WO2016075226A1 - Plasmonic biosensor based on molecular conformation - Google Patents

Plasmonic biosensor based on molecular conformation Download PDF

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Publication number
WO2016075226A1
WO2016075226A1 PCT/EP2015/076413 EP2015076413W WO2016075226A1 WO 2016075226 A1 WO2016075226 A1 WO 2016075226A1 EP 2015076413 W EP2015076413 W EP 2015076413W WO 2016075226 A1 WO2016075226 A1 WO 2016075226A1
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analyte
protrusions
protrusion
plasmon resonance
particles
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PCT/EP2015/076413
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French (fr)
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Peter Zijlstra
Menno Willem José PRINS
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Technische Universiteit Eindhoven
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Priority to US62/078,870 priority
Priority to US201462092763P priority
Priority to US62/092,763 priority
Priority to US62/132,096 priority
Priority to US201562132096P priority
Application filed by Technische Universiteit Eindhoven filed Critical Technische Universiteit Eindhoven
Publication of WO2016075226A1 publication Critical patent/WO2016075226A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366Apparatus specially adapted for solid-phase testing
    • G01N33/54373Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6816Hybridisation assays characterised by the detection means
    • C12Q1/6825Nucleic acid detection involving sensors
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55Specular reflectivity
    • G01N21/552Attenuated total reflection
    • G01N21/553Attenuated total reflection and using surface plasmons
    • G01N21/554Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346Nanoparticles
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/5436Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase

Abstract

A target analyte in a matrix is sensed using a sensor device having protrusions [500] such as e.g. nanorods, containing free charge carriers. Conformational molecules [504, 506] are bound at a first end to the protrusions, and bound at a second end to a label [502] e.g. a nanoparticle, that is free to move relative to the protrusions. The conformational molecule changes its conformation when bound to the analyte, thereby changing the distance and/or the relative orientation of the label to the protrusion. Energy [510] is used to excite free electrons in the protrusion near a plasmon resonance and resulting optical radiation [514] at wavelengths near the plasmon resonance wavelength is detected [516] and analyzed [518] to determined the presence/concentration of the analyte.

Description

PLASMONIC BIOSENSOR BASED ON MOLECULAR CONFORMATION

FIELD OF THE INVENTION

The present invention relates generally to biochemical detection and monitoring. More particularly, the invention relates to a biosensing device for the detection of analytes.

BACKGROUND OF THE INVENTION

Most biosensing principles for biochemical markers have been developed for use in in-vitro diagnostics, where a sample is taken (e.g., blood or saliva) and is transferred to an artificial device (e.g., a plastic disposable) outside a living organism. In such biosensing assays, a wide range of sample pre-treatment steps can be applied (e.g., separation or dilution steps) and multiple reagents can be introduced in the assay (e.g., for target amplification, signal amplification, or washing steps), often resulting in waste materials. Examples of in-vitro biosensing assays are: immunoassays, nucleic acid tests, tests for electrolytes and metabolites, electrochemical assays, enzyme activity assays, cell-based assays, etc.

In in-vivo biochemical sensing, at least a part of the sensor system remains connected to or is inserted in the human body, e.g., on the skin, or in the skin, or below the skin, or on or in or below another part of the body. Due to the contact between the biosensor and the living organism, in-vivo biochemical sensing sets high requirements on biocompatibility (e.g., inflammation processes should be minimized) and the sensor system should operate reliably within the complex environment of the living organism. For monitoring applications, the system should be able to perform more than one measurement over time and the system should be robust and easy to wear.

An important application of in-vivo biochemical sensing is continuous glucose monitoring (CGM). A disadvantage of present-day CGM systems is that the sensor response shows drift, and therefore the systems require regular recalibration by an in-vitro blood glucose test. Continuous glucose monitoring is generally based on enzymatic electrochemical sensing. Current sensors show drift and need regular recalibration. Single-molecule sensitivity is not achieved.

SUMMARY OF THE INVENTION

The current invention is a new biosensing technology for in-vivo biochemical monitoring, where the sensing principle is designed to be sensitive, specific, stable, and biocompatible. The invention is also relevant for in-vitro diagnostic testing, particularly for point-of-care testing, where it is advantageous if a specific molecular binding process leads to a signal that is detectable without further chemical/biochemical/fluidic processing. The current invention provides a reliable and easy to use biochemical monitoring and detection technique that is suitable, for example, for continuous glucose monitoring, which is very relevant for diabetic patients; electrolyte and metabolite monitoring, which is relevant for patients that may become unstable, e.g., in critical care; electrolyte measurements, which are helpful to monitor kidney function, e.g., in cardiac patients; protein measurements, which can be helpful to monitor, e.g., cardiac function (e.g., BNP as an important marker for heart failure) or inflammation; drug and/or drug metabolite measurements, which are helpful to monitor drug intake (compliance) and pharmacokinetics (aiming to keep the drug within the desired concentration window); or drug response measurements, which is helpful to monitor drug effectiveness. The current invention provides advantages that include stability in that there is no bleaching and no blinking, and reliability/specificity, where the plasmon field relies on free charge carriers (e.g., electrons), which are present in high numbers. Therefore the plasmonic transduction principle is quite insensitive to the chemical conditions of the fluid. The invention allows for the real-time probing of analyte concentrations in complex fluids (e.g., blood, saliva, interstitial skin fluid). Single-molecule resolution is achievable for high sensitivity. Furthermore, high specificity can be reached by isolating specific from non-specific interactions by performing signal processing on collected data, e.g., on a molecule-by-molecule basis. This allows direct real-time series of measurements in complex fluids without repeated sample taking or intermediate filtering steps. The current invention is useful for in-vivo biosensing and for in-vitro biosensing, where the biosensors are based on particle labels and can have single-particle and single-molecule resolution. The current invention may also find application in biological, biomedical and pharmaceutical research, e.g. to monitor assays with live cells, tissue, organs, etc.

Applications of the current invention that would benefit from a reliable and easy to use biochemical monitoring system include continuous glucose monitoring for diabetic patients, without the need to regularly recalibrate the sensor system, electrolyte and metabolite monitoring, which is important for patients that may become unstable, e.g., in critical care for patient monitoring systems; electrolyte measurements for monitoring kidney function, for example in cardiac patients; protein measurements for monitoring cardiac function (for example BNP is a key marker for heart failure) or inflammation; drug and/or drug metabolite measurements are helpful to monitor drug intake, such as for compliance, and pharmacokinetics directed to keeping the drug within the desired concentration window; and drug response measurements for monitoring drug effectiveness. Analytes can be electrolytes, small molecules, lipids, carbohydrates, peptides, hormones, proteins, oligonucleotides, DNA, RNA, etc.

In one aspect, the present invention provides a method for sensing an analyte. The method includes bringing a matrix containing the analyte into contact with a sensor device having protrusions containing free charge carriers. Conformational molecules are bound at a first end to the protrusions, and bound at a second end to a label, where the label and protrusion can move with respect to each other. In some embodiments, the protrusion is static and the label is mobile. In other embodiments, the protrusion is mobile and the label is static. The conformational molecule changes its conformation and/or size and/or shape and/or orientation, when bound to the analyte, thereby changing the distance and/or the relative orientation of the label to the protrusion. The method includes exciting free charge carriers (e.g., electrons) in the protrusion and detecting optical radiation at wavelengths from the protrusions, where the exciting and/or detecting is performed at a wavelength near the plasmon resonance wavelength of the protrusion while the matrix containing the analyte is in contact with the protrusions of the sensor device. The presence/concentration of the analyte is determined from changes in the detected optical radiation while the matrix containing the analyte is in contact with the protrusions of the sensor device. In some embodiments, bringing the analyte into contact with the protrusions of the sensor device comprises bringing a fluid containing the analyte or an analyte-permeable matrix containing the analyte into contact with the protrusions of the sensor device.

In some embodiments, the protrusions have a long axis that is smaller than 200 nm, and at least one short axis that has a length that is shorter than 80% of the long axis. Alternatively, the protrusions have a cross-sectional area of which the long axis is smaller than 200 nm while the height that protrudes out of the cross-sectional area is at least 0.75 times the long axis of the cross-sectional area.

In some embodiments, the protrusions are non-spherical metallic particles, e.g., nanorods or bipyramids. In another aspect, the invention provides a system for sensing an analyte. The system includes a sensing device that includes at least two objects connected by at least one conformation- modulation molecule (CMM). The CMM is attached to a protrusion of at least one of the two objects. The CMM is selected such that the binding of at least one target analyte to the CMM changes the molecular conformation of the CMM, and thereby changes the distance between the at least two objects and/or the relative orientation of the at least two objects. The protrusion is a convex structure that has in two dimensions a radius of curvature of less than 100 nm, such that the protrusion may generate a localized electromagnetic field when excited.

In another aspect, the protrusion contains free electrons that may be excited near a plasmon resonance, for example by optical or electrical excitation. Detection of changes of distance and/or relative orientation can occur via excitation and/or detection near the plasmon

wavelength of the protrusion. For example, plasmon excitation can occur by irradiating with light at a wavelength near the plasmon wavelength, and detection can occur also near the plasmon wavelength. Alternatively, plasmons can be excited by electrical means and light can be detected near the plasmon wavelength. Or optical excitation can occur near the plasmon wavelength and optical detection can occur away from the plasmon wavelength, e.g., when using two-photon luminescence, 2nd harmonic generation, or higher order techniques. Or optical excitation can occur away from the plasmon wavelength and optical detection near the plasmon wavelength, e.g., in case of one-photon luminescence with excitation at short wavelengths and detection near the plasmon. The system includes a detector that is capable of detecting optical radiation, such as elastic or inelastic emission effects from the excited plasmons. The system also includes a processor for determining the presence and/or concentration of the target analyte from changes of plasmon resonance properties indicated by the detected optical radiation. The detector may be realized as a camera with sufficient dynamic range and wavelength sensitivity to achieve single-molecule resolution. In a further embodiment, the system may include an optical probe (e.g., optical fibre) to allow for measurements to be conducted directly in complex biological environments. In some embodiments, at least one of the two objects is a plasmonic nanorod, e.g., containing silver or gold. In some embodiments, at least one of the two objects is anchored to a body that has a dimension that is larger than the largest dimension of the at least two objects such as a surface, a matrix, a scaffold, a network, or a polymer. In another aspect, the nanoparticles are embedded in a matrix that has a filtering function such as being open for target analyte.

According to a further aspect of the invention, the CMM may be a natural, semi-synthetic, or synthetic molecule or a molecular construct with modular buildup, containing a member of one of the following classes of molecules: (bio)polymers, aptamers, nucleic acids, peptides, carbohydrates, or proteins.

In another aspect of the invention, at least one of the two objects may have a size on a nanometer scale, for low viscous drag, for high speed, for rapid measurement of conformation changes, in order to record binding/unbinding events with single-molecule resolution, and in order to increase specificity. Low viscous drag and rapid measurements will be particularly useful if the affinity between the CMM and the analyte is low. In that case the CMM undergoes short and rapid conformational changes, which can only be resolved if one of the objects is of nanometer dimensions and can follow conformational change without being slowed by drag. In yet another aspect of the invention, the processor may perform histogram and/or histogram processing (e.g., filtering) to suppress background noise and enhance specificity. For example, short-lived events (that are dominantly weak and non-specific interactions), and long-lived events (that are dominantly specific interactions), can be discriminated on a molecule-by- molecule basis. This increases sensing specificity and allows for the continuous measurement of analyte concentrations in very complex environments, such as in-vivo or directly in body fluids such as blood. In yet another aspect of the invention, multiple objects connected by a CMM are detected and monitored in parallel. This increases the statistics and allows for multiplexed measurements (e.g., multiple objects, multiple object types, multiple CMM types). E.g., controls can be included, and data can be collected for multiple analyte types. According to another aspect of the invention, the analyte is a member of one of the following classes of molecules: electrolytes, metabolites, small molecules, drugs, peptides, proteins, hormones, or nucleic acids.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A and IB are schematic diagrams of a sensing device showing unfolded and folded states, respectively, according to an embodiment of the invention.

FIG. 1C is a graph of scattering cross-section vs wavelength for unfolded and folded states of a sensing device, showing a detected shift in wavelength of a plasmon resonance peak, according to an embodiment of the present invention.

FIG. 2 is a schematic diagram of system for sensing an analyte in a fluid, according to an embodiment of the present invention.

FIG. 3 is a graph of plasmon shift vs. separation distance for a rod- sphere dimer in water, according to an embodiment of the present invention.

FIG. 4 is a schematic overview of a possible pathway for the preparation of a rod-sphere dimer bridged by an oligonucleotide, according to an embodiment of the present invention. FIG. 5 is a schematic diagram of a system for sensing the presence of a target analyte using optical excitation and detection of plasmon resonance shifts in dimers, according to an embodiment of the present invention.

FIGS. 6A-B illustrate two implementations of a combined microscopy and spectroscopy method, according to embodiments of the present invention.

FIG. 7 is a graph of mean binding rate vs. analyte concentration according to a power law, according to an embodiment of the present invention.

DETAILED DESCRIPTION

Upon irradiation with light, the free conduction electrons of metallic nanostructures oscillate collectively, known as surface plasmon, because of its interaction with the light's electric field, with a resonant frequency that depends on the nanoparticles' size, shape, and composition. When the resonance conditions are met, the incident photon resonates with the surface plasmon, which leads to the absorbance and scattering of the incident light. Resonance and, hence, the wavelength of absorption depends on the nature of the metal, size of the nanostructure, its aspect ratio and the local dielectric environment around the nanostructure, which can be summed as the refractive index of the surrounding medium. For example, 13-nm gold nanoparticles show one absorption band in water at 520nm. Other metallic nanostructures such as gold and silver nanorods and other shapes show two peaks, one peak due to resonance between lateral modes with light and one for resonance due to the longitudinal modes that is tunable from the visible through the near infrared, depending on the nanorod aspect ratio.

Like surface plasmon resonance (SPR), localized surface plasmon resonance (LSPR) can measure the changes in the local refractive index, which results in the concomitant shift in the wavelength peak in the extinction spectra. Unlike SPR, LSPR has a higher sensitivity to refractive index changes very close to the surface, within a range of 5-15 nm from the nanostructure surface, compared with approximately 200-300nm from metal film surface in SPR. In both cases, the sensitivity decreases exponentially from the surface. Thus, the sensitivity of a LSPR biosensor can be increased by using a small bioreceptor near the surface. The present inventors have discovered a technique for increasing sensitivity through the use of a label attached to a biomolecule, such as aptamers, that undergoes a large conformational change when a target molecule binds to it. Aptamers are oligonucleotides that are specifically selected to bind to its target molecules by an in vitro combinatorial process called SELEX, Systematic Evolution of Ligands by Exponential Enrichment. Aptamers are either RNA or DNA and normally contains 15-60 nucleotide sequences. Aptamers show high affinity and selectivity that is traditionally associated with proteins such as antibodies. These artificial antibodies are small compared with antibodies and can immobilize directly on the gold nanostructures via end functional group in a particular orientation using a number of bioconjugation routes. These small receptors increase the sensitivity of the LSPR compared with antibody-based LSPR sensors by binding the target close to the gold surface.

Aptamers or other similar biomolecules immobilized on the surfaces of gold nanorod and gold bipyramid nanostructures allow LSPR sensing of target analyte with significant increase in sensitivity. Because of the compact structure of such biomolecules, capture of the target protein is positioned optimally for maximum LSPR signal affording detection of e.g. picomolar concentrations of target analytes.

FIGS. 1A and IB are schematic diagrams of a sensing device according to an embodiment of the invention. The sensing device includes a protrusion 100 which may be attached to a substrate 102. A conformation modulation molecule (CMM) 104 is bound at a first end to the protrusion, and bound at a second end to a label 106 whose motion is constrained by the state of the conformational molecule but is otherwise free to move relative to the protrusion. The conformational molecule 104 changes its conformation when bound to a target analyte, thereby changing the distance and/or the relative orientation of the label 104 to the protrusion 100. FIG. 1A shows the unfolded state of the CMM when the analyte is not present, while FIG. IB shows the folded state of the CMM when the analyte is present. The protrusion 100 has free electrons which may be excited near a plasmon resonance while the matrix containing the analyte is in contact with the protrusions of the sensor device. The conformation change of the CMM in response to the presence of the analyte thus modulates the inter-particle distance, resulting in a change of the plasmon resonance (e.g., a color change or a spectral shift) which may be detected optically or electrically. FIG. 1C is a graph of the detected shift in wavelength of a plasmon resonance peak. In a preferred embodiment, the protrusion is a gold nanorod. The narrow line width and bright optical response of a single gold nanorod enables high sensitivity, e.g., single- molecule sensitivity.

FIGS. 1A and IB show an embodiment in which a nanorod with protrusion 100 is attached to a substrate 102 and where label 106 is free to move, so that the label and the protrusion can move with respect to each other. Alternatively, the label can be attached to a substrate and the nanorod with protrusion can be free to move, allowing the label and the protrusion to move with respect to each other. For example, a gold nanosphere label is attached to a glass substrate, and the nanorod is free to move in solution with respect to the label, with a motion that is restricted by the CMM. The function of the label is to alter the plasmon resonance in the nanorod, in dependence of the relative position of the label with respect to the nanorod. Alternatively, a substrate can act as a label, e.g., a nanorod is attached to a substrate via a CMM, and a change of the CMM causes a change of proximity of the nanorod to the substrate. The change of proximity to the substrate causes a change of the plasmon resonance of the nanorod. The change of plasmon resonance is detected and serves to determine the presence/concentration of the analyte from changes in the detected optical signal.

According to one embodiment, the protrusion 100 is preferably a convex structure that has, in two dimensions, a radius of curvature of less than 100 nm, where the protrusion is capable of producing a localized electromagnetic field when it is excited, such that only a small volume of fluid or matrix near the protrusion is exposed to the electromagnetic field. Free electrons in the protrusion are excited near a plasmon resonance by optical or electrical excitation. The plasmons excited in the protrusion may be detected by optical radiation, which can include elastic or inelastic effects. The localized electromagnetic field causes a high sensitivity of the plasmon to inter-object distance and/or orientation. The presence and/or concentration of the target analyte is then detected by the change of plasmon resonance properties of the device.

In one embodiment, the protrusion 100 is a gold nanorod with a small (less than 100 nm) radius of curvature at its tip. The spectral breadth of the nanorod is 3-5 fold narrower compared to commonly used spherical particles. Moreover, their brightness and their plasmon shift (i.e., color changes) are more than ten times larger compared to a sphere of equal volume. This narrow spectral line width combined with increased sensitivity enables the detection of the binding of a single target molecule to the CMM, i.e., single-particle sensitivity. These changes in color can be detected in an optical setup with wavelength sensitivity.

In one embodiment, the target 106 is a metal (e.g., silver or gold) nanoparticle that strongly absorbs a certain part of the optical spectrum due to its plasmon resonance. The plasmon resonance changes when the distance and/or orientation of the particle with respect to the surface protrusion 100 changes. The metal nanoparticles, for example, may be made of gold, which strongly absorbs a certain part of the optical spectrum due to its plasmon resonance, where it has a strong absorption peak as a specific wavelength, or color. This color peak changes its wavelength when the distance between the target and protrusion change by as little as 10 nm, depending on the size of the particles. The target size is preferably on a nanometer scale, for low viscous drag, for high speed, for rapid measurement of conformation changes, in order to record binding/unbinding events with single-molecule resolution, and in order to increase specificity. Low viscous drag and rapid measurements will be particularly useful if the affinity between the CMM and the analyte is low. In that case the CMM undergoes short and rapid conformational changes, which can only be resolved if one of the objects is of nanometer dimensions and can follow conformational change without being slowed by drag.

The CMM 104 may be an aptamer or other biomolecule that undergoes a large conformational change when a target molecule binds. The CMM 104 binds the protrusion 100 and target 106 to form a dimer. The CMM is preferably a natural, semi-synthetic, or synthetic molecule, such as a member of one of the following classes of molecules: (bio)polymers, aptamers, nucleic acids, peptides, or proteins. The protrusion 100 is preferably anchored to a body 102 that has a dimension that is larger than the largest dimension of the dimer. The body may be a substrate, a matrix, a scaffold, a network, or a polymer. In another aspect, the protrusion and target may be embedded in a matrix that has a filtering function such as being permeable to the target analyte. In some embodiments, multiple targets 106 may be connected by multiple CMMs 104 to multiple protrusions 100, allowing detection at many sites in parallel. This increases the statistics and allows for multiplexed measurements. These may include multiple distinct types of CCMs, targets, and protrusions, designed for detection of distinct analytes. Controls can be included, and data can be collected for multiple analyte types. The analytes may be a member of one of the following classes of molecules: electrolytes, metabolites, small molecules, drugs, peptides, proteins, hormones, or nucleic acids. Analytes can be measured directly, i.e., by binding directly to the CMM. Alternatively, the analyte can be measured indirectly, e.g., by being converted (e.g., by an enzyme) into a product that then binds to the CMM. Yet alternatively, the analyte can be measured indirectly by a competitive process, e.g., by competing with an analyte-analogue for binding to the CMM. Thus, in the present description, when referring to an analyte that binds to the CMM, this can refer to the analyte product or analyte analogue that serves as an indirect indicator of the target analyte.

An optical or electrical detector may be coupled with the sensing device of FIGS. 1A-B to detect changes in the plasmon resonance peak. A processor connected to the detector may then process the detected changes to determine the presence of the analyte. In one embodiment, histogram and/or histogram processing (e.g., filtering) are used to suppress background noise and enhance specificity. For example, short-lived events (that are dominantly weak and nonspecific interactions), and long-lived events (that are dominantly specific interactions), can be discriminated on a molecule-by-molecule basis. This increases sensing specificity and allows for the continuous measurement of analyte concentrations in very complex environments, such as in-vivo or directly in body fluids such as blood.

The sensing device of FIGS. 1A-B may be used as a component of a system for detecting analytes. According to one embodiment of the invention, such a system is connected in a closed- loop with a treatment system such as a device that doses a drug, such as insulin in the case of diabetes, or a device that otherwise influences the body, for example a device that provides an organ with a physical stimulation, such as electrical. More generally, the sensing device may be used for in-vivo, ex-vivo, or in-vitro applications. It may be used for applications on human subjects, or on non-human subjects, e.g., in veterinary applications or for testing of other biological systems.

The sensing device may be part of a disposable probe that is in contact with the subject or with the biological system, or it may be part of a disposable cartridge, e.g., a lab-on-a-chip cartridge. Such a probe or cartridge may be attached to an instrument or an analyzer in order to power and/or actuate and/or read out the probe or cartridge. Such an instrument is suited for processing signals from the probe or cartridge, and/or for communicating data between the instrument and the probe or cartridge, and/or for communicating data between the instrument and e.g., an information system or communication network.

Sensor devices according to some embodiments of the invention may be fabricated as follows. A localized surface plasmon resonance (LSPR) sensor surface may be fabricated by the deposition of gold nanorods on a glass substrate and subsequent immobilization of the DNA aptamer, which specifically bind to thrombin. This LSPR aptamer sensor has a response of 6-nm wavelength shift for protein binding with the detection limit of at least 10 pM.

A LSPR sensor may also be fabricated using gold bipyramid, which has higher refractive index sensitivity than the gold nanorods, but the overall response of gold bipyramid sensor appears to be 25% less than that of the gold nanorod substrate, despite the approximately twofold higher refractive index sensitivity. This is due to the low surface density of aptamers on the gold bipyramid compared with gold nanorods. The small size of aptamers relative to other bioreceptors is the key to achieving high sensitivity by biosensors on the basis of LSPR. Gold nanorods may be grown from a gold nanoparticle seed solution prepared using sodium borohydride reduction of hydrogen tetrachloroaurate (III) in the presence of the surfactant CTAB. This seed solution is added to a nanorod growth solution consisting of 425 ml of 100 mM CTAB, 18 ml of lOmM hydrogen tetrachloroaurate (III), 2.7ml of lOmM silver nitrate, and 2.9 ml of 100 mM ascorbic acid.

The gold bipyramid may be prepared using sodium citrate-stabilized gold seed particles. Bipyramids are grown using 0.5 ml of 10 mM hydrogen tetrachloroaurate (III) and 10ml of lOOmM CTAB mixed with 0.1ml of lOmM silver nitrate solution. Sequentially, 0.2ml of 1.0M HC1 and 0.08ml of lOOmM ascorbic acid are added to the solution, and the seed solution is added. The volume of seed solution is varied between 15 and 50ml to synthesize different sizes of gold bipyramids. These solutions are kept at 28 C for several hours. During this time, the color changes gradually from almost clear to dark pink, with most of the color change occurring in the first hour.

The gold nanorods are deposited on the clean glass microscopic slide. First, the clean glass slides were immersed overnight in a 5-mM APTES solution in ethanol, rinsed with water, and dried under the flow of dry nitrogen. The APTES coated glass slides are then immersed in a PEGylated nanorod solution overnight. Then, the slides are rinsed and dried, and a uniform layer of gold nanorods remained on the surface with an absorbance of approximately 0.1 at the LSPR peak wavelength. The gold bipyramid substrates are also prepared using the same procedure. The CTAB-stabilized gold nanostructures (nanorods and bipyramids) were PEGylated by suspension in thiolated PEG solution for 12 hr.

Glass substrates coated with gold nanorod as well as gold bipyramid are subjected to an oxygen plasma cleaner at low power for 30 sec in 200 mT oxygen then rinsed with ethanol, water and briefly dried in a stream of dry nitrogen. The cleaned LSPR substrates are immersed in ImM solutions of an aptamer thiol solution in 1 M potassium phosphate solution (pH 8) for 2 hr, then rinsed with deionized water and briefly dried in a stream of dry nitrogen. Immersion of these substrates in 10 mM of HS(CH2)1 l(OCH2CH2)30H (EG3-OH) solution in ethanol for 2 hr, rinsing with ethanol and then water, and drying in a stream of dry nitrogen covers the remaining bare gold with EG3-OH.

An analyte may be sensed in a fluid using a system as shown in FIG. 2. A fluid containing an unknown quantity of a target analyte is provided in vessel 200 from which it flows through sensing device 202 in the form of a closed flow cell. An interior wall of the cell 202 is a substrate with protrusion, CMM, and label, as described above in relation to FIGS. 1A-B. In one specific realization, a glass slide is coated with gold nanorod substrate and functionalized with mixed aptamer monolayer on one side and a clean glass slide on the other side separated with a 1.5-mm-thick polydimethylsiloxane (PDMS). Two holes are drilled to connect the input and output flows of fluid through the cell, and the PDMS sheet provides a 1cm 2cm slot that serves as the flow volume. This flow cell 202 is mounted vertically on an optical bench in between a quartz-tungsten- halogen light source 204 with collimating lens and a portable spectrometer 206. A flow rate of 400 ml/min is controlled by a syringe pump. After equilibration with PBS buffer, the fluid with target analyte passes over the sensor device surface under flow conditions. Absorbance spectra are collected continuously with averaging every 30 sec and recorded. Each spectrum is then analyzed by processor 208 to monitor the peak wavelength, height, and width versus time.

The utility of aptamers for LSPR applications is amplified by the high sensitivity to protein binding, which can be attributed to the relatively compact size of the aptamer. Thus, the integration of aptamers into LSPR sensors can provide the highest sensitivity achievable by a plasmon-based biosensor and the detection limits with a wider range of analyte concentrations versus gold nanoparticle aggregation assays. An equally appealing aspect of LSPR-based sensors is that detection is carried out by measurements using a basic hand-held UV-vis spectrophotometer, versus the high cost of equipment and supplies normally associated with SPR. Although SPR spectroscopy provides much higher sensitivity to changes in the bulk refractive index than LSPR spectroscopy, the response of the two techniques becomes comparable when measuring short-range changes in the refractive index due to a molecular adsorption layer. This is a result of the much smaller sensing volume presented by LSPR sensors, because the EM-field-decay length is 40-50 times shorter than that of the SPR sensors. Hence, the sensitivity of LSPR is comparable to SPR for the detection of adsorbed molecules in a 5-15-nm region above the substrate surface. Because of its low detection volume, LSPR measurement is not very sensitive to temperature changes as is the case with SPR. Thus, biosensors based on an LSPR format like the one described here can be used at varying ambient temperatures usually found in laboratory, hospital, or field settings.

Biopolymers such as proteins and nucleic acids fold into a three-dimensional structure within microseconds. The probing of these transition paths on single molecules remains a formidable challenge due to the photophysics (blinking, bleaching) of fluorescent labels. Embodiments of the present invention employ a new approach to probe conformational dynamics based on the detection of plasmon shifts caused by the distance-dependent coupling between two objects, e.g., gold nanorod and gold nanosphere. The large optical cross section of a plasmonic nanoparticle enables microsecond integration times, whereas the photostability allows us to detect many conformational changes of the same molecule.

Of particular interest is the folding of single aptamers, i.e., single-stranded oligonucleotides that fold into a compact conformation in the presence of a target. The conformational behavior of aptamers is readily extended to proteins and other biomolecules. The transition path time (i.e., the folding "speed") of aptamers is 10-100 μβ.

Proteins are the biological workhorses that carry out vital functions in every cell. To carry out their task, proteins fold into complex three-dimensional conformations. Once folded, their intrinsic flexibility allows them to dynamically change their conformation in response to temperature, pH, ionic strength, or ligand-binding.

Recent fluorescence measurements estimate of the transition path time of ~10 for a small protein. However, averaging over several hundred single-molecule trajectories was required to overcome the limited brightness and photostability of the fluorophores. Conformational dynamics of biopolymers have also been studied using mechanical probes such as optical and magnetic tweezers or atomic force microscopes. However, the micron-sized beads or cantilevers that are used preclude microsecond timescales because their dynamics is slowed by hydrodynamic drag.

Embodiments of the present invention use a plasmonic two-particle system to measure the conformational dynamics of a single interconnecting molecule on microsecond timescales and in real-time. A conformational change of the interconnecting molecule will modulate the interparticle distance and will result in a shift of the plasmon resonance of the dimer. This approach enables microsecond time resolution.

Dimers of plasmonic nanospheres have been employed to measure the presence of analyte in a biosensing assay. However, plasmon shifts of a gold-sphere dimer cannot be monitored with microsecond integration times due to their broad plasmon resonance and their relatively low optical cross section per unit volume. Instead of a dimer of nanospheres, preferred embodiments of the present invention use a dimer composed of a gold nanorod and a gold nanosphere. A rod- sphere dimer has two key advantages over previous approaches:

1. Microsecond integration times: The nanorod's optical response is narrow, bright and not easily saturated. This enables us to probe plasmon shifts with microsecond integration times, which we will use to study the transition path time of a single molecule that folds in real-time.

2. Observation times of hours: The optical response of a plasmonic particle does not blink or bleach, enabling