WO2008140620A2 - Détection de biomolécules ultrasensibles à l'aide de nanoparticules d'or cochargées par de l'adn double brin et de molécules de capture co-immobilisées - Google Patents

Détection de biomolécules ultrasensibles à l'aide de nanoparticules d'or cochargées par de l'adn double brin et de molécules de capture co-immobilisées Download PDF

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Publication number
WO2008140620A2
WO2008140620A2 PCT/US2007/088621 US2007088621W WO2008140620A2 WO 2008140620 A2 WO2008140620 A2 WO 2008140620A2 US 2007088621 W US2007088621 W US 2007088621W WO 2008140620 A2 WO2008140620 A2 WO 2008140620A2
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WIPO (PCT)
Prior art keywords
oligonucleotide
capture
substrate
probe
amplifier
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PCT/US2007/088621
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English (en)
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WO2008140620A3 (fr
Inventor
Martin Huber
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Nanosphere, Inc.
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Publication date
Application filed by Nanosphere, Inc. filed Critical Nanosphere, Inc.
Publication of WO2008140620A2 publication Critical patent/WO2008140620A2/fr
Publication of WO2008140620A3 publication Critical patent/WO2008140620A3/fr

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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
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6823—Release of bound markers

Definitions

  • the present invention relates to detection methods and devices for detecting for the presence or absence of one or more target analytes such as proteins, nucleic acids, or other compounds derived from biological samples.
  • the present invention relates to a method, and substrate and kit therefore, comprising capture probes and capture oligonucleotide co-immobilized on discrete spots on the substrate to achieve ultra-sensitive level of detection for target analytes such as proteins, DNA, peptides, and small molecules
  • Ultra sensitive biomolecule detection plays an important role in modern diagnostics. Detecting biomarkers at very low levels may allow for diagnosis of early onset of certain diseases which in turn will improve the outcome of medical treatment.
  • the present invention provides reliable diagnostic systems that are both simple to fabricate and able to detect targets with a high degree of sensitivity.
  • the described invention overcomes current low detection sensitivity by employing a double- stranded DNA functionalized gold nanoparticle probe co-loaded with Antibodies (Protein detection) or single-stranded oligonucleotides (DNA detection) as well as locally co-immobilized capture molecules for the target and the amplifier oligonucleotides.
  • Upon capturing a target biomolecule on a solid substrate the captured molecules are tagged with the co-loaded nanoparticles.
  • a method for detecting at least one target analyte with at least two binding sites in a sample comprises steps of (a) providing a substrate comprising at least one type of capture probe and at least one type of capture oligonucleotide attached thereto, wherein said capture probe binds to a first binding site of the target analyte, and wherein the capture probe and the capture oligonucleotide are co-immobilized on discrete spots on the substrate; (b) providing a detector probe comprising at least one type of amplifier oligonucleotide and at least one type of binding complement, wherein said binding complement binds to a second binding site of the target analyte, and wherein the amplifier oligonucleotide hybridizes to at least a portion of the capture oligonucleotide; (c) contacting the sample with the substrate and the detector probe under conditions that are effective for the capture probe, the detector probe, and the target analyte
  • the capture probe and the capture oligonucleotide are co-immobilized on the discrete spots on the substrate at a ratio of from about 1 : 100 to about 100:1 with a most preferable molar ratio of 1 : 1.
  • the method of claim 2 wherein the capture oligonucleotide is co-immobilized with the capture probe on the discrete spots on the substrate at a density of from about 1 ⁇ M to about 1 mM.
  • the method further comprises steps of (e) releasing the amplifier oligonucleotide from the complex; and (f) capturing the released amplifier oligonucleotide with the capture oligonucleotide on the substrate under conditions that are effective for the amplifier oligonucleotide to bind to at least a portion of the capture oligonucleotide.
  • the amplifier oligonucleotide is released by a chemical agent.
  • the detector probe further comprises a complementary oligonucleotide that hybridizes to at least a portion of the amplifier oligonucleotide to form a double-stranded oligonucleotide.
  • the method further comprising a step of releasing the amplifier oligonucleotide from the complex by disrupting the double-stranded oligonucleotide.
  • the oligonucleotide is released from the double-stranded oligonucleotide by heat denaturation.
  • the method further comprises a step of providing a detection probe, said detection probe comprising a detection oligonucleotide that hybridizes to at least a portion of the amplifier oligonucleotide.
  • the detection probe further comprises a particle, wherein the detection oligonucleotide is attached to the particle.
  • the particle could be a microparticle or a nanoparticle.
  • the particle is a gold nanoparticle, and the complex is detected by silver staining the substrate.
  • the amplifier oligonucleotide further comprises a reporter group.
  • the reporter group comprises a fluorophore, a chromophor, a redox-active group, a group with an electrical signature, radioactive group, a biotin group, a catalytic group, or Raman label.
  • the complex can then be detected by the detection of the reporter group.
  • the detector probe further comprises a particle, where the particle can be a microparticle or a nanoparticle. In one embodiment, the particle is a gold nanoparticle, and the complex can be detected by silver staining the substrate.
  • the capture probe and the detector probe comprises an antibody, an antibody fragment, an aptamer, a peptide, or a nucleic acid.
  • the substrate comprises at least two capture probes, each capture probe binding to the same or different target analytes.
  • a substrate having at least one type of capture probe and at least one type of capture oligonucleotide bound thereto is provided, where the capture oligonucleotide and the capture probe are co-immobilized on discrete spots on the substrate.
  • the capture oligonucleotide and the capture probe are co-immobilized on discrete spots on the substrate at a ratio of from about 1 :100 to about 100:1 with a most preferable molar ratio of 1 :1.
  • the capture oligonucleotide is co-immobilized with the capture probe on discrete spots on the substrate at a density of from about 1 ⁇ M to about 1 mM.
  • a kit for detecting a target analyte is provided.
  • the kit provides a substrate having at least one type of capture probe and at least one type of capture oligonucleotide bound thereto, wherein the capture oligonucleotide and the capture probe are co-immobilized on discrete spots on the substrate.
  • the substrate provided in the kit contains the capture oligonucleotide and the capture probe co -immobilized on discrete spots on the substrate at a ratio of from about 1 :100 to about 100:1 with a most preferable molar ratio of 1 :1.
  • the capture oligonucleotide is co-immobilized with the capture probe on discrete spots on the substrate at a density of from about 1 ⁇ M to about 1 mM.
  • the kit further comprises a detector probe, said detector probe comprising at least one amplifier oligonucleotide and at least one type of target analyte-binding molecule.
  • Figure 1 shows a schematic of one embodiment of the invention for protein detection.
  • Figure 2 shows a schematic of one embodiment of the invention for DNA detection.
  • Figure 3 shows a schematic of the method as described in the application
  • FIG 4 shows data for the ultrasensitive detection of total PSA (tPSA).
  • FIG. 5 shows data for the ultrasensitive detection of total PSA (tPSA) in comparison with the method described in the application USSN 11/506,280.
  • the present invention comprises methods of detection that have many advantages over the prior art.
  • the described format allows for ultra-sensitive detection of biomolecules utilizing Antibody/DNA co-loaded gold nanoparticle as well as co- immobilized capture molecules for target and amplifier oligonucleotides, respectively.
  • the target biomolecule is sandwiched between a capture molecule (e.g. Antibody, DNA oligonucleotide) and a detector (e.g. Antibody, DNA oligonucleotide) attached to a nanoparticle probe.
  • the nanoparticle is loaded with double stranded DNA fragments of which one strand is used as an amplifier oligonucleotide. After the sandwich is formed the amplifier oligonucleotide is released and captured on the solid surface.
  • the conversion of a captured protein into a detected DNA signal allows for detection of proteins directly from Serum, Plasma or whole blood.
  • the use of blood or blood components usually causes high unspecific background signal due to extremely high concentration of proteins, carbohydrates, lipids and other cellular components.
  • ionic detergents like SDS
  • DNA hybrids are not prone to denaturation by ionic detergents and can withstand high concentrations of SDS.
  • the herein presented approach is able to detect proteins directly in Serum which is an important aspect of a clinical test set-up.
  • an Antibody, or a Fab fragment, or an aptamer (DNA, RNA) together with a DNA Oligonucleotide complementary to the amplifier oligonucleotide is immobilized on a solid support.
  • a pair of Oligonucleotides is immobilized, one serving as capture oligonucleotide for nucleic acid detection, the other being the complement capture of the amplifier oligonucleotide. After capturing the corresponding target a co-loaded gold nanoparticle is reacted with the complex.
  • the co-loaded gold nanoparticle harbors multiple double-stranded (ds) amplifier DNA fragments as well as a secondary Antibody, or a secondary Fab fragment, or a secondary Aptamer (for Protein detection) or a target specific oligonucleotide (DNA detection).
  • ds double-stranded
  • a secondary Antibody or a secondary Fab fragment, or a secondary Aptamer (for Protein detection) or a target specific oligonucleotide (DNA detection).
  • a detector probe is hybridized to the amplifier oligonucleotide which is detected by applying an evanescent wave in the planar glass slide. This wave creates light scattering at the gold nanoparticle which is measured by a photosensor. Additional ways of detecting the amplifier oligonucleotide are via light reflection, fluorescence, radioactivity, quantum dots, chemiluminescence, mass spectrometry just to name a few.
  • the term “particle” refers to a small piece of matter that can preferably be composed of metals, silica, silicon-oxide, or polystyrene.
  • a “particle” can be any shape, such as spherical or rod-shaped.
  • the term “particle” as used herein specifically encompasses both nanoparticles and microparticles as defined and described hereinbelow.
  • Nanoparticles useful in the practice of the invention include metal (e.g., gold, silver, copper and platinum), semiconductor (e.g., CdSe, CdS, and CdS or CdSe coated with ZnS) and magnetic (e.g., ferromagnetite) colloidal materials.
  • Other nanoparticles useful in the practice of the invention include ZnS, ZnO, TiO 2 , AgI, AgBr, HgI 2 , PbS, PbSe, ZnTe, CdTe, In 2 S 3 , In 2 Se 3 , Cd 3 P 2 , Cd 3 As 2 , InAs, and GaAs.
  • the size of the nanoparticles is preferably from about 5 nm to about 150 nm (mean diameter), more preferably from about 5 to about 50 nm, most preferably from about 10 to about 30 nm.
  • the nanoparticles may also be rods.
  • Suitable nanoparticles are also commercially available from, e.g., Ted Pella, Inc. (gold), Amersham Corporation (gold), Nanoprobes, Inc. (gold), and Quantom Dot Inc. (core-shell semiconductor particles such as CdSe/ZnS).
  • a universal nanoparticle or universal nanoparticle probe is use that comprises a nanoparticle and a member of a specific binding pair attached to the nanoparticle.
  • the specific binding pair can be one or a plurality of ligands.
  • the ligands can be any molecule or biomolecule such as peptides, proteins or nucleic acids.
  • the ligand is a nucleic acid molecule that comprises complementary region to the mediator probes.
  • the a surface density of the attached ligand on the nanoparticle may range from between about 8.9 x 10 11 and about 6.4 x 10 12 molecules/cm 2 is useful. Nanoparticles of differing size and composition can be used in the invention to produce the universal nanoparticle probe.
  • nanoparticles include those described U.S. Patent No. 6,506,564; International Patent Application No. PCT/US02/16382; U.S. Patent Application Serial No. 10/431,341 filed May 7, 2003; and International Patent Application No. PCT/US03/14100; all of which are hereby incorporated by reference in their entirety.
  • the diameter of the nanoparticle ranges from 5-100 nm, preferably, the diameter is from 10-50 nm. In one preferred embodiment, the nanoparticle is 13 nm in diameter and made of gold.
  • a “biomolecule” refers to one or more molecules derived from a biological sample.
  • a “sample” refers to any quantity of a substance that comprises nucleic acids, proteins, lipids, carbohydrates, fats or any biological substance, and that can be used in a method of the invention.
  • the sample can be a biological sample or can be extracted from a biological sample derived from humans, animals, plants, fungi, yeast, bacteria, viruses, tissue cultures or viral cultures, or a combination of the above. They may contain or be extracted from solid tissues (e.g. bone marrow, lymph nodes, brain, skin), body fluids (e.g. serum, blood, urine, sputum, seminal or lymph fluids), skeletal tissues, or individual cells.
  • solid tissues e.g. bone marrow, lymph nodes, brain, skin
  • body fluids e.g. serum, blood, urine, sputum, seminal or lymph fluids
  • the sample can comprise purified or partially purified nucleic acid molecules and, for example, buffers and/or reagents that are used to generate appropriate conditions for successfully performing a method of the invention.
  • target refers to substances to be detected or assayed by the method of the invention.
  • Typical analytes may include, but are not limited to proteins, peptides, nucleic acid segments, molecules, cells, microorganisms and fragments and products thereof, or any substance for which attachment sites, binding members or receptors (such as antibodies) can be developed.
  • the analytes have at least one binding site, preferably at least two binding sites, e.g., epitopes, that can be targeted by a capture probe and a detection probe, e.g. antibodies or aptamers or both.
  • a "target,” “analyte” or “target analyte” is a compound or composition to be detected, including drugs, metabolites, pesticides, pollutants, and the like.
  • the analyte can be comprised of a member of a specific binding pair (sbp) and may be a ligand, which is monovalent (monoepitopic) or polyvalent (polyepitopic), preferably antigenic or haptenic, and is a single compound or plurality of compounds, which share at least one common epitopic or determinant site.
  • the analyte can be a part of a cell such as bacteria or a cell bearing a blood group antigen such as A, B, D, etc., or an HLA antigen or a microorganism, e.g., bacterium, fungus, protozoan, or virus.
  • the analyte can be further modified, e.g. chemically, to provide one or more additional binding sites.
  • the analyte has at least two binding sites.
  • the monoepitopic ligand analytes will generally be from about 100 to 2,000 molecular weight, more usually from 125 to 1,000 molecular weight.
  • the polyvalent ligand analytes will normally be larger organic compounds, often of polymeric nature, such as polypeptides and proteins, polysaccharides, nucleic acids, and combinations thereof. Such combinations include components of bacteria, viruses, chromosomes, genes, mitochondria, nuclei, cell membranes and the like.
  • the polyepitopic ligand analytes to which the subject invention can be applied will have a molecular weight of at least about 5,000, more usually at least about 10,000.
  • the polymers of interest will generally be from about 5,000 to 5,000,000 molecular weight, more usually from about 20,000 to 1,000,000 molecular weight; among the hormones of interest, the molecular weights will usually range from about 5,000 to 60,000 molecular weight.
  • proteins may be considered as belonging to the family of proteins having similar structural features, proteins having particular biological functions, proteins related to specific microorganisms, particularly disease causing microorganisms, etc.
  • proteins include, for example, immunoglobulins, cytokines, enzymes, hormones, cancer antigens, nutritional markers, tissue specific antigens, etc.
  • the types of proteins, blood clotting factors, protein hormones, antigenic polysaccharides, microorganisms and other pathogens of interest in the present invention are specifically disclosed in U.S. Pat. No. 4,650,770, the disclosure of which is incorporated by reference herein in its entirety.
  • the analyte may be a molecule found directly in a sample such as a body fluid from a host.
  • the sample can be examined directly or may be pretreated to render the analyte more readily detectible.
  • the analyte of interest may be determined by detecting an agent probative of the analyte of interest such as a specific binding pair member complementary to the analyte of interest, whose presence will be detected only when the analyte of interest is present in a sample.
  • the agent probative of the analyte becomes the analyte that is detected in an assay.
  • the body fluid can be, for example, urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, and the like.
  • a "nucleic acid sequence,” a “nucleic acid molecule,” or “nucleic acids” refers to one or more oligonucleotides or polynucleotides as defined herein.
  • polynucleotide as referred to herein means single-stranded or double-stranded nucleic acid polymers of at least 10 bases in length.
  • the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
  • Said modifications include base modifications such as bromouridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.
  • base modifications such as bromouridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.
  • polynucleotide specifically includes single and double stranded forms of DNA.
  • oligonucleotide includes naturally occurring, and modified nucleotides linked together by naturally occurring, and/or non-naturally occurring oligonucleotide linkages.
  • Oligonucleotides are a polynucleotide subset comprising members that are generally single-stranded and have a length of 200 bases or fewer. In certain embodiments, oligonucleotides are 10 to 60 bases in length. In certain embodiments, oligonucleotides are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides may be single stranded or double stranded, e.g. for use in the construction of a gene mutant. Oligonucleotides of the invention may be sense or antisense oligonucleotides with reference to a protein-coding sequence.
  • capture probe of "detector probe” used herein refers to a probe comprising a binding molecule that binds to the target analyte.
  • the binding molecule includes, but is not limited to, an antibody, an antibody fragment, an aptamer, a peptide, or a nucleic acid.
  • a “substrate” or “solid support” is any surface capable of having capture probes bound thereto. Such surfaces include, but are not limited to, glass, quartz, ceramic, metal, plastic, or materials coated with a functional group designed for binding of the probes.
  • Example 1 Shown in Figure 4 is an example for ultrasensitiv detection of total PSA
  • tPSA-prostate specific antigen with and without release of the amplifier oligonucleotides.
  • Various amounts of tPSA is captured by immobilized Antibodies (AB) on a microarray.
  • One part of the array contains immobilized ABs plus co- immobilized captures for the amplifier oligonucleotide (AB+Oligo) and as a control Antibody only spots (AB).
  • An additional control in the same experiment was the use of a single-stranded co-loaded Probe (ss coload) where no amplifier oligo is present and therefore no signal amplification possible. The actual signal amplification occurs when using a double-stranded co-loaded probe (ds coload) in combination with immobilized AB+Oligo.
  • Figure 4 summarizes the data of a signal amplification reaction (AB+ Oligo and ds coload).
  • the limit of detection (LOD) is between 500ag and 5fg tPSA in a 50 uL reaction whereas the LOD for a control reaction without release of the amplifier oligonucleotides is at 50 fg tPSA (in 5OuL).
  • Figure 5 shows the Net signal intensities of a target titration experiment.
  • a . dilution series of PSA was bound by a surface immobilized Antibody and subsequently reacted with a DNA/coloaded gold probe.
  • the control experiments were silver amplified at this point and the scatter signal intensities measured (light grey bar).
  • the scatter signal from these control experiments originated from the light scattering of the bound co-loaded probes.
  • No release of the amplifier oligonucleotides was performed during the control experiments.
  • the dark grey bars show the signal intensity after release of the amplifier oligonucleotide and hybridization to the corresponding capture oligonucleotide (physically separated in the approach as described in the application USSN 11/506,280 vs.
  • IxTBST • A second, static wash in IxTBST is followed by a IxPBS and Ix wash D wash. Spin dry and attach a new 1 Owe 11 Hybridization unit.
  • IxTBST • A second, static wash in IxTBST is followed by a IxPBS and Ix wash D wash. Spin dry and attach a new 1 Owe 11 Hybridization unit.
  • a second, static wash in wash A is followed by a wash B wash and a wash D. Spin dry.

Abstract

L'invention concerne un procédé de détection, un dispositif pour détecter la présence ou l'absence d'un ou plusieurs analytes cibles tels que des protéines, des acides nucléiques ou autres composés issus d'échantillons biologiques. Le procédé, le substrat et le coffret comprennent des sondes de capture et un oligonucléotide de capture co-immobilisé sur des points discrets sur le substrat pour obtenir un niveau ultrasensible de détection pour des analytes cibles tels que des protéines, de l'ADN, des peptides et des petites molécules.
PCT/US2007/088621 2006-12-21 2007-12-21 Détection de biomolécules ultrasensibles à l'aide de nanoparticules d'or cochargées par de l'adn double brin et de molécules de capture co-immobilisées WO2008140620A2 (fr)

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US87645306P 2006-12-21 2006-12-21
US60/876,453 2006-12-21

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Cited By (4)

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WO2011070333A3 (fr) * 2009-12-10 2011-09-01 Trillion Genomics Limited Sondes
CN108410952A (zh) * 2018-05-11 2018-08-17 重庆出入境检验检疫局检验检疫技术中心 单核细胞增生李斯特氏菌夹心dna杂交快速检测用探针、试剂盒和检测方法
CN111004836A (zh) * 2019-12-24 2020-04-14 山东大学 双向放大的比率型电化学适体传感器及其应用
WO2021007099A1 (fr) * 2019-07-05 2021-01-14 Ultivue, Inc. Procédé de multiplexage amélioré

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011070333A3 (fr) * 2009-12-10 2011-09-01 Trillion Genomics Limited Sondes
CN108410952A (zh) * 2018-05-11 2018-08-17 重庆出入境检验检疫局检验检疫技术中心 单核细胞增生李斯特氏菌夹心dna杂交快速检测用探针、试剂盒和检测方法
WO2021007099A1 (fr) * 2019-07-05 2021-01-14 Ultivue, Inc. Procédé de multiplexage amélioré
CN111004836A (zh) * 2019-12-24 2020-04-14 山东大学 双向放大的比率型电化学适体传感器及其应用

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