WO2004102160A2 - Plate-forme d'imagerie de detection de nanoparticules appliquee a une analyse d'interactions biomoleculaires par resonance plasmonique de surface - Google Patents

Plate-forme d'imagerie de detection de nanoparticules appliquee a une analyse d'interactions biomoleculaires par resonance plasmonique de surface Download PDF

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WO2004102160A2
WO2004102160A2 PCT/US2004/006006 US2004006006W WO2004102160A2 WO 2004102160 A2 WO2004102160 A2 WO 2004102160A2 US 2004006006 W US2004006006 W US 2004006006W WO 2004102160 A2 WO2004102160 A2 WO 2004102160A2
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spr
fluid
objects
flow imaging
imaging system
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PCT/US2004/006006
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WO2004102160A3 (fr
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Ralph Jorgenson
David Basiji
William Ortyn
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Amnis Corporation
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Publication of WO2004102160A3 publication Critical patent/WO2004102160A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons
    • G01N21/554—Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Definitions

  • the present invention generally relates to the field of imaging objects entrained in a flow of fluid, and more specifically, relates to imaging objects such as ⁇ individual nanoparticles that are entrained in a flow of fluid to obtain surface plasmon resonance (SPR) spectra corresponding to the objects.
  • SPR surface plasmon resonance
  • SPR is the resonant excitation of oscillating free charges at the interface of a metal and a dielectric.
  • Whe SPR* spectra are generated and collected, they can be used to determine specificity, kinetics, affinity, and concentration with respect to the interactions between two or more molecules, where one of the molecules is attached to a solid sensing surface. Reaction kinetics correspond to both an association and a dissociation rate at which an analyte interacts with the bound detection molecule.
  • Affinity refers to the strength with which an analyte binds to the detecting molecule.
  • Specificity refers to the propensity of a molecule to bind to the detecting molecule to the exclusion of other molecules.
  • SPR spectra have been used in studies involving many types of molecules including proteins, peptides, nucleic acids, carbohydrates, lipids, and low molecular weight substances (e.g., hormones and pharmaceuticals).
  • SPR based bio-sensing One analytical technique, known as SPR based bio-sensing, has been developed to enable direct measurements of the association of ligands with receptors, been used in studies involving many types of molecules including proteins, peptides, nucleic acids, carbohydrates, lipids, and low molecular weight substances (e.g., hormones and pharmaceuticals).
  • SPR based bio-sensing One analytical technique, known as SPR based bio-sensing, has been developed to enable direct measurements of the association of ligands with receptors, without the use of indirect labels, such as fluorescent markers and radioactive molecular tags.
  • This label free direct sensing technique reduces the time and workload required to perform assays, and minimizes the risk of producing misleading results caused by molecular changes induced by the use of indirect labels.
  • Another important aspect of the bio-sensing technique is that SPR based bio-sensing enables bio-molecular interactions to be measured continuously and in real-time, thereby enabling the determination of association and dissociation kinetic data in contrast to traditional "end point" analytical methods.
  • Nanoparticles are particles that are less than 100 nanometers in diameter. They display large absorbance bands in the visible wavelength spectrum yielding colorful colloidal suspensions. The physical origin of the light absorbance is due to incident light energy coupling to a coherent oscillation of the conduction band electrons on the metallic nanoparticle.
  • This coupling of incident light is unique to discrete nanoparticles and films formed of nanoparticles (referred to as metallic island films). Achieving SPR with ordinary bulk materials requires the use of a prism, grating, or optical fiber to increase the horizontal component of the incident light wave vector (i.e., to achieve the required coupling).
  • FIGURES 1A and IB schematically illustrate a simplified version of a prior art SPR detection device including a single channel SPR bulk optic prism based sensor 10, which includes a prism 12 .
  • the base of prism 12 is covered with a layer 14 of gold about 550 Angstroms thick.
  • the gold film is pre- functionalized with a defined detecting molecule 16, such as an antigen.
  • a biological fluid sample containing a corresponding analyte 32 (such as an antibody) is brought into contact with gold layer 14 and detecting molecules 16 by introducing the sample fluid into a flow cell 22 (note gold layer 14 and detecting molecules 16 together represent a sensor surface 20, which is in fluid communication with flow cell 22).
  • a range of angles of monochromatic light 24 are directed towards and reflected from sensor surface 20.
  • SPR arises through the coupling of energy between the incident photons of light with free electron oscillations ("plasmons") occurring at a gold film/liquid chemical sample interface at sensor surface 20.
  • This interaction can cause a reduction in an intensity of reflected light 28 for a given angle 30, resulting in an absorbance or "resonance" dip 31 in a measured reflectance spectrum 34 (see FIGURE 1C).
  • This resonance can also be observed in the wavelength domain if white light is introduced at an optimal fixed angle of incidence.
  • an analyte 32 binds to immobilized detecting molecule 16 on sensor surface 20 (see analyte 32a and molecule 16a of FIGURE IB)
  • the local mass concentration of molecules changes, which causes a corresponding change in the local refractive index close to sensor surface 20.
  • the resultant increase in the refractive index causes a shift in the resonance angle, from angle 30 as illustrated in FIGUREl A, to an angle 33 in FIGURE IB.
  • Angle 33 results in a "resonance" dip 35 in a measured reflectance spectrum 37, which is readily distinguishable from spectrum 34 (no binding event, so the reflectance angle is unchanged).
  • Sensor 10 enables data collection to be performed continuously and in real-time, and some systems enable the user to observe the binding events in real time on a personal computer.
  • FIGURE ID graphically illustrates a typical SPR response curve 38 based on the association and dissociation of two bio-molecules.
  • Curve 38 can be separated into four well-defined segments, each relating to a specific portion of an association/disassociation cycle. The portion of the cycle corresponding to segment A is schematically illustrated in FIGURE IE.
  • Flow cell 22 is in fluid communication with detecting molecules 16, which are bound to gold layer 14. While no prism is shown in conjunction with FIGURES 1E-1H, it should be understood that the flow cells of FIGURES 1E-1H are used in with a prism, as shown in sensor 10 of FIGURES 1 A and IB.
  • FIGURE IF (corresponding to portion B of spectrum 38), molecules of analyte 32 are introduced into flow cell 22 (i.e., a sample fluid containing the analyte is introduced into the flow cell). Some of the molecules of analyte 32 bind to detecting molecules 16, and the angle of incidence changes over time.
  • Response curve 38 of FIGURE ID typically represents a time period of about 5 to 20 minutes in duration. This response level indicates the baseline response. During this "association" period, the analyte binds to the surface, thereby increasing the refractive index, causing the SPR resonant angle to increase (note the rise in portion B of spectrum 38).
  • FIGURE 1G (corresponding to portion C of response curve 38), no additional analyte is introduced into the flow cell. Instead, the flow cell is flushed with a buffer solution. This step results in analytes being released from detection molecules 16, as the bound analytes 32 attempt to reach an equilibrium with the buffer solution. The decrease in the amount of bound analyte is reflected in a dip in spectrum 38.
  • FIGURE 1G corresponding to portion D of spectrum 38
  • the flow cell is flushed with an acidic solution, which ensures that any residual bound analytes are removed from the detection molecules, thereby regenerating the sensor surface.
  • This "regeneration" step enables the sensor surface to be returned to its original baseline configuration, so that further analyses can be performed.
  • the data collected during portions A-D of spectrum 38 (often referred to as a Sensorgram) enable the user to determine kinetics, concentration, binding specificity, and affinity.
  • FIGURE 2 shows a different prior art technique that has been developed, which also involves exciting and detecting SPR on gold and silver nanoparticles.
  • Chemical and biological sensing applications using nanoparticles have been performed primarily by measuring a transmitted light intensity 80 through a high concentration of suspended particles 82.
  • the resultant spectrum 84 has an absorbance dip 86 due to the excitation of SPR at a certain coupling wavelength 88, as shown in FIGURE 2.
  • the exact position and shape of the SPR spectrum is a function of such factors as the metal used, the bulk solution and adsorbed film complex refractive indices, the adsorbed film thickness, the nanoparticle morphology (size and shape), and inter-particle coupling effects (e.g. the concentration and proximity of nanoparticles to one another).
  • the extinction cross section for the nanoparticles can be approximated as indicated in Eq. 1, as follows:
  • V is the spherical nanoparticle volume
  • c is the speed of light
  • ⁇ is the angular frequency of the incident light
  • e & is the permittivity of the surrounding bulk dielectric medium (assumed to be relatively independent of the frequency of light)
  • the maximum absorbance wavelength, p ⁇ (SPR coupling wavelength) dependence on refractive index is not as sensitive as the bulk thin film SPR measurements. Sensitivity of a 75 nanometer shift in the SPR coupling wavelength per refractive index unit (RIU) is reported, as compared to 3000 nanometer shift per RIU for bulk film SPR devices. Thus, gold nanoparticle SPR measurement based methods are 40 times less sensitive. However, additional geometries, including gold/silver alloy nanoparticles, ellipsoidal nanoparticles, triangular nanoparticles, and hollow nanoshells have been reported as having increased sensitivities up to six fold (400 nm wavelength shift per RIU).
  • the nanoparticles have an advantage with respect to the sensitivity of adsorption of molecules to the gold surface. Specifically, the decay length of the electric field extending from the gold/chemical sample interface is approximately 20 times shorter for that of nanoparticle colloidal gold versus bulk thin gold film. Therefore, because nanoparticles have more energy confined closer to the gold surface, these particles are more surface sensitive and will yield a larger signal during receptor/ligand interactions.
  • FIGURES 1 A and IB Current commercial SPR instrumentation uses a fixed sensor having a gold layer capable of supporting SPR, such as the traditional SPR bulk optic prism based sensor shown in FIGURES 1 A and IB.
  • the bio-molecular receptor molecules attached to the gold layer are analyte specific.
  • the analyte is brought to this sensor surface via fluidics, and the analyte associates with the bound receptor molecules on ,the sensor surface.
  • Current planar embodiments are severely mass transport limited by diffusion to time scales on the order of 16 to 160 minutes for analytes at bulk concentrations less than 10 "7 M.
  • a first aspect of the invention is directed to an SPR biomolecular interaction method that uses flow imaging systems, which can combine the speed, sample handling, and cell sorting capabilities of flow cytometry with the imagery, sensitivity, and resolution of multiple forms of microscopy and full visible/near infrared spectral analysis of detector technology to collect and analyze SPR spectra from objects entrained in a flow of fluid that emit an SPR spectra.
  • This method includes the steps of placing gold or silver nanoparticles or beads that have a detector molecule attached to them in a container, adding a solution of analyte or ligand molecules to the container, and introducing a portion of the sample into a flow imaging system.
  • the flow imaging system provides up to a thousand-fold increase in signal collection over conventional SPR instrument designs and allows for maintaining particles in suspension to ensure optimal free solution conditions for association and dissociation of bio-molecular species. This approach is thus not severely mass transport limited, like planar embodiments.
  • a peak absorbance wavelength can then be measured using detector technology. Since full spectral SPR data can be collected with this detector technology, the entire angular or wavelength spectrum is measured, providing a very precise measurement of the coupling angle or wavelength.
  • this approach has the ability to measure libraries of different bead receptors. Also, this method includes repeating these steps on the portion of the sample that remains in the container.
  • this buffered portion of the remaining sample can be introduced into the flow imaging system, and disassociation rates can be studied.
  • an optional step can be employed, wherein a low pH wash is used to remove the bound ligands from the receptors attached to the nanoparticles, and repeated measurements can then be made.
  • corrections can be made to a nanoparticle response curve that exhibits a non-linear response. Specifically, larger nanoparticles may be used to increase the curve's linearity, and calibration corrections can be made for the non-linear response.
  • the preferred flow imaging systems can be used to analyze white light spectral scatter analysis of gold nanoparticles and nanoparticle-coated microbeads using prism dispersion. A prism or grating is employed to disperse laterally high resolution white light spectral scatter spectra of the nano or micro particles being analyzed.
  • Yet another step of this method involves collecting simultaneous imaging of absorbed, scattered, and fluorescent light from microbeads.
  • the prism is removed from the preferred flow imaging system and a focusing spherical lens is replaced with a cylindrical lens, the high resolution scattered angular spatial intensity of the nano or micro particle can be measured under monochromatic side illumination.
  • Another aspect of this invention provides for multi-spectral darkfield scattering to analyze particles.
  • the size of such particle can be determined by measuring the relative light scattering intensity across multiple wavelengths.
  • the ratio of the scattered light intensities at given wavelengths is a function of the size of the particle, based upon Raleigh scattering.
  • a holographic notch filter can be used with the preferred imaging system to filter out the excitation laser light signal to detect surface enhanced Raman spectroscopy.
  • FIGURE 1A schematically illustrates a prior art SPR sensor including a thin layer of gold and a prism
  • FIGURE IB schematically illustrates analytes being attracted to specific detection molecules attached to the gold film in the prior art SPR sensor of FIGIRE 1A
  • FIGURE 1C is a graph illustrating the relationship between an intensity of light reflected from the gold layer through the prism and an angle of incidence with which such light is reflected using the prior art SPR sensor of FIGIRE 1 A ;
  • FIGURE ID is a graph illustrating a characteristic relationship between a 5 resonant angle and time based on a preferred analytical technique using the prior art SPR sensor of FIGURE 1A;
  • FIGURE IE schematically illustrates a first step in a preferred prior art technique for using the prior art SPR sensor of FIGURE 1 A;
  • FIGURE IF schematically illustrates a second step in a preferred prior art 10 technique for using the prior art SPR sensor of FIGURE 1A;
  • FIGURE 1G schematically illustrates a third step in a preferred prior art technique for using the prior art SPR sensor of FIGURE 1 A;
  • FIGURE 1H schematically illustrates a fourth step in a preferred prior art technique for using the prior art SPR sensor of FIGURE 1 A; 15.
  • FIGURE 2 schematically illustrates a prior art technique for obtaining a transmission measurement from a bulk solution of gold nanoparticles;
  • FIGURE 3 is a schematic illustration of a preferred flow imaging system used in accord with the present invention.
  • FIGURE 4 is a schematic illustration of a readout provided by a time delay 20 integrated (TDI) detector employed in a preferred imaging system used in accord with the present invention
  • FIGURES 5A-5G schematically illustrate a method for using the flow imaging system of FIGURE 3 and the detector technology of FIGURE 4 for biomolecular interaction analysis, in connection with nanoparticle or micro particle 25 SPR substrates;
  • FIGURE 6 is a flow chart showing the steps of the method illustrated in FIGURE 5A-5G, including an additional step for facilitating repeated measurements;
  • FIGURE 7 is a schematic illustration of a theoretical SPR Coupling 30 Wavelength shift for a bulk gold film and a gold nanoparticles as a function of adsorbed film thickness
  • FIGURE 8A is a schematic illustration of yet another preferred imaging system, which incorporates a prism for full spectrum analysis;
  • FIGURE 8B schematically illustrates data segments collected using the imaging system of FIGURE 8 A
  • FIGURE 9 schematically illustrates additional data segments collected using the imaging system of FIGURE 8 A
  • FIGURE 10A is a schematic side elevational view of an imaging system including a system of lenses in place of the prism employed in the imaging system of FIGURE 9;
  • FIGURE 10B is a schematic plan view of an imaging system including a system of lenses in place of the prism employed in the imaging system of FIGURE 9;
  • FIGURE 11 is a multi-spectral darkf ⁇ eld scatter analysis often continuous segments, 350 nm diameter beads; and FIGURE 12 is a schematic illustration of yet another preferred imaging system including a prism, which is particularly well adapted to be employed for surface enhanced Raman spectroscopy detection.
  • the present invention encompasses a method of using flow imaging systems that can combine the speed, sample handling, and cell sorting capabilities of flow cytometry with the imagery, sensitivity, and resolution of multiple forms of microscopy and full visible/near infrared spectral analysis to collect and analyze SPR spectra from objects entrained in a flow of fluid that emit an SPR spectra.
  • Conventional methods of collecting and analyzing SPR spectra either employ a fixed sensor that emits SPR spectra as a solution of particles interacts with a fixed sensor, or emits a combined spectra from a bulk solution of particles that individually emit SPR spectra.
  • the fixed sensor embodiment is widely used, but has a limited throughput.
  • the spectra collected from the bulk solution does not enable spectra from individual particles to be discerned.
  • the present invention enables SPR spectra from individual particles to be collected with a much greater throughput than achievable using the fixed sensor prior art techniques (discussed above in connection with FIGURES 1A-1H).
  • FIGURE 3 illustrates the key components of an optical system employed to project light from objects in flow onto a detector (FIGURE 4) that employs an exemplary readout for any particle.
  • Objects are hydrodynamically focused into a single-file line in the fluidic system, forming a tall but narrow field of view. This approach enables the lateral dimension of the detector to be used for signal decomposition.
  • objects 99 are hydrodynamically focused in a flow of fluid directed into a flow cuvette 116 and illuminated from one or more sides using light sources 98 and 100.
  • Light is collected from the objects with a high numerical aperture objective 102, and the collected light is directed along a light path including lenses 103 and slit 105.
  • a fraction of the collected light is transmitted to an auto focus subsystem 104 and a velocity detection system 106.
  • velocity detection system 106 is important to ensure the data acquired by the detection system, which are integrated over to time, increase the signal and are properly synchronized to the flow of fluid through the imaging system.
  • the objects are nanoparticles and micro particles including gold/and or silver film to enable SPR spectra to be collected.
  • the imaging system can be used to image a wide variety of object types, including but not limited to biological cells and beads.
  • the majority of the light is passed to a spectral decomposition element 108.
  • the decomposition element employs a fan-configuration of dichroic mirrors 110 to direct different spectral bands laterally onto different regions of a TDI detector 114.
  • the imaging system is able to decompose the image of a single object 118 into multiple sub-images 120 across detector 114, each sub- image corresponding to a different spectral component.
  • detector 114 has been enlarged and is shown separately to highlight its elements.
  • FIGURE 3 illustrates a typical flow-based embodiment of a flow imaging system.
  • inset 101 in this Figure illustrates a plate-based embodiment of an imaging system that can be used in place of the flow-based embodiment.
  • the flow imaging system can employ a prism (as shown in
  • FIGURE 8A or a grating oriented to disperse light laterally with regard to the axis of flow prior to the final focusing optics, for spectral analysis of each object's intrinsic fluorescence.
  • a cylindrical final focusing lens (see FIGURE 10A) can be employed to image a Fourier plane on the detector in the cross-flow axis, enabling analysis of the light scatter angle.
  • These techniques for multi-spectral imaging, flow spectroscopy, and Fourier plane scatter angle analysis can be employed simultaneously by splitting the collected light into separate collection paths, with appropriate optics in each light path.
  • a second imaging objective and collection train can be used to image the particles through an orthogonal facet of the flow cuvette 116, thereby viewing the objects in stereoscopic perspective with no loss of speed or sensitivity.
  • detector 114 of the flow imaging system shown in FIGURE 3 uses a TDI that performs high throughput imaging with high sensitivity.
  • the image on the TDI detector is read out one row of pixels at a time from the bottom of the detector. After each row is read out, the signals in the remaining detector pixels are shifted down by one row.
  • the readout/shift process repeats continuously, causing latent image 142 to translate down the detector during readout (note the movement of latent image 142 through frames T1-T6). If the readout rate of the detector is matched to the velocity of the object being imaged, the image does not blur as it moves down the TDI detector.
  • the TDI detector electronically "pans" to track the motion of an object being imaged.
  • the key to this technique is to accurately measure the velocity of the objects being imaged and to employ that measurement in feedback control of the TDI readout rate.
  • accurate velocity detection for objects moving in flow enables the TDI imaging to be implemented properly.
  • a preferred flow imaging system used in connection with the present invention includes a TDI detector that has 512 rows of pixels, giving rise to a commensurate 500X increase in signal integration time. This increase enables the detection of even faint fluorescent probes within cell images and intrinsic auto fluorescence of cells acquired at a high-throughput.
  • TDI detector When applied to nanoparticles in suspension in a cuvette ,116, real-time triggering and isolation of certain nanoparticle receptor/ligand combinations for post capture analysis can be performed. For example, selective retrieval of proteins from a complex biological sample in real time can be monitored. By isolating the nanoparticle receptor/ligand combination, mass spectroscopy can be used for identity confirmation of the affinity retrained analyte via its unique molecular mass.
  • TDI detector increases measured signal intensities up to a thousand fold, representing over a 30 fold improvement in signal-to-noise ratio compared to other approaches in the prior art.
  • This increased signal intensity enables individual particles to be optically addressed, providing high resolution measurement of either scattered spectral intensity of white light or scattered angular analysis of monochromatic light.
  • the ability to optically address individual particles, without requiring a prism to be disposed immediately adjacent to a thin metal film significantly distinguishes the use of the preferred imaging system of FIGURE 3 from the prior art SPR sensor of FIGURE 1.
  • the sensor component in FIGURE 1 (the gold layer and the detector molecule) are fixed, whereas the method of the present invention employs a flow imaging system similar to that illustrated in FIGURE 3, so that the "sensor” providing the SPR spectra is the gold or silver film deposited individually on particles imaged by the system.
  • this technique dramatically reduces the size of the detector surface, enabling more accurate data collection to be achieved.
  • a flow imaging system used in the present invention can be configured for multi-spectral imaging and can operate with six spectral channels: DAPI fluorescence (400-460 nm), Darkfield (460 - 500 nm), FITC fluorescence (500- 560 nm), PE fluorescence (560 - 595 nm), Brightfield (595 - 650 nm), and Deep Red (650 - 700 nm).
  • the TDI detector can provide 10 bit digital resolution per pixel.
  • the numeric aperture of the imaging system used with this invention is typically 0.75, with a pixel size of approximately 0.5 microns. However, those skilled in the art will recognize that this flow imaging system is neither limited to six spectral channels nor limited to either the stated aperture size or pixel size and resolution.
  • the SPR biomolecular interaction method of the present invention which uses an imaging system (or a substantially similar imaging system), as described above, to image nanoparticles and larger particles having a metal film will now be described in detail.
  • the method of the present invention benefits from the ability of this preferred flow imaging system to optically address and measure individual SPR spectra of nanoparticles and larger sized particles in flow, resulting in up to a thousand-fold increase in signal collection over conventional SPR instrument designs.
  • the steps involved in this method are schematically illustrated in FIGURES 5A-5H, and these same steps are shown as a flow chart in FIGURE 6. Note that the flow chart of FIGURE 6 includes an optional step that enables a user to repeat measurements, if so desired.
  • a solution 162 of gold coated nanoparticles 168 is introduced into a container 164.
  • the gold coated nanoparticles have been functionalized with a "detecting" or receptor molecule attached to the surface of the gold nanoparticles.
  • gold island microbead films can be employed.
  • Gold is preferred, but silver coated microbeads and nanoparticles are also known to enable SPR spectra to be generated and can instead be used with this invention. Combinations of gold and silver films can also be employed.
  • container 164 has a minimal amount of a buffer solution, so as to minimize the dilution of solution 162.
  • any buffer solution added to container 164 should be chemically consistent with solution 162 (i.e., if solution 162 is a saline solution, any additional buffer should be a saline solution) to minimize bulk refractive index effects.
  • container 164 which includes solution 162 (with functionalized gold nanoparticles, or larger particles including receptors molecules), generally corresponds to flow cell 22 in FIGURE IE.
  • Flow cell 22 in FIGURE IE includes a functionalized gold sensor, but no analytes.
  • container 164 includes a plurality of individual particles, each particle acting as an individual sensor, while flow cell 22 of FIGURE IE includes only a single sensor.
  • container 164 may be of any type and size capable of holding the solution, including but not limited to a beaker or test tube.
  • FIGURES 5A-5H are not drawn to scale. For example, the amount of solution shown in the vial and its size may vary.
  • an analyte solution 170 i.e., a solution including a concentration of analyte or ligand molecules that will associate with the detection molecules bound to the individual particles in solution 162 contained in a second container 173 is added to solution 162 in container 164.
  • solution 170 is shown as being added as drops dispensed by a dropper, it should be understood that the present invention in not limited to that specific implementation for transferring solution 170 to container 164.
  • solution 170 may be transferred into container 164 simply by pouring it, or by a more controllable technique, such as by utilizing a micro pipette to transfer the solution.
  • Solutions 162 and 170 combine in container 164 to form a sample solution 180, which includes gold covered nanoparticles, analyte molecules, and detector molecules bound to the nanoparticles.
  • a sample solution 180 which includes gold covered nanoparticles, analyte molecules, and detector molecules bound to the nanoparticles.
  • the removed solution 180 is introduced into a flow imaging system, such as the imaging system discussed in detail above with respect to FIGURE 3.
  • the removed portion of solution 180 is introduced into a rotating syringe suspension pump (not separately shown).
  • Such a pump serves to keep the particles in suspension via rotation, as well as enabling a precisely metering amount of sample to be introduced into the flow imaging system (i.e., into cuvette 116 as shown in FIGURE 3).
  • a preferred imaging system requires small volume injection and very precise injection rates in order to maintain synchronization between the particulate flow rate and the TDI detector read out rate.
  • a preferred syringe pump not only rotates the sample to maintain the particles in suspension, but also provides constant volume pumping with low pulsatility.
  • Maintaining particles in suspension enables optimal free solution conditions for association and dissociation of bio-molecular species (i.e., the analyte molecules in solution 170) to the receptors/detector molecules on the gold nanoparticles in solution 162.
  • Achieving such free solution conditions is a major advantage over prior art planar embodiments (i.e., as shown FIGURES 1A-1H and discussed above), which are severely mass transport limited by diffusion, to time scales on the order of 16 to 160 minutes for analytes at bulk concentrations less than 10 "7 M.
  • the flow imaging system preferably employed uses hydrodynamic focusing (i.e., uses a sheath fluid) to confine a sample fluid (solution 180) to the central portion of a cuvette 116, as indicated in FIGURE 3.
  • hydrodynamic focusing i.e., uses a sheath fluid
  • the sheath flow improves the precision with which the sample solution can be positioned in an observation region, enabling particles entrained in the flow of sample fluid to be more precisely imaged.
  • FIGURE 5D schematically illustrates an output of this data processing, in which SPR coupling wavelength 186 is plotted as a function of time 188, during the association time period (typically between 10 and 20 minutes).
  • the method of the present invention uses the flow imaging system's capability for the collection of full spectral SPR data, the entire angular or wavelength spectrum is measured, which provides a very precise measurement of the coupling angle or wavelength. This benefit is a clear advantage over the prior art, where only a single angle or single wavelength intensity can be measured. Additionally, throughput rates for imaging of macroscopic objects using this preferred imaging system are approximately 100 objects per second. When such a flow imaging system is operated in either spectral or angular dispersion mode for nanoparticles (scattered light or fluoresced light), these rates can be increased to achieve imaging of over a thousand particles per second.
  • a library set of nanoparticles having different SPR absorption spectra can be created. This step can be carried out by using alloy nanoparticles composed of silver and gold. By adjusting the mole fractions of the alloy, up to a 150 nm separation can be achieved. Given that the kinetic association and dissociation rates are continuous, this approach enables the encoding of nanoparticles that have relatively close absorbance spectra separation (e.g., about 5 nm), so that a library of 30 beads can readily be created.
  • bead on bead labeling can be used to encode a bead library numbering in the millions, using multiple fluorescent channel imagery.
  • the SPR spectrum can be measured in the angular domain by using spatial light scattering.
  • spectral data and darkfield image 190 shown in FIGURE 5D is exemplary of images collected from fluorescent data, and while image 190 does not illustrate SPR reflection spectra, similar images based on SPR data can be obtained using this preferred flow imaging system.
  • the sensor area can be significantly reduced, which as noted above, is advantageous over prior art SPR sensors having larger surface areas, since a large sensor area limits the analyte sensitivity, because the SPR signal is proportional to the density of binding.
  • a total of 180,000 beads would allow a bead library of 100 different receptor beads, and a sub-population of 1,800 beads per receptor. .
  • This preferred flow imaging system enables one bead to be read per second over a 30 minute association/dissociation observation period.
  • the portion of solution 180 that remains in container 164 (the portion that was not introduced into the flow imaging system) is used to measure the dissociation rate kinetics.
  • solution 180 remaining in container 164 also undergoes the same association kinetics as occurred in the portion of solution 180 that was introduced into the flow imaging system. Therefore, this bead population may be used to study the dissociation rate kinetics.
  • solution 180 remaining in the container is concentrated into a small portion, by centrifuging. The supernatant (i.e., the portion of the solution containing little or no particles) is removed to achieve a concentrated solution 180a of gold nanoparticles.
  • a buffer solution 194 is added to the concentrated solution in container 164, to achieve a solution 195 that is a mixture of buffer solution 194 and concentrated solution 180a.
  • solution 195 is removed from container 164 and introduced into the flow imaging system discussed in detail above, as schematically illustrated in FIGURE 5F.
  • solution 195 is preferably introduced into the flow imaging system using a rotating syringe pump.
  • the flow imaging system generates absorption and/or reflected spectra data for each individual nanoparticles 168 in solution 195, generating spectral data as shown in FIGURE 5G.
  • the peak absorbance wavelength is determined and plotted as a function of time during the dissociation time period (typically between 5 and 15 minutes).
  • a further optional step (not shown in FIGURE 5) is necessary if the user wants to repeat the measurement.
  • the user may utilize a low pH wash step in order to remove the bound ligands (i.e., the analytes) from the receptors attached to the gold nanoparticles, so that the nanoparticles can be used again.
  • FIGURE 6 is a flow chart of the method schematically illustrated in FIGURES5A-5G.
  • the method begins in a block 200 where a container is filled with functionalized gold or silver particles.
  • a functionalized particle is a particle in which a receptor or detector molecule (such as an antibody) is attached to a metal layer on the particle, thereby enabling SPR spectra to be generated.
  • the particles are gold-plated nanoparticles.
  • silver-coated nanoparticles, or nanoparticles coated with mixtures of gold and silver can alternatively be employed.
  • larger sized particles supporting metal island films also be alternatively employed.
  • an analyte to be studied is added to the functionalized gold nanoparticle solution in a block 202.
  • Half of the sample is aspirated by the preferred flow imaging system's rotational suspension pump for kinetic association analysis in a block 204. It should be understood that either more or less than half of the solution can be used in this step; using about half of the solution ensures that some solution is left to study dissociation kinetics, as described above. Further, if desired, all of the solution can be used to study association kinetics, if no data are desired from disassociation kinetics measurements.
  • the preferred flow imaging system performs continuous spectral analysis of individual particles. It should be understood that modifications can be made to the preferred imaging system described in FIGURE 3, so long as the desired SPR spectral data are obtained by the resulting flow imaging system. Thus, it will be clear that the flow imaging system of FIGURE 3 is merely exemplary of a system that is suitable and capable of obtaining the desired spectral data, but is not intended to be limiting of this invention.
  • the maximum absorbance wavelength versus time is plotted. While such a plot is useful, it should be understood that the method does not require the data be thus processed immediately. Instead, the raw data can be collected for review and processing at a later time.
  • the sample solution remaining in the container to which the analyte was added is concentrated, and an additional buffer solution is added (as discussed in relation to FIGURES ID and IF, the additional buffer is required to induce the disassociation because equilibrium drives the analytes attached to the receptor molecules bound to the nanoparticles and metal films into the buffer solution).
  • a block 212 the remaining concentrated sample and the buffer solution (see FIGURE 5E and 5F) is introduced into the preferred flow imaging system for kinetic disassociation analysis.
  • the preferred flow imaging system performs continuous spectral analysis of individual particles in a block 214.
  • the maximum absorbance wavelength versus time is optionally plotted in a block 216.
  • a decision block 218 determines if the functionalized gold nanoparticles will be reused for further analysis of additional analytes. If so, the gold nanoparticles that have been analyzed by the flow imaging system are collected and rinsed with acid in a block 220 to remove any analyte molecules that remain bound to receptor molecules on the nanoparticles (see FIGURES ID, portion D, and FIGURE 1H).
  • FIGURE 7 graphically illustrates the SPR coupling wavelength shift 230 as a function of the adsorbed film thickness 232 for a bulk gold film 234 (solid line) and nanoparticle 236 (dashed line) configurations, respectively, showing that the sensitivity of the two SPR configurations are within a factor of two of one another.
  • Nanoparticle response curve 236 exhibits a non-linear response 240, since it represents the entire dynamic range of film thickness, and the non- linearity is due to the exponential decay of the electric field.
  • the bulk SPR sensor generates a substantially linear response.
  • this nanoparticle response curve should not be considered as limiting the present invention.
  • the non-linearity and abbreviated dynamic range of nanoparticles response curve 236 should not be understood to mean that nanoparticle SPR spectra are not useful. Larger nanoparticles may be employed to increase the linearity of the curve, and calibration corrections can be made for the non-linear response. Significantly, most SPR sensorgrams do not utilize more than 40 nm in their response, but, as long as a generally linear response in that range is achieved, such spectra are useful. .
  • the calculations employed to generate the response curves of FIGURE 7 assume a bulk solution refractive index of 1.3336 (for water) and an adsorbed film refractive index of 1.45 (for proteins).
  • the data calculated for bulk gold film assume a prism material of BK-7, incident angle of 82 degrees, and a gold film thickness of 550 Angstroms and were calculated with a software program using a matrix form of the Fresnel reflection coefficients.
  • the data calculated for nanoparticle assume an enhanced nanoshell (exhibiting six-times enhanced signal shift) and employ an approximation of the SPR coupling wavelength dependence described by Eq (2.0), as follows:
  • FIGURE 8A schematically illustrates how the preferred flow imaging system discussed in connection with FIGURE 3 can be modified to be used for white light spectral scatter analysis using prism dispersion. Such a modification enables detailed spectral characterization of both gold nanoparticles and nanoparticle coated microbeads.
  • spectral decomposition filter stack element 108 shown in FIGURE 3
  • prism 290 or grating oriented to disperse laterally as shown in FIGURE 8 A
  • Exemplary data obtained from the imaging system of FIGURE 8A are shown in FIGURE 9.
  • FIGURE 8B illustrates continously segmented data 292 from eighteen 1 micron diameter fluorescently labelled particles.
  • the range of wavelength detection is from 488 nm to 750 nm.
  • Each particle is imaged in the darkfield at the 488 nm excitation wavelength, and additionally, the fluorescent spectral emision from each particle is determined.
  • the bead set analyzed contained three separate populations with different fluorescent emision spectra, as illustrated by the top three data segments 296 of the data buffer.
  • Data segment 298 (the 6 segment from the top) and data segment 300 (the 16 th segment form the top) indicate the prescence of two clumped beads.
  • Data segment 302 (the 11 th segment) in the fluorescent spectrum indicates the presence of three clumped beads.
  • the preferred flow imaging system of FIGURE 3 can be readily modified as indicated in FIGRE 8A to achieve a tool for individually measuring the SPR spectra of nanoparticles.
  • FIGURE 9 schematically illustrates exemplary data collected from the preferred imaging system of FIGURE 3, modified as indicated in FIGURE 8 A, where the resulting flow imaging system enables simultaneous imaging of absorbed, scattered, and fluorescent light from objects in flow. While this configuration cannot image objects as small as nanoparticles (as opposed to collecting spectral data from such small particles), the configuration of the imaging system in FIGURE 8A does allow image data from microbeads to be analyzed. As indicated above, metal island films exhibiting the SPR phenomena can be supported on micron-sized beads. Thus, the imaging system shown in FIGURE 8 A can be used to decode bead-on-bead libraries of metal island micron- sized beads.
  • FIGURE 9 shows exemplary data 268 from a multispectral image data set of fluorescent calibration beads generated by the flow imaging system of FIGURE 8A. Images of each bead 260 appear in a brightfield channel 262 and a darkfield channel 264, along with fluorescence images in a channel 266 in the channel corresponding to the dye present on each of the fluorescent calibration beads. This imagery was gathered at a magnification of 20X, corresponding to a pixel size of approximately 0.65 microns at the object. The darkfield 264 imagery shows the lensing effect of each bead 260 due to its large index of refraction relative to the buffer solution. A scatter plot 270 in FIGURE 9 shows the discrimination power and sensitivity of the system.
  • the TDI detector is linear in its response, high dynamic range is achieved, because each image covers more than 50 pixels, each of which is digitized with 8-bit resolution. With typical cell samples and the 10-bit per pixel resolution of the TDI detector, over four decades of dynamic range is achievable.
  • FIGURE 10A is a side view
  • FIGURE 10B is a plan view, showing how the imaging system of FIGURE 8 A can be modified to enable scattered light angular analysis of the biomolecular interaction analysis using nanoparticle SPR substrates described in connection with FIGURES 5A-5H and FIGURE 6.
  • These modifications enable the detailed monochromatic angular scattered light intensity characteristic of both gold nanoparticles and nanoparticle coated microbeads.
  • monochromatic light 306 from a laser source 98 (FIGURE 8A) is used to illuminate a center 308 of cuvette 116.
  • a nanoparticle 99 enters the field of view, the laser light is scattered along light paths 310, and the scattered light is collected and collimated by a sperical collection lens 312 to achieve collimated light 314.
  • the collimated light is then focused by a cylindrical lens 304 upon TDI detector 114.
  • the readout rate of the TDI detector is synchronized with the flow speed of nanoparticle 99, enabling up to a thousand fold increase in signal intensity.
  • the scattered angle spatial intensity distribution is measured along the pixelated row of TDI detector 114
  • FIGURE 11 illustrates exemplary data collected from a early version of the flow imaging system of FIGURE 3, indicating how multispectral darkfield scattering can be used to analyze particles. For particles equal in size or smaller than the pixel size in the image plane, the size of such particles can be determined by measuring the relative light scattering intensity across multiple wavelengths. The ratio of the scattered light intensities at given wavelengths is a function of the size of the particle based upon Raleigh scattering.
  • FIGURE 11 illustrates continuously segmented data from ten unlabelled beads 350, each about 350 nm in diameter. The orthogonal scattered light images were collected using 488, 532, 670, and 780 nm laser excitation.
  • Imagery in channel 352 was collected using a 455 nm laser excitation wavelength; imagery in channel 354 was collected using a 532 nm laser excitation wavelength; imagery in channel 356 was collected using a 760 nm laser excitation wavelength; and imagery in channel 358 was collected using a 780 nm laser excitation wavelength.
  • Current implementations of the flow imaging system of FIGURE 3 have been optimized for excitation wavelengths from 400-750 nm. For nanoparticles that are small relative to the 0.25 micron pixel size of a flow imaging system, the image acts as a spatial noise filter, excluding the pixels outside the boundaries of the nanoparticles from integrated intensity calculations, thereby enhancing the signal-to-noise ratio.
  • the measurement of absorbance intensity from a nanoparticle that spans three pixels in a fluorescence image will have approximately 100 times less background than a non-imaging system employing a 20 ⁇ m laser spot.
  • FIGURE 12 illustrates how the flow imaging system of FIGURE 8 A can be modified to detect surface enhanced Raman spectroscopy.
  • An optional holographic notch filter 309 is used to filter out the excitation laser light signal.

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Abstract

L'invention concerne un système d'imagerie d'écoulements utilisé dans la détection par résonance plasmonique de surface pour étudier les interactions biomoléculaires. Ce système d'imagerie est utilisé pour capturer des spectres d'absorption de résonance plasmonique de surface d'un grand nombre d'objets, chaque objet comprenant à la fois un film métallique pouvant présenter une résonance plasmonique de surface et des molécules de détection. Des molécules d'analyte sont ajoutées à une solution constituée par lesdits objets, et le résultat est introduit dans le système d'imagerie d'écoulements qui rassemble toutes les données de spectre de résonance plasmonique de surface des objets individuels. Ces données de spectre peuvent être ensuite utilisées pour déterminer la spécificité, la cinétique, l'affinité et la concentration par rapport aux interactions entre les molécules de détection et les molécules d'analyte.
PCT/US2004/006006 2003-02-27 2004-02-27 Plate-forme d'imagerie de detection de nanoparticules appliquee a une analyse d'interactions biomoleculaires par resonance plasmonique de surface WO2004102160A2 (fr)

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WO2019020975A1 (fr) * 2017-07-24 2019-01-31 University College Cardiff Consultants Ltd Analyse de nano-objets
CN110907643A (zh) * 2019-12-02 2020-03-24 中国科学院重庆绿色智能技术研究院 一种大肠杆菌检测芯片的制备方法及检测芯片

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US5991048A (en) * 1995-10-25 1999-11-23 University Of Washington Surface plasmon resonance light pipe sensor

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US5991048A (en) * 1995-10-25 1999-11-23 University Of Washington Surface plasmon resonance light pipe sensor

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019020975A1 (fr) * 2017-07-24 2019-01-31 University College Cardiff Consultants Ltd Analyse de nano-objets
US10996159B2 (en) 2017-07-24 2021-05-04 University College Cardiff Consultants Limited Analysing nano-objects
CN110907643A (zh) * 2019-12-02 2020-03-24 中国科学院重庆绿色智能技术研究院 一种大肠杆菌检测芯片的制备方法及检测芯片

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