WO2010115147A2 - Biomolecular sensing with metal-nanostructures - Google Patents

Abstract

Systems and methods for providing biomolecule interaction sites are disclosed. In certain embodiments, coupling of two or more different metal-nanoparticles can provide a significant increase in flexibility in controlling parameters associated with excitation of fluorophores attached to the biomolecules. Types and number of metal-nanoparticles and spatial arrangement of such nanoparticles are some of the parameters that can be adjusted to control fluorescence parameters such as excitation wavelengths and intensity of fluorescence. In certain embodiments, enhanced fluorescence effect can be confined to a sufficiently small region so as to allow sensing of one biomolecule at a time. Combined with enhanced fluorescence intensity, such small enhancement region can be used to perform processes such as real-time DNA sequencing.

Description

BIOMOLECULAR SENSING WITH METAL-NANOSTRUCTURES

Field

[0001] The present disclosure generally relates to the field of nanostructures, and more particularly, to systems and methods for sensing biomolecules using metal- nanoparticles.

Background

[0002] Biological molecules such as DNA base molecules can be detected by attaching detectable labels such as fluorophores. Such labeled base molecules can bind to specific complementary bases in a DNA strand. Thus, identification of the labeled base molecules allows determination of the sequence of the complementary bases in the DNA strand being analyzed.

[0003] When excitation energy such as a laser is introduced to the labeled molecules such as fluorescence labeled molecules, the fluorescence labels can fluoresce and yield detectable signal. Detection of such signals can provide both qualitative and quantitative information about various properties of labeled molecules.

[0004] For analyses based on such fluorescence or other detectable signals, it is generally desirable to have increased resolution, as well as an ability to monitor various processes in real time.

SUMMARY

[0005] At least some of the foregoing concerns can be addressed by systems and methods for providing biomolecule interaction sites. In certain embodiments, coupling of two or more different metal-nanoparticles can provide a significant increase in flexibility in controlling parameters associated with excitation of fluorophores attached to the biomolecules. Types and number of metal-nanoparticles and spatial arrangement of such nanoparticles are some of the parameters that can be adjusted to control fluorescence parameters such as excitation wavelengths and intensity of fluorescence. In certain embodiments, an enhanced fluorescence effect can be confined to a sufficiently small region so as to allow sensing of one biomolecule at a time. Combined with enhanced fluorescence intensity, such a small enhancement region can be used to perform processes such as real-time DNA sequencing.

[0006] In certain embodiments, the present disclosure relates to a system for sensing biological molecules. The system includes a detection zone having one or more metal-nanostructure assemblies such as metal-nanoparticle assemblies. Each of the metal-nanostructure assemblies includes a plurality of metal-nanostructures coupled via one or more linkers such as DNA strands, where at least one of the metal- nanostructures is can be different than at least one other metal-nanostructure. Each of the metal-structure assemblies further includes an interaction site, such as a polymerase molecule or polymerase-based structure, disposed relative to the plurality of metal-nanostructures and configured to preferentially provide a location for detecting a detectable biological molecule such as a dNTP molecule labeled with a fluorophore, and optionally with quencher. The system further includes a detector configured to detect a signal emitted by the detectable biological molecule. The system further includes an optics assembly configured to direct signal from the biological molecule to the detector.

[0007] In certain embodiments, the present disclosure relates to an apparatus for capturing a biological molecule. The apparatus includes a first metal-nanostructure such as a metal-nanoparticle coupled to a first linker such as an oligonucleotide strand or an enzyme. The apparatus further includes a second metal-nanostructure that is different than the first metal-nanostructure and coupled to a second linker. The first and second metal-nanostructures are coupled to each other via the first and second linkers. The apparatus further includes an interaction site coupled to either of the first and second linkers, so as to be positioned relative to the first and second metal- nanostructures. The interaction site can be configured to facilitate preferential binding of the biological molecule with a template molecule such as a single strand DNA.

[0008] In certain embodiments, the present disclosure relates to an assembly of metal-nanoparticle based structures. The assembly includes a substrate that defines a surface. The assembly further includes a plurality of metal-nanoparticle based structures coupled to the surface. The coupling can include immobilization, or can include a long DNA attached to surface, with the metal-nanoparticle floating substantially free. Each of the structures includes a plurality of metal-nanoparticles, where each of the metal-nanoparticles is coupled to at least one other metal- nanoparticle. At least one of the metal-nanoparticles is different than at least one other metal-nanoparticle. Each of the structures further includes an interaction site disposed relative to the plurality of metal-nanoparticles and configured to provide a catalyst functionality for a reaction involving a detectable biological molecule. The interaction site is positioned so as to experience a metal-enhancement effect provided by the plurality of metal-nanoparticles.

[0009] In certain embodiments, the present disclosure relates to a composition that includes a plurality of metal-nanoparticle based structures. Each structure includes a plurality of metal-nanoparticles, with each of the metal-nanoparticles being coupled to at least one other metal-nanoparticle. At least one of the metal- nanoparticles is different than at least one other metal-nanoparticle. Each structure further includes an interaction site disposed relative to the plurality of metal- nanoparticles and configured to provide a catalyst functionality for a reaction involving a detectable biological molecule. The interaction site is positioned so as to experience a metal-enhancement effect provided by the plurality of metal-nanoparticles.

[001O] In certain embodiments, the present disclosure relates to a method for sensing biological molecules. The method includes providing one or more metal- nanoparticle assemblies, with each of the assemblies having an arrangement of at least two types of metal-nanoparticles and an interaction site disposed relative to the metal-nanoparticles, such that the interaction site experiences a metal plasmon- enhanced fluorescence effect. The method further includes introducing one or more fluorophore labeled biological molecules to the one or more metal-nanoparticle assemblies such that a fluorophore labeled biological molecule positioned at the interaction site experiences the metal plasmon-enhanced fluorescence effect. The method further includes providing excitation energy to the one or more metal- nanoparticle assemblies so as to excite one or more of the labeled biological molecules positioned at the interaction sites. The method further includes detecting fluorescence signal emitted by the excited one or more labeled biological molecules. - -

[0011] In certain embodiments, the present disclosure relates to a method for fabricating a sensor for sensing of biological molecules. The method includes providing a plurality of first type of metal-nanoparticles with one or more linkers such as DNA strands attached thereto. The method further includes providing a plurality of second type of metal-nanoparticles with one or more linkers attached thereto, where the second type is different than the first type. The method further includes coupling at least some of the first type of metal-nanoparticles with a corresponding number of the second type of metal-nanoparticles. The coupling can be achieved via respective one or more linkers of the first and second types of metal-nanoparticles. The method further includes providing an interaction site for each of the coupled assembly of first and second types of metal-nanoparticles such that the interaction site is disposed relative to the first and second metal-nanoparticles. The interaction site is positioned so as to experience a metal-enhanced fluorescence effect.

[0012] In certain embodiments, the present disclosure relates to a method for sensing biological molecules. The method includes providing one or more hosting sites for one or more biological molecules tagged with fluorophores. Each of the hosting sites includes at least two different metal-nanostructures that are coupled, and an interaction site disposed relative to the at least two different metal-nanostructures, such as a metal-nanoparticle and a nanostructure. The interaction site is configured to receive one of the one or more biological molecules. The method further includes providing a far-field excitation beam to the hosting site such that location of the biological molecule attached to the interaction site is within a near-field excitation region induced by interaction of the excitation beam with the at least two different metal-nanostructures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 A shows a block diagram of one embodiment of a system configured to sense molecules associated with biological processes;

[0014] Figure 1 B shows an example system configured to perform sensing of biological molecules via one or more metal-nanoparticle based components; - -

[0015] Figure 2 shows that in certain embodiments, the metal-nanoparticle based assembly can provide an interaction site configured to detect single base molecules undergoing hybridization with complementary bases in a single-strand DNA sample being analyzed, where the metal-nanoparticle based assembly can further provide a metal-enhanced effect such as a metal-enhanced fluorescence effect to facilitate such single-molecule detection;

[0016] Figure 3 shows that in certain embodiments, a metal-nanoparticle assembly can have two coupled metal-nanoparticles and an interaction site disposed relative to the two nanoparticles so as to provide a metal-enhanced effect such as a metal enhanced fluorescence effect at the interaction site;

[0017] Figures 4A - 4C show an example process for preparing one or more of the metal-nanoparticle assembly of Figure 3;

[0018] Figure 5 shows that in certain embodiments, a metal-nanoparticle assembly can have more than two coupled metal-nanoparticles and an interaction site disposed relative to the nanoparticles, so as to facilitate greater flexibility in selecting one or more features provided by the metal-enhanced effect;

[0019] Figure 6 shows an example process for preparing the example metal- nanoparticle assembly of Figure 5;

[0020] Figures 7A - 7C show non-limiting examples of metal-nanoparticle shapes that can be used in various metal-nanoparticle assemblies;

[0021] Figure 8A shows that in certain embodiments, two triangular shaped metal-nanoparticles can be used to for the example assembly of Figure 3, so as to provide a "bow-tie" configuration for providing the metal-enhanced effect;

[0022] Figure 8B shows another example configuration for a four-particle assembly where each metal-nanoparticle can have a triangular shape;

[0023] Figures 9A - 9C show non-limiting examples of metal-nanoparticle assemblies that can be arranged in two or three dimensions;

[0024] Figure 1 OA shows that in certain embodiments, one or more metal- nanoparticle assemblies can provide the metal-enhancement effect as substantially free bodies not bound to a substrate; [0025] Figure 1 OB shows that in certain embodiments, one or more metal- nanoparticle assemblies can be immobilized on a surface of a substrate and provide the metal-enhancement effect;

[0026] Figure 1 1 shows that in certain embodiments, one or more metal- nanoparticle assemblies can be immobilized in a well having a selected dimension;

[0027] Figure 12 shows that in certain embodiments, one or more metal- nanoparticle assemblies can be immobilized in a waveguide structure such as a zero- mode waveguide;

[0028] Figure 13 shows that in certain embodiments, a plurality of metal- nanoparticle assemblies can be arranged in an array on a substrate;

[0029] Figure 14 shows that in certain embodiments, a plurality of metal- nanoparticle assemblies can be arranged in a substantially random manner on a substrate;

[0030] Figure 15 shows a non-limiting use of the metal-nanoparticle assembly, where an interaction site such as polymerase or polymerase-based structure can catalyze reactions involving labeled biological molecules such as dNTPs so as to facilitate a biological analysis such as real-time sequencing;

[0031] Figures 16A and 16B show example data that demonstrate how metal- enhanced fluorescence can be controlled for different biological molecules; and

[0032] Figures 17A - 17C show example simulation results that demonstrate how a metal-nanoparticle assembly can be configured to allow control of excitation at different wavelengths.

[0033] These and other aspects, advantages, and novel features of the present disclosure will become apparent upon reading the following detailed description and upon reference to the accompanying drawings. In the drawings, similar elements have similar reference numerals.

DETAILED DESCRIPTION

[0034] The present disclosure generally relates to systems and methods for detecting biological molecules. In certain embodiments, such systems and methods can be used to detect single biological molecules. In many situations, such biological molecules can include deoxyribonucleotides dATP, dCTP, dGTP and dTTP (and analogs) that are involved in DNA-related processes. As described herein, efficient detection of such molecules can facilitate, for example, real-time DNA sequencing.

[0035] In certain situations, presence and/or involvement of such molecules and other DNA fragments can be detected by tagging them with detectable labels such as fluorescent labels. Excitation of the tagged labels (such as fluorophores) attached to the molecules of interest can result in detectable signals (such as fluorescent light) being emitted and detected.

[0036] In certain embodiments, the detectable signals can be collected and sent to an imaging detector such as a charge-coupled-device (CCD), a complementary- metal-oxide-semiconductor (CMOS), an electron-multiplying CCD (EMCCD), a scanned avalanche photodiode detector (APD) array, or a black silicon array. Thus, accurate imaging of the signals from the molecules of interest can be an important feature.

[0037] Various parameters associated with emission of detectable signals (such as fluorescence emission) by the labels (such as fluorophores) can play important roles in determining the quality and resolution limits of detection of the biological molecules. By way of non-limiting examples, quantum yield, excitation wavelength and other fluorescence effects such as quenching are important features that can determine the quality of the detected signals. Sufficient quantum yield per tagged molecule can allow detection of a single molecule. In certain situations, however, quenching can offset the beneficial effect of such increase of excitation rate, and even provide a detrimental effect. In certain embodiments, such balancing of enhanced excitation versus metal quenching can be addressed by selecting an appropriate spacing of the fluorescence location as described herein. For at least these reasons, it can be desirable to be able to adjust different fluorescence-related parameters in measurement situations such as in single-molecule detection. Various features that can provide such adjustments, or facilitate such adjustments, are described herein.

[0038] Figure 1 A shows an example schematic diagram for a biological analyzer 100 capable of characterizing (e.g., sequence determination or fragment analysis) biological samples (such as nucleic acid samples) or processes (such as real-time DNA sequencing). In various embodiments, the analyzer 100 may include one or more components or devices that can be used for labeling and identification of the sample and may provide features for performing sequence analysis. In certain embodiments, the various components of the analyzer 100 can include separate components or a singular integrated system. The present disclosure may be applied to both automatic and semi-automatic sequence analysis systems as well as to methodologies wherein some of the sequence analysis operations are manually performed. Additionally, systems and methods described herein may be applied to other biological analysis platforms to improve the overall quality of the analysis.

[0039] In various embodiments, the methods and systems of the present disclosure may be applied to numerous different types and classes of photo and signal detection methodologies and are not necessarily limited to CCD, CMOS, EMCCD, APD, or black silicon array based detectors. Additionally, although various embodiments of the present disclosure are described in the context of sequence analysis, these methods may be readily adapted to other devices/instrumentation and used for purposes other than biological analysis.

[004O] In various embodiments, the methods and systems of the present disclosure may be applied to numerous different types and classes of excitation methodologies and are not necessarily limited to laser based excitation systems.

[0041] In the context of sequence analysis, the example sequence analyzer 100 may include a reaction component 102 wherein amplification or reaction sequencing (for example, through label incorporation) of various constituent molecules contained in the sample can be performed. Using these amplification techniques, a label or tag, such as a fluorescent label or tag may be introduced into the sample constituents resulting in the production of a collection of nucleotide fragments of varying sequence lengths..

[0042] In certain embodiments, and as described herein, a label or tag, such as a fluorescent label or tag may be introduced into single-molecule sample constituents such as base molecules dATP, dCTP, dGTP and dTTP (and analogs). As described herein, interactions of such single-molecules with their respective complementary bases in a DNA sample can be facilitated by an interaction site. By providing such an interaction site with metal-enhancement effect(s) as provided herein, single molecules involved in such interactions can be distinguished over other base-pair interactions that may be occurring without the metal-enhancement effect.

[0043] For the purpose of description herein, an "interaction site" can include a structure or a molecule that can facilitate a reaction (e.g., as a catalyst) between one molecule with another molecule. By way of an example, interaction site can include an enzyme such as polymerase that facilitates hybridization reactions between bases of a single strand DNA and the bases' complementary base molecules. In such an example context, the interaction site can include the polymerase molecule, or some other structure that includes the polymerase molecule.

[0044] For the purpose of description, reactions facilitated by the interaction site can reside in the interaction site in a static manner, or reside in the interaction site temporarily such as in hybridization of the single strand DNA into a double strand DNA.

[0045] For the purpose of description, the interaction site can also include a molecule of a structure that can be immobilized with respect to one or more metal- nanostructures, where the molecule or structure can be configured to bind preferentially with a molecule of interest. If such molecule of interest is fluorescence labeled, binding to the interaction site can allow more effective detection of fluorescence due to the metal-enhanced fluorescence effect provided by the metal- nanostructures.

[0046] In certain embodiments, a detector component 106 can be configured to detect and facilitate identification of interactions between the labeled molecules and the sample based on the presence of the incorporated label or tag. In certain embodiments, such detection of a labeled molecule may be performed by generation of a detectable signal produced by a fluorescent label that is excited by a laser tuned to the label's absorption wavelength.

[0047] Figure 1 B shows an example detection configuration 110 where various components of the detector 106 (Figure 1 A) may be used to acquire the signal associated with one or more labeled molecules or particles present in a detection zone 1 12. As previously indicated, the labeled fragments in the detection zone 1 12 may be resolved by measuring, for example, the wavelength of fluorescence or emitted energy generated when the labels are subjected to excitation energy of appropriate wavelength and intensity. In certain embodiments, such excitation energy can be provided by an energy source such as a laser.

[0048] In certain embodiments, the energy emissions produced by the labeled molecules in the detection zone 1 12 may be detected using a detector 1 16, such as a CCD or a CMOS based detector. As described herein, other types or detectors can also be used. In certain embodiments, such detector 1 16 can have a plurality of energy detecting elements (e.g., pixels) that capture at least a portion of the emitted energy from the labeled molecules.

[0049] In certain embodiments, signals generated by the detector 1 16 can yield a spectral distribution of light emitted from a given labeled molecule. Such spectral distribution can have one or more peaks that may be of interest in identifying the labeled molecule. Because different labeled molecules can yield different spectra under fluorescence, being able to detect and distinguish such labels based on the spectra can be an important tool in various biological analyses such as DNA sequencing. As described herein, various features of the present disclosure can provide flexibility in providing enhancement properties for different fluorescing labels and/or enhancement for different wavelengths for a given label.

[005O] In certain embodiments, a signal processor 1 18 can be configured to perform signal sampling operations to acquire the signal generated by the detector 1 16 in response to the signals from the detection zone 1 12.

[0051] In various embodiments, some of the information that may be determined through signal sampling operations may include determination of, for example, the detected molecule's spectra, kinetics, and binding/incorporation properties. Evaluation of the signals may also be used to determine the sequence or composition of the sample using the sequence of detected molecules.

[0052] In various embodiments, the analysis of the signal representative of the aforementioned example data may be advantageously performed by the signal processor 1 18. The signal processor 1 18 may further be configured to operate in conjunction with one or more processors. The signal processor's components may include, but are not limited to, software or hardware components, modules such as software modules, object-oriented software components, class components and task components, processes methods, functions, attributes, procedures, subroutines, segments of program code, drivers, firmware, microcode, circuitry, data, databases, data structures, tables, arrays, and variables. The signal processor may also include or be a part of, for example, computer, DSP, GPU, FPGA, and the like. Furthermore, the signal processor 1 18 may output a processed signal or analysis results to other devices or instrumentation where further processing may take place.

[0053] As shown in Figure 1 B, imaging of the detection zone 1 12 can be facilitated by an assembly of optics 1 14. In certain embodiments, the optics component 1 14 can be configured to direct at least a portion of the signals from the detection zone 1 12 to the detector 1 16. In certain embodiments, the optics component 1 14 can include one or more elements that can facilitate processes such as total internal reflection fluorescence (TIRF) excitation.

[0054] In certain embodiments, one or more metal-nanoparticle (MNP) based assemblies 120 can be present in the detection zone 1 12, or be incorporated as a part of the detection zone 1 12. Various non-limiting examples of such MNP assemblies 120 and their implementations are described herein in greater detail.

[0055] Figure 2 shows a non-limiting example of how one or more of the MNP assemblies 500 can be utilized. As described herein, the MNP assembly 500 can include a plurality of MNPs (502a, 502b) that can be coupled via a linker 504. The MNP assembly 500 can further include an interaction site 506 disposed relative to the MNPs 502.

[0056] In certain embodiments, the interaction site 506 can be selected so as to facilitate interactions such as hybridization of labeled base molecules 520 with their corresponding bases in a single strand DNA sample 510 being analyzed. In Figure 2, the base molecule 514 is depicted as being matched and undergoing binding to its corresponding base in the single strand DNA 510. Repetition of such interactions can yield a hybridized double strand DNA 516.

[0057] The labeled base molecules 520 can include base molecules 522 such as deoxyribonucleotides dATP, dCTP, dGTP, dTTP and analogs. The labeled base molecules 520 can further include labels 524 such as different types of fluorophores. For example, the base molecules A, C, G, and T can be labeled with different fluorophores to allow detection of the type of base molecule 514 that in undergoing interaction via the interaction site 506.

[0058] In certain embodiments, the interaction site 506 can include an enzyme such as a polymerase that can facilitate the example hybridization process. The polymerase can be positioned relative to the MNPs 502 via attachment to the linker 504. The polymerase can be attached either directly to the linker 504, or via an intervening structure.

[0059] For the purpose of description, it will be understood that interaction site can include structures such as enzymes (e.g., polymerase). The interaction site can be positioned relative to the MNPs via one or more linkers, either directly or via one or more intervening structures. The interaction site can also be positioned relative to the MNPs via structures other than the linkers.

[006O] In certain embodiments, the interaction site can facilitate selective binding of a labeled molecule (e.g., labeled base molecule) with another molecule (e.g., single- strand DNA molecule). In certain embodiments, the interaction site can also be configured to be a receptor to allow selective binding of a labeled molecule to the receptor.

[0061] In certain embodiments, the interaction site can be positioned relative to the MNPs so as to allow an interacting labeled molecule to experience metal- enhancement effect such as metal-enhanced fluorescence. Such interacting labeled molecule can include a labeled base molecule undergoing binding or a bound base molecule substantially within the metal-enhancement region.

[0062] As depicted in Figure 2, metal-enhanced fluorescence can yield a signal 530 having an increased signal-to-noise ratio. In comparison, an example signal 532 emitted from a base molecule that has been incorporated into the double-strand 516 and outside of the metal-enhancement zone. The example signal 532 is depicted as being less than that of the enhanced signal 530. Similarly, an example signal 534 emitted from a free base molecule outside of the metal-enhancement zone is depicted as being less than that of the enhance signal 530.

[0063] The example signals 530, 532, and 534 are depicted as example distributions 540 that can be obtained from detection and measurement. As shown, the enhance signal 530 (solid line) can yield a signal distribution 542 (solid line) where a peak of interest 550 can be enhanced. In comparison, the un-enhanced signal 532 (dotted line) can yield a signal distribution 544 (dotted line) where a similar peak of interest is depicted as being substantially lesser than that of the enhanced peak 550. For the purpose of this particular example, the example signals 530 and 532 can be assumed to result from same type of fluorophores attached to same type of base molecules.

[0064] As further shown in Figure 2, the un-enhanced signal 534 (dashed line) can yield a signal distribution 546 (dashed line) that also has a relatively low signal-to- noise ratio. The signal 534 can be an example of a fluorescence resulting from a different type of fluorophore attached to a different type of base molecules (than those associated with the signals 530 and 532).

[0065]As shown in Figure 2, enhancement of the fluorescence signal 530 can allow determination of the base molecule 514 undergoing the interaction. In certain embodiments, such calling of single interacting base molecules can be performed in substantially real time, such that the corresponding sequencing of the single strand DNA sample can also be in substantially real time as the hybridization process progresses.

[0066] As described herein, various embodiments of the present disclosure can provide a functionality such as metal-enhancement of fluorescence via MNPs. In certain embodiments, such enhancement of fluorescence can be performed in a selective manner. For example, the example signal distribution 542 (Figure 2) is depicted as having peaks (550 and 552). For the example described herein in reference to Figure 2, the peak 550 is designated as the peak of interest. In certain situations, however, it may be desirable to enhance the other peak 552. As described herein, flexibility provided by various possible configurations can allow such enhancement to occur.

[0067] Figure 3 shows a block diagram of an example assembly 130 of metal- nanoparticles. For the purpose of description, a first metal-nanoparticle (MNP) 132 is also designated as MNP "A"; and a second MNP 134 is designated as MNP "B." In certain embodiments, MNPs A and B can be based on different metal atoms. - -

[0068] In certain embodiments, the MNPs 132 and 134 can be coupled via a coupling link 136. In certain embodiments, the coupling link 136 can be formed by attaching a first oligonucleotide strand to the first MNP 132 and a second oligonucleotide strand to the second MNP 134, where at least portions of the first and second strands are complementary to each other so as to allow formation of double strands. In certain embodiments, the overlapping portion of the linker 136 can form a double stranded DNA such as native DNA or other types of DNA.

[0069] In certain embodiments, the length between the first and second MNPs 132 and 134, and/or strength of the coupling link 136 can be adjusted by selecting the lengths of the first and second strands and/or the amount of complementary portions of the first and second strands. In certain embodiments, more than two strands can be used to form the linker 136 to provide, for example, an increased stiffness. Other types of linkers are also possible. In certain embodiments, configuring of the strands to achieve MNP parameters such as coupling length and strength can be achieved using known techniques.

[007O] In certain embodiments, the assembly 130 can further include an interaction site 140 disposed relative to the MNPs (132, 134). In certain embodiments, the interaction site 140 can be positioned as such by being immobilized to a portion of the coupling link 136. For example, the interaction site 140 can be immobilized to one of the first and second oligonucleotide strands. In certain embodiments, the interaction site 140 can provide one or more functionalities as described in reference to Figure 2.

[0071] For the purpose of description, terms such as "immobilize," "attach" and "bind" can refer to customarily understood meanings associated with molecular biology and/or nanotechnology.

[0072] Figures 4A - 4C show an example of how the assembly 130 of MNPs can be fabricated. In certain embodiments, one or more first sub-assemblies 152 can be immobilized on a substrate surface 160 such as a surface of a quartz coverslip. As shown in Figure 4A, the first sub-assembly 152 can include a first MNP 154 attached to a first strand of oligonucleotide 156. In certain embodiments, the first MNPs 154 can be immobilized to the substrate surface 160. In certain embodiments as described below by way of example, the MNP/strand(s) sub-assembly can be immobilized to the - -

surface via a strand of oligonucleotide. Other attachment forms can include, for example, preferential attachment, gridding, attachment of both MNPs with preferential orientation from flow, laser tweezers and the like, orientation to facilitate lighting with polarization aligned in a desirable manner.

[0073] As shown in Figure 4A, one or more second sub-assemblies 162 can be introduced to the first assemblies 152. The second sub-assembly 162 can include a second MNP 164 attached to a second strand of oligonucleotide 166. In certain embodiments, an interaction site 168 can be immobilized to the second strand 166. In certain embodiments, the interaction site 168 can be immobilized to the first strand 156.

[0074] In certain embodiments, the first strand 156 can be a single strand of oligonucleotides having a first length, and the second strand 166 can be a single strand of oligonucleotides having a second length. At least some portions of the first and second strands (156, 166) can be substantially complementary to each other so as to allow formation of a double strand. In certain embodiments, the separation distance between the first and second MNPs 154 and 164 can be controlled by selecting the respective lengths of the first and second strands 156 and 166, and/or their complementary portions. In certain embodiments, the location of the interaction site's immobilization on the first or second strands can be selected to control the location of the interaction that is being monitored via the enhancement provided by the first and second MNPs.

[0075] Figure 4B depicts the first sub-assemblies 152 and second sub- assemblies 162 having hybridized and coupled so as to form one or more assemblies 172 of MNPs.

[0076] Figure 4C shows that for each assembly 172 of MNPs, a desired labeled particle 182 can either bind to the corresponding interaction site 168, or bind to another structure such as a DNA strand being analyzed (not shown) via the interaction site 168. In either situation, the presence of the labeled particle 182 at or in close proximity to the interaction site 168 can allow labeled particle 182 to benefit from the enhancement effect provided by the MNPs. As described herein, the labeled particle 182 can include a molecule (such as one of base molecules A, C, G, and T) labeled with a detectable tag such as a fluorophore. In situations where fluorophores are used as detectable tags, the MNPs can enhance the local electric field at or about the fluorophore's location when subjected to excitation energy such as a laser. Such fluorescence enhancement is believed to occur due to a resonance effect of plasmons on or about the surface of the MNPs upon appropriate excitation. Additional details of fluorescence enhancement via MNPs can be found in, for example, the following references: Enhancement and Quenching of Single-Molecule Fluorescence, Anger, P., et al, Phys. Rev. Let. (2006), 96:1 13002; Metal-Enhanced Fluorescence: An Emerging Tool in Biotechnology, Asian, K., et al, Curr. Opin. Biotechnol. (2005), 16:55-62; Single- Molecule Studies on Fluorescently Labeled Silver Particles: Effects of Particle Size, Zhang, J., et al, J. Phys. Chem. C (2008), 1 12, 18-26; Systematic Computational Study of the Effect of Silver Nanoparticle Dimers on the Coupled Emission from Nearby Fluorophores, Chowdhury, M., et al, J. Phys. Chem. C (2008), 1 12, 1 1236-1 1249; and Metal-Enhanced Single Molecule Fluorescence on Silver Particle Monomer and Dimer: Coupling Effect Between Metal Particles, Zhang, J., et al, Nano Letters (2007), 7, 2101 - 2107.

[0077] Additional details about metal-enhanced fluorescence can be found in, for example, the following references: Fluorescence Emission at Dielectric and Metal-Film Interfaces, Hellen, E., et al, J. Opt. Soc. Am. B (1987), 4:337-350; and Single-Molecule Studies of Enhanced Fluorescence on Silver Island Films, Fu, Y., et al, Plasmonics (2007), 2(1 ), 1 -4.

[0078] In certain embodiments, the type of metal particle can influence the electric field enhancement, and thus the fluorescence properties of the fluorophore. Thus, use of different metal particles for the first and second MNPs can facilitate a greater flexibility in the types of fluorophores that can be used, as well as excitations at different wavelengths. For example, an aluminum MNP is shown enhance the Alexa- 488 emission but not the Cy5 dye emission (excited at approximately 633 nm). In another example, a gold MNP can enhance the emission of Cy5 but not Alexa-488.

[0079] In certain embodiments, enhancement of fluorescence, and wavelength of enhancement can also depend strongly on factors such as sizes, shapes, and arrangements of the MNPs. As described herein, such factors can be adjusted separately or in some combination to achieve a desired enhancement effect. Aside from the example MNP structures described herein, other nano-structures such as nanorice, nanoshells, nanospheres, nanorods, and nanostars can also provide similar functionalities. Additional details about such dependencies can be found in, for example, Single-Molecule Studies on Fluorescently Labeled Silver Particles: Effects of Particle Size, Zhang, J., et al, J. Phys. Chem. C (2008), 1 12, 18-26. Non-limiting examples of variations in sizes and shapes of the MNPs are described herein in greater detail.

[008O] In certain embodiments, the ability to enhance selected wavelengths or ranges of wavelength can provide a significant improvement in flexibility in the manner in which analyses can be performed. For example, a limited number of different types of fluorophores used for different base molecules can compete for distinguishable peaks in a limited range of the fluorescence spectrum. By providing the ability to select the wavelength (or range of wavelength) of enhancement, ambiguities resulting from such competing effects can be reduced or removed, thereby allowing an improved determination of the detected labeled molecule.

[0081] Figure 5 shows that in certain embodiments, more than two MNPs can be incorporated into an assembly to enhance the flexibility in the fluorescence effect and/or the selectivity in the wavelength of enhancement. In an example assembly 190, four MNPs 192, 194, 196, and 198 are designated as "A," "B," "C," and "D," respectively. In certain embodiments, there can be at least two types of MNPs among the example four.

[0082] In the example configuration 190, the MNPs 192 and 194 are depicted as being coupled via a link indicated as 200 and 202. In certain embodiments, the coupling between the MNPs 192 and 194 can be similar to that described in reference to Figure 3.

[0083] The example configuration 190 is shown to have the MNP 196 coupled to the assembly 190 via a link 204, and the MNP 198 coupled to the assembly 190 via a link 206. An example of how such links can be implemented is described in greater detail in reference to Figure 6. [0084] The example configuration 190 is shown to have an interaction site 208 positioned as part of the assembly 190. In the example shown, the interaction site 208 can be immobilized to the link between the MNPs 192 and 194. In certain embodiments, the interaction site 208 can be immobilized at other locations along links 202, 204, and 206. Further, it will be understood that coupling of the example four MNPs can be arranged in a number of other ways; thus, the example shown in Figure 5 should be viewed as a non-limiting example. In certain embodiments, the links can be different sequences of DNA without homology. Other types of linkers are also possible.

[0085] By providing the four example MNPs in proximity to the interaction site 208, a greater degree of flexibility in various fluorescence related parameters can be achieved for fluorescence processes occurring at or near the interaction site 208. For example, MNP types, MNP sizes, distances among the MNPs, and the general arrangement of the MNPs are some of the parameters whose degrees of adjustability can be increased from that associated with the example two-MNP configuration.

[0086] Figure 6 shows an example of how the assembly 190 of Figure 5 can be fabricated. In certain embodiments, a first sub-assembly 210 having a first MNP 212 can be immobilized to a substrate surface 230 (such as a surface of a quartz coverslip) via an oligonucleotide strand 214. The example MNP 212 is shown to have another oligonucleotide strand 216 attached thereto. In certain embodiments, the strand 216 can be the same type of strand as the strand 214; and that strand 216 can be attached to the MNP 212 at, for example, midpoint). As shown, an interaction site 220 can be immobilized on the strand 216. Also on the strand 216, another strand 218 can be immobilized so as to form a branch.

[0087] As shown in Figure 6, one or more of second sub-assemblies 240 can be introduced to one or more of the first sub-assemblies 210. The example second sub- assembly 240 can include an oligonucleotide strand 244 attached to a second MNP 242; and another strand 246 is shown to branch out from the strand 244.

[0088] In certain embodiments, the example strands 216 and 244 can include complementary portions so as to allow formation of double strands, thereby forming a two-MNP configuration 250 immobilized to the substrate surface 230 via the strand 214.

[0089] As shown in Figure 6, one or more of third sub-assemblies 260 can be introduced to one or more of the two-MNP assemblies 250. The example third sub- assembly 260 can include an oligonucleotide strand 264 attached to a third MNP 262.

[009O] In certain embodiments, the example strands 264 (of the sub-assembly 260) and 218 (of the sub-assembly 210) can include complementary portions so as to allow formation of double strands, thereby forming a three-MNP configuration 270.

[0091] As shown in Figure 6, one or more of fourth sub-assemblies 280 can be introduced to one or more of the three-MNP assemblies 270. The example fourth sub- assembly 280 can include an oligonucleotide strand 284 attached to a fourth MNP 282.

[0092] In certain embodiments, the example strands 284 (of the sub-assembly 280) and 246 (of the sub-assembly 240) can include complementary portions so as to allow formation of double strands, thereby forming a four-MNP configuration 290. As described herein, certain embodiments of the linkers can include more than two strands to provide, for example, greater stiffness and strength. Having one or more additional strands can also facilitate features such as having more than one interaction sites disposed relative to the MNPs.

[0093] Assemblies having other numbers of MNPs are also possible. It will be appreciated that a number of different arrangements are possible by forming branched MNPs to achieve various desired configurations. It will also be appreciated that the branch-based fabrication of MNP-assemblies can allow formation of desired configurations that are very difficult to form in traditional semiconductor-based methods.

[0094]As shown in the example of Figure 6, the MNP-assembly 290 can be immobilized to the substrate surface 230 via the example strand 214. As with other links in the assembly 290, the length of the strand 214 can be selected so as to control the separation of the interaction site 220 from the surface 230. In certain situation, such controlled separation can be advantageous. By way of an example, surface interactions of certain dye-labeled biological molecules can be problematic. By suspending the interaction site 220 away from the surface 230, such surface effects can be reduced or eliminated.

[0095] In certain embodiments, a given MNP can have different shapes. Figures 7 A - 7C show non-limiting examples of some of the shapes that the MNP can be configured as. As shown in Figure 7A, a sub-assembly 300 can include a strand 304 immobilized to a spherical shaped MNP 302.

[0096] Figure 7B shows that in certain embodiments, a sub-assembly 310 can include a strand 314 immobilized to an MNP 312 that has a triangular shape in at least one sectional view. An MNP such as a pyramid shaped MNP 322 (Figure 7C) can be an example of the triangular shaped MNP 312. In Figure 7C, a strand 324 is depicted as being attached to the pyramid shaped MNP 322 so as to form a sub-assembly 320.

[0097] Other shapes of MNPs are also possible. As described herein non- limiting examples of structures such as nanorice, nanoshells, nanospheres, nanorods, and nanostars can be implemented using one or more features of the present disclosure. Further, strands such as oligonucleotide strands can be attached to different locations of the MNPs than shown in the example configurations of Figures 7A - 7C.

[0098] In certain embodiments, determining various locations for attachments associated with MNP assemblies can be achieved via photo-attachment technique. For example, a location for attaching an interaction site on a oligonucleotide strand can be determined by such a technique so as to yield a maxima in enhancement of desired properties in fluorescence occurring at or near the interaction site. In certain situations, such a maxima can include a maxima in the plasmon resonance induced electric field strength. In certain situations, such a maxima can include a balance of electric field strength and quenching effect associated with the increased field. In certain embodiments, such selection of interaction site positioning can be based on one or more areas of MNP assembly structures with highest enhancement(s). In certain embodiments, such selection based on enhancement can also include wavelength- specific enhancement.

[0099] Figures 8A and 8B show examples of configurations where triangular shaped MNPs can be implemented in the example two-MNP and four-MNP assemblies described herein in reference to Figures 3 - 6. In certain embodiments, a two-MNP assembly 330 can include first and second triangular shaped MNPs 332 and 334 coupled via a link 336. In the example, the link 336 is shown to be attached to the MNPs 332 and 334 at their respective vertices. Such a configuration can yield a "bowtie" or dipole shaped assembly that can provide a greater field enhancement than that associated with spherical shaped MNPs. In certain embodiments, such structures can be formed on a surface of a substrate.

[00100] In certain embodiments, the first and second MNPs 332 and 334 can be based on different metal particles. For example such different metal particles can be based on different metal atoms, on coatings of different metals, or on different sizes of structures. As described herein, use of such different MNPs can provide flexibility in controlling various fluorescence related parameters.

[00101] In certain embodiments, as shown in Figure 8B, a four-MNP assembly can include four MNPs 342, 344, 346, and 348 coupled via respective links depicted as 350, 352, 354, and 356. In certain embodiments, at least two different types of MNPs can be used among the four, so as to provide flexibility in controlling various fluorescence related parameters. In certain embodiments, an interaction site (not shown) immobilized with respect to the example double bowtie arrangement 340 can experience a greater amount of field enhancement and/or provide a greater degree of flexibility in such enhancement than the example single bowtie arrangement 330 of Figure 8A.

[00102] Figures 9A - 9C show non-limiting examples of different types of MNP arrangements that can be implemented. In certain embodiments, as shown in Figure 9A, an arrangement 600 of MNPs can be such that a plurality of MNPs 602 are positioned in a two-dimensional manner. As shown, the MNPs 602 are depicted as being arranged in a plane of the paper, and coupled via a linker structure indicated as 604. As described herein, an interaction site 606 can be positioned relative to the MNPs.

[00103] In certain embodiments, as shown in Figure 9B, another example configuration 610 can include a plurality of MNPs 612 arranged and coupled (via linkers 614) so as to form a ring structure. The example ring structure 610 may be - -

substantially two-dimensional; however, a three-dimensional arrangement is also possible. As shown, an interaction site 616 can be disposed relative to the MNPs 612 so as to provide an enhancement effect. In the example shown, the interaction site 616 is depicted as being couple via a linker 618. It will be understood that the interaction site 616 can be positioned relative to the MNPs in a number of ways.

[00104] In certain embodiments, as shown in Figure 9C, another example configuration 620 can include a plurality of MNPs 622 arranged and coupled (via linkers 624) so as to form a three-dimensional structure. As shown, an interaction site 626 can be disposed relative to the MNPs 622 so as to provide an enhancement effect. In the example shown, the interaction site 626 is depicted as being couple via a linker 628. It will be understood that the interaction site 626 can be positioned relative to the MNPs in a number of ways.

[00105] Figures 10 - 12 show non-limiting examples of implementation of one or more MNP assemblies on different settings. In certain situations, as shown in Figure 1 OA, one or more MNP assemblies 652 may be implemented in a substantially free configuration 650. For example, the MNP assemblies 652 can be suspended in a solution containing the particles to be detected.

[00106] In one example configuration 360, as shown in Figure 1 OB, one or more MNP assemblies 362 can be disposed on a substrate surface 364. As describe in reference to Figures 4 and 6, a layer of quartz can be an example of such substrate, and the MNP assemblies can be disposed on its surface. In certain embodiments, positioning of MNP assemblies can be guided by the structure of the MNP assembly itself, and/or the type of the substrate surface. For example, attachment to certain metal spots which can support enhancement may allow only room for one MNP assembly. In such a situation, a two-MNP-assembly can be positioned on the surface so that one MNP is supported by the surface, and the other MNP is positioned above the first MNP (similar to the example configuration of Figure 4B).

[00107] In another example configuration 370, as shown in Figure 1 1 , one or more MNP assemblies 376 can be disposed on a surface that is part of a well 374 defined by a substrate 372. In certain embodiments, the well can be formed from substrate and metal, or metal and high index dielectric. In certain embodiments, the dimension 378 of the well 374 can be selected to provide a desired functionality. For example, the dimension 378 can be selected to be greater than approximately half of the wavelength of a given excitation and/or fluorescence light, so as to facilitate reduction in background fluorescence levels.

[00108] In another example configuration 380, as shown in Figure 12, one or more MNP assemblies 388 can be disposed in a waveguide 386 such as a zero-mode waveguide. Such zero-mode waveguide can include a metal layer 382 defining an aperture, disposed on a substrate such as silicon dioxide, to thereby define the waveguide 380 cavity 386. Additional details about the zero-mode waveguide and its uses can be found in literatures associated with Pacific Biosciences of Menlo Park, California.

[00109] In certain embodiments, placement of MNP-based assembly in the zero-mode waveguide can further enhance and/or allow control of fluorescence parameters beyond those provided by the waveguide. For example, real-time sensing of a labeled molecule 390 (such as a dNTP molecule) can be enhanced by inclusion of the MNP-based assembly 388. Such detection of individual labeled molecules can facilitate determination of sequence of complementary bases in a DNA strand (not shown) being analyzed.

[00110] Figures 13 and 14 show that MNP-based assemblies can be arranged on substrates in different ways. In an example configuration 400, as shown in Figure 13, a plurality of MNP assemblies 402 can be arranged in an array on a substrate surface 404. In certain embodiments, the assemblies 402 can be one or more types.

[00111] In certain situations, however, the array type of arrangement may not be necessary. In an example configuration 410, as shown in Figure 14, a plurality of MNP assemblies 412 can be arranged on a substrate surface 414 in a substantially random manner. In certain embodiments, a cluster of the randomly arranged MNP assemblies 412 can be of the same type; and there can be different clusters (such as spots or macro-sized beads) having different MNP assemblies. In certain embodiments, such attachment of MNP assemblies to beads or substrates can be in a random or non- random manner. Such a configuration can be used for testing clonal populations and - -

for hybridization ligation assays, permitting different molecules to be enhanced with different wavelengths of excitation and/or fluorescence.

[00112] Various other non-limiting features can be implemented in formation of and/or use of MNP assemblies. In certain embodiments, an MNP can be based on one or more of the same metal atoms. Alternatively, an MNP can be based on alloys having two or more different metal atoms. Such variation of the composition within the MNP can provide further flexibility than that associated with, for example, different compositions among the MNP assemblies.

[00113] In certain embodiments, MNP assemblies can be sensitive to polarization of the excitation beam (such as a laser beam). In such situations, use of an unpolarized laser may be preferred so as to benefit from an average enhancement effect rather than suffer from a possible reduction in enhancement due to an undesired polarization.

[00114] In certain embodiments, collection and detection of fluorescence light from labeled particles present at MNP assemblies can be facilitated via a configuration associated with total internal reflection fluorescence (TIRF) techniques. In certain situations, TIRF based collection and detection can provide reduced background levels.

[00115] In certain embodiments, MNPs can be combined with other features and/or techniques. For example, MNPs can be combined with Qdots® or upconverting phosphors for either single molecule or clonal assays. In certain situations, the Qdots and/or upconverting phosphors can be used to further reduce background. In certain situations, multiple Qdots of different wavelengths can be used with different MNPs to provide multiple wavelengths from a single excitation laser or on a single MNP. MNPs can enhance energy transfer between donor fluors (which may be attached on or near the MNPs) to acceptor fluors with greater efficiency and over greater distances using plasmons. When using MNPs in such a situation, it may be desirable to have the MNPs float substantially freely, as opposed to being attached a surface. Such free floating configuration may be further advantageous for kinetics assays.

[00116] In certain embodiments, one or more of the MNP assemblies disclosed herein can be used for sensing of single dNTP molecules. In certain embodiments, such sensing can be performed in real or substantially real time. Such single-molecule sensing and/or real time sensing of molecules are now described by way of a non- limiting example.

[00117] Figure 15 shows an example configuration 420 where a MNP assembly 422 is depicted as being disposed on a substrate surface 424. The example assembly 422 is a two-MNP assembly; however, it will be understood that assemblies having other number of MNPs, or different arrangements, can also be used.

[00118] As shown in Figure 15, an interaction site 426 can be disposed on the coupling link between the MNPs. The example interaction site 426 is depicted as providing a binding preference for a dNTP molecule 430 also indicated with single hash marks. Such preference can be due to, for example, the interaction site processing a portion of the DNA sample (not shown) that complements the dNTP molecule 430. Thus, among a plurality of other types of dNTP molecules 434, 434, and 436, the interaction site 426 can facilitate binding of a single molecule 430 to the DNA sample. As described herein such sensing of the single dNTP molecule 430 can allow real-time sequencing of the DNA sample.

[00119] As shown in Figure 15, excitation energy 440 can be provided to or to an area near the interaction site 426 so as to excite the corresponding molecule present at the interaction site 426. Due to the enhancement of fluorescence from the MNPs, detection of the bound or attached single molecule (430 in this example) can be facilitated.

[00120] In certain embodiments, the interaction site 426 can include a polymerase molecule or a structure having a polymerase molecule, and such interaction site 426 can be immobilized between the two MNPs according to the following example. In certain embodiments, a biotin molecule can be attached to the coupling link (for example, a DNA strand) by using a biotin derived nucleotide. A biotinylated polymerase can then be immobilized to the DNA strand through streptavidin. By this example process, a single polymerase molecule can be immobilized relative to the MNPs. Distances between the polymerase and the MNPs can be controlled by adjusting the lengths of the DNS strands associated with the MNPs. In certain situations, the benefit of enhancing the electric field increase may need to be balanced with the increased likelihood of quenching of the fluorophores.

[00121] Referring to Figure 15, and for the purpose of description, it will be assumed that the dNTPs have already been labeled with appropriate fluorophores at their bases and/or phosphate terminus. In certain embodiments, the plasmon resonance on or about the MNP surfaces can be excited by far-field excitation. In certain embodiments, the resulting enhancement can be near-field, thereby providing an excitation volume that can be confined to a relatively small volume. In certain embodiments, distances between MNPs can be on the order of 20nm, with the fluorophore approximately half way between. Such selection can be based on, for example, typical quenching due to proximity to metal occurring typically at 5nm or less, and some nanostructures being able to extend significant enhancement over larger distances, such as 50nm, 100nm, 200nm or more. In certain embodiments, the MNP separation distance can be selected so as to avoid quenching (e.g., greater than 5nm), but keeping the distance relatively small, since background can increase with the separation distance.

[00122] By using such a small excitation volume, detection of a single molecule among high concentration of labeled molecules can be possible. In certain embodiments, such single-molecule detection can be monitored in real or substantially real time, to thereby facilitate desirable processes such as real time DNA sequencing and step-wise DNA sequencing.

[00123] In certain embodiments, the fluorescence enhancement provided by the MNPs can allow reduction in the power requirement of the excitation laser. Further, as described herein, use of different excitation wavelengths can be made more flexible via use of different MNPs.

[00124] An example of wavelength dependence of enhancement is shown in Figures 16A and 16B. A confocal scanning image 450 of dATP-Fam (excited at approximately 488 nm) and dCTP-Cy5 (excited at approximately 633 nm) disposed on a quartz surface is shown in Figure 16A. The image 450 shows generally red patches. The same type of labeled molecules disposed on aluminum nanowells and excited in the same manner results in an image 460 shown in Figure 16B. The image 460 shows well defined and generally bright green dots. The concentration for both dyes is approximately 1 M, and the excitation power for both wavelengths (488 nm and 633 nm) is approximately 10 W.

[00125] Based on the example image 450 of Figure 16A, it is readily apparent that dATP-Fam molecules yield much weaker signals than that of dCTP-Cy5 molecules when both compounds are passively bound to the quartz surface and not being subject to metal enhancement of fluorescence. On the other hand, the example image 460 of Figure 16B shows that dATP-Fam molecules yield much stronger signals than that of dCTP-Cy5 molecules when both compounds are passively bound to in aluminum nanowells thereby being subject to metal plasmonic enhancement of fluorescence.

[00126] The foregoing observation demonstrates that aluminum nanostructure can selectively enhance the Fam fluorophore so as to yield efficient excitation at 488 nm. It has been demonstrated in various experiments that the emission of Cy5 can be enhanced by gold or silver nanostructures. Thus, if enhanced emissions of different fluorophores are desired, it would be advantageous to have different metals assembled in nanostructures so as to cover a wide range of excitation spectra.

[00127] To see whether selective field enhancement can be achieved using two different MNPs, the following example simulation was performed. The example simulation was performed using a finite-difference time-domain (FDTD) technique that can implement Maxwell's time-dependent curl equations for approximating the temporal variation of electromagnetic waves within a finite space that contains a target of arbitrary shape. For the example simulation, an example two-MNP assembly as shown in Figure 17A was defined. An aluminum MNP and a gold MNP, each having an effective size of approximately 50 nm, were separated in air by approximately 50 nm. The resulting MNP assembly 470 was subjected to a linearly polarized plane wave excitation light at appropriate orientation.

[00128] Figure 17B shows an example image 480 when the MNP assembly 470 was subjected to excitation light at approximately 488 nm; and Figure 17B shows an example image 490 when the MNP assembly 470 was subjected to excitation light at approximately 633 nm. The comparison of the two example images 480 and 490 shows that enhancement at 488 nm primarily occurs from the aluminum MNP, while enhancement at 633 nm occurs primarily from the gold MNP, thus demonstrating that an assembly having different MNPs can facilitate selective fluorescence excitation at different wavelengths.

[00129] In the example simulation in reference to Figures 17A - 17C, the images 480 and 490 were obtained with the plane wave (parallel to the plane of the paper) of the excitation light propagating through the center of the MNPs. It was observed that rotating the polarization orientation of the excitation light by 90 degrees results in substantially eliminating the enhancement effect between the MNPs. It was also observed that rotating the orientation of the MNP assembly (to a vertical orientation) can result in the MNP assembly being insensitive to polarization orientation.

[00130] Although the above-disclosed embodiments have shown, described, and pointed out the fundamental novel features of the invention as applied to the above-disclosed embodiments, it should be understood that various omissions, substitutions, and changes in the form of the detail of the devices, systems, and/or methods shown may be made by those skilled in the art without departing from the scope of the invention. Consequently, the scope of the invention should not be limited to the foregoing description, but should be defined by the appended claims.

[00131] All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A system for sensing biological molecules, comprising: a detection zone having one or more metal-nanostructure assemblies, each of said metal-nanostructure assemblies comprising: a plurality of metal-nanostructures coupled via one or more linkers, at least one of said metal-nanostructures being different than at least one other metal-nanostructure; and an interaction site disposed relative to said plurality of metal- nanostructures and configured to preferentially provide a location for detecting a detectable biological molecule; a detector configured to detect a signal emitted by said detectable biological molecule; and an optics assembly configured to direct signal from said biological molecule to said detector.

2. The system of Claim 1 , wherein said detectable biological molecule comprises a biological molecule labeled with a fluorophore, such that said fluorophore experiences a metal-enhanced fluorescence effect when positioned at said location and subjected to excitation energy.

3. The system of Claim 2, wherein said signal comprises a fluorescent signal emitted by said fluorophore.

4. The system of Claim 3, wherein said interaction site comprises a polymerase molecule.

5. The system of Claim 4, wherein the position of said polymerase relative to said plurality of metal-nanostructures can be controlled by adjusting length of said one or more linkers.

6. The system of Claim 5, wherein said adjusted length is selected so as to provide near-field enhancement of fluorescence induced by said plurality of metal- nanostructures.

7. The system of Claim 2, wherein types and arrangements of said plurality of metal- nanostructures relative to said fluorophore can be selected to obtain one or more desired excitation wavelengths of said excitation energy.

8. The system of Claim 2, wherein said metal-enhanced fluorescence effect allows use of lower intensity of said excitation energy.

9. The system of Claim 1 , wherein said location comprises a base of a single strand DNA template, said base being positioned at said interaction site.

10. The system of Claim 1 , wherein said metal-nanostructure comprises a metal- nanoparticle.

1 1. The system of Claim 1 , wherein said one or more linkers comprises one or more DNA strands.

12. An apparatus for capturing a biological molecule, comprising: a first metal-nanostructure coupled to a first linker; a second metal-nanostructure that is different than said first metal- nanostructure and coupled to a second linker, said first and second metal- nanostructures coupled to each other via said first and second linkers; and an interaction site coupled to either of said first and second linkers so as to be positioned relative to said first and second metal-nanostructures, said interaction site configured to facilitate preferential binding of said biological molecule with a template molecule.

13. The apparatus of Claim 12, wherein said first and second linkers comprise first and second oligonucleotide strands.

14. The apparatus of claim 13, wherein at least a portion of said first oligonucleotide strand is complementary to at least a portion of said second oligonucleotide strand to thereby form a double strand at said complementary portions of said first and second oligonucleotide strands.

15. The apparatus of Claim 14, wherein lengths of said first and second oligonucleotide strands and locations of said complementary portions are selected based at least in part on desired separation of said first and second metal-nanostructures.

16. The apparatus of Claim 12, wherein said biological molecule comprises a detectable biological molecule.

17. The apparatus of Claim 16, wherein said detectable biological molecule comprises a fluorophore labeled biological molecule, such that said fluorophore of said labeled biological molecule bound to said template molecule experiences a metal-enhanced fluorescence effect when subjected to an excitation energy.

18. The apparatus of Claim 17, wherein said first and second metal-nanostructures being different facilitates selected enhancement of emissions from said fluorophore.

19. The apparatus of Claim 12, further comprising one or more additional metal- nanostructures coupled to said first and second metal-nanostructures.

20. The apparatus of Claim 12, wherein said first and second metal-nanostructures comprise first and second metal-nanoparticles.

21. The apparatus of Claim 20, wherein each of said first and second metal- nanoparticles is comprises a gold atom, a silver atom, a copper atom, an aluminum atom, or an alloy particle.

22. The apparatus of Claim 12, wherein said interaction site comprises a polymerase.

23. The apparatus of Claim 22, wherein said polymerase is coupled to said first or second linker via an anchor molecule.

24. The apparatus of Claim 23, wherein said anchor molecule comprises a biotin molecule.

25. The apparatus of Claim 24, wherein biotin molecule is coupled to said first or second linker via a biotin derived nucleotide.

26. The apparatus of Claim 24, wherein said polymerase comprises a biotinylated polymerase.

27. The apparatus of Claim 26, wherein said biotinylated polymerase is coupled to said biotin molecule via a streptavidin.

28. The apparatus of Claim 22, said biological molecule comprises a dNTP molecule labeled with a fluorophore.

29. The apparatus of Claim 28, wherein said fluorophore is attached to said dNTP molecule at either or both of base and phosphate terminus of said dNTP molecule.

30. The apparatus of Claim 12, wherein said template molecule comprises a single strand DNA having a plurality of bases such that said interaction site facilitates binding of said bases with said biological molecules comprising complementary base molecules.

31. An assembly of metal-nanoparticle based structures, comprising: a substrate that defines a surface; and a plurality of metal-nanoparticle based structures coupled to said surface, each structure comprising: a plurality of metal-nanoparticles, each of said metal-nanoparticles coupled to at least one other metal-nanoparticle, at least one of said metal- nanoparticles being different than at least one other metal-nanoparticle; and an interaction site disposed relative to said plurality of metal- nanoparticles and configured to provide a catalyst functionality for a reaction involving a detectable biological molecule, said interaction site positioned so as to experience a metal-enhancement effect provided by said plurality of metal-nanoparticles.

32. The assembly of Claim 31 , wherein said structures are arranged in an array on said surface.

33. The assembly of Claim 31 , wherein said structures are arranged in a substantially random manner on said surface.

34. The assembly of Claim 31 , wherein said substrate defines a well such that said surface is part of said well.

35. The assembly of Claim 34, wherein said detectable biological molecule includes a fluorophore that emits fluorescent light in response to excitation energy.

36. The assembly of Claim 31 , wherein said interaction site comprises an enzyme that facilitates hybridization reactions between detectable biological molecules comprising labeled base molecules and their complementary bases in a single strand DNA.

37. A method for sensing biological molecules, comprising: providing one or more metal-nanoparticle assemblies, each of said assemblies having an arrangement of at least two types of metal-nanoparticles and an interaction site disposed relative to said metal-nanoparticles, such that said interaction site experiences a metal-enhanced fluorescence effect; introducing one or more fluorophore labeled biological molecules to said one or more metal-nanoparticle assemblies such that a fluorophore labeled - -

biological molecule positioned at said interaction site experiences said metal- enhanced fluorescence effect; providing excitation energy to said one or more metal-nanoparticle assemblies so as to excite one or more of said labeled biological molecules positioned at said interaction sites; and detecting enhanced fluorescence signal emitted by said excited one or more labeled biological molecules.

38. A method for fabricating a sensor for sensing of biological molecules, comprising: providing a plurality of first type of metal-nanoparticles with one or more DNA strands attached thereto; providing a plurality of second type of metal-nanoparticles with one or more DNA strands attached thereto, said second type being different than said first type; coupling at least some of said first type of metal-nanoparticles with corresponding number of said second type of metal-nanoparticles, said coupling being achieved via respective one or more DNA strands of said first and second types of metal-nanoparticles; and providing an interaction site for each of said coupled assembly of first and second types of metal-nanoparticles such that said interaction site is disposed relative to said first and second metal-nanoparticles and experience a metal- enhanced fluorescence effect.

39. The method of Claim 38, further comprising providing and coupling additional one or more types of metal-nanoparticles having respective one or more DNA strands attached thereto to said coupled assembly of first and second types of metal-nanoparticles.

40. The method of Claim 38, wherein said interaction site is immobilized to a DNA strand associated with one of said first and second types of metal-nanoparticles.

41. The method of Claim 40, wherein said interaction site immobilization is performed prior to coupling of said first and second types of metal-nanoparticles.

42. The method of Claim 40, wherein said interaction site immobilization is performed after coupling of said first and second types of metal-nanoparticles.

43. The method of Claim 38, further comprising immobilizing at least one of said first and second types of metal-nanoparticles to a substrate;

44. The method of Claim 43, wherein said immobilizing to said substrate comprises immobilizing metal-nanoparticles to said substrate;

45. The method of Claim 43, wherein said immobilizing to said substrate comprises immobilizing one of said one or more DNA strands to said substrate.