Patent Application
20060100787
1. Field of the Invention
The invention relates generally to detection and analysis of macromolecules, and more specifically to detection and analysis of macromolecules using scanning probe microscopy of molecular barcodes (nanocodes).
2. Background Information
Detection and/or identification of biomolecules are of use for a variety of applications in medical diagnostics, forensics, toxicology, pathology, biological warfare, public health and numerous other fields. Although the principle classes of biomolecules studied are nucleic acids and proteins, examination of other biomolecules such as carbohydrates, lipids, polysaccharides, fatty acids and others also can be informative. A need exists for rapid, reliable and cost effective methods of identification of biomolecules, methods of distinguishing between similar biomolecules and of analyzing macromolecular complexes such as pathogenic spores or microorganisms.
Standard methods for nucleic acid detection, such as Southern blotting, northern blotting or binding to nucleic acid chips, rely on hybridization of a fluorescent, chemiluminescetit or radioactive probe molecule with a target nucleic acid molecule. In oligonucleotide hybridization-based assays, a labeled oligonucleotide probe that is complementary in sequence to a target nucleic acid is used to bind to and detect the nucleic acid. More recently, DNA (deoxyribonucleic acid) chips have been designed that can contain hundreds or thousands of attached oligonucleotide probes for binding to target nucleic acids. Unfortunately, problems with sensitivity and/or specificity may result from nucleic acid hybridization between sequences that are not completely complementary, and the presence of low levels of a target nucleic acid in a sample may not be detected, thus limiting the usefulness of such chip based analysis.
A variety of techniques are available for identifying proteins, polypeptides and peptides. Commonly, these techniques involve binding and detection of antibodies. Although antibody-based identification is fairly rapid, such assays occasionally show high levels of false positives or false negatives. In addition, the cost of performing immunoassays is high, and simultaneous assaying of more than one target can be difficult. Further, the methods require that an antibody be prepared against the target protein of interest before an assay can be performed.
A number of applications in molecular biology, genetics, disease diagnosis and prediction of drug responsiveness involve identification of nucleic acid sequence variants. Existing methods for nucleic acid sequencing, including dideoxy sequencing and sequencing by hybridization, tend to be relatively slow, expensive, and labor intensive, and can involve use of radioactive tags or other toxic chemicals that require special precautions for storage and handling, and must be disposed of as hazardous waste. Existing nucleic acid sequencing methods are also limited as to the amount of sequence information that can be obtained in a single assay, typically less than about 1000 bases. Thus, a need exists for more rapid and cost-effective methods of nucleic acid and other biomolecule analysis, particularly methods suitable to automation.
Embodiments of the present invention are based, in part, on the development of nucleic acid based nanocodes that can be detected using scanning probe microscopy (SPM). As such, methods of using SPM to detect a molecular probe operably linked to such a nanocode are provided, as are methods of using SPM to detect the presence of a target nucleic acid in a sample by detecting specific binding of such a nanocode tagged molecular probe. In various embodiment, molecular identification systems, which include one or more nanocode tagged molecular probes, also are provided, wherein the molecular probe (and a target molecule bound thereto) can be identified by detecting the surface property of the nanocode. In one embodiment, the molecular identification system includes a solid support (e.g., a wafer, chip, glass slide or bead) to which one or a plurality of same and/or different nanocode tagged molecular probe(s) is (are) immobilized (for example, a plurality of different nanocode tagged molecular probes, which can be arranged in an array (e.g., an addressable array) on the solid support).
As used herein, the term “a” or “an” means one or more than one of an item. As used herein, the term “multiplicity” or “plurality” of an item means two or more of the item or of different items, which can be related (e.g., two or more nanocodes, two or more molecular probes, two or more molecular probes, each of which is operably linked to a nanocode, wherein the nanocodes can be the same or different).
The present invention provides methods of using nanocodes for identifying a molecular probe. In one embodiment, a method is provided to identify a molecular probe, which is operably linked to a nanocode having a surface property, by contacting the molecular probe with an SPM probe, and detecting the surface property of the nanocode. The surface property provides a code that is detectable by SPM and is unique to a specified nanocode, wherein the code is defined by subfeatures, including, for example, by the relative thickness (diameter) of regions of the nanocode, by the relative size (e.g., height or length) of the nanocode or portions of the nanocode (e.g., branches or bubbles), by the presence (or absence) of one or more objects (tags, moieties), which can be the same or different objects, operably associated with the nanocode, by the length of the nanocode, or by any other surface property that can be detected using SPM (see Examples 1 to 4). By detecting the surface property of a nanocode operably linked to a molecular probe, and having knowledge of the particular nanocode, the molecular probe can be identified, as can a target molecule specifically bound by the molecular probe.
The present nanocodes, which are suitable for detection by SPM, are exemplified by mosaic nanocodes that include at least one nucleic acid component and at least one polypeptide component (see
A nanocode that is a mosaic biomolecule containing one or more nucleic acid regions and one or more peptide regions can have a surface property defined by the relative diameters of the nucleic acid and peptide regions, as well as by the relative lengths of each such region. Since a double stranded nucleic acid sequence has a smaller diameter than a double stranded peptide region (see
A nanocode generally contains at least two (e.g., 2, 3, 4, 5) organic polymer strands (e.g., a double stranded nucleic acid nanocode), although the nanocodes can further contain single stranded regions (e.g., at a terminus of the full length structure or a terminus of a branch; see, e.g.,
Nanocodes also can be modified to contain one or more objects that affect the surface property of the nanocode such that the presence or absence or relative abundance of the modifying object can be identified by SPM. Such modifying objects, which can provide an additional level of diversity to the nanocodes, include nanoparticles such as silica, alumina, metal ions (e.g., gold or silver), or metal ion complexes (e.g., ferritin), which can be linked to one or more positions of a nanocode, including, for example, to one or more branches (see, e.g.,
A nanocode can be operably linked to a molecular probe, thus allowing detection and identification of the molecular probe by SPM. The term “molecular probe” is used herein to refer to any molecule that exhibits selective and/or specific binding to one or more targets. In one embodiment, a molecular probe is operably linked to a nanocode, which is distinguishable based on its surface property as detected by SPM, and each molecular probe of a plurality of molecular probes is operably linked to one or more nanocodes, wherein individual molecular probes of the plurality are distinguishable based on the nanocode operably linked thereto. As such, binding of a particular molecular probe among a population of different molecular probes (e.g., to a target molecule) can be detected by determining a surface property of the operably linked nanocode using an SPM method.
As disclosed herein, any molecular probe, including, for example, a nucleic acid (e.g., an oligonucleotide probe or PCR primer), a peptide ( e.g., an antibody or antigen binding fragment of an antibody, a binding protein, or a receptor), a lectin, or other molecule that can act, for example, as a substrate, inhibitor, activator, or analyte (e.g., a hormone, a cytokine, a growth factor, etc.), can be operably linked to a nanocode, thus rendering the molecular probe identifiable by SPM. The term “analyte” refers to any atom, chemical, molecule, compound, composition or aggregate of interest for detection and/or identification. Non-limiting examples of analytes include an amino acid, peptide, polypeptide, protein, glycoprotein, lipoprotein, nucleoside, nucleotide, oligonucleotide, nucleic acid, sugar, carbohydrate, oligosaccharide, polysaccharide, fatty acid, lipid, hormone, metabolite, cytokine, chemokine, receptor, neurotransmitter, antigen, allergen, antibody, substrate, metabolite, cofactor, inhibitor, drug, pharmaceutical, nutrient, prion, toxin, poison, explosive, pesticide, chemical warfare agent, biohazardous agent, radioisotope, vitamin, heterocyclic aromatic compound, carcinogen, mutagen, narcotic, amphetamine, barbiturate, hallucinogen, waste product and/or contaminant.
As used herein, the term “operably coupled” or “operably linked” or “operably associated” means that there is an interaction between two or more units of a molecule, a complex, an apparatus and/or a system, wherein each component retains a function characteristic of the individual component. For example, an SPM probe can be operably coupled to a computer if the computer can obtain, process, store and/or transmit data on SPM signals detected by the detector. In another example, a surface property modifying object (e.g., a gold particle) can be operably associated with a nanocode, wherein the object is bound such that it remains associated with the nanocode under conditions to which the complex (nanocode-gold particle) is to be exposed (e.g., physiological conditions). In still another example, a molecular probe can be operably associated with nanocode having a surface property, wherein the molecular probe can specifically interact with a target molecule and the surface property of the nanocode can be identified. A molecular probe that is operably linked to a nanocode can be any type of molecule that specifically interacts with a target molecule, including, for example, a peptide (e.g., an antibody or a peptide comprising an epitope that is specifically bound by an antibody, an enzyme or a substrate acted upon by an enzyme), or a nucleic acid (e.g,. an oligonucleotide probe that specifically hybridizes to a target nucleic acid molecule, or a nucleic acid comprising a protein binding site such as a transcriptional regulatory element that is specifically bound by a transcription factor (or the protein component, e.g., a DNA binding domain of a transcription factor can be operably linked to the nanocode, thus allowing for detection by SPM of a nucleic acid regulatory element specifically bound by the transcription factor).
The term “binds specifically” or “specific binding activity” or “specifically interacts” or the like, when used in reference to a specific binding pair (e.g., a molecular probe and a target molecule), means that the molecular probe can associate with the target molecule with sufficient specificity such that the probe can be used to detect the presence of the target molecule. In general, a molecular probe that specifically binds a target molecule does not substantially interact with a second (or other molecule), including a second molecule that is structurally and/or functionally similar to the target molecule. However, a molecular probe also can be designed to have a more relaxed, but specific, binding activity (e.g., such that the molecular probe can identify related members of a family of target proteins or target nucleic acids (e.g., genes)), such relaxed but specific binding being encompassed within embodiments of the invention.
Specific binding can be identified using methods as disclosed herein or otherwise known in the art, and will depend, for example, on the phyico-chemical properties of the molecular probe and target molecule, including, for example, on whether the interacting molecules are nucleic acid, peptides (proteins), glycoproteins, etc. Similarly, conditions suitable for obtaining specific binding of a members of a specific binding pair are known in the art and will depend, for example, on the chemical nature of the interacting molecules, and whether the specific binding pair members are contacted in vitro (e.g., using purified or substantially purified components), or in a cell, which can be a cell in culture, a cell ex vivo, or a cell in situ (e.g., in vivo).
It is recognized that members of a specific binding pair can crossreact with related or unrelated molecules due, for example, to similar primary, secondary and/or tertiary structures present in such related or unrelated molecules as compared to a specific binding pair member. However, such crossreacting (non-specific) binding (e.g., cross-hybridization of nucleic acid probes, or cross-reactivity of antibodies), when not desired, can be identified using appropriate controls, and can be minimized or avoided by selecting conditions particularly favorable for the specific interaction (e.g., highly stringent nucleic acid hybridization conditions). For example, blocking agents (e.g., bovine serum albumin, Denhardt's reagent) can be included in an incubation medium such that non-specific binding of one or more reagents (e.g., molecular probe linked to nanocode) to a surface contacted by the reagents.
Specific binding of proteins (e.g., an antibody and its cognate epitope; or first and second interacting proteins in a biological pathway; or an enzyme and its substrate) can be identified, for example, by determining that the proteins have a dissociation constant of at least about 1×10−6 M, generally at least about 1×10−7 M, usually at least about 1×10−8 M, and particularly at least about 1×10−9 M or 1×10−10 M or less. For antibodies, specific binding also can be detected more qualitatively, for example, by detecting that an antiserum or other preparation containing the antibody has a relatively high titer for binding a specific protein (target molecule).
The term “antibody” is used herein in its broadest sense to include polyclonal and monoclonal antibodies, as well as antigen binding fragments of such antibodies. As such, whole antibodies and functional fragments thereof are contemplated, including intact antibody molecules as well as fragments thereof such as Fab and F(ab′)2, Fv and SCA fragments that can specifically bind an epitopic determinant. An Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain. An Fab′ fragment of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab′ fragments are obtained per antibody molecule treated in this manner. An (Fab′)2 fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A (Fab′)2 fragment is a dimer of two Fab′ fragments, held together by two disulfide bonds. An Fv fragment is defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains. A single chain antibody (“SCA”) is a genetically engineered single chain molecule containing the variable region of a light chain and the variable region of a heavy chain, linked by a suitable, flexible polypeptide linker.
The term “antibody” as used herein includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof. Such non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains (see Huse et al., Science 246:1275-1281, 1989). These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14:243-246, 1993; Ward et al., Nature 341:544-546, 1989; Harlow and Lane, “Antibodies: A laboratory manual” (Cold Spring Harbor Laboratory Press, 1988); Hilyard et al., “Protein Engineering: A practical approach” (IRL Press 1992); Borrabeck, “Antibody Engineering” 2d ed. (Oxford University Press 1995)). Modified or derivatized antibodies such as pegylated (polyethylene glycol modified) antibodies provide an additional example of antibodies.
Methods for raising polyclonal antibodies, for example, in a rabbit, goat, mouse or other mammal, are well known in the art (see, for example, Green et al., “Production of Polyclonal Antisera,” in Immunochemical Protocols (Manson, ed., Humana Press 1992), pages 1-5; Coligan et al., “Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters,” in Curr. Protocols Immunol. (1992), section 2.4.1). In addition, monoclonal antibodies can be obtained using methods that are well known and routine in the art (Harlow and Lane, supra, 1988).
Specific binding of an antibody molecular probe to a target molecule can be detecting using immunoassays, including competitive and non-competitive immunoassays in a direct or indirect format, as are known in the art. Such immunoassays include, for example, enzyme linked immunosorbent assays (ELISA), radioimmunoassays, and the like. In particular, where a molecular probe is operably linked to a nanocode, specific binding of the molecular probe to a target molecule can be detected using a scanning probe microscopy (SPM) method, wherein SPM is used to detect the surface property of the nanocode linked to the molecular probe.
A receptor (e.g., a cell surface receptor such as a T cell receptor, or an intracellular receptor such as a G-protein coupled receptor) provides another example of a molecular probe, which can be operably linked to a nanocode, or a target molecule. Receptors are well known in the art, and include receptors that are made up of a single protein (polypeptide), and receptor complexes, which can include two or more of the same or different protein subunits (e.g., homodimers, heterotrimers, and the like). A receptor generally is characterized in that it specifically binds a particular molecule (i.e., a ligand), which can be a protein, nucleic acid, or small organic molecule, or other molecule. Generally, binding of a ligand to a receptor triggers a specific response, for example, a cellular response.
As such, it will be recognized that a nanocode suitable for detection by SPM can be operably linked to any polypeptide, particularly a polypeptide that specifically interacts with a second (or more) molecule (e.g., a second polypeptide such as an enzyme, or a nucleic acid molecule such as a gene regulatory element). The term “polypeptide” or “peptide” is used broadly herein to mean two or more amino acids linked by a peptide bond. As such, the term “polypeptide” or “peptide” is not used herein to suggest a particular size or number of amino acids comprising the molecule, and, therefore, can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or more (e.g., 20, 40, 60, 80, 100) amino acid residues or more. A protein is a polypeptide that is produced in a cell and can be modified, for example, by a post-translational modification to include chemical moieties other than amino acids, including, for example, one or more phosphate groups (phosphoprotein), carbohydrate moieties (glycoproteins), or nucleic acid molecules (nucleoprotein).
A nucleic acid molecular probe operably linked to a nanocode also can be detected by SPM according to embodiments of the present invention. Specific binding of nucleic acids (e.g., an oligonucleotide probe to a target nucleotide sequence) can be identified by detecting binding under stringent hybridization and/or washing conditions. An oligonucleotide molecular probe is of a sufficient length, generally at least about 15 bases in length (e.g., 15, 16, 17, 18, 19, 20, 21, 25, 30, 40, 50, or more) such that the oligonucleotide selectively hybridizes to a target molecule. As used herein, the term “selective hybridization” or “selectively hybridize” refers to hybridization under moderately stringent or highly stringent physiological conditions, which can distinguish related nucleotide sequences from unrelated nucleotide sequences.
In nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids (i.e., molecular probe and target nucleic acid molecule) being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (for example, relative GC:AT content), and nucleic acid type, i.e., whether the oligonucleotide or the target nucleic acid molecule is DNA or RNA, can be considered in selecting hybridization conditions (see, e.g., Sambrook et al., “Molecular Cloning: A laboratory manual” (Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y. 1989). An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter. Methods for selecting appropriate stringency conditions can be determined empirically or estimated using various formulas, and are well known in the art (see, for example, Sambrook et al., supra, 1989). An example of progressively higher stringency conditions is as follows: 2×SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2×SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2×SSC/0.1% SDS at about 42° C. (moderate stringency conditions); and 0.1×SSC at about 68° C. (high stringency conditions). Washing can be carried out using only one of these conditions, for example, high stringency conditions, or each of the conditions can be used, for example, for 10 to 15 minutes each, in the order listed above, repeating any or all of the steps listed. Hybridization conditions that include formamide (e.g., 50% formamide), wherein hybridization is performed at a lower temperature (e.g., 40-50° C.), also can be used as is known in the art.
The term “nucleic acid” or “polynucleotide” is used broadly herein to mean a sequence of two or more deoxyribonucleotides or ribonucleotides that are linked together by a covalent bond. As such, the term “nucleic acid” or “polynucleotide” includes RNA and DNA, which can be a gene or a portion thereof, a cDNA, a synthetic polydeoxyribonucleic acid sequence, or the like, and can be single stranded, double stranded, triple stranded, tetra-stranded, or more, as well as DNA/RNA hybrids. A nucleic acid can be a naturally occurring polynucleotide, which can be isolated from a cell, or a synthetic molecule, which can be prepared, for example, by methods of chemical synthesis or by enzymatic methods such as by the polymerase chain reaction (PCR).
It should be recognized that a nucleic acid can be a target molecule, a molecular probe (which can be operably linked to a nanocode), and/or a component of a nanocode. As such, the nucleic acid can contain nucleoside or nucleotide analogs, or a backbone bond other than a phosphodiester bond. Thus, the nucleotides comprising a nucleic acid can include naturally occurring deoxyribonucleotides, such as adenine, cytosine, guanine or thymine linked to 2′-deoxyribose, or ribonucleotides such as adenine, cytosine, guanine or uracil linked to ribose, and/or can contain nucleotide analogs, including non-naturally occurring synthetic nucleotides or modified naturally occurring nucleotides. Such nucleotide analogs are well known in the art and commercially available, as are polynucleotides containing such nucleotide analogs (Lin et al., Nucl. Acids Res. 22:5220-5234, 1994; Jellinek et al., Biochemistry 34:11363-11372, 1995; Pagratis et al., Nature Biotechnol. 15:68-73, 1997).
The covalent bond linking the nucleotides of a polynucleotide can be a phosphodiester bond, or can be any of numerous other bonds, including a thiodiester bond, a phosphorothioate bond, a peptide-like bond or any other bond known to those in the art as useful for linking nucleotides to produce synthetic polynucleotides (see, for example, Tam et al., Nucl. Acids Res. 22:977-986, 1994; Ecker and Crooke, BioTechnology 13:351360, 1995). The incorporation of non-naturally occurring nucleotide analogs or bonds linking the nucleotides or analogs can be particularly useful where the nucleic acid (e.g., molecular probe and/or nanocode) is to be exposed to an environment that can contain a nucleolytic activity, including, for example, a tissue culture medium or an in vivo environment, since the modified polynucleotides can be less susceptible to degradation.
A nucleic acid containing naturally occurring nucleotides and phosphodiester bonds can be chemically synthesized or can be produced using recombinant DNA methods, using an appropriate polynucleotide as a template. In comparison, a nucleic acid comprising nucleotide analogs or covalent bonds other than phosphodiester bonds generally are chemically synthesized, although an enzyme such as T7 polymerase can incorporate certain types of nucleotide analogs into a polynucleotide and, therefore, can be used to produce such a polynucleotide recombinantly from an appropriate template (Jellinek et al., supra, 1995).
Depending on whether the nucleic acid comprises a target molecule, a molecular probe, a nanocode, or a component thereof, it can be of any length of interest, including about 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, 30,000, 40,000, 50,000, 75,000, 100,000, 150,000, 200,000, 500,000, 1,000,000, 1,500,000, 2,000,000, 5,000,000, or more bases or base pairs in length, up to a full length chromosomal DNA molecule. For example, a nucleic acid molecular probe comprising a transcription factor binding site (e.g., a gene regulatory element such as a promoter or enhancer) can be from about 5 to 1000 nucleotides (e.g., about 8 to 100, or 10 to 50), whereas a molecular probe comprising a hybridizing oligonucleotide can be from about 15 to 1000 (or more) nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 25, 30, 40, or more nucleotides). A nucleic acid component of a nanocode can vary, for example, from about 5 to 500 nucleotides (e.g., 10 to 100). In comparison, a target nucleic acid molecule can be from a few nucleotides in length (e.g., where the target is a gene regulatory element) to a full length chromosome (e.g., where an in situ hybridization procedure using a molecular probe operably linked to a nanocode is being used to identify a gene locus (or the absence thereof) on a chromosome). As such, the nucleic acids can be referred to, without limitation, as oligonucleotides and/or polynucleotides.
A specific binding pair can include two different types of molecules, e.g., a nucleic acid molecule and a polypeptide, either of which can be operably linked to a nanocode and serve as a molecular probe. For example, transcription factors are proteins that include a DNA binding domain, which specifically binds a target nucleic acid sequence (a transcriptional regulatory element), and a transcriptional activation (trans-activation) domain, which specifically binds other transcription factors and/or accessory proteins (e.g., eukaryotic initiation factors). As such, transcription factors, or a domain thereof, e.g., a DNA binding domain, can be operably linked to a nanocode and used as a molecular probe for SPM to identify gene regulatory element(s) specifically bound by the transcription factor. Alternatively, oligonucleotides that are designed to have a characteristic of a transcriptional regulatory element, or that are based on an upstream nucleotide sequence of a gene (i.e., 5′ to the coding sequence), can be operably linked to a nanocode and used as a molecular probe for SPM to identify a transcription factor that specifically binds to and regulates the expression of a particular gene. Methods for detecting specific binding of a protein and a nucleic acid are well known in the art and include, for example, gel electrophoresis mobility shift assays and affinity chromatography assays.
Accordingly, methods are provided for detecting a target molecule by contacting a sample, which contains or is suspected of containing (or which is known not to contain and is being used as a control)) the target molecule with a molecular probe, which is operably linked to a nanocode having a surface property, under conditions suitable for specific binding of the molecular probe to the target molecule; and detecting binding of the nanocode to the target molecule, when present, by SPM. According to the present methods, SPM is used to identify a molecular probe specifically bound to a target molecule, wherein a change in a signal due to a nanocode operably linked to the molecular probe in the presence of the target molecule as compared with the signal in the absence of the target molecule is indicative of specific binding of the molecular probe to the target molecule. As such, the present methods can be used to identify the presence (or absence) of a target molecule (e.g., in a sample such as a biological sample), and can further be used to determine the relative amount of a target molecule in a sample. Quantification of a target molecule can be performed, for example, by measuring the signal intensity of bound nanocode and comparing the signal to a calibration curve prepared using known amounts of a nanocode standardized preparation. By identifying the surface property of the nanocode using SPM, and knowing the specific molecular probe to which a nanocode having the particular surface property is operably associated, the identity of the molecular probe, as well as the identity of the target molecule to which the molecular probe is specifically bound, can be determined.
A target molecule can be any molecule that can be specifically bound by a molecular probe, including, for example, a biomolecule (e.g., a molecule that is produced in a living organism as part of a normal or abnormal biological process) or an environmental molecule (e.g., a pollutant or contaminant such as an industrial product,), which also can be a biomolecule. As such, the target molecule can be a protein, a peptide, a glycoprotein, a lipoprotein, a prion, a nucleic acid, a polynucleotide, an oligonucleotide, a lipid, a fatty acid, a carbohydrate, a glycolipid, a phospholipid, a sphingolipid, a lipopolysaccharide, a polysaccharide, a eukaryotic cell, a prokaryotic cell, a bacterium, a phage, a virus, a pathogen, or any organic or non-organic molecule for which a molecular probe (i.e., specific binding partner) is available (e.g., dioxin, which can be specifically bound by an aromatic hydrocarbon (dioxin) receptor (AhR) molecular probe).
In one embodiment, the method allows for the identification of a target molecule in a biological sample. The term “biological sample” is used herein to refer to any specimen comprising and/or obtained from a living organism. In certain embodiments, a biological sample can be a sample obtained from a subject of interest (e.g., a human patient suffering from an unknown disorder, or a patient believed to be suffering from a particular disorder), including, for example, a urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, cell, tissue, or organ sample. In further embodiments, the biological sample is a sample obtained from a mammalian subject, including, for example, a feline, canine, ovine, bovine, porcine, murine, or human subject.
A biological sample generally, though not necessarily, comprises a sample that contains or is suspected of containing a target molecule (i.e., a second specific binding pair member), and that allows for, or can be modified (e.g., diluted, concentrated, or fractionated) to allow for, detection by SPM of a nanocode operably linked to a molecular probe. The biological sample can be a cell, tissue or organ sample (e.g., a biopsy sample) comprising at least one (e.g., 1 to 10,000,000; 1000 to 10,000,000; or 1,000,000 to 10,000,000) somatic cell and/or germ cell, and can contain one or more biologic fluids that may normally be associated with the cell, tissue or organ (e.g., blood, lymph, or interstitial fluid). The biological sample also can be a cell, tissue or organ extract (or an aliquot or fraction thereof), which can, but need not, contain intact cells. As mentioned above, the biological sample also can be a biological fluid (e.g., urine, saliva, sputum, blood serum, cerebrospinal fluid, or semen.). Where the sample is a biological sample obtained from a mammalian subject (e.g., a human), it can be obtained by any method typically used to obtain the particular type of sample, including, for example, by surgery, biopsy, swab, stool, or other collection method. The sample also can be a forensic sample obtained, for example, at a crime scene or in association (or suspected association) with a crime, including, for example, a biological sample obtained from a weapon (e.g., a knife), clothes, or a wall or floor. In other embodiments, the biological sample contains, or is suspected of containing, a pathogen, for example a viral, fungal, or bacterial pathogen, or is obtained from a subject at risk of containing the pathogen (e.g., to monitor the susceptible subject for the presence of the pathogen). The biological sample also can be sample of a plant.
The nanocodes useful for preparing compositions and practicing embodiments of the present methods provide surface properties that are particularly suitable for detection by SPM. As such, SPM is used to detect nanocodes, particularly the surface properties (subfeatures) of nanocodes, that are operably linked to a molecular probe. SPM utilizes a variety of instruments that can measure the physical properties of objects on a nanometer-to-micrometer scale. Different modalities of SPM technology are available, any of which can be used for detection and/or identification of a nanocode according to embodiments of the invention. In general, an SPM instrument uses a laser beam or a very small, pointed probe in very close proximity to a surface to measure the properties of objects (see
Scanning tunneling microscopy (STM) is an SPM technique that relies on the existence of quantum mechanical electron tunneling between the STM probe tip and the sample surface. The tip is sharpened to a single atom point and is raster scanned across the surface, maintaining a probe-surface gap distance of a few angstroms without actually contacting the surface. A small electrical voltage difference (on the order of millivolts to a few volts) is applied between the probe tip and sample and the tunneling current between tip and sample is determined. As the tip scans across the surfaces, differences in the electrical and topographic properties of the sample cause variations in the amount of tunneling current. The relative height of the tip can be controlled by piezoelectric elements with feed-back control, interfaced with a computer. The computer can monitor the current intensity in real time and move the tip up or down to maintain a relatively constant current. In different embodiments, the height of the tip and/or current intensity may be processed by the computer to develop an image of the scanned surface.
Because STM measures the electrical properties of the sample as well as the sample topography, it can distinguish between different types of conductive material such as different types of metal (e.g., metal modifying agents linked to a nanocode). STM also can measure local electron density. Because the tunneling conductance is proportional to the local density of states (DOS), STM can be used to distinguish surface properties that vary in their electronic properties depending on the diameter and/or length of a subfeature. As such, STM can be used to detect and/or identify, as well as distinguish, any nanocodes that differ in their electrical properties. Where the nanocode is operably linked to a molecular probe, the identification of a particular nanocode consequently identifies the molecular probe and, where present, a target molecule specifically bound by the molecular probe.
Another modality of SPM is atomic force microscopy (AFM). Methods of biomolecule analysis by AFM are well known (see, e.g., Uchihashi et al., “Application of Noncontact-Mode Atomic Force Microscopy to Molecular Imaging”, on the world wide web, at URL “http://www.foresight.org/Conferences/MNT7/Abstracts/Uchihashi”). In AFM microscopy, the probe is attached to a spring-loaded or flexible cantilever that is in contact with the surface to be analyzed. Contact is made within the molecular force range (i.e., within the range of interaction of van der Waals forces).
Within AFM, different modes of operation are possible, including contact mode, non-contact mode and TappingMode™ mode. In contact mode, the atomic force between probe tip and sample surface is measured by keeping the tip-to-sample distance constant and measuring the deflection of the cantilever, typically by reflecting a laser off the cantilever onto a position sensitive detector. Cantilever deflection results in a change in position of the reflected laser beam. As in STM, the height of the probe tip in AFM can be computer controlled using piezoelectric elements with feedback control. In an embodiment of the invention, a relatively constant degree of deflection is maintained by raising or lowering the probe tip. Because the probe tip may be in actual (van der Waals) contact with the sample, contact mode AFM tends to deform non-rigid samples. In non-contact mode, the tip is maintained between about 50 to 150 angstrom above the sample surface and the tip is oscillated. Van der Waals interactions between the tip and sample surface are reflected in changes in the phase, amplitude or frequency of tip oscillation. The resolution achieved in non-contact mode is relatively low.
In TappingMode™ mode, the cantilever is oscillated at or near its resonant frequency using piezoelectric elements. The AFM tip periodically contacts (taps) the sample surface, at a frequency of about 50,000 to 500,000 cycles per second in air and a lower frequency in liquids. As the tip begins to contact the sample surface, the amplitude of the oscillation decreases. Changes in amplitude are used to determine topographic properties of the sample. Because AFM analysis does not depend on electrical conductance, it can be used to analyze the topological properties of non-conductive materials. The nanocodes disclosed herein, including, for example, mosaic nanocodes and/or nanocodes containing bubbles and/or branches, and that can, but need not contain modifying objects, differ in their topological properties and, therefore, can be detected and/or identified using AFM techniques.
In alternative modes of AFM, information in addition to the topological profile of the sample can be obtained. For example, in lateral force microscopy (LFM), the probe is scanned perpendicular to its length and the degree of torsion of the cantilever is determined. Cantilever torsion will be dependent on the frictional characteristics of the surface. Since the frictional characteristics of coded probes may vary depending on their composition, LFM can be useful to detect and identify different nanocodes.
Another variation of AFM is chemical force microscopy (CFM), in which the probe tip is functionalized with a chemical species and scanned over a sample to detect adhesion forces between the chemical species and the sample (Frisbie et al., Science 265:2071-2074, 1994). Chemicals with differing affinities for nanocode materials (e.g., gold or silver) can be incorporated into an AFM probe tip and scanned across a surface to detect and identify nanocodes. Another SPM mode of potential use is force modulation imaging (Maivald et al., Nanotechnology 2:103, 1991). Uchihashi et al. (supra) disclose a method of biomolecule imaging using frequency modulation in non-contact mode AFM.
Other SPM modes that can be used to detect and/or identify nanocodes as disclosed herein include magnetic force microscopy (MFM), high frequency MFM, magnetoresistive sensitivity mapping (MSM), electric force microscopy (EFM), scanning capacitance microscopy (SCM), scanning spreading resistance microscopy (SSRM), tunneling AFM, and conductive AFM. In certain of these modalities, magnetic properties of a sample can be determined, for example, of nanocodes modified to contain appropriate metal ions. SPM instruments of use for coded probe detection and/or identification are commercially available (e.g., Veeco Instruments, Inc., Plainview N.Y.; Digital Instruments, Oakland, Calif.). Alternatively, a custom designed SPM instrument can be used.
In another embodiment, a molecular characterization system is provided for biomolecule analysis. A molecular characterization system 1400 is illustrated in
In the characterization process, certain molecular probes 438 of molecular probe/nanocodes 430 specifically bind (e.g., selectively hybridize) with the sample molecule 420. If a known molecular probe 438 hybridizes at a specific location on the sample molecule 420, an inference can be made about characteristics of the sample molecule (e.g., that the sample molecule 420 comprises a target molecule that is specifically bound by the molecular probe 438). In the characterization process, other molecular probes 448 associated with other molecular probe/nanocodes 440 will not specifically bind the sample molecule 420 because the sample molecule 420 does not comprise a target molecule. Such molecular probe/nanocodes 440 can be removed (e.g., eluted) from the reaction chamber 410 at a chamber outlet 414.
Referring to
As illustrated in
A molecular characterization system can be operably linked, for example, to an information processing and control system, which can be used to analyze data obtained from an SPM instrument and/or to control the movement of the SPM probe tip, the modality of SPM imaging used and the precise technique by which SPM data is obtained. An exemplary information processing system may incorporate a computer comprising a bus for communicating information and a processor for processing information.
Referring to
For run-time operation, the system 100 further includes an operating system program 16. In one embodiment, the operating system program 16 resides in a non-volatile storage device 18, such as a hard disk drive or compact disk (CD) ROM. In a second embodiment, the operating system program 16 does not reside on the system 1000. Instead, the system 1000 is accessible to a network (not shown) by a network interface card 26. Once the network connection is made, the operating system software 16 can be downloaded to the system 1000. In one embodiment of system 1000 containing processor 10a and optional processor 10b, the BIOS program 14 retains control of a portion of the system 1000 instead of relinquishing full control of the system 1000 to the operating system 16. This enables the BIOS program 14 to minimally configure and test the system 1000 such that the operating system can be booted more expeditiously. Further, the portion of the system that was not relinquished to the operating system 16 can be fully initialized and tested during runtime, e.g., after the system 1000 is fully capable. Accordingly, as illustrated in
In one embodiment, the system 1000 further includes a configuration table 22, which is accessible to both the BIOS program 14 and the operating system program 16. Configuration table 22 includes information about resources within and connected to the system 1000. In particular, configuration table 22 supplies the operating system 16 with the amount of memory 20 available in the system 1000 as well as the number and type of processor(s) 10. In one embodiment, the processor(s) of a system is selected from the Pentium® family of processors, including without limitation the Pentium® II family, the Pentium® III family and the Pentium® 4 family of processors available from Intel Corp. (Santa Clara, Calif.). In alternative embodiments, the processor can be a Celeron® processor, an Itanium® processor, an X-Scale® processor, or a Pentium Xeon® processor (Intel Corp., Santa Clara, Calif.). In various other embodiments of the invention, the processor may be based on Intel® architecture, such as Intel® IA-32 or Intel® IA-64 architecture. Other processors also can be used.
As discussed above, the information processing system (computer) can further include a random access memory (RAM) or other dynamic storage device, a read only memory (ROM) or other static storage and a data storage device such as a magnetic disk or optical disc and its corresponding drive. The information processing system also can include other peripheral devices known in the art, including, for example, a display device (e.g., cathode ray tube or liquid crystal display), an alphanumeric input device (e.g., keyboard), a cursor control device (e.g., mouse, trackball, or cursor direction keys) and/or a communication device (e.g., modem, network interface card, or interface device used for coupling to an Ethernete link, token ring, or other type of network).
In particular embodiments of the invention, an SPM unit can be connected to the information processing system. Data from the SPM can be processed by the processor and data stored in the main memory. The processor can analyze the data from the SPM to identify a nanocode and, therefore, a molecular probe and/or target molecule associated therewith.
It is appreciated that a differently equipped information processing system can be used for certain implementations and, therefore, that the configuration of the system can vary. For example, while the processes disclosed herein can be performed under the control of a programmed processor, in alternative embodiments, the processes can be fully or partially implemented by any programmable or hardcoded logic, such as Field Programmable Gate Arrays (FPGAs), TTL logic, or Application Specific Integrated Circuits (ASICs), and the like. Additionally, the disclosed methods can be performed by any combination of programmed general purpose computer components and/or custom hardware components.
In certain embodiments of the invention, custom designed software packages can be used to analyze the data obtained from an SPM. In other embodiments, data analysis can be performed, for example, using an information processing system and publicly available software packages. Non-limiting examples of available software for DNA sequence analysis include the PRISM™ DNA Sequencing Analysis Software (Applied Biosystems; Foster City Calif.), the Sequencher™ package (Gene Codes; Ann Arbor Mich.), and a variety of software packages available through the National Biotechnology Information Facility on the worldwide web at the URL “nbif.org/links/1.4.1.php”.
Accordingly, in one embodiment, a molecular characterization system is provided, wherein the molecular characterization system includes a reaction chamber, which can contain at least one sample, and at least one molecular probe operably linked to a nanocode. In another embodiment, the reaction chamber comprises a substrate, which contains the sample or a plurality of samples, which can be the same or different or a combination thereof. In still another embodiment, the molecular characterization system comprises a plurality of molecular probes, wherein each molecular probe of the plurality is operably linked to a nanocode, wherein molecular probes of the plurality are the same or different, and wherein each of different molecular probes is operably linked to the same or different nanocodes. In one aspect, each of different molecular probes is operably linked to each of different nanocodes, wherein the nanocode identifies the molecular probe.
In another embodiment, an apparatus is provided, wherein the apparatus includes a substrate, which comprises a molecular probe operably linked to a nanocode; an SPM probe, which can detect a surface property of the nanocode, wherein the SPM probe is in operable association with the substrate; and a detector operably coupled to the SPM probe, wherein the detector provides a signal representative of a surface property of the nanocode. Such an apparatus can further include a processor, which can detect specific binding of the molecular probe comprising the nanocode to a target molecule. In various embodiments, the nanocode can be a mosaic biomolecule comprising a nucleic acid bound to a polypeptide; a nucleic acid molecule comprising regions of differing strandedness; a branched nucleic acid molecule; a nucleic acid molecule comprising an object that affects the surface property of the nanocode; or a combination thereof.
In still other embodiments, solutions containing one or more nanocodes as disclosed herein can be applied to property (e.g., personal property or commercial property) for security tracking purposes, as is known in the art. For example, SmartWater Ltd., a British company, has developed methods to mark valuables with fluids containing strands of digital DNA. The DNA is virtually impossible to wash off of the article and can be used to uniquely identify expensive items or heirlooms. The DNA can be detected by any forensic laboratory. Such methods can also be utilized to mark items with the molecular nanocodes disclosed herein. In such applications, detection of the nanocode would not require forensic analysis based on DNA sequence.
An apparatus for barcode preparation, use, and/or detection can be incorporated into a larger apparatus and/or system. In certain embodiments, the apparatus can include a micro-electro-mechanical system (MEMS). MEMS are integrated systems including mechanical elements, sensors, actuators, and electronics, all of which can, but need not, be manufactured by microfabrication techniques on a common chip, of a silicon-based or equivalent substrate (see, e.g., Voldman et al., Ann. Rev. Biomed. Eng. 1:401-425, 1999). The sensor components of MEMS can be used to measure mechanical, thermal, biological, chemical, optical and/or magnetic phenomena to detect barcodes. The electronics can process the information from the sensors and control actuator components such as pumps, valves, heaters, and the like, thereby controlling the function of the MEMS.
The tapered portion 20 of the beam includes a proximal end 30 and a distal end 32. The proximal end 30 is attached to the anchor 12, while the distal end 32 is attached to the actuation portion 22. The tapered portion 20 of the beam is vertically offset relative to the anchor 12 to provide a space 34 between the actuation portion 22 and the actuation electrode 26. The tapered portion 20 of the beam can be relatively thick (approximately 6 μm), and can be made of a highly conductive material such as gold, or combination of materials (e.g., a composite construction). The gap 34 between the actuation electrode 26 and the actuation portion of the beam generally is small (e.g., approximately 5 μm), although, in various embodiments, a greater or lesser gap can be used.
The actuation portion 22 is mounted to the distal end 32 of the tapered portion 20 of the beam. The actuation portion 22 is relatively wide compared to the tapered portion 20, to provide a greater area over which the force applied by the activation of the actuation electrode 26 can act. Since actuation force is proportional to the area of the actuation portion 22, the wider and longer actuation portion 22 of the beam causes a larger force to be applied to the beam when the actuation electrode 26 is activated, resulting in a faster switch response. Like the tapered portion 20, the actuation portion 22 also can be made of a highly conductive material or combination of materials.
A tip 24 is attached to the actuation portion 22 of the beam opposite from where the tapered portion 20 is attached. On the lower side of the tip 24 is a contact dimple 36, which functions to make contact with the electrode 29 when the cantilever beam 18 deflects in response to a charge applied to the actuation electrode 26. The tip 24 is vertically offset from the actuation area, much like the tapered portion 20 is offset vertically from the anchor 12; the vertical offset of the tip 24 relative to the actuation area 22 reduces capacitive coupling between the beam 18 and the second portion 29 of the signal line.
In operation of the switch 10, the anchor 12 is in electrical contact with, and forms part of, a first portion 28 of a signal line carrying an electrical signal. Opposite the first portion 28 of the signal line is a second portion 29 of the signal line. To activate the switch 10 and make the signal line continuous, such that a signal traveling down the first portion 28 of the signal line will travel through the switch 10 and into the second portion 29 of the signal line, the actuation electrode 26 is activated by inducing a charge in it. When the actuation electrode 26 becomes electrically charged, because of the small gap between the actuation electrode and the actuation portion 22 of the beam, the actuation portion of the beam is drawn toward the electrode such that the beam 18 deflects downward, bringing the contact dimple 36 in contact with the second electrode 29, thus completing the signal line and allowing a signal to pass from the first portion 28 of the signal line to the second portion 29 of the signal line.
The electronic components of MEMS can be fabricated using integrated circuit (IC) processes (e.g., CMOS or Bipolar processes), and can be patterned using photolithographic and etching methods for computer chip manufacture. The micromechanical components can be fabricated using compatible “micromachining” processes that selectively etch away parts of the silicon wafer or add new structural layers to form the mechanical and/or electromechanical components. Basic techniques in MEMS manufacture include depositing thin films of material on a substrate, applying a patterned mask on top of the films by some lithographic methods, and selectively etching the films. A thin film can be in the range of a few nanometers to 100 micrometers. Deposition techniques of use can include chemical procedures such as chemical vapor deposition (CVD), electrodeposition, epitaxy and thermal oxidation and physical procedures like physical vapor deposition (PVD) and casting. Methods for manufacture of nanoelectromechanical systems can also be used (see, e.g., Craighead, Science 290:1532-36, 2000).
In some embodiments, apparatus and/or detectors can be connected to various fluid filled compartments, for example microfluidic channels or nanochannels. These and other components of the apparatus can be formed as a single unit, for example, in the form of a chip (e.g. semiconductor chips) and/or microcapillary or microfluidic chips. Alternatively, individual components can be separately fabricated and attached together. Any material known for use in such chips can be used in the disclosed apparatus, including, for example, silicon (e.g., nanocrystalline silicon), silicon dioxide, polydimethyl siloxane (PDMS), polymethylmethacrylate (PMMA), plastic, glass, quartz, etc. As used herein, the term “nanocrystalline silicon” refers to silicon that comprises nanometer-scale silicon crystals, typically in the size range from 1 to 100 nanometers (nm). The term “porous silicon” refers to silicon that has been etched or otherwise treated to form a porous structure.
Techniques for batch fabrication of chips are well known in computer chip manufacture and/or microcapillary chip manufacture. Such chips can be manufactured by any method known in the art, such as by photolithography and etching, laser ablation, injection molding, casting, molecular beam epitaxy, dip-pen nanolithography, chemical vapor deposition (CVD) fabrication, electron beam or focused ion beam technology or imprinting techniques. Non-limiting examples include conventional molding, dry etching of silicon dioxide; and electron beam lithography. Methods for manufacture of nanoelectromechanical systems can be used for certain embodiments (see Craighead, supra, 2000.) Various forms of microfabricated chips are commercially available (e.g., Caliper Technologies Inc., Mountain View Calif.; ACLARA BioSciences Inc., Mountain View).
For fluid-filled compartments that can be exposed to various analytes, for example, nucleic acids, proteins and the like, the surfaces exposed to such molecules can be modified by coating, for example, to transform a surface from a hydrophobic to a hydrophilic surface and/or to decrease adsorption of molecules to a surface. Surface modification of common chip materials such as glass, silicon, quartz and/or PDMS is known (e.g., U.S. Pat. No. 6,263,286). Such modifications can include, for example, coating with commercially available capillary coatings (Supelco; Bellafonte Pa.), silanes with various functional (e.g. polyethyleneoxide or acrylamide, etc.).
In certain embodiments, such MEMS apparatus can be use to prepare nanocodes, to separate formed nanocodes from unincorporated components, to expose nanocodes or compositions containing the nanocodes (e.g., a molecular probe having an nanocode operably linked thereto) to targets, and/or to detect nanocodes bound to target molecules.
In other embodiments, kits are provided that include at least one (e.g., 1, 2, 3, 4, 5, etc.) or a plurality of nanocode(s), which can, but need not, be operably linked to a molecular probe, or can be in a form such that one or more conveniently can be operably linked to molecular probes comprising any of various chemical compositions (e.g., peptide molecular probes, nucleic acid molecular probes, etc.). As such, the nanocode(s) of the kit can contain a reactive group suitable for operably linking the nanocode to a particular type of molecular probe (e.g., a peptide molecular probe or a nucleic acid molecular probe), or the nanocodes can be in a form such that they can be modified such that, upon contact with the molecular probe and appropriate reagents, the nanocode is operably linked to the molecular probe. In one embodiment, the kit contains a plurality of different nanocodes, each of which can be operably linked to a molecular probe. In another embodiment, the nanocode(s) of the kit is (are) immobilized on a solid support, particularly in a form such that a molecular probe can be operably linked to the nanocode. In another embodiment, the kit contains a plurality of molecular probes (e.g., oligonucleotide probes such as a plurality of randomized oligonucleotides; ligands such as a plurality of receptor binding ligands; or antibodies such as a plurality of monoclonal antibodies each of which is specific for a target molecule), each probe of which is (or can be) operably linked to a particular nanocode.
In another embodiment of the invention, biomolecular probes, which include a molecular probe operably associated with a nanocode, are provided, wherein the molecular probe can specifically bind a biological material (target molecule). Also provided is a molecular identification assembly, which includes a nanocode, and a molecular probe operably associated with the nanocode. The molecular probe can be any molecular probe as disclosed herein or otherwise known in the art (e.g., a nucleic acid molecule or a polypeptide), and the nanocode can, but need not, further contain an operably linked object having a surface property detectable by SPM (e.g., a nanoparticle).
A plurality of molecular identification assemblies is provided, including a plurality of molecular identification assemblies in which two or more molecular identification assemblies of the plurality are the same or different. Molecular identification assemblies of a plurality can include same molecular probes operably linked to same nanocodes, same molecular probes operably linked to different nanocodes, different molecular probes operably linked to same nanocodes, different molecular probes operably linked to different nanocodes, and combinations thereof
In another embodiment, a molecular identification assembly, or each molecular identification assembly of a plurality, is immobilized onto a solid substrate (e.g., a chip, wafer, or bead). Where a plurality of molecular identification assemblies is immobilized on the solid substrate, they can be immobilized in an array, which can be an addressable array. An addressable array allows for conveniently practicing the methods of the invention in a high throughput format, wherein one or more, including all, steps of the method are performed automatically (e.g., using robotic devices that dispense and/or remove reagents from specified positions in the array at or after specified times, or that perform SPM to detect the surface property of a nanocode in a specified position). In another embodiment, the presence of multiple target molecules in a sample can be determined in a single (multiplex) reaction because different nanocodes linked to different molecular probes can be distinguishably detected.
A solid substrate provides a surface for immobilizing a nanocode and associated molecular probe, or performing a reaction. For example, the surface of the substrate can be modified to contain wells in which a reaction can be performed. Such modification can be performed, for example, using photolithography, stamping techniques, molding techniques or microetching techniques, the particular method being selected, for example, based on the composition and shape of the substrate. The surface of a substrate also can be modified to contain chemically derived sites that can be used to attach a molecular probe comprising an operably linked nanocode, or other material as desired, on the substrate. The addition of a pattern of chemical functional groups, such as amino groups, carboxy groups, oxo groups and thiol groups can be used to covalently attach molecules having the corresponding reactive functional groups or linker molecules, to which the desired molecules, in turn, can be bound.
The following examples are intended to illustrate but not limit the invention;
This example provides methods for synthesizing nanocode subunits and nanocode, and methods for modifying the subunits and nanocodes using tags such as gold particles and ferritin.
Precision Biology (PB) nanocode DNA structures were designed and prepared by scientists at Intel Precision Biology Group. Nanocodes were constructed from double stranded and branched DNA molecules derived from the pBK-CMV plasmid (Stratagene Corp., La Jolla Calif.; see
Different versions of a nanocode structure, designated “PB2”, are shown in
Modified versions of the PB2C nanocode containing non-DNA tags attached to the branches were constructed (designated “PB3”) as shown in
Nanocode XPB2, which containing two linked nanocode PB2 structures, was constructed as shown
Nanocode PB2ex was constructed as shown in
These results demonstrate that basic subunits can be constructed for use in generating a variety of nanocodes, which can be modified and/or further used as building blocks of additional nanocodes.
This example demonstrates that DNA based nanocodes can be detected using a scanning probe microscopy probe.
Scanning probe microscopy (SPM) was performed using a Digital Instruments Dimension™ 5000 instrument (VEECO Instruments; Fremont Calif.) and TappingMode™ imaging. Nanosensors™ Silicon non-contact cantilevers having a nominal frequency of about 300 kHz (corresponding to 125 μm long cantilevers) were used. Scan dimensions depended on the sample, and ranged nominally from 0.5 μm to 5 μm, with scan rates of 0.5 Hz to 1 Hz.
Control experiments examining imaging of different sized gold particles and different substrates were performed. Gold particles having sizes of 50 nm, 10 nm, 5 nm and 2 nm particles were purchased from Ted Pella, Inc. (Redding Calif.), and used as imaging controls on two different substrates, poly-L-lysine coated glass coverslips and 3-aminopropyltriethoxysilane (AP) treated mica (vapor phase silylation of freshly cleaved mica for 2 hr with 3-aminopropyltriethoxysilane). For the poly-L-lysine coated glass coverslips, 10 μl of gold colloidal solution was placed on the coverslips and allowed to dry. For the AP-mica substrate, 100 μl of gold colloidal solution was placed on the substrate for 15 min, then wicked off using a KIMWIPE tissue.
Atomic force microscopy (AFM) was performed using a poly-L-lysine coated coverslip (control), and on poly-L-lysine coated coverslips containing 50 nm gold nanoparticles, 10 nm gold nanoparticles, or 5 nm gold nanoparticles. In the control, features as high as 10 nm were observed. Poly-L-lysine was suitable for immobilization of 50 nm gold nanoparticles (see
In another experiment, lambda-DNA (New England Biolabs, Inc.) was imaged as a control in AP-mica. Ten μl of lambda-DNA solution (10 ng/μl in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA) was incubated on AP-mica for 15 min, then rinsed off gently with NANOPURE deionized (DI) water and blow-dried with N2. Imaging near the center of the DNA spot revealed that the concentration of DNA was too high, whereas imaging obtained at the edges of the spot, where alignment had occurred due to rinsing, revealed the lambda DNA structures.
These experiments demonstrate that AP-mica is a suitable substrate for resolving the sizes of nanostructures by SPM (e.g., 2 nm nanoparticles can be distinguished from 5 nm nanoparticles by height).
In order to optimize imaging of the nanocodes, various nanocodes, including tagged nanocodes, were placed on the AP-mica substrate under different conditions, and the samples were imaged. Ten μl of PB2C (see
Following deposition of optimal concentrations of DNA solution, sub-features were observed in the strands of PB2C. In order to confirm that true sub-features were being detected, and not artifacts, a mixture of a 2.8 kb linearized plasmid double stranded DNA and PB2C was imaged. When the DNA solutions were premixed before deposition, the imaging did not yield good results, as the DNA tended to aggregate in some regions and was very sparse in other regions. To overcome this difficulty, the DNA solutions of linearized plasmid and PB2C were spotted alongside each other and the substrate was gently shaken for 10 sec to induce diffusion of smaller PB2C DNA. AFM imaging of the interface between nanocode PB2 and 2.8 kb linearized plasmid DNA revealed that sub-features of nanocode PB2 were clearly evident (
In another experiment, PB2C was intercalated with ethidium bromide to determine whether sub-features were enhanced. PB2C was intercalated with ethidium bromide (EtBr) at 1 μl (1 mg/ml concentration EtBr solution) DNA to 10 μl of PB2C stock solution (100 ng/μl). AFM images were obtained of nanocode PB2 intercalated with ethidium bromide, and sub-features were detected.
PB3-Fe was prepared by adding 2 μl of PB2C solution (100 ng/μl) to 2 μl of ferritin stock solution, and diluting it with 16 μl of 1×TE buffer (final concentration of PB3-Fe was 10 ng/μl). The solution was further diluted to 1 ng/μl, then 20 μl was incubated on AP-mica for 10 min, dipped and rinsed off gently in NANOPURE DI water, and blow-dried with N2. Different parts of the substrate were observed to have different concentrations of ferritin, DNA, and ferritin-labeled DNA (see
PB3-G was prepared by labeling PB2C, which contained biotin sites, with different sizes of gold (Au) particles. Different barcodes were synthesized by attaching 5 nm and 1 nm Au nanoparticles to PB2C (5115, 1551, etc.) to obtain PB3-G-5115, PB3-G-1551, etc. Initial imaging was performed on samples in which the ligation buffer contained dithiothreitol (DTT), and blind tests were performed with samples that were synthesized using ligation buffer that was free of DTT to determine whether DTT affected binding of the gold particles to the DNA nanocode. The stock solution of PB3-G (approx. 1.2 ng/μl) was diluted to different concentrations in 1×TE Buffer at dilutions of 1:5, 1:50 and 1:100. Images were obtained for PB3-G-5115 (synthesized with buffer containing DTT containing ligation buffer) at different dilutions, and, in blind test, in the absence of DTT. AFM imaging of nanocode PB35115 on AP-mica was obtained at a 1:5 dilution of a 1.2 ng/μl solution and at a 1:100 dilution of a 1.2 ng/μl solution, and of nanocode PB3 deposited on the substrate in the presence or absence of dithiothreitol. Although attachment and ligation appeared to be incomplete and the efficiency was low under either condition (with or without DTT), partially labeled strands of DNA at low concentrations along with the gold particles were imaged.
PB3-B was prepared by tagging PB2B with gold (Au) nanoparticles using a PNA based strategy. The stock solution of PB3B (approx. 3.5 ng/μl) was diluted to different concentrations in 1×TE buffer at dilutions of 1:5, 1:50 and 1:100, and imaged. AFM imaging of PB3B-G at a 1:100 dilution of a 3.5 ng/μl stock solution showed individual DNA strands that appeared to be partially labeled (
PB2ex, which is a modified PB2 nanocode containing longer branches and different sub-features, was purified by gel isolation and imaged. One set of samples showed a high background, whereas a second set of samples had a clean background, though the images showed the DNA strands to be coiled.
These results demonstrate that imaging of DNA-based nanocodes can be optimized by varying the concentration of samples comprising the nanocodes and by varying the conditions used for depositing samples comprising the nanocodes on an imaging substrate.
This example provides methods for producing nanocodes that contain bubbles having specified sizes in specified positions.
Nanocodes containing bubbles can be constructed of a single type of polymer or of different types of polymers (mosaic nanocodes).
To construct a mosaic nanocode containing bubbles as shown in
To construct a nanocode containing bubbles, and that is composed of a single type of polymer such as a nucleic acid as exemplified in
This example provides methods for producing nanocodes that contain branches having specified sizes in specified positions.
To construct a branched nanocode as exemplified in
To construct a modified branched nanocode as exemplified in
Additional nanocodes can be constructed by combining the methods described in Examples 1 to 4, for example, to produce nanocodes containing bubbles and branches.
Although the invention has been described with reference to the above example, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.
