Patent Application
20120172249
The invention relates to a method for detection and quantification of biomolecules, such as DNA, RNA or proteins, using isotope labeling and secondary ion mass spectrometry, and arrays designed for carrying out said method.
In the early sixties, Castaing and Slodzian developed mass-filtered emission ion microscopy using secondary ions, which is part of a technique later named secondary ion mass spectrometry (SIMS). With this technique, a beam of ions (the primary ion beam) is used as a probe to sputter the surface atomic layers of a sample into atoms or atomic clusters, a small fraction of which are ionized. In a SIMS instrument, these secondary ions are separated according to mass and are then used to measure a secondary ion current to create, for example, a quantitative atomic mass image of the analyzed surface.
SIMS has become a major tool in semiconductor and surface science studies, geochemistry, the characterization of organic material, and cosmochemistry. However, ion microscopy has been for a long time considered only as a marginal method for solving problems in life sciences, due mainly to poor lateral resolution (1-0.5 μm) and insufficient mass separation power.
Technological and conceptual improvements led to significant progress in both lateral resolving power and mass resolution, in particular due to the use of a finely focused primary ion beam. SIMS microscopy has therefore become a very powerful imaging tool. For example, Lechene et al. were able using the SIMS technique to image individual stereocilia, the mechanosensory organelles of the inner cells of the cochlea (Lechene at al. Journal of Biology, 2006, 5:20). In another experiment, they were able to study the nitrogen fixation in bacteria cultured in a 15N atmosphere. The use of SIMS technique also allowed Lechene et al. to localize, quantify and compare nitrogen fixation in single cells and subcellular structures (Lechene et al. Science 2007, 317:1563). Thus, SIMS technology is now widely used for imaging cells or tissues, and is a powerful tool for diagnostic.
SIMS technique was also used to detect hybridization of unlabelled DNA to microarrays of peptide nucleic acids (PNA) (Brandt et al, 2003, Nucleic Acids Research, 31: 19). In these experiments, PNA/DNA or PNA/RNA duplexes were visualized by SIMS detecting the phosphates that are an integral part of the nucleic acids but are completely missing in PNA.
The invention aims to provide a method for detecting and quantifying the presence or absence of a number of biomolecules in a sample using the SIMS technique. The method described in Brandt et al. presents the following drawbacks: (i) it can only be applied with PNA probes or probes that do not contain phosphates and (ii) it does not allow quantification of the interaction probe/target. In a previous patent application, the Applicant aimed to provide a universal method that can be applied to a great number of samples for the detection and the quantification of a great number of interaction probe/targets in each sample using the SIMS technique, the detection and the quantification of said interactions being determined by the calculation of the isotopic ratio probe/target (see
The present invention relates to an array comprising a substantially planar substrate having a conducting surface and a number of discrete areas containing probes being labeled with at least one rare, stable or unstable isotope or exogenous isotope, wherein the array or the discrete areas are coated with a hydrophilic polymer that does not contain the common secondary ions corresponding to the rare or exogenous isotope used for labeling the probes. According to one embodiment, said substantially planar substrate having a conducting surface is a silicon wafer.
According to one embodiment, said hydrophilic polymer that does not contain nitrogen is selected in the group comprising monosaccharide, disaccharide, polysaccharide, and synthetic hydrophilic polymer that does not contain nitrogen.
According to one embodiment, said synthetic hydrophilic polymer is polyethylene glycol.
According to one embodiment, said probes are labeled with at least one heavy stable isotope selected in the group 2H, 13C, 15N, 17O, 18O, 33S, 34S and 36S, or with at least one unstable isotope selected in the group 3H and 14C or with at least one exogenous isotope selected in the group of 79Br and 81Br.
According to one embodiment, said array is a microarray wherein each discrete area has one of the dimensions length, width or diameter being from 1 μm to 1000 μm.
According to one embodiment, each discrete area is a microwell that comprises probes being labeled with at least one rare, stable or unstable isotope or exogenous isotope.
According to one embodiment, said array is a nanoarray wherein each discrete area has one of the dimensions length, width or diameter being from 1 nm to 1000 nm.
According to one embodiment, each discrete area is a nanowell that comprises probes being labeled with at least one rare, stable or unstable isotope or exogenous isotope.
According to one embodiment, each microwell comprises a number of nanowells, and said nanowells comprise the probes being labeled with at least one rare, stable or unstable isotope or exogenous isotope.
The present invention relates to a method for detecting and quantifying in at least one sample the presence or absence of at least one biomolecule, comprising:
According to one embodiment, each sample to be tested is contacted with one or more discrete area of the array.
According to one embodiment, said sample to be tested is a single cell.
According to one embodiment, said method is for determining a molecular atlas of the sample tested, wherein said molecular atlas is the determination of the transcriptome, proteome, lipidome, metabolome, glycome and/or interactome of said sample.
According to one embodiment, said method is for predicting a predisposition to a disease, or for diagnosing a disease in a subject in need thereof.
According to one embodiment, said method is for monitoring the efficacy of a therapeutic agent administrated to a subject to treat a disease.
According to one embodiment, said method is for screening therapeutic agents.
While carrying out the method of the invention with labeling the probes with 15N, the inventors found that coating the array with a hydrophilic polymer that does not contain the common secondary ions corresponding to the rare isotope used for labeling the probes, in this case nitrogen (14N), gives an additional advantage to the method of the invention, as it prevents the non-specific adsorption of any hydrophobic molecules such as nucleic acids, peptides, or proteins present in the sample to be tested and thus minimizes the background. Indeed, in order to minimize background, the person skilled in the art would have coated the array with bovine serum albumin (BSA) or casein as in conventional methods. However, the inventors found that coating the array with BSA or casein enhances the background in the method of the invention as it contains nitrogen. Thus, they found another means to minimize the background using a hydrophilic polymer that does not contain the common secondary ions corresponding to the rare isotope used for labeling the probes such as for example polyethylene glycol. Without willing to be bound by a theory, the inventors state that the high number of water molecules bound to the hydrophilic polymer prevents the non specific binding of hydrophobic molecules, thus minimizing the background.
One object of the invention is an array comprising a substantially planar substrate having a conducting surface and a number of discrete areas, which may or may not be in the form of wells, containing probes being labeled with at least one rare isotope (e.g. stable heavy or light isotope or unstable isotope) or with at least one exogenous isotope (e.g. 79Br, 81Br), wherein the array or discrete areas are coated with a hydrophilic polymer that does not contain the common secondary ions corresponding to the rare or exogenous isotope used for labeling the probes.
According to the invention, the rare or exogenous isotope used for labeling the probes is the means for detecting the target in the sample to be tested; thus, the term “a hydrophilic polymer that does not contain the common secondary ions corresponding to the rare or exogenous isotope used for labeling the probes” is equivalent to “a hydrophilic polymer that does not contain the common secondary ions corresponding to the rare or exogenous isotope used for detecting the target”.
According to the invention, a substantially planar substrate having a conducting surface is a silicon wafer or any other solid or semi-solid surface made of gold, silver, aluminum, copper, platinum, palladium or other metal, or semiconductors such as GaAs, InP, or other material treated to make the surface conducting e.g. polymer material, polymer-coated material, superconducting material, ceramics, metal oxides, silicon oxide, etc.
In one embodiment of the invention, said substantially planar substrate having a conducting surface is compatible with SIMS.
In a preferred embodiment of the invention, said SIMS-compatible substantially planar substrate having a conducting surface is a silicon wafer.
According to the invention, the term “a polymer that does not contain the common secondary ions corresponding to the rare or exogenous isotope used for labeling the probes” refers for example to a polymer that does not contain nitrogen, in particular 14N, when the probes are labeled with 15N. In another example, it refers to a polymer that does not contain sulfur, in particular 32S, when the probes are labeled with 33S, 34S or 36S. Examples of common secondary ions include 1H, 12C, 14N, 32S, 80Br, 16O . . .
In one embodiment of the invention, said hydrophilic polymer according to the invention (i.e. which does not contain the common secondary ions corresponding to the rare or exogenous isotope used for labeling the probes) contains at least one of said rare stable isotopes or exogenous isotopes used for labeling the probes. For example, said hydrophilic polymer may comprise 13C as carbon atoms only (and no 12C atom, as it corresponds to a common secondary ion), and/or 2H as hydrogen atoms only (and no 1H atom, as it corresponds to a common secondary ion), and/or 15N as nitrogen atoms only (and no 14N atom, as it corresponds to a common secondary ion).
According to one embodiment of the invention, said hydrophilic polymer that does not contain the common secondary ions corresponding to the rare or exogenous isotope used for labeling the probes can be selected in the group comprising monosaccharide, disaccharide, polysaccharide such as dextran, chemically modified polysaccharide such as hydroxymethylcellulose and synthetic hydrophilic polymer such as linear or branched polyvinylalcohols. Said hydrophilic polymer may also be a protein, which is natural or chemically modified, like for example protein A.
According to one embodiment of the invention, said synthetic hydrophilic polymer is polyethylene glycol. Polyethylene glycol presents the advantage in particular of not containing nitrogen or sulfur, and thus can be used preferably when probe labeling is done with 15N or 33S, 34S and 36S.
According to one embodiment of the invention, said hydrophilic polymer is coated onto the entire surface of the substantially planar substrate. According to this embodiment, the non-specific binding of molecules present in the sample to be tested is prevented on the whole surface, thus avoiding background and non-specific binding of the molecule to be tested.
Another object of the invention is a microwell array being the microarray as described here above, wherein the discrete areas are microwells that contain probes labeled with at least one rare, stable or unstable isotope. The microwells may be any shape, for example dots, lines, circles, squares or triangles, and may be arranged in any larger pattern, for example rows and columns, lattices, grids etc. These microwells contain probes labeled with at least one rare, stable or unstable isotope.
In one embodiment of the invention, the microwell array of the invention comprises from 10 to 100000 microwells, in another embodiment from 10 to 25000 microwells, in another embodiment from 100 to 10000 microwells and in another embodiment from 1000 to 5000 microwells.
In one embodiment of the invention, the shape and the size of the microwells are suitably determined to store a single cell in each microwell.
In one embodiment, each microwell has one of the dimensions length, width or diameter in the range from 1 μm to 1000 μm, in another embodiment from 1 μm to 500 μm, in another embodiment from 1 μm to 200 μm and in another embodiment from 1 to 100 μm.
In one embodiment, each microwell has a depth from 1 μm to 100 μm, in another embodiment from 1 μm to 50 μm, in another embodiment from 5 μm to 20 μm.
The distance between each microwell may be from 25 to 5000 μm, in another embodiment from 100 to 1000 μm and in another embodiment from 50 μm to 150 μm.
The microwell may be of any shape: for example it can be cylindrical, non-cylindrical such as a polyhedron with multiple faces (a parallelepiped, hexagonal column, octagonal column), an inverted cone, an inverted pyramid, or it may have a shape combining two or more of these shapes. For conical and parallelepiped shapes, the bottom of the microwell is normally flat, but curved surfaces (convex or concave) are also possible.
In one embodiment of the invention, the substantially planar conducting surface carrying the set of microwells may be shaped as a rectangular solid or a disc (although other shapes are possible), having a length of 1 cm, a width of 1 cm and a thickness of about 250 μm.
Another object of the invention is a nanowell array being the nanoarray as described here above, wherein the discrete areas are nanowells that contain probes being labeled with at least one rare, stable or unstable isotope. The nanowells may be any shape, for example dots, lines, circles, squares or triangles, and may be arranged in any larger pattern, for example rows and columns, lattices, grids etc. These nanowells contain probes labeled with at least one rare, stable or unstable isotope.
In one embodiment of the invention, the nanowell array of the invention comprises from 10 to 100000 nanowells, in another embodiment from 10 to 25000 nanowells, in another embodiment from 100 to 10000 nanowells and in another embodiment from 1000 to 5000 nanowells.
In one embodiment, each nanowell has one of the dimensions length, width or diameter being from 1 nm to 1000 nm, in another embodiment from 5 nm to 500 nm, in another embodiment from 10 nm to 200 nm and in another embodiment from 50 to 100 nm.
In one embodiment, each nanowell has a depth from 1 nm to 100 nm, in another embodiment from 1 nm to 50 nm, in another embodiment from 5 nm to 20 nm.
The distance between each nanowell may be from 10 to 1000 nm, in another embodiment from 50 to 500 nm and in another embodiment from 100 nm to 200 nm.
The nanowell may be of any shape: for example it can be cylindrical, noncylindrical such as a polyhedron with multiple faces (a parallelepiped, hexagonal column, octagonal column), an inverted cone, an inverted pyramid, or it may have a shape combining two or more of these shapes. For conical and parallelepiped shapes, the bottom of the nanowell is normally flat, but curved surfaces (convex or concave) are also possible.
In one embodiment of the invention, the substantially planar conducting surface carrying the set of nanowells may be shaped as a rectangular solid or a disc (although other shapes are possible), having a length of 1 cm, a width of 1 cm and a thickness of about 250 μm.
Another object of the invention is an array being an arrangement of a set of discrete areas, or pattern units, forming a larger pattern on a substrate. The discrete areas or pattern units may be any shape, for example dots, lines, circles, squares or triangles, and may be arranged in any larger pattern, for example rows and columns, lattices, grids etc. These discrete areas contain probes labeled with at least one rare, stable (or unstable) isotope.
In one embodiment of the invention, the array of the invention comprises from 10 to 100000 discrete areas, in another embodiment from 10 to 25000 discrete areas, in another embodiment from 100 to 10000 discrete areas and in another embodiment from 1000 to 5000 discrete areas.
In one embodiment of the invention, the array of the invention is a microarray. In this embodiment, each discrete area has one of the dimensions length, width or diameter being from 1 μm to 1000 μm, in another embodiment from 5 μm to 500 μm, in another embodiment from 10 μm to 200 μm and in another embodiment from 50 to 100 μm. The distance between each discrete area may be from 10 to 1000 μm, in another embodiment from 50 to 500 μm and in another embodiment from 100 μm to 200 μm.
In one embodiment of the invention, the array of the invention is a nanoarray. In this embodiment each discrete area has one of the dimensions length, width or diameter being from 1 nm to 1000 nm, in another embodiment from 5 nm to 500 nm, in another embodiment from 10 nm to 200 nm and in another embodiment from 50 to 100 nm. The distance between each discrete area may be from 10 to 1000 nm, in another embodiment from 50 nm to 500 nm and in another embodiment from 100 μm to 200 nm.
In one embodiment of the invention, the membrane or silicon wafer carrying the set(s) of discrete areas may be shaped as a rectangular solid or a disc (although other shapes are possible), having for example a length of 1 cm, a width of 1 cm and a thickness of about 250
In this embodiment, the probes, which bind to targets (usually the biomolecules that constitute cells but even viruses, organelles or cells themselves), may be made of any molecules (biological or non-biological) such as nucleic acids (oligonucleotides, DNA, RNA, PNA, aptamers), peptides or proteins (antibodies, enzymes), ligands (an antigen, enzyme substrate, receptor or ligand for the receptor), glycans, lipids, polyamines, molecularly imprinted polymers, phages, viruses, or combination of these molecules.
Preferably, the probes are molecules which are able to specifically bind to their target(s) molecule(s) (for example via a non covalent bond), or specifically interact with their target(s) molecule(s) (for example via hybridization, as is the case for nucleic acids).
According to the invention, the probes contain at least one rare, stable or unstable isotope: in one embodiment, probes contain at least one heavy stable isotope such as 2H, 13C, 15N, 17O, 18O, 33S, 34S and 36S.
in another embodiment, probes contain at least one unstable isotope such as 3H and 14C,
in another embodiment, probes contain at least one exogenous isotope such as 79Br and 81Br.
The attachment or grafting of probes to the array is achieved by techniques well-known in the art. The probes may be adsorbed, physisorbed, chemisorbed, or covalently attached to the arrays. Lithography printing may also be used to allow probes to be transferred and adsorbed directly or indirectly to surfaces in a patterned fashion.
For example, attachment of probes may be achieved by introducing functional groups onto the surface for chemical reaction between the surface and the probe to be grafted.
The carboxyl group (COOH) is one of the best-known functional groups for grafting. Chemical bonds are produced between amino-groups from proteins and carboxyl functional groups. Acrylic acid or copolymerised vinylsilane and maleic anhydride acid can also be used to generate silicon-COOH substrates that act as spacers to graft proteins onto the surface using e.g. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride.
In one embodiment of the invention, the attachment or grafting of probes to the array is achieved via functionalization of the hydrophilic polymer coated onto the array. For example, in one embodiment of the invention, the array is coated with polyethylene glycol which is functionalized with N-hydroxysuccimide (NHS) to allow the grafting of probes. This grafting presents the advantage of being very simple to carry out.
In one embodiment in which a diversity of different probes is attached to the array, the probes are identified via the position or coordinates of the discrete area containing the probes.
According to the method of the invention, the sample to be tested may be isolated from cells, tissue, organ, body fluid such as for instance sera, plasma, seminal fluid, synovial fluid, cerebrospinal fluid, blood or urine, a cell culture, water such as sewage water, freshwater, marine coastal water, ground water . . .
According to the method of the invention, the sample to be tested may comprise nucleic acids (oligonucleotides, DNA, RNA, PNA), peptides or proteins (antibodies, enzymes), ligand (an antigen, enzyme substrate, receptor or ligand for the receptor), glycans, lipids, polyamines, phages, viruses or a combination thereof. Thus the biomolecules to be detected may be nucleic acids (oligonucleotides, DNA, RNA, PNA), peptides or proteins (antibodies, enzymes, prions), ligand (an antigen, enzyme substrate, receptor or ligand for the receptor), glycans, lipids, polyamines, phages, viruses or a combination thereof.
According to the method of the invention, the probes or the targets, i.e. the biomolecules present in the sample(s) to be tested, are labeled with rare, stable or unstable isotopes or exogenous isotopes.
In one embodiment, probes or targets may be labeled with at least one rare, stable isotope such as 2H, 13C, 15N, 17O, 18O, 33S, 34S and 36S.
In another embodiment, probes or targets may be labeled with at least one unstable isotope such as 3H and 14C.
In another embodiment, probes or targets may be labeled with at least one exogenous isotope such as 79Br and 81Br.
Labeled probes can be obtained by two techniques, in vivo and in vitro:
In the case of in vitro labeling, 1) the polymerase chain reaction is used to produce labeled oligonucleotides, 2) peptide synthesis is used to produce labeled peptides, 3) in vitro transcription/translation is used to produce labeled RNA and labeled proteins, 4) reverse transcription is used to produce labeled cDNA, 5) chemical synthesis is used to produce labeled PNA.
In the case of in vivo labeling, either prokaryotic or eukaryotic cells are grown on media containing nutrients (such as NH4Cl, glucose and amino acids) labeled with combinations of 2H, 13C, 15N, 17O, 18O, 33S, 34S and 36S or with combinations of 3H, 14C, or other unstable isotopes with long half-lives. These cells thus produce labeled probes (such as antibodies or bacteriophage or nucleic acids or proteins or sugars or other cellular constituents). Where the targets, i.e. the biomolecules present in the sample(s) to be tested, are to be labeled, the cells are generally grown on media containing nutrients (such as NH4Cl, glucose and amino acids) labeled with combinations of 2H, 13C, 15N, 17O, 18O, 33S, 34S and 36S or with combinations of 3H and 14C. These cells thus produce labeled biomolecules.
In one embodiment of the method of the invention, the sample to be tested is put in contact with the array (microarray or nanoarray) containing a number of diverse probes.
In another embodiment, where a number of samples is to be tested, each sample is put in contact with one or more microwells of a microwell array.
In another embodiment, where the number and diversity of samples to be tested corresponds to the number and diversity of single cells, the contents of each single cell, obtained via an appropriate method, is put into contact with one microwell of a microwell array.
The sample(s) is/are then contacted with the array under conditions that allow the probes present onto the array to interact with the target biomolecules.
The binding of probes to their targets is then performed in a variety of buffers from which, typically, the common 12C and 14N isotopes are absent (such as Phosphate Buffered Saline).
After careful washing with pure water (preferably at low temperature to limit the dissociation of probes from their targets) to eliminate salts (which can form crystals at the drying step), the unbound molecules and the big cellular debris, the array is dried in a dust-free atmosphere either in an oven under vacuum or by freeze-drying.
According to the method of the invention, counting the numbers of rare and common secondary ions so as to obtain the Detection Isotopic Ratio allows detection of the duplexes probe/target.
In one embodiment, the duplexes probe/target are detected by Dynamic Secondary Ion Mass Spectrometry (D-SIMS). In another embodiment, the duplexes probe/target are detected by Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS).
Secondary Ion Mass Spectrometry (SIMS) allows the analysis of the surface composition of inorganic and organic materials based on mass spectral analysis of secondary ions extracted from the surface (1 nm-1 μm depth) of a solid sample under the impact of an energetic beam of primary ions. Molecules are fragmented by the primary ion beam in D-SIMS either totally into their constituent atoms by dynamic D-SIMS or partially into molecular fragments such as amino acids by ToF-SIMS. SIMS-related technologies such as Laser SNMS may also be used to detect probe/target interactions.
In one embodiment of the method of the invention, the probes that are attached to the array are labeled with rare, stable or unstable isotopes or exogenous isotopes. In this embodiment, the sample(s) to be tested have biomolecules (targets) that only contain these rare isotopes in their natural proportions (or even lower than these proportions).
The presence of an interaction probe/target is determined by the comparison of the Detection Isotopic Ratio obtained from each discrete area, microwell or nanowell to the background Detection Isotopic Ratio obtained from the reference discrete area, microwell or nanowell or from discrete areas in which there is no specific interaction between labeled probe and target (i.e. the target is not present in the sample to be analyzed) or in which there is a specific interaction between unlabeled probe and unlabeled target.
Therefore, in the case where probes are labeled and targets are unlabeled, the presence of an interaction probe/target is revealed if the Detection Isotopic Ratio obtained from one discrete area, microwell or nanowell is significantly superior to the Detection Isotopic Ratio obtained from the reference discrete area, microwell or nanowell. For example, in the case where probes are labeled with 13C, the presence of an interaction probe/target is revealed if the Detection Isotopic Ratio of 12C/(12C+13C) (or alternatively 12C14N/(12C14N+12C15N)) obtained from one discrete area, microwell or nanowell is superior to the Detection Isotopic Ratio obtained from the reference discrete area, microwell or nanowell, for the same instrumental settings.
Therefore, in the case where probes are unlabeled and targets are labeled, the presence of an interaction probe/target is revealed if the Detection Isotopic Ratio obtained from one discrete area, microwell or nanowell is significantly inferior to the Detection Isotopic Ratio obtained from the reference discrete area, microwell or nanowell. For example, in the case where probes are labeled with 13C, the presence of an interaction probe/target is revealed if the ratio of 12C/(12C+13C) secondary ions (or alternatively 12C14N/(12C14N+12C15N)) obtained from one discrete area, microwell or nanowell is inferior to this ratio of secondary ions obtained from the reference discrete area, microwell or nanowell, for the same instrumental settings.
Therefore, in the case where both probes and targets are labeled with the same isotope/isotopes, the presence of an interaction probe/target is revealed if the Detection Isotopic Ratio obtained from one discrete area, microwell or nanowell is significantly different from the Detection Isotopic Ratio obtained from the reference discrete area, microwell or nanowell.
According to the invention, the probes or targets may also be labeled with more than one rare, stable or unstable isotopes or exogenous isotopes of their constituent elements. For example, probes or targets may be labeled with 13C and 15N. Multiple labeling such as 13C15N of the probes is particularly useful to minimize the background.
According to the invention, where the targets are labeled with rare, stable (or unstable) isotopes, various experimental conditions (for example, with and without drug treatments) may be differentiated by a differential labeling of each condition.
One object of the invention is a method for detecting and quantifying in at least one sample the presence or absence of at least one biomolecule, comprising:
This allows the determination of the DIR.
The DIR obtained from each discrete area, microwell or nanowell is then compared to the DIR obtained from the reference discrete area, microwell or nanowell; a significant change of the DIR (see
According to another embodiment, said method for detecting and quantifying in at least one sample the presence or absence of at least one biomolecule, allows the determination of a molecular atlas of said sample, which means the determination of the transcriptome, proteome, lipidome, metabolome, glycome or interactome of said sample, or allows the determination of biomolecules and their interactions in a single cell.
According to one embodiment of the invention, the method for detecting and quantifying in a sample the presence or absence of at least one biomolecule, is intended for providing a molecular atlas of said sample.
In one embodiment, the diversity of biomolecules to be detected and quantified is genomic sequences, allowing the determination of genomic variation in said sample.
In another embodiment, the diversity of biomolecules to be detected and quantified is RNA, allowing the determination of the transcriptome of said sample.
In another embodiment, the diversity of biomolecules to be detected and quantified is proteins, allowing the determination of the proteome of said sample.
In another embodiment, the diversity of biomolecules to be detected and quantified is lipids, allowing the determination of the lipidome of said sample.
In another embodiment, the diversity of biomolecules to be detected and quantified is metabolites, allowing the determination of the metabolome of said sample.
In another embodiment, the diversity of biomolecules to be detected and quantified is glycosylated proteins, allowing the determination of the glycome of said sample.
In another embodiment, the diversity of biomolecules to be detected and quantified is proteins that interact with at least one specific probe, allowing the determination of the interactome of said sample.
According to another embodiment of the invention, the method for detecting and quantifying at a single cell level the presence or absence of at least one biomolecule, is intended for providing a molecular atlas of a single cell level.
In one embodiment, the diversity of biomolecules to be detected and quantified is genomic sequences, allowing the determination of genomic variation at the single cell level.
In another embodiment, the diversity of biomolecules to be detected and quantified is RNA, allowing the determination of the transcriptome at the single cell level.
In another embodiment, the diversity of biomolecules to be detected and quantified is proteins, allowing the determination of the proteome at the single cell level.
In another embodiment, the diversity of biomolecules to be detected and quantified is lipids, allowing the determination of the lipidome at the single cell level.
In another embodiment, the diversity of biomolecules to be detected and quantified is metabolites, allowing the determination of the metabolome at the single cell level.
In another embodiment, the diversity of biomolecules to be detected and quantified is glycosylated proteins, allowing the determination of the glycome at the single cell level.
In another embodiment, the diversity of biomolecules to be detected and quantified is proteins that interact with at least one specific probe, allowing the determination of the interactome at the single cell level.
Another object of the invention is the use of said method for detecting and quantifying at least one biomolecule in a sample or at a single cell level for:
Another object of the invention is a method for predicting a predisposition to a disease, or for diagnosing a disease in a subject. This comprises:
In one embodiment, the subject is a mammal. In a preferred embodiment, the subject is a human being.
According to the method of the invention, the sample to be tested may be isolated from cells, tissue, organ, or body fluid such as for instance sera, plasma, seminal fluid, synovial fluid, cerebrospinal fluid, blood or urine, from the subject.
The sample may be derived from diseased cells or tissues. For example, the cells or tissues may be infected by a pathogen such as HIV, influenza, malaria, hepatitis, cytomegalovirus, herpes simplex virus. In one embodiment, the cells or tissues are infected by a viral or a bacterial pathogen. In another embodiment, the disease is cancer. In another embodiment, the disease is a neurodegenerative disease such as Parkinson, Alzheimer or Multiple Sclerosis.
In one embodiment of the invention, said method is intended to predict a predisposition to a cancer, or for diagnosing a cancer in a subject.
In another embodiment of the invention, said method is intended to predict a predisposition or to diagnose a bacterial disease.
In another embodiment of the invention, said method is intended to predict a predisposition or to diagnose a viral disease.
Another object of the invention is a method for screening therapeutic agents, comprising:
Another object of the invention is a method for monitoring efficacy of a therapeutic agent administrated to a subject to treat a disease, said method comprising:
In the following description, all experiments for which no detailed protocol is given are performed according to standard protocol.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
This experiment shows that the SIMS technique allows a quantitative detection of the proportion of the 13C isotope in a mixture containing isotopically labeled proteins and a different amount of unlabeled molecules.
Isotope-labeled molecules are generally supplied in a buffer containing protectants and preservatives such as glycerol (a source of 12C) and sodium azide (a source of 14N). These agents are used to preserve dialysis membranes (as supplied by Millipore). The aim of the following experiments is to determine the conditions of dialysis needed to remove them.
Proteins were extracted from E. coli grown in a minimal medium in which the only available nitrogen was 15N. Using SIMS, the isotopic fraction of these proteins was found to be 97.8%±0.02%. 20 μg of these labeled proteins were dialysed for 6 hours in water on Millipore dialysis membranes which either were unwashed or had been washed previously.
The level of the isotopic fraction of the washed membrane was similar to that of the control (97.9%±0.02%) whilst that of the unwashed membrane (containing contaminant nitrogen) was lower (92.1%±1.4%). Hence, prewashing the dialysis membrane restored the initial isotopic fraction.
Proteins were also extracted from bacteria grown in a medium in which the only available carbon was 13C. The isotopic fraction of these proteins as measured by SIMS was 86%±0.5%. These proteins were then dissolved in water and a second solution of them was made to a final concentration of 10% glycerol.
The addition of glycerol reduced the isotopic fraction to 78%±2% but the initial fraction (86%±2%) was recovered by the subsequent dialysis which eliminated the glycerol.
This experiment shows that the isotopically labeled oligonucleotide (probes) can be prepared using the PCR technique and an unconventional buffer that does not contain nitrogen compounds (which, as contaminants, might perturb the DIR measurement described in the section “Definitions”).
Many methods in biology use Tris-based buffers. This organic molecule is an excellent buffer and, moreover, does not lead to the precipitation of calcium or magnesium salts. For our method, however, Tris has the disadvantage of containing 14N and of having a strong affinity for DNA (and other macromolecules). This disadvantage arises because this 14N is an important contaminant in our method which depends on measuring accurately the 15N/(14N+15N) ratio resulting from the hybridization of a 15N probe with a 14N target.
The two approaches to making DNA without contaminant nitrogen are either i) using Tris in the buffers and then removing it once the DNA has been made or ii) using Tris-free buffers. Since the first method entails purification methods that are time-consuming, wasteful and expensive, we adopted the second solution to provide the right pH and concentration conditions for PCR (polymerase chain reaction). Although, to our knowledge, no literature describes the use of borax buffers for PCR, we used a borax buffer, an inorganic buffer that does not contain nitrogen and that maintains the same pH values as Tris buffers.
A 200 bp sequence of DNA from the PML gene was amplified. The template itself was made using only one round of amplification (to avoid error generation). Either 20 cycles of PCR were performed to determine quantitatively the effect of the borax buffer (and limit error generation) or 60 cycles were performed to consume the limiting substrate and obtain maximal amplification. The amplicons were analyzed by electrophoresis on 1% agarose gels. The results below are shown for a Tris buffer and for a borax buffer.
In the case of 20 cycles of amplification, the borax buffer gives half the yield of the Tris buffer and, significantly, the borax buffer did not generate amplicons of different sizes. This means that the polymerase continued replication to the end of the templates. In the case of 60 cycles of amplification, both Tris and borax buffers gave similar results.
A borax buffer is perfectly suitable for reactions such as PCR amplification and hence for the synthesis of oligonucleotide probes. Hence this buffer can be used to avoid contaminant nitrogen as required in the SIMS analyses proposed here.
Protein arrays were prepared for detection by SIMS.
Wafers were coated with PEG functionalized with N Hydroxy Succinimide (NHS) to allow the anti-CD34 antibody enriched in 15N to be linked to the PEG coat of a silicon wafer. Three identical anti-CD34 wafers were prepared. The first wafer was incubated with the target protein (Wtap for target protein), CD34 whilst, as negative controls, the second wafer was incubated with BSA (Wbsa for BSA) and the third wafer was incubated in the same solution but without protein (Wabo for antibody only).
The three wafers were then analyzed using a NanoSIMS 50.
1 μL of a solution of 0.22 μg/μL of an anti-CD34 antibody (supplied by Biosynergy, Cambridge UK) enriched with 15N (Detection Isotopic Ratio was 58±11%) was deposited on a silicon wafer coated with PEG functionalized with NHS (Microsurfaces Inc., Minneapolis, USA). After 1 h at room temperature (25° C.), the surface was washed 5× for 5 minutes with a solution of PBS pH 7.4 Tween-20 (0.1% v/v) then for 30 minutes with a solution of PBS pH 8 Tween-20 (0.1% v/v) and finally for 5 minutes with water (milli-Q).
Incubation with Target Protein (CD34) and with Control (BSA)
To be sure to saturate the antibodies with the target protein, the ratio of target protein to antibody (linked to the wafer in a functional orientation for target binding) was chosen to be 100:1. We estimated from the data of the supplier (Proteomics 2005, 5, 416-419) that the surface concentration of the antibody in a functional orientation was of the order of 1012 proteins cm−2 and hence in the area (spot) on the wafer to which the antibody was linked (0.02 cm−2) there were 2×1010 functional antibodies. We therefore chose to use a solution that would provide 0.1 μg of CD34 (Mr 35 kDa) which contains around 2×1012 proteins.
10 μL of a solution of PBS-Tween-20 pH 7.4 (Ab+no protein), or of a solution of PBS-Tween-20 pH 7.4 containing 0.01 μg/μL of the recombinant protein CD-34 (Assay Designs, Ann Arbor, USA) (sample Ab+CD34), or of a solution of PBS-Tween-20 pH 7.4 containing 0.01 μg/μL of BSA (Sigma) (Ab+BSA) were put on the surface of the wafer. After incubation for 1 h at room temperature, the surface of the wafer was washed 4× for 5 minutes with a solution of PBS pH 7.4 Tween-20 (0.1% v/v) then 2× for 5 minutes with water (milli-Q). The wafers were then dried under a vacuum and covered with gold for 60 seconds using a Cressington sputter coater 108 auto.
The surface of the wafers was analyzed using a NanoSIMS 50 in the imaging mode (field: 20×20 μm2, 128×128 pixels, 10 ms/pixel diaphragm D1−). This entailed sampling in four to five different places within the area where the antibody was linked; 12 successive scans were performed at each of these places. These scans were performed with settings that were estimated to give a sputtering rate of 0.3 nm/scan after removal of the gold layer which is sputtered out rapidly (around 1.5 nm/scan). The sum of the average number of counts of secondary ions 12C14N, ñ(12C14N), and of 12C15N, ñ(12C15N), of scans 2 to 12 were used to obtain the Detection Isotopic Ratio in each of the 3 samples using the formula:
Detection Isotopic Ratio=(Σñ12C15N)/(Σñ12C15N+Σñ12C14N).
The Detection Isotopic Ratio obtained from the wafer with antibody only (Wabo) shows a monotonous increase from a value close to that of the natural isotopic ratio of 15N (0.0037) to a plateau of 0.22 (
The possibility of using the capacity of the NanoSIMS 50 to obtain successive scans opens up the possibility of obtaining a 3 dimensional image of the structure of regions containing probes and targets and contaminants. Clearly, this capacity may then used to further refine the technique.
Finally, the control with BSA has a Detection Isotopic Ratio similar to that of the control without protein. This shows that the materials and methods we used for incubating target proteins with the antibodies and for subsequent washing steps do not result in the non-specific adsorption of proteins to the surface.
In particular, the coating of wafers with polyethylene glycol did prevent non-specific adsorption of proteins to the surface, while coating of wafers with BSA or casein resulted in high background (data not shown).
When the antibody linked to the wafer is incubated with the target protein (Wtar) there is a significant drop in the Detection Isotopic Ratio with respect to 15N in comparison with the negative controls (Wabo, absence of target protein; Wbsa, presence of Bovine Serum Albumin). This drop is significant as shown by the P value of 0.0012 (i.e. a certainty of 99.88%).
A prototype of protein chip technology compatible with SIMS has been developed to study the interaction of protein A (S. aureus) synthesized in a recombinant system E. coli and a commercial antibody.
The support consists of a silicon wafer covered with a film of PEG functionalized with N-hydroxysuccinimide (NHS); the 15N-labeled protein A (probe) is thus covalently attached to PEG (see
A production strain E. coli BL21 (DE3) was transformed with the expression vector pGTPc500—6His8PSA (32-327) with selection on LB agar+kanamycin.
An isolated clone is then used to perform the production:
1 μL of a solution of protein A at 0.33 μg/μL enriched in 15N (isotopic percentage of 88±4%) is deposited on a silicon wafer functionalized with PEG-NHS (Microsurfaces Inc., Minneapolis, USA). After 1 hour at room temperature (25° C.), the surface is cleaned 5 times for 5 minutes with a solution of PBS pH 7.4 Tween-20 (0.1% v/v), and then 30 minutes with a solution of PBS pH 8 Tween-20 (0.1% v/v) and finally for 5 minutes with water (milli-Q).
Incubation with the Target Protein (Antibody)
To establish a dilution range of the target molecule (antibody), we decided to do a solution with 50%, 100%, 200% and 1000% of target molecules, for a constant amount (in number of molecules) of probes (protein A).
10 μL of a solution of PBS-Tween-20 pH 7.4 containing 16.5 μg/μL of antibody (Sigma, Aldrich, Rockford, USA) was diluted with PBS-Tween-20 pH 7.4. Each solution was deposited on a spot of protein A and incubated for 1 h at room temperature, the surface of the wafer being cleaned 4 times for 5 minutes with a solution of PBS pH 7.4 Tween-20 (0.1% v/v) and 2 times for 5 minutes with water (milli-Q). The wafers are then dried under vacuum and coated with gold for 60 seconds with a Cressington sputter coater 108 auto.
The surface of the wafers is analyzed with the NanoSIMS 50 in isotope ratio mode (fields 200×200 μm2, diaphragm D2). An incision was made to determine the location of the spot of protein A, a dozen of analysis are performed on each spot. The scan includes 600 measures, and weights of 12C14N and 12C15N are studied in parallel. For obtaining the isotopic fraction, we use the following formula:
Isotopic fraction=(Σn12C15N)/(Σn12C15N+Σn12C14N).
By performing the curve of the isotopic fraction as a function of the amount of target molecules, there is a quick decrease of the fraction 12C15N: it rises from 52.93%±6% to less than 10% (9.17%±3.8%) when protein A interacts with a quantity of antibodies equal to 50% of the quantity of protein A. For comparable amounts of targets and probes, the isotopic fraction is significantly higher than the natural fraction: 2.89%±1.6% against 0.366%, whereas for higher concentrations of the target molecule, 200% and 1000%, the signal is very close to the natural fraction, respectively 0.43%±0.03% and 0.4%±0.01%. This strong decrease of the isotopic fraction is explained by the fact that the target molecule, the antibody, is a molecule of a size considerably greater than the probe molecule, respectively 150 KDa cons 33 KDa. Whenever an interaction occurs, the nitrogen amount will be greatly changed from 425 atoms of nitrogen for protein A to about 2500 for the protein A/antibody complex. This demonstrates that the sensitivity of SIMS NADs chips are highly dependent on analyzed molecules, and this sensitivity will be higher when the target biomolecule is substantial in size vis-à-vis the probe biomolecule.
| Number | Date | Country | Kind |
|---|---|---|---|
| 09305855.0 | Sep 2009 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2010/063569 | 9/15/2010 | WO | 00 | 3/9/2012 |
