diseases or the identification of tumor markers for the diagnosis of tumors.

[0048] The way in which this integration of the oscillator circuit and the SAW circuit will take place within a single package, in the case of the biosensor, will be through the use of components with SMD technology (Surface-Mount Devices).

[0049] In a first object, the present invention presents an integrated system comprising:

- at least one Surface Acoustic Wave biosensor comprising:

- at least one metal film;

- at least one substrate of piezoelectric material; and

- at least one binding element;

- at least one oscillator circuit board, and

- at least one microfluidic cell plate, volumetric (only for containing liquid volumes) or functional (with channels or not, and / or combined with other materials with functions for handling / displacement / separation of components from the analyzed biological sample);

wherein said linking element comprises at least one aptamer. [0050] In one embodiment of the integrated system, the linker consists of molecules of aptamers.

[0051] In one embodiment of the integrated system, the metal film comprises noble metal.

[0052] In one embodiment of the integrated system, the noble metal is gold.

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11/22 [0053] In an embodiment of the integrated system, the Surface Acoustic Wave biosensor has a working frequency in the range of 30 MHz to 3GHz. [0054] In an embodiment of the integrated system, the oscillator circuit board has a working frequency in the range of 70 to 160 MHz.

[0055] In one embodiment of the integrated system, the oscillator circuit board has stabilized oscillators with at least one Surface Acoustic Wave delay line.

[0056] In a second object, the present invention discloses a method for detecting the presence of antigen or tumor marker, comprising the following steps:

- contact of a sample containing the antigen or tumor marker with the Surface Acoustic Wave biosensor of the integrated system

- interaction of said antigen or tumor marker with aptamer of the integrated system

- collection of data from said sample by an integrated system incorporated into the reading circuit.

- data transmission to an interface.

[0057] In one embodiment of the method, the antigen is selected from the group consisting of DEN-1, DEN-2, DEN-3, DEN-4 or combinations thereof.

[0058] In one embodiment of the method, the reading circuit has analog / digital converters.

[0059] In one embodiment of the method, the reading circuit has high-resolution analog / digital converters.

[0060] In one embodiment of the method, the reading circuit obtains the signals from said integrated system.

[0061] In a third object, the present invention presents a method of diagnosing diseases comprising the following steps:

- contact of a biological fluid sample with the Surface Acoustic Wave biosensor of said integrated system

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12/22

- collection of data from said sample by the integrated system incorporated into the reading circuit.

- transmission of data to an interface, and

- data analysis to determine the presence of components related to the disease and, thus, determining the disease.

- Data recording in a local or remote database [0062] In one embodiment of the method, the disease is dengue.

[0063] In one embodiment of the method, the reading circuit has analog / digital converters.

[0064] In one embodiment of the method, the reading circuit has high-resolution analog / digital converters.

[0065] In one embodiment of the method, the reading circuit obtains the signals from said integrated system.

[0066] In one embodiment of the method, data transmission occurs through a microcontrolled wireless communication circuit.

[0067] The main advantage of this invention is the use of aptamers in biosensors, which are endowed with characteristics that surpass the use of antibodies, mainly related to their specificity, and are being used both for therapy and for the diagnosis and development of drugs .

[0068] Another advantage in the use of this technology is the question of cost, because, due to its reduced encapsulation, it needs less raw material to be manufactured. This helps a lot in the fact that the biosensor is disposable, as it maintains the low cost characteristics of the device, thus reducing expenses for medical examinations. And, even if all the electronics are embedded in the biosensor system, it has as an additional advantage the reliability of the results due to the fact that each SAW is connected to its own oscillator circuit, which avoids coupling problems due to the use of connectors, and in future makes room even for this new package to become a single circuit

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13/22 integrated, further reducing your costs.

[0069] In this document the term SAW is understood as the Surface Acoustic Wave technology.

Examples - Embodiments [0070] The examples shown here are intended only to exemplify one of the countless ways of carrying out the invention, however without limiting its scope.

Example 1 - Production of the biosensor device based on SAW technology for use with aptamers.

1. SAW sensor [0071] The designed sensor consists of two oscillators, each of which is composed of a SAW delay line (figure 1) manufactured to operate at a frequency of 70MHz, the delay lines were manufactured in LiNb3 cut xxxx . The upper delay line is covered with a sensitive film that makes the frequency of this device dependent on the concentration of the analyte. The change in the total mass loading of the crystal surface is monitored by changing the oscillation frequency.

- Manufacture of masks for sensors [0072] To allow the manufacture of sensor devices, a photolithographic mask was manufactured using laser lithography.

[0073] For the manufacture of this mask, a 5 x 5 inch borosilicate glass substrate covered with a 500 nm film of oxidized chromium and with AZ 1518 resists, suitable for the laser wavelength, will be used.

[0074] The equipment used in the generation of the mask is a DWL 66FS laser configurations generator from Heidelbergh. This equipment allows the manufacture of masks with a smaller fabricated size of 0.6 um. For the manufacture of the sensor mask, the writing head with a focus distance of 4 mm was chosen, which allows manufacturing with a resolution of 400 nm.

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14/22 [0075] The mask manufacturing process initially consists of data preparation, where a device drawing file, which was generated in the design phase, is converted from the original format, in this case DXF, to the writing format of the laser pattern generator.

[0076] After writing the mask on the DWL 66FS equipment, the substrate is processed using the photolithographic laser mask manufacturing process that consists of developing the AZ1518 resistor, chrome corrosion, cleaning and substrate inspection. This whole process is carried out in the clean room environment of the Micro Systems Division - DMS of CTI.

[0077] The photolithographic mask can be seen in figure 2.

3-Manufacturing of sensors on piezoelectric substrate [0078] A set of sensors were manufactured and assembled to enable aptamer testing. The sensors were manufactured on a piezoelectric substrate following the process developed for the manufacture of SAW devices.

[0079] In this manufacturing process, a piezoelectric substrate is covered with aluminum with a thickness of 300 nm by a sputtering deposition process.

[0080] After the deposition of aluminum, the lithographic process is done to record the device's structures on the substrate.

[0081] In this process the structure of the device is transferred from the mask to the substrate by a lithographic process.

[0082] Tamarack 155 lithographic copying equipment was used for this transfer [0083] For the lithography stage of the aluminum layer, the resistor AZ5214 is used, which is deposited on the slide after a dehumidification and deposition process. HMDS adhesion promoter. Both the resist deposition stage and the HMDS deposition performed by the resist deposition technique via spin coating.

[0084] The aluminum attack and reveal step is performed in the

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15/22 chemical processing equipment CPP-8 Convac.

4- Gold Film Deposition [0085] This deposition process is preceded by a lithographic step to define the sensing region where the film will be deposited. This lithographic process is known as a lift-off. In this process the blade is completely covered with negative resite, in this case the AZ5214 resist is used with an image reversal process. After the resist is developed, gold is deposited by an electron gun evaporation process.

[0086] In this process a gold sample is heated by the incidence of a high-power electron beam, which causes the gold to evaporate and deposit on the substrate.

[0087] In this process, the gold will deposit on the areas protected by the resist and on the substrate in the areas where the resist was removed after development. After the deposition of gold by evaporation, the substrate is immersed in an acetone bath that removes the remaining resist, leaving only the gold in the sensing areas.

[0088] The lift-off process requires the manufacture of a second level of mask for the sensing region that is aligned with the sensor structures already defined by the aluminum lithography stage. This alignment process is performed on the MJB3 Mask Aligner equipment.

5- Development of aptamer deposition process [0089] After the manufacturing process, the sensor devices were mounted on a PCI card. The sensor delay line was glued to a gold-plated plate and the transducer bus was micro-welded to that plate. The board on which the electronic components are located is separate from the board on which the delay line is located. The connection between the plates was made using retractable pins and the fixation was made with screws and nuts.

[0090] For deposition tests, microfluidic cells were constructed that consist of two circular cavities of dimensions

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16/22 identical. The dimensions of the wells were calculated for an internal volume of 3 microliters. These cavities were deposited with gold.

[0091] For the aptamer characterization tests, it was decided to deposit these films by pipetting directly into the cavity. In figure 3 we show the sensor test plate with the sensor mounted and the microfluidic structure already mounted on the sensor.

[0092] In this testing phase, a process for depositing aptamers by direct deposition in the cavity by pipetting was used.

[0093] In this process a volume of 3 microliters of aptamer was placed in the cell. After fifteen minutes, the cell was emptied and washed.

[0094] This process, although simple, provided satisfactory results since it was possible to evaluate the presence of the aptamer film in the sensor after deposition by confirming the functioning of the sensor in the presence of the anti-NS1 protein.

6-Characterization of deposited films [0095] As previously reported, aptamer films were indirectly characterized by confirming the sensor's functioning in the presence of anti-NS1 protein [0096] For the purpose of this test, a sensor structure composed of two cells one reference and one sensor. The reference structure is identical to the sensor structure and works differently, that is, any disturbance that affects both structures at the same time is not felt by the sensor, as both the sensor and the reference cell will have the same response. Disturbances that act differently between the sensor and the reference cell will be felt.

[0097] In this test, only the phase shift of the oscillator output signal formed by the sensor delay line was verified. When there is a loading of the sensor surface caused by the presence of an analyte, a phase shift proportional to the loading of the analyte will occur. In this case, it is not necessary to use the reference oscillator because

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17/22 we can record the test image before and after loading.

[0098] For the test, the aptamer was prepared with heating on a hot plate at 95 ° C and cooling in a melting ice bath. This process is carried out to unfold the aptamer.

[0099] Then the test was performed with the sensor cell.

[0100] The film was deposited in the microfluidic cell by pipetting and after 15 minutes it was washed. The phase change was recorded due to the loading of the sensor surface by the aptamer.

[0101] Then the microfluidic cell was filled with 1.5 microliters with deionized water and the phase shift due to loading was recorded (figure 4).

[0102] After filling the cell, an additional 1.5 microliters of water was added to check the behavior of the sensor when the sample does not have anti NS1. It was found that there was no phase change in the sensor.

[0103] Then 1.5 microliters of water was removed from the cell and adjusted and the phase was recorded.

[0104] In the sequence 1.5 microliters of Anti NS1 solution was added. The phase shift of the signal caused by the capture of the anti NS1 protein by the aptamer was observed. (figure 5)

Example 2 - Development of the Integrated System with electronics embedded in the biosensor [0105] The company has been developing a solution based on nanotechnology and microelectronics based on SAW (Surface Acoustic Wave) or Surface Acoustic Waves. In the current stage of development there is a functional prototype (Figure 6) where the RF circuit is incorporated in the reader device, and only the SAW on the chip, and the signals are preferably read through wireless communication technologies, such as grps, wifi, Bluetooth and others on a mobile device, where analysis is performed

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Held 18/22.

[0106] A sensor box was assembled, which the electronics ships the sensitive SAW board (Figure 7).

[0107] Reading circuit and sensor data transmission: A microcontrolled circuit was developed with wireless communication, such as grps, wifi, Bluetooth and others, and with high-resolution analog / digital converters already dimensioned to receive the signals from the SAW sensor. and transmit data via Bluetooth (Figure 8).

[0108] A platform was developed formed by a SAW sensor reader device with communication with the application on the mobile device, and the SAW sensor itself sensitized with biological material directed to the disease to be diagnosed. Its operation consists of connecting the sensor to the reader equipment, placing a sample of the patient's biological sample (for example blood) in the biosensor, and thus a 16-bit microcontroller, which manages all equipment, will also read the sensor and will transmit the data via Bluetooth to the smartphone. For the smartphone, a mobile application was developed which will serve as an interface with the user and will carry out some processing with this data, which will then be saved in the cloud. (Figure 9).

Example 3 - Aptamer immobilization test on SAW Biosensor

1. Synthesis, cloning, expression and purification of recombinant protein

NS1 of the Dengue virus [0109] The first step in the selection of aptamers is the acquisition of the chosen target in quantity and quality. The strategy adopted for the project was the development of recombinant proteins. Thus, after in silico studies of the genomic sequences of the Dengue virus, the nucleotide sequence for the NS1 protein coding was chosen and synthesized. This sequence was amplified in propagation vectors and subcloned into expression vectors also containing the 6 amino acid coding sequence histidine in E. Coli bacteria. After protein overexpression

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19/22 recombinants in culture medium for bacterial growth and expression induction by IPTG, the proteins expressed on a large scale were purified by chromatography on the AKTA Pure apparatus (GE Healthcare) in affinity columns for Histidine. The purified proteins were dialyzed, concentrated and the samples, corresponding to the peak of purification, verified in SDSPAGE, quantified and used as a protein target for the selection of aptamers.

2. Selection of anti-NS1 aptamers [0110] After protein purification and quantification in a spectrophotometer, aptamer selection processes were started based on the SELEX methodology Systematic Evolution of Ligands 2 by EXponential enrichment described by Teuerk and Gold (1990) with modifications implemented by DNAPTA biotechnology. After 10 rounds of selection against the NS1 protein, the product of the last round of selection, referring to aptamers that bind with high affinity and enriched by the methodology, was amplified and cloned into plasmid vectors. The products were linked in the vector pJET 1.2 (Kit CloneJet - Fermentas), following the manufacturer's recommendations. The transformation of the bacteria was performed using the TransformAid Bacterial Transformation Kit (Thermo Scientific) also following the manufacturer's recommendations and plated in a selective solid culture medium. For the identification of the colonies that presented the insert of interest (aptamers), a PCR (screening) was performed using the Bio-Rad MyCyler Thermal cycle thermocycler and the primers of the vector pJET1.2 Reverse sequencing, of the polymerase enzyme (TrueStart Hot Sart Taq DNA polymerase - Fermentas, USA). The reactions were performed on and positive amplicons were visualized on a 1% agarose gel.

[0111] The products of the positive clones were subjected to automatic sequencing performed on the ABI 3130 Genetic Analyzer (Applied Biossystems) using the Big Dye Terminator Kit v3.1 (Applied Biosystems), according to the manufacturer's recommendations.

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20/22 [0112] The aptameric sequences obtained were analyzed with the aid of the MEME software (Multiple Em for Motif Elicitation, http://meme.nbcr.net) for the identification of the binding motifs. Likewise, the secondary structure of the selected aptamers was determined with the aid of the secondary software mfold (http://mfold.rna.albany.edu/?q=mfold) and Oligoanalyser 3.1 (http://www.idtdna.com/ analyzer / applications / oligoanalyzer /).

3. Binding Tests [0113] Based on the analysis of the sequences and on the conformation analysis of the secondary structures, 18 aptamers were selected for the NS-1 protein binding tests. These tests were carried out using the ELONA experimental methodology (Enzime-Linked oligonucleotide Assay). The tests revealed that the aptamer D106 was one of those that showed the highest binding capacity to the NS-1 protein (Figure 10) and was the one selected for the CHIP immobilization tests.

[0114] Aptamer D106, selected against the recombinant protein NS1 of the dengue virus, was used for tests to assess the immobilization of aptamers to the gold (Au) surface of the biosensor. For this, a biotin molecule was added to the 5'end of the aptamer functional band. In addition, in order to enable the visualization of the aptamer on the surface of the biosensor by fluorescence microscopy, changes in the aptameric structure were necessary by inserting fluorescent molecules in the 3 'region on one of its DNA strands. Thus, tests were carried out with three different labeling constructs of aptamers, therefore the fluorophores Cy5, FAM as well as the intercalating DNA molecule GelRed (5.5 'iodide (6.22-dioxo-11,14,17-trioxa-7,21 -diazaheptacosane-1,27-diyl) bis (3,8-diamino-6phenylphenanthridin-5-ium) to the double stranded DNA of the aptameric sequence. [0115] The experimental procedures were initiated by fixing the chips on a glass slide for microscopy on a double-sided tape (Figure 11 a and b). 1 µl of the aptamer solution (1 ng / ul) was added directly

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21/22 on the gold surface of the chip. (Figure 11c). The chip was then incubated in the dark for 1h (Figures 11d and 11e) and after the incubation period the chip was washed twice with 2 μl of milli-Q water and after drying in RT in the dark, subjected to microscopy viewing fluorescent.

[0116] The slides were observed in a Leica BS 400 inverted fluorescence microscope with LP425, BP 525 and BP 645 filters and Leica Application Suite V3 acquisition software. The results obtained presented in Figure 12 demonstrate the fluorescent signals with the labeling for the fluorophore FAM (Figure 12C) on the chips with the immobilized aptamers and not on the chips treated with the control solution 5 (without the fluorophores) Figure 12B, indicating the immobilization of the aptamers on the chip.

[0117] Although the results obtained with fluorescent microscopy have revealed the success in the immobilization of aptamers to the biosensor, we have chosen to refine the observation by multiphotonic microscopy, which provides greater accuracy in detection. In this way the slides were analyzed with the aid of the LSM 7MP (Carl Zeiss) image system, which is extremely efficient for excitations using the multifoton laser (fentoseconds) in the infrared spectrum. The images were obtained at the AxioObserver Microscope with W N-Achroplan 10X / 0.3, W Plan-Apochromatica 20X / 1.0, W Plan-Apochromatica 63X71.0 objectives. NDD detectors, 1 BigGasp Detector in reflected light, 3 channels of external detectors in transmitted light.

[0118] The high resolution images obtained are shown in Figure 13. Observation of the image obtained in the gold region in the control biosensor, ie, where only the initiators were immobilized (primers with the fluorophore FAM, do not show fluorescent signal (Fig 13A On the other hand, in the image obtained with the aptamers modified by the addition of FAM in its structure, it clearly shows (Fig 13B and detail) the presence of fluorescent signals thus confirming the immobilization of the aptamers to the Biosensor.

[0119] Additionally, tests that prove the detection of

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22/22 tumor markers were also performed.

[0120] Those skilled in the art will value the knowledge presented here and will be able to reproduce the invention in the modalities presented and in other variants, covered by the scope of the attached claims.

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Claims (24)

Claims 1. Integrated system characterized by comprising: - at least one Surface Acoustic Wave biosensor comprising: - at least one metal film; - at least one substrate of piezoelectric material; and - at least one binding element; - at least one oscillator circuit, and - at least one microfluidic cell, volumetric or functional; wherein said linker comprises at least one aptamer. Integrated system according to claim 1, characterized in that the binding element consists of aptamers. Integrated system according to any one of claims 1 to 2, characterized in that the metal film comprises noble metal. 4. Integrated system according to claim 3, characterized by the noble metal being gold. 5. Integrated system according to any one of claims 1 to 4, characterized in that the Surface Acoustic Wave biosensor comprises a working frequency in the range of 30 MHz to 3GHz. 6. Integrated system according to any one of claims 1 to 5, characterized in that the oscillator circuit board comprises Radio Frequency. Integrated system according to any one of claims 1 to 6, characterized in that the oscillator circuit board comprises a working frequency in the range of 70 to 160 MHz. Integrated system according to any one of claims 1 to 7, characterized in that the oscillator circuit board comprises oscillators stabilized with at least one Surface Acoustic Wave delay line. Integrated system according to any one of claims 1 to 8, characterized in that the oscillator circuit board is attached adjacent to the reading and / or data transmission circuit. Petition 870160046322, of 08/24/2016, p. 26/41 2/3 10. Integrated system according to any one of claims 1 to 9, characterized in that the oscillator circuit board is shipped adjacent to the biosensor. 11. Integrated system according to any one of claims 1 to 10, characterized in that the microfluidic cell plate comprises two Surface Acoustic Wave delay lines 12. Method for detecting the presence of antigen or tumor marker characterized by comprising the following steps: - contact of a sample containing the tumor antigen or marker with the Surface Acoustic Wave biosensor of the integrated system as defined in any of claims 1 to 11; - interaction of said antigen or tumor marker with aptamer of the integrated system as defined in any one of claims 1 to 11; - collection of data from said sample by an integrated system as defined in any one of claims 1 to 11 incorporated into the reading circuit; - data transmission to an interface through or without a microcontrolled wireless communication circuit. Method according to claim 12 characterized in that the antigen is selected from the group consisting of DEN-1, DEN-2, DEN-3, DEN-4 or combinations thereof. Method according to claim 12 or 13, characterized in that the reading circuit comprises analog / digital converters. Method according to any one of claims 12 to 14, characterized in that the reading circuit comprises high-resolution analog / digital converters. Method according to any one of claims 12 to 15, characterized in that the reading circuit comprises the signals from the integrated system as defined in any one of claims 1 to 11. Petition 870160046322, of 08/24/2016, p. 27/41 3/3 17. Method according to any one of claims 12 to 16, characterized in that it additionally comprises a step of recording the data carried out in a local or remote database. 18. Disease diagnosis method characterized by comprising the following steps: - contact of a biological fluid sample with the Surface Acoustic Wave biosensor of the integrated system as defined in any one of claims 1 to 11; - collection of data from said sample by an integrated system as defined in any of claims 1 to 11 incorporated into the reading circuit; - transmission of data to an interface; - analysis of data to determine the presence of components related to diseases and, thus, determining the disease. 19. Method according to claim 18, characterized in that the disease is dengue. 20. Method according to claim 18 or 19, characterized in that the reading circuit comprises analog / digital converters. 21. Method according to any one of claims 18 to 20, characterized in that the reading circuit comprises high-resolution analog / digital converters. 22. The method of any one of claims 18 to 21, characterized in that the reading circuit comprises the signals from the integrated system as defined in any one of claims 1 to 11. 23. The method of any one of claims 18 to 22, characterized in that the data transmission takes place via a microcontrolled wireless communication circuit. 24. Method according to any of claims 18 to 23, characterized in that it additionally comprises a step of recording the data carried out in a local or remote database. Petition 870160046322, of 08/24/2016, p. 28/41 1/9 FIGURES