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WO2016113622A1 - Récupération d'une séquence de nucléotides à l'aide de nanocomposites magnétiques - Google Patents

Récupération d'une séquence de nucléotides à l'aide de nanocomposites magnétiques Download PDF

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Publication number
WO2016113622A1
WO2016113622A1 PCT/IB2016/000011 IB2016000011W WO2016113622A1 WO 2016113622 A1 WO2016113622 A1 WO 2016113622A1 IB 2016000011 W IB2016000011 W IB 2016000011W WO 2016113622 A1 WO2016113622 A1 WO 2016113622A1
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Prior art keywords
solution
nucleotide sequence
dna
set forth
nanocomposites
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PCT/IB2016/000011
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English (en)
Inventor
Juan Carlos MEDINA-LLAMAS
Alicia Elizabeth CHÁVEZ-GUAJARDO
Cesar Augusto SOUZA DE ANDRADE
Kleber Goncalves BEZERRA ALVES
Celso Pinto De Melo
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Nanomark Pesquisa E Desenvolvimento Tecnológico Ltda
Universidade Federal De Pernambuco - Ufpe
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Priority claimed from US14/594,845 external-priority patent/US9738888B2/en
Application filed by Nanomark Pesquisa E Desenvolvimento Tecnológico Ltda, Universidade Federal De Pernambuco - Ufpe filed Critical Nanomark Pesquisa E Desenvolvimento Tecnológico Ltda
Publication of WO2016113622A1 publication Critical patent/WO2016113622A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/06—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising oxides or hydroxides of metals not provided for in group B01J20/04
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/262—Synthetic macromolecular compounds obtained otherwise than by reactions only involving carbon to carbon unsaturated bonds, e.g. obtained by polycondensation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28009—Magnetic properties
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3204—Inorganic carriers, supports or substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention refers to fluorescent nanoparticle composites. More specifically, it refers to the composites themselves, to the process of preparing such composites, to systems for rapid diagnosis (as "kits") containing such composites, and to the use of such composites. Besides that, the composites of the present invention have an affinity for biological molecules, such as DNA and RNA, also providing for applications in the medical and veterinarian fields, and in the diagnosis of genetic diseases as well as those caused by several pathogens. The invention also pertains to the use of magnetism in nanoparticles for the retrieval of DNA, and for rapidly providing the DNA for the diagnosis.
  • the molecular diagnosis of diseases and genetic traits is an emerging field, particularly in the area of clinical analysis. Generally, it uses techniques from molecular biology for the study of DNA/RNA, of infectious agents, or of genetic changes in the organism itself, aiding in the diagnosis and prognosis of infectious and genetic diseases.
  • PCR enzymatic amplification of the DNA
  • restriction enzymes restriction enzymes
  • electrophoretic separation of the DNA or of the PCR product hybridization of the DNA or PCR fragments with oligonucleotide probes
  • DHPLC cytogenetic methods.
  • cytogenetic methods based on the microscopic observation of normal and abnormal chromosomes, allow the construction of cytogenetic maps of the genomes of many species.
  • the FISH (fluorescent in situ hybridization) cytogenetic method is the most direct means of locating molecular and genetic markers in the cytogenetic map, allowing the integration between genetic and molecular markers.
  • Probes are widely used for diagnosis, such as cosmid probes, which are unique sequences connected in small segments of certain chromosomes, being useful for the study of _microdeletions._ Other probes are used to detect-transloeations and- highly repetitive sequences. However, one should point out that some of these techniques still have some limitations, such as false-positive signals, that can lead to an error in diagnosis.
  • a well-known molecular diagnosis system is the ELISA (Enzyme-Linked Immunosorbent Assay).
  • ELISA Enzyme-Linked Immunosorbent Assay
  • This immune-enzymatic test allows the detection of specific antibodies in the serum of patients, being the first-line test in the diagnosis of HIV (human immunodeficiency virus) infection.
  • the method for performing the test is based on the antibody-antigen interaction, with this test also being capable of detecting other substances, such as hormones.
  • the present invention refers to the fluorescent nanoparticle composites themselves, method for the preparation of these composites, system for rapid diagnosis (as "kits") containing such compounds, and functioning of said "kits".
  • the composites of the present invention have specific characteristics regarding size and fluorescence, and have an affinity for biological molecules, such as DNA, RNA, and also proteins.
  • the method for the preparation of these compounds is also described in the present invention. Plus, the present invention describes the method of preparation for an adequate probe (named here as "support”) containing biological material of the organism one wishes to study.
  • the fluorescent nanoparticle composites and the patient's biological material are added, comprising a diagnostic system, designated here as the ELINOR (from "Enhanced Luminescence from Inorganic/Organic nanocomposites”) test, for the diagnosis of diseases caused by several pathogens and/or genetic diseases, amongst other things.
  • ELINOR from "Enhanced Luminescence from Inorganic/Organic nanocomposites” test, for the diagnosis of diseases caused by several pathogens and/or genetic diseases, amongst other things.
  • the present invention has application mainly in the medical and veterinarian fields.
  • the present invention differs from all of those documents by not requiring a step of amplification (such as the one performed in the PCR technique) in order to perform the molecular diagnosis.
  • Document US 2007/0059693 describes a biosensor containing a fluorescent surface, molecules of nucleic acid, and a fluophore.
  • the fluorescent surface may be a metal, including gold.
  • the molecules of nucleic acid must have one of the ends bound to the fluorescent surface and the other end to a fluophore.
  • This molecule of nucleic acid may also have internal hybridization regions that, when hybridized, form a "staple". In these cases, the fluophore will be close to the fluorescent surface, allowing fluorescence to occur.
  • the present invention differs from that document due to the support surface not being necessarily fluorescent or metallic, and not requiring that the molecules of nucleic acid form a "staple" in order to emit fluorescence.
  • Document US 2005/0196876 describes a method for the analysis of the content of a biological sample through the contact of the sample with a nanoporous biosensor.
  • This biosensor contains probes that bind to the samples forming complexes that will be bound to a second probe. That probe will be illuminated so as to send a specific fluorescent signal.
  • this biosensor may have a layer of gold.
  • the present invention differs from the aforementioned document by dealing with fluorescent nanoparticles containing gold, there being no need to bind to more than one probe.
  • Document U.S. Pat. No. 7,083,928 describes the detection of negatively charged polymers using water-soluble cationic polythiophenes.
  • the negatively charged polymers include biological molecules such as nucleic acid. This polymer may be bound to a conductive support, such as a gold surface. When the polymer is detected, there is a change in the electronic load, fluorescence, or color,
  • the present invention differs from the aforementioned document by dealing with nanoparticles of gold covered by polymers that interact with the biological molecules, with the gold not being part of the adequate support that will immobilize the biological molecules.
  • Extraction of DNA from cells and biological samples and its purification are of primary importance to the fields of molecular diagnostics, biotechnology and forensics. Further, extraction and purification of DNA are generally the first steps in the analysis and manipulation of DNA that allow scientists to detect genetic disorders, produce DNA fingerprints of individuals and prepare for therapeutical solutions.
  • Magnetic separation is attained by adding magnetic nanoparticles (MNPs) to a solution containing the target molecule and allowing enough time for interaction between the target molecules and the MNPs to occur. Subsequently, the MNPs are spatially confined by the application of a non-uniform external magnetic field, resulting in a concentration of the target molecules.
  • MNPs magnetic nanoparticles
  • an embodiment of the present disclosure provides a process for retrieval of nucleotide sequence.
  • the process comprises in solution, mixing iron chloride tetrahydrate with iron (III) chloride hexahydrate; adding ammonium hydroxide to the mixture and stirring to form maghemite nanoparticles; stirring the maghemite nanoparticles in a solution with an inorganic acid, a surfactant and a monomer precursor of a conducting polymer; initiating polymerization of the monomer by adding the inorganic acid and an oxidizing agent to the stirred solution and further stirring to yield Polyaniline/maghemite nanocomposites; adding the nanocomposites to an first aqueous solution of the nucleotide sequence and stirring so as to electrostatically interact the nanocomposites with the nucleotide sequence; and weakening the electrostatic interaction between the nanocomposite and the nucleotide sequence to recover the nanocomposite independently of the nucleotide sequence.
  • mixing the iron chloride tetrahydrate with iron chloride hexahydrate comprises mixing in a molar ratio of 1 :2.
  • the process further comprises doping the nanocomposites with one or more acids.
  • the nanocomposite is washed and acid doped then added to a second aqueous solution of nucleotide sequence and stirred so as to electrostatically interact the nanocomposite with the nucleotide sequence of the second solution.
  • stirring the maghemite nanoparticles in a solution with hydrochloric acid, sodium dodecyl sulfate and a monomer further comprises stirring the maghemite nanoparticles in a solution with hydrochloric acid, sodium dodecyl sulfate and aniline.
  • adding the inorganic acid and an oxidizing agent to the stirred solution further comprises adding ammonium persulfate to the stirred solution.
  • stirring the maghemite nanoparticles in a solution with an inorganic acid, a surfactant and a monomer precursor of a conducting polymer further comprises stirring in a solution with sodium dodecyl sulfate.
  • stirring the maghemite nanoparticles in a solution with an inorganic acid comprises stirring with hydrochloric acid.
  • weakening the electrostatic interaction between the nanocomposite and the nucleotide sequence to recover the nanocomposite independently of the nucleotide sequence comprises weakening with a solution of an alkali salt.
  • weakening with a solution of an alkali salt comprises weakening with a solution of sodium hydroxide.
  • electrostatically interacting the nanocomposites with the nucleotide sequence further comprises electrostatically interacting with DNA or RNA.
  • an embodiment of the present disclosure provides a biological diagnosis kit for rapid patient diagnosis.
  • the kit comprises at least one composite; at least one short nucleotide sequence; an appropriate substrate for the immobilization of the short nucleotide sequence; and a genetic sample of the patient.
  • the composite of the biological diagnosis kit further comprises at least one magnetic nanoparticle and at least one conducting polymer.
  • the magnetic nanoparticle of the biological diagnosis kit comprises maghemite.
  • the at least one conducting polymer of the biological diagnosis kit comprises polyaniline.
  • the short nucleotide sequence of the biological diagnosis kit comprises RNA or single-stranded DNA.
  • the substrate of the biological diagnosis kit comprises a glass slide, paper and/or a polymer strip.
  • the at least one composite comprises a fluorescent composite.
  • an embodiment of the present disclosure provides a DNA- bonding nanocomposite.
  • the DNA-bonding nanocomposite comprises at least one oxidizing agent; at least one stabilizer agent; and at least one conducting polymer.
  • the oxidizing agent of the DNA-bonding nanocomposite comprises iron chloride tetrahydrate with iron (III) chloride hexahydrate.
  • the conducting polymer of the DNA-bonding nanocomposite is polyaniline (Pani).
  • FIGURE. 1 is a schematic representation of the preparation route to obtain the fluorescent composites ((Au nanoparticles)/(conducting polymer)).
  • FIGURE. 2 is the UV-Visible absorption spectrum of the gold/polyaniline (Au/PANI) composites, where the plasmon band (associated to the reduced gold forming metallic nanoparticles) and the polaron band (associated to the monomer oxidation process leading to the formation of the polymer).
  • FIGURE. 3 is a scanning electron microscope image of the fluorescent composites (Au nanoparticles)/(conducting polymer). (Magnifying factor of 4,000x).
  • FIGURE. 4 is a scanning electron microscope image of the fluorescent composites (Au nanoparticles)/(conducting polymer). (Magnifying factor of 10,000*).
  • FIGURE. 5 is a transmission electron microscope image of the fluorescent composites (Au nanoparticles)/(conducting polymer).
  • FIGURE. 6 is a transmission electron microscope image in dark field of the fluorescent composites (Au nanoparticles)/(conducting polymer). The lighter regions indicate the presence of metallic nanoaggregates enveloped by the polymeric chains.
  • FIGURE. 7 is a transmission electron microscope image of the fluorescent composites (Au nanoparticles)/(conducting polymer) where the metallic nanoaggregates can be seen.
  • FIGURE. 8 is a high-resolution transmission electron microscope image of the fluorescent composites (Au nanoparticles)/(conducting polymer).
  • FIGURE. 9 is a X-ray diffraction image of the fluorescent composites (Au nanoparticles)/(conducting polymer).
  • FIGURE 10A is a Transmission Electron Microscopy (TEM) micrographs of y- Fe 2 03 nanoparticles (NPs) (a) and PANI/y-Fe20 3 maghemite nanocomposite (MNC).
  • TEM Transmission Electron Microscopy
  • NPs y- Fe 2 03 nanoparticles
  • MNC maghemite nanocomposite
  • FIGURE 10B is inset: Energy Dispersive Spectroscopy (EDS) spectrum of the MNC, specifically, histogram of the particle size of the y-Fe203 NPs obtained after estimating the diameter of 300 particles depicted in several TEM micrographs.
  • EDS Energy Dispersive Spectroscopy
  • FIGURE 10C is a histogram of the particle size of the Pani/y-Fe20 3 MNC obtained through Dynamical Light Scattering (DLS).
  • DLS Dynamical Light Scattering
  • FIGURE 10D is another histogram of the particle size of the Pani/y-Fe 2 03 MNC obtained through DLS.
  • FIGURE 1 1 is the room temperature magnetization curves of -Fe203 NPs (a) and Pani/y-Fe203 MNC (b).
  • FIGURE 12 is Fourier Transform Infrared Spectroscopy (FTIR) spectra of Pani/y-Fe203 MNC (a), Pani (b) and y-Fe203 NPs (c).
  • FTIR Fourier Transform Infrared Spectroscopy
  • FIGURE 13 is a schematic diagram of different steps involved in the procedure used to achieve DNA retrieval and desorption.
  • FIGURE 14 is effect of interaction time on the DNA retrieval by the Pani/y- Fe 2 03 MNC.
  • FIGURE 15 is effect of the DNA concentration on the retrieval capacity of the
  • FIGURE 16 is DNA desorption process of the Pani/y-Fe203 MNC as a function of the pH.
  • FIGURE 17 is a table representing Langmuir and Freundlich isotherms parameters for SS_DNA adsorption onto Pani/ -Fe2C»3 MNC.
  • FIGURE 18 is DNA adsorption isotherm onto Pani/ -Fe203 MNC of the experimental values (special dot) and Langmuir model (line).
  • FIGURE 19 is a table representing a comparison of characteristics of various DNA adsorbents including Pani/ -Fe203.
  • the composites of the invention are useful for different applications, including: the preparation of photovoltaic devices, such as solar cells, and electroluminescent devices, as organic LEDs, leading in both cases to a substantial increase in their quantum efficiency; the increase in the lighting efficiency of fluorescent lamps; the preparation of reagents and consumable items for diagnosis procedures, amongst other applications.
  • the composites of the present invention provide, among other advantages, the absorption of incident light in the ultraviolet or visible regions and the emission of light in the ultraviolet and visible region, inclusive in the "deep blue” and/or green colors, providing a special advantage in their use in photovoltaic devices, such as solar cells, or in electroluminescent devices, as organic LEDs, or for the increase in the quantum yield of lighting systems, such as fluorescent lamps.
  • the composites of the present invention provide an environmentally friendlier and more energy efficient alternative to the phosphors presently used in the internal layer of coverage of fluorescent lamps to assure the ultraviolet quantum cut-off and that are a source of pollution when not properly discarded.
  • the composites of the invention can be prepared so as to provide emission in different colors and with wide-range adjusting intensities, according to the tuning of their composition and preparation manner.
  • the composites of the present invention have affinity for biological molecules, such as DNA, RNA, or proteins, providing also applications in the areas of human and animal health and in the diagnosis tests for diseases caused by different pathogenic agents.
  • the following examples do not have the purpose of limiting the range of the invention, but rather only illustrate one of the innumerable manners of realizing the invention.
  • biological material the group of compounds that comprises, but it is not limited to, DNAs, RNAs, proteins, lipids, peptides, non- codifying RNAs, and/or any other biological material that could be represented by a single chain or single strand.
  • biological material of the patient the group of biological material that comprises, but it is not limited to, the biological material of any organism that could be present in a small amount of blood or obtained from a simple collection of epithelial or mucosa cells, and/or from secretions and/or excretions of the patient.
  • oxidizing agent is a salt in which the cation is selected from the group comprising metals chosen from groups 1 B to 8B of the periodic table.
  • This group of compounds comprises, but it is not limited to, to gold compounds, such as HAuCU. Preferentially, the gold atom is in the 3+ oxidation state.
  • monomer any compound that can be polymerized by the oxidizing agent. Namely, it is chosen from the group that comprises, but it is not limited to, the smallest repetitive unit of a polymer, as those derived from aniline (C6H5NH2), thiophene (C4H4S), pyrrole (C4H5N), or precursor molecules of the respective polymers, PANI, PEDOT ((poly(3,4-ethylenedioxythiphene)poly (styrenesulfonate)), PTAA (polythiophene acetic acid) and polypyrrole, and/or a mixture of these.
  • stabilizing agent the group of compounds that comprises, but it is not limited to, silanes, such as (3-mercaptopropyl) trimethoxy silane (MPS), (3-mercaptopropyl) methyldimethoxysilane, (3-mercaptopropyl) triethoxysilane e (3-mercaptoethyl)trimethoxysilane and/or a mixture of them.
  • silanes such as (3-mercaptopropyl) trimethoxy silane (MPS), (3-mercaptopropyl) methyldimethoxysilane, (3-mercaptopropyl) triethoxysilane e (3-mercaptoethyl)trimethoxysilane and/or a mixture of them.
  • alcohol the group that comprises, but it is not limited to methanol, ethanol, propanol, butanol, glycerol, ethylene glucol and/or a mixture of them.
  • Photoluminescence properties were measured by use of a quartz cuvette (1 cm and 5 mL) in a PC1 (ISS, USA) spectrofluorimeter at (20 ⁇ 1 )°C.
  • the samples were monitored at different pH values by use of two luminescence matrices: (1 ) in the 200 to 360 nm excitation range and emission in the 370 to 600 nm interval; and (2) in the 270 to 330 nm excitation range and emission in the 280 to 600 nm interval.
  • Morphological analyses were performed by scanning electron microscopy (SEM), by use of a JSM-5900 (JEOL, Japan) electron microscope. The samples were placed atop a glass substrate and fixed by a carbon tape.
  • the samples were covered by a thin gold layer by use of a sputtering (BalTec SCD 050).
  • the size of the particles was determined by a light-scattering method by use of a Zetasizer Nano- ZS90 instrument (Malvern).
  • Gold nanoparticies with diameters of the order of ⁇ 5 nm exhibit in their absorption spectrum a surface plasmon (SP) band centred in 525 nm.
  • the UV-Vis spectrum of the composites is shown in FIGURE 2, where once can observe the strong presence of a SP band at 560 nm.
  • the wavelength and the intensity of the SP band vary according to the size, shape and the "interparticle" dielectric medium, and that it is also sensitive to the relative molar fraction (stabilizing agent)/Au [J. Am. Chem. Soc. 2003, 125, 9906].
  • PAN I exhibit two characteristic absorption bands (324 nm and 625 nm) in the UV- Vis region.
  • the gold containing compound acts as an oxidizing agent, i.e., the trigger of the aniline polymerization, while a mercapthosilane is included as a co-stabilizer of the formed metallic nanoparticies.
  • oxidizing agent i.e., the trigger of the aniline polymerization
  • mercapthosilane is included as a co-stabilizer of the formed metallic nanoparticies.
  • the fluorescence matrix of the PANi— Au sample one can verify that the composite exhibit luminescent properties in the visible region, since the composite presents a peak of photoluminescence centred close to 400 nm when excited in the ultraviolet (350 nm) region.
  • the method used has allowed the inventors to prepare gold nanoparticies with sizes of the order of 5 nm (or less), enveloped by a "shell" of conducting polymers, whose dielectric properties can be changed by varying either their oxidation state and/or the pH of the medium where they are dispersed.
  • the emission wavelength of the composite at least in principle one can tune the emission wavelength of the composite by properly adjusting the dielectric properties of the medium.
  • Measurements of the quantum yield of the first samples of the composites have indicated values in the 1.5 to 7.5% interval; however, modifications in the method of preparation already implemented have allowed the inventors to increase the quantum yield, as well as emission of the same system in different wavelengths.
  • FIGURE 7 reveals that there is a monodisperse distribution of nanoparticles of sizes varying from 2 to 5 nm, even though in some cases formation of geminal particles— a well-known characteristic of gold nanoparticles— could be identified.
  • a High Resolution Transmission Electron Microscopy (HRTEM) image of the hybrid gold/(conducting polymer) nanocomposite reveals the presence of crystalline structures (FIGURE 8), an observation that is confirmed by examining the corresponding X-ray diffraction (XRD) pattern (FIGURE 9).
  • HRTEM High Resolution Transmission Electron Microscopy
  • the specifity towards the presence of a given pathogenic agent is determined by the nature of the fragment of the biological material (such as a DNA single strand) immobilized in the probe, so that the technique do not is limited on that regard, and can be used for the identification of any organism for which a specific short sequence of biological material, such as DNA, can be obtained;
  • the technology is of general use for the diagnosis of any disease: whose origin can be: a) attributed to a known pathogenic agent, or b) associated to the presence of a specific sequence of biological material (such as DNA or RNA), even if human (and so it opens the possibility of using the technology for the investigation not only of diseases already installed but also for the analysis of genetic tendency of patients with regard to the future development of hereditary pathologies;
  • the amount of biological material to be used in the diagnosis assays is extremely small (e.g., a volume of 1 ⁇ _ of a 100 pmol solution of biological material, such as DNA);
  • the preparation of the probes containing the sequence of the biological materials is a step that can be adapted to large scale production, once again at a very low cost;
  • the result of the diagnosis assay has a conclusive character (i.e., positive/negative) and it can be obtained in a matter of minutes, with no need of using any kind of culture medium;
  • the result of the diagnosis assay is based in the observation of the intensity of the fluorescence signal, indicating the presence or absence of the nucleotide sequence of interest;
  • the assay probe in the case of existence of genetic variation of the pathogenic agent in different subtypes (as in the case of the dengue virus, for example), can be prepared in such manner as to contain biological material of each subtype to be investigated, and hence a single test can provide a conclusive answer with regard to the presence of any variety of the pathogen in the genetic sample provided by the patient;
  • the probe in the case in which the symptoms exhibited by the patient can be attributed to a limited number of possible pathogenic agents (as, for example, in the case of hospital acquired infections, or in the case of victims of accidents with deep perforations and wounds), the probe can be prepared in such manner as to contain biological materials (nucleotide sequence) of each one of the agents, so that in a single and rapid exam the diagnosis can be conclusive for the presence of any of them;
  • the rapid diagnosis kit here proposed can be used, but is not limited, to the diagnosis of: dengue virus: ii) tuberculosis; iii) hepatitis C; iv) human papillomavirus (HPV), v) leishmaniasis, vi) rapid identification (from within a pre-selected range of options) of the cause of hospital acquired infections; vii) rapid identification of meningococcus infections; viii) bioterrorism hazards, besides ix) genetic screening of hereditary diseases (such as Tay-Sachs, phenylketonuria, breast cancer, among others). A few examples are discussed below.
  • the diagnosis procedure uses a short sequence of a single nucleotide strand consisting of 20 bases of the variety 16 of HPV.
  • the quality of the response can be attested when a negative answer was obtained whenever the probe was exposed to a double strand of the variety 18 of HPV with circa of 500 base pairs and a positive answer only when the probe was exposed to double strand with 500 bases pairs of the variety 16 of HPV.
  • the diagnosis procedure uses a short single strand consisting of 22 bases of the subtype 2 of the dengue virus.
  • the quality of the response is associated to a negative answer when the probe was exposed to a double strand non- complementary to the original sequence used and to a positive diagnosis when the probe was exposed to a double strand containing 22 base pairs of the subtype 2 of the virus dengue.
  • the diagnosis procedure uses short single strand sequences of 19 (MBL54mt) and 22 (MBL57mt) bases corresponding to human lectin responses to different HPV varieties, some of them containing mutations in specific positions that could block the hybridization of the DNA chains of the pathogenic agent present in the material of the patient.
  • the type of response (positive or negative answer) obtained, respectively, for homozygous and heterozygous patients define the sensitiveness of the technology as excellent.
  • the total DNA In case of existence of genetic material of the pathogenic agent in the biological material obtained from the patient (the "total DNA"), a long nucleotide strand of the pathogenic agent will hybridize to the immobilized short sequence, and retain a larger amount of fluorescent composite: a "positive” answer will then arise. If the hybridization did not occur, only a smaller amount of the composite Will remain attached to the short immobilized sequence of nucleotide, and as a consequence the fluorescence signal will be minimum (basal): the "negative" answer.
  • the composites of the present invention can act as "nanoluminol"; a fairly recent publication of the University of San Diego, available in http://www.topnews.in/health/handheld-dna-detector-may-soon-be- reality-2141 1 , shows that DNA portable detectors may offer substantial advantages over the present technology. Even though the technology adopted in such reference is much more complex and expensive (ion-selective field-effect transistor -ISFET) than that discussed in the present invention, it is an important example of the actual need of new developments this area of expertise.
  • the present disclosure provides a DNA-bonding nanocomposite for DNA retrieval.
  • the DNA-bonding nanocomposite is composed of an oxidizing agent, a stabilizing agent and a conducting polymer.
  • the oxidizing agent is iron chloride tetrahydrate (FeCl2-4H20) with iron (III) chloride hexahydrate (FeCl3-6H20).
  • the conducting polymer includes PANI.
  • maghemite (gamma-iron oxide; Y-Fe203) nanoparticles may be prepared as follows: 50 ml_ of FeCl2-4H20 and FeCl3-6H20 solution is made in a molar ratio of 1 :2, and then mixed in a 250 mL round-bottom flask under vigorous stirring for 10 min. After that, 125 mL of an aqueous solution of Nh OH (50 vol%) may be added quickly and the resulting solution stirred for 2 h. Next, the freshly formed NPs may be decanted with the help of a handheld magnet.
  • the resulting material may be washed with deionized water, and the magnet used to once more decant the y-Fe203. Repeating this process a number of times, for example four, minimizes contamination by any non-magnetic impurity. Finally, the NPs are dried in a vacuum oven at 60 C for 48 h to obtain a brown powder.
  • MNC DNA-bonding, magnetic nanocomposite, Pani/ v-Fe203
  • 100 mL of a 0.1 M HCI solution, 60.7 mM of sodium dodecyl sulfate, 0.06 g of y-Fe203 NPs and 1.5 mM of aniline added to a flask are stirred for 15 min.
  • 20 mL of a 0.1 M of HCI and 1.5 mM of ammonium persulfate (APS) may be slowly added to initiate polymerization. Allowing polymerization to proceed for 24 h at room temperature under stirring results in a green solution.
  • APS ammonium persulfate
  • the obtained product may be magnetically decanted, washed with HCI (0.1 M) to assure the acidic doping of the polymer, and then dried in a vacuum oven at 40 C for 24 h.
  • HCI 0.1 M
  • Particle diameters are represented in the histogram plot of FIGURE 10c, where it may be observed that the y-Fe203 NPs have a size distribution of 5.5-28.0 nm, with an average diameter value of (14.0 ⁇ 7.5) nm.
  • OSC as a contrast agent [Scanning Electron Microscopy and X-Ray Microanalysis, Klumer Academic/Plenum, 2003] followed by staining the polymer, makes it possible to observe the presence of a pattern of repeating dark and light regions.
  • the dark regions appear to have one or more y-Fe203 NPs, with the lighter regions corresponding to the Pani.
  • an Energy Dispersive Spectroscopy EDS analysis confirms the presence of Fe and O in a sample.
  • the micrographs reveal not only that the Pani chains envelope the y- Fe20 3 NPs but also that the size of the Pani/y-Fe203 MNC is in the nanoscale.
  • the composite diameters are represented in the histogram plot of FIGURE 10d, where it may be observed that the Pani/y-Fe203 MNCs have diameters ranging from 50 nm to 142 nm with an average diameter value of (87 ⁇ 32) nm, as determined from DLS measurements.
  • FIGURE 12 a Fourier Transform Infrared Spectroscopy (FTIR) analysis for use as an auxiliary technique for determining the composition of the y-Fe203 NPs and Pani/y-Fe203 MNC samples is presented in FIGURE 12. It may be observed that the Pani/y-Fe20 3 MNC spectrum (curve a) exhibits the same characteristic peaks observed in pure spectra of Pani samples (curve b) and in the -Fe203 (curve c). This was taken as evidence of the presence of the two species (iron oxide and Pani) in the MNC, as follows: (i) the peak at 3420 cm "1 correspond to the NAH stretching vibration of PANI [J. Magn. Magn. Mater.
  • FTIR Fourier Transform Infrared Spectroscopy
  • the present disclosure further relates to a method for retrieval of a nucleotide sequence such as RNA or DNA with the magnetic nanocomposite.
  • the process includes, mixing iron chloride tetrahydrate with iron (III) chloride hexahydrate in solution; adding ammonium hydroxide (NH OH) to the mixture and stirring to form y-Fe203 nanoparticles; stirring the y-Fe20 3 nanoparticles in a solution with an inorganic acid, a surfactant and a monomer precursor of a conducting polymer; initiating polymerization of the monomer by adding the inorganic acid and an oxidizing agent to the stirred solution and further stirring to yield PANI/y-Fe203 nanocomposites; adding the nanocomposites to an first aqueous solution of the nucleotide sequence and stirring so as to electrostatically interact- the— nanocomposites with the nucleotide sequence; and weakening the electrostatic interaction between the nanocomposite and the nucleotide sequence to
  • iron chloride tetrahydrate is mixed with iron chloride hexahydrate in a molar ratio of 1 :2.
  • the process includes doping the nanocomposites with one or more acids.
  • stirring the Y-Fe20 3 nanoparticles in a solution with hydrochloric acid, sodium dodecyl sulfate and a monomer further includes stirring the Y-Fe20 3 nanoparticles in a solution with hydrochloric acid, sodium dodecyl sulfate and aniline.
  • adding the inorganic acid and an oxidizing agent to the stirred solution further includes adding ammonium persulfate to the stirred solution.
  • stirring the y-Fe203 nanoparticles in a solution with an inorganic acid, a surfactant and a monomer precursor of a conducting polymer further includes stirring in a solution with sodium dodecyl sulfate.
  • stirring the y-Fe 2 03 nanoparticles in a solution with an inorganic acid comprises stirring with hydrochloric acid.
  • the nanocomposite is washed and acid doped then added to a second aqueous solution of nucleotide sequence and stirred so as to electrostatically interact the nanocomposite with the nucleotide sequence of the second solution.
  • electrostatically interacting the nanocomposites with the nucleotide sequence further comprises electrostatically interacting with DNA or RNA.
  • weakening the electrostatic interaction between the nanocomposite and the nucleotide sequence to recover the nanocomposite independently of the nucleotide sequence comprises weakening with a solution of an alkali salt. Further, weakening with a solution of an alkali salt comprises weakening with a solution of sodium hydroxide.
  • 10 imL of a 50 mg/L sperm salmon DNA solution in a glass flask may be agitated using an orbital shaker operating at 230 rpm to achieve a good interaction between the MNC and DNA.
  • the DNA concentrations may be determined by measuring absorbance at 260 nm.
  • MNC 1 mg, 2 mg, 3 mg and 4 mg
  • 10 mL of a 50 mg/L SS-DNA Different doses of MNC (1 mg, 2 mg, 3 mg and 4 mg) in 10 mL of a 50 mg/L SS-DNA, emphasize the capacity of the Pani/v-Fe203 MNC for DNA retrieval as a function of the interaction time (5, 10, 30, 60, 120, 180 and 240 min).
  • the adsorption capacity of the MNC as a function of the concentration (5, 7.5, 10, 12.5, 15, 20, 25, 30, 40 and 50 mg/L) of SS-DNA in the solution may be evaluated.
  • Co and Ce are the initial and final DNA concentration (mg/L) in the solution, respectively.
  • adsorption is a consequence of minimization of the surface energy of the particles.
  • the exact nature of the DNA/MNP bonding depends on the details of the species involved.
  • adsorption isotherms may be constructed to fit experimental data to the Langmuir and Freundlich models [Synth. Met. 160 (2010) 762-767; and , J. Ind. Eng. Chem. 18 (2012) 948-956.].
  • the adsorption capacity of the MNC may be calculated as
  • q e is the amount of DNA adsorbed per MNC mass unit (mg/g)
  • V is the volume of the solution
  • Co is the initial (mg/L) concentration of the DNA solution
  • C e is the equilibrium concentration of DNA solution (mg/L)
  • m is the mass (in g) of the MNC used.
  • the MNCs prepared in accordance with the present methods exhibit a high DNA adsorption capacity.
  • Introducing different amounts (1 , 2, 3, and 4 mg) of Pani/y- Fe20 3 MNC in a flask containing 10 mL of 50 mg/L solution of single stranded (SS)- DNA and thereafter adjusting the pH to 3.8 results in different rates of electrostatic interaction between the Pani chains and the double-stranded SS-DNA.
  • the DNA-loaded MNC was magnetically decanted and the 260 nm absorbance of the now DNA-depleted solution was measured at varying exposure times to establish fractional adsorption as a function of interaction time.
  • FIGURE 14 A plot of the relations for the four different amounts of Pani/ y-Fe203 is represented by FIGURE 14. It may be observed that with increasing interaction time, the DNA adsorption increases until reaching a maximum when the MNC becomes completely saturated with DNA, i.e. the system reaches an equilibrium where the MNC is not able to adsorb additional DNA from the solution. When 1 mg of the Pani/y-Fe203 MNC is added (curve a), the removal process takes longer, since a time of 80 min is required for the MNC to reach its maximum adsorption (22%).
  • Adsorption of DNA onto the nanocomposite is related to the concentration of DNA solution.
  • 4 mg of Pani/y-Fe203 MNC may be used to determine the effect of DNA concentration upon % adsorption,
  • a fixed amount of 4 mg of the MNC may be added to various RNA solutions or various solutions of SS-DNA having varying concentrations in the 5-50 mg/L range and the corresponding degree of DNA captured.
  • the data indicate that full retrieval was achieved for cases in which DNA solution concentrations are equal to or less than 25 mg/L.
  • concentrations are equal to or less than 25 mg/L.
  • concentrations are equal to 30 mg/L, 40 mg/L and 50 mg/L, nominal capabilities of 82%, 68%, 64% may be observed, indicating that the saturation limit of the MNC may have been reached before removal of all DNA chains present in the solution was possible.
  • the protonated Pani chains have several positively charged active sites that can interact with the laterally positioned negative phosphate groups in the DNA double-chains. After the addition of NaOH, however, these sites become deprotonated, leading to a drastic reduction in the number of active sites in the MNC still able to continue to interact with the DNA molecules. As a whole, the desorption process proved to be simple and fast, taking about 2 min for all DNA to become desorbed from the Pani/ -Fe203 MNC.
  • Isotherm adsorption describes the amount of DNA adsorbed at the MNC surface as a function of the DNA present in the solution. Collected data for the SS- DNA adsorption isotherms on Pani/v-Fe203 MNC may be fitted to both the Langmuir and Freundlich isotherm models.
  • the Langmuir isotherm model valid for a monolayer adsorption of a species onto a surface containing a finite number of identical adsorption sites, may be expressed in a linear form as:
  • q e is the amount (in mg) of DNA adsorbed per mass unit (g) of MNC
  • Ce is the final concentration of DNA (mg/L) in the solution after that the MNC was completely saturated (i.e. its maximum adsorption capacity was reached)
  • b and q m are constants.
  • b given in L/mg
  • q m given in mg/g
  • a plot of Ce/qe vs. Ce would give a straight line that intercepts the C e /qe- axis in 1/(bq m ) and has a slope of 1/q m .
  • the Freundlich isotherm model which assumes that the adsorbent consists of a heterogeneous surface composed of different classes of adsorption sites can be expressed in its linearized logarithmic form as:
  • the data indicate that the adsorption of SS- DNA adsorption onto the Pani/v-Fe203 MNC may be better described by the Langmuir isotherm model, which estimates the maximum adsorption capacity q m as 75.2 mg DNA/g MNC.
  • MNC cost-effectiveness of MNC as presently disclosed is additionally improved by its ability to be desorbed and reused successfully after at least three regeneration steps without important losses in its adsorption capacity. This is described in detail in the following examples.
  • Pani/Y-Fe203 MNC is reusable in a series of nucleotide sequence retrievals.
  • an amount of the disclosed MNC used in a previous desorption process and without DNA may be collected, washed with deionized water and HCI, and then added to 10 mL of a fresh 50 mg/L solution of SS-DNA.
  • this process is repeated three times and the q m for each cycle of adsorption-desorption determined, where q m was the maximum adsorption capacity, the value of q m observed in the second cycle is 96.5% of the total value obtained in the first cycle and in the third cycle is 90%.
  • Pani/y-Fe20 3 MNC may be reused successfully after at least three regeneration steps, without significant loss in its adsorption capacity.
  • enriching the DNA content in an originally very dilute DNA solution may be achieved, by following several cycles of capture, magnetic separation and subsequent release of DNA in a smaller recipient solution.
  • MNC/DNA zeta potential ( ⁇ ) measurements were performed for the MNC dispersed in water, the pure DNA solution and the DNA solution after the interaction with the MNC. In the case of MNC dissolved in water, a ⁇ value of 4.2 mV (pH 3.8) was obtained, while the much more stable DNA solution had a ⁇ value of 54 mV (pH 6).
  • the ⁇ value of the DNA solution decreased to 3 mV (pH 3.8) after the interaction had taken place and the magnetic decantation of the MNC had been implemented.
  • This reduction in the ⁇ value implies that when the DNA chains were captured, there was a decrease in the total number of negative charges per dissolved particle. This is entirely consistent with the idea that the interaction mechanism corresponds to the electrostatic attraction between the positive charges present in the Pani chains of the MNC and the negatively charged phosphate groups of the DNA molecules.
  • the present disclosure also relates to a biological diagnosis kit for rapid patient diagnosis employing the DNA-bonding nanocomposite for DNA retrieval within the kit.
  • the kit includes at least one composite, at least one short nucleotide sequence, and an appropriate substrate for the immobilization of the short nucleotide sequence such as RNA or a single-stranded DNA as well as a genetic sample of the patient.
  • the composite includes at least one magnetic nanoparticle such as maghemite and at least one conducting polymer such as PANI.
  • the at least one composite include a fluorescent composite.
  • a substrate in the form of a glass slide, paper and/or a polymer strip may also be provided.
  • the oxidizing agents iron chloride tetrahydrate and iron (III) chloride hexahydrate contribute oxidation to the DNA-bonding nanocomposite.
  • the DNA bonding nanocomposite bonds with the DNA.
  • the DNA content in an originally very dilute DNA solution can be enriched, by following several cycles of capture, magnetic separation and posterior release of DNA in a smaller recipient. It is to be understood that, the procedure of molecular diagnosis may be sharpened with a thus enriched DNA solution.
  • a fluorescent composite may facilitate the biological analysis.
  • the presently disclosed MNC has an estimated adsorption capacity significantly higher than many other adsorbents.
  • Table 2 of FIGURE 19 presents data related to adsorption characteristics of the presently disclosed MNC relative to other known adsorbents. While magnetic mesoporous silica has been reported as having a q m value as high as 121.6 mg/g, this maximum amount of DNA adsorption did not occur until 1200 min. In contrast, the MNC of the present disclosure required just 10 minutes to reach maximum DNA adsorption. It should be noted that a smaller particle size resulting in a larger surface area available for adsorption may offer significant contribution to reaching a high maximum adsorption capacity in a short amount of time. Thus, in an embodiment, the disclosed MNC may include individual particles with sizes within the nano-scale range. For example, the MNC may have an average diameter value of approximately 14nm.

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Abstract

Cette invention concerne un procédé de récupération d'une séquence de nucléotides. Le procédé comprend les étapes de mélange d'un tétrahydrate de chlorure de fer avec un hexahydrate de chlorure de fer (III) en solution ; ajout d'hydroxyde d'ammonium au mélange et agitation pour former des nanoparticules de maghémite ; agitation des nanoparticules de maghémite en solution avec un acide inorganique, un tensioactif et un précurseur monomère de polymère conducteur ; initiation de la polymérisation du monomère par ajout de l'acide inorganique et d'un agent oxydant à la solution sous agitation et poursuite de l'agitation pour obtenir des nanocomposites de polyaniline/maghémite ; ajout des nanocomposites à une première solution aqueuse de la séquence de nucléotides et agitation pour provoquer une interaction électrostatique des nanocomposites avec la séquence de nucléotides ; et affaiblissement de l'interaction électrostatique entre le nanocomposite et la séquence de nucléotides pour récupérer le nanocomposite indépendamment de la séquence de nucléotides.
PCT/IB2016/000011 2015-01-12 2016-01-12 Récupération d'une séquence de nucléotides à l'aide de nanocomposites magnétiques WO2016113622A1 (fr)

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