WO2015139028A1 - Using optical forces to measure the property of a substance - Google Patents

Abstract

The present invention provides devices, methods, and systems for measuring a property of a substance. In certain aspects, the invention provides devices, and methods, and systems for separating substances based upon a property of the substance. The present invention makes use of one or more optical waveguides and the observations of substance-waveguide interactions to measure a property of the substance.

Description

TITLE OF THE INVENTION Using Optical Forces to Measure the Property of a Substance

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. provisional application No.

61/952,922 filed on March 14, 2014, and U.S. provisional application No. 62/001,304 filed on May 21, 2014, each of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with government support under REB015797A awarded by the National Institute of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Nanoparticles represent the largest commercialization of nanotechnology (Matsui, 2005, Journal of Chemical Engineering of Japan, 38: 535-46; Salata, 2004, J Nanobiotechnology, 2:3; Doll et al, 2013, J R Soc Interface, 10: 20120740; Jia et al, 2013, J Control Release, 172: 1020-34; Prasad, 2009, Safety of Nanoparticles, Springer, New York, 89-109) and are used, for example, in medicine, electronics, batteries and household products. Despite the recent manufacturing advances that enable synthesis and manufacture of a huge variety of nanoparticles, there remain significant measurement challenges. For example, in biomedical applications, nanoparticles are often highly reactive, display complicated size-dependent interfacial properties and are applied in complex biological systems with often unclear and ambiguous results (Grainger et al, 2008, Advanced Materials, 20: 867-77; Naahidi et al, 2013, J Control Release, 166: 182- 84; Dreaden et al, 2012, Ther Deliv, 3: 457-78; Blanco et al, 2011, Cancer Sci, 102: 1247-52). Specifically, the vastly increased surface area and high surface energy of nanoparticle dispersions result in performance that is strongly mediated by surface interactions. There is a pressing demand for improved nanoparticle surface analysis (Grainger et al, 2008, Advanced Materials, 20: 867-77; Grassian et al, 2008, J Phys Chem C, 112: 18303-13; Baer et al, 2010, Anal Bioanal Chem, 396: 983-1002; Verma et al, 2010, Small, 6: 12-21; Ventola, 2012, P T, 37: 512-25). For example, in

nanomedicine, there is a lack of accurate nanoparticle surface analysis methods for predicting long term stability, an FDA requirement. Further, it is critical to be able to ascertain the homogeneity or heterogeneity of a population of manufactured products, either in terms of individual particle size, shape, and/or surface property. Thus there is a need in the art for devices and methods to effectively evaluate the properties of individual nanoparticles in a population. The present invention satisfies this unmet need.

SUMMARY OF THE INVENTION

In one embodiment, the invention is a method of measuring at least one property of a substance including the steps of positioning a substance in the vicinity of one or more optical waveguides, such that the substance is captured by a waveguide and travels along the waveguide; measuring one or more metrics of the interaction between the substance and the waveguide; and determining at least one property of the substance based upon the measured metric.

In another embodiment, the invention is a method of separating a plurality of substances within a population into one or more sub-populations including the steps of positioning each substance in the vicinity of one or more optical waveguides, such that the substance is captured by a waveguide and travels along the waveguide, where a property of the substance determines the distance which each substance travels along the waveguide before the forces acting upon the substance force the release of the substance from the waveguide, thereby separating the plurality of substances into sub-populations based upon the location at which each substance is released.

In yet another embodiment, the invention is a device for measuring the property of a substance including one or more optical waveguides; one or more fluidic channels; where at least one of the one or more fluidic channels is in communication with one or more optical waveguides; at least one light source operably connected to the one or more waveguides to provide optical power to the one or more waveguides. In yet another embodiment, the invention is a device for separating multiple substances in a population into one or more sub -populations including one or more optical waveguides; one or more fluidic channels; where at least one of the one or more fluidic channels is in communication with one or more optical waveguides; at least one light source operably connected to the one or more waveguides to provide optical power to the one or more waveguides.

In yet another embodiment, the invention is a system for measuring a property of a substance including a device including one or more optical waveguides; one or more fluidic channels; wherein at least one of the one or more fluidic channels is in communication with one or more optical waveguides; at least one light source operably connected to the one or more waveguides to provide optical power to the one or more waveguides; a fluidic delivery system in communication with at least one fluidic channel of the device.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figure 1 depicts a schematic illustrating the characterization and sorting of differently coated particles. Optical forces generated by the waveguides separate a mixture of coated particles flowing into the chamber into distinct streams based on coating thickness/quality. The degree of deflection for each individually particle type can be measured by observing the particle scatter along a waveguide with a charge-coupled device (CCD).

Figure 2, comprising Figures 2A and 2B, is a set of illustrations demonstrating how the mobility of a particle is determined by the properties of the particle. Particles are held close to the waveguide by optical trapping. Laser scattering forces push them along the waveguide. Particles progress along the waveguide according to their surface adsorption properties. An exemplary system of the invention and an experimental image are depicted in Figure 2A. A conceptual explanation is shown in Figure 2B.

Figure 3 depicts an exemplary ovular set of waveguides or concentric waveguide racetracks.

Figure 4, comprising Figures 4A and Figure 4B, depict illustrations of exemplary waveguide designs of the present invention. Figure 4A depicts a spiral waveguide coupled to a single laser input. Figure 4B depicts a design having a shallower angle of attack, multiple laser inputs and a bifurcation light splitting technique.

Figure 5, comprising Figures 5A through 5C, is a set of illustrations depicting an exemplary device comprising a Y-shaped input channel (Figure 5A), a microscope mount (Figure 5B), and fluidic delivery system (Figure 5C).

Figure 6, comprising Figures 6A through Figure 6C, depicts the results of simulations of separation assay and predicted single deflection separation and

downstream separation distance as a function of waveguide power and angle of attack

(Figure 6 A) Simulation domain comparison between 300nm and 350nm particles (Figure 6B) and 500nm and 550nm particles (Figure 6C). Flow speed in all cases is 200 mm/s.

Figure 7, comprising Figures 7A through Figure 7C, depicts the results of experiments demonstrating the use of optical waveguides to analyze particle size and shape. Figure7A: A variety of different 200-250 nm particles measured via optical scatter on waveguides. Figure 7B: Sizing data generated from the scattered light of the particles in (Figure 7A). Figure 7C: Shape analysis of protein aggregates. The scatter pattern for amorphous nanoparticles is dramatically different compared to that of spherical particles, which remain constant and circular.

Figure 8, comprising Figures 8 A and 8B, depicts the results of experiments using waveguides to capture and measure active pharmaceutical ingredients (APIs). Figure 8A shows the difference in appearance of the APIs on the system compared to polystyrene nanoparticles. This histogram in Figure 8B demonstrates the ability to size the APIs.

Figure 9, comprising Figures 9A and 9B, depicts the results of experiments demonstrating the differing velocity of differently sized particles. Figure 9A: Compiled data for 100-5000 nm nanoparticles run in this system show a clear

relationship between size and velocity. Figure 9B: Semi-processed data showing a 40 seconds (400 frames) of compressed video. The streaks represent 500 nm (artificially colored red or dark) and 200 nm (artificially colored green or light). The lower the slope, the faster the particle motion. The different trajectories are stark and highlight the significance and novelty of an aspect of the invention.

Figure 10, comprising Figures 10A and 10B, depicts the results of experiments demonstrating that particle motion decreases with Debye length. Debye length was reduced by increasing salt concentration. This allows for more

adsorption/attraction to the waveguide, thus slowing the particles. Figure 10A:

Relationship of Debye to velocity. Figure 10B: Raw data with artificial color added for visualization.

Figure 11, comprising Figures 11A and 1 IB, depicts predicted results of experiments examining the use of coated waveguides. Functional waveguides are expected to provide higher sensitivity towards particle coating extent than bare waveguides, as shown in the graph of Figure 11A. Waveguide coatings are applied to the waveguide via silane coupling chemistry. Example chemistries for functional and bare waveguides are shown in Figure 1 IB.

Figure 12 is an exemplary histogram depicting the frequency of intensity observations of the scattered light from a particle traveling along a waveguide. When particles penetrate the near field of the waveguide, they form a refractive index defect that transforms near field light into far field scattered light that is collected and routed to a detector/camera.

Figure 13 is an exemplary plot depicting a potential energy well calculated from intensity observations of the scattered light from a particle traveling along a waveguide. In this plot, both surface and optical forces are present.

Figure 14 is an exemplary plot depicting a potential energy well, as well as the calculation of the components of potential energy well due to optical energy and surface interactions.

Figure 15, comprising Figures 15A through Figure 15C, depicts the results of experiments evaluating the relationship of aspect ratio on observed velocity and observed intensity of optical scattering. The velocity and intensity of a variety of differently shaped particles are plotted in linear scale (Figure 15 A) and log scale (Figure 15B). The slopes of velocity/intensity (V/I) are plotted for a given aspect ratio of the tested particles (Figure 15C). The data demonstrates that the slope of V/I has a relationship to aspect ratio, which can thus be used to evaluate the shape of an unknown particle.

Figure 16, comprising Figures 16A through 16E, depicts the results of surface properties measured from a PVP-coated silver nanoparticles sample. Figure 16A is a data summary of the analysis. Figure 16B is a plot of intensity data for all particles. Figure 16C is histogram depicting the frequency of intensity observations of the scattered light from a particle traveling along a waveguide. Figure 16D is a plot depicting a potential energy well and the calculation of the components of potential energy well due to optical energy and surface interactions. Figure 16E is a plot of surface force for each particle.

Figure 17, comprising Figures 17A and 17B, show Raman spectra for submicrometer latex (Figure 17A) and sun block particles (Figure 17B) using a waveguide as both the trapping and excitation source.

Figure 18 shows Raman spectra for <1 μιη particles.

Figure 19, comprising Figures 19A through 19C, depict a system and steps for particle surface analysis. Figure 19A is a diagram for a waveguide-based optical trapping and visualization system. Figure 19B is a diagram showing particles near a waveguide, illustrating how particles move around an equilibrium height determined by the balance of surface repulsion and optical trapping (i.e. they "sit" at the bottom of a potential well). Figure 19C are resulting force plots of a technique used to analyze a batch of 10 particles.

Figure 20, comprising Figures 20A through 20C, depicts how features can be determined. Figure 20A is a plot as a histogram of the intensity of a particle. Figure 20B is a plot showing the potential energy well. Figure 20C is a plot showing the surface energy component. Accordingly, potential wells (Figure 20B) can be determined from the intensity histograms (Figure 20A) of each particle, and surface energy plots (independent of particle size) (Figure 20C) can be extracted by removing the optical component of the well (Figure 20B).

DETAILED DESCRIPTION

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

As used herein, each of the following terms has the meaning associated with it in this section.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

"About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

As used herein, "near field light" refers to the passage of light in sub wavelength dimensions. In some instances the near field effect discussed herein is known as the "evanescence wave" or the "evanescent field."

As used herein, a "photonic waveguide" refers to a light guide patterned in microfabricated material that has microscale, nanoscale, or subwavelength dimensions.

As used herein, a "slot waveguide" refers to an optical waveguide that guides strongly confined light in a sub-wavelength-scale low refractive index region by total internal reflection. In certain embodiments, a slot- waveguide comprises two strips or slabs of high-refractive-index separated by a sub-wavelength-scale low- refractive -index slot region of a lower refractive index material. In some embodiments, the lower index material is an aqueous solution or buffer. Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description

The present invention provides devices, systems, and methods for analyzing a property of a substance using optical forces. For example, the present invention allows for interrogation of a biological, chemical, and/or physical property of a substance of interest. In certain embodiments, the present invention exploits the interaction of the substance of interest with an optical waveguide surface in order to determine or measure a property of the substance. In certain embodiments, the method comprises measuring one or more metric of a substance's interaction with a waveguide, including for example, the attraction/repulsion of the substance to the waveguide. For example, in certain embodiments, a property of the substance may be determined by measuring one or more metric of the motion of the substance along the waveguide, including but not limited to the substance residence time, velocity, variation in velocity, waveguide position, distance of the substance to the waveguide, and deflection distance. In one embodiment, a property of the substance may be determined by measuring one or more properties of the light scattered by the substance, including but not limited to the intensity, shape, pattern, spectral characteristics, and wavelength of the scattered light. In certain embodiments, a property of the substance may be determined by measuring the affinity towards other substances and/or surfaces.

The present invention can be used, for example, to study the size of substance, the structure of a substance, the shape of the substance, the surface coating of the substance, the surface property of a substance, the solubility of a substance, the interaction affinity of the substance to like and non-like substances, the composition of the substance, and the like. In certain embodiments, the invention may be used to study the interaction between one or more substances, for example the binding affinity of a first substance to a second substance.

Exemplary substances analyzed by way of the present invention include, but are not limited to, foams, emulsions, sol/colloids, nanoparticles, microparticles, nanotubes, nanocrystals, liposomes, exosomes, polymer shell nanoparticles, core shell, contrast agents, dendrimers, wax particles, subvisible particles, quantum dots, lipoparticles, vesicles, oil droplets, bioparticles, biomolecules, nucleic acid molecules, proteins, enzymes, antibodies, viruses, bacteria, cells, small molecules, protein complexes, carbohydrates and the like.

In certain embodiments, the substance comprises any material, including, but not limited to, metals, plastics, polymers, alloys, glass, and the like. In one embodiment, the substance comprises a surface coating. Additionally, viruses, virus-like particles, bacteria, vectors, cells, liposomes, which may be referred to as bioparticles, and the like can also be analyzed according to the methods described herein. Other types of substances that can be analyzed using the present invention include liposomes or liposomal structures. The present invention is not limited to any particular type of substance. Rather the present invention encompasses the analysis of any substance whose interaction with an optical wave guide may be measured.

Thus, the term "substance" refers to any molecule, compound,

composition, particle, or biological composition that can be optically manipulated and analyzed as described herein and elsewhere.

Previously, it has been shown that the location and movement of particles can be manipulated using optical forces. For example, U.S. Patent Application

Publication Nos. 2011/0039730 and 2012/033915 and International Application

Publication Nos. WO/2012/048220 and WO/2013/172976 describe various devices and methods that can be used to manipulate the location and movement of particles, each of which is hereby incorporated by reference in its entirety. The present invention utilizes optical forces to attract a substance of interest and to propel the substance of interest along a length of an optical waveguide. For example, optical gradient forces attract the substance to the waveguide, while optical scattering forces propels the substance along the waveguide (Figure 1). In certain embodiments, the motion of the substance as it travels along the waveguide is measured, which is used to evaluate one or more properties of the substance. In certain

embodiments, the particle is captured by the waveguide and scatters light as the particle interacts with the near field light of the waveguide. That is, in certain embodiments, the waveguide is not just a trap but also acts as a light excitation source that makes the particles light up. Thus, in certain embodiments, the system analyzes scattered light using a near field excitation source, and the scattered light signatures are used to determine coating and composition, etc.

In certain embodiments, one or more properties of the substance is determined by analyzing the potential energy well of the substance as it interacts with the waveguide. For example, the shape and depth of the potential energy well depends on the type and strength of both attractive and repulsive forces present between the substance and the waveguide.

In one embodiment, the invention allows for the analysis of individual substances within a population. For example, in certain embodiments, the present invention provides for a way to evaluate the homogeneity or heterogeneity of a property of interest within a population. In one embodiment, the method comprises evaluating the property, or properties, of interest of a plurality of substances within a population, thus giving a measure of homogeneity of the property within the population.

In one embodiment, each substance is deflected along the waveguide until the forces acting on the substance, including for example, fluid drag, Brownian motion, thermal energy, and the like, cause the substance to be released. The properties of each substance dictate the distance along the waveguide the substance travels and the overall deflection distance. In certain embodiments, the variation in the deflection distance within the substance population provides a measure of population heterogeneity.

In one embodiment, the invention comprises separation of individual substances within a population. For example, in certain embodiments, individual substances within a population are separated into sub-populations, each sub-population having a distinct value of the property of interest. In one embodiment, the differential distance in which the substances are propelled or deflected along the waveguide allows for the separation of substances (Figure 1). For example, the differentially deflected substances can be released from the waveguide into individual flow streams or channels. In certain instances the presence of the separated sub-populations is observed

downstream of the waveguide, using known detection systems and methods (e.g.

microscopy, spectroscopy, and the like) (Figure 1). In certain embodiments, the different flow streams allow for the collection of uniform or near-uniform substances in each sub- population.

In one embodiment, the present invention provides for a measurement of the surface property of an individual substance, including for example a nanoparticle. As the size of substance shrinks, the surface properties of the substance grow in importance (Ventola, 2012, P T, 37: 512-25; Chan, 2006, Regul Toxicol Pharacol, 46: 218-24; Sesai, 2012, AAPS J, 14: 282-95). For example, the surface of a nanoparticle is a key determinant of its properties and performance. Further, the synthesis of nanoparticle dispersions almost always utilizes a surface treatment/coating to yield dispersions with both chemical and colloidal stability for subsequent applications (Grassian et al, 2008, J Phys Chem C, 112: 18303-13; Shaw, 1992, Introduction to colloid and surface chemistry, Oxford, Boston; Prieve and Lin, 1982, Journal of Colloid and Interface Science, 86: 17- 25). Yet evaluating the surface properties and predicting long-term stability is not possible with current commercial techniques despite the need to be evaluated (Ventola, 2012, P T, 37: 512-25; Chan, 2006, Regul Toxicol Pharacol, 46: 218-24; Sesai, 2012, AAPS J, 14: 282-95; Prieve and Lin, 1982, Journal of Colloid and Interface Science, 86: 17-25). The most common methods for measuring stability are observing changes in size, absorbance or turbidity in the presence of different salt concentrations (Santander-Ortega et al, 2006, J Colloid Interface Sci, 302: 522-9; Davalos-Patoja et al, 2001, Colloids Surf B Biointerfaces, 20: 165-75; Riley et al, 1999, Colloids Surf B Biointerfaces, 16: 147-57; Vera et al, 1996, Journal of Colloid and Interface Science, 177: 553-60). These methods are useful as a first pass for detecting gross or incipient instability due to electrostatic interactions, but fail to capture long-term instability mechanisms, such as dissociation or chemical degradation of the coating. Accordingly, it is routine practice to assess long- term stability by observing sedimentation, accelerated by aggregate formation, over the duration of the stability lifetime (Kakran et al., 2012, J Pharm Pharmacol, 64: 1394-402; Cauda et al, 2010, J Mater Chem, 20: 8693-9; Fang et al, 2009, Small, 5: 1637-41; Hwang et al, 2008, Powder Technology, 186: 145-53). For particles of intermediate stability, this can be on the order of weeks or months. This low throughput, coupled with minimal data yielded (stable or not stable) can prevent systematic and rapid optimization of coating protocols. Existing methods for measuring compositional and morphological qualities are generally high vacuum systems not suitable for colloids. These include X- ray photoelectron spectroscopy (XPS), Auger electron spectroscopy, mass

spectrometries, and others (Grainger and Cashier, 2008, 20: 867-77). Instead, most liquid-based nanoparticle measurement systems are used for measuring size (dynamic light scattering, nanoparticle tracking, environmental SEM) whereas there are limited techniques for measuring surface properties in liquid. Zeta sizers measure only bulk colloidal surface charge and don't yield meaningful results for uncharged particles such as those with a stabilizer coating, and AFM, while applicable to measuring surface properties of individual particles in thin fluid films, suffer from low throughput insufficient for routine analysis as well as is limited to force resolutions typically in excess of 10 pN due to thermal noise. A variety of authors (Grainger and Castner, 2008, 20: 867-77; Grassian 2008, J Phys Chem C, 112: 18303-13; Baer et al, 2010, Anal Bioanal Chem, 396: 983-1002; Verma and Stellacci, 2010, Small, 6: 12-21; Ventola, 2012, P T, 37: 512-25) and regulatory agencies (Nanomedicines Drafting Group, 2013, European Medicines Agency, Committee for Medicinal Products for Human Use (CHMP); Baer, 2012, Surface and Interface Analysis, 44: 1305-8) have specifically cited the need for improved methods to analyze nanoparticle surfaces.

In DLVO theory (Shaw, 1992, Introduction to colloid and surface chemistry, Oxford; Boston), the classical explanation of the stability of colloidal suspensions, colloidal stability for uncoated particles, naturally charged in solution, is generally only achieved at low electrolyte concentrations. In order to extend colloidal stability beyond low electrolyte concentration media, particles are routinely coated with adsorbing polymers or surfactants, which self-assemble to form a monomolecular film or micelle-like aggregates on the particle surface. The charge on the particle surface itself is eliminated, and colloidal stability is dictated by the steric and/or charge-repulsive interactions of these coatings, extending stability to significantly higher salt

concentrations.

Other technology used for colloidal stability measurements measure one of size, turbidity/absorbance, or zeta potential. Size-based measurement systems like dynamic light scattering (DLS) or nanoparticle tracking see the increase in average particle size when aggregation occurs due to measured changes in Brownian motion. Turbidity/absorbance measurements observe bulk changes in scattered light intensity due to aggregate formation. Zeta sizers measure the average zeta potential, indicative of surface charge, of nanoparticles. It is an ensemble measurement and thus does not provide information on charge (and therefore coating) variability across particles in a sample. The difference in long term stability between samples containing uniform or heterogeneous surface charge/coating across particles is stark. Continuous flow size sorters reported in the literature are not yet able to handle particles less than several micrometers in size (Dielectrophoresis (DEP) (Cheng et al, 2009, Lab Chip, 9: 3193- 201; Srivastava et al, 2011, J Chromatogr A, 1218: 1780-9), optical (MacDonald et al, 2003, Nature, 426: 421-4; Grujic et al, 2005, Opt Express, 13: 1-7; Wang, 2005, Nature Biotechnol, 23: 83-7; Paterson et al, 2007, J Biomed Opt, 12: 054017; Lin et al, 2008, Biomed Microdevices, 10: 55-63) or mechanical/acoustic (Hatta et al., 2004, Chem

Commun, 2772-3; Yamada and Seki, 2006, Anal Chem, 78: 1357-62; Chen et al, 2009, Biomed Microdevices, 11 : 1223-31; Adams and Soh, 2010, Appl Phys Lett, 97).

All current separation systems typically use one of the techniques described in Table 1. Two major advantages exist for using optical principles to analyze substance properties and/or sort substances based upon substance properties. The first advantage over the state of the art comes in the »a regime where propulsive velocity is proportional to a⁵ (λ: wavelength of light, a: substance radius). A small change in substance radius, then, translates into a dramatic change in velocity. The other techniques have at most an a² dependence on velocity. The second major advantage is that nanoparticle substances are closer in size to binding entities (polymer coatings, surfactant molecules, biomolecules) and so the extent of coating will have a dramatically larger effect per particle than for micron sized beads (usually millions of times larger in volume). The first advantage indicates the potential for a high performance analysis tool and sorter while the second advantage lends this system for use as a sensitive technique for the measurement of substance properties (e.g. surface coatings or surface properties). The modulation of attractive forces between a substance and the waveguide through a substance coating will further amplify differences in observed velocities. That is, a well- coated substance will be both larger and less tightly bound to the waveguide than an uncoated substance, resulting in an even faster velocity than predicted by size

consideration alone.

Table 1 : BioPrism comparison with other chromatography and separation techniques

e ectr c e , : zeta potent a , p: ens ty, : pressure

* Free solution electrophoresis. Gel electrophoresis exhibits a non-linear drag that increase the dependence of V_ep on a.

** Approximate, based on assumption of proportionality between hydrodynamic volume and molecular weight.

In terms of the ability of the present invention to sort or separate different substances, a common way to quantify separation resolutions is to compare the spatial resolution between two species at some point downstream and divide the difference between the widths of the concentration profiles. Comparing optical separation resolution R₀p with electrophoretic separation R_ep we can derive the following:

R_op = (1 + Δα/α)⁵ - 1, R_ep = 1 - o/(o + Δα) (1), (2) where a is particle radius (see Erickson et al, 2011, Lap Chip, 11 : 995- 1009 for details). For a 1% and 10% size difference (from the addition of proteins on the surface of a bead) a 500% and 680% improvement in fractionalization efficiency is obtained in the present optical separation technique, compared to the state-of-the-art. More details on these calculations are available in Erickson et al. (Erickson et al., 2011, Lap Chip, 11 : 995-1009) Furthermore, although there is some small contribution to the separation velocity due to refractive index change (squared relationship), the vast majority of the force dependency will be on substance radius change (fifth power relationship). Using polystyrene nanoparticles with a refractive index of around 1.57 and doubling the particle volume with high refractive index protein, gives the following breakdown: 4.7%> change in force due to refractive index and 438%) change due to radius increase. Refractive index in this case contributes, therefore to about 1% of the total force.

In the present invention, properties of a substance, for example the size, shape, or surface property of the substance, are analyzed through observing and measuring substance-waveguide interactions as individual substances are deflected along the waveguide. The surface property of the substance may be the natural properties of a material of the substance, or a surface coating applied to a substance.

The surface coatings are applied to various substances for various reasons, which depend on the use of the substance and or the field of use of the substance. For example, in the pharmaceutical industry, active pharmaceutical ingredients (APIs) are often coated to prevent agglomeration, adsorption of biomolecules, and/or to prevent toxic side-effects. Exemplary types of surface coatings for such pharmaceutical substances include, but are not limited to, polyethylene glycol (PEG), albumin, polyvinylpyrrolidone (PVP), dextran, and pluronic block polymers. In some instances, a substance is coated with an antibody, or antibody fragment, to provide a targeting moiety to the substance.

Additional exemplary surface coatings include charged coatings such as carboxylic acid and amines as well as self-assembled monolayers (SAMs). An example of SAMs include coatings of silane monomers. Exemplary silane-based coatings are discussed elsewhere herein. The present invention provides for the evaluation of the surface property of a substance by evaluating the interaction of the substance with a waveguide or with another substance already interacting with the waveguide. Such interactions can be determined by measuring metrics such as the residence time, velocity, variation of velocity, deflection distance, position along the waveguide, the intensity of scattered light, pattern of scattered light, shape of the scattered light, wavelength(s) of scattered light, and the like. In one embodiment, the interaction of the substance and the waveguide is dependent upon the adsorption of the substance to the waveguide. In certain embodiments, the adsorption between the substance and the waveguide is dependent upon the presence of a surface coating of the substance, the presence of a surface coating on the waveguide, the nature of the surface coating of the substance, the nature of the surface coating of the waveguide, and the Debye length.

For example, if the substance comprises a hydrophilic coating, while the waveguide comprises a hydrophobic coating, a well coated substance would not exhibit strong adsorption to the waveguide, and thus will travel faster along the waveguide. In contrast, if the substance has a non-uniform hydrophilic coating, or for some reason is missing a hydrophilic coating, it would experience a strong adsorption force, as compared to the well coated substance, and would thus travel slower. In certain embodiments, instead of using velocity as a measurement vehicle, a potential well can also be used as a measurement vehicle. If there is a favorable interaction (e.g., A hydrophobic particle with hydrophobic surface), there isn't lower surface energy, and this can be measured that way directly through potential well analysis. Potential well analysis measures the distance of the particles in the z-direction from the waveguide as opposed to how fast it is traveling.

In general, substances that are coated with materials that have strong affinity to the waveguide coating will exhibit slower motion on the waveguide. For example, substances coated with a positive charge will adsorb better and travel faster onto a waveguide with a negative charge than waveguides with a positive charge. Polar substance coatings will adsorb well onto polar waveguide coatings and travel slower than a polar with non-polar pair. Hydrophobic substance coatings will not adsorb well onto hydrophilic waveguide coatings and will travel faster than substances with a hydrophilic coating. While the foregoing examples described "coated" substances, they are equally applicable to substances which may not necessarily comprise a coating, but rather had a surface property inherent to the substance itself.

As described above, in certain instances well-coated substances will glide smoothly along the waveguide due to the repulsive interaction between the waveguide and substance (Figure 2). Nanoparticles with poor, incomplete, or heterogeneous surface coatings will experience increased attractive interactions with the waveguide, and as a result will travel along the waveguide more slowly. Similarly, the size of a substance can be analyzed, as larger substances will travel faster along the waveguide than smaller substances. Further, the aspect ratio of a substance also influences the motion, as well as observed intensity, of the substance.

Forces acting upon the substance, which therefore governs the substance interaction with the waveguide, motion along the waveguide, and, in certain instances, the release of the substance from the waveguide, are described elsewhere herein.

In one embodiment, the device of the present invention comprises at least one fluidic channel (i.e. a microchannel) and at least one optical waveguide, coupled to at least one light source (i.e. laser). Individual substances, to be analyzed by the device and method of the present invention, are administered to the fluidic channel, which carries the substances toward the at least one waveguide. Upon approaching the at least one waveguide, optical forces generated by the light within the waveguide capture a substance and propels the substance along the waveguide. For example, as light travels down a waveguide, a portion of the energy resides in the liquid as an evanescent field. This field interacts with substances in the fluidic channel and causes an attractive force (to the waveguide) and a propulsive force (along the waveguide). As shown in the conceptual portion of Figure 2, optical scatter causes a force in the direction of the traveling light. This scattering based waveguide transport is demonstrated in data presented herein and on numerous occations in the literature (Yang et al., 2009, Nature, 457: 71-5; Schmidt et al, 2007, Opt Express, 14: 14322-34; Yang and Erickson, 2010, Lab Chip, 10: 769-74; Li et al, 2012, Opt Express, 20: 24160-6; Cai and Poon, 2012, Lab Chip, 12: 3803-9; Cai and poon, 2010, Opt Lett, 34: 2855-7). The motion of each substance along the waveguide is opposed by the adsorption forces between the waveguide and the substance as well as the fluidic drag forces between the substance and the fluid. The residence time, velocity, substance position, distance to which the substance travels, intensity of scattered light, and/or pattern of scattered light is a function of the size, shape, and surface property of the substance. In one embodiment, the motion of the substance and the scattered light produced by the substance are directly measured while the substance is traveling along the waveguide, thereby providing a mechanism to analyze one or more properties of the substance. For example, the motion of the substance may be measured by measuring one or more of residence time of the substance, velocity of the substance, variation in velocity of the substance, substance position, and distance to which the substance travels. In certain embodiments, substance position is the position of the substance along a waveguide at any given time point. In one embodiment, substance position comprises historical data of all the positions of the substance over a defined time frame.

For example, in certain instances, substances travel along the waveguide for a residence time one the order of seconds on each waveguide, their natural Brownian motion will allow the waveguide to sample each part of the substance surface. Therefore, substances with incomplete or heterogeneous coatings exhibit variations in velocity, which can be quantified and correlated to quality and/or extent of substance coating or surface property.

Figure 1 shows that particles that have a thicker coating experience a greater optical force due to increase in volume, with larger particles exhibiting larger optical forces. The conceptual image in Figure 2 shows a particle that is well coated and another that is poorly coated. The property of the coating in Figure 2 is such that it prevents adsorption of the particle onto the waveguide surface (e.g., coating is

hydrophobic and waveguide surface is hydrophilic). Particles that are poorly coated will therefore exhibit stronger adsorption to the surface (if particle core is hydrophilic) and experience a large surface friction force which lowers its optical propagation velocity (mobility). This has been observed to be consistent with experimental results. A scenario opposite of that in Figure 2 could easily be envisioned (if you flip the particle core chemistry and the particle coating chemistry and keep same waveguide chemistry), where particles with poor coating could actually be further separated from the waveguide and travel faster.

In certain embodiments, the method comprises analyzing the potential energy well in which the substance resides. For example, the amount of light scattered by the substance during its interaction with the waveguide can indicate the distance of the substance from the waveguide surface. Due to the decay of the evanescent field emanating from the waveguide, a particle attracted towards the waveguide will scatter more or less light if it moves closer to or farther away from the waveguide respectively. Thus, in certain embodiments, continuous instantaneous measurements of scattered light intensity can be translated into fluctuations in the substance position relative to the waveguide surface. In one embodiment, after many such measurements of an individual substance, the equilibrium separation distance of the substance from the waveguide surface can be accurately calculated. The distribution of intensity measurements can be used to accurately calculate the potential energy well in which the substance resides.

In one embodiment, the substance analyzed by way of the device and method of the present invention has a size of size less than ΙΟΟμιη, in any one dimension. In one embodiment, the substance analyzed by way of the device and method of the present invention has a size of size less than 1 μιη, in any one dimension. In one embodiment, the substance analyzed by way of the device and method of the present invention has a size of size less than 500nm, in any one dimension. In one embodiment, the substance analyzed by way of the device and method of the present invention has a size of size less than lOOnm, in any one dimension. In one embodiment, the substance analyzed by way of the device and method of the present invention has a size of size less than lOnm, in any one dimension.

In one embodiment, the device comprises one or more slot waveguides

(see for example, U.S. Application Publication No. US2012/0033915). In brief, a slot waveguide comprises a nanoscale slot having a relatively low refractive index, sandwiched between two walls of significantly higher refractive index. The low refractive index material of the slot can also consist of the surrounding fluid. A laser provides light to the waveguide and therefore within the slot, which produces an optical force to immobilize a molecule or particle within or on the sides of the slot. While particular embodiments are exemplified herein using slot waveguides, one skilled in the art would recognize that any suitable waveguides can be utilized in the method of the present invention. Exemplary optical waveguides, include, but are not limited to straight waveguide, a slot waveguide, and a liquid core waveguide. In certain embodiments, the waveguide contains a resonator, including, for example, a ring resonator, fabry-perot cavity, ID photonic crystal resonators, and the like.

The optical waveguide may have any suitable geometry. For example, in certain embodiments, the waveguide may comprise a region that is curved. In one embodiment, the waveguide may have a circular, ovular, or spiral shape. In certain embodiments, the waveguide is branched or bifurcated. In certain embodiments, the cross-section of the waveguide is varied. For example, the cross-section may be varied in shape, size, or both. In certain embodiments, the waveguide is configured to support different modes of light (multi or single mode).

In certain embodiments, the slot of the slot waveguide may range in width from 10 nm to 800 nm.

In certain embodiments, the waveguide is manufactured of materials such as silicon, silicon nitride (Si₃N₄), SU-8 (MicroChem Corp.), silicon oxide, polymethyl methacrylate, glass, silicon carbide, Polydimethyl Sulfoxide (PDMS), benzocyclobutene (BCB), aluminum gallium arsenide, sapphire, and the like.

In certain embodiments, the waveguide comprises a surface coating. For example, in one embodiment, the waveguide is modified with a surface coating. As described elsewhere herein, in certain instances, the interaction between a substance of interest and the waveguide is dependent upon the presence and nature of a surface coating on the waveguide. Thus, in certain instances, the one or more waveguides of the device is coated to either enhance or lessen a substance-waveguide interaction or to increase the selectivity between mixtures of different substances. For example, surface coating of a waveguide may be hydrophobic, hydrophilic, positively charged, neutrally charged, negatively charged, and the like.

In certain instances, application of a functional surface coating to the one or more waveguides modulates the retention time of the substance of interest. In certain instances this may improve the sensitivity of the present invention towards small changes in the properties of the substance of interest. For example, with respect to separation of substances based on substance properties, increasing the sensitivity of the device through waveguide coatings would improve the separation efficiency. Further, surface coatings may result in higher reproducibility, as silicon nitride surfaces are subject to variability in absolute and relative concentrations of native active groups (silanol and amine) depending on cleaning history and solution conditions (Stine et al, 2007, Langmuir, 23: 4400-4; Brow and Pantano, 1986, Journal of the American Ceramic Society, 69: 314-6) or decrease fouling.

In one embodiment, the waveguide is functionalized with one or more functional groups to provide a surface coating. For example, in one embodiment, the coating comprises self-assembled monolayers (SAMs). An example of SAMs are coatings of silane monomers. Silanes with a wide variety of functional groups are commercially available and translatable to Si3N4 (Diao et al, 2005, Anal Biochem, 343: 322-8; Wu et al, 2006, Biosens Bioelectron, 21 : 1252-63; Manning and Redmond, 2005, Langmuir, 21 : 395-402; Colic et al., 1998, Journal of the American Ceramic Society, 81 : 2157-63). Exemplary silane functional groups include, but are not limited to

octadecyltrimethoxysilane (ODS), octyltrimethoxysilane (OS), aminopropyl- trimethoxysilane (APS), and (3-chloro)propyl-trimethoxysilane (CPS). Surfaces comprising ODS and/or OS would render the waveguide hydorophobic, while APS and CPS render the waveguide positively or negatively charged, respectively. In addition to charged interactions, hydrophilic silane coatings (e.g. 2-cyanoethyltrichlorosilane (CES), 2-(carbomethoxy)ethyltrichlorosilane (CMS), and 3 -methoxypropyltrimethoxy silane (MPS).)which are polar or participate in hydrogen bonding can be used to further tune the interactions on coated waveguides. Other silanes which are appropriate include, but are not limited to, polyethylene glycol/oxide-like silanes (e.g. 2-

[methoxy(polyethylenoxy)propyl]trichlorosilane) to discourage non-specific binding of materials to the waveguide. Dipodal silanes may be employed to improve long term stability of a silane coating. A blend of two or more silanes may also be employed to tailor the surface properties of the waveguide.

Coating the waveguide with SAMs may be performed using any methodology known in the art. For example, in one embodiment, a surface coating of the waveguide is applied using vapor-phase deposition (Zhang et al, 2010, Langmuir, 26: 14648-54; Popat, 2002, Surface and Coatings Technology, 154: 253-61). SAMs deposition can also be carried out via solution phase deposition, for example by deposition from aqueous alcohol solutions, aqueous solutions, alcohol solutions, anhydrous liquids. SAMs can also be deposited by spincoat/spincast methods, spray application, micro-contact printing, soft lithography, and dip-pen lithograhpy,

In another embodiment, the coating consists of polymeric materials.

Coating the waveguide with polymeric materials may be performed using any

methodology known in the art. For example, single or multilayer polyelectrolytes can be deposited from aqueous solutions to coat the waveguide and render the waveguide with a positive or negative charge. Other techniques of polymer deposition include, but are not limited to, plasma polymerization, spin coating, and dip coating.

In another embodiment, the coating consists of inorganic material, for example, a sub-micron oxide coating or metallic thin film. Coating the waveguide with an inorganic material can be achieved, for example, by physical vapor deposition, including sputtering, evaporation, or laser ablation, or chemical vapor deposition, or electroplating. An inorganic coating may be used to alter the surface properties of the waveguide itself, or to enable the application of a secondary coating. For example, a sub 10 nm thick layer of gold can enable certain depositions of SAMs with affinity to a gold substrate, for example thiol-containing molecules.

In another embodiment, the coating consists of biological or biomimetic materials, including, but not limited to, polysaccharides (e.g cellulose,dextran), proteins, peptides, or proteinaceous coatings, nucleic acids, or lipids. Methods for deposition include but are not limited to, adsorprtion, bionconjugation, and langmuir-blodgett deposition,

In another embodiment, the coating consists of surfactant molecules, for example, Pluronic surfactants. Surfactants can be adsorbed by methods including, but not limited to, solution deposition, langmuir-blodgett technique, spray coating, and dip coating.

In one embodiment, one or more waveguides of the device, or different regions of a single waveguide, are differentially coated. For example, the device may comprise one waveguide coated with a hydrophobic coating and another waveguide coated with a positively charged coating. A device comprising a plurality of different coatings on different regions may allow for improved characterization of the properties of the substance.

As described elsewhere herein, the at least one waveguide of the device is optically coupled to at least one light source. The light source acts a powering system, providing the optical force. The light source can be a laser or other type of optical force. In one embodiment, the power of the laser is configured to be between 1-lOOOmW. In another embodiment, the power of the laser is configured to be between 10-lOOmW. In certain embodiments, the power of the laser, the wavelength of the light, and/or polarization of the light determines the size, size range, or refractive index range of the substance being analyzed. For example, in one embodiment, the power of the laser is tuned such that only substances of a particular size and/or refractive index range are attracted and manipulated by the waveguide, while substances that are larger than the range or smaller than the size range and/or outside of the refractive index range flow past the waveguide.

In certain embodiments, the light source is coupled with one or more lenses, light guides, apertures, annuli, optical fibers, fiber optics, or the like.

The light source may be configured for any mode of light delivery, including, but not limited to single wavelength, multiple wavelengths, different polarizations, constant power, modulated power, pulsed. The light source may be a single source or multiple sources. In certain embodiments, the light source is a single mode source or a multimode broad band source.

In certain embodiments, the one or more waveguides of the device are oriented at a given angle relative to the flow within the channel. For example, in one embodiment, the waveguide is oriented such that the light within the waveguide is substantially perpendicular (90°) to the flow. In another embodiment, the waveguide is oriented such that the light within the waveguide is substantially parallel to the flow. In another embodiment, the waveguide is oriented such that the waveguide is at an angle of about 30° to about 45° to the flow. It should be appreciated that there is no limitation to the waveguide angle used. In one embodiment, the forces acting upon the substance, while traveling along the waveguide, eventually deflect the substance off of the waveguide and into a flow stream. As the substance properties dictate the motion of the substance along the waveguide, substances having different properties are deflected at different distances, thereby effectively separating the substances into sub-populations. In certain instances, the optical power of the light source and/or the angle of the waveguide in relation to the flow in the channel can influence the deflection distance of substance, or the separation of deflection distances between two or more different substance types. In one embodiment, substances having different deflection distances are separated into different flow streams. In one embodiment, the degree of deflection for each substance is measured, for example by visualizing the deflected substances after they have been released from the waveguide. If for example, it is observed that all the substances had about the same deflection distance, it can be inferred that the population of substances was relatively homogenous. However, if it is observed that there are several observed sub-populations of substances, each having a different deflection distance, it can be inferred that the original population was heterogeneous, and that there are multiple types of substances within the population or that the material properties, such as size, shape, coating, coating thickness, coating uniformity, or the like are heterogeneous. The device and method of the invention can thus separate the substances based on a given property, for example, size, shape, and/or surface property. The system is very similar to adsorption chromatography which separates mixtures of chemical species by passing them through a column filled with porous media. Each species has a characteristic mobility that comes about by adsorption with the solid phase which slows the progression through the column depending on the strength of interaction.

In certain embodiments, the waveguide is shaped such that the forces acting upon a substance are altered as the substance travels along the waveguide. For example, in one embodiment, the one or more waveguides of the device has at least one curved region (Figure 3 and Figure 4A). As the waveguide curves, the orientation of the optical forces changes and ultimately loses out to the fluid drag force, which rips the substance away from the waveguide.

Figure 4 depicts a pair of exemplary waveguide designs for the device of the present invention. In one embodiment, the one or more waveguide of the device is shaped such that the substances may interact with the same waveguide more than once. For example, in certain embodiments, the device comprises a waveguide having a circular or spiral shape, which allows a single light source to power numerous sites of substance-waveguide interaction. For example, Figure 4A depicts a spiral design, wherein a single waveguide is coupled to a single light source.

In one embodiment, the device comprises a plurality of distinct or interconnected waveguides. For example, Figure 4B depicts a design comprising a plurality of branched or bifurcated waveguides, each coupled to its own light source. The plurality of waveguides may be optically coupled to one or more light sources. The use of more than one substance-waveguide interaction, either by way of a single waveguide or a plurality of waveguides, allows for numerous deflections of individual substances, which in some instances, enhances the separation between substance types in the population. This method of applying deflection forces to a mixture of particles as they progress through space in order to measure material properties is often called flow field fractionation.

In one embodiment, the design is optimized to provide an increased interaction surface. An increase in active surface area allows more interaction and better separation. An analogy is the use of activated carbon, which has a high degree of surface area, and is used in gas masks to adsorb chemical threats. Increased interaction surface may be accomplished, for example, by increasing the number and density of waveguides or substance-waveguide interaction sites along the device. In certain embodiments, the design of the one or more waveguides of the device is optimized for increased

functionality. For example, in certain aspects the design is optimized for increase separation efficiency. In one embodiment, the device is designed to provide a longer interaction length between the substance and the one or more waveguides of the device. Prolonged interaction would allow for improved separation of different substances, similar to how longer chromatography columns increase separation efficiency. Increase interaction length may be devised by increasing the number of interaction sites within the device. In one embodiment, the device is modified to provide higher optical power. In certain instances, higher optical power directly influences the motion of a substance and the amount of deflection. Optical power may be increased, for example, by using multiple lasers per waveguide or by using higher powered lasers. In certain embodiments, the device is designed to optimize the deflection angle. For example, as shown in Figure 6, a shallower deflection angle allows longer interaction with each waveguide and increases the downstream interaction length.

In one embodiment, the device comprises one or more fluidic channels. In one embodiment, the one or more fluidic channels are used to carry a fluidic sample comprising the substance of interest to and from the one or more waveguides of the device. The one or more fluidic channels may be of any suitable size and shape which is able to carry a fluidic sample. In one embodiment, and as is shown in Figure 5, the device comprises one or more distinct fluidic channels which connect to form a single channel at some point upstream from the one or more waveguides. For example, the one or more channels may comprise a sample channel and a buffer channel. This allows the sample to enter at a particular location such that the substance of interest is captured on the waveguide at or near a predetermined location. In certain instances, this allows for substances to effectively be deflected into different flow streams.

The present invention includes a system for substance analysis and/or separation comprising the device described elsewhere herein. In one embodiment, the system comprises the device combined with a fluidic delivery system such that one or more substances are delivered to the device. For example a fluidic delivery system can deliver the one or more substances to the channel of the device, which in turn delivers the substance toward the one or more waveguides. This can be done with a fluidic channel and/or flow cell. Any fluidics system can be used. For example, a syringe can be used to apply the substance to the device. In one embodiment, a fluid droplet can be transferred to the surface of the device without any flow cell or chamber. In one embodiment, a stream of gas can be directed towards the surface of the device. However, much more sophisticated systems can be used such that the system can be automated. In one embodiment, the fluidic delivery system and device are included on a single chip. In another embodiment, the device is applied to a chip, comprising a fluidic delivery system.

In one embodiment, the fluidic delivery system comprises a microscope mount. In certain instances, the mount comprises one or more devices of the present invention. For example, the mount may hold one or more re -usable or disposable devices. The devices may be implemented on cassettes or chips that may be housed within the mount. The mount can be designed to mount to any microscope or microscope stage known in the art. In certain embodiments, the mount comprises one or more fluid inlets and/or one or more fluid outlets, which deliver the fluidic sample and other buffers to and from the one or more devices.

In one embodiment, the fluidic delivery system comprises a pump, used to pump one or more fluids, including for example a fluidic sample and other buffers, from containers or reservoirs to the one or more devices. Exemplary pumps are known in the art and are commercially available. Exemplary pumps include pneumatic pumps, syringe driven pumps, peristaltic pumps, membrane pumps or the like.

In one embodiment, the fluidic delivery system comprises a flow control system. In certain embodiments, the flow control system monitors and controls the flow rate of one or more fluids, including for example a fluidic sample and other buffers, being delivered to the device. In one embodiment, the flow control system comprises a sensor, which is used to monitor the delivered flow rate of the one or more fluids. In one embodiment, the sensor and pump of the delivery system work together to provide a built-in feedback of flow rate control.

In one embodiment, the system comprises a measurement system or measurement detection device. For example, the device of the invention may be combined with known devices and techniques to interrogate the substance of interest to determine its size, shape, surface property, surface coating, binding affinity, and the like. Exemplary devices include, but are not limited to, fluorescence microscopes,

fluorescence detectors, fluorescence spectrometers, light scattering detectors, optical sensors, Raman microscopes, Raman spectrometers, spectrometers, photodiodes, charged coupled devices (CCDs), Complementary metal-oxide-semiconductor (CMOS) cameras, spectrum analyzers, interferometers, ellipsometers, integrating spheres, and

photomultipliers, zeta sizers, particle tracking analysis systems (e.g. a NanoSight instrument).

In certain embodiments, the system of the invention comprises at least one of a power supply, fiber-coupled semiconductor laser, an optical isolator to protect the laser from back scatter, photodiode signal digitizer, syringe pump, computer interface hardware, AC/DC power supply for USB ports for computer interface, a single mode polarization maintaining fiber optic that connects to a silicon nitride waveguide on a silicon chip. The chip sits in a plastic carrier which assists handling and fits into a holder that connects the fluid lines from the syringe pump to the chip. The chip has a laser cut adhesive gasket that defines a microfluidic channel which is completed with an optical cover slip. The chip and mount are placed under an objective lens of a microscope. .

The present invention provides a method to analyze a property of an individual substance. For example, in certain embodiments, the property is at least one of the structure of a substance, the shape of the substance, the surface coating of the substance, the surface property of a substance, the solubility of a substance, the interaction affinity of a substance, the flocculation state of a substance, the aggregation of a substance and the like. In one embodiment, the method allows for the interrogation of a property of an individual substance within a population. For example, the method allows for the determination of the homogeneity or heterogeneity of a population, based upon the detected properties of the individual substances.

Substances analyzed by way of the present method include, but are not lmited to, nanoparticles, microparticles, nanotubes, nanocrystals, exosomes, liposomes, polymer shell nanoparticles, nano contrast agents, nano wax particles, subvisible particles, quantum dots, lipoparticles, vesicles, oil droplets, bioparticles, biomolecules, nucleic acid molecules, proteins, enzymes, antibodies, viruses, bacteria, cells, small molecules, protein complexes, carbohydrates and the like.

In one embodiment, the substance analyzed by way of the device and method of the present invention has a size of size less than 1 μιη, in any one dimension. In one embodiment, the substance analyzed by way of the device and method of the present invention has a size of size less than 500nm, in any one dimension. In one embodiment, the substance analyzed by way of the device and method of the present invention has a size of size less than lOOnm, in any one dimension. In one embodiment, the substance analyzed by way of the device and method of the present invention has a size of size less than lOnm, in any one dimension.

In some embodiments, a substance of interest is administered to the device of the invention, as described elsewhere herein. For example, the substance may be in a fluidic sample delivered to the one or more fluidic channels of the device. In one embodiment, the substance is contacted with the waveguide by allowing the substance to flow over the waveguide. Any method can be used to allow the substance to flow over the waveguide. The light source may be turned on prior to or after the substance is contacted with the device.

In some embodiments, the flow rate of the fluid carrying the substance can be modulated (e.g. increased or decreased), which can facilitate the capture, motion, and/or release of the substance from the waveguide. Additionally, different substances can be contacted to the device using either the same channel, or alternatively by using separate channels. In one embodiment, the flow can be used to influence which specific type of substance, from a fluid comprising a plurality of substances, is captured by the waveguide. For example, in one embodiment, the flow is tuned such that only substances of a particular range are captured and manipulated by the optical trap, while substances that are larger than the range or smaller than the range flow past the trap.

A number of different flow schemes can be used, including but not limited to, pressure driven flow, electromagnetically driven flow, electrokinetically driven flow, capillary driven flow, flow focusing, flow contacting, varying channel geometries to affect how the substance of interest is manipulated.

In some embodiments, the fluid delivery scheme can comprise of one or more inlets and one or more outlets. Additionally, a micro fluidic circuit can be established to precisely meter and deliver different fluids to the one or more waveguides. Each inlet and channel can deliver separate reagents or process reagents, for example to microfluidically generate a concentration gradient.

As described herein, the waveguide can be powered before introducing the sample that contains the substance of interest or the waveguide can be turned on after the sample has been introduced. The waveguide can also be pulsed or modulated such that there is a controllable duty cycle. That is, the power can be turned on and off rapidly according to some periodicity. Thus, the waveguide will alternate between an on state and an off state with some frequency. The power of the waveguide can also be modulated by simply reducing the optical power delivered to the waveguide, by adjusting the polarization of the light or by changing the wavelength of the light. The power of the waveguide can also be adjusted in order to manipulate smaller objects. The amount of force that is necessary can be determined by the skilled artisan in view of the present disclosure. In some embodiments, the power can be pulsed to prevent substances from sticking to the surface (e.g. to avoid surface charges and other surface effects).

In some embodiments, after, prior, or simultaneously to the substance being administered to the device, additional reagents can be introduced to interact with the device or to the substance itself. These reagents can be any reagent that can be used in the device. Non-limiting examples, including, blocking buffers to passivate the surface so that future reagents do not non-specifically bind to a surface or substance, additional substances that may or may not bind to the substance of interest, a washing buffer to remove the substance, a new buffer or solution, a continuously changing buffer (varying in concentration of salts, pH, concentration of buffer constituent), a solution of standardized particles used to calibrate the system, a solution of particles that are initially captured within a waveguide for measuring their interaction with subsequent substances, and the like.

In one embodiment, the method comprises application of an additional reagent comprising one or more additional substances (e.g. nanoparticles, bioparticles, proteins, organic molecules, inorganic molecules, nucleotide molecules, and the like) to evaluate their influence on the substance of interest. In one embodiment, the additional reagent comprises one or more additional substance that may or may not influence the size, shape, or surface property of the substance of interest. In one embodiment, these additional substances can bind to the substance of interest and form temporary or permanent complexes or aggregates. In some embodiments, the additional substances can cause the substance of interest to aggregate/form complexes or cause the substance of interest to become disassociated into smaller components or subunits.

For example, in one embodiment, the method of the invention comprises a screening method, where additional reagent comprise a test compound from a library of compounds, and the device is used to evaluate whether or not the test compound influences the properties of a substance of interest.

In some embodiments, the substance of interest is released from the waveguide. In certain embodiments, as described elsewhere herein, the substance of interest travels along the waveguide until the flow forces the substance to be released. In one embodiment, the substance of interest is released from the waveguide by turning off the power source, modulating the power of the power source, modulating the polarization of the light, or tuning the laser of the power source to a different wavelength. The released substance can then be analyzed or otherwise manipulated.

In some embodiments, the environment of the substance of interest is altered. In some embodiments, the temperature of the substance, sample, or device modified. In some embodiments, the substance is exposed to light (any wavelength), radiation, electric field, gasses, or other reagents to change the substance's environment. In certain embodiments, the pH, salt concentration, viscosity, or another modifiable property of a solution carrying the particle, is altered. As described elsewhere herein, in certain embodiments, the environment of the substance is altered by administering one or more additional substances to the solution.

In one embodiment, the method comprises evaluating the motion of a substance as it travels along the waveguide. For example, in certain embodiments, the residence time, velocity, and/or deflection distance of the substance is measured. In one embodiment, the method comprises visualizing the motion of the substance, using known visualizing systems and methods, including for example, a camera that observes the emitted light produced, scattered or altered by the substances. In one embodiment, the motion of the substance is recorded, for example with use of a camera, to allow for future analysis. The residence time, velocity, and deflection distance of the substance may be computed from the visualized and/or recorded motion of the substance using known techniques and/or software programs.

In one embodiment, the observed velocity of an individual substance is associated with one or more properties of the individual substance. In one embodiment, the observed variation in velocity between multiple individual substances within a population indicates the homogeneity or heterogeneity of the population. In one embodiment, an observed variation in the velocity of an individual substance (e.g. a nonuniform velocity, multiple starts and stops, etc.) as it travels along the waveguide is indicative a non-uniform property of the individual substance. In certain embodiments, the observed scattered light of an individual substance is measured. For example, the intensity, shape, pattern, wavelength(s) of the scattered light of a substance as it travels along the waveguide may be observed and/or recorded using known systems, including for example a CCD or CMOS camera, photodiode, avalanche photodiode, single photon counters, integrating spheres, photodiode array, spectrometer, thermal absorbers, pyroelectric energy sensors. In certain embodiments, the light can be collected using waveguides, fiber optics, light pipes, index matching fluid, lenses, integrating spheres, and optical filters.

In certain instances, one or more properties of the scattered light may be associated with one or more properties of the individual substance. For example, in certain embodiments, the intensity and/or pattern of scattered light indicates the size, shape, and/or surface property of the substance.

In certain aspects, the pattern of scattered light or the polarization state of the scattered light are used to characterize the substance of interest. For example, the near field nature of the interrogating light may provide sub-wavelength analysis of the substance, allowing for morphological information for features smaller than the wavelength of light used. In certain instances substance attributes may alter the polarization state of the light that is scattered, for example morphological, chemical or material properties.

In certain aspects, both the observed intensity and observed velocity are used together to characterize the substance of interest. For example, in certain instances, an aspect ratio of an individual substance of interest can be determined by measuring the velocity of the substance and intensity of scattered light produced by the substance, thereby providing a measure of the shape of the substance. For example, it is described herein that in certain embodiments, the observed velocity over the observed intensity (V/I) can be used to determine the aspect ratio of a substance. It other instances, the aspect ratio of a substance may be ascertained from a different function of the observed velocity and scattered light.

In certain embodiments, the method of the invention comprises observation of separated sub-populations of substances after the substances have been released from the waveguide. As described elsewhere herein, substances having differing properties exhibit differing deflection distances which can thus result in separation of substances into distinct flow streams that correspond to sub-populations, each sub- population comprising substances with the same or similar properties. In one

embodiment, the method comprises detecting the presence, number, and/or location of substance-containing flow streams downstream of the waveguide. Such detection may be carried out using any known detection systems of methodology (e.g., microscopy, spectroscopy, or the like).

In certain embodiments, the method comprises collecting each flow stream downstream of the waveguide, thereby collecting each separated sub-population. For example, in one embodiment, the device of the invention comprises one or more collecting channels, each designed and positioned to collect a flow stream comprising a sub -population of substances having the same or similar properties.

In some embodiments, a control is used. Control measurements can also be taken of other devices that have different substances or other control measures.

Measurements can be taken of other locations on the chip for background or control purposes.

Embodiments of the invention have numerous benefits and advantages, as described in detail throughout. Some of the benefits and advantages of the system, especially with respect to particle surface analysis and near field light scattering to analyze nanoparticle coatings, include the following: the system can measure individual particle surface properties, the system can probe a variety of different surface interactions (charged, steric, etc.), the system operates in the particle's native environment, measurement sensitivity is in the piconewton range, the system can operate on a wide variety of particle material types, and in certain exemplary embodiments, the system can operate on particles from 10 nm up to 5 μιη.

The presently described methods can be used in conjunction with other detection methods. The detection methods can be used to measure substance size, molecule composition, binding affinities, kinetics, inhibition or activation of an enzyme or other process, and the like. Examples of other detection methods include, but are not limited to, fluorescence, chemiluminescence, optical scattering, Raman spectroscopy, colorimetric, electrochemical methods and Surface Plasmon Resonance and spectroscopy. Such exemplary methods may be integrated onto devices or systems comprising a waveguide (i.e. on-chip), or alternatively be externally coupled (i.e. off- chip).

EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Example 1 : Simulations of substance interaction with a waveguide

As shown in Figure 6, when a substance interacts with a waveguide at an angle, Θ, to the flow it experiences the following forces.

∑Fx = 0 = F_scat + Ff_lowsin0 - F_fric, (3a) ∑F_y = 0 = F_trap - F_flowcos6 (3b)

Where FJJ_OW is the drag force on the substance due to the imposed flow and Ftrap and F_scat are the optical forces incident on the substance both perpendicular and along the length of the waveguide. Fd_rag is the resistive friction force on the substance which impedes it motion along the length of the waveguide and is a combination of hydrodynamic drag and friction with the surface. Consistent with low Reynolds number flow and linear friction model, it is assumed that Ff_ric is proportional to the propulsive velocity of the substance, v_p. Details of the numerical simulation methodology and complete theoretical analysis are provided in Yang and Erickson (Yang and Erickson, 2008, Nanotechnology, 19: 045704). Briefly by solving the wave equation in a computation domain that comprises the waveguide and a particle one can extract out F_scat and Ftrap from the x and y components of the surface integral of the time averaged Maxwell stress tensor and Ffl_ow is computed by solving the low Reynolds number stokes equations within the same computation domain and integrating the fluid stress tensor over the particle surface.

The average deflection distance for a substance interacting with the waveguide, X in Figure 6, can be calculated with a knowledge of v_p and the average residence time on the waveguide, t, via X = v_ptcos6. v_p can be computed by solving Eq. (3 a). The average resistance time can be determined by estimating depth of the potential well in which the particle is trapped, U_trap, or equivalently the amount of thermodynamic work required to remove the particle from a stably trapped position. This work is given by Eq. (4).

Utrap = ^{F L} (! + /? ( Ι^η β) - 1)) (4)

where γ is the force decay rate as the substance is removed from the waveguide (roughly equivalent to the decay length of the evanescent field) and β is Ffl_owcos Θ/Ftrap- Given that U_tra_P can be computed from the numerical results presented above, the average resistance time of the substance on the waveguide can be estimated from Eq. (5) where the reference time t_Q is obtained from experiments conducted under conditions for which U_tra_P,o is known.

The results of these computations are shown in Figure 6B and Figure 6C for two set of particle sizes (300/350nm particle separation in (a) and 500/550nm particle separation in (b)). The downstream separation distance is indicative of the ideal single deflection spatial separation between the particles. As can be seen the predicted separations distances are relatively large and increase as a function of angle of attack and increasing optical power. These simulations provide further evidence of the potential sensitivity of the present method backing up the experiments and theory presented elsewhere herein. However, as stated elsewhere herein, the present invention is not limited to measuring the deflection distance of a substance. Rather, in certain embodiments, a property of the substance can be measured by observing the resident time and/or velocity of the substance while traveling along the waveguide.

Example 2: Analysis of substance properties using scattered light

Experiments were conducted to examine whether the properties of various substances can be elucidated by observing and measuring the optical scatter of substances as they interact with a waveguide. As shown in Figure 7A and Figure 7B, BaTi, API, polystyrene, PMMA, and silica particles were contacted to a waveguide. It is observed that each substance produced a distinct optical scatter pattern (Figure 7A). Further, the size distribution of each of the particle types was able to be ascertained from their interaction with the waveguide (Figure 7B).

Further, Figure 8 depicts the results of experiments comparing a 250nm pharmaceutical nanoparticle with a 200nm polystyrene particle, again demonstrating that they produce different optical scattering patterns when contacted to the waveguide.

Further, the size distribution of a population of pharmaceutical nanoparticles was able to be determined using both the intensity of the scattered light as well as the velocity of the nanoparticles using the device of the present invention.

It was also examined, whether the shape of a particle can influence the optical scatter. To do so, protein aggregates, having amorphous shape, were contacted to a waveguide, and the optical scatter pattern of these aggregates was compared to the pattern produced by spherical particles. As can be seen in Figure 7C, the optical scatter pattern of the amorphous protein aggregates was dramatically different than that of spherical particles. Example 3 : Determining particle size by measuring velocity

Experiments were conducted to examine whether the size of a particle influences the velocity at which the particle travels along a waveguide.

The waveguide design of the presently described device was tested using different sized particles standards. Data was collected using different populations of nanoparticles, each population having a particle size of 100-5000nm. In each case, the velocities of individual nanoparticles were observed over time as a result of the optical forces. In general, larger nanoparticles exhibit much faster and more consistent velocities than the smaller nanoparticles (Figure 9). As seen in Figure 9, it was observed that the observed velocity of a particle increases as the size of the particle increases. Figure 9 depicts semi-processed data showing the position of a 500nm particle and a 200nm particle over time, again demonstrating that the velocity of the larger 500nm particle was greater than that of the smaller 200nm particle.

The smaller particles appear to periodically stop followed by continued movement. While not wishing to be bound by any particular theory, one explanation for this is that there was a heterogeneity in surface coatings on the smaller nanoparticles (Velegol, 2001, Langmuir, 7687-93; Feick et al, 2004, Langmuir, 20: 3090-5; Feick et al, 2004, Ind Eng Chem Res, 43: 3478-83). Due to their smaller size, surface charge heterogeneity impacts particle motion on the waveguide much more dramatically. When, during their natural Brownian motion, poorly coated regions happen to face the waveguide, the increase in adsorption forces opposes the optical forces and slows down the nanoparticle. As described above, it should also be pointed out that different sized particles can be easily distinguished, not only by their velocity, but also by the amount of scattered light.

Example 4: Particle motion is influenced by particle-waveguide interactions

Experiments were conducted to examine particle motion in response to altered Debye length. Salt concentration was altered to tune the Debye length and hence strength of particle-waveguide attractive forces. A smaller Debye length allows the particles to get closer to the waveguide and experience stronger attraction/adsorption forces. NaCl was varied from 1 mM to 15 mM with carboxylate modified latex, 200 nm nanoparticles using bare waveguides. As the Debye length decreases, particle velocity decreases significantly on the waveguide (Figure 10). Additionally, the variation in mean particle velocity increases, suggesting heterogeneity in carboxylate modification, and hence surface charge, across particles. Introducing a positively-charged polyelectrolyte waveguide coating increases the attractive force between the negatively charged particles and the waveguide, resulting in nearly stationary particles. Example 5 : Waveguide coatings

Experiments are conducted to examine the influence of applying functional surface coatings to the waveguide in order to modulate the retention time of particles. A reproducible and robust method for functionalizing S1₃N₄ surfaces with desired functional groups through a vapor-phase silane deposition technique is developed. Vapor-phase deposition methods are the preferred approach for achieving silane monolayers (Zhang et al, 2010, Langmuir, 26: 14648-54; Popat et al., 2002, Surface and Coatings Technology, 154: 253-61), and silanes with a wide variety of functional groups are commercially available and translatable to S1₃N₄ (Diao et al., 2005, Anal Biochem, 343: 322-8; Wu et al., 2006, Biosens Bioelectron, 21 : 1252-63; Manning and Redmond, 2005, Langmuir, 21 : 395-402; Colic et al., 1998, Journal of the American Ceramic Society, 81 : 2157-63). It is first attempted to deposit, on blanket S1₃N₄ substrates, four silane monomers currently used in functional silica gels for adsorption chromatography (Dash et al, 2008, Adv Colloid Interface Sci, 140: 77-94; Sucia and Auler, 2005, Journal of Chromatography A, 147-53; Dai et all, 2003, Journal of

Chromatography A, 1005: 63-82) in order to obtain a variety of surface properties. These are: octadecyltrimethoxysilane (ODS) and octyltrimethoxysilane (OS), which will render the waveguide hydrophobic, and aminopropyl-trimethoxysilane (APS) and (3- chloro)propyl-trimethoxysilane (CPS), which will render the waveguide positively or negatively charged, respectively. Standard recipes from literature are followed (Zhang et al, 2010, Langmuir, 26: 14648-54; Popat et al, 2002, Surface and Coatings Technology, 154: 253-61). Characterization of the coating is carried out via water contact angle measurements and XPS to ensure uniform coverage and covalent attachment of silane monolayers.

Experiments are conducted to determine whether the thickness and wetting properties of the introduced functional layer prevent normal device operation (e.g. insufficient capture or non-specific binding) or significantly alter experimental conditions (laser power, flow pressure) optimal for the uncoated waveguide case.

Polystyrene nanospheres are used as standard samples. Microspheres are either uncoated (bare polystyrene), carboxylate-modified (negative surface charge), amine-modified (positive surface charge), stabilized with an ionic surfactant, or coated with PVP. Each particle coating type is tested with each waveguide functionalization and compared with the un-coated waveguide, to screen for significant differences in capture behavior, or cases in which the laser power needs to be modulated to increase or decrease trapping tendency if altered by the functional coating.

Experiments are conducted to quantify the improvement in sensitivity gained through functional waveguide coatings and identify which coatings offer the largest improvement for a specific API particle-coating system. Model hydrophobic and hydrophilic API's (e.g. paclitaxel, megestrol acetate) are prepared and coated (by means of a known high-stability coating protocol), an intermediate (particle partially

destabilized), and an uncoated state. The particles in each three states are tested with uncoated waveguides and compared with waveguides functionalized with the coatings listed above. Both the chemical nature of the coating employed and the stability of the particle are expected to influence the interaction, and hence retention time, of the particle with the waveguide in both the functional and uncoated waveguide case. For example, hydrophobic particles stabilized with a hydrophilic coating are expected to experience diminished interaction for the coated state, and hence shorter residence times on the waveguides (coated or uncoated), whereas particles in the uncoated state experience enhanced interaction and increased residence times on waveguides with a hydrophobic coating (ODS or OS) (Figure 14). Functional waveguides are expected to provide higher sensitivity towards particle coating extent than bare waveguides. Waveguide coatings are applied to the waveguide via silane coupling chemistry. Comparable tests are conducted on a variety of both APIs and particle coating types to determine the gain in sensitivity towards particle stability and/or coating degradation.

Example 6: Particle-waveguide interaction as measured by calculation of a potential energy well

In certain instances, the interaction between the particle surface and waveguide surface is precisely measured by mathematically calculating a potential energy well. A particle that is confined to a region close to the waveguide, due to an optical attractive force, but not actually touching the waveguide, due to surface repulsion, can be said to reside in a potential energy well. The shape and depth of the potential energy well depends on the type and strength of both attractive and repulsive forces present. This example calculation demonstrates how the potential energy well and surface repulsion force can be measured by observing a particle's motion on a waveguide.

A particle's natural thermal motion will cause a particle to oscillate about an equilibrium distance from the waveguide surface in a characteristic way. Due to the decay of the evanescent field emanating from the waveguide, a particle attracted towards the waveguide will scatter more or less light if it moves closer to or farther away from the waveguide respectively. Instantaneous measurements of scattered light intensity can be translated into fluctuations in a particle's position relative to the waveguide surface. After many such measurements of an individual particle, the particle's equilibrium separation distance can be accurately calculated, and the distribution of intensity measurements can be used to accurately calculate the potential energy well in which the particle resides.

The first step in describing the potential energy well is to generate a histogram of the distribution of scattered intensity measurements (Figure 12).

Alternatively, a continuous distribution function can be fit to the experimental data to generate expected frequencies and perform the calculations described below. When using histograms, the bin with the highest frequency is assumed to represent the equilibrium intensity, I_0t of the particle.

Experiments were conducted by administering 200 nm polsytrene particles in aqueous 4 nM sodium chloride solution toward a waveguide. The waveguide was uncoated and operated at 200 mW of power. Imaging was acquired using a CMOS microscope camera operating at 500 frames per second and with an exposure time of approximately 1 millisecond. The observed scattered intensity was then used to generate a histogram of the distribution of scattered intensity measurements (Figure 12).

Intensity values are translated to position values by using the known profile of the evanescent field intensity and by taking the ratio of each intensity measurement to the equilibrium intensity value, using:

= ln ( ¾) (6)

where h is the instantaneous separation between the waveguide surface and the particle center, h₀ is the equilibrium separation between the waveguide surface and particle center (corresponding to measured intensity, / equal to I₀), and <i is a characteristic decay length of the waveguide's evanescent field, which is calculated using the known material parameters of the waveguide and surrounding medium.

Once instantaneous intensity values are translated into relative

displacement values, the potential energy well of the particle-waveguide interaction be calculated using the Boltzmann distribution:

where V(h) is the potential energy at a height above the waveguide surface h, ks is the Boltzman constant, is temperature, 1(h) is the intensity of scattered light at a given height h, and N[I(h)J is the number of measurements in a histogram bin obtained at height h.

Figure 13 depicts an exemplary potential energy well. The potential energy well represents the sum of all attractive and repulsive energies present between the particle and waveguide in solution. Once the potential energy well is obtained, the optical component can be calculated analytically or numerically and subtracted from the total potential energy well to yield the component due to surface interactions. In this calculation, it is assumed that the dominant attractive interaction acting on a particle at long particle-waveguide separation is the optical trapping energy. The relationship of the optical energy with respect to distance is well understood to be an exponential decay of the form

where x is a constant, d is the distance away from the waveguide and λ is a decay rate. As shown in Figure 14, an exponential curve is fitted to the data to the right side of the potential well and is an estimate of the optical potential energy. This optical energy curve is subtracted from the potential well to yield the potential energy specifically due to surface interactions. Differentiating the energy curves yield force-distance relationships for both surface and optical components. Parameters from force-distance relationships can be extracted for quantification and comparison of particle-surface interactions. While the data shows displacement relative to the equilibrium separation, h_a, absolute displacement values can be obtained via calibration of intensity with a bound/stuck particle.

This example calculation can be used to measure a wide variety of particles with different surface treatments or surface conditions. Applying a surface treatment to a microparticle or nanoparticle will almost always change some surface property of the particle which can be measured for quality control purposes or to gauge the success of the treatment. For example, adding PEG, an antibody, albumin, charged chemical groups, adjusting the hydrophobicity, should all result in alterations to the potential well and force-distance relationship between the particle and the surface.

Applying a surface coating to the waveguide itself is one method of increasing the sensitivity of this technique. The coating itself may be intelligently chosen to intensify the difference between two types of particles. For example, if two particles are known to have differences in hydrophobicity or charge, a waveguide coating may be selected to elucidate those differences.

Example 7: Aspect Ratio

Experiments were conducted to examine substances with varied aspect ratios and their motion along a waveguide. It was observed that substances with given aspect ratios exhibit a linear relationship between velocity and intensity. Five different types of gold nanoparticles were examined with length and radius respectively approximately equal to, 162nm & 63nm, lOOnm & 33nm, 106nm & 48nm, 92nm & 21nm, or 200nm & 14nm. The observed velocity and observed intensity of optical scatter for each particle was plotted, and the data for each population of particles was fitted with a linear fit. Data plotted on a linear scale is shown in Figure 15A and data plotted on a log scale is shown in Figure 15B. As can be seen from Figure 15, each population exhibited a distinct slope of velocity/intensity (V/I). The relationship of aspect ratio and the V/I slope is shown in Figure 15C, demonstrating that V/I slope has a relationship to the long/short aspect ratio. It suggests that the larger the aspect ratio, the faster the particle moves for a given intensity of scattered light.

Example 8: Surface properties of PVP-coated silver nanoparticles sample An experiment was devised to demonstrate the ability to measure surface properties of nanoparticles. Polyvinylpyrrolidone Coated Silver Nanoparticles (Ag-PVP) with a nominal diameter of 75 nm were acquired from the US National Institute of Standards and Technology (NIST). The Ag-PVP nanoparticles, were subjected to a ligand exchange with thiol-glutathione. The thiol binds more strongly to the silver nanoparticles than PVP but results in a less stable particle. The surface properties of the particles were measured by potential well analysis at the initial condition (with PVP on the surface) and after 1 and 24 hours of ligand exchange. Results are shown in Figures 16A-16E. Intensity data for all particles is shown in the plot of Figure 16B, while a data summary is shown in Figure 16A. The stability index is a measurement of how stable a particle formulation is. It is the integral under the force curve from the equilibrium height to equilibrium height - particle radius. It represents how much energy needs to be imparted to make a particle stick on a surface. It can be correlated to how much energy is required to make a particle crash out of solution, with higher stability index indicating a more stable formulation. The stability index shows that the initial particles, and the 1 and 24 hour ligand exchanged particles measured 18.6, 8.4 and 7.3 kT respectively. This indicates that the ligand exchanged particles were less stable and were continuing to destabilize over time. These ligand exchanged particles became aggregated and crashed out of solution within a few weeks, verifying the usefulness of the measurement.

Example data for a single particle is shown in Figures 16C and 16D. The observed scattered intensity was used to generate a histogram of the distribution of scattered intensity measurements, shown in Figure 16C. A plot depicting a potential energy well, as well as the calculation of the components of potential energy well due to optical energy and surface interactions is shown in Figure 16D. Figure 16E plots surface force for each particle, averaged over all particles in each population.

Example 9: Single submicron Raman spectra for submicrometer latex and sun block particles using waveguide as both the trapping and excitation source

Commercially available latex nanoparticles and sunblock loaded wax particles were measured using a Raman spectroscopy configuration. A 785 nm laser was coupled directly into a waveguide. This light was used both to trap the particles and served as the excitation light for the Raman spectroscopy. Additional optical filters were used (as is often done for Raman spectroscopy applications) to improve the quality of the excitation light. The light was collected with a microscope objective lens and the Raman- shifted light was passed into a spectrometer. These signals appear in blue in Figures 17A and 17B. For comparison, a standard "far field" Raman excitation configuration was used to measure the Raman spectrum of bulk latex material and bulk wax material. These bulk samples were simply dried mounds of the nanoparticles. These spectra are shown in red in Figures 17A and 17B. Example 10: Particle Identification: Chemical analysis using near- field

Raman spectroscopy

Raman Spectroscopy is a powerful analytical technique that can obtain a chemical signature from a material. The interrogating light (a single wavelength) interacts with the chemical bonds in the material causing an energy shift depending on the type of bond. These shifts show up as peaks in a Raman spectrum. The spectrum is unique to a material (glass, polystyrene, titanium dioxide, etc.) and can be considered a fingerprint (see for example Figure 18). While Raman Spectroscopy can allow researchers to extract molecular fingerprints, Raman signals are typically very weak and difficult to obtain. Traditionally, a laser is focused on a bulk piece of material, powder, or solution making it harder to separate the material of interest from the background and characterize it accurately. Conventional Raman systems can only be used to look at particles if they are stationary (not in solution) and visible (greater than a few microns).

Unlike conventional Raman microscopes, which can only provide information about microparticles or larger, the embodiments of the invention can trap, visualize, and obtain Raman spectra from true nanoparticles. Rather than an external laser focused onto a substrate, it uses near field light leaking out of a waveguide to optically excite and trap the particles in their native environments. This is the key breakthrough that enables the performance increase. The intense light in the form of an evanescent field leads to heightened signal and less background than traditional illumination systems. Additionally, because the particles are temporarily trapped during the analysis, an arbitrarily long exposure time can be acquired. Benefits of such systems according to embodiments of the invention disclosed herein include (1) near field Raman is useful for key areas of R&D including quality control, environmental analysis, drug delivery, and nanotoxicity; (2) identification of sub-micron particles in their native solution

environment; (3) the microfluidic flow cell enables straightforward sample handling using small volumes; and (4) permanent optical connections eliminate the need for complicated laser alignment

Example 11 : Particle Surface Analysis: Near field light scattering to analyze nanoparticle coatings

As already discussed in certain detail above, the surface properties of nanoparticles are an important determining factor in the overall behavior of nanoparticles and influences colloidal and chemical stability, biodistribution, biological function and toxicity. Despite the widespread and rapidly growing use of nanoparticles in large sectors of our economy, effectively measuring particle interfacial forces in liquid is notoriously challenging and largely ineffective with existing commercial equipment.

Embodiments of the invention directly probes the mechanical properties of nanoparticle coatings by monitoring how they interact with a surface - an approach made possible by the disclosed waveguide -based measurement technology. This method differs from adsorption chromatography, since in this case, rather than chemical separations through an adsorption column, optical forces on a waveguide surface are used to measure individual nanoparticles. The use of waveguide-based trapping using near field optical forces allows for imaging submicron particles in solution and increases the interaction time between surfaces so that thousands of measurements can be taken of each nanoparticle.

In one aspect, the waveguide-based optical trapping and visualization system (see Figures 19A-19C) can be understood as follows: (1) Particles "latch" on to a waveguide by the optical gradient forces. (2) They progress along the waveguide (like a moving train) due to optical scattering forces. (3) Their presence within the evanescent field scatters light making them appear as bright spots against a black background. (4) High speed imaging records the brightness and position of each nanoparticle. (5) This process is repeated many times as the particles interact with the waveguide. A particle trapped on the waveguide can be said to reside in a potential well. The potential well is created by both surface repulsion and the attractive force of the optical trap. The particle "likes to sit" at the bottom of the potential well (at a particular height above the waveguide surface) but moves around with Brownian motion and will escape the well if the trapping force is insufficient (Figure 19B). The shape of the potential well reveals the nature of both the optical and surface forces. It can be calculated with a knowledge of the particle's position (height) over time. This is accomplished by using the known intensity profile of the waveguide's exponentially decaying evanescent field. As the particle fluctuates around the equilibrium point, snapshots are taken with the microscope camera. Brighter instances indicate closer proximity to the waveguide (more deeply bathed in the evanescent field) and dimmer instances indicate greater distance. Over the course of 2-4 seconds, several thousands of measurements are taken and a smooth potential well is calculated. Surface measurements are generated by processing many images of the same particle as it moves about within the potential well. The well understood optical forces are subtracted from the potential well revealing the particle waveguide surface force profile independent of particle size or material properties. This technique was used to analyze a batch of 10 particles and the resulting force plots are shown in Figure 19C.

Intensity measurements of individual particles captured by the waveguide can be used to study particle-surface interactions, independent of a particle's size or optical properties. This approach builds upon evanescent wave dynamic light scattering, an established method used in total internal reflection microscopy for studying particle- surface interactions. Total internal reflection microscopy is an optical technique for monitoring the instantaneous separation distance between a particle and a light guiding surface. To determine the instantaneous separation distance, one measures the intensity of light scattered by the particle when it is illuminate by an evanescent wave. In this approach, an optically trapped particle fluctuates, due to thermal energy, about an equilibrium height determined by the balance of its attractive and repulsive interactions with the waveguide surface. The intensity of a particle is recorded using a high-speed camera (>500 fps) and plotted as a histogram (Figure 20A). Using the known intensity profile of the waveguide's exponentially decaying evanescent field, fluctuations in particle-waveguide separation can be calculated from fluctuations in measured intensity using equation 6, where h is the instantaneous height of the particle above the waveguide surface, ho is the equilibrium/most frequent height observed, / is the measured intensity of the particle, Io is the most frequent intensity value (corresponding to the intensity when h=ho), and <i is a decay length of the waveguide's evanescent field, which is calculated using known material parameters of the waveguide and surrounding medium. Using Boltzmann statistics, whereby the potential energy of a state is proportional to its observed occupancy, we assume the most frequent height corresponds to a potential energy minimum, and the potential energy well governing particle-waveguide separation (shown in Figure 20B) is calculated from the histogram of intensity measurements using: equation 7, where V(h) is the potential energy at a height above the waveguide surface h, kB is the Boltzman constant, is temperature, and N[I(h)] is the frequency of the intensity histogram bin corresponding to height h. The optical energy component of a particle in the evanescent field is known to be of the form Eoptical=_^4*exp(- /z) where A is a constant, h is the distance away from the waveguide and λ is a decay rate. The optical component is calculated and subtracted from the potential energy well, leaving the surface energy component (Figure 20C). The shape of the surface energy-distance relationship is dependent on the magnitude and type of surface energies present between the particle and the waveguide. Furthermore, following subtraction of the optical component, the resulting surface energy is independent of the optical effects of size and refractive index. By functionalizing waveguide surfaces this method can be applied to study specific surface interactions between particles and waveguides with well- characterized surfaces.

While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed:

1. A method of measuring at least one property of a substance comprising: positioning a substance in the vicinity of one or more optical waveguides, such that the substance is captured by a waveguide and travels along the waveguide;

measuring one or more metrics of the interaction between the substance and the waveguide; and

determining at least one property of the substance based upon the measured metric.

2. The method of claim 1, wherein at least one of the one or more optical waveguides is used as both a substance excitation and a substance attraction source.

3. The method of claim 1, wherein each of a plurality of the one or more optical waveguides are used as both a substance excitation and a substance attraction source.

4. The method of claim 1, further comprising detecting one or more properties of the light scattered by the substance.

5. The method of claim 4, wherein the one or more properties of the scattered light is at least one selected from the group consisting of the amount, the intensity, the wavelength, the shape, the pattern, and spectral characteristics.

6. The method of claim 1, wherein the substance is a substance selected from the group consisting of a foam, emulsion, sol/colloid, nanoparticle, microparticle, nanotube, nanocrystal, exosome, liposome, polymer shell nanoparticle, core shell, contrast agent, dendrimer, micelle, wax particle, subvisible particle, quantum dot, lipoparticle, vesicle, oil droplet, bioparticle, biomolecule, nucleic acid molecule, protein, enzyme, antibody, virus, bacterium, cell, small molecule, protein complex, and carbohydrate.

7. The method of claim 1, wherein the at least one property of the substance is a property selected from the group consisting of size, shape, surface property, the presence of surface coating, the extent of surface coating, presence of a surface modification, extent of surface modification, the aggregation of the substance, and the interaction affinity of the substance toward another substance.

8. The method of claim 1, wherein the one or more metrics of the interaction between the substance and the waveguide comprises one or more metrics of the motion of the substance along the waveguide.

9. The method of claim 8, wherein the one or more metrics of motion is at least one selected from the group consisting of residence time, velocity, variation in velocity, position, and deflection distance.

10. The method of claim 1, wherein the one or more metrics of the interaction between the substance and the waveguide comprises one or more metrics of the attraction between the substance and the waveguide.

11. The method of claim 10, wherein the one or more metrics of the attraction between the substance and the waveguide comprises the distance between the substance and waveguide surface.

12. The method of claim 10, wherein the step of determining is further based on analyzing a potential energy well.

13. The method of claim 1, wherein measuring the metric is qualitative or quantitative.

14. The method of claim 1, further comprising altering the local environment of the substance.

15. The method of claim 1, wherein at least one of the one or more optical waveguides is curved.

16. The method of claim 1, wherein at least one of the one or more optical waveguides comprises a circular, ovular, or spiral shape.

17. The method of claim 1, wherein positioning of the substance comprises administering a fluid comprising the substance to a fluidic channel, wherein the flow in the fluidic channel positions the substance in the vicinity of the one or more optical waveguides.

18. The method of claim 17, wherein a region of at least one of the one or more waveguides transverses the flow in the fluidic channel at an angle of about 0°-90°.

19. The method of claim 17, wherein a region of at least one of the one or more waveguides transverses the flow in the fluidic channel at an angle of about 10°-60°.

20. The method of claim 17, wherein at least one of the one or more waveguides comprises an engineered surface coating.

21. A method of separating a plurality of substances within a population into one or more sub-populations comprising;

positioning each substance in the vicinity of one or more optical waveguides, such that the substance is captured by a waveguide and travels along the waveguide, wherein a property of the substance determines the distance which each substance travels along the waveguide before the forces acting upon the substance force the release of the substance from the waveguide, thereby separating the plurality of substances into sub-populations based upon the location at which each substance is released.

22. The method of claim 21, wherein each substance is released into a flow-stream, wherein all substances of the same sub-population are released into the same flow stream.

23. The method of claim 21, wherein each substance is a substance selected from the group consisting of a nanoparticle, microparticle, nanotube, nanocrystal, exosome, liposome, polymer shell nanoparticle, core shell, contrast agent, dendrimer, micelle, wax particle, subvisible particle, quantum dot, lipoparticle, vesicle, oil droplet, bioparticle, biomolecule, nucleic acid molecule, protein, enzyme, antibody, virus, bacterium, cell, small molecule, protein complex, and carbohydrate.

24. The method of claim 21, wherein the at least one property of each substance is a property selected from the group consisting of size, shape, surface property, the presence of surface coating, the extent of surface coating, presence of a surface modification, extent of surface modification, the aggregation of the substance, and the interaction affinity of the substance toward another substance.

25. The method of claim 21, wherein at least one of the one or more optical waveguides is curved.

26. The method of claim 21, wherein at least one of the one or more optical waveguides comprises a circular, ovular, or spiral shape.

27. The method of claim 21, wherein positioning of each substance comprises administering a fluid comprising the plurality of substances to a fluidic channel, wherein the flow in the fluidic channel positions each substance in the vicinity of the one or more optical waveguides.

28. The method of claim 27, wherein a region of at least one of the one or more waveguides transverses the flow in the fluidic channel at an angle of about 0°-90°.

29. The method of claim 27, wherein a region of at least one of the one or more waveguides transverses the flow in the fluidic channel at an angle of about 10°-60°.

30. The method of claim 21, wherein at least one of the one or more waveguides comprises an engineered surface coating.

31. The method of claim 21 , wherein at least one of the one or more optical waveguides is used as both a substance excitation and a substance attraction source.

32. The method of claim 21, wherein each of a plurality of the one or more optical waveguides are used as both a substance excitation and a substance attraction source.

33. A device for measuring the property of a substance comprising one or more optical waveguides;

one or more fluidic channels; wherein at least one of the one or more fluidic channels is in communication with one or more optical waveguides;

at least one light source operably connected to the one or more

waveguides to provide optical power to the one or more waveguides.

34. The device of claim 33, wherein the one or more fluidic channels comprises at least one sample channel configured to deliver a sample comprising a substance to the one or more optical waveguides.

35. The device of claim 33, wherein at least one of the one or more optical waveguides is curved.

36. The device of claim 33, wherein at least one of the one or more optical waveguides comprises a circular, ovular, or spiral shape.

37. The device of claim 33, wherein a region of at least one of the one or more waveguides transverses the flow in at least one of the one or more fluidic channels at an angle of about 0°-90°.

38. The device of claim 33, wherein a region of at least one of the one or more waveguides transverses the flow in at least one of the one or more fluidic channels at an angle of about 10°-60°.

39. The device of claim 33, wherein at least one of the one or more waveguides comprises an engineered surface coating.

40. The device of claim 33, wherein at least one of the one or more optical waveguides is used as both a substance excitation and a substance attraction source.

41. The device of claim 33, wherein each of a plurality of the one or more optical waveguides are used as both a substance excitation and a substance attraction source.

42. A device for separating a plurality of substances in a population into one or more sub-populations comprising

one or more optical waveguides;

one or more fluidic channels; wherein at least one of the one or more fluidic channels is in communication with one or more optical waveguides;

at least one light source operably connected to the one or more

waveguides to provide optical power to the one or more waveguides.

43. The device of claim 42, wherein the one or more fluidic channels comprises at least one sample channel configured to deliver a sample comprising a substance of the population to the one or more optical waveguides.

44. The device of claim 42, wherein at least one of the one or more optical waveguides is curved.

45. The device of claim 42, wherein at least one of the one or more optical waveguides comprises a circular, ovular, or spiral shape.

46. The device of claim 42, wherein a region of at least one of the one or more waveguides transverses the flow in at least one of the one or more fluidic channels at an angle of about 0°-90°.

47. The device of claim 42, wherein a region of at least one of the one or more waveguides transverses the flow in at least one of the one or more fluidic channels at an angle of about 10°-60°.

48. The device of claim 42, wherein at least one of the one or optical waveguides comprises an engineered surface coating.

49. The device of claim 42, wherein the one or more fluidic channels comprises one more collecting channels, wherein each collecting channel is positioned downstream of the one or more optical waveguides to collect a flow stream comprising a sub-population of substances separated from the population.

50. The device of claim 42, wherein at least one of the one or more optical waveguides is used as both a substance excitation and a substance attraction source.

51. The device of claim 42, wherein each of a plurality of the one or more optical waveguides are used as both a substance excitation and a substance attraction source.

52. A system for measuring a property of a substance comprising:

a device comprising:

one or more optical waveguides;

one or more fluidic channels; wherein at least one of the one or more fluidic channels is in communication with one or more optical waveguides;

at least one light source operably connected to the one or more waveguides to provide optical power to the one or more waveguides; and

a fluidic delivery system in communication with at least one fluidic channel of the device.

53. The system of claim 52, further comprising a detector for observing the motion of the substance as it travels along the one or more waveguides.

54. The system of claim 52, wherein at least one of the one or more optical waveguides is used as both a substance excitation and a substance attraction source.

55. The system of claim 52, wherein each of a plurality of the one or more optical waveguides are used as both a substance excitation and a substance attraction source.