Method of determining lipoprotein concentration in solution using light scattering

Abstract

To provide a method of overcoming disadvantages (being time-consuming or expensive, providing incomplete information on lipoprotein particles, and / or having limited application to clinical settings) of methods for detection of lipoproteins.SOLUTION: The invention relates to the use of single particle light scattering, preferably interferometric scattering microscopy (also referred to herein as iSCAT), to measure the concentration of particles in a solution. The invention furthermore relates to the use of light scattering to detect lipoprotein particles in a sample, and to related diagnostic and treatment methods.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] The present invention relates to the use of single particle light scattering, preferably interference scattering microscopy (also referred to herein as iSCAT), to measure the concentration of particles in solution. The particles can be in simple or complex solutions, such as biological or environmental samples. The present invention can be used to determine the absolute concentration of particles in solution, and this ability provides a means for studying the stoichiometric relationships between different particles in solution. As the inventors have previously demonstrated, iSCAT also provides a means for robust and accurate detection, mass quantification, imaging, and characterization of particles as small as single molecules; therefore, the ability to use the same technique for concentration measurements would be highly beneficial.

[0002] The present invention further relates to the use of light scattering, preferably interference scattering microscopy (also referred to herein as iSCAT), to detect lipoprotein particles in a sample, and to related diagnostic and treatment methods. The ability of the methods of the present invention to measure the concentration of various lipoproteins in a solution is of particular interest. [Background technology]

[0003] iSCAT has been realized as a powerful approach for both single particle tracking with unique spatiotemporal resolution and label-free sensitivity down to the single molecule level. Interference scattering microscopy provides information about the relative distribution of particles of different masses in a solution without the need for added labels. Adding absolute or relative concentrations of particles provides valuable additional information about the sample being analyzed, especially when the solution is a biological or environmental sample.

[0004] Prior to the applicant's earlier work, widespread application of iSCAT was limited by the need for custom microscopes, unconventional cameras, and complex sample illumination, which limited iSCAT's ability to robustly and accurately detect, image, and characterize particles as small as single molecules. However, improvements to the instrumentation have progressed, resulting in the technology evolving into a powerful means for viewing single objects. Exemplary designs of iSCAT instruments are described in Cole et al. ACS Photonics, 2017, 4 (2), pp. 211-216 and Arroyo et al. Nat Protocols 2016, 617-633, both of which are incorporated herein by reference. Further details regarding the instrumentation are provided in the applicant's prior application, WO 2018 / 011591, which is incorporated herein by reference.

[0005] iSCAT has previously been described for the detection of purified single proteins (Cole et al. (ACS Photonics, 2017, 4(2), pp 211-216)), but not for the detection of particles in complex solutions. The detection and quantification of lipoproteins has not previously been explored because it was not considered feasible.

[0006] Diagnostic assays for cardiovascular disease may include the detection of lipoproteins (also referred to as lipoprotein particles). Various methods for detecting lipoproteins are reviewed in Circulation. 2009 May 5; 119(17): 2396-2404; Curr Opin Lipidol. 2017 June; 28(3): 261-266. These methods have the disadvantages of being time-consuming, expensive, providing incomplete information about lipoprotein particles, and / or having limited application in clinical settings. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2018 / 011591 [Non-patent literature]

[0008] [Non-Patent Document 1] Cole et al ACS Photonics, 2017, 4 (2), pp 211-216 [Non-patent document 2] Arroyo et al. Nat Protocols 2016, 617-633 [Non-patent document 3] Cole et al (ACS Photonics, 2017, 4(2), pp 211-216) [Non-patent document 4] Circulation. 2009 May 5; 119(17): 2396-2404 [Non-patent document 5] Curr Opin Lipidol. 2017 Jun;28(3):261-266 Summary of the Invention

[0009] The present inventors have surprisingly confirmed that it is possible to determine the concentration of particles in a solution using single particle light scattering, preferably single particle coherent scattering microscopy. In addition, the present inventors have surprisingly demonstrated that it is possible to determine the concentration of particles directly from biological samples (e.g., blood or plasma) by single particle light scattering (e.g., by using iSCAT) without separating the particles from other components, despite the background signal provided by these other components. This has proven to be a particularly useful technique, for example, for lipoproteins in biological samples.

[0010] The present invention provides a method for determining the concentration of particles in a solution, comprising contacting the solution with a surface and detecting binding of the particles to the surface using light scattering. The light scattering method for detecting particle binding is preferably iSCAT. The method can be preferably performed using a suitable microscope. The concentration is preferably an absolute concentration. The binding of particles to the surface can be detected or visualized using single particle light scattering.

[0011] The present invention provides a method for determining the concentration of particles in a solution, comprising contacting said solution with a measurement solution and a surface, and detecting binding of particles to said surface using light scattering.

[0012] This feature of the invention is particularly useful in situations where the solution is considered concentrated. A volume of solution containing particles can be included with a volume of measurement solution in the sample holder prior to detection of particle binding to the surface. The advantage of this aspect is that it ensures that the measured concentration represents the state of the particles in the original, more concentrated solution.

[0013] The present invention provides a method for determining the concentration of particles in a solution, comprising contacting the solution with a surface in the presence of a calibrant and detecting binding of the particles to the surface using light scattering.

[0014] Thus, the present invention allows for the measurement of the concentration of particles in a solution by detecting the rate of binding of said particles to a surface. Furthermore, the present invention allows for the measurement of absolute concentrations of particles in solution by analysis of the change or decay over time in the rate of particle binding to a surface as detected by light scattering.

[0015] Absolute concentrations in solution can be determined by measuring binding events between particles in a sample and a surface and can be calibrated against binding events observed with samples of known concentrations of particles. The surface can be part of a sample holder. In sample holders with a high surface area to volume ratio, particle binding to the surface decreases over time as a function of the remaining number of particles in solution and the remaining number of available sites for binding. The initial binding rates for a range of concentrations can be extrapolated from the binding data and used to generate a standard curve, allowing conversion of the measured initial binding rates in the samples to the respective absolute concentrations of particles in solution.

[0016] Binding of the particles to the surface can be detected or visualized using light scattering, preferably iSCAT. Alternatively, the concentration in solution can be determined by using a passivated or activated surface, so that the binding rate of the particles can be controlled by controlling the binding activity between the surface and the particles. In this setting, surface activation or passivation can increase the binding affinity, thus increasing the measurement range to lower concentrations, or decrease the binding affinity, thus allowing measurements at higher concentrations.

[0017] The surface preferably forms part of a sample holder for said solution. The sample holder may be an element of a light scattering microscope. The sample holder may be a high surface area to volume chamber.

[0018] To calculate the concentration, it may be necessary to include a correction for the diffusion rate, which can be easily calculated based on known properties of the particles such as mass and shape.

[0019] Accordingly, the present invention provides a method for measuring the concentration of particles in a solution, the method comprising the steps of: i) contacting the solution with the surface; ii) detecting particle binding to the surface visualized by light scattering; iii) repeating the detection process and calculating the change in particle binding rate to the surface and initial binding rate over time; iv) providing a calibration curve of initial binding rate versus concentration based on data from solutions of known particle concentrations; v) Using the calibration curve in step (iv), convert the initial binding rate recorded for the sample in step (iii) into a concentration of particles in solution.

[0020] Preferably, the concentrations are absolute concentrations. In some variations, the solution can be pretreated, for example with a measurement solution or calibrant, before or simultaneously with contacting the surface.

[0021] The particles in the solution can be brought into contact with the measurement solution and the surface. The advantage of this aspect is that it ensures that the measured concentration represents the state of the particles in the original, more concentrated solution.

[0022] The measurement solution can be a buffer solution. Generally, the volume of the measurement solution is known to allow calculation of the dilution effect. Various techniques may be required to prepare a solution for concentration determination. For example, a volume of solution can be placed in a sample holder, preferably with a known geometry. This allows the introduction of a volume of measurement solution (or the introduction of a particle solution into the measurement solution), and using a dilution action, this allows the detected binding rate of particles to the surface to be converted into the particle concentration.

[0023] The detection of particle binding to the surface can be performed at one or more different time intervals following the introduction of the measurement solution. If the detection step is repeated at different time intervals after the introduction of the measurement solution, this will allow correction for dilution effects such as dissociation. If measurements are performed at different time points after dilution, changes in species distribution, for example, if particles dissociate or cluster at lower concentrations, can be visualized. This approach therefore allows a more detailed view of the particles and allows for the validation of concentration measurements.

[0024] Alternatively or additionally, the volume of one or both of the solutions (particles or measurement) can be changed and / or the geometry of the sample holder can be changed. Both allow for correction of dilution effects. If the detection process is repeated in one or more different solution volumes and / or in one or more sample holder geometries, this can also correct for dilution effects such as dissociation.

[0025] Accordingly, the present invention provides a method for determining the concentration of particles in a solution, the method comprising the steps of: i) contacting the solution with a surface and a volume of measurement solution in a sample holder having a certain geometric shape; ii) detecting particle binding to the surface visualized by light scattering; iii) repeating the detection process and calculating the change in particle binding rate to the surface and initial binding rate over time; iv) providing a calibration curve of initial binding rate versus concentration based on data from solutions of known particle concentrations; v) Using the calibration curve in step (iv), converting the initial binding rate recorded for the sample in step (iii) into an absolute concentration of particles in solution.

[0026] The method may further comprise repeating steps (i)-(ii) using one or more sample holders of different geometries or using different volumes of measurement solution to verify the determined concentrations.

[0027] Alternatively, particles in solution can be contacted with the surface in the presence of a calibrant. The calibrant in solution has a predetermined concentration and mass. Once introduced into the particle solution, the binding rates of the calibrant and particles to the surface are detected separately but simultaneously. Repeated measurements of both particle and calibrant binding to the surface can be performed, which allows for the determination of the initial binding rate for the particles and the initial binding rate for the calibrant. The initial binding rate of a calibrant of known concentration allows for the conversion of the initial binding rate of particles to the concentration of the particles. In one embodiment, the calibrant is selected to resemble the particles in terms of their characteristics in binding to the surface. For example, it is desirable to select a calibrant with similar surface properties (e.g., charge, hydrophobicity, hydrophilicity). Such selection allows for more accurate measurements across a variety of particles. In practice, the calibrant and particles should have the same affinity for the surface, and therefore, determining the binding rate of a calibrant of known concentration allows for the conversion of the binding rate of particles to concentration.

[0028] Accordingly, the present invention provides a method for measuring the concentration of particles in a solution, the method comprising the steps of: i) contacting the solution with the surface in the presence of the calibrant; ii) detecting particle binding to the surface visualized by light scattering; iii) simultaneously detecting calibrant binding to the surface visualized by light scattering; iv) repeating detection steps (ii) and (iii) and calculating the change over time in the binding rate and initial binding rate of the particles and calibrant to the surface; v) Converting the initial binding rate of particles calculated in step (iv) into a concentration of particles in solution using the initial binding rate calculated in step (iv) for the calibrators.

[0029] Preferably, the calibrant is of known concentration and has similar characteristics to the particles, particularly with respect to binding to the surface. Furthermore, the inventors have surprisingly found that lipoprotein particles, which represent heterogeneous mixtures of different biomolecules containing various non-protein components, can be detected by light scattering, particularly using interference scattering microscopy. Single lipoprotein particles can be optically visualized. This allows the number and size of lipoprotein particles (and of different classes of lipoprotein particles) to be determined directly in a sample. The relative proportions of different lipoprotein particles in a sample can also be determined in a single measurement. Thus, the detection method of the present invention provides a rapid and simple means of detecting lipoprotein particles and provides extensive information regarding the nature and distribution of lipoprotein particles in a sample. The optical detection performed is also relatively inexpensive, advantageously suitable for clinical settings, and avoids the complex processing protocols associated with previous methods for lipoprotein detection.

[0030] In addition, the inventors have surprisingly demonstrated that it is possible to detect lipoprotein particles by light scattering (e.g., by using iSCAT) directly from biological samples (e.g., blood or plasma) without purifying the particles away from other components, despite the background signal provided by these other components.

[0031] The methods of detection of the present invention are advantageously used in clinical settings for the detection of lipoprotein particles from patient samples to aid in the diagnosis and treatment of diseases and conditions associated with lipoprotein particle size and / or number.

[0032] Thus, the present invention provides a method for the detection of lipoprotein particles in a sample, which method comprises detecting said particles by light scattering, preferably by interference scattering microscopy. The present invention further provides a method for diagnosing a disease or condition in an individual which is associated with the size and / or number of lipoprotein particles, or for determining the risk that an individual will develop said disease or condition, which method comprises detecting the size and / or number of lipoprotein particles in a sample from said individual by interference scattering microscopy.

[0033] The present invention further provides a method for selecting an individual to whom a substance or composition should be administered or a therapeutic regimen should be prescribed, wherein said substance or composition or therapeutic regimen is suitable for treating or preventing a disease or condition associated with lipoprotein particle size and / or number, the method comprising detecting the size and / or number of lipoprotein particles in a sample from said individual by a method for detection of the present invention, and selecting said patient for said administration or said therapeutic regimen if the detected lipoprotein particle size and / or number indicates the presence of, or risk of, said disease or condition.

[0034] The present invention also provides a method of treating or preventing a disease or condition associated with lipoprotein particle size and / or number in an individual, which method comprises diagnosing or determining the risk of said disease or condition in said individual, or selecting said individual by a method of the invention, and administering to said individual a substance or composition, or administering to said individual a therapeutic regimen, that is effective to treat or prevent said disease or condition in that individual.

[0035] The present invention further provides a substance or composition for use in a method of treating or preventing a disease or condition associated with lipoprotein particle size and / or number in an individual, wherein said individual has been diagnosed or determined to be at risk by the methods of the invention. be selected or

[0036] The invention also provides the use of a substance or composition in the manufacture of a medicament for the prophylactic treatment of a disease or condition associated with lipoprotein particle size and / or number in an individual, wherein said individual is diagnosed or determined to be at risk or selected by the methods of the invention. [Brief explanation of the drawings]

[0037] [Figure 1] Figure 1 provides a diagram depicting the different types of lipoprotein particles present in human blood and their respective diameters (nm) and densities (g / ml). The table also provides information on the relative proportions of protein, cholesterol, phospholipids, and triglycerides in each type of particle. [Figure 2] FIG. 2 shows an exemplary scheme for detection of lipoprotein particles by iSCAT from a blood sample. [Figure 3] Figure 3 is a schematic diagram of an iSCAT microscope incorporating a spatial filter. [Figure 4] Figure 4 shows captured images of HDL and LDL lipoprotein particles from a purified lipoprotein sample. The scale bar indicates the iSCAT signal, defined as (Is-Ip) / Is, where Is is the reflection intensity from the glass surface in the absence of particles and Ip is the same scale in the presence of particles. Images of HDL and LDL lipoprotein particles are shown along with single particle histograms plotting the iSCAT signal against the number of each particle detected from all HDL and LDL images. [Figure 5] FIG. 5 provides images and single particle histograms corresponding to those shown in FIG. 4 for HDL and LDL lipoproteins detected by iSCAT from serum samples. [Figure 6-1]Figure 6 provides single particle histograms for HDL, LDL, and VLDL lipoprotein particles detected by iSCAT from whole blood samples. Figures 6A and 6B provide histograms from non-fasting and fasting samples, respectively. [Figure 6-2] Figure 6 provides single particle histograms for HDL, LDL, and VLDL lipoprotein particles detected by iSCAT from whole blood samples. Figures 6A and 6B provide histograms from non-fasting and fasting samples, respectively. [Figure 7] Figure 7 shows the calibration of iSCAT measurements using lipoproteins of known mass / concentration. The left panel plots particle binding events against incubation time (seconds). The right panel plots the initial binding rate determined from the binding data against concentration, providing a calibration curve that allows conversion of the measured initial binding rate to the concentration of particles in the sample. [Figure 8a] Figure 8a shows an example frame of a movie of 300 nM actin landing in a gasket using the dilution method described in Example 4, recorded at a frame rate of 1 kHz, effective frame rate: 25 Hz. [Figure 8b] Figure 8b for comparison with Figure 8a shows exemplary frames from a movie of landing of 300 nM actin in a flow chamber under similar conditions, recorded at a frame rate of 1 kHz, effective frame rate: 25 Hz. [Figure 9] FIG. 9 shows the resulting mass histogram compiled from four repeats of the experiment described in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention allows for the measurement of the concentration of particles in a solution by using light scattering to detect the binding of particles in a sample to a surface. The use of a light scattering microscope is preferred. The present inventors have specifically identified the ability to directly detect single lipoprotein particles by light scattering, and in particular by the use of coherent scattering microscopy. The method can be used to detect all forms of lipoprotein particles, including different forms of lipoproteins.

[0039] particle Particles that can be detected according to the methods of the present invention can be any particle in solution, from single molecules to biological macromolecules through oligomeric assemblies. Examples of suitable particles are single molecules, proteins, polypeptides, peptides, amino acids, monosaccharides, carbohydrates, oligosaccharides, polysaccharides, glycopeptides, glycoproteins, lipids, fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, phospholipids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides, glycolipids, lipoproteins, nucleic acids, nucleotides, polynucleic acids, molecular clusters, assemblies, aggregates, protein / protein interactions, protein / small molecule interactions, protein-nucleic acid interactions, and / or oligomeric assemblies.

[0040] For determining the concentration of particles in a solution, it is not necessary to label the particles prior to detection: light scattering microscopy can determine the mass of a particle, which can be identified simply by mass.

[0041] Lipoprotein particles Lipoprotein particles that can be detected according to the present invention can be any lipoprotein particle present in a sample. Lipoprotein particles can be of any size or present in any number. Lipoprotein particles are typically selected from lipoprotein particles present in human or animal blood. Lipoprotein particles typically contain triglycerides, cholesterol, phospholipids, and one or more proteins, such as one or more apolipoproteins. Lipoprotein particles can have varying ratios and / or densities of lipids and proteins. Apolipoproteins can be peripheral apolipoproteins or integral apolipoproteins. Apolipoproteins can be selected from any class or subclass of apolipoproteins. Apolipoproteins can have any genetic polymorphism. Apolipoprotein(s) can be selected from the A, B, C, D, E, and / or H classes. Class A apolipoproteins can be selected from apo AI, apo A-II, apo A-IV, and apo AV; class B apolipoproteins can be selected from apo B48 and apo B100; and class C apolipoproteins can be selected from apo CI, apo C-II, apo C-III, and apo C-IV. Class B apolipoproteins are present in LDL lipoprotein particles, while the other classes of apolipoproteins are typically associated with HDL lipoprotein particles.

[0042] Lipoprotein particles typically have a diameter of at least 1 nm and can have diameters up to 1000 nm or more. Specific types of lipoprotein particles that can be detected according to the present invention include chylomicrons (also referred to as ultra-low density lipoproteins, or ULDLs), very low-density lipoproteins (VLDLs), intermediate low-density lipoproteins (IDLs), low-density lipoproteins (LDLs), and high-density lipoproteins (HDLs). Exemplary particle sizes, densities, and lipid:protein ratios for the above lipoprotein particles are provided in FIG. 1. Lipoprotein particles detected according to the present invention can have any particle size, density, or lipid:protein ratio shown in FIG. 1. Thus, for example, HDL lipoprotein particles can have a particle size in the range of about 5 to about 15 nm. LDL lipoprotein particles can have a particle size in the range of about 18 to about 28 nm. IDL lipoprotein particles can have a particle size in the range of about 25 to about 30 nm. They can have a particle size of about 50 nm. VLDL lipoprotein particles can have a particle size of about 30 to about 80 nm. Chylomicrons can have a particle size of about 100 to about 1000 nm. HDL lipoprotein particles can have a molecular weight in the range of about 200 to about 500 kDa. LDL lipoproteins can have a molecular weight of about 3 MDA.

[0043] Preferably, the methods of the present invention detect HDL and / or LDL lipoprotein particles. The methods of the present invention can include detecting VLDL lipoprotein particles. The methods of the present invention can include detecting HDL and LDL; HDL and VLDL; HDL and IDL; HDL and UDL; LDL and VLDL; LDL and IDL; or LDL and UDL lipoprotein particles. The methods of the present invention can include detecting HDL, LDL, and VLDL; HDL, LDL, and IDL; or HDL, LDL, and UDL lipoprotein particles. The methods of the present invention can include detecting HDL, LDL, IDL, VLDL, and ULDL lipoprotein particles. Detection of IDL and VLDL particles, which have particle sizes overlapping with other types of lipoprotein particles, can include an additional step (e.g., using fluorescently labeled antibodies) to detect specific proteins present in such particles.

[0044] Particles in solution The solution can be any solution of particles in a liquid solvent. The solution can be simple, such as particles dispersed in water (aqueous solution of particles), or it can be a complex solution, such as particles and one or more other solutes. The solution can be a sample. The sample can be taken from a commercially prepared solution to be tested for concentration. Body fluids are examples of complex solutions in which numerous solutes are present, including electrolytes, sugars, and urea. The sample can be taken from any source, including biological samples or environmental samples. If the sample is a biological sample, it can be taken or obtained from a human or animal body or individual, such as blood, serum, plasma, urine, saliva, lymph, sweat, amniotic fluid, cerebrospinal fluid, breast milk, tears, secretions, synovial fluid, semen, bile, or mucus. If the sample is an environmental sample, it can be taken from any source, such as water (e.g., wells, streams, rivers, lakes, rainwater, seawater, etc.), food and drink (e.g., beverages), agricultural samples, or liquid samples from factories and manufacturing processes.

[0045] It is not necessary to prepare the solution or sample before contacting it with the surface, but if the solution is considered concentrated, it can be contacted with the measurement solution as discussed herein.

[0046] surface The surface used to bind particles in order to determine their concentration is preferably a detector surface and may form part of a sample holder for a light scattering microscope.

[0047] The surface is preferably glass, sapphire, or made from a transparent polymer. A sample is contacted with the surface to determine the concentration. The sample can be placed in a sample holder that includes the surface. As described above, the microscope includes a sample holder for holding the sample at the sample position. The sample can be a liquid sample containing particles to be imaged, which is described in more detail below. The sample holder can take any form suitable for holding a sample. Typically, the sample holder holds the sample on a surface that forms an interface between the sample holder and the sample. For example, the sample holder can be a cover glass and / or made from glass. The sample can be provided on the sample holder in a simple manner, for example, using a micropipette or an automated dispensing system.

[0048] The sample holder can take any suitable form. In some forms, the sample holder will allow a high surface area to volume ratio such that access to the surface does not limit particle binding in solution. In other forms, the ratio between surface area and volume can be reduced such that there is a low surface area to volume ratio that may limit particle access to the surface. It will be apparent to those skilled in the art that the selection of the surface area to volume of the sample holder will change the way concentration can be measured. If the sample holder has a low surface area to volume ratio, for example, the particle binding rate will not decrease over time. In this situation, the binding rate at any instant detected using light scattering can be used to calculate the concentration.

[0049] The surface used in the present invention can be any suitable surface. For example, the surface can be a passivated surface. Passivation is the process of treating or coating a surface to enhance or reduce chemical reactivity, and thus increase or decrease the number of binding events.

[0050] Alternatively, the surface can be an activated, coated, derivatized, or treated surface. The surface can be derivatized by immobilizing an entity such as silane on the surface. In Example 4, the surface is coated with APTES, which allows for silanization, the functionalization of the surface with alkoxysilane molecules. This is advantageous for some particles because it results in a positively charged surface. The coating process with APTES results in the formation of a covalent bond between the surface and the silane. The protonated amine groups align themselves in free space, creating positive docking sites for any negatively charged groups on the particle (e.g., sialic acids, carboxyl, and sulfate ester groups).

[0051] The surface can be coated with a specific binding entity or partner for the particle, allowing for the selection of the particle from a concentrated or complex mixture.

[0052] Calculating concentrations in this scenario requires that the binding constants for the particle and the specific binding entity be known. If the surface is modified, it is important that these modifications do not cause any change in the ability of the particles to be bound to be detected by light scattering.

[0053] Contacting the sample with the surface The solution is contacted with a surface to determine the concentration of particles in said solution, the particles bind to the surface, and the binding to the surface is visualized or detected using light scattering, preferably iSCAT, preferably using a microscope.

[0054] Binding of particles to a surface can be non-specific, or binding can be specific, depending on the nature of the surface used, as discussed above. If the surface is glass (i.e., a glass coverslip), binding will be non-specific. If the surface is treated, binding can be specific. The rate of binding to a surface can be calculated by repeating the detection step one or more times, or can be obtained from a single measurement.

[0055] The binding and / or number of particles bound to the surface can be detected using light scattering. This can then be repeated one or more times to determine the rate of binding and / or change in the initial binding rate of particles to the surface. The binding at each time point can be plotted to establish the binding rate.

[0056] If the surface area to volume ratio of the solution is low, a single measurement may be sufficient to determine the concentration, since the binding rate at any given time represents the number of particles. The binding rate is constant.

[0057] In some cases, it may be desirable to repeat the detection of bound particles to determine whether the binding rate is constant or has changed over time. If there is no change in the binding rate (constant rate), the binding rate recorded at any time point represents the absolute concentration. If there are changes in the binding rate, these changes can be plotted and the data can be extrapolated to the zero time point to calculate the concentration.

[0058] If there is a change in binding rate from a constant binding rate or from an initial binding rate, a decay or decline in the binding rate of particles to the surface can be calculated. The decline in binding rate can be correlated to the concentration of particles in solution. For example, particle binding to a surface (visualized by iSCAT) decreases over time as a function of the remaining number of particles in solution and the remaining number of accessible sites for binding.

[0059] Detection of particle binding to the surface can be repeated over time, with the interval between measurements being expected to depend on the nature of the particles and solution requiring measurement, and will vary from solution to solution.

[0060] Illustratively, measurements can be taken immediately after the sample contacts the surface, e.g., effectively within one or a fraction of a second of contact, and then measurements can be taken every few seconds thereafter, e.g., at intervals between measurements of 1-60 seconds, 1-30 seconds, 1-5 seconds, 5-10 seconds, 10-15 seconds, 15-20 seconds, 20-25 seconds, 25-30 seconds, 30-35 seconds, 35-40 seconds, 45-50 seconds, 55-60 seconds after contact. Measurements can be repeated as often as needed to determine the initial binding rate.

[0061] It will be understood that the interval between measurements can be related to the particle under investigation and therefore can be longer, i.e., the time between measurements could be from a few minutes to a few hours. Thus, measurements can be taken every few minutes after the first measurement, for example, at intervals between measurements of 1-60 minutes, 1-30 minutes, 1-5 minutes, 5-10 minutes, 10-15 minutes, 15-20 minutes, 20-25 minutes, 25-30 minutes, 30-35 minutes, 35-40 minutes, 45-50 minutes, or 55-60 minutes after contact. Alternatively, or in addition, measurements can be taken every few hours after the first measurement, for example, at intervals between measurements of 1-24 hours, 1-12 hours, 1-6 hours, 1-5 hours, 1-4 hours, 1-3 hours, 1-2 hours, or 1 hour after contact.

[0062] A longer interval would provide further evidence that there is no dissociation within a few minutes of the actual measurement, as it would be possible to show that it takes much longer to occur.

[0063] In addition, the time interval can be varied within an assay. For example, the first few measurements can be taken every few seconds, and further measurements can be taken at longer time intervals of several minutes. The interval can be varied as long as the time of detection is recorded.

[0064] Preferably, binding is detected at least once following the initial detection. Preferably, binding of particles to the surface is detected two or more times, and even more preferably three, four, five, six, seven, eight, nine, or more times. Each separate detection event of binding of particles to the surface is separated by a time interval as previously discussed, which may be The time intervals between the detection events can be the same or can vary.

[0065] To detect the rate of binding, it may be useful to record / video the binding to the surface, as shown in Example 4. Concentration determination To determine the concentration of particles in a solution, a constant or initial binding rate can be compared against a constant or initial binding rate for a known concentration of particles in the solution, allowing the method to be calibrated because it has been previously performed on particles of known concentration.

[0066] A series of known concentrations of initial binding rates can be extrapolated from the binding data and used to generate a standard curve that allows for the conversion of measured initial binding rates to the respective absolute concentrations of particles in a sample. In one embodiment, particles of known mass / concentration can be used to calibrate concentration measurements made using light scattering microscopy. An example is shown in FIG. 7, where the particles are lipoproteins. The left panel plots particle binding events versus incubation time (seconds). The right panel plots the initial binding rate determined from the binding data versus concentration, providing a calibration curve that allows for the conversion of measured initial binding rates to particle concentrations in a sample. In this case, the binding rate is the rate of particle binding to a surface. This surface is present in the sample holder in this embodiment where the solution is held.

[0067] Analysis of the decay or decrease in the rate of particle binding to the detected surface over time can provide the basis for enabling concentration determination. It may be necessary to use the binding rates of known concentrations of particles to calibrate the method.

[0068] To calculate the concentration, it may be necessary to include a correction for the diffusivity, which can be easily calculated based on known properties of the particles such as mass and shape.

[0069] Alternatively, the assay can be calibrated using an internal calibrator. The calibrator has similar characteristics to the particles with respect to their ability to bind to a surface (e.g., charge, hydrophobicity, hydrophilicity), and is therefore selected based on the properties of the particles in solution. The calibrator has a known mass and concentration. The calibrator is introduced to the particles in solution and the surface substantially simultaneously (i.e., at the same time). Detecting the binding of the calibrator to the surface using light scattering allows for the determination of the initial binding rate of the calibrator. Simultaneously, the initial binding rate of the particles to the surface is determined using light scattering. The initial binding rate of the particles can be compared to the initial binding rate of a known concentration of calibrator, which allows for the calculation of the concentration of the particles.

[0070] As discussed in previous publications, single particle light scattering can be used to determine the mass of particles, thus determining the binding rate of a particular particle type and using mass to identify particles of interest. Particles of interest or calibrants can be selected using contrast related to mass. Thus, it is possible to detect binding of only particles or calibrants to a surface and ignore binding of other solutes to the surface.

[0071] Concentrated solution If the solution is suspected to be concentrated, this can make measuring the concentration more complicated. To ensure that measurements determined using the method of the present invention are correct, the inventors have developed a method using a measurement solution that is introduced into the particle solution substantially simultaneously with the surface. do.

[0072] The measurement solution can be a buffer solution. Generally, the volume of the measurement solution is known to allow calculation of the dilution effect. A volume of particle solution can be placed in a sample holder, preferably with known geometry, which allows the introduction of a volume of measurement solution (or the introduction of particle solution into measurement solution) and, using a dilution action, this allows the detected binding rate of particles to the surface to be converted into a particle concentration.

[0073] Detection of particle binding to the surface can be performed at one or more different time intervals following the introduction of the measurement solution, as detailed above. If the detection step is repeated at different time intervals after the introduction of the measurement solution, this will allow for correction for dilution effects such as dissociation. If measurements are performed at different times after addition (and thus dilution), changes in species distribution, for example, when particles dissociate or cluster at lower concentrations, can be visualized. This approach therefore allows for a more detailed observation of the particles and allows for the validation of concentration measurements.

[0074] Alternatively or additionally, the volume of one or both of the solutions (particles or measurement) can be changed and / or the geometry of the sample holder can be changed. Both allow for correction of dilution effects. If the detection process is repeated in one or more different solution volumes and / or in one or more sample holder geometries, this can also correct for dilution effects such as dissociation.

[0075] method process As described herein, the present invention provides a method for measuring the concentration of particles in a solution, the method comprising the steps of: i) contacting the solution with the surface; ii) detecting particle binding to the surface visualized by light scattering; iii) repeating the detection step and calculating the change in the binding rate and / or initial binding rate of the particles to the surface over time; iv) providing a calibration curve of initial binding rate versus concentration based on data from solutions of known particle concentrations; v) Using the calibration curve in step (iv), convert the initial binding rate recorded for the sample in step (iii) into a concentration of particles in solution.

[0076] The method may further comprise repeating steps (i)-(ii) using one or more sample holders of different geometries or using different volumes of measurement solution to verify the determined concentrations.

[0077] Alternatively, the present invention provides a method for measuring the concentration of particles in a solution, the method comprising the steps of: i) contacting the solution with the surface; ii) detecting particle binding to the surface visualized by light scattering; iii) repeating the detection step and calculating the binding rate of particles to the surface and / or the change in the constant binding rate over time; iv) providing a calibration curve of constant binding rate versus concentration based on data from solutions of known particle concentrations; v) Using the calibration curve in step (iv), convert the constant binding rate recorded for the sample in step (iii) into a concentration of particles in solution.

[0078] The method may further include repeating steps (i)-(ii) using one or more sample holders of different geometries or using different volumes of measurement solution to verify the determined concentrations.

[0079] When an internal calibrant is used in the method of the invention, the invention provides a method for measuring the concentration of particles in a solution, the method comprising the steps of: i) contacting the solution with the surface in the presence of the calibrant; ii) detecting particle binding to the surface visualized by light scattering; iii) simultaneously detecting calibrant binding to the surface visualized by light scattering; iv) repeating detection steps (ii) and (iii) and calculating the change over time in the binding rate and / or initial binding rate of the particles and calibrant to the surface; v) Converting the initial particle binding rate calculated in step (iv) into a concentration of particles in solution using the binding rate calculated in step (iv) for the calibrator.

[0080] In all variations, it is the detection of particle binding to the surface that allows for the determination of the concentration of particles in solution. The rate of particle binding to the surface can be constant, in which case the constant rate of binding can be compared to a constant rate of binding for the same particles at a known concentration. The rate of binding can vary, so an initial rate of binding can be calculated for comparison to the initial rate of binding for the same particles at a known concentration. As previously discussed, the properties of the sample holder, in terms of surface area to volume ratio, can alter the concentration calculation.

[0081] Lipoprotein measurement As discussed above, the methods of the present invention allow for the direct measurement of the size and number of one or more lipoprotein particles in a sample, as well as the ratio of one or more different lipoprotein particles. The methods can include simultaneously or in parallel determining the number, size, or ratio of two or more different types of lipoprotein particles. The methods can include determining the total number of lipoprotein particles of a particular class in a sample, such as the total number of HDL and / or LDL lipoprotein particles. The methods can include determining the number of lipoprotein particles of a particular size in a sample. The methods can include determining the size distribution for a particular type of lipoprotein particle in a sample. The methods preferably include determining the ratio of HDL to LDL lipoprotein particles in a sample. The ratio of one or more other lipoprotein particles in a sample, such as HDL and / or LDL to VLDL lipoprotein particles, can also be determined. Thus, the relative number of particles of each detected lipoprotein type or fraction can be determined.

[0082] The method of the present invention can include obtaining a single particle histogram for one or more lipoprotein particles in a sample, including the number of particles of each different size detected for a given type of lipoprotein particle. Thus, in contrast to previous detection methods that characterize the bulk total lipoprotein content, each particle in a sample is detected separately. Thus, in contrast to previous detection methods that rely on algorithms, the distribution of lipoproteins within a population can be directly detected. The method preferably includes obtaining a single particle histogram for HDL and / or LDL lipoprotein particles.

[0083] The method can include determining the mass of lipoproteins in a sample. The method can include determining the absolute concentration of one or more types of lipoproteins in a sample. The mass and / or concentration of the lipoproteins can be determined by calibration to an appropriate standard. Thus, a range of known masses of lipoproteins (e.g., lipid nanodiscs) can be detected by light scattering, e.g., by iSCAT. Suitable purified lipoprotein preparations can be prepared, for example, as described in the Examples by Lee B et al. Available from iosolutions.

[0084] Thus, a calibration curve can be generated that has the relationship between lipoprotein mass and iSCAT signal. The iSCAT signals obtained for particles detected in a sample of interest can then be correlated with the calibration curve to identify their masses.

[0085] Absolute concentrations in solution can be determined by measuring binding events between lipoprotein particles and a surface in a sample calibrated against binding events observed in samples of known lipoprotein concentrations. For example, particle binding to a surface (visualized by iSCAT) decreases over time as a function of the remaining number of particles in solution and the remaining number of accessible sites for binding. Initial binding rates for a range of concentrations can be extrapolated from the binding data and used to generate a standard curve, allowing conversion of the measured initial binding rates in a sample to the respective absolute concentrations of lipoproteins. Alternatively, a constant binding rate at a given concentration can be determined by constant feeding of the sample in a flow chamber or by using a passivated surface that allows only transient binding of particles.

[0086] Exemplary calibration for determining the mass and concentration of HDL and LDL lipoproteins in sample is described in Examples.It should be understood that the method of the present invention can also not include any calibration with known standard.Instead, the comparison of particle size and distribution can be carried out using the representative particle size / distribution of target lipoprotein fraction.

[0087] The method can also include determining one or more other parameters of lipoprotein particles in the sample. The method can include detecting cholesterol, triglyceride, and / or apolipoprotein levels in the sample. The levels of these lipoprotein components can be determined by methods known in the art, as discussed below, or can be determined from knowledge of the average number of molecules of a given type in the lipoprotein particles of interest. Thus, the number of lipoprotein particles of a given type detected by light scattering, e.g., iSCAT, can be multiplied by the known average number of molecules of the lipoprotein component of interest in such lipoprotein particles to calculate the total level of that component. For example, LDL or VLDL particles contain only one apoB protein per particle (apoB protein is not present in HDL particles).

[0088] The level of a protein of interest in lipoproteins in a sample can be determined by immunoassay, for example, an immunoassay for apoB100. The presence of a particular protein in the detected lipoprotein particles can be determined by using a detectably labeled agent that specifically binds to the protein. The agent can include any suitable detectable label. The agent can be an antibody that specifically binds to the protein. Preferably, the detectable label allows for detection of the protein simultaneously or in parallel with detection of the particles by light scattering, for example, by iSCAT. Preferably, the detectable label is a fluorescent label (thus, for example, the agent is a fluorescently labeled antibody), and particles incorporating the protein are detected by fluorescence. Thus, the method of the present invention can include detection of lipoprotein particles by light scattering (e.g., by iSCAT) and detection of lipoprotein particles by fluorescence.

[0089] The methods of the invention can also include performing one or more other lipoprotein detection methods. Other lipoprotein detection methods include polyacrylamide gradient gel electrophoresis (see, e.g., U.S. Pat. No. 5,925,229 A), gradient density ultracentrifugation (see, e.g., U.S. Patent Application Publication No. 20140049775 A1), nuclear magnetic resonance (see, e.g., U.S. Pat. No. 5,343,389 A), and the like. (See, e.g., U.S. Pat. No. 7,259,018 B2), and ion mobility analysis (see, e.g., U.S. Pat. No. 7,259,018 B2). The method can further include performing a standard Friedewald assay to determine one or more of total glyceride (TG); total cholesterol (TC); HDL-cholesterol (HDL-C); and LDL-cholesterol (LDL-C) levels. The Friedewald assay requires precipitating VLDL and LDL to measure LDL-cholesterol levels, which are calculated by the following formula: TC - (HDL-C + TG / 5). A limitation of this assay is its limited accuracy when TG > 400 mg / ml or when the VLDL TG / Chol ratio deviates from 5:1. Also, fasting of the individual from whom the sample for lipoprotein measurement is obtained is required.

[0090] Results obtained by light scattering (e.g., by iSCAT) can be compared with results obtained by other method(s), or other method(s) can be used to provide additional information. Thus, for example, iSCAT can be used to provide information regarding particle size and number, and the total concentration of one or more lipoprotein components can be determined by other methods.

[0091] detection Detection of particles, including lipoprotein particles, according to the claimed methods is carried out using light scattering, preferably using interferometric scattering microscopy (iSCAT), a technique reviewed, for example, in Kukura et al., Nature Methods 2009 6:923-935 and Ortega-Arroyo et al., Physical Chemistry Chemical Physics 2012 14: 15625-15636.

[0092] iSCAT involves determining the interference between light scattered by an object in a sample and light reflected from a location on the sample. The interference depends on the scattering amplitude of the object (and in turn its volume) and is measured as an iSCAT signal. Thus, the iSCAT signal generated by a lipoprotein particle can be related to its volume and diameter, allowing the type of lipoprotein particle present in the sample to be identified. In calibration, the iSCAT signal can also be used to estimate the mass / concentration of the particle, as described below. Thus, the method of the present invention typically involves determining an iSCAT signal. The iSCAT signal can be described as the ratio of light detected in the presence and absence of the particle. More specifically, it is expressed as (I s -I p ) / I s where I s is the reflected intensity from the sample position (e.g., glass surface) in the absence of particles, and I p is the same measure in the presence of particles.

[0093] The method can include using an interference scattering microscope including: a sample holder for holding a sample at a sample position; an illumination light source arranged to provide illumination light; a detector; and optics arranged to direct the illumination light onto the sample position and to collect reflected output light (the output light including both light scattered from the sample position and illumination light reflected from the sample position) and direct the output light to the detector. The microscope can further include a spatial filter arranged to filter the output light, the spatial filter arranged to pass the output light but at a reduced intensity within a predetermined numerical aperture than at larger numerical apertures. Such a spatial filter advantageously maximizes image contrast, as described in PCT / GB2017 / 052070 and Cole et al. (ACS Photonics, 2017, 4(2), pp. 211-216), each of which is incorporated herein by reference.

[0094] The light used can be the following: ultraviolet light (which can be defined herein as having a wavelength in the range of 10 nm to 380 nm); visible light (which can be defined herein as having a wavelength in the range of 380 nm to 740 nm); infrared light (which can be defined herein as having a wavelength in the range of 740 nm to 300 μm). (This can be done by using a fluorescently labeled antibody.) The light is preferably visible light. Blue light is preferred due to the high sensitivity of the detection of lipoprotein particles. Red light can also be used advantageously to allow the detection of particles by iSCAT to be combined with the detection of specific lipoprotein components (e.g., specific proteins of interest) by fluorescence, for example, using a fluorescently labeled antibody that binds to the protein of interest. The light can be a mixture of wavelengths. The illumination light can be coherent light, for example provided by a laser.

[0095] 3 illustrates an iSCAT microscope 1 that may be utilized in the present invention, arranged as follows (and configured with a spatial filter as discussed above): While a spatial filter is advantageous for enhancing contrast for the reasons discussed, the methods of the present invention may alternatively utilize an iSCAT microscope that does not have a spatial filter.

[0096] The microscope 1 includes the following components, with the exception of a spatial filter, which will be described in more detail below, having a configuration commonly used in the field of microscopy: The microscope 1 includes a sample holder 2 for holding a sample 3 at a sample position. The sample 3 can be a liquid sample containing the object to be imaged, which is described in more detail below. The sample holder 2 can take any form suitable for holding the sample 3. Typically, the sample holder 2 holds the sample 3 on a surface that forms an interface between the sample holder 2 and the sample 3. For example, the sample holder 2 can be a coverslip and / or can be made of glass. The sample 3 can be provided on the sample holder 2 in a simple manner, for example, using a micropipette.

[0097] The microscope 1 further comprises an illumination source 4 and a detector 5 . The illumination light source 4 is arranged to provide illumination light. The illumination light can be coherent light. For example, the illumination light source 4 can be a laser. The wavelength of the illumination light can be selected depending on the nature of the sample 3 and / or the properties to be investigated. In one example, the illumination light has a wavelength of 405 nm.

[0098] In some cases, the illumination light may be, for example, a light source as described in Kukura et al., “High-speed nanoscopic tracking of the position and orientation of a single virus,” Nature Methods 2009 6:923-935 As detailed in

[10] , the illumination can be spatially modulated to eliminate speckle patterns arising from the coherent nature of the illumination and laser noise.

[0099] Detector 5 receives output light reflected from the sample location. Typically, microscope 1 can operate in a wide field mode, in which case detector 5 can be an image sensor that captures an image of sample 3. Alternatively, microscope 1 can operate in a confocal mode, in which case detector 5 can be an image sensor or a point-like detector such as a photodiode, in which case a scanning arrangement can be used to scan an area of ​​sample 3 to construct an image. Examples of image sensors that can be utilized as detector 5 include a CMOS (complementary metal-oxide semiconductor) image sensor or a CCD (charge-coupled device).

[0100] Microscope 1 further includes an optical system 10 disposed between sample holder 2, illumination source 4, and detector 5. Optical system 10 is arranged to direct illumination light onto a sample location to illuminate sample 3, and to collect output light reflected from the sample location and direct the output light to detector 5, as follows:

[0101] The optical system 10 includes an objective lens 11, which is a lens system arranged in front of the sample holder 2. The optical system 10 also includes a condenser lens 12 and a tube lens 13. The condenser lens 12 condenses illumination light (shown by a solid line in FIG. 1) from the light source 11 onto the sample 3 at the sample position through the objective lens 11.

[0102] The objective lens 11 collects output light that includes both (a) illumination light reflected from the sample position (shown by solid lines in FIG. 1 ) and (b) light scattered from the sample 3 at the sample position (shown by dotted lines in FIG. 1 ). The reflected light is primarily reflected from the interface between the sample holder 2 and the sample 3. Typically, this is a relatively weak reflection, such as a glass-water reflection. For example, the intensity of the reflected illumination light can be about 0.5% of the intensity of the incident illumination light. The scattered light is scattered by objects in the sample 3.

[0103] In a manner similar to conventional iSCAT, scattered light from objects at or near the surface of the sample constructively interferes with the reflected light and is therefore visible in the image captured by detector 5. This effect differs from microscopes operating in transmission, where the illumination light reaching the detector is transmitted through the depth of the sample, which results in much less imaging contrast.

[0104] 1, the reflected illumination light and the scattered light have different directionality. In particular, the reflected illumination light has a numerical aperture due to the geometry of the beam of light output by the light source 4 and the optical system 6. The scattered light is scattered over a large range of angles and therefore satisfies a larger numerical aperture than the reflected illumination light.

[0105] Tube lens 13 focuses the output light from objective lens 11 onto detector 5 . The optical system 6 also includes a beam splitter 14 positioned to split the optical path for the illumination light from the light source 4 and the output light directed to the detector 5. Except for a spatial filter, as described below, the beam splitter 14 can have a conventional structure that provides partial reflection and partial transmission of the light incident thereon. For example, the beam splitter 14 can typically be a plate with a film, which can be metallic or dielectric, positioned at 45° to the optical path. Alternatively, the beam splitter 14 can be a cube beam splitter formed by a matched pair of prisms with a partially reflective film at the interface between the prisms. Alternatively, the beam splitter 14 can be a polarizing beam splitter used in combination with a quarter-wave plate between the beam splitter 14 and the sample 3.

[0106] In the example shown in FIG. 1 , light source 4 is offset from the optical path of objective lens 11 so that illumination light from light source 4 is reflected into objective lens 11 by beam splitter 14, and conversely, detector 5 is aligned with the optical path of objective lens 11 so that output light from the sample position is transmitted through beam splitter 14 towards detector 5.

[0107] In addition to the above components, which can be of conventional construction, microscope 1 includes spatial filter 20. In the example shown in FIG. 1 , spatial filter 20 is formed on beam splitter 14, thereby positioning it behind the back aperture of objective lens 11 and thus directly behind the back focal plane 15 of objective lens 11. Thus, spatial filter 20 can be implemented without entering the objective lens, as in phase-contrast microscopy. Positioning the spatial filter directly behind the objective lens entrance aperture rather than in a conjugate plane (e.g., as described below) has the distinct advantage of strongly suppressing back reflections resulting from the multiple lenses in high-numerical-aperture microscope objectives. This, in turn, reduces imaging noise, lowers incoherent background, reduces experimental complexity, the number of optics, and optical path length, and leads to increased stability of the optical setup and, therefore, improved image quality.

[0108] However, this location is not essential and a spatial filter with equivalent functionality can be provided elsewhere, as described below. Spatial filter 20 is positioned to filter the output light passing therethrough to detector 5. Thus, in the example shown in Figure 1, where detector 5 is aligned with the optical path of objective lens 11, spatial filter 20 is of the transmissive type.

[0109] The spatial filter 20 is partially transmissive, thus passing the output light, including the reflected illumination light, but at a reduced intensity. The spatial filter 20 is also aligned with the optical axis and has a predetermined aperture such that it provides the intensity reduction within a predetermined numerical aperture. Numerical aperture is defined herein in its usual way as a dimensionless quantity that characterizes the range of angles relative to the sample position over which the output light originates. Specifically, the numerical aperture NA can be defined by the equation NA = n·sin(θ), where θ is half the collection angle and n is the refractive index of the material (e.g., the material of a component of the optical system 6) through which the output light passes.

[0110] Spatial filter 20 does not provide an intensity reduction outside the range of the predetermined numerical aperture, or, in principle, spatial filter 20 could provide a reduction in intensity outside the range of its predetermined numerical aperture (albeit a smaller reduction in intensity than the reduction in intensity within the range of the predetermined numerical aperture), but this would be undesirable.

[0111] The spatial filter 20 can be formed in any suitable manner and typically comprises a layer of deposited material, which can be a metal such as silver, for example. The deposition can be carried out using any suitable technique.

[0112] Because sub-diffraction sized objects near an interface preferentially scatter light to larger numerical apertures than to reflected illumination light, the reduction in intensity provided by spatial filter 20 preferentially reduces the intensity in the detection of reflected illumination light over scattered light. Thus, the intensity reduction provided by spatial filter 20 at low numerical apertures primarily affects reflected illumination light and has minimal effect on scattered light, thereby maximizing contrast in the captured image. The increased imaging contrast enables high-contrast detection of objects that are weak scatterers.

[0113] The contrast enhancement can be understood as follows: Because the spatial filter 20 passes a portion of the output light at a predetermined numerical aperture (i.e., is partially transmissive in this example), a fraction of the illumination and scattered light fields reaches the detector and interferes with the fully coherent illumination source. The intensity I of the light reaching the detector is then det I det =|E inc | 2 {r 2 t 2 +|s| 2 +2rt|s|cosΦ}, where E inc is the incident light field, and r 2 is the reflectivity of the interface, and t 2is the transmittance of the spatial filter 20, s is the scattering amplitude of the object, and Φ is the phase difference between the transmitted illumination light and the scattered light. Thus, the scattering contrast is enhanced, even at the expense of the total number of detected photons.

[0114] Contrast is thus provided in a manner similar to conventional iSCAT, but is additionally controlled by the transmittance of the spatial filter 20, t, as opposed to being fixed by the reflectance of the glass-water interface as in standard iSCAT. 2 If the spatial filter 20 is a layer of deposited material, the transmittance t 2 can be selected by the choice of material and / or layer thickness. Such adjustments can be made depending on, for example, the scattering objects of interest, the full well capacity of the camera, and the magnification.

[0115] To maximize these beneficial effects for iSCAT, the predetermined numerical aperture can be, but is not required to be, the numerical aperture of the reflected illumination light in the output light. For example, benefits of a similar nature can be achieved if the predetermined numerical aperture is slightly smaller or slightly larger than the numerical aperture of the reflected illumination light.

[0116] Lipoprotein-containing sample The sample can be any sample containing lipoprotein particles. The particles are typically lipoprotein particles produced in vivo, for example, in humans or animals. Thus, lipoprotein particles are typically present in biological samples. However, lipoprotein particles produced in vitro and / or lipoprotein particles provided in purified form can also be detected, for example, for calibration purposes as discussed above. The sample can contain multiple different types of lipoproteins in purified form. The sample can also be a complex mixture or polydisperse solution of various components, for example, a solution containing multiple different ions, proteins, and lipoproteins.

[0117] When the sample is a biological sample obtained from a human or animal, it can be a clinical sample. As described below in the context of the treatment and diagnosis methods of the present invention, the sample can be a clinical sample from any subject. It can be a sample of any body fluid or tissue containing lipoprotein particles. Typically, the sample is blood or a blood component, such as plasma or serum. The sample is capillary, venous, or arterial blood, or plasma or serum derived therefrom. Sample collection can be performed by any means, including finger prick or venipuncture.

[0118] As discussed above, detection of lipoproteins according to the present invention is advantageously achieved without separation or purification of the lipoproteins from other components of the sample. Separation and purification, as required in previous detection methods, can be time-consuming and can artificially alter the specific characteristics of lipoprotein particles. However, it should be understood that biological samples, such as blood, will typically be diluted to reduce particle density and facilitate optimal resolution of different types of lipoprotein particles. The sample can be diluted in any suitable buffer. Typically, a suitable buffer has a physiological pH and salt concentration. Non-physiological buffer conditions can also be used to examine the effect of these conditions on lipoprotein particles.

[0119] Those skilled in the art can select an appropriate dilution level for a given sample for detecting a specific lipoprotein particle of interest through routine experimentation. Depending on the imaging speed and sensitivity of the detection device, dilution can be performed empirically until a dilution level that allows for the detection of a single particle is identified. The dilution factor can then be taken into account to extrapolate the number of lipoprotein particles in the undiluted sample.

[0120] If the sample is blood or plasma, it can be diluted 10,000-fold or more in a suitable buffer to allow optimal detection of HDL (which at high concentrations is relatively dense compared to LDL) or simultaneous detection of HDL and LDL degradation. Lower dilutions (1-5,000-fold) can be used for detection of LDL.

[0121] The amount of sample required for detection of lipoproteins by iSCAT is minimal and can be as little as a microliter, depending on the type of sample and the means of collection. A finger prick allows very small amounts of blood to be collected. The sample can be provided in any suitable sample chamber. The sample chamber can be provided by a gasket on a coverslip, for example, as described in the Examples. Alternatively, the sample chamber can be provided by a flow chamber or a microfluidic device or chip, such as a For example, it can be a capillary tip.

[0122] Methods of diagnosis or risk determination The present invention has particular utility in diagnostic applications based on the correlation between the size, number, distribution, and ratio of different types of lipoprotein particles and disease. Accordingly, the present invention provides a method for diagnosing a disease or condition associated with the size and / or number of lipoprotein particles in an individual, or for determining an individual's risk of developing said disease or condition, comprising detecting the size and / or number of lipoprotein particles in a sample from said individual by interference scattering microscopy. Detection can be performed for any type of lipoprotein particle(s) as described above, using any of the methods described above in connection with the detection methods of the present invention. Thus, the method can comprise detecting the size and / or number of one or more different types of lipoprotein particles, or the ratio of different types of lipoprotein particles, in said sample. The method preferably comprises detecting high-density lipoprotein (HDL) particles and / or low-density lipoprotein (LDL) particles. The method can particularly comprise determining the ratio of HDL particles to LDL particles in said sample. The sample can be any of the biological or clinical samples described above, preferably blood, serum, or plasma. The individual can be an animal, a mammal, or a human.

[0123] The level, distribution, and size of specific lipoprotein particles may be correlated with disease. HDL, described in the art as "good cholesterol," serves as a transit point for cholesterol from the body's blood, tissues, and organs to the liver. Therefore, normal HDL levels provide adequate control of blood cholesterol levels. Changes in HDL levels / redistribution of cholesterol to other lipoprotein particles may be associated with disease or increased disease risk. For example, having an HDL cholesterol (concentration of cholesterol carried by HDL particles) level below 40 mg / dL for men and below 50 mg / dL for women is a major risk factor for heart disease (Mayo Clinic, 2016). HDL particles are normally the most frequent marker in the blood. It is a high lipoprotein fraction.

[0124] In contrast, LDL, the next most common lipoprotein fraction in normal blood, is often considered "bad cholesterol" due to its role in the biodistribution of cholesterol to the body's tissues and organs. Having high levels of LDL can lead to plaque formation in arteries, which likely increases the risk of heart disease and stroke (American Heart Association, 2014). LDL size has also been shown to be related to cardiovascular health. Normal LDL cholesterol (the concentration of cholesterol carried by LDL particles) levels range from 100 to 129 mg / dL. Smaller LDL particles are more closely associated with cardiovascular events (Foroutan, MS, RDN, 2015).

[0125] Other lipoprotein fractions (VLDL, IDL, UDL) are 10-100 times less frequent than LDL. Therefore, accurate measurement of the number, size, and ratio of HDL and / or LDL (and similar parameters for other lipoprotein particles), as obtained by detection according to the present invention, provides diagnostically valuable information for diseases or conditions associated with the size and / or number of lipoprotein particles. In particular, measurement of the number / size of HDL and LDL is of diagnostic value for cardiovascular disease. The direct homogeneous assay provided according to the present invention can also improve lipoprotein analysis and enable more accurate LDL values, particularly for patients suffering from hyperlipidemia, rather than determining LDL-C values ​​based on the Friedewald calculation (Nauck, Wamick, & Rifai, 2002). Also, while determination of total serum cholesterol levels is routinely used as a diagnostic tool, it is not always possible to determine the LDL-C value for patients suffering from symptomatic coronary artery disease. Approximately half of patients with this condition have normal LDL-cholesterol concentrations measured using standard methods. Thus, there is likely a hidden risk undetected by conventional clinical laboratory measurements of cholesterol, which is advantageously avoided by direct detection of lipoprotein particle number and size according to the present invention.

[0126] The disease or condition to be diagnosed or the risk thereof to be determined may be any disease or condition associated with the size and / or number of lipoprotein particles. The disease or condition may be associated with the size and / or number of HDL and / or LDL lipoprotein particles. The disease or condition may be associated with the ratio of HDL to LDL lipoprotein particles. Alternatively or additionally, the disease or condition may be associated with the size and / or number of VLDL, IDL, and / or UDL lipoprotein particles, or the ratio of VLDL, IDL, and / or UDL to HDL and / or LDL. The disease or condition may be associated with abnormal lipoprotein distribution or size.

[0127] The disease or condition may be one in which a decreased number of HDL particles are present compared to normal (control or reference) levels. Therefore, a decreased number of HDL particles compared to a control or reference sample / level may indicate that an individual has the disease or condition, or is at increased risk for developing the disease or condition. The disease or condition may involve a blood HDL cholesterol concentration of less than 40 mg / dL for men or less than 50 mg / dL for women. Blood HDL cholesterol concentrations of 40-50 mg / dL (1.0-1.3 mmol / L) for men and 50-59 mg / dL (1.3-1.5 mmol / L) for women are associated with an average risk of heart disease. Based on many epidemiological studies, a blood HDL cholesterol concentration of 60 mg / dL (1.55 mmol / L) is associated with an average risk of heart disease. EndA blood HDL cholesterol concentration of 0.01 mmol / L is associated with a lower than average risk of heart disease. Normal HDL cholesterol levels can more commonly be defined as levels greater than 1 mmol / L.

[0128] Alternatively, or in addition, the disease or condition may be one in which an increased number of LDL particles or an increased number of smaller-sized LDL particles are present compared to normal levels. Thus, an increased number and / or size of LDL particles compared to a control or reference sample / level indicates that an individual has the disease or condition or is at increased risk for developing the disease or condition. The disease or condition may involve a blood LDL cholesterol concentration greater than 3 mmol / L. If a person does not have other risk factors for cardiovascular disease, a blood LDL cholesterol level of less than 100 mg / dL (2.59 mmol / L) can be considered optimal; a level of 100-129 mg / dL (2.59-3.34 mmol / L) can be considered near-optimal / superoptimal, and a level of 130-159 mg / dL (3.37-4.12 mmol / L) can be considered borderline high.

[0129] The HDL:LDL ratio may be decreased in diseases or conditions. The normal number and / or size for a given lipoprotein particle is determined by reference to the baseline number and / or size for this type of particle in an individual who does not have any conditions associated with lipoprotein particle size and / or number, such as those discussed below. Such an individual thus provides a control or reference number and / or size of one or more lipoprotein particle(s) that can be compared with the number and / or size of one or more lipoprotein particle(s) detected by iSCAT in a sample from the individual on whom the diagnostic method is performed. The control or reference size / number can be an average of values ​​from multiple normal individuals. The individual has normal total blood cholesterol levels, preferably normal HDL blood cholesterol, as discussed above. A person can have medium and LDL blood cholesterol levels. A normal (healthy) total blood cholesterol level is less than 5 mmol / L.

[0130] Therefore, the methods of diagnosing or determining risk of the present invention typically include comparing the number and / or size of one or more lipoprotein particle(s) in a sample from an individual with a control number and / or size of said lipoprotein particle(s). An increased or decreased number and / or size of lipoprotein particle(s) may indicate the presence of a disease or condition or a risk thereof, as discussed further below. It should be understood that the control or reference level can be previously determined by iSCAT or by any other method of detecting lipoprotein particles, such as any other known detection method described herein. Alternatively, the number and / or size of lipoprotein particles can be detected simultaneously with the cholesterol level or distribution in a control sample by iSCAT. The individual can have any cholesterol-related condition. The disease or condition can be associated with increased or high levels of total blood cholesterol, HDL cholesterol, and / or LDL cholesterol. Normal total cholesterol and HDL and LDL cholesterol levels are discussed above. The disease or condition may be associated with increased or high levels of triglycerides. The disease or condition is preferably a cardiovascular disease. The disease or condition may be a vascular disease.

[0131] Cardiovascular disease (CVD) refers to a class of diseases involving the heart or blood vessels. Cardiovascular diseases involving blood vessels are also known as vascular diseases. The disease or condition can be any vascular disease. The disease or condition can be selected from coronary artery disease, coronary heart disease, ischemic heart disease, peripheral artery disease, cerebrovascular disease, stroke, ministroke, renal artery stenosis, and aortic aneurysm. The disease or condition can be any cardiovascular disease involving the heart. The disease or condition can be selected from hypertensive heart disease secondary to hypertension, hypertension, heart failure, pulmonary heart disease, cardiac arrhythmia, abnormal heart rhythm, inflammatory heart disease, endocarditis, inflammatory cardiac hypertrophy, myocarditis, valvular heart disease, and rheumatic heart disease. The disease or condition can be hypercholesterolemia, e.g., familial hypercholesterolemia. The disease or condition can be any non-cardiovascular disease or condition associated with elevated cholesterol or triglycerides, such that determining the number and / or size of lipoprotein particles can aid in the diagnosis (and treatment) of this aspect of the disease or condition. Examples of such diseases and conditions include diabetes, kidney disease, thyroid dysfunction, or an inflamed pancreas (pancreatitis).

[0132] The individual can have previously been characterized as being at risk for cardiovascular disease and / or been recommended for testing of blood cholesterol levels. The individual can have previously been diagnosed with cardiovascular disease, including any of the diseases listed above. In this aspect, the present invention provides for more accurate determination of the number and / or size of lipoprotein particles in an individual.

[0133] Individuals can be selected for diagnosis or risk assessment based on any risk factors, such as gender, age, family history, weight, body mass index, diet, or lifestyle. The individual can be over 40 years old; according to NHS guidelines, people over 40 should have their CVD risk estimates periodically reassessed. The individual can have a family history of early cardiovascular disease—for example, a father or brother who developed heart disease or had a heart attack or stroke before age 55, or a mother or sister who had such a condition before age 65. The individual can have a family member, typically a close relative, with a cholesterol-related condition, such as familial hypercholesterolemia. The individual may be overweight or obese. The individual may have high blood pressure or diabetes.

[0134] The present invention further provides a method for selecting an individual to whom a substance or composition or therapeutic regimen should be administered, said substance or composition or therapeutic regimen being suitable for treating or preventing a disease or condition associated with lipoprotein particle size and / or number, comprising detecting the size and / or number of lipoprotein particles in a sample from said individual by interference scattering microscopy according to the detection method of the invention, and selecting said patient for said administration or said therapeutic regimen if the detected lipoprotein particle size and / or number indicates the presence of, or risk of, said disease or condition. The method may further include selecting an individual based on any of the above risk factors.

[0135] Suitable substances and compositions for administration are described below. An appropriate treatment regimen may include changes in lifestyle, diet, or exercise. The present invention further provides kits providing means suitable for use in the methods of detecting lipoprotein particles or methods of diagnosing or determining risk according to the present invention. The kits can include components suitable for detecting lipoprotein particles by interference scattering microscopy. The kits can include instructions for using the kits according to the methods of the present invention. The instructions can provide reference levels for the number and size of one or more lipoprotein particles and reference single particle histograms for one or more lipoprotein particles. The kits can also include details regarding the individuals on whom the methods can be performed. The kits can include one or more components of a sample chamber, such as a coverslip and gasket, a flow cell, a microfluidic device or chip, such as a capillary chip. The kits can include means for obtaining a sample (typically a blood sample) from an individual, such as a capillary blood collection device, a fingerprick blood collection device, or any instrument including a needle. The kits can include one or more standard lipoprotein samples (e.g., nanodiscs described herein) that provide calibration of lipoprotein particle measurements according to the present invention. The kits can further include means for measuring other laboratory or clinical parameters. Procedures using these kits can be performed by clinical laboratories, laboratories, medical professionals, or private individuals. The present invention further provides the use of a component suitable for detecting lipoprotein particles by interference scattering microscopy in the manufacture of a test kit for the diagnosis or risk determination of a disease or condition associated with lipoprotein particle size and / or number. The component can be a component of a sample chamber as described above. The test kit can include instructions, means, or a standard sample as described above.

[0136] Methods of treatment and medical uses The present invention further provides a method of treating or preventing a disease or condition associated with lipoprotein particle size and / or number in an individual, the method comprising selecting said individual, or diagnosing or determining said individual's risk of said disease or condition by the above-described method, and administering to said individual a substance or composition, or carrying out a therapeutic regimen thereon, which is effective to treat or prevent said disease or condition in said individual.

[0137] The present invention also provides substances or compositions for use in methods of treating or preventing a disease or condition associated with lipoprotein particle size and / or number in an individual, wherein said individual is diagnosed or determined to be at risk or selected by the methods of the invention described above.

[0138] The present invention further provides the use of a substance or composition in the manufacture of a medicament for the prophylactic treatment of a disease or condition associated with lipoprotein particle size and / or number in an individual, wherein said individual is diagnosed or determined to be at risk or selected by the above-described methods of the invention.

[0139] The individual can be any of the individuals described above, preferably a human individual. The individual can have or be at risk of any of the diseases or conditions described above, and can have any of the risk factors described above. Preferably, the individual can have or be at risk of cardiovascular disease, which can be any of the specific cardiovascular diseases described above.

[0140] The substance or composition is administered in an amount effective for preventing or treating the disease. The therapeutic regimen (e.g., lifestyle changes, diet, or exercise) is typically implemented for a period effective for preventing or treating the disease. An appropriate therapeutic regimen may include weight loss, reducing or ceasing smoking or alcohol intake, increasing exercise, and / or a healthy diet. The therapeutic regimen may also include surgery, such as coronary angioplasty or coronary artery bypass surgery. Surgery may include the introduction of a stent. Disease prevention or treatment may be determined by reference to preventing or delaying the onset of the disease, or by reducing or eliminating one or more symptoms of the disease. Prevention or treatment may induce or prolong remission of the disease or condition, or delay recurrence. Administration of an effective amount or implementation of an effective therapeutic regimen may be determined by measuring the number, size, or ratio of normal lipoprotein particles (or a shift toward the number, size, or ratio of normal lipoprotein particles) in a sample from the individual after administration or the therapeutic regimen.

[0141] The substance or composition can be any substance or composition suitable for treating or preventing a disease or condition associated with the size and / or number of lipoprotein particles by any means. The substance or composition can lower LDL concentration or reduce the size of LDL lipoprotein particles. The substance or composition can increase HDL concentration. The substance or composition can lower blood cholesterol levels, including total blood cholesterol levels or total LDL blood cholesterol levels. The substance or composition can lower blood pressure or widen arteries. The drug can be a vasodilator. The substance or composition can include any known drug for preventing or treating cardiovascular disease or lowering blood pressure. The drug can be a statin. Statins (also known as HMG-CoA reductase inhibitors) are a group of drugs that can lower blood LDL cholesterol levels and have been found to reduce cardiovascular disease and mortality in high-risk individuals. The drug can be a beta-blocker, a nitrate, an ACE (angiotensin converting enzyme) or angiotensin receptor II inhibitor, a calcium channel blocker or a diuretic.

[0142] The substance or composition can be a small molecule inhibitor, a peptide, a protein, an antibody, a polynucleotide, an oligonucleotide, an antisense RNA, a small interfering RNA (siRNA) or a small hairpin RNA (shRNA).

[0143] A polynucleotide, e.g., a nucleic acid, is a polymer containing two or more nucleotides. Nucleotides can be naturally occurring or artificial. Nucleotides typically contain a nucleobase, a sugar, and at least one linking group, such as a phosphate, 2'O-methyl, 2'methoxy-ethyl, phosphoramidate, methylphosphonate, or phosphorothioate group. Nucleobases are typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, more specifically adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C). The sugar is not limited. The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The nucleotide can be any nucleotide or modified nucleotide, typically a ribonucleotide or deoxyribonucleotide, or a modified version thereof. The nucleotide typically contains a monophosphate, diphosphate, or triphosphate. The phosphate can be attached to the 5' or 3' side of the nucleotide. The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). It can be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), morpholino nucleic acid, or other synthetic polymers with nucleotide side chains. The polynucleotide can be single-stranded or double-stranded.

[0144] The polynucleotide sequence can be cloned into any suitable expression vector. In an expression vector, the polynucleotide sequence encoding the construct is typically operably linked to a control sequence that can provide expression of the coding sequence by the host cell. Such an expression vector can be used to express the construct. Antisense and RNA interference (RNAi) techniques for knocking down protein expression are well known in the art, and standard methods can be used to knock down the expression of a molecule of interest. Both antisense and siRNA techniques interfere with mRNA. Antisense oligonucleotides interfere with mRNA by binding to (hybridizing with) a certain section of mRNA. RNAi involves the use of double-stranded RNA, such as small interfering RNA (siRNA) or short hairpin RNA (shRNA), which can bind to mRNA and inhibit protein expression.

[0145] An oligonucleotide "specifically hybridizes" to a target sequence if it hybridizes preferentially or with high affinity to the target sequence but does not substantially hybridize, does not hybridize, or hybridizes only with low affinity to other sequences. Conditions that allow hybridization are well known in the art (e.g., Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rd ed., Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995)). Hybridization conditions can be stringent conditions as described in the art.

[0146] An antibody can specifically bind to any target molecule (typically a protein). The target molecule can be a component of a lipoprotein particle, such as an apolipoprotein. An antibody "specifically binds" to a protein if it binds preferentially or with high affinity to that protein but does not substantially bind, does not bind, or binds only with low affinity to other proteins. For example, an antibody "specifically binds" to a target molecule if it binds preferentially or with high affinity to that target but does not substantially bind, does not bind, or binds only with low affinity to other human proteins.

[0147] The antibody is 1 x 10 -7 M or less, preferably 5×10 -8 M or less, preferably 1×10 -8 M or less, or more preferably 5 × 10 -9 K below M d An antibody binds with preferential or high affinity if it binds at 1 x 10 -6 M or more, preferably 1×10 -5 M or more, preferably 1×10 -4 M or more, preferably 1×10 -3 M or more, and even more preferably 1×10 -2 K over M d When it binds to the ATP, it does so with low affinity.

[0148] The antibody can be, for example, a monoclonal antibody, a polyclonal antibody, a single-chain antibody, a chimeric antibody, a bispecific antibody, a CDR-grafted antibody, or a humanized antibody. The antibody can be an intact immunoglobulin molecule or a fragment thereof, such as a Fab, F(ab')2, or Fv fragment.

[0149] The particular route of administration, dosage and method of administration of the therapeutic agents described herein can be routinely determined by a medical practitioner. The agents for use in the methods of treatment described herein can be formulated into pharmaceutical compositions. These compositions can contain, in addition to the therapeutically active ingredient(s), pharmaceutically acceptable excipients, carriers, diluents, buffers, stabilizers, or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the effectiveness of the active ingredients. The pharmaceutical carrier or diluent can be, for example, an isotonic solution.

[0150] The precise nature of the carrier or other material may depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, and intraperitoneal. Examples of suitable compositions and administration methods are provided in Esseku and Adeyeye (2011) and Van den Mooter G. (2006). For example, solid oral forms may contain, together with the active ingredient, diluents such as lactose, dextrose, saccharose, cellulose, corn starch, or potato starch; lubricants such as silica, talc, stearic acid, magnesium or calcium stearate, and / or polyethylene glycols; binders such as starch, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, or polyvinylpyrrolidone; disaggregating agents such as starch, alginic acid, alginates, or sodium starch glycolate; effervescent mixtures; dyes; sweeteners; wetting agents such as lecithin, polysorbates, lauryl sulfate; and non-toxic and pharmacologically inert substances commonly used in pharmaceutical formulations. Such pharmaceutical preparations can be produced in known manner, for example, by mixing, granulating, tableting, sugar-coating, or film-coating processes.

[0151] Oral formulations contain commonly employed excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders and contain 10% to 95%, preferably 25% to 70%, of the active ingredient. If the pharmaceutical composition is lyophilized, the lyophilized material can be reconstituted (e.g., as a suspension) prior to administration. Reconstitution is preferably accomplished in a buffer solution.

[0152] Capsules, tablets and pills for oral administration to an individual can be provided with an enteric coating comprising, for example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose acetate phthalate, or hydroxypropyl methylcellulose.

[0153] Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain as carrier, for example, sucrose or sucrose with glycerine and / or mannitol and / or sorbitol.

[0154] Suspensions and emulsions may contain as a carrier, for example, natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. Suspensions or solutions for intramuscular injections may contain the active substance together with a pharmaceutically acceptable carrier, for example, sterile water, olive oil, oleic acid, or the like. The composition may contain ethyl acetate, glycols such as propylene glycol, and an appropriate amount of lidocaine hydrochloride if desired.

[0155] The solutions for intravenous administration or infusion may contain as a carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

[0156] For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.

[0157] The dosage can be determined depending on various parameters, particularly the substance used; the age, weight, and condition of the individual to be treated; the route of administration; and the required therapeutic regimen. A physician will be able to determine the required route of administration and dosage for any particular individual. A typical daily dose is about 0.1 to 50 mg / kg body weight, depending on the above conditions. The dosage can be provided as a single dose or as multiple doses, for example, 2, 3, or 4 doses administered at regular intervals, for example, every hour.

[0158] Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, Vol. Edition, 2000, pub. Lippincott, Williams & Wilkins. The substances or compositions used in the above methods and medical applications can be administered alone or in combination with other therapeutic substances, compositions or treatments, e.g., as adjunctive therapy. The other therapeutic compositions or treatments can be administered either simultaneously with or sequentially to the substances, compositions or treatments of the present invention. [Example]

[0159] Example 1 - Detection of purified lipoprotein particles by iSCAT Materials / methods Preparation of supports and measurements Borosilicate glass coverslips (No. 1.5, 24 x 50 mm, VWR) were washed by sequential rinsing with MilliQ water, followed by ethanol, and then MilliQ water again. They were then dried under a stream of dry nitrogen. A CultureWell silicone gasket (Grace Bio-Labs) was cut and placed on the freshly cleaned coverslip, providing four independent 30-50 μl sample chambers on the same support.

[0160] Purified HDL and LDL samples were obtained from Lee Biosystems (LDL catalog number 360-10; HDL catalog number 361-10) and were identified as follows: https: / / www.leebio.com / product / 984 / low-density-lipoprotein-ldl-human-serum-360-10 HDL and LDL were separated by ultracentrifugation. The samples were diluted 500,000 and 50,000 times, respectively.

[0161] Data acquisition and analysis were then performed by iSCAT, imaging and recording nonspecific binding of lipoproteins to the glass support. The experimental setup was identical to that described in Figure 4 by Cole et al. (2012). Images were acquired over 30 seconds (at 100 frames / s) and consisted of 512 x 512 pixels with a pixel size of 23.4 nm. Images were pixel binned 3 x 3 before storage, giving a final pixel size of 70.2 nm.

[0162] Ratiometric image stacks were extracted from raw movies as described in Cole et al. Particles that landed on the glass surface were identified in the ratiometric images by an automated spot detection routine based on 2D Gaussian fitting of the point spread function.

[0163] The results are shown in Figure 4, which provides images of HDL and LDL detected by iSCAT and the corresponding histograms obtained from the complete series of images. Example 2 - Detection of purified lipoprotein particles from blood and serum by iSCAT The iSCAT method described in Example 1 was performed on serum and blood samples from human individuals. To obtain serum, blood was allowed to clot in an upright position for at least 30 minutes and then centrifuged (30 minutes, 1500 x g). Serum was transferred to plastic screw-cap vials. Blood and serum samples were prepared as follows: 1 μl of finger-prick blood or serum sample was diluted 2000-fold in HEPES / KCl buffer containing 5 mM EDTA (to prevent clotting) (see below). 10 μl of the diluted sample was added to 40 μl of 25 mM HEPES buffer (pH 7.4) containing 100 mM KCl, resulting in a final 10,000-fold dilution of the blood.

[0164] Figure 5 shows the detection of HDL and LDL in plasma samples. Imaging of the plasma samples simultaneously revealed signals corresponding to both HDL and LDL. As expected, HDL particles were more frequent than LDL (see histogram below).

[0165] Figures 6A and 6B show the detection of HDL and LDL in whole blood, with results similar to those in Figure 5. We also used both "fasting samples" and samples collected after a meal. The difference between fasting and non-fasting conditions can be noticed in the regime of higher iSCAT signals (inserts) corresponding to larger lipoproteins, such as VLDL. As expected, these appear after a meal. As previously discussed, the presence of a VLDL fraction is the main reason why patients must fast before standard cholesterol testing. VLDL contains cholesterol but is primarily triglycerides (TGs), and if TG levels are too high, standard (Friedewald) methods cannot be used. Thus, detection of lipoproteins (including HDL, LDL, and VLDL) without any fasting requirement in the individual providing the sample is possible according to the present invention.

[0166] Example 3 - Calibration for calculating HDL and LDL concentrations To enable calculation of the mass / concentration of HDL and LDL in the blood samples of Example 2, we established a calibration curve, where proteins of known mass / concentration were measured, giving a linear relationship between molecular weight and iSCAT contrast.

[0167] For concentration calibration, we used known concentrations of MSP1D1 DMPC lipid nanodiscs. Nanodiscs are a synthetic model membrane system composed of a lipid bilayer of phospholipids with a hydrophobic tail screened by two amphipathic proteins. These proteins, called membrane skeletal proteins (MSPs), are arranged in a double-belt configuration. Nanodisc samples were diluted to the nM range and added to buffer. Nonspecific binding of nanodiscs to glass was recorded using iSCAT, and the results are shown in Figure 7. The frequency of binding decreases as the number of particles in solution and the number of accessible sites for binding decrease (as shown in the left panel: number of binding events as a function of time for four different concentrations). We fitted an exponential decay, knowing when the sample was added (t = 0), and extrapolated the initial binding rate.

[0168] The initial binding rate was then plotted as a function of concentration (right panel). The slope of the linear fit is a conversion factor that can be used to determine the absolute concentration of the sample using the following relationship: [Sample concentration] = [measured initial rate] / [conversion factor] x [sample dilution] Using this calibration curve, HDL and LDL lipoprotein particle concentrations were calculated from whole blood samples in preliminary experiments and found to correlate with expected values.

[0169] Example 4 - Concentration measurements on actin solutions General steps taken in calculating concentrations for this example: 1. Place the sample into the sample holder (which is a high surface area to volume chamber).

[0170] 2. As shown in FIG. 7, after sample application, recording of individual binding events begins at a fixed, well-controlled time delay. 3. The frequency of binding is calculated as a function of time after addition of the sample.

[0171] 4. Fit the observed decay in binding frequency as a function of time to a single exponential decay function. 5. A time zero section is drawn to determine the frequency of binding upon addition of sample.

[0172] 6. The procedure is repeated as a function of sample concentration. 7. Fit the observed initial binding frequency versus sample concentration to obtain a linear correlation. Once that correlation is established, for any given sample, the concentration can be estimated by repeating steps 1-5 to establish an initial binding frequency, which can be converted to sample concentration from the relationship in step 7.

[0173] The slope of the linear fit is a conversion factor that can be used to determine the absolute concentration of the sample using the following relationship: [Sample concentration] = [measured initial rate] / [conversion factor] x [sample dilution] Protocol for the Titration Dilution Method for Actin: Prepare (3-aminopropyl)triethoxysilane (APTES) coated coverslips as described elsewhere (these coverslips are specific for actin; for other proteins, use simple untreated cleaned glass).

[0174] · A 2x2 hole was cut in a 3mm diameter PDMS gasket (Grace Bio-Labs GBL103250, available from Sigma Aldrich) with a clean scalpel.

[0175] The gasket was rinsed with milliQ (MQ) water, isopropanol, MQ water, isopropanol, MQ water and then blown dry with a stream of nitrogen. Attach the gasket to the center of the APTES-coated coverslip (contact of the PDMS with the glass surface is sufficient for it to be attached).

[0176] Under the microscope, a 37ul droplet of F-actin buffer was placed on one of the gaskets on the APTES coverslip and the focus was adjusted to the glass surface in the buffer-filled gasket.

[0177] A 11.4 uM stock solution of actin (a 42 kDa protein that polymerizes into filaments) was diluted to 333.33 nM in G-actin buffer. 90ul of 333.33nM actin stock to 10ul of F-actin buffer Mixed with 0x concentrate (meaning to initiate polymerization specific to actin experiments) → 300 nM final actin concentration.

[0178] After 15 minutes of polymerization, a 3ul aliquot of the 300nM actin solution was pipetted and extruded to form a small droplet at the tip of the pipette, and the actin droplet was then fused with the buffer droplet in the gasket.

[0179] Immediately after one minute of perturbations, recording of landing molecules began. This method works well for systems where the oligomerization equilibrium changes slowly compared to the time it takes to dilute the sample and record the movie.

[0180] buffer solution G-actin buffer 2mM tris(hydroxymethyl)aminomethane (Tris base) 0.2mM CaCl2 0.2mM adenosine triphosphate (ATP) 2mM dithiothreitol (DTT) The pH was adjusted to 8.0 with HCl.

[0181] F-actin buffer 10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 100mM KCl 2mM MgCl2 1mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) The pH was adjusted to 7.5 with KOH.

[0182] The results are shown in Figures 8A and 8B. 8A shows a frame from an example video of 300 nM actin landing in a gasket using the dilution method, recorded at a frame rate of 1 kHz and an effective frame rate of 25 Hz. For comparison, 8B shows a frame from an example video of 300 nM actin landing in a flow chamber under similar conditions, recorded at a frame rate of 1 kHz and an effective frame rate of 25 Hz.

[0183] The experiment was repeated four times and the resulting accumulated mass histogram is shown as FIG.

Claims

1. A method for measuring the concentration of particles in a solution, comprising contacting the solution with a surface and detecting binding of the particles to the surface using light scattering.

2. 10. The method of claim 1, wherein the surface is passivated, activated, coated, treated or derivatized.

3. 3. The method of claim 1 or claim 2, wherein the detection of binding of the particles to the surface is repeated after one or more time intervals.

4. 4. The method of claim 3, wherein the repeated measurements allow calculation of the initial binding rate of the particles to the surface.

5. 5. The method of claim 4, wherein the initial binding rate of the particles is compared against initial binding rates for known concentrations of the particles.

6. 4. The method of claim 1, wherein the measurement allows calculation of a constant binding rate of the particles, which is optionally compared to a constant binding rate for a known concentration of the particles.

7. 10. The method of claim 1 for measuring the concentration of particles in a solution, said method comprising the steps of: i) contacting the solution with a surface; ii) detecting binding of the particles to the surface visualized by light scattering; iii) repeating the detection step and calculating the change in the binding rate and / or initial binding rate of the particles to the surface over time; iv) providing a calibration curve of initial binding rate versus concentration based on data from solutions of known particle concentration; v) using the calibration curve in step (iv) to convert the initial binding rate recorded for the sample in step (iii) to a concentration of the particles in solution; The method comprising:

8. 10. The method of any preceding claim, wherein the concentration is an absolute concentration.

9. 10. The method of any preceding claim, wherein the particles are also contacted with a measurement solution.

10. 5. The method of claim 1, wherein the solution is contacted with the surface in the presence of a calibrant, and binding of the calibrant to the surface is detected using light scattering.

11. 1. A method for the detection of lipoprotein particles in a sample, comprising detecting said particles by light scattering, optionally interference scattering microscopy.

12. 12. The method of claim 11, wherein said detecting comprises determining the size and / or number of said particles.

13. 13. The method of claim 11 or 12, wherein more than one different type of nucleotides in the sample are present. detecting the size and / or number of lipoprotein particles in said sample and optionally detecting the ratio of different types of lipoprotein particles in said sample.

14. The method according to any one of claims 11 to 13, comprising detecting high density lipoprotein (HDL) particles and / or low density lipoprotein (LDL) particles.

15. 15. The method of any one of claims 11 to 14, comprising determining the ratio of HDL particles to LDL particles in said sample.

16. 1. A method for diagnosing a disease or condition in an individual that is associated with the size and / or number of lipoprotein particles, or for determining the individual's risk of developing said disease or condition, the method comprising detecting the size and / or number of lipoprotein particles in a sample from said individual by light scattering, optionally by coherent scattering microscopy.

17. 17. The method of claim 16, comprising detecting the size and / or number of more than one different type of lipoprotein particle, or the ratio of different types of lipoprotein particle, in the sample.

18. 18. A method according to claim 16 or claim 17, comprising detecting high density lipoprotein (HDL) particles and / or low density lipoprotein (LDL) particles in the sample, and optionally determining the ratio of HDL particles to LDL particles.

19. 19. The method of claim 17 or claim 18, wherein a decreased number of HDL particles compared to a control sample or reference sample / level indicates that the individual has the disease or condition or has an increased risk of developing the disease or condition.

20. A method according to claim 17 or claim 18, wherein an increased number and / or decreased size of LDL particles compared to a control or reference sample / level indicates that the individual has the disease or condition or is at increased risk of developing the disease or condition.

21. The method according to any one of claims 16 to 19, wherein the disease is a cardiovascular disease.

22. The method according to any one of claims 1 to 21, wherein said solution or sample is a biological sample, optionally obtained from a human individual.

23. 23. The method of claim 22, wherein the biological sample is blood, plasma or serum, optionally obtained from a human individual.

24. 16. A method for selecting an individual to whom a substance or composition should be administered or a therapeutic regimen should be prescribed, said substance or composition or therapeutic regimen being suitable for treating or preventing a disease or condition associated with lipoprotein particle size and / or number, the method comprising detecting the size and / or number of lipoprotein particles in a sample from said individual by a method according to any one of claims 11 to 15, and selecting said patient for said administration or said therapeutic regimen if the detected lipoprotein particle size and / or number indicates the presence of, or risk of, said disease or condition.

25. 24. A method of treating or preventing a disease or condition associated with the size and / or number of lipoprotein particles in an individual, the method comprising diagnosing or determining the risk of said disease or condition in said individual, or selecting said individual by the method of any one of claims 16 to 23, and administering to said individual a substance or composition, or carrying out a therapeutic regimen thereon, that is effective for treating or preventing said disease or condition in said individual.

26. 24. A substance or composition for use in a method for treating or preventing a disease or condition associated with lipoprotein particle size and / or number in an individual, wherein said individual is diagnosed or determined to be at risk or selected by the method of any one of claims 16 to 23.

27. 24. Use of a substance or composition in the manufacture of a medicament for the prophylactic treatment of a disease or condition associated with lipoprotein particle size and / or number in an individual, wherein said individual has been diagnosed or determined to be at risk or selected by the method of any one of claims 16 to 23.

Images