WO2016205971A1 - Méthode ultrasensible de détection de biomarqueur du cancer de l'estomac - Google Patents

Méthode ultrasensible de détection de biomarqueur du cancer de l'estomac Download PDF

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WO2016205971A1
WO2016205971A1 PCT/CL2016/050035 CL2016050035W WO2016205971A1 WO 2016205971 A1 WO2016205971 A1 WO 2016205971A1 CL 2016050035 W CL2016050035 W CL 2016050035W WO 2016205971 A1 WO2016205971 A1 WO 2016205971A1
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nanoparticles
methylated
rprm
dna
raman
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Alejandro CORVALÁN
Marcelo Kogan
Leda GUZMÁN
Ariel GUERRERO
María José MARCHANT
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Pontificia Universidad Catolica De Chile
Pontificia Universidad Católica De Valparaíso
Universidad De Chile
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated

Definitions

  • Gastric cancer is the fifth most common cancer and the third one related to cause of death in the world.
  • the prognosis is negative since it is frequently detected in invasive stages.
  • the survival rate is 95% at 5 years.
  • the survival of patients drops substantially to 10 to 20% at 5 years.
  • gastric atrophy precursor lesion of gastric cancer
  • pepsinogen l / ll the marker of gastric atrophy
  • gastric atrophy has a risk of developing gastric cancer between 5 to 10%, which implies that these patients will require special surveillance with invasive methods such as radiology and endoscopy.
  • gastric atrophy in addition to being the precursor lesion of gastric cancer is an injury associated with aging. Therefore, its predictive value is lost as the age of the population under evaluation increases.
  • the Reprimo gene (official RPRM symbol) is located on chromosome 2q23.3. Repress is induced after X-ray irradiation in a p53-dependent manner, causing cell cycle arrest in G2 phase (1). Additionally, the ectopic expression of RPRM also induces G2 arrest, where an inhibition of both cdc2 activity and cyclin -Bl nuclear translocation has been observed. These antecedents suggest the participation of Reprimo in the regulation pathway of the cdc2 / cyclin-Bl complex. DNA-level analyzes indicate that RPRM is contained in a single 393 bp exon, which codes for a 109 amino acid protein. The region i Reprimo promoter contains 56 CpG dinucleotides around the transcription start site (STT).
  • the invention now provides a method of detecting this ultrasensitive and specific biomarker, which can be applied in blood tests of the patient.
  • the invention is based on the specificity of oligonucleotides that detect the promoter region of the methylated RPRM gene, combined with the sensitivity of the use of nanoparticles, which comprise these oligonucleotides.
  • the sensitivity of the method can be increased by the optional use of fluorescence or Raman markers in the nanoparticles.
  • the properties of the nanoparticles allow amplifying the signals of this type of markers, which further increases the sensitivity of the method.
  • WO 211035453 (Corvalán, A. 31.03.2011), of one of the inventors of the present invention, the utility of evaluating the methylation of the promoter region of the RPRM gene as a gastric cancer biomarker is established and a reaction is proposed PCR as a detection method.
  • the present invention is a continuation of this work, where splitters other than oligonucleotides used in WO 211035453 are used and a new ultrasensitive method is provided to detect said marker. Subsequently, the inventors developed a method of early detection of gastric cancer disclosed in WO 2015/181804 (Corvalán A.
  • 24.06.2014 discloses a method based on nanostructures attached to a metal surface and an anchor probe. Where 2 specific probes are designed for a particular white DNA, one attached to a fluorescent marker and the other attached to a molecule that binds to said first anchor probe that is attached to the metal structure, thus markings Fluorescents would be attached to the metal surface in the presence of white nucleic acid.
  • the method of the invention is simpler than that of US Patent 8,759,110 B2, since nanoparticle hybrids are formed in solution and separated by simple centrifugation; Additionally, the method of the invention can be carried out with or without fluorescent probes, since hybrids of nanoparticles and methylated RPRM DNA present in the sample can be detected even by molecular absorption: UV-Visible spectroscopy.
  • the inventors have designed an ultrasensitive and selective method of detecting the promoter region of the methylated RPRM gene, mediating the use of functionalized metal nanoparticles with specific recognition oligonucleotides for the promoter region of the methylated RPRM gene .
  • This system designed in this way, is what makes it possible to improve the sensitivity and selectivity of current techniques, in order to carry out an early stage diagnosis of Gastric Cancer (GC).
  • GC Gastric Cancer
  • the method of the invention comprises using two nanoparticles functionalized with each of the oligonucleotides of the invention, a metal nanoparticle of between 40 and 80 nm is required that contains on its surface several copies of a primer that recognizes methylated RPRM, chosen from the SEQ ID N ⁇ l and SEQ ID N ⁇ 2 and a second metal nanoparticle between 8 to 20 nm (or a quantum point between 2 and 10 nm) that contains on its surface several copies of a primer that recognizes methylated RPRM chosen from the SEQ ID N ⁇ l and SEQ ID N ⁇ 2, different from that included in the first nanoparticle.
  • This oligonucleotide-coupled nanoparticle system recognizes the methylated RPRM sequence, forming a hybrid with both nanoparticles, as outlined in Figure 1.
  • the nanoparticles can be coupled to a Raman spectroscopy marker, fluorescence marker, or quantum dots.
  • the marker used is fluorescent, it allows a plasmonic effect known as fluorescence amplified by nanoparticles (surface-enhanced fluorescence, SEF) to occur, giving greater sensitivity to the method.
  • Figure 1 Scheme of the method of the invention, showing each of the oligonucleotides of the invention attached to each nanoparticle. Said nanoparticles in the presence of methylated RPRM, form a hybrid for base complementarity.
  • the oligonucleotides have a fluorescent probe attached (- * ⁇ )
  • the large nanoparticle can be gold, silver or copper
  • the small nanoparticle can be gold, silver, copper or quantum dots or quantum dots of cadmium-tellurium or Cadmium Selenium
  • Figure 2 Scheme describing the SEF effect.
  • a fluorophore located on the surface of the nanoparticle undergoes fluorescence quenching, which is much greater than the amplification of the fluorescence provided by the electric field of the nanoparticle at that point. Both switching off and amplification decrease with distance; however, as the distance increases, the quenching decreases much faster than the amplification, reaching a point where the quenching is minimal and the molecule undergoes effective amplification of the fluorescence.
  • FIG. 1 Transmission electron microscopy (TEM) images of the 12 and 55 nm gold nanoparticle (AuNPs) hybridization reaction with different samples.
  • FIG. 6 UV-Vis spectra of the hybridization reaction between AuNPs-DNA.
  • A Hybridization reaction. In the presence of DNA there is a greater absorption at 533 nm with respect to the reaction without DNA. This increase to 533 nm accounts for the formation of a hybrid with the specific sequence for Reprimo. A change can also be observed in the area near the UV (350 nm). This increase is attributable to the presence of DNA and the formation of hybrids, which are observed by TEM.
  • B UV-Vis spectrum of control reaction supernatants.
  • C UV-Vis spectrum of the reaction supernatants with Reprimo DNA.
  • Figure 7 Graph showing an increase in absorption at 533 nm when a hybrid is formed with the nanoparticles of the invention with different concentrations of methylated RPRM tempered DNA expressed as nanograms of DNA, where concentrations of the order of 0.001 ng of Methylated RPRM DNA is detected by the method of the invention, giving absorbances on the negative control without DNA, or with a DNA that does not contain the sequence for repression. DETAILED DESCRIPTION OF THE INVENTION.
  • the present invention describes an ultrasensitive and selective method of detecting the promoter region of the methylated Reprimo gene (RPRM), mediating the use of functionalized metal nanoparticles with specific recognition oligonucleotides for said promoter region of the methylated Repress gene, hereinafter simply Repressed methylated or methylated RPRM, which is a biomarker of gastric cancer.
  • RPRM methylated Reprimo gene
  • the ultrasensitive detection of these modified sequences in the blood of a patient allows an early detection of Gastric Cancer through a non-invasive method that allows to investigate patients with gastric cancer in very early stages of the disease, even asymptomatic.
  • the invention discloses a new ultrasensitive method of detecting the promoter region of the RPRM gene methylated in a DNA sample, which comprises the following steps: a) treating the DNA sample with sodium bisulfite so that unmethylated cytosines become in uracil, while methylated cytosines remain intact;
  • b) provide a metal nanoparticle between 40 and 80 nm, which comprises on its surface several copies of an oligonucleotide that recognizes methylated RPRM chosen between SEQ ID N ⁇ 1 and SEQ ID N ⁇ 2 and optionally a fluorescence or Raman marker;
  • a second metal nanoparticle between 8 to 20 nm or is a quantum point of cadmium-tellurium or cadmium-selenium of 2 to 10 nm, which comprises on its surface several copies of an oligonucleotide that recognizes methylated RPRM chosen from the SEQ ID N ⁇ l and SEQ ID N ⁇ 2, other than that included in the first nanoparticle and optionally a fluorescence or Raman marker;
  • oligonucleotide sequences are as follows:
  • the metal nanoparticles of both sizes used in the invention can be gold, silver or copper. If necessary, the small nanoparticle can be replaced by a quantum dot or quantum dot of cadmium-tellurium or cadmium-selenium from 2 to 10 nm.
  • Both nanoparticles are coated by silica; organic polymers including (but not limited to): polyethylene glycol (ethylene polyoxide), polylactoglyconic acid (PLGA), polyvinyl alcohol, polystyrene sulfonate, polymethacrylate, polymethylmethacrylate, polycaprolactone, poloxamers; natural and synthetic polysaccharides including: chitosan and derivatives, alginates, ursolic acid, etc; polypeptide chains including (but not limited to): peptides and proteins; aptamers (long chains of nucleic acids), hydrocarbons including (but not limited to): alkanothiols, functionalized alkanothiols and combinations of any of the foregoing.
  • organic polymers including (but not limited to): polyethylene glycol (ethylene polyoxide), polylactoglyconic acid (PLGA), polyvinyl alcohol, polystyrene sulfonate, polymethacrylate, polymethylmethacryl
  • the nanoparticles may be coupled to marker compounds, which may be attached to the coating or oligonucleotides of the invention.
  • markers can be fluorescent probes or Raman reporters.
  • the hybrid complexes can be detected by molecular absorption, or if it uses fluorescent probes or if the small nanoparticle is a quantum dot or quantum dot of Cadmium Telurium the hybrid complexes can be detected by fluorescence spectroscopy; or by Raman spectroscopy in the case of having these devices.
  • fluorescent markers that can be used in the method of the invention are: Rhodamine and derivatives, fluorescein and derivatives, cyanine derivatives, anthracenes, pyrenes and other fluorescent dyes.
  • Raman markers that can be used in the method of the invention are: violet crystal, methylene blue, p-mercaptobenzoic acid, 5,5'-dithiobis (succinimidyl-2-nitrobenzoate) (DSNB), rhodamine and derivatives , and other dyes.
  • the conditions under which DNA hybridization of the samples to be evaluated with the method of the invention is carried out and the nanoparticles functionalized with the oligonucleotides of the invention first include a suitable buffer, such as for example PBS buffer IX. pH 7.4, but the nature of the buffer is not critical and any other suitable buffer available in the art can be used.
  • a suitable buffer such as for example PBS buffer IX. pH 7.4, but the nature of the buffer is not critical and any other suitable buffer available in the art can be used.
  • the mixture of the sample and the functionalized nanoparticles of the invention is incubated at 95 ° C for 5 to 10 minutes, and then allowed to stir at 37 ° C at 300 rpm for at least 2 hours, and preferably overnight.
  • hybrids formed in the presence of methylated RPRM are centrifuged at speeds between 2500 to 10500 g for 5 to 20 minutes, where the hybrid is in the sediment, which is resuspended in buffer, for example PBS IX pH 7.4.
  • the sensitivity of the method can be favored by the characteristics of the nanoparticles used, which amplify the detection signals.
  • Nanotechnology in biomedicine has had an important development since the beginning of the 21st century since it has provided new tools for the therapy and diagnosis of various diseases such as cancer (3) or Alzheimer's (4).
  • AuNP gold
  • AgNP silver
  • CuNP copper
  • LSPR local surface plasmon resonance
  • This term is defined as the collective oscillation of the electrons on the surface of the nanoparticle, and is the origin of the particular optical properties of metals such as gold, silver and copper, which in the nanometric scale absorb light causing these solutions acquire very striking colorations (eg AuNP of 20 nm are deep red).
  • metals such as gold, silver and copper
  • they have the property of scattering light (scattering) in all directions, at wavelengths close to which these particles absorb, which can be used in diagnosis for the detection of tumor cells.
  • nanoparticles in particular those of gold, silver and copper, since it allows the functionalization with biomolecules that contain thiol groups through a process of chemisorption forming stable Au-S bonds , Ag-S and Cu-S, respectively.
  • the nanoparticles can be coated with biomolecules.
  • EAP plasmon-amplified spectroscopy
  • SERS surface-enhanced Raman spectroscopy
  • Another useful concept for this description is that of plasmon coupling that modifies the effective cross-section of the particles.
  • two plasmonic particles approach the combined effect of both is observed and not that of each separated.
  • a large gold nanoparticle is coupled with several small ones, they absorb as a single large particle, increasing the effective absorption section.
  • EAP techniques are used for the detection of cancer-associated biomarkers and also of tumor cells, using as functionalized AuNP, AgNP or CuNP probes.
  • an ultrasensitive methodology has been developed for the detection of biomarkers, specifically methylated RPRM.
  • EAP Plasmon amplified spectroscopy
  • EAP plasmon-amplified spectroscopy
  • SERS surface-enhanced Raman scattering
  • SEF surface-enhanced fluorescence spectroscopy
  • SEF refers to amplified fluorescence (surface-enhanced fluorescence, also called metal-enhanced fluorescence, or more recently plasmon-enhanced fluorescence, MEF and PEF respectively).
  • SERS and SEF depend on the distance between the target molecule and the nanoparticle. In this regard, it should be noted that such distances have optimal values for amplification. In the case of the SERS, said distance must be the smallest possible with respect to the surface of the nanoparticle being the same independent of its size, while for the SEF said distance is dependent on the size of the nanoparticle, see Figure 2.
  • metal nanoparticles with plasmon must be used. This is related to the power of nanoparticles to absorb and scatter light from light at certain wavelengths. These processes reach their maximum at the wavelengths in which the so-called resonance condition occurs, which is related to the optical properties of the material with which the nanoparticle is made in accordance with the dielectric function £ (complex type) of the material from which the particle is made:
  • the real part of the dielectric function of the material from which the nanoparticles are made must be close to -2 and the imaginary part must be close to zero, for the incident light of a certain wavelength (expressed as angular frequency ⁇ in the formula above).
  • Silver fulfills this condition in the visible very well, and gold and copper to a lesser extent (its imaginary part is more significant), and for this reason they are the most used metals for EAP.
  • Alkali metals also fulfill this property, but given their chemical reactivity they are not practical for use. What interests us most about nanoparticles, with a view to amplification of fluorescence, absorbance and Raman signals, is their power to scatter light (scattering).
  • the nanoparticles must exceed 40 nm in diameter for a spherical particle, but remain below 100 nm since the contributions of the higher order poles (quadrupoles, octapoles, etc., beyond 100 nm). ) begins to be significant, and this reduces the power of plasmon that is dipole in character, turning it off and lowering the amplification values.
  • Stamplecoskie and Scaiano have experimentally determined an optimal size for silver nanospheres between 50-60 nm towards SERS, and Hong and Li (12) have obtained very similar values for gold nanospheres. According to the above, silver provides better SERS and SEF amplification values than gold, for particles of equal size, but also causes greater reactivity on the surface of the nanoparticle, and that is why gold nanoparticles are often preferred .
  • quantum dots or quantum dots which are nanoparticles made of semiconductor materials such as cadmium, tellurium and selenium. These materials, being of very small sizes, have the effect of quantum confinement, which makes them fluorescent such as traditional fluorophores molecules, but they are not molecules but nanoparticles, functionalizable and large surface area, which makes them particularly useful for fluorescence detection.
  • the inventors have determined that a large nanoparticle of gold, silver or copper between 40 and 80 nm (in diameter), which provides plasmonic amplification, and a small nanoparticle of gold, silver or copper between 8 and 20 nm, or a quantum dot or quantum dot of cadmium-tellurium or cadmium-selenium between 2 and 10 nm are the most appropriate for carrying out the invention.
  • the nanoparticles used in the method of the invention are of vital importance in terms of the sensitivity of the method, another critical aspect is the specificity thereof, so that the results obtained are of diagnostic utility.
  • the specificity of the method is given by the oligonucleotides used, which allow detecting DNA sequences from the promoter region of the methylated RPRM gene, which are biomarkers of gastric cancer.
  • a previous treatment of the total DNA to be analyzed with sodium bisulfite is required. DNA reacts with this reagent and unmethylated cytosines become uracil, while methylated cytosines remain intact.
  • Example 1 Obtaining Nanoparticles of the invention.
  • concentration of added sodium citrate determines the size of the nanoparticles.
  • concentration of added sodium citrate determines the size of the nanoparticles.
  • 500 ⁇ of 29.4 mM aqueous HAuCU solution and 500 ⁇ of 38.8 mM trisodium citrate aqueous solution were used.
  • 1,709 mL of 29.4 mM aqueous HAuCU solution and 5 mL of 38.8 mM trisodium citrate aqueous solution were used.
  • Oligonucleotides to detect methylated RPRM 2 oligonucleotides were used that are at a distance of approximately 100 base pairs in the promoter region of the Reprimo gene. They were sent to Synthesize Integrated ADN Technologies, Inc., with a modification of an amino group at one end of the oligonucleotide. Once received, they were prepared at a final concentration of 100 ⁇ in TE buffer pH 8.0.
  • the oligonucleotide sequences are as follows:
  • oligonucleotide coupling reaction was carried out by reaction with EDC (l-ethyl-3- (3-dimethylaminopropyl) carbodiimide).
  • EDC l-ethyl-3- (3-dimethylaminopropyl) carbodiimide
  • 1 ⁇ of oligonucleotide was diluted in 2.4 mL of PBS IX.
  • 2.5 mL of peeled nanoparticles previously washed with PBS IX
  • 0.8 mg of EDC dissolved in 100 ⁇ of PBS IX was added.
  • the reaction was left for 3 hours at 25 ⁇ C at 300 rpm of agitation.
  • nanoparticles of the invention were washed 3 times with PBS IX pH 7.4, and stored at 4 ⁇ C.
  • the hybridization assay is performed by incubating nanoparticles of the invention, obtained in example 1, ie AuNPs of 12 and 55 nm functionalized with the oligonucleotides of the invention; in the presence of 100 and 50 ng DNA containing the promoter region of methylated RPRM, in PBS buffer IX pH 7.4. The mixture is incubated at 95 ° C for 5 minutes, and then left under stirring at 300 rpm at 37 ° C overnight to favor the formation of hybrids. The samples are washed with PBS IX and subjected to centrifugations at 2200 g for 10 minutes, and the supernatants are separated from the sediment.
  • Hybrids due to their higher molecular weight are expected to be in the sediment, while unreacted nanoparticles remain in the supernatant.
  • Each sample - supernatant and sediment - is made UV-Vis spectra, and analyzed by TEM.
  • Figure 6 shows these results
  • Figure 6 A shows the absorption resulting from the Hybridization Reaction of the AuNPs nanoparticles obtained in example 1 and white DNA, that is, from the promoter region of the methylated RPRM gene, where it is observed that in presence of DNA there is a greater absorption at 533 nm with respect to the reaction without DNA.
  • This increase to 533 nm accounts for the formation of a hybrid with the specific sequence for Reprimo.
  • a change can also be observed in the area near UV (350 nm). This increase is attributable to the presence of DNA and the formation of hybrids, which are observed by TEM.
  • Figure 6 B shows the UV-Vis spectrum of the control reaction supernatants.
  • Figure 6 C shows the UV-Vis spectrum of the supernatants of the reaction with Reprimo DNA. It is observed that there is an absorption close to 520 nm greater in graph B, compared to the graph in C. The 12 nm AuNPs when the hybrid does not form (because there is no DNA (control reaction)), are suspended, the which are separated by centrifugations. On the contrary, in the presence of a DNA containing the methylated region of Reprimo, hybridization occurs between both nanoparticles, forming a triad (Fig. 5 B and C).
  • oligonucleotide-coupled gold nanoparticles system allows us to specifically determine a methylated RPRM DNA sequence and form a hybrid, which can be detected through the use of molecular absorption spectroscopy.
  • the increase in absorption at 533 nm is mainly due to the coupling of plasmons described at the beginning of this invention.
  • the technique also allows the determination of concentrations of the order of 0.001 ng of methylated RPRM DNA.
  • This technique requires a bisulfite treated DNA sample and a detection system based on molecular absorption.
  • our system does not require amplifications of a sample by PCR, which decreases costs, obtaining better results, since the sensitivity of the method is higher.
  • Nanomaterials in combating cancer Therapeutic appl ications and develo pments.
  • Nanomedicine Nanotechnology, Biology and Medicine. 2014; 10 (1): 19-34.

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Abstract

L'invention concerne une méthode ultrasensible de détection de biomarqueur du cancer de l'estomac, le gène Reprimo (RPRM) méthylé dans un échantillon d'ADN. Cette méthode consiste à traiter l'échantillon d'ADN avec du bisulfite de sodium ; à utiliser une nanoparticule métallique de 40 à 80 nm, qui comprend dans sa surface un oligonucléotide qui reconnaît le gène RPRM méthylé sélectionné entre la SEQ ID Nº1 et la SEQ ID Nº2 et éventuellement un marqueur pour la spectroscopie de fluorescence, ou la spectroscopie Raman ; à utiliser une deuxième nanoparticule métallique de 8 à 20 nm ou un point quantique de 2 à 10 nm, qui comprend dans sa surface un oligonucléotide qui reconnaît le gène RPRM méthylé sélectionné entre la SEQ ID Nº1 et la SEQ ID Nº2, différent de celui présent dans la première nanoparticule et éventuellement un marqueur pour la spectroscopie de fluorescence, ou la spectroscopie Raman ; à permettre l'hybridation de l'échantillon et des deux nanoparticules, à centrifuger et détecter dans le sédiment remis en suspension la présence de complexes hybrides de nanoparticules et d'ADN. La détection peut être effectuée par absorption moléculaire ou éventuellement dans un cas donné, par spectroscopie de fluorescence, ou par spectroscopie Raman.
PCT/CL2016/050035 2015-06-26 2016-06-24 Méthode ultrasensible de détection de biomarqueur du cancer de l'estomac WO2016205971A1 (fr)

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CN108827935A (zh) * 2018-06-08 2018-11-16 南京师范大学 一种基于金纳米孔阵列的dna甲基化表面增强拉曼散射光谱检测方法及其应用
CN109929919A (zh) * 2018-09-14 2019-06-25 深圳市晋百慧生物有限公司 Dna甲基化检测方法及相关应用
RU2723160C1 (ru) * 2019-08-15 2020-06-09 Федеральное государственное бюджетное образовательное учреждение высшего образования "Московский государственный университет имени М.В. Ломоносова" (МГУ) Способ обнаружения и определения днк с заданной последовательностью методом спектроскопии гигантского комбинационного рассеяния
US11746387B2 (en) 2018-06-20 2023-09-05 Pontificia Universidad Catolica De Chile Non-invasive detection of gastric cancer by detecting the methylation of Reprimo-like in the blood

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