WO2018035593A1 - Système intégré, méthode de détection d'antigène et méthode de diagnostic de maladies - Google Patents

Système intégré, méthode de détection d'antigène et méthode de diagnostic de maladies Download PDF

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Publication number
WO2018035593A1
WO2018035593A1 PCT/BR2017/050240 BR2017050240W WO2018035593A1 WO 2018035593 A1 WO2018035593 A1 WO 2018035593A1 BR 2017050240 W BR2017050240 W BR 2017050240W WO 2018035593 A1 WO2018035593 A1 WO 2018035593A1
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WIPO (PCT)
Prior art keywords
integrated system
biosensor
integrated
procedure
antigen
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PCT/BR2017/050240
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English (en)
Portuguese (pt)
Inventor
Luiz Eduardo Dos Santos TAVARES
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Taquion Desenvolvimento De Produtos E Serviços Inovadores Ltda
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Publication of WO2018035593A1 publication Critical patent/WO2018035593A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
    • G01N29/02—Analysing fluids
    • G01N29/022—Fluid sensors based on microsensors, e.g. quartz crystal-microbalance [QCM], surface acoustic wave [SAW] devices, tuning forks, cantilevers, flexural plate wave [FPW] devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Definitions

  • the present invention describes an integrated system comprising biosensors utilizing Surface Acoustic Wave technology comprising aptamer molecules integrated with the required microelectronics and a management system. Further, the present invention describes an antigen detection method and a rapid disease diagnosis method. The present invention is in the nanoelectronic, microelectronic, microfluidic, nanofluidic and biomedicine fields.
  • Dengue, Zika and other tropical diseases have now become an international problem, and one of the major shortcomings is the country's ability and autonomy to produce rapid tests that enable low cost, accurate and rapid diagnostics.
  • Today, health plans are already required to cover rapid tests for dengue, but these are still scarce in the market either because of the cost, time taken for analysis, or even low effectiveness.
  • Point Of Care (POC) examinations which were initially performed at the patient's bedside and provide rapid qualitative or semi-quantitative results, gained new space in the laboratory world, allowing answers to be given within minutes of testing.
  • the patient is, that is, far from the confines of traditional laboratories, however there are still no widespread technological solutions in day-to-day consultations by non-costly or highly proprietary healthcare professionals such as offered by GE, SIEMENS, ROCHE and other major players in the international market.
  • OAS Superficial Acoustic Waves
  • OAS sensors are a class of microelectronic systems based on acoustic wave modulation to detect any physical phenomenon. These sensors are widely used in electronic equipment, such as mobile phones, and can assume filter, direct current to DC (DC / DC), and other functions.
  • DC / DC direct current to DC
  • the senor Because it is a piezoelectric material, the sensor works through resonant waves that travel across its surface. For use in biosensors, it is necessary that the resonance pattern on the sensor surface change for any disease-linked element to be diagnosed, this element may be an antibody or antigen.
  • 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, because it will dictate how often the OAS will oscillate.
  • the problem is precisely that the integration between the oscillator circuit and the complete system can cause problems related to impedance and structural differences of waveforms, which can cause measurement errors and errors. consequently, misdiagnosis.
  • Aptamers are small single stranded nucleic acid molecules that adopt a vast number of complex three-dimensional structures virtually capable of binding tightly and specifically to any target molecule, from ions to large proteins (Li et al, 2013). Aptamers may be considered analogous to monoclonal antibodies, but have no immunogenic activity, are obtained using conventional nucleic acid synthesis methods and have high stability.
  • aptamers due to their high specificity and relative simplicity of the molecule (nucleotide sequence), they can be associated with more complex molecules, such as chemotherapy drugs, and direct the treatment specifically to cells that express the chosen target (Li et al., 2013).
  • recent literature describes various forms of conjugation of molecules to aptamers such as by non-covalent conjugation, covalent chemical conjugation as well as the different nanoparticles (NP) among them gold, liposomes, carbon nanotubes, chitosans and polyethylene glycol.
  • nanoparticle-functionalized aptamers such 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. Al, 2013).
  • aptamers can structurally alter the target protein by modifying the conformation of its functional sites and thereby inactivating its molecular function, thereby interfering with metabolic pathways essential for development and progression. of diseases.
  • 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 for monoclonal antibodies and can distinguish between chiral molecules and are able to recognize a distinct epitope of a target molecule (Vant-Hull et al 1998; Michaud et al, 2003).
  • PM 106312 discloses aptamers which exhibit considerable affinity and specificity for purinergic receptor binding and are relevant in the diagnostic process. However, this document among other technical reasons, does not disclose a diagnosis made quickly and affordably and does not use OAS technology.
  • BR102013024319 discloses a dengue virus detector microdevice in a biological sample, which has an inert substrate, metal film, adherent film and a receptor molecule.
  • this document does not use a A rapid diagnostic method does not use OAS, 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.
  • WO02095398 discloses a method of aptamer sensitized biosensor analytical detection using Surface Acoustic Waves technology.
  • this document does not solve the technical problem present in the state of the art as this biosensor must be later coupled to the oscillator circuit and furthermore the document does not disclose an antigen detection method or a diagnostic method for said oscillator. biosensor, only for detection of cigarette components in atmospheric air.
  • WO2015088446 discloses a sensor with Surface Acoustic Waves technology to detect Influenza A virus.
  • this biosensor is not applied in an integrated analysis system, leaving open the technical problem present in the state of the art of integrating the complete solution by coupling all required modules.
  • the present invention aims to solve the constant problems in the state of the art from the vessel of all necessary electronics in a single integrated system with its own management system compatible with the parameters of signal acquisition and analysis, for example for medical diagnostic purposes, which would be an innovation that would solve the commercial issue of developing technology.
  • the advantage of using this technology is its portability, ease of use and low cost because, due to its reduced encapsulation and its functions, it allows the entire diagnostic cycle (collection, analysis and report) to be made. in one place, resulting in up to twenty minutes. In addition, it requires less raw material to be manufactured. This greatly assists in the fact that the biosensor is disposable as it maintains the low cost features of the system and is a platform for multiple exams where only the disposable sensor component is replaced, reducing expenses for conducting medical examinations. In addition to the main advantage of having the entire system integrated.
  • the present invention discloses an integrated system comprising:
  • At least one Surface Acoustic Wave biosensor comprising at least one aptamer
  • control board is associated with the biosensor and the oscillator circuit board
  • management system is implemented on the control board; wherein the biosensor comprises interaction with the oscillator circuit board.
  • the present invention discloses a method for detecting the presence of antigen comprising the following steps:
  • the present invention provides a method of diagnosing diseases comprising the following steps:
  • said integrated biosensor system comprises at least one type of aptamer.
  • Figure 1 shows the functional prototype of the integrated system of the present invention.
  • Figure 2 shows the topology of the developed OAS sensor based Point of Care device and its components integrated by the management system is represented by the image and its block diagram.
  • FIG. 3 shows the original Radio Frequency Circuit (oscillator circuit): Radio Frequency board image.
  • the red color corresponds to the top level
  • the blue color corresponds to the bottom level
  • the green elements are components of Radio Frequency.
  • Figure 4 shows the developed sensor chip
  • Figure 5 shows the unique mechanical design to which the circuit of
  • RF oscilillator
  • control board read and transmit circuit
  • Figure 6 shows the georeferenced information in real time when using the integrated system in an antigen detection method.
  • Figure 7 shows NS-1 protein binding assays by the experimental ELONA methodology (Enzyme-Linked oligonucleotide Assay).
  • Figure 8 shows the immobilization of aptamer D106 in the biosensor by all experimental procedures: glass slide for fluorescent microscopy with double sided tape for immobilization of the chip; b- fixation of the biosensor to the slide; and d adding the aptamer solution to the biosensor; e- incubation of the biosensor in the dark.
  • Figure 9 shows the results obtained by observing the slides under a Leica BS 400 inverted fluorescence microscope.
  • Figure 10 shows the immobilization images of the high resolution D106 FAM aptamer obtained by Multiphoton Microscopy.
  • Figure 11 shows the adsorption of antigen on the sensor surface, where the horizontal axis represents time in milliseconds and the vertical axis represents frequency.
  • the peak represented by (a) corresponds to the insertion of the PBS and the peak represented by (b) corresponds to the insertion of the aptamer.
  • the saturation process occurred.
  • Figure 12 shows the adsorption of antigen on the sensor surface to the saturation moment, where the horizontal axis represents time in milliseconds and the vertical axis represents frequency.
  • Figure 13 shows the placement of 4 microliters of PBS (time:
  • Figure 14 shows the procedure performed by the firmware.
  • Figure 15 shows a commonly used bench system.
  • the present invention describes an integrated disposable embedded microelectronics biosensor system, an antigen detection method and a rapid disease diagnosis method.
  • the present invention utilizes OAS (Surface Acoustic Waves) biosensors with aptamers.
  • the present invention describes a system integrated with the vessel of all necessary electronics, which would be an innovation that would solve the commercial issue of the technology under development.
  • the present invention discloses an integrated system comprising:
  • linker comprises aptamer molecules
  • control board is associated with the biosensor and the oscillator circuit board
  • management system is implemented on the control board; wherein the biosensor comprises interaction with the oscillator circuit board.
  • the binder element consists of aptamer molecules.
  • the metal film is noble metal.
  • the noble metal is gold.
  • the biosensor of the integrated system the biosensor of the integrated system
  • the biosensor of the integrated system the biosensor of the integrated system
  • the oscillator circuit board has a working frequency in the range of 70 to 160 MHz.
  • the oscillator circuit board will have stabilized oscillators with at least one Surface Acoustic Wave delay line.
  • the microfluidic cell board will have two Surface Acoustic Wave delay lines.
  • the management system comprises input and output control.
  • the management system will be firmware adapted for aptamers as shown in Figure 14.
  • the integrated system further comprises at least one microfluidic cell plate, comprising:
  • microfluidic cell plate is over the biosensor or the biosensor components are integrated into the plate
  • the microfluidic plate further comprises an air bubble removal mechanism.
  • the microfluidic plate comprises separation of blood plasma.
  • the oscillator circuit board comprises Radio Frequency.
  • the oscillator circuit board comprises working frequency in the range of 70 to 160 MHz.
  • the oscillator circuit board comprises stabilized oscillators with at least one Surface Acoustic Wave delay line.
  • the microfluidic cell plate comprises two Surface Acoustic Wave delay lines.
  • the management system operates at a low level.
  • the present invention discloses a method for detecting the presence of antigen comprising the following steps:
  • data management comprises the following steps:
  • the reading circuit will comprise analog / digital converters.
  • the reading circuit will comprise high resolution analog / digital converters.
  • the reading circuit comprises the signals of the integrated system.
  • the present invention provides a method of diagnosing diseases comprising the following steps:
  • the data management comprises the following steps:
  • the advantage of using this technology is the cost issue because, due to its reduced encapsulation, it needs less raw material to be manufactured. This greatly assists in the fact that the biosensor is disposable, as it maintains the low cost features of the device, thus reducing the expenses for performing medical examinations. Besides the main advantage of having the whole system integrated, which is the reliability of the results by the fact that each OAS is connected to its own oscillator circuit, which avoids coupling problems due to the use of connectors, and in the future even makes room This new package will become a single integrated circuit, further reducing costs and ensuring easy transport and usability.
  • OAS is understood as Surface Acoustic Waves technology.
  • firmware adapted for aptamers means firmware adapted for the collection and interpretation of biosensor input and output signals having aptamers as their identifier.
  • Radio Frequency Circuit An RF board for Une delay OAS sensors has been developed.
  • the board is designed to work in the 70-160 MHz frequency range. It has two stabilized oscillators with OAS delay lines. One of the oscillators is used as the reference channel and another - with measurement channel ( Figure 3).
  • the oscillator circuit must be shielded to prevent interference with transmitted signals.
  • a sensitive OAS board has been developed that has two OAS delay lines with a microfluidic cell coupled with it ( Figure 4).
  • a platform has been developed consisting of an OAS sensor reader device, with embedded management system, communicating with the application on the mobile device, and the OAS sensor itself sensitized with disease-directed biological material to be diagnosed. Its operation consists of connecting the sensor to the reader equipment, placing a patient's blood sample on the biosensor, and thus a 16-bit microcontroller that manages the entire The device will also read the sensor and transmit the data wirelessly to the smarthphone.
  • a mobile application which will serve as a user interface and perform some processing with this data, which will then be saved in the cloud generating georeferenced information in real time. (Figure 6).
  • the first step in selecting aptamers is the acquisition of the chosen target in quantity and quality.
  • the strategy adopted for the project was the development of recombinant proteins.
  • 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 histidine coding sequence in E. coli bacteria.
  • the large scale expressed proteins were purified by chromatography on the AKTA Pure apparatus (GE Healthcare) on Histidine affinity columns.
  • the purified proteins were dialyzed, concentrated and the samples corresponding to the peak of purification checked on SDSPAGE, quantified and used as a protein target for aptamer selection. affinity and enriched by the methodology, was amplified and cloned into plasmid vectors. The products were ligated into pJET 1 vector
  • aptamer selection processes were initiated based on the methodology of SELEX 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, relating to the high affinity binding aptamers enriched by the methodology, was amplified and cloned into plasmid vectors. Products were ligated in the pJET 1 .2 vector (CloneJet Kit - Fermentas) following the manufacturer's recommendations.
  • Bacterial transformation was performed using the TransformAid Bacterial Transformation Kit (Thermo Scientific) also following the manufacturer's recommendations and plated in selective solid culture medium.
  • a PCR screening was performed using the Bio-Rad MyCyler Thermal cycle thermal cycler and pJET1 .2 Reverse sequencing enzyme primers (TrueStart Hot Sart Taq). DNA polymerase - Fermentas, USA). Reactions were performed on and positive amplicons were visualized on 1% agarose gel.
  • the obtained aptameric sequences were analyzed with the aid of MEME software (Multiple Em for Motif Elicitation, http://meme.nbcr.net) for the identification of the binding motifs.
  • MEME software Multiple Em for Motif Elicitation, http://meme.nbcr.net
  • aptamers were selected for the NS-1 protein binding assays. These tests were performed by ELONA (Enzyme-Linked oligonucleotide Assay) experimental methodology. The tests revealed that aptamer D106 was one of those with the highest NS-1 protein binding capacity ( Figure 7) and was selected for CHIP immobilization tests.
  • ELONA Enzyme-Linked oligonucleotide Assay
  • Aptamer D106 selected against the recombinant Dengue virus NS1 protein was used for the evaluation tests of immobilization of the biosensor gold surface (Au) aptamers. To this end a biotin molecule was added to the 5 'end of the aptamer functional tape. In addition, in order to enable the visualization of the aptamer on the biosensor surface by fluorescence microscopy, changes in the aptameric structure were required by the insertion of fluorescent molecules in the 3 'region in one of its DNA strands.
  • Example 3 Functionalization of sensor chip using integrated system / platform
  • the graph in Figure 13 shows the placement of 4 microliters of PBS (time: 140ms-151ms) in the reference channel and 4 microliters of anti-NS1 antigen checking binding and onset of saturation within 15 minutes, confirming the antigen detection through the integrated platform.
  • Linear sources are designed for DC power from the Oscillator and control circuit thus reducing noise and harmonics as one of the main advantages of linear sources is their low noise and harmonic emission.
  • the impedance matching on the RF circuit is critical to the operation of the OAS circuit. This circuit development process determines the stability of the generated signal so that measurement is performed correctly. In designing an oscillator circuit we use both empirical and analytical methods. For each new PCB layout, it is necessary to adjust the values of the passive components of the circuit, and this process consists of changing directly on the board and then measuring with a network analyzer or spectrum analyzer. This is a procedure that needs a professional familiar with this type of hardware design.
  • Frequency Counting There are two fundamental types of frequency counting: direct counting mode and reciprocal counting.
  • the direct counting method was used, which consists of recording the number of occurrences of the measured signal edge in a defined time frame. By setting this time 1 second, the value recorded would be directly the frequency in hertz. The method has some stability and reads the frequency roughly.
  • reciprocai counting was tested. Which consists of using a reference clock and counting how many clock cycles occur between two rising edges of the measured signal. By dividing the frequency by the number of counts, we find the frequency in hertz. The method is quite accurate when the reference clock is 10 times higher than the measured signal. But below that, the error gradually increases. So the possibility of using this algorithm has been ruled out for this particular case.
  • the direct counting method is used, which is sufficient for the case in point.
  • Bluetooth Integration The Bluetooth used was version 4.0, or Bluetooth Low Energy. With this version of Bluetooth the consumption is lower compared to previous versions and it is possible to communicate with mobile devices of different brands with Android operating system and newer iPhones different from Bluetooth version 2.0, which is not compatible with newer iPhones. If reader equipment needs to communicate with other medical equipment or external systems, there is a suggested standard called ISO 1 1073 - Health informatics - Medical / health device communication standards, which suggests protocols and communication standards, allowing a standard to already exist. known by the two communicating devices. In order to use this standard, it is necessary to implement both the Bluetooth module firmware and the reader communication application. When using Bluetooth 2.0 there are Profiles specific to health equipment such as heart rate monitor and glucose meter. The rapid diagnostic equipment in question does not fit these Profiles. Using Bluetooth 4.0, the implementation of Profile for diagnostic equipment becomes more flexible, enabling the developing this equipment. The implementation of this standard will still be developed.
  • Biometrics Integration The biometrics that is used is the fingerprint of the user's finger. Recognition of this fingerprint requires a biometric reader to capture this image and save it to the system so that it can be compared at another time. In the case of this system, the reader equipment would have an integrated digital reader, which would capture the digital image and send it to the smartphone via Bluetooth, which successively sends it to the API on the WEB. So that sending the file containing the fingerprint via Bluetooth does not take long in the process of saving and verifying the biometrics, it would be better to send a template, a more compact file with information about the user's biometrics characteristics, instead of the image.

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  • Engineering & Computer Science (AREA)
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  • Investigating Or Analyzing Materials By The Use Of Ultrasonic Waves (AREA)
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Abstract

La présente invention concerne une solution pour la détection d'antigènes et/ou pour le diagnostic rapide de maladies, consistant à utiliser un système intégré. Plus particulièrement, la présente invention comprend un système intégré comportant des biocapteurs utilisant la technologie des ondes acoustiques de surface, faisant intervenir des molécules d'aptamère intégrées dans la microélectronique nécessaire et dans un système de gestion. La présente invention concerne également une méthode de détection d'antigène et une méthode de diagnostic rapide de maladies. La présente invention trouve une application dans les domaines de la nanoélectronique, de la microélectronique, de la microfluidique, de la nanofluidique et de la biomédecine.
PCT/BR2017/050240 2016-08-24 2017-08-22 Système intégré, méthode de détection d'antigène et méthode de diagnostic de maladies WO2018035593A1 (fr)

Applications Claiming Priority (2)

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BR102016019574-8 2016-08-24
BR102016019574-8A BR102016019574A2 (pt) 2016-08-24 2016-08-24 Sistema integrado, método de detecção de antígeno ou marcador tumoral e método de diagnóstico de doenças

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006010206A1 (fr) * 2004-07-29 2006-02-02 Mnt Innovations Pty Ltd Capteur biologique saw
US20080015435A1 (en) * 2002-02-20 2008-01-17 Liposonix, Inc. Ultrasonic treatment and imaging of adipose tissue
WO2010138871A1 (fr) * 2009-05-29 2010-12-02 Aviana Molecular Technologies, Llc Système de capteur à ondes acoustiques de surface à biopuce intégrée pour détecter des agents infectieux

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080015435A1 (en) * 2002-02-20 2008-01-17 Liposonix, Inc. Ultrasonic treatment and imaging of adipose tissue
WO2006010206A1 (fr) * 2004-07-29 2006-02-02 Mnt Innovations Pty Ltd Capteur biologique saw
WO2010138871A1 (fr) * 2009-05-29 2010-12-02 Aviana Molecular Technologies, Llc Système de capteur à ondes acoustiques de surface à biopuce intégrée pour détecter des agents infectieux

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