Patent Application
20120208174
Not applicable.
1. Field of the Invention
The present invention relates to the fields of surface plasmonic sensing compositions, methods and devices for the detection of molecular binding on membrane surfaces.
2. Related Art
Metal nanostructures can be used for the label-free optical detection of molecular binding to surfaces. This is due to strong, environmentally-sensitive light scattering caused by the localized surface plasmon resonance (LSPR) of electrons at the metal surface. (Kelly, K.; Coronado, E.; Zhao, L.; Schatz, G. Journal of Physical Chemistry B 2003, 107, 668-677). Characteristic LSPR spectra exist for a variety of shapes and configurations of particles, (See Baciu, C. L.; Becker, J.; Janshoff, A.; Sonnichsen, C. Nano Letters 2008, 8, 1724-1728; Yonzon, C. R.; Jeoungf, E.; Zou, S. L.; Schatz, G. C.; Mrksich, M.; Van Duyne, R. P. Journal of the American Chemical Society 2004, 126, 12669-12676; Prikulis, J.; Hanarp, P.; Olofsson, L.; Sutherland, D.; Kall, M. Nano Letters 2004, 4, 1003-1007; Tao, A.; Sinsermsuksakul, P.; Yang, P. D. Angewandte Chemie-International Edition 2006, 45, 4597-4601; and Tao, A.; Sinsermsuksakul, P.; Yang, P. Nature Nanotechnology 2007, 2, 435-440) and similar to conventional surface plasmon resonance (SPR) spectrometry, the scattering spectra of the nanostructures are dependent upon the refractive index of the surrounding medium, which enables the detection of molecular binding to or near the nanostructure surface.(Zhao, J.; Das, A.; Zhang, X.; Schatz, G.; Sligar, S.; VanDuyne, R. Journal of the American Chemical Society 2006, 128, 11004-11005, Haes, A.; Chang, L.; Klein, W.; VanDuyne, R. Journal of the American Chemical Society 2005, 127, 2264-2271).
There is a pronounced need for analytical technology capable of probing molecular interactions in a cell membrane environment. Most biochemical processes involve membranes at some point and, correspondingly, over half of the one hundred best selling marketed drugs target cellular membrane-associated proteins[Drews, J. Science 2000, 287, 1960-1964, Yildirim, M. A.; Goh, K.-I.; Cusick, M. E.; Barabasi, A.-L.; Vidal, M. Nat Biotech 2007, 25, 1119-1126]. To address this need, there has been significant interest in supported lipid bilayers[Sackmann, E. Science 1996, 271, 43-48, Groves, J. T. Current Opinion in Drug Discovery and Development 2002, 5, 606-612, and Tanaka, M.; Sackmann, E. Nature 2005, 437, 656-663], which share many of the same properties as cellular membranes. In particular, supported membranes retain lateral fluidity, and allow membrane components to rearrange naturally in response to molecular interactions. Furthermore, membrane proteins are notoriously difficult to work with outside of the membrane environment; supported membranes offer a strategy to handle these. G-protein coupled receptors provide a case in point, and have been screened in a supported membrane microarray format[Bieri, C.; Ernst, O. P.; Heyse, S.; Hofmann, K. P.; Vogel, H. Nat Biotech 1999, /7, 1105-1108, Fang, Y.; Frutos, A. G.; Lahiri, J. ChemBioChem 2002, 3, 987-991]. Supported membranes have also been used to reconstitute protein-protein signaling at membrane surfaces[Gureasko, J.; Galush, W. J.; Boykevisch, S.; Sondermann, H.; Bar-Sagi, D.; Groves, J. T.; Kuriyan, J. Nat Struct Mol Biol 2008, 15, 452-461] and to imitate one face of cell-cell junctions at T-cell synapses[Groves, J. T.; Dustin, M. L. Journal of Immunological Methods 2003, 278, 19-32, Mossman, K. D.; Campi, G.; Groves, J. T.; Dustin, M. L. Science 2005, 310, 1191-1193].
Many drug targets are membrane-resident, so detecting binding at membranes is important for drug discovery, as well as biological research more generally. There are no commercial platforms that provide a way to screen these kinds of interactions in a phospholipid membrane environment.
The most common, comparable, way of probing molecular interactions for drug discovery in vitro is surface plasmon resonance (SPR), which involves very expensive instrumentation and consumables. Surface plasmon resonance is also not compatible with phospholipid membranes. There is a relative lack of techniques for measuring interfacial binding at membrane surfaces, and especially a lack of techniques which are label-free. The most common surface binding tools, such as surface plasmon resonance (SPR), are generally not compatible with membranes. In fact, there is no commercially standard technique for measuring binding at bilayer membrane surfaces. While there are academic studies that use surface-based noble metal nanostructures as membrane binding sensors, many require burdensome technical requirements such as micropatterning of substrates.
Thus there is a need for parallel high-throughput applications for the detection of binding at membrane surfaces which require only simple readout as distinguished from conventional SPR and related nanomaterial-based sensors.
The present invention provides a sensor for detecting the binding of molecules to membrane surfaces. In one embodiment, the sensor comprising nanoscale silver cubes deposited on a glass surface and which are embedded in a phospholipid membrane that coats the entire surface of the device.
In another aspect, the present invention provides a composition comprising, a substrate having a continuous membrane coating, wherein the substrate features nanoparticles disposed between the membrane coating and the substrate. In some embodiments, the substrate is planar, spherical or a wall of a microfluidic channel. The membrane coating over the substrate is a supported lipid bilayer and the membrane coating over the nanoparticles is a hybrid lipid bilayer. The nanoparticles comprises nanopolyhedras. In one embodiment, the nanopolyhedra is a nanocube. The nanoparticles can comprise a metal, a semiconductor material, multi-layers of metals, a metal oxide, an alloy, a polymer, or carbon nanomaterials. In one embodiment, the nanoparticles comprise metal such as gold or silver.
In a further aspect, to form the hybrid lipid bilayer, the nanoparticles are chemically modified to display a self-assembled monolayer. In one embodiment, the membrane coating further comprising a ligand within the membrane. In another embodiment, the sensor further comprising an analyte molecule possibly capable of binding the ligand, wherein the analyte is a cell-surface protein or a functionalized lipid headgroup.
One object of the invention is to provide a nano-plasmonic sensing device having simplicity of fabrication and of readout. In one embodiment, the manufacture of the basic sensor surface is based on a series of solution-based deposition and wash steps, and the readout is using simple absorbance spectrophotometry in an off-the-shelf instrument. The device presented herein is potentially easily parallelized for high-throughput applications, which distinguishes it from conventional SPR and related nanomaterial-based sensors.
Thus the invention also provides a method comprising: (a) contacting a target molecule with a substrate having a continuous membrane coating a plurality of nanoparticles disposed between the membrane and the substrate, (b) applying a molecule possibly capable of binding the target molecule, and (c) detecting plasmon generated phenomena at a nanoparticle.
In one embodiment, the plasmon-generated phenomena is optically detectable. In another embodiment, the step of detecting plasmon-generated phenomena comprises detecting light selected from absorbed light, reflected light, scattered light, or any combination thereof, and further wherein the method of detection comprises any combination selected from imaging, spectral characterization, intensity measurement, interferometry, and interference fringe analysis.
In another embodiment, the method further comprising: detecting a spectral shift in the known spectra of the nanoparticles, wherein such a spectral shift indicates the presence of the molecule possibly capable of binding the target molecule.
In one embodiment, the target molecule is a cell-membrane protein or a functionalized lipid headgroup.
In one embodiment, a sensor comprising a substrate having nanoparticles embedded on said substrate and a continuous supported lipid membrane coating said substrate and nanoparticles, wherein the nanoparticles are chemically modified to display a self-assembled monolayer such that subsequent exposure of the surface to lipid vesicles results in formation of a continuous lipid membrane coating the nanoparticles and the supporting substrate.
In another embodiment, a method for detecting an analyte of interest comprising the steps of: (a) providing a substrate having a continuous membrane coating, wherein the substrate features nanoparticles disposed between the membrane coating and the substrate, wherein the nanoparticles have a known spectra, and wherein the continuous membrane displays a ligand for the analyte of interest; (b) applying a sample suspected of containing a target analyte of interest to the substrate; (c) detecting plasmon generated phenomena at the nanoparticles, whereby a spectral shift in the known spectra of the nanoparticles indicates that the target analyte is bound to the ligand.
The ligand within the membrane can be oligonucleotides, ribonucleic acid residues, deoxyribonucleic acid residues, polypeptides, proteins, receptors, carbohydrates, a lipid-linked small molecule, thyroxine binding globulin, antibodies, enzymes, Fab fragments, lectins, nucleic acids, nucleic acid aptamers, avidin, protein A, barsar, complement component C1q, or other organic or inorganic molecules having a binding affinity for an analyte of interest.
Analytes of interest that can be detected include nucleic acid molecules, proteins, peptides, haptens, metal ions, drugs, metabolites, pesticides, pollutants, toxins, hormones, enzymes, lectins, proteins, signaling molecules, inorganic or organic molecules, antibodies, contaminants, viruses, bacteria, other pathogenic organisms, idiotopes and cell surface markers.
The present invention provides a sensor for detecting the binding of molecules to membrane surfaces. In one embodiment, the sensor comprising a substrate having a continuous membrane coating, wherein the substrate features nanoparticles disposed between the membrane coating and the substrate. The nano-plasmonic sensing device is intended to have simplicity of fabrication and of readout. In one embodiment, the manufacture of the basic sensor surface is based on a series of solution-based deposition and wash steps, and the readout is using simple absorbance spectrophotometry in an off-the-shelf instrument. The sensor presented herein is potentially easily parallelized for high-throughput applications, which distinguishes it from conventional SPR and related nanomaterial-based sensors.
In one embodiment, a multiplexable, label-free sensor device to measure interfacial binding of an analyte at a phospholipid membrane surface. Referring now to
Referring now to
While there are academic studies that use surface-based noble metal nanostructures as membrane binding sensors, the present embodiment lacks their burdensome technical requirements such as micropatterning of substrates. In contrast, nanocubes can be synthesized en masse and easily deposited over large areas. This means that this system is potentially easily multiplexed/parallelized and automated. For example, this could be achieved by using our basic technique adapted to a glass-bottomed 96-well plate and read in a plate reader absorbance spectrophotometer. Thus, in another embodiment, the present device provides for methods for detecting an analyte of interest or assays for biodetection.
An instrumental development that enables this invention is the capability of producing defect-free, fluid lipid bilayers that coat the nanoparticles and surrounding glass surface alike. Bilayers will form on glass, mica and some hydrophilic surfaces under a specific range of conditions, and not at all on bare or polymer-coated silver. Any surface contamination on the glass can have detrimental effects on the bilayer. The implementation of this device has required developing a method that allows both the nanoparticle and the glass to be covered with a continuous bilayer (hybrid bilayer on the nanoparticles; standard bilayer over the glass), preserves the environmental sensitivity of the nanoparticles' spectrum, and also allows the LSPR spectrum to be easily interrogated. Additionally, mathematical fitting of the quadripolar LSPR peak allows the accurate determination of the peak maximum beyond the resolution limit of the spectrophotometer. This is necessary for monitoring small shifts in the nanoparticle spectrum. Finally, a small, continuous-flow chamber to contain our experiments enables fluid exchange over the slide surface during data collection, though not all applications may require it.
The substrate for the sensor may comprise materials such as glass, mica, quartz, polydimethylsiloxane (PDMS), polystyrene, silica, SiO2, MgF2, CaF2, polyacrylamide, and various polysaccharides including dextran, agarose, cellulose and modified, crosslinked and derivatized embodiments thereof, and any other materials with constant spectra or any lipid-compatible material, i.e., a bilayer will form on the surface. For example, polymers like PDMS, or substrates like glass that have been decorated with biomolecules which can support lipid membranes (e.g. polymer supported bilayers) {See Tanaka, M.; Sackmann, E. Nature 2005, 437, 656-663, Sackmann, E. Science 1996, 271, 43-48} and can be suitable substrates. SiO2 is a particularly effective substrate material, and is readily available in the form of glass, quartz, fused silica, or oxidized silicon wafers. These surfaces can be readily created on a variety of substrates, and patterned using a wide range of micro- and nano-fabrication processes including: photolithography, micro-contact printing, electron beam lithography, scanning probe lithography and traditional material deposition and etching techniques.
In another embodiment, the nanoparticles are other polyhedra including but not limited to, nanopyramids, nanobowties, nanorods, nanocrescents, nanotubes, nanowontons, nanodisks, layered nanodisks with an alternating shielding layer, and other nanoscale polyhedra.
The nanoparticles can comprise a metal, a semiconductor material, multi-layers of metals, a metal oxide, an alloy, a polymer, or carbon nanomaterials. In certain embodiments the nanoparticle comprises a metal selected from the group consisting of Ga, Au, Ag, Cu, Al, Ta, Ti, Ru, Ir, Pt, Pd, Os, Mn, Hf, Zr, V, Nb, La, Y, Gd, Sr, Ba, Cs, Cr, Co, Ni, Zn, Ga, In, Cd, Rh, Re, W, Mo, and oxides, and/or alloys, and/or mixtures, and/or nitrides, and/or sintered matrix thereof.
In one embodiment the nanoparticles are silver or gold nanocubes. The remarkably sharp quadripolar resonance peak of silver nanocubes allows us to resolve more subtle variations in the spectrum compared with the very broad scattering signatures of other nanoparticles.
In one embodiment, the nanoparticles can be made according to the methods described in A. Tao, P. Sinsermsuksakul, and P. Yang. Tunable plasmonic lattices of silver nanocrystals. Nature Nanotechnology, 2(7):435-440, July 2007 and A. Tao, P. Sinsermsuksakul, and P. D. Yang. Polyhedral silver nanocrystals with distinct scattering signatures. Angewandte Chemie-International Edition, 45(28):4597-4601, 2006, both of which are hereby incorporated by reference.
Co-pending U.S. patent application Ser. No. 12/151,553, filed on Jul. 21, 2008, entitled, “A Fluid Membrane-Based Ligand Display System for Live Cell Assays and Disease Diagnosis Applications,” hereby incorporated by reference in its entirety, discloses detection of cell phenotypes in an soluble lipid bilayer (SLB) assay using soluble signaling ligands attached to the lipid bilayers. Other SLB assays are described in U.S. Pat. No. 6,228,326, which is incorporated by reference in its entirety. Co-pending U.S. patent application Ser. No. 10/076,727, incorporated by reference in its entirety, describes use of SLB assays to effect and modulate cell adhesion. All these related publications and patent applications are incorporated by reference in their entirety, especially for the purposes of enabling and exemplifying aspects of the present invention that had been developed in previous work conducted by some of the same inventors.
The supported bilayer of the assay system comprises a lipid bilayer wherein the primary ingredient is an egg-phosphatidylcholine (PC) membrane. In the absence of dopants, cells do not adhere to this membrane. Other suitable lipids that do not permit cell adhesion include pure phosphatidylcholine membranes such as dimyrstoyl-phosphatidylcholine or dipalmitoylphosphatidylcholine. Another suitable primary lipid component is phosphatidylethanolamine (PE), which is also, in addition to PC, a primary component.
The lipid composition in the supported lipid bilayer can comprise dopants to vary bilayer properties. Preferred dopant lipids are a negatively, positively or neutrally charged lipid. In one embodiment, the dopant lipid is the negatively charged lipid phosphatidylserine (PS). Other potential dopants can be dipalmitoylphosphatidic acid (PA), distearoylphosphatidylglycerol (PG), phosphatidylinositol,1,2-dioleoyl-3-dimethylamonnium-propane, 1,2 dioleoyl-3-trimethylammonium-propane (DAP), dimethyldioctadecylammonium bromide (DDAB), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (ethyl-PC), N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine ammonium salt (NDB-PE). Suitable neutral lipid dopants include cerebrosides and ceramides. The amount of the dopant is selected based on the property of the dopant. For a lipid dopant, 2 to 10%, up to 20% is preferred.
In a preferred embodiment, the device comprising a glass slide with a randomly ordered array of ˜100 nm wide silver nanocubes {Tao, Angewandte Chemie-International Edition 2006, 45, 4597-4601, Tao, Nature Nanotechnology 2007, 2, 435-440} at ˜10 to 100 cubes/μm2 density, coated by a hybrid lipid bilayer and surrounded by a normal lipid bilayer surface (see
In contrast to other metal nanoparticle-based systems, the data collection technique described measures a signal derived from large populations of nanoparticles, which means particle-to-particle variation in LSPR response is averaged. This helps ensure the comparability of one device to another. Thus, in some embodiments, a population density of ˜10-100 particles/μm2 density on a surface is more preferred.
To facilitate simple fabrication of the sensors, a method for fabrication was designed. In one embodiment, the manufacture of the basic sensor surface is based on a series of solution-based deposition and wash steps, and the readout is using simple absorbance spectrophotometry in an off-the-shelf instrument.
Referring now to FIG. 1D(1), a substrate and polymer-coated nanoparticles are provided. In one embodiment, the polymer-coated nanoparticles are coated with a polymer such as polyvinylpyrrolidone and its derivatives. The polymer-coated nanoparticles are dried onto the substrate in a solvent.
In FIG. 1D(2), the polymer coating on the polymer-coated nanoparticles is replaced by a self-assembled monolayer (SAM) by such techniques known in the art as solution deposition, physical vapor depositions, electrodeposition, adsorption or silanization. The nanoparticles are coated with the SAM to provide a chemical link between the nanoparticle surface and the surrounding supported bilayer. In one embodiment, the nanoparticles are coated with an alkanethiol SAM. Other SAM linking molecules are known in the art and can include such molecules as chlorosilanes, disulfides, amines, alcohols, carboxylic acids and phosphonic acids.
In one embodiment, the planar surface is a glass slide, a microfluidic device, or glass surface having a flow chamber to allow the sample suspected of containing an analyte to interact with the membrane-coated device. In another embodiment, rather than the flow chamber, the surface of a glass-bottomed multi-well plate could be used, and thus allowing the assay to be multiplexed and enabling a readout in a plate reader or spectrophotometer.
The nanoparticles may be adsorbed onto other surfaces instead of a substantially planar surface. In one embodiment, the surface is a bead similar to that in copending U.S. patent application Ser. No. 10/581,371, the contents of which are herein incorporated by reference. Specific examples of the particles include polystyrene, cellulose, dextran crosslinked with bisacrylamide (Biogel™, Bio-Rad, U.S.A.), agar, glass beads and latex beads. The beads may be nanometer to micrometer scale in diameter. This would enable LSPR readout of surfaces from suspension rather than on a monolithic surface (e.g., in a cuvette).
Referring now to FIG. 1D(3), the substrate featuring the SAM-covered nanoparticles are exposed to lipid vesicles in buffer, and the vesicles are allowed to rupture to form a hybrid bilayer over the nanoparticles and a conventional supported bilayer over the substrate (FIG. 1D(4)) to create the sensor to detect binding at membrane surfaces. The membranes can be formed by solution deposition of the nanoparticles on the substrate or by other methods known in the art including Langmuir-Blodgett or Langmuir-Schaeffer methods. In one embodiment, the planar supported membranes are formed by fusion of small unilamellar vesicles (SUV) with clean silica substrates according to the methods described in Salafsky, J., J. T. Groves, and S. G. Boxer, Architecture and function of membrane phospholipids in erythrocytes as factor in adherence to endothelial cells in proteins, Biochemistry, 1996, 35: 14773-14781, and U.S. Pat. No. 6,228,326, both of which are hereby incorporated in their entirety.
In another embodiment, a lipid solution in chloroform is evaporated onto the walls of a round bottom flask that is then evacuated overnight. Lipids are resuspended in distilled water by vortexing moderately for several minutes. The lipid concentration at this point should be around 3 mg/ml. The lipid dispersion is then probe sonicated to clarity on ice, yielding small unilamellar vesicles (SUV). The SUVs are purified from other lipid structures by ultracentrifugation for 2 hours at 192,000 g. SUVs are stored at 4° C. and typically are stable for a few weeks to several months. The SUVs are fused onto the aqueous phase on the substrate. The vesicles spontaneously assemble in a matter of seconds to form a continuous single bilayer on the substrate. Excess vesicles are rinsed away while maintaining the membrane bilayer under bulk aqueous solution at all times. In another embodiment, monodisperse lipid vesicles are made by extrusion through a porous filter. For example, vesicles can be prepared by drying lipids dissolved in CHCl3 in a round bottom flask, then suspending the dried lipid film in water, and repeatedly passing the suspension through a 100 nm pore filter in a high pressure extruder at 50° C. to form the lipid vesicles.
After forming the sensor, the present device can be used in sensing and detection methods. In one embodiment, a method comprising: contacting a target molecule with a substrate having a continuous membrane coating a plurality of nanoparticles disposed between the membrane and the substrate, applying a molecule possibly capable of binding the target molecule, and detecting plasmon generated phenomena at a nanoparticle.
In another embodiment, a method for detecting an analyte of interest comprising the steps of: (a) providing a substrate having a continuous membrane coating, wherein the substrate features nanoparticles disposed between the membrane coating and the substrate, wherein the nanoparticles have a known spectra, and wherein the continuous membrane displays a ligand for the analyte of interest; (b) applying a sample suspected of containing a target analyte of interest to the substrate; (c) detecting plasmon generated phenomena at the nanoparticles, whereby a spectral shift in the known spectra of the nanoparticles indicates that the target analyte is bound to the ligand.
Referring now to FIG. 1D(5), in one embodiment, a protein binds to the membrane and alters LSPR scattering spectrum of substrate. Binding occurs via functional lipid headgroup in this case.
The term “analyte”, “analyte of interest”, or “target analyte” refers to the compound or composition to be detected, including drugs, metabolites, pesticides, pollutants, and the like. The analyte can be comprised of a member of a specific binding pair (sbp) and may be a ligand, which is monovalent (monoepitopic) or polyvalent (polyepitopic), preferably antigenic or haptenic, and is a single compound or plurality of compounds, which share at least one common epitopic or determinant site. The analyte can be a part of a cell such as bacteria or a cell bearing a blood group antigen such as A, B, D, etc., or an HLA antigen or a microorganism, e.g., bacterium, fungus, protozoan, or virus. If the analyte is monoepitopic, the analyte can be further modified, e.g. chemically, to provide one or more additional binding sites. In practicing this invention, the analyte has at least two binding sites.
The term “ligand” refers to any organic compound for which a receptor naturally exists or can be prepared. The term ligand also includes ligand analogs, which are modified ligands, usually an organic radical or analyte analog, usually of a molecular weight greater than 100, which can compete with the analogous ligand for a receptor, the modification providing means to join the ligand analog to another molecule. The ligand analog will usually differ from the ligand by more than replacement of a hydrogen with a bond, which links the ligand analog to a hub or label, but need not. The ligand analog can bind to the receptor in a manner similar to the ligand. The analog could be, for example, an antibody directed against the idiotype of an antibody to the ligand.
The term “receptor” or “antiligand” refers to any compound or composition capable of recognizing a particular spatial and polar organization of a molecule, e.g., epitopic or determinant site. Illustrative receptors include naturally occurring receptors, e.g., thyroxine binding globulin, antibodies, enzymes, Fab fragments, lectins, nucleic acids, nucleic acid aptamers, avidin, protein A, barsar, complement component C1q, and the like. Avidin is intended to include egg white avidin and biotin binding proteins from other sources, such as streptavidin.
The ligand may be an oligonucleotide of ribonucleic acid residues, deoxyribonucleic acid residues, polypeptides, proteins, receptors, carbohydrates, thyroxine binding globulin, antibodies, enzymes, Fab fragments, lectins, nucleic acids, nucleic acid aptamers, avidin, protein A, barsar, complement component C1q, organic or inorganic molecules having a binding affinity for an analyte of interest, or lipid-linked small molecules that are displayed, bound or otherwise attached to the membrane coating the sensor.
The term “specific binding pair (sbp) member” refers to one of two different molecules, which specifically binds to and can be defined as complementary with a particular spatial and/or polar organization of the other molecule. The members of the specific binding pair can be referred to as ligand and receptor (antiligand). These will usually be members of an immunological pair such as antigen-antibody, although other specific binding pairs such as biotin-avidin, enzyme-substrate, enzyme-antagonist, enzyme-agonist, drug-target molecule, hormones-hormone receptors, nucleic acid duplexes, IgG-protein A/protein G, polynucleotide pairs such as DNA-DNA, DNA-RNA, protein-DNA, lipid-DNA, lipid-protein, polysaccharide-lipid, protein-polysaccharide, nucleic acid aptamers and associated target ligands (e.g., small organic compounds, nucleic acids, proteins, peptides, viruses, cells, etc.), and the like are not immunological pairs but are included in the invention and the definition of sbp member. A member of a specific binding pair can be the entire molecule, or only a portion of the molecule so long as the member specifically binds to the binding site on the target analyte to form a specific binding pair.
The term “specific binding” refers to the specific recognition of one of two different molecules for the other compared to substantially less recognition of other molecules. Generally, the molecules have areas on their surfaces or in cavities giving rise to specific recognition between the two molecules. Exemplary of specific binding are antibody-antigen interactions, enzyme-substrate interactions, polynucleotide interactions, and so forth.
The analyte of interest may be nucleic acid molecules, proteins, peptides, haptens, metal ions, drugs, metabolites, pesticide or pollutant. The method can be used to detect the presence of such analytes as toxins, hormones, enzymes, lectins, proteins, signaling molecules, inorganic or organic molecules, antibodies, contaminants, viruses, bacteria, other pathogenic organisms, idiotopes or other cell surface markers. It is intended that the present method can be used to detect the presence or absence of an analyte of interest in a sample suspected of containing the analyte of interest.
In some embodiments, the target analyte is comprised of a nucleic acid and the specific binding complement is an oligonucleotide. Alternatively, the target analyte is a protein or hapten and the specific binding complement is an antibody comprising a monoclonal or polyclonal antibody. Alternatively, the target analyte is a sequence from a genomic DNA sample and the specific binding complement are oligonucleotides, the oligonucleotides having a sequence that is complementary to at least a portion of the genomic sequence. The genomic DNA may be eukaryotic, bacterial, fungal or viral DNA.
In one embodiment, detection of a particular cytokine can be used for diagnosis of cancer. Specific analytes of interest include cytokines, such as IL-2 as shown in the examples. Cytokines are important analytes of interest in that cytokines play a central role in the regulation of hematopoiesis; mediating the differentiation, migration, activation and proliferation of phenotypically diverse cells. Improved detection limits of cytokines will allow for earlier and more accurate diagnosis and treatments of cancers and immunodeficiency-related diseases and lead to an increased understanding of cytokine-related diseases and biology, because cytokines are signature biomarkers when humans are infected by foreign antigens.
Chemokines are another important class of analytes of interest. Chemokines are released from a wide variety of cells in response to bacterial infection, viruses and agents that cause physical damage such as silica or the urate crystals. They function mainly as chemoattractants for leukocytes, recruiting monocytes, neutrophils and other effector cells from the blood to sites of infection or damage. They can be released by many different cell types and serve to guide cells involved in innate immunity and also the lymphocytes of the adaptive immune system. Thus, improved detection limits of chemokines will allow for earlier and more accurate diagnosis and treatments, i.e. for bacterial infections and viral infections.
In some embodiments, the target analyte may be a variety of pathogenic organisms including, but not limited to, sialic acid to detect HIV, Chlamydia, Neisseria meningitides, Streptococcus suis, Salmonella, mumps, newcastle, and various viruses, including reovirus, sendai virus, and myxovirus; and 9-OAC sialic acid to detect coronavirus, encephalomyelitis virus, and rotavirus; non-sialic acid glycoproteins to detect cytomegalovirus and measles virus; CD4, vasoactive intestinal peptide, and peptide T to detect HIV; epidermal growth factor to detect vaccinia; acetylcholine receptor to detect rabies; Cd3 complement receptor to detect Epstein-Barr virus; β-adrenergic receptor to detect reovirus; ICAM-1, N-CAM, and myelin-associated glycoprotein MAb to detect rhinovirus; polio virus receptor to detect polio virus; fibroblast growth factor receptor to detect herpes virus; oligomannose to detect Escherichia coli; ganglioside GM1 to detect Neisseria meningitides; and antibodies to detect a broad variety of pathogens (e.g., Neisseria gonorrhoeae, V. vulnificus, V. parahaemolyticus, V. cholerae, and V. alginolyticus).
In some embodiments, multiple analytes of interest can be detected by utilizing multiple ligands specific to different analytes of interest and utilizing distinct barcode oligonucleotides corresponding to each analyte of interest.
The analyte of interest may be found directly in a sample such as a body fluid from a host. The host may be a mammal, reptile, bird, amphibian, fish, or insect. In a preferred embodiment, the host is a human. The body fluid can be, for example, urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, pus, phlegm, and the like. The particles can be mixed with live cells or samples containing live cells.
Where the sample is live cells or samples containing live cells, a cell surface protein or other molecule may serve as the analyte of interest. This allows for the detection of cell activation and proliferation events, cellular interactions, multiplexing, and other physiologically relevant events
The target molecule binding as well as target molecule adhesion to a cell can be detected by any method of detection including but not limited to detection by absorbed light, reflected light, scattered light, back reflected interference fringes, or scattered reflected intergerence fringes, light from resonant energy transfer energy of the plasmonic field coupled to flourophores (like flourescence resonance energy transfer).
In another embodiment, the sensor can be an array of individually addressable regions of substrate (e.g., wells in a microwell plate, or channels in a microfluidic chip) to form a multiplex assay that allows testing different events in different wells, or channels.
In one embodiment, absorbance or reflectance spectra of the entire substrate is measured. The image and spectrum of the sensor can be acquired using a dark-field microscopy system with a true-color imaging camera and a spectrometer. For example, the microscopy system can consist of a Carl Zeiss Axiovert 200 inverted microscope (Carl Zeiss, Germany) equipped with a darkfield condenser (1.2<NA<1.4), a true-color digital camera (CoolSNAP cf, Roper Scientific, NJ), and a 300 mm focal-length and 300 grooves/mm monochromator (Acton Research, MA) with a 1024×256-pixel cooled spectrograph CCD camera (Roper Scientific, NJ). After photobleaching the fluorescence, the true-color scattering images of the nanoparticles are taken using a 60× objective lens (NA=0.8) and the true-color camera with a white light illumination from a 100 W halogen lamp.
In another embodiment, rather than measuring the absorbance spectrum of the entire substrate, interrogation of individual nanoparticles or regions/clusters of nanoparticles is contemplated. Moreover the sensor could record scattered light instead of an absorbance spectrum. The scattering spectra of the nanoparticles can be taken using the same optics, but they are routed to the monochromator and spectrograph CCD. Furthermore, a 2 μm-wide aperture can be placed in front of the entrance slit of the monochromator to keep only a single nanoparticle in the region of interest.
Raw spectra are normalized with respect to the spectrum of a non-resonant nanoparticle (i.e., polystyrene) after the background subtraction. In the spectroscopy experiments, the nanoparticle-immobilized glass slide can be mounted on a transparent ITO heater with an external thermostat. The immobilized membranes and nanoparticles are immersed in a drop of buffer solution which also serves as the contact fluid for the dark-field condenser. The distance between the condenser and nanoparticles can be ˜1-2 mm. The sample suspected of containing an analyte to be detected can be loaded by pipette into the contact fluid and the continuous spectrum acquisition started simultaneously. The microscopy system can be completely covered by a dark shield, which prevents ambient light interference and serious evaporation of the sample.
Normalized fluorescence recovery of lipids over a nanocube or over glass is shown in
Application of wash steps or an agent to cause unbinding of the analyte from the ligand can also be performed to allow monitoring of LSPR peak shift. Observed shift in λmax position compared with t=0 for a nanoparticle-embedded bilayer with or without the analyte bound or with or without the ligands present in the lipid membrane. In one embodiment, the line is a least-squares fit of the equation y=A*exp [−t/a]+B*exp [−t/b]+y0 to the data where y is the shift in λmax and t is time. The observed peak shift upon addition of an agent which removes all remaining remaining analyte and defines y0. The remaining terms are found by the fitting procedure.
To measure small shifts in the LSPR-derived absorbance spectrum of the sensor resulting from molecular binding, the quadripolar absorbance peak is fit to a 5th order polynomial function over a consistent sampling range. This allows the precise determination of peak maximum position, and in the present implementation at least 0.02 nm resolution is achieved, as discussed below. In contrast to previous examples of this approach, changes in the peak maximum position (λmax,
In one embodiment, the analyte density is calculated by considering the fluorescence of the analyte bound to identical bilayers as herein described and in Galush et al. Biophys J, 2008, which is hereby incorporated by reference, and demonstrated by the Examples infra. Furthermore, other ways to calibrate the analyte density can be employed. For example, instead of fluorescence, one could use mass standards. In one instance, another protein binding in known amounts to the same or identical substrate can be calculated.
In another embodiment, sensor response could be measured by localizing the spectrum peak by position of maximum signal, position of centroid, or absolute intensity (spectrum height). The sensor response could be measured by monitoring the increase in fluorescence emission of the analyte upon binding to the membrane. This is what accounts for the bright appearance of the nanoparticles in
In yet another embodiment, darkfield microscopy of the whole substrate, portions of the substrate, or individual particles could be used as the readout.
In another embodiment, for real-time plasmon resonance sensing of molecular binding or interactions, the continuous acquisition of the scattering spectrum of a selected nanoparticle starts in synchronization with the introduction of the sample suspected of containing the analyte. For example, one spectrum is taken every minute with a 10-second integration time. The plasmon resonance wavelength data exhibits a first-order exponential decay. Calibration curves generated by plasmon resonance sensing of multiple analytes can be generated and typical scattering spectra and plasmon resonance peak wavelengths of the nanoparticle after the interactions and reactions with multiple analytes can be acquired. In one embodiment, the curve is fit from a semi-empirical model using a Langevin-type dependence of the refractive index vs. amount of unbound ligand or analyte.
And in another embodiment, surface enhanced Raman spectroscopy (SERS) can be used to perform the detection and the readout instead of absorbance (see {McFarland:2005, Porter:2008}). A typical SERS experimental system configuration comprising a microscopy system with Raman spectrometer used to acquire Raman scattering spectra from single tagged nanoplasmonic resonators. In a preferred embodiment, the system is comprised of inverted microscope equipped with a digital camera and a monochromator with a spectrograph CCD camera, a laser source and an optical lens. In one embodiment, Raman spectra can be measured using a modified inverted microscope, such as the Carl Zeiss Axiovert 200 (Carl Zeiss, Germany), with a 50× objective in a backscattering configuration. The laser wavelength can be in the visible and near infrared region. In a preferred embodiment, a 785 nm semiconductor laser is used as the excitation source of Raman scattering, and the laser beam is focused by a 40× objective lens on the NPR. The 785 nm or other near infrared light source can assure less absorption by the biological tissue and lower fluorescence background. However, for certain applications, lower wavelength excitation light might be more advantageous, and even UV light excitation can be used for applications. The excitation power can also be measured by a photometer to insure an output of ˜0.5 to 1.0 mW. The Raman scattering light is then collected through the same optical pathway through a long-pass filter and analyzed by the spectrometer. The Raman spectrometer is preferably linked to a computer whereby the spectrometer can be controlled and the spectra can be obtained and a spectrograph can be observed. The spectral detection can be done with ordinary spectral polychrometer and cooled CCD camera. In an embodiment where the ligands and analytes are nucleotides, the monitored wavenumbers of Raman peaks can range from 400 cm−1 to 2000 cm−1.
In one embodiment, the sensor is incubated with a sample suspected of containing the biomolecule to be detected, preferably in a closed transparent microchamber. The microchamber is mounted on a 37° C. thermal plate on an inverted Raman microscope with darkfield illumination for nanoparticle visualization. The nanoparticles are visualized using the darkfield illumination from oblique angles as the bright dots. The excitation laser is focused on the nanoparticles by a microscopy objective lens. A SERS signal is collected by the same objective lens and analyzed by a spectrometer.
In some embodiments, the sensor can be used to measure supported bilayer formation or change in supported bilayer physical properties, in aggregate or on a microscopic scale.
In another embodiment, the sensor can be used to quantify cell adhesion to the substrate mediated by a membrane-resident molecule. As cells tightly bind to the surface and closely adhere, this should change the LSPR scattering signature. In another embodiment, the sensor can be used to monitor lipid vesicle/micelle/bicelle binding.
In some embodiments, using a microscope, we could address different regions of the substrate independently. This could be on the single- or multi-nanoparticle scale. This could be done using darkfield microscopy, or localized illumination or scattering sensor to see the LSPR signature. Notably, SPR is not spatially resolved, whereas our technique can be.
The present sensor is not bound by the described applications but is contemplated to find use in sensing and detection in various SPR methods and devices.
A multiplexable, label-free sensor device to measure interfacial binding of an analyte at a phospholipid membrane surface was made comprising a glass slide with a randomly ordered array of ˜100 nm wide silver nanocubes{Tao:2006,Tao:2007} (at ˜10-100 cubes/μm2 density), coated by a hybrid lipid bilayer and surrounded by a normal lipid bilayer surface (see
The device measures binding by exploiting the optical absorbance due to localized surface plasmon resonance (LSPR) scattering by the silver nanocubes. The LSPR scattering spectrum of nanocubes has sharply defined peaks, the positions of which are dependent on the refractive index of the surrounding environment, and hence to analyte bound to the membrane. Silver nanocubes exhibit a sharp quadripolar LSPR peak that provides a sensitive gauge of the refractive index in the immediately surrounding environment, and have been characterized both experimentally and theoretically[Tao, A. et al, Angewandte Chemie-International Edition 2006, 45, 4597-4601, Tao, A. et al., Nature Nanotechnology 2007, 2, 435-440, Sherry, L. J.; Chang, S. H.; Schatz, G. C.; Van Duyne, R. P.; Wiley, B. J.; Xia, Y. N. Nano Letters 2005, 5, 2034-2038]. At the quadripolar LSPR wavelength, the electromagnetic field exhibits localized hot spots of amplified intensity which extend approximately 10 nm beyond the metal surface, with the field being strongest along the edges and corners of the cube[Sherry, L. J.; et al., Nano Letters 2005, 5, 2034-2038]. This results in less influence from solution components when compared with conventional surface plasmon resonance, which has far longer (200 nm) field penetration depths [Jung, L.; Campbell, C.; Chinowsky, T.; Mar, M.; Yee, S. Langmuir 1998, 14, 5636-5648, Zhou, Y.; Xu, H.; Dahlin, A. B.; Vallkil, J.; Borrebaeck, C. A. K.; Wingren, C.; Liedberg, B.; Hook, F. Biointerphases 2007, 2, 6-15]. The nanocube LSPR field still extends beyond the approximately 5 nm thickness of the hybrid bilayer[Leonenko, Z. V.; Finot, E.; Ma, H.; Dahms, T. E. S.; Cramb, D. T. Biophysical Journal 2004, 86, 3783-3793] to allow probing of binding at the membrane surface
Spectral shifts of the peaks indicate binding or unbinding of the analyte to the bilayer surface. The device is easily realized as a simple flow chamber that may be placed in an absorbance spectrophotometer, where the nanoparticle scattering registers as a distinct absorbance spectrum. Experiments have shown that this device is capable of collecting binding kinetics data as well as specificity measurements, all without depending on potentially disruptive analyte labeling.
The construction of the biosensor begins with drying a solution of silver nanocubes onto a glass substrate. The nanocubes are synthesized using the polyol method[Tao, A.; Sinsermsuksakul, P.; Yang, P. D. Angewandte Chemie-International Edition 2006, 45, 4597-4601; Fievet, F.; Lagier, J. P.; Blin, B.; Beaudoin, B.; Figlarz, M. Solid State Ionics 1989, 32-33, 198-205, and Sun, Y.; Xia, Y. Science 2002, 298, 2176-2179], capped with poly(vinylpyrrolidone) (PVP), and stored in ethylene glycol for extended periods of time (up to months) before use. Nanocubes are first washed extensively with ethanol to remove residual synthetic reagents. A small droplet of the colloidal suspension is spread onto a glass microscope slide, which has been previously cleaned in a 1:4 30% H2O2:H2SO4 mixture of piranha solution (extremely reactive, use caution). The droplet is allowed to dry under a N2 atmosphere for ten minutes; air exposure is minimized to avoid silver oxidation. Slides are then incubated in a hexane solution with 3 mM 1-octanethiol for at least 12 hours to form an alkanethiol self assembled monolayer (SAM) over the metal. The slides are subsequently rinsed by immersion in acetone, isopropanol, and twice in deionized water. After drying under N2 for 30 minutes, the nanoparticle-covered slides are assembled into a flow chamber using a silicone gasket (Invitrogen) and a second slide with holes cut to allow solution exchange within the device (
The extinction spectrum of a nanocube-decorated substrate, as monitored by a standard spectrophotometer (Cary 100), is illustrated in
It is possible to directly measure the response of SAM-coated nanocubes to changes in the surrounding refractive index (RI) by exposing the sensor to aqueous solutions of glycerol and tracking the shift in position of λmax relative to its initial wavelength in water alone. A plot of the shift of the peak versus the refractive index of the surrounding glycerol/water solution yields a sensitivity of 165 nm RI−1 (
To form a phospholipid membrane on the nanocube-coated substrate, the flow chamber is filled with 50 mM Tris, 200 mM NaCl, pH 7.5 buffer and allowed to incubate for 30 minutes before rinsing with further buffer to remove loosely adhered particles. A solution of lipid vesicles is injected into the flow chamber in the Tris buffer and allowed to incubate for an additional 30 minutes (shorter incubations than these are also likely sufficient). During this time, vesicles rupture to form a supported phospholipid bilayer over the bare glass regions and a phospholipid monolayer over the alkanethiol-modified nanocubes (
Coating the nanocubes with an alkanethiol SAM is required to create a laterally fluid, continuous membrane over the glass and metal substrate surface. This is demonstrated by fluorescence recovery after photobleaching (FRAP) experiments, where fluorophores in a small region of the substrate surface are bleached under high intensity illumination in a microscope and recover with time due to the lateral diffusion of membrane components (
The nanocubes are seen clearly in fluorescence microscopy images as objects that appear brighter than the surrounding fluorescent supported bilayer (
To measure small shifts in the LSPR-derived absorbance spectrum of the sensor resulting from molecular binding, the quadripolar absorbance peak is fit to a 5th order polynomial function over a consistent sampling range. This allows the precise determination of peak maximum position, and in the present implementation at least 0.02 nm resolution is achieved, as discussed below. In contrast to previous examples of this approach, changes in the peak maximum position (λmax,
As
whereIx,y is the local intensity of each pixel over a n×m region that completely encompasses the emission of the nanocube, and Ibackground is the average intensity of the bilayer in an area with no nanocubes.
The fractional excess surface area of membrane contained in the pixel in question, Aexcess is,
where Ananocube is the area of the face of an individual nanocube and Apixel is the calibrated size of each pixel. The excess area is thus the sum of the five exposed faces of the nanocube modified for the fact each has only half the number of lipids as a bilayer, less the area of the glass substrate being occupied by the nanocube. This is expressed as a fraction of the area of the pixel containing the nanocube.
The excess brightness associated with the nanocube is Iexcess/Aexcess, which is found to be 4±1 for the ≈100 nm nanocubes used here. This represents the average of 1694 individual nanocubes from multiple sample substrates. Notably, even if all fluorophores were localized to the upper leaflet of the membrane, there is still an excess of fluorescence (2-fold, in this case) compared with what would be expected in this analysis.
The level of nonspecific binding to the membrane-nanocube substrates is extremely low compared with some previous reports of LSPR-based membrane binding sensors[Baciu, C. L.; Becker, J.; Janshoff, A.; Sonnichsen, C. Nano Letters 2008, 8, 1724-1728]. As seen in FIG. 2c, coating an alkanethiol-modified nanocube substrate with phospholipids (96.5% DOPC, 3% biotin-cap-PE, 0.5% TR-DHPE) results in a 2.40 nm shift in the quadripolar peak. Subsequent addition of 0.03 mg ml−1 bovine serum albumin barely shifts the peak position by a further 0.03 nm. Conversely, the addition of neutravidin, which specifically binds to biotin-headgroup lipids incorporated into this membrane composition, results in a 1.26 nm shift. This constitutes a signal/noise ratio of 42 over nonspecific binding.
Molecular binding to the membrane surface can also be monitored dynamically, enabling kinetic analyses. In the example considered here, DOGS-NTA-Ni lipids provide the binding functionality—a membrane receptor for these purposes—in a membrane mixture of 89.5% DOPC, 10% DOGS-NTA-Ni, and 0.5% TR-DHPE. The DOGS-NTA-Ni lipids bind to a hexahistidine tag at the C-terminus of yellow fluorescent protein (YFP)[ Ormö, M.; Cubitt, A. B.; Kallio, K.; Gross, L. A.; Tsien, R. Y.; Remington, S. J. Science 1996, 273, 1392-1395]. Other membrane-associated species including membrane proteins, DNA/RNA, or lipid-conjugated small molecules can also be readily used in this configuration[Salafsky, J.; Groves, J. T.; Boxer, S. G. Biochemistry 1996, 35, 14773-14781, Yoshina-Ishii, C.; Boxer, S. G. Journal of the American Chemical Society 2003, 125, 3696-3697, and Parthasarathy, R.; Groves, J. T. Proceedings of the National Academy of Sciences of the United States of America 2004, 101, 12798-12803]. The graph in
The LSPR-based measurements are compared to fluorescence from YFP on the membrane surface, which is directly monitored by microscopy in a glass-bottomed 96 well plate format (Nalge-Nunc). This configuration is chosen to compare the data from the nanocube hybrid membranes to conventional supported membranes without nanocubes. Membranes are of the same compositions as those used with the LSPR measurements, and are formed similarly as described elsewhere[Nye, J. A.; Groves, J. T. Langmuir 2008, 24, 4145-4149]. After loading with YFP, fluorescence microscopy images are taken of different regions of several replicate bilayers over the course of time, with manual rinsing of wells between each image acquisition (Nikon TE-300 equipped with a high pressure Hg lamp and Chroma 31001 filter set). The intensity of the fluorescence microscopy images is proportional to the amount of YFP on the surface. These data show that YFP desorbs from the membrane biexponentially with halflives of 7±1 and 80±8 min−1 (
The fluorescence microscopy images used above also provide a direct way to estimate the amount of protein bound to the membrane, and thus the sensitivity of the LSPR assay. The absolute surface density of protein can be measured using a set of bilayer calibration standards containing varying concentrations of BODIPY-DHPE lipid (Invitrogen), which provide the relationship between fluorescence intensity and surface density of fluorophore. The intensity of YFP can be scaled to be directly comparable to that of BODIPY-DHPE, which allows the density of YFP to be inferred[Galush, W. J.; Nye, J. A.; Groves, J. T. Biophysical Journal 2008, 95, 2512-2519]. This analysis shows that initial protein density on the bilayer is approximately 21,000 μm−2, and decreases over the course of the experiment to approximately 2,000 μm−2 (
An estimate of sensor noise is found by considering data from the negative control bilayer (without DOGS-NTA-Ni), shown in
Many implementations of nanostructure-based sensors require complicated nanostructured templates and device fabrication. Realization of this sensor only requires simple-to-manufacture, self-assembled nanocube/bilayer detection surfaces, along with a standard absorbance spectrophotometer. The membrane-coated nanocube substrates are also potentially very easy to multiplex. Rather than a dedicated flow chamber as used here, it should be possible to realize the same basic system using glass-bottomed 96 well plates and an optical plate reader (e.g. high resolution models from Molecular Devices, BMGLabtech, and Biotek, among others). This allows easy multiplexing and scalability of the technique, since nanocube deposition, modification, and membrane coating could all be performed in an individual well whose spectrum is read out independently and analyzed as above. The membrane functionality of this technique allows readout of binding in an environment very different than that in solution or provided in most standard SPR formats. Some applications may not require the membrane environment itself, but membrane resistance to nonspecific binding (especially of proteins) may still prove useful as a scaffold for monitoring natively soluble proteins interacting with each other.
The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, databases, and patents cited herein are hereby incorporated by reference for all purposes.
This application claims priority to PCT International Application No. PCT/US2010/023375, filed on Feb. 5, 2010, hereby incorporated by reference in its entirety, which claims priority to U.S. Provisional Patent Application, 61/150,680, filed on Feb. 6, 2009, and U.S. Provisional Patent Application, 61/174,855, filed on May 1, 2009, each of which is incorporated by reference in its entirety.
This invention was made with government support under Contract No. DE-AC02-05CH11231 awarded by the U.S. Department of Energy. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61150680 | Feb 2009 | US | |
| 61174855 | May 2009 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2010/023375 | Feb 2010 | US |
| Child | 13204506 | US |
