• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Method and device for the analysis of nucleic acids. Constituted from a set of systems for reception and processing of samples, capture, concentration and purification of target molecules marked by application of a magnetic field, excitation of the marking particles for the generation of a light and acquisition signal and digital processing of the recorded signal to convert it into a qualitative variable that indicates the presence or absence of the target molecule in a test sample through a light, a screen, any other digital interface or through any other system that allows visualization of the results obtained. In the same way, it is possible to carry out a quantitative analysis of the concentration of the target molecule in the analyzed sample thanks to the comparison of the recorded signal intensity with previously calibrated reference values. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2702432A1
    申请号:ES201700707
    申请日:2017-08-31
    公开日:2019-02-28
    发明作者:Venegas Pedro Manuel Medina；Villalon Ignacio Calvo
    申请人:Venegas Pedro Manuel Medina；Villalon Ignacio Calvo；
    IPC主号:
    专利说明:

    [0001]
    [0002] Method and device for the analysis of nucleic acids.
    [0003]
    [0004] Object of the invention
    [0005]
    [0006] The present invention relates to a device and a procedure to be carried out with it that allow the analysis of nucleic acids present in all types of samples.
    [0007]
    [0008] Currently, nucleic acid electrophoresis is a routine procedure in clinics, hospitals and analytical, forensic and research laboratories. This separation technique is carried out after the amplification of the genetic material and is the most used method for the analysis of nucleic acids. The test is based on the differential migration of said genetic material through a polymeric agarose or polyacrylamide gel by applying an electric field, which gives rise to different bands according to the size of the fragments analyzed.
    [0009] This analysis allows the identification of the sequences of interest after the staining of the generated bands, as well as the recovery of the desired fragments for subsequent tests and verifications, although with a fairly low yield. In any case, electrophoresis is a very versatile technique and able to adapt to the analytical needs of the user, although it has certain limitations:
    [0010]
    [0011] - The bands obtained after the separation of the fragments can be the result of the superposition of different sequences, be unspecific bands of the same size or present a high degree of diffusion, which will prevent a correct recognition.
    [0012]
    [0013] - In some cases, complementary tests may be necessary, such as the analysis of restriction maps, with the consequent increase in costs and time for obtaining conclusive results.
    [0014]
    [0015] - Every so often, the buffer used in the electrophoresis loses its pH-regulating capacity and can alter the load of the sample, compromising the analytical result.
    [0016]
    [0017] - It is necessary to use a molecular weight marker to determine the size of the band to be analyzed. In addition, the type of polymer and its concentration in the gel must be adjusted, as well as the migration time, in order to achieve a correct identification of the target sequence, which can generate problems related to the impossibility of separating certain combinations of size sequences similar or depolymerization of the gel due to high exposure times.
    [0018]
    [0019] - In certain types of analysis by gel electrophoresis, polyacrylamide is used, which is neurotoxic for humans, while, in the case of agarose, ethidium bromide, a substance with carcinogenic potential, is usually used as a clarifier in the sample. These aspects tend to be limiting in laboratories with a high sensitivity to harmful agents and space limitations, since the use of ethidium bromide, for example, requires having a delimited and specifically conditioned area to avoid contamination of other areas of the environment. laboratory.
    [0020]
    [0021] There is also another usual procedure for the recognition of products amplified by PCR or other amplification techniques, carried out by laboratories specialized in this type of services: sequencing. This analytical process encompasses a set of very varied techniques that have evolved considerably in recent years, although for the Processing of amplification products is still resorting to traditional techniques such as Sanger sequencing. In any case, the use of sequencing services may have certain disadvantages for users:
    [0022] - There is a temporary limitation that does not allow the immediate obtaining of results, since it is necessary to outsource the service to specialized laboratories that take approximately 5-7 days to execute the analyzes.
    [0023] - A series of procedures and previous controls must be carried out in the sample for the correct processing of the sample without interferences by subproducts. In addition, a minimum amount of 20ng of DNA is required per 100 bases of length of the sequence to be analyzed, always maintaining a minimum concentration of 10ng / pL. To estimate these variables, nucleic acid quantification instruments are commonly used, such as the NanoDrop ™ spectrophotometer, which has a series of significant limitations: it does not allow to verify the integrity of the sample as it is not able to distinguish degraded nucleic acids, it has a low analytical sensitivity , offers results that may be affected by interference due to the simultaneous presence of DNA and RNA and, in addition, its sale price is very high.
    [0024] - Generally, the length of the sequence to be analyzed must be indicated and, in the case of the necessary use of specific primers for sequencing, these must be provided by the client under specific conditions of concentration and solvent.
    [0025] Due to the needs and requirements previously mentioned, the unsuccessful sequencing of fragments of interest is quite common, which affects the continuity of the daily work in the laboratories and causes them additional costs that they must assume.
    [0026] On the other hand, the main advantages of the proposed invention are the following:
    [0027] - The proposed device can be used as a pre-screen for electrophoresis of fragments amplified by PCR or other amplification techniques and, in addition, can replace electrophoresis in all types of nucleic acid analysis.
    [0028] - The use of the proposed device can avoid unsuccessful results during the identification of the target fragment by sequencing due to problems with the specific primers or the amount of DNA, since the developed technology can be used as a pre-sequencing test to obtain information that may be subsequently used for the satisfactory identification of the target sequence. Furthermore, it is possible to use the proposed device as an alternative to nucleic acid sequencing thanks to the reliability of the specific sequence analysis that can be performed with it.
    [0029] The proposed invention makes it possible to carry out direct analysis of the target molecule with sufficiently powerful sensors, without the need for any pretreatment of the samples, such as, for example, the purification or amplification of the target molecule.
    [0030] - There are no potentially carcinogenic compounds involved in the use of the proposed device, unlike certain substances used in electrophoresis, such as ethidium bromide or acrylamide.
    [0031] - The analysis time is drastically reduced, going up to several hours in the case of electrophoresis and the previous amplification within a few minutes with the proposed device.
    [0032]
    [0033] - The analysis costs are lower with the use of the proposed device, since electrophoresis uses many elements that are not reusable, have a reduced useful life or a high price. Thus, the proposed device drastically reduces the costs associated with a PCR test with electrophoresis and subsequent sequencing simply by avoiding the reading by electrophoresis, although, if this cost reduction is added the corresponding one to avoid sequencing and / or Previous amplification of the target molecule, the economic savings is remarkably substantial.
    [0034]
    [0035] - In sequence-specific nucleic acid analysis, the proposed device is more reliable than electrophoresis, since multiple identification is performed per sequence and not only by size. With this, the main limitation of electrophoresis is overcome and a much more reliable and accurate pre-sequencing analysis can be carried out, and it is even possible to dispense with this confirmation test.
    [0036] - The useful life of the proposed device is equivalent to that of the electrophoresis and sequencing equipment, while the fungible elements have a set number of tests that can be performed and there is no need to estimate or verify their viability, as is the case with certain components of the electrophoresis analysis or sequencing.
    [0037]
    [0038] - The reading of the results obtained after sequencing requires specific programs that can be difficult to use for non-specialized personnel. However, with the developed device a simple and easily interpretable result is obtained by all kinds of technical personnel.
    [0039]
    [0040] - The proposed device allows to reduce the needs of highly qualified personnel, a saving due to the simplicity of the treatment of the samples, since it is a faster and simpler procedure that contributes to eliminate, at the same time, the risk of losing samples when loading the channels of the electrophoresis gels, the failures produced during the transport of said samples for sequencing or the errors occurred during this process.
    [0041]
    [0042] - The proposed device represents an affordable alternative for the diagnosis of infectious diseases or of genetic origin, reducing costs to the final client without this implying a reduction of benefits for system users.
    [0043]
    [0044] - The proposed invention allows a complete analysis to be carried out without the need for accessory or complementary technology, so, by not requiring any additional equipment, it makes it possible to carry out analyzes in situ or in the field and not only in specialized facilities, reducing both the time needed to obtain results and the costs derived from the transport and storage of the samples.
    [0045]
    [0046] - The proposed device allows the automation of the analytical process, which significantly increases the number of analysable samples per unit of time and, therefore, optimizes laboratory work and reduces costs per test.
    [0047]
    [0048] - The proposed device allows to perform both qualitative analyzes that indicate the presence or absence of the target molecule in test samples and the quantitative analysis of said target molecule in order to determine its concentration in samples problem through the digital processing of the recorded signal and its comparison with previously calibrated reference values.
    [0049]
    [0050] - Using the proposed device and method of analysis, it is possible to analyze not only nucleic acids, but also other types of biological macromolecules -especially proteins-, for which it is only necessary to adapt the probes with which the capture and functional particles are functionalized. Marking of the target molecule.
    [0051]
    [0052] - In the case of protein analysis, the proposed invention also allows to significantly reduce the costs and analysis time corresponding to the use of standard techniques such as protein electrophoresis or western blot.
    [0053]
    [0054] - The industrial application of this invention is part of the analysis procedures used, among others, in the biotechnological and health sectors, as well as in biological science research.
    [0055]
    [0056] BACKGROUND OF THE INVENTION
    [0057]
    [0058] Although no invention identical to that described has been found, the documents found that reflect the state of the art related thereto are set forth below.
    [0059]
    [0060] Thus, document ES2607753T3 refers to a method for diagnosing the susceptibility of an individual suffering from a disease to treatment with an HDAC inhibitor, the method comprising evaluating the level of expression or activity of a gene or its expression products, or the sequence of a gene, in a tissue sample of a patient and comparing said level of expression or activity or sequence with a reference, wherein the level of expression or activity or sequence that is different from said reference is indicative of an altered susceptibility to the treatment with the HDAC inhibitor in relation to the reference state, and wherein said gene is Myd88 (primary response gene of myeloid differentiation 88). In any case, the cited invention does not describe a procedure or a device similar to those proposed for the marking of the target molecule and obtaining analytical results.
    [0061]
    [0062] EP0447154A3 discloses a device for performing a heterogeneous assay, comprising: a porous member comprising a plurality of pores for physically entrapping, in said porous member, at least one target ligand of a fluid sample, said porous member being adapted to receive a reagent labeled to detect the presence or amount of, at least, said target ligand; and a non-absorbent member having a textured surface, said non-absorbent member in liquid communication with said porous member, said non-absorbent member being directly in contact and forming a network of capillary channels with said porous member, wherein said liquid communication in said capillary channels is from said porous member to said non-absorbent member. In any case, said invention does not share either the method or the device that describes the proposed invention for the analysis of nucleic acids.
    [0063]
    [0064] W02012013731A1 refers to a method for simultaneously detecting and quantifying a microbial nucleic acid in a biological sample, said method comprising: a) isolating and purifying said microbial nucleic acid, b) providing a reaction mixture comprising a first and a second nucleic acid control in different concentrations in which the first control nucleic acid is a quantitative standard nucleic acid present at a concentration of 20 to 5,000 times the detection limit of said microbial nucleic acid, and the second control nucleic acid is a qualitative internal control nucleic acid present in a concentration of 1 to 10 times the detection limit of said microbial nucleic acid, one or more pairs of primers that specifically hybridize with different sequence portions of said microbial nucleic acid and with different sequence portions of said control nucleic acids, and probes that hybridize specifically with each of the sequences amplified by said one or more pairs of primers, wherein said microbial nucleic acid and said first control nucleic acid and said second control nucleic acid hybridize with different probes carrying different labels, c) add the isolated and purified microbial nucleic acid to said reaction mixture, d) perform a or more cycling steps, wherein a cycling step comprises an amplification step, said amplification step comprising the production of one or more amplification products derived from said microbial nucleic acid if present in said sample and the production of a product of amplification derived from said first nucleic acid with trol and said second control nucleic acid, and wherein a cyclization step comprises a hybridization step, said hybridization step comprising hybridizing the sequences amplified by said primer pair with said probes, wherein the probes are labeled with a fluorescent moiety donor and a corresponding fluorescent acceptor moiety and each of the probes carries a different fluorescent dye; e) detect and measure fluorescent signals generated by the amplification products of said first control nucleic acid and said microbial nucleic acid and which are proportional to their concentration , and simultaneously detect fluorescent signals generated by said amplification product of said second control nucleic acid, wherein the presence of an amplification product of said second control nucleic acid is indicative of an amplification that occurs in the reaction mixture even in the absence of an amplification product for said microbial nucleic acid, and determining the amount of said microbial nucleic acid in said biological sample compared to the signals generated by said microbial nucleic acid and said first control nucleic acid. In any case, the method described is far from the one proposed in the proposed invention for the analysis of nucleic acids and no reference is made to an analysis device similar to that proposed in the present invention.
    [0065]
    [0066] ES2603380A1 discloses a protozoan detection equipment and method that integrates in a single assembly a sampling device and a protozoon detection kit, preferably housed in a portable enclosure, the sampling device admitting two variants, one of them, more complete , for taking samples for a longer time, of the order of days or weeks, while the other, simpler, is indicated for taking samples in a short time, of the order of hours at most. The protozoa detection kit includes reactive strips along with portable equipment and reagents to perform in situ DNA extraction processes (deoxyribonucleic acid), amplification by PCR (polymerase chain reaction), and hybridization / development . These test strips present the result of the analysis through specific markings of the amplified products. In any case, the method proposed differs from that described in the main invention and, moreover, no analysis device with the characteristics of the proposed invention is mentioned.
    [0067]
    [0068] CN 2010 101935704 B shows a nucleic acid detection system based on two double hybridizations carried out in two phases: in the first, magnetic microspheres and gold nanoparticles are used, functionalized with a specific sequence probe that will only be retained when applying a field magnetic field in the presence of the target DNA and, subsequently, said sequence (bar code) is marked with functionalized quantum dots. In any case, the characteristics of a procedure or an analysis device similar to those proposed in the present invention are not mentioned, but rather those of a reaction chamber where nucleic acids are amplified and detected.
    [0069]
    [0070] EP 20112270202 A1 presents a Mycobacterium detection kit based on a double hybridization system of the target molecule with a capture probe attached to a magnetic microsphere (via streptavidin-biotin linkage) and a labeling probe attached to a quantum dot Tables with the specific sequences used are provided and a minimum homology of 90% is required. In any case, the cited invention does not mention an analysis device similar to the one proposed, but only the reagents included in the detection kit.
    [0071]
    [0072] EP 2012 1502101 B1 describes a method of detecting cells and viruses in all types of samples-pretreated or not-using label probes consisting of both oligonucleotides and antibodies conjugated with any signaling molecule, including quantum dots. In parallel, marked cells are isolated by centrifugation or application of a magnetic field after being captured, among other proposed methods. However, the cited invention does not mention an analysis device with the characteristics of the proposed invention.
    [0073]
    [0074] EP 2012 1747295 B1 presents a method of general detection of analytes consisting of its capture with specific probes - protein or nucleotide - conjugated with magnetic microspheres and in their labeling with functionalized nanoparticles with probes of binding to the target molecule and barcode DNA sequences marked with signaling structures. After double hybridization, the DNA barcodes are released from the structure by thermal or chemical treatment and finally analyzed through the signaling group to determine the presence or absence of the target molecule. In any case, a device similar to the invention proposed for the processing or analysis of the generated signal is not described nor are the characteristics that the equipment needed to carry out the test must be detailed.
    [0075]
    [0076] EP 2014 2616557 B1 proposes a double hybridization nucleic acid detection system with capture and labeling of the target molecule in which a nuclease captured by one of the probes linked to said target molecule intervenes. In this case, the positive result is obtained by reducing the nuclease activity in the reaction mixture with respect to the negative control without target molecule, or, by generating nuclease activity after the release of the retained enzymes in the presence of the target molecule . In any case, a detection kit is patented with the reagents necessary to carry out the test, but an analysis device similar to the proposed invention is not mentioned.
    [0077]
    [0078] US 2002 0028457 A1 is based on a detection method comprising the labeling of the target molecule with complementary probes functionalized with quantum dots, said target molecule being optionally amplified to couple certain chemical groups - for example, biotin - allowing its immobilization on a support solid. However, no reference is made to an integrated analysis device similar to the proposed invention nor is the use of magnetic capture particles considered.
    [0079]
    [0080] US 20070259359 A1 consists of a method of detecting genetic sequences captured on a solid support based on the labeling of the target molecule with probes functionalized with signaling groups, so that the conformation of the probe only makes possible its degradation and the release of the group signaling once it is attached to the target molecule. The detection would be carried out using structures constituted by electrodes coupled to a substrate in which complementary probes are provided to reporters released by the action of a nuclease, which generates a signal due to the variations in electrical potential recorded. In addition, methods of signal amplification and array-like structures are presented to carry out the test, but there are no references to magnetic capture systems or to an analysis device with the characteristics of the proposed invention.
    [0081]
    [0082] US 2007 0269852 A1 proposes a device for the detection of airborne pathogens by means of a system of marking with biocomplexes with quantum dots. Said device allows the injection, filtration and centrifugation of the sample, as well as the obtaining of a fluorescent signal, although magnetic capture systems are not employed, nor is the system for generating and detecting said fluorescent signal described, nor does the mentioned device have the characteristics of the proposed invention.
    [0083]
    [0084] US 2009 0156428 A1 discloses a detection method based on an array-type structure with molecular probes of all types arranged in any conformation. Different labeling techniques of the probes complementary to the target molecule and possible conformations of the assay device that could incorporate this analysis structure are mentioned, but their characteristics are not detailed nor are magnetic capture systems of the target molecule mentioned.
    [0085]
    [0086] US 2010 0086993 A1 consists of a cell detection system based on its capture with binding ligands to conjugated receptors with magnetic microspheres and their labeling with binding ligands to quantum dots conjugated receptors. Reference is also made to the instruments that could be used for the detection of the generated fluorescent signal and the characteristics of the reagents used (composition of the quantum dots, reaction pH ...) are exhaustively defined, but no device is described. analysis with the characteristics of the proposed invention.
    [0087]
    [0088] US 20107799554 B2 proposes a test for the detection of nucleic acids based on the use of a lateral flow device that has a reaction zone and a visualization zone. In addition, there are labeling probes with a signaling group that is only activated in the presence of the analyte to be detected and capture probes that retain the analyte in the display area. However, an analysis device with the characteristics of the proposed invention is not mentioned nor are the details of its operation described.
    [0089]
    [0090] US 2011 0152111 A1 proposes a simultaneous detection kit for multiple target sequences based on a selective amplification system of the target molecules present in different types of samples. In this case, there are tagging probes functionalized with different chromophores, but no analysis device similar to the proposed invention is proposed, nor are capture and concentration systems of the target molecules mentioned.
    [0091]
    [0092] US 2011 0160090 A1 consists of a lateral flow microarray for the detection of single-stranded nucleic acids that has a microporous membrane through which a fluid sample is displaced by capillarity. In addition, there is a labeling area where a complementary probe functionalized with quantum dots and a capture zone with immobilized probes are attached to the target molecule. However, magnetic capture particles are not used throughout the process nor is an analysis device with the characteristics of the proposed invention mentioned.
    [0093]
    [0094] US 2011 0171749 A1 presents a nucleic acid detection system consisting of the use of labeling nanoparticles functionalized with specific probes for each target molecule (single-stranded pathogenic DNA) and magnetic particles functionalized with capture probes. In this case, the sequences of the different probes and the size and characteristics of the nanoparticles used are detailed. In addition, different generated signal processing systems are mentioned, which are based on the measurement of the concentration of the marking particles through electrodes once the excess of free nanoparticles has been washed. However, light signal generation and detection systems are not employed nor are the characteristics of an analysis device similar to the proposed invention detailed.
    [0095] US 2011 7955802 B2 describes a nucleic acid amplification and detection method employing primers functionalized with labeling agents and capture probes attached to magnetic spheres attracted by an electromagnet adjacent to the reaction chamber. In this way, functionalized amplicons are generated with said labeling agent which are concentrated by the action of a magnetic field. However, the characteristics of the excitation system of the marking agent and of the detection and processing system of the generated signal are not described. In addition, the detection process is not carried out without the previous step of amplification or an analysis device with the characteristics of the proposed invention is mentioned.
    [0096]
    [0097] US 2012 0071330 A1 proposes a nucleic acid detection method based on a double hybridization with capture and labeling of the target molecule, so that the capture probes are immobilized on a solid support. In addition, the use of nested probes is proposed for the amplification of the generated signal and a series of procedures are described that allow maximizing the proportion of target molecules. However, mechanisms of concentration of said target molecules by the application of magnetic fields are not mentioned, nor are the detection mechanisms of the signal generated by the signaling groups of the labeling probes detailed, nor is there mentioned an integrated analysis device similar to the proposed invention.
    [0098]
    [0099] US 2012 8298765 B2 proposes a nucleic acid detection system based on the hybridization of the target molecule with nucleotide monomers capable of polymerizing with each other by binding to said target molecule. In this case, the monomers are marked with quantum dots that emit fluorescence and some characteristics of the excitatory source responsible for the polymerization and generation of the fluorescent signal are detailed, but no capture mechanisms of the target molecules are mentioned based on the application of magnetic fields or a device with the characteristics of the proposed invention is mentioned.
    [0100] US 2013 0023433 A1 proposes a system for the identification of nucleic acids from complex hybridizations involving their capture and labeling. Furthermore, the possibility of incorporating molecular amplifiers into the system and detecting SNP-type mutations is mentioned, but the nature of the capture and labeling probes employed is not described nor is a device with the characteristics of the proposed invention mentioned.
    [0101]
    [0102] US 2013 0085078 A1 consists of a nucleic acid detection system through labeling with probes immobilized on a solid support equipped with a signaling group and hybridization with a primer that allows a polymerase with exonuclease activity to release the signaling group from the probe bound to the target molecule. In this case, the positive result may be due to an increase or decrease in the signal by comparison with the initial signal or with unhybridized reference probes. In addition, the possibility of amplifying the target molecule before detecting the generated signal is mentioned, but the characteristics of the generation, processing and detection system of the signal are not detailed, nor are capture mechanisms mentioned by application of magnetic fields or described an analysis device with the characteristics of the proposed invention.
    [0103]
    [0104] US 2013 0123145 A1 proposes a test kit, applicable to diverse analytical techniques, based on quantum dots of different characteristics linked to specific probes of different nature and directed against specific cellular markers. However, an analysis device capable of generating and processing a detectable signal with the characteristics of the proposed invention is not described, nor are capture mechanisms based on the application of magnetic fields mentioned.
    [0105]
    [0106] US 2013 0172211 A1 consists in a method for detecting genetic sequences of interest based on the generation and subsequent amplification and sequencing of a ligation product resulting from the union of the different complementary fragments to the target molecule, hybridized consecutively and sequentially in the presence Of the same. In addition, it is mentioned that the amplification and detection process may involve labeling with fluorophores, but not the capture mechanisms of the target molecule are detailed, as well as the characteristics of an analysis device similar to the proposed invention.
    [0107]
    [0108] US 20138609337 B2 consists of a method of preparation of signaling particles where a hydrogel microsphere is functionalized with a set of anchoring and signaling probes that allow the identification and detection of specific molecules. In this case, a marking system based on quantum dots is proposed, but neither molecular capture mechanisms nor the characteristics of an analysis device similar to the proposed invention are mentioned.
    [0109]
    [0110] US 2014 0024024 A1 proposes a detection system capable of sequentially identifying RNA, proteins and DNA in cell and tissue samples, as well as obtaining images of each one of the analytical results generated throughout the process. In this case, a signal amplification process is contemplated prior to the detection thereof, so that the intensity of said signal correlates positively with the concentration of the target molecule present in the sample. In addition, the possible fluorescent nature of the signal is mentioned, but molecular capture mechanisms are not detailed nor is an analysis device capable of generating and detecting the signal with the characteristics of the proposed invention described.
    [0111]
    [0112] US 20140332407 A1 consists of a detection kit based on a sensor constituted by a pair of electrodes between which a structure is arranged capable of immobilizing a series of receptor molecules that retain target molecules of different nature in their path. Subsequently, said target molecules are marked with signaling molecules with conductive properties or capable of depositing metal ions in the area between the electrodes. In any case, although it is mentioned that the nature of the detected signal can be both optical and electrical, molecular capture mechanisms based on the application of magnetic fields are not detailed nor is an analysis device capable of generating and detecting the signal with the characteristics of the proposed invention.
    [0113]
    [0114] US 2014 8691500 B2 proposes a system of cameras where to perform the processing and subsequent analysis of biological samples for the detection of specific analytes. In this case, the target molecule is captured with a magnetic particle and marked with a label - whatever its origin - that would allow its subsequent detection. However, there is no mention of a signal generation and detection module in the system or an integrated analysis device with the characteristics of the proposed invention.
    [0115]
    [0116] US 2015 0004598 A1 proposes different methods to identify or quantify analytes in various samples using a nucleic acid as a link between the analyte to be identified and the label particle, be it a fluorophore, a magnetic sphere, a quantum dot, an antibody, etc. However, a device similar to the proposed invention is not mentioned, since the aforementioned patent has the objective of integrating the method in techniques such as immunohistochemistry, western blot, in situ hybridization, etc.
    [0117]
    [0118] US 2016 0231324 A1 proposes an instrument for the detection of molecular markers by means of a microfluidic system where the sample is embedded in microdroplets and where a labeling system based on antibodies, enzymes, aptamers and fluorescence sensors is used. In any case, it is a detection device similar to a flow cytometer, which lacks systems for processing the samples and which does not have the characteristics of the proposed invention.
    [0119]
    [0120] US 20160265036 A1 proposes a method of identification and quantification of specific nucleic acids in biological samples by double hybridization with complementary strands: one of them fixed to one surface and the other, to a phosphate group that, linked to an enzyme catalytic, generates a detectable reaction product. However, an integrated and functional analysis device similar to the proposed invention is not disclosed.
    [0121]
    [0122] US 20160298179 A1 proposes a microchip to detect target molecules by joining signaling groups with luminophores. It is a structure similar to a microarray, although it uses a different system for the marking of the target molecule. In fact, it is intended to use the microarray analysis instruments to carry out the described test and, therefore, no device is mentioned to be patented as in the case of the proposed invention.
    [0123]
    [0124] US 20169274077 B2 has as its object a system for detecting interactions and bonds between molecules applicable, in addition, to the sequencing of polynucleotides. Said system is based on the use of electrodes and / or an electromagnetic force, but does not share the characteristics of the integrated analysis device object of the invention.
    [0125]
    [0126] US 20169482662 B2 discloses a biomolecule detection method that involves labeling them with different probes and using a well system adapted for the visualization of said biomolecules under the microscope, either using white light or generating fluorescence. However, an integrated and functional analysis device similar to the proposed invention is not mentioned nor is reference made to the processing of the samples analyzed.
    [0127]
    [0128] US 2017 9536041 B2 proposes a method of sequencing simple molecules of DNA based on a fluorescent, chemical, electrical or electromagnetic signal and in a multichannel system that also allows detecting the epigenetic pattern. However, a device for the generation and acquisition of the signal or for the processing of the samples with the characteristics of the proposed invention is not described.
    [0129]
    [0130] WO 1998033939 A1 presents a system for sequencing simple DNA molecules that uses magnetic microspheres to fix the target molecules to a support. Subsequently, once these target molecules are captured, the incorporation, by action of a polymerase, of identifiable modified nucleotides is produced thanks to the reflection of the ultraviolet radiation that is incised on the sample. However, an analysis device similar to the invention proposed to carry out this procedure is not described.
    [0131]
    [0132] WO 2003 025540 A2 describes a nucleic acid analysis method that employs up to three molecular tags, be they luminescent or fluorescent molecules, enzymes, radioisotopes, biotins, avidins, semiconductors, colloidal gold, quantum dots, antibodies, aptans, lipids, etc. In this way, the signal is amplified in order to increase the resolution of the analysis and achieve a more precise identification of the target molecule, but an integrated and functional analysis device similar to the proposed invention is not described in the cited patent.
    [0133] WO 2005 107818 A2 proposes a method to perform imaging in vivo using quantum dots conjugated with molecular labels, whether nucleic acids, antibodies, antigens or lipids, in living samples such as tissues or cells of different types. However, an analysis device with the characteristics of the invention proposed to carry out the test is not described.
    [0134]
    [0135] WO 2009012343 A2 proposes an array for detecting a target molecule in a sample; in particular, a reading system that allows the analysis in a microfluidic environment using an injection mechanism. However, an analysis device with the characteristics of the invention proposed to carry out the test is not described.
    [0136] WO 2010 057264 A1 proposes a method for the rapid detection of analytes -either nucleic acids, proteins, lipids, antibodies, viruses, bacteria or cells- in aqueous samples using fluorophores or quantum dots. In certain types of analysis, the aforementioned system requires a double-stranded DNA digestion process in order to detect the signal generated later. In any case, after the labels are linked to the analyte, the spectrum of the recorded signal will be analyzed, but no device is described for the generation and detection of the signal or for the processing of the samples with the characteristics of the proposed invention.
    [0137]
    [0138] WO 2011 038403 A1 refers to a method of molecular detection based on hybridization processes under different conditions, but an integrated device similar to the proposed invention, a generating system or signal detector, or the nature of the generated signal is not described. nor the molecular mechanisms of capture of the target molecule.
    [0139]
    [0140] WO 2012 016357 A1 refers to an array that allows analyzing the interaction between different molecules, whether polypeptides, antibodies, carbohydrates, etc. To do this, a marking particle - a magnetic microsphere, a fluorochrome or a quantum dot - is conjugated to the first problem molecule and the second problem molecule is fixed to a substrate of different origin. Thus, if the interaction between the two molecules takes place, a detectable signal will be generated when applying a magnetic field, an electric field or an electromagnetic radiation, but an integrated device similar to the proposed invention that allows carrying out this analysis.
    [0141]
    [0142] WO 2016 005517 A1 describes a method of detecting Leishmaniosis by amplifying and labeling the target molecule with colloidal gold. In this case, a direct labeling of the amplicons of the target molecule is carried out instead of being mediated by probes, but no analysis device similar to the invention proposed to carry out the assay is mentioned.
    [0143]
    [0144] WO 2016 065192 A1 proposes a method for detecting nucleic acids based on the release of a marker after the digestion of double-stranded DNA by the action of nucleases on an immobilized and labeled sample. However, an analysis device similar to the invention proposed to carry out the test is not described.
    [0145]
    [0146] WO 2017 008177 A1 proposes a microarray that detects mutations in specific genes by means of a procedure that involves the use of magnetic marking particles, quantums dot, fluorescent proteins, molecular dyes, etc. However, an integrated and functional analysis device similar to the proposed invention is not mentioned.
    [0147]
    [0148] Conclusion: As can be seen from the research carried out, none of the documents found solve the problems raised as the proposed invention does.
    [0149]
    [0150] Description of the invention
    [0151]
    [0152] The device for the analysis of nucleic acids object of the present invention is constituted from an integrated structure that has the following modules:
    [0153]
    [0154] - Sample reception system comprising a set of structures for the coupling of the containers containing said samples, as well as the containers in which said samples are deposited for carrying out the analysis.
    [0155] - Sample processing system equipped with an adjustable thermal source that allows to modulate and control the temperature of said samples so that the denaturation of the target molecules and hybridization of said target molecules with the labeling probes and the capture probes incorporated into the samples.
    [0156]
    [0157] - System of capture, concentration and purification of target molecules marked by application of a magnetic field. In this case, there is a system capable of generating, modulating and applying a magnetic field on the coupling structures with the containers containing the samples. This system allows to capture, concentrate and purify the target molecules attached to the capture and labeling probes and, subsequently, allows to remove the excess of labeling probes and remove the capture probes to avoid interference during the acquisition of the light signal. In addition, this system has a denaturing agent applied manually or automatically to the contents of the containers where the samples are processed to release the labeling probes and the capture probes retained after the purification step, which allows the withdrawal of said capture probes and the subsequent generation of the light signal by excitation of the particles attached to the labeling probes.
    [0158]
    [0159] - System of excitation of marking particles for the generation of the light signal. This module consists of a set of LEDs capable of emitting ultraviolet radiation or any other lighting system capable of emitting ultraviolet radiation or capable of emitting any other type of electromagnetic radiation capable of exciting the marking particles, causing said radiation to influence the coupling structures with the containers where the maceage probes are released after capture and purification of the labeled target molecules. On the other hand, this system has a series of antireflective materials and structures and optical filters arranged in such a way that they minimize the light pollution and, with it, the interference on the generated signal.
    [0160]
    [0161] - System of acquisition and processing of the light signal through photosensors, amplification structures, digital processing elements and systems for the visualization of results. This module is constituted by a set of photosensors capable of detecting light from the visible spectrum or any other type of light signal and whose position and orientation with respect to the coupling structures with the containers in which the released labeling probes are located allow recording optimally the signal generated by the excitation of the marking particles. Additionally, structures of amplification of the registered signal -like, for example, photomultiplier tubes, voltage amplifiers or optical systems- and elements of digital processing that convert the registered signal into a qualitative variable that indicate the presence or absence of the target molecule in the sample analyzed through a light, a screen, any other digital interface or through any other system that allows the visualization of the results obtained. In the same way, it is possible to carry out a quantitative analysis of the concentration of the target molecule in the analyzed sample thanks to the comparison of the recorded signal intensity with previously calibrated reference values.
    [0162]
    [0163] - Auxiliary systems:
    [0164]
    [0165] - Insulating structures that prevent the heat generated by the regulated thermal source from affecting the operation of other components of the device and, on the other hand, keep the photosensors protected to guarantee the stability, reliability and robustness of the measurements made by said photosensors.
    [0166] - Power systems, electrical resistances, switches and any other electrical or electronic component of the device.
    [0167]
    [0168] - Portable protective housing that houses the set of modules and systems described above in an integrated and functional way.
    [0169]
    [0170] As for the analysis carried out using the proposed invention, the assay is based on the molecular principles shown in Figure 1, which allow the capture and labeling of the target molecule.
    [0171]
    [0172] On the other hand, the procedure to carry out the analysis of nucleic acids that makes use of the proposed device is developed in the following stages:
    [0173]
    [0174] i. Take, as a starting point, a sample problem that may contain the target molecule.
    [0175]
    [0176] ii. Transfer the test sample to the initial container and add the marking particles and the magnetic capture particles functionalized with the corresponding probes.
    [0177] iii. Attach the initial container in the initial receptacle, activate and regulate the thermal source so that the problem sample reaches the denaturation temperature, wait a few minutes once that temperature is reached, regulate the thermal source so that the test sample reaches the hybridization temperature and wait a few minutes once reached that temperature.
    [0178]
    [0179] iv. Deactivate the thermal source, activate the magnetic capture system, wait a few seconds, remove the supernatant from the initial container, deactivate the magnetic capture system, apply the denaturing agent to the contents of the initial container, shake gently for a few seconds, activate the Magnetic capture and wait a few seconds.
    [0180]
    [0181] v. Take, without uncoupling the initial container from the initial receptacle, the volume contained in said initial container, free of magnetic capture particles, and transfer it to the final container.
    [0182]
    [0183] saw. Deactivate the magnetic capture system, activate the signal generation system and connect the final container in the final receptacle to visualize the result obtained by the system of acquisition and processing of the generated signal.
    [0184]
    [0185] In a different embodiment, a sample pumping system or other sample injection systems may be employed instead of the device having structures enabled for the reception of the containers with said samples. In any case, the samples analyzed would continue to undergo the same treatment; it would only change the way of passing them through the device.
    [0186]
    [0187] BRIEF DESCRIPTION OF THE DRAWINGS
    [0188]
    [0189] For a better understanding of the present description some drawings representing a preferred embodiment of the present invention are attached:
    [0190]
    [0191] Figure 1: Schematic of the molecular principles on which the nucleic acid analysis carried out using the proposed device is based, with double hybridization between the target molecule (central horizontal line) and the probe with the capture particle (left) and the probe with the marking particle (right).
    [0192] Figure 2: Development of the steps comprising the nucleic acid analysis procedure carried out using the device object of the present invention.
    [0193] The numerical references that appear in said figures correspond to the following constituent elements of the invention:
    [0194] 1. Analysis device
    [0195] 2. Initial container
    [0196] 3. Marking particles
    [0197] 4. Magnetic capture particles
    [0198] 5. Initial receptacle
    [0199] 6. Adjustable thermal source
    [0200] 7. Magnetic capture system
    [0201] 8. Denaturing agent
    [0202] 9. Final container
    [0203] 10. Final receptacle
    [0204] 11. Signal generation system
    [0205] 12. Results visualization system
    [0206] Description of a preferred embodiment
    [0207] A preferred embodiment of the device for analyzing nucleic acids object of the present invention can be based on an integrated structure that has the following modules:
    [0208] - Sample reception system comprising a set of structures for the coupling of the containers containing said samples, as well as the containers in which said samples are deposited for carrying out the analysis. - Sample processing system equipped with an adjustable thermal source that allows to modulate and control the temperature of said samples so that denaturation of the target molecules takes place and the hybridization of said target molecules with the tag probes and the capture probes incorporated to the samples.
    [0209] - System of capture, concentration and purification of target molecules marked by application of a magnetic field. In this case, there is a system capable of generating, modulating and applying a magnetic field on the coupling structures with the containers containing the samples. This system allows to capture, concentrate and purify the target molecules attached to the capture and labeling probes and, subsequently, allows to remove the excess of labeling probes and remove the capture probes to avoid interference during the acquisition of the light signal. In addition, this system has a denaturing agent applied manually or automatically to the contents of the containers where the samples are processed to release the labeling probes and the capture probes retained after the purification step, which allows the withdrawal of said capture probes and the subsequent generation of the light signal by excitation of the particles attached to the labeling probes.
    [0210]
    [0211] - System of excitation of marking particles for the generation of the light signal. This module consists of a set of LEDs capable of emitting ultraviolet radiation or any other lighting system capable of emitting ultraviolet radiation or capable of emitting any other type of electromagnetic radiation capable of exciting the marking particles, causing said radiation to influence the coupling structures with the containers where the labeling probes released after capture and purification of the labeled target molecules are located. On the other hand, this system has a series of antireflective materials and structures and optical filters arranged in such a way that they minimize the light pollution and, with it, the interference on the generated signal.
    [0212]
    [0213] - System of acquisition and processing of the light signal through photosensors, amplification structures, digital processing elements and systems for the visualization of results. This module is constituted by a set of photosensors capable of detecting light from the visible spectrum or any other type of light signal and whose position and orientation with respect to the coupling structures with the containers in which the released labeling probes are located allow recording optimally the signal generated by the excitation of the marking particles. Additionally, structures of amplification of the registered signal -like, for example, photomultiplier tubes, voltage amplifiers or optical systems- and elements of digital processing that convert the registered signal into a qualitative variable that indicate the presence or absence of the target molecule in the sample analyzed through a light, a screen, any other digital interface or through any other system that allows the visualization of the results obtained. In the same way, it is possible to carry out a quantitative analysis of the concentration of the target molecule in the analyzed sample thanks to the comparison of the recorded signal intensity with previously calibrated reference values.
    [0214]
    [0215] - Auxiliary systems:
    [0216]
    [0217] - Insulating structures that prevent the heat generated by the regulated thermal source from affecting the operation of other components of the device and, on the other hand, keep the photosensors protected to guarantee the stability, reliability and robustness of the measurements made by said photosensors.
    [0218]
    [0219] - Power systems, electrical resistances, switches and any other electrical or electronic component of the device.
    [0220]
    [0221] - Portable protective housing that houses the set of modules and systems described above in an integrated and functional way.
    [0222]
    [0223] On the other hand, the procedure for carrying out the nucleic acid analysis using the proposed device, with reference to the numerical references, is carried out in the following stages:
    [0224]
    [0225] i. Take, as a starting point, a sample problem that may contain the target molecule.
    [0226] ii. Transfer the test sample to the initial container (2) and add the marking particles (3) and the magnetic capture particles (4) functionalized with the corresponding probes.
    [0227] iii. Attach the initial container (2) in the initial receptacle (5), activate and regulate the thermal source (6) so that the test sample reaches the denaturation temperature, wait a few minutes once that temperature is reached, regulate the thermal source (6). ) so that the problem sample reaches the hybridization temperature and wait a few minutes once that temperature is reached.
    [0228] iv. Deactivate the thermal source (6), activate the magnetic capture system (7), wait a few seconds, remove the supernatant from the initial container (2), deactivate the magnetic capture system (7), apply the denaturing agent (8) to the contents of the initial container (2), shake gently for a few seconds, activate the magnetic capture system (7) and wait a few seconds.
    [0229] v. Take, without uncoupling the initial container (2) from the initial receptacle (5), the volume contained in said initial container (2), free of magnetic capture particles (4), and transfer it to the final container (9).
    [0230] saw. Deactivate the magnetic capture system (7), activate the signal generation system (11) and attach the final container (9) in the final receptacle (10) to visualize the result obtained by the system of acquisition and processing of the generated signal (12).
    权利要求:
    Claims (17)
    [1]
    1. Method for the analysis of nucleic acids characterized in that it comprises the following stages:
    i. - Take, as a starting point, a sample problem that may contain the target molecule.
    ii. - Transfer the test sample to the initial container (2) and add the marking particles (3) and the magnetic capture particles (4) functionalized with the corresponding probes.
    iii. - Attach the initial container (2) in the initial receptacle (5), activate and regulate the thermal source (6) so that the test sample reaches the denaturation temperature, wait a few minutes once that temperature is reached, regulate the thermal source ( 6) so that the test sample reaches the hybridization temperature and wait a few minutes once that temperature is reached.
    iv. - Deactivate the thermal source (6), activate the magnetic capture system (7), wait a few seconds, remove the supernatant from the initial container (2), deactivate the magnetic capture system (7), apply the denaturing agent (8) to the contents of the initial container (2), shake gently for a few seconds, activate the magnetic capture system (7) and wait a few seconds.
    v. - Take, without uncoupling the initial container (2) from the initial receptacle (5), the volume contained in said initial container (2), free of magnetic capture particles (4), and transfer it to the final container (9).
    saw. - Deactivating the magnetic capture system (7), activating the signal generation system (11) and coupling the final container (9) in the final receptacle (10) to visualize the result obtained by the system of acquisition and processing of the generated signal (12).
    [2]
    2. Method for the analysis of nucleic acids, according to claim 1, characterized in that, after removing the non-retained supernatant by the action of the magnetic capture system, it is required to add a certain volume of a buffer solution before applying the denaturing agent.
    [3]
    3. Method for the analysis of nucleic acids, according to claims 1 and 2, characterized in that the analysis does not need to transfer the supernatant from one container to another, it being possible to carry out the test in a single compartment as the different modules are integrated and systems comprising the device used in carrying out the analysis, so that the processing of the sample and the generation, acquisition and processing of the signal are carried out optimally and without interference of any kind.
    [4]
    4. Method for the analysis of nucleic acids, according to claims 1 to 3, characterized in that, when analyzing samples that may contain nucleic acids, it will be possible to establish the necessary negative and positive controls to guarantee reliability, robustness, sensitivity, specificity, repeatability and reproducibility of the trial.
    [5]
    Method for the analysis of nucleic acids, according to claims 1 to 4, characterized in that the simultaneous analysis of a plurality of samples can be performed using a device that has a plurality of sample receiving systems with structures enabled for this purpose and comprises a plurality of processing systems samples and a plurality of systems for generating, acquiring and processing the signal or, at least, comprising a plurality of some of the structures that compose them.
    [6]
    Method for the analysis of nucleic acids, according to claims 1 to 5, characterized in that the analysis of all types of test samples can be carried out, whether or not they have undergone any type of previous treatment, such as, for example, the purification or amplification of the target molecule.
    [7]
    7. Method for the analysis of nucleic acids, according to claims 1 to 6, characterized in that it allows to perform the analysis of the target molecule regardless of the nature, size and sequence of said target molecule and whether or not the sequence of said molecule is known. target molecule.
    [8]
    8. Method for the analysis of nucleic acids, according to claims 1 to 7, characterized in that it allows to perform the analysis of the target molecule whatever the nature of the probes, the marking particles and the capture particles used in the embodiment of that analysis.
    [9]
    9. Method for the analysis of nucleic acids, according to claims 1 to 8, characterized in that said method can be applied to the analysis of proteins by adapting only the probes with which the capture and labeling particles of the target molecule are functionalized.
    [10]
    10. Device for the analysis of nucleic acids characterized in that it forms part of the execution of the procedure described in the previous claims and comprises the following systems:
    - Sample reception system comprising a set of structures for the coupling of the containers containing said samples, as well as the containers in which said samples are deposited for carrying out the analysis.
    - Sample processing system equipped with an adjustable thermal source that allows to modulate and control the temperature of the samples to produce the denaturation of the target molecules and the hybridization of said target molecules with the tag probes and the capture probes incorporated to the samples.
    - Capture, concentration and purification system of the labeled target molecules that allows to generate, modulate and apply a magnetic field on the coupling structures with the vessels containing the samples to capture, concentrate and purify the target molecules attached to the capture probes and of marking and, later, removing the excess of labeling probes and removing the capture probes to avoid interference during the acquisition of the light signal.
    - System of excitation of marking particles for the generation of the light signal comprising a set of LEDs capable of emitting ultraviolet radiation or any other lighting system capable of emitting ultraviolet radiation or capable of emitting any other type of electromagnetic radiation capable of emitting ultraviolet radiation. excite the marking particles, causing said radiation to influence the coupling structures with the containers where the labeling probes are released after the capture and purification of the labeled target molecules. In addition, this system also comprises a series of antireflective materials and structures and optical filters arranged in such a way as to minimize light pollution and, therefore, interference with the generated signal.
    - Light signal acquisition system comprising a set of photosensors capable of detecting light from the visible spectrum or any other type of light signal and whose position and orientation with respect to the coupling structures with the vessels in which the probes are located Released markings allow the signal generated by the excitation of the marking particles to be optimally recorded.
    [11]
    Device for the analysis of nucleic acids, according to claim 10, characterized in that, additionally, structures of amplification of the registered signal-such as, for example, photomultiplier tubes, voltage amplifiers or systems-are coupled to the system for acquiring the light signal. optical- and digital processing elements that convert said registered signal into a qualitative variable that indicates the presence or absence of the target molecule in the sample analyzed through a light, a screen, any other digital interface or through any other system that allows the visualization of the obtained results.
    [12]
    Device for the analysis of nucleic acids, according to claims 10 and 11, characterized in that it allows carrying out a quantitative analysis of the concentration of the target molecule in the analyzed sample thanks to the digital processing of the intensity of the recorded signal and its comparison with values of reference previously calibrated.
    [13]
    Device for the analysis of nucleic acids, according to claims 10 to 12, characterized in that it comprises the following auxiliary systems: insulating structures that prevent the heat generated by the regulated thermal source from affecting the operation of other components of the device and, on the other hand, part, they keep the photosensors protected to guarantee the stability, reliability and robustness of the measurements made by said photosensors; power systems, electrical resistances, switches and any other electrical or electronic component of the device. In addition, said device also comprises a portable protective housing that houses the set of modules and systems described in previous claims in an integrated and functional manner.
    [14]
    14. Device for the analysis of nucleic acids, according to claims 10 to 13, characterized in that the samples are provided by a pumping or injection system that makes them pass through the device for the analysis instead of being transferred to containers that can be coupled in structures enabled for this purpose.
    [15]
    15. Device for the analysis of nucleic acids, according to claims 10 to 14, characterized in that it allows to perform the analysis of the target molecule directly and without the need for accessory or complementary technology, which makes it possible to perform analyzes both in situ or in the field. in laboratories and in any other type of facilities equipped and equipped for molecular analysis.
    [16]
    16. Device for the analysis of nucleic acids, according to claims 10 to 15, characterized in that the analytical procedure carried out with said device can be automated using additional software and hardware elements.
    [17]
    17. Device for the analysis of nucleic acids, according to claims 10 to 16, characterized in that said device can be applied to the analysis of proteins adapting only the probes with which the capture and labeling particles of the target molecule are functionalized.
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    同族专利:
    公开号 | 公开日
    WO2019043277A1|2019-03-07|
    JP2020532306A|2020-11-12|
    US20200354776A1|2020-11-12|
    KR20200042511A|2020-04-23|
    MA50044A|2020-07-08|
    ES2702432B2|2019-08-05|
    EP3677690A4|2021-06-30|
    EP3677690A1|2020-07-08|
    CN111051527A|2020-04-21|
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    申请号 | 申请日 | 专利标题
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    PCT/ES2018/070564| WO2019043277A1|2017-08-31|2018-08-20|Method and device for analysing nucleic acids|
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    EP18851994.6A| EP3677690A4|2017-08-31|2018-08-20|Method and device for analysing nucleic acids|
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    US16/640,697| US20200354776A1|2017-08-31|2018-08-20|Method and device for analysing nucleic acids|
    KR1020207007807A| KR20200042511A|2017-08-31|2018-08-20|Method and apparatus for analyzing nucleic acid|
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