Method and system for determining presence of multiple fluorophore-labeled detection molecules

Abstract

Methods and systems for determining the presence of a plurality of fluorophore-labeled detection molecules are disclosed. The detection molecules bind to a plurality of particles (110) disposed on a planar surface (122). Thus, a sample containing particles suspended in a liquid medium (115) is provided (12) and the particles are exposed (14) to an alternating magnetic field (B) to reduce the tendency of the particles to aggregate on a planar surface. The particles are allowed to settle (16) onto the planar surface, thereby irradiating (18) them and detecting (20) photons emitted from the irradiated detection molecules. The presence of the detection molecules is then determined (22) based at least in part on the detected photons.

Description

Technical Field

[0001] The present invention relates to research techniques such as assays, and in particular to immunoassays using magnetic microspheres. Background Art

[0002] Immunoassays are widely used in medical diagnosis and research to detect and quantify specific analytes in samples, such as proteins or other molecules, including hormones, antibodies, antigens and other biomarkers of health or disease. Typically, immunoassays involve combining analytes with analyte-specific capture molecules to form capture molecule-analyte complexes. Unbound material can then be separated from the complex before detection and quantification of the complex. Detection and quantification typically include a secondary reaction that produces a detectable signal, which is typically generated by fluorescent emission from a detection molecule bound to the complex. The signal intensity is proportional to the concentration of the analyte in the sample.

[0003] In a flow-based assay, capture molecules can be attached to the surface of particles commonly referred to as microspheres. Once a complex is formed, the microspheres are passed through a flow cytometer. The instrument typically uses a laser to excite the fluorescent detection molecules and the stained particles themselves, allowing the emitted fluorescence to be measured for each microsphere that passes through. Although flow-based assays can be powerful tools for bioanalysis, they also have disadvantages. One disadvantage relates to the complexity of the instrument, which is expensive to maintain and requires skilled technicians and training to operate. Another disadvantage relates to the preparation and handling of samples, as samples tend to clog the cell counter over time and will inherently contaminate the instrument with potentially dangerous pathogens.

[0004] Therefore, there is a need for improved and more efficient techniques for detecting and quantifying analytes in samples using microspheres. Summary of the invention

[0005] It is an object of the present invention to provide a method and a system that solves at least some of the above mentioned disadvantages. This object is achieved by the present invention.

[0006] According to a first aspect of the present invention, a method for determining the presence of a plurality of fluorophore-labeled detection molecules bound to a plurality of particles arranged on a planar surface is provided. The method comprises providing a sample comprising particles suspended in a liquid medium, exposing the particles to an alternating magnetic field to reduce the tendency of the particles to aggregate on the planar surface, and allowing the particles to settle on the planar surface. Irradiating the settled particles, detecting photons emitted from the irradiated detection molecules, and determining the presence of the detection molecules based at least in part on the detected photons.

[0007] According to a second aspect, a system for determining the presence of a plurality of fluorophore-labeled detection molecules bound to a plurality of particles arranged on a flat surface is provided. The system includes a magnetic field generator configured to expose a sample comprising particles suspended in a liquid medium to an alternating magnetic field to reduce the tendency of the particles to aggregate on the flat surface. The system also includes: an irradiation tool for irradiating the particles deposited on the flat surface; a detector configured to generate a signal indicating photons emitted from the irradiated detection molecules; and a processor configured to determine the presence of the detection molecules based at least in part on the signal from the detector.

[0008] The present invention relates to so-called image-based assays or planar assays, in which fluorescent signals from detection molecules are collected when particles are arranged on a planar surface. The particles can be randomly arranged on a planar surface, and their positions can be determined by additional fluorescent signals emitted from one or more dyes of the particles. This additional signal can also be used to determine the identity or type of the particles, as will be described below with reference to some embodiments of the present invention.

[0009] Compared to flow-based assays, the present invention provides several advantages. First, image-based assays may be more accessible and easier to use than flow-based assays, which typically require more trained operators to handle and maintain. Samples are arranged on a flat surface, such as on a porous plate, which typically makes handling easier than techniques in which samples are provided in the form of a fluid through a circulation pool. Secondly, the present invention allows non-contact reading of samples, which inherently protects the instrument from pathogen contamination and eliminates clogging problems. Third, arranging samples on a flat surface typically allows samples to be stored and subsequently reanalyzed. This is typically not the case in flow-based assays, which typically analyze samples immediately.

[0010] In addition, the inventors have discovered a previously unrecognized problem of particle aggregation on flat surfaces. It is believed that the aggregation may be caused by magnetic interactions between particles, which may be due to the residual magnetization of the particles. It has been observed that at least some particles can aggregate on flat surfaces when they are deposited on the surface and over time, that is, aggregate together, agglomerate, agglomerate or form larger structures. This problem has also been observed for particles that, according to the particle provider, are said to exhibit superparamagnetic properties. Aggregation can interfere with the analysis of the sample because it may be difficult to distinguish adjacent particles and their respective fluorescence signals. In addition, when determining the co-localized analyte signal specific to the irradiated detection molecule in the fluorescence image, aggregation can complicate the determination of the particle center, which is a key parameter in the image-based analysis using multiple dyed particles.

[0011] The inventors have realised that if the particles are exposed to an alternating magnetic field before they settle onto a flat surface, aggregation can be reduced. Exposing the particles to an alternating magnetic field can be referred to as "demagnetising" or "demagnetising" the particles, which can be understood as a reduction or substantially complete elimination of the magnetisation of the particles.

[0012] The particles can be exposed to an alternating magnetic field associated with the stirring of the sample. Stirring is generally performed to reduce any aggregation and form a suspension in which the particles are substantially uniformly distributed in the liquid medium. The particles can be exposed to the magnetic field before, during, or after stirring. In some instances, the particles can be at least partially stirred by exposure to a magnetic field.

[0013] After exposure to the magnetic field, the particles can be allowed to settle on the flat surface. This can be achieved by allowing the sample to rest while relying on the influence of gravity to cause the particles to sink toward the flat surface. However, sedimentation can be aided by applying an external force, such as the centrifugal force generated by a centrifuge.

[0014] The magnetic field may be generated by one or more electromagnets that can be operated to alternate or change the direction of the magnetic field to which the particles are exposed. The magnetic field may also be generated by one or more permanent magnets that can be moved to alternate or change the field direction.

[0015] In some embodiments, each of the plurality of particles can include a magnetic material, such as a ferromagnetic material. For example, the ferromagnetic material can be provided in the form of magnetic particles dispersed in the material forming the particles. Advantageously, the magnetic material can improve the ease and speed of separation. By applying a magnetic field, the particles (together with any combined analytes) can be taken out from the solution faster and separated from the unbound components while reducing sample loss. Compared with conventional centrifugation or filtering methods, this can simplify the cleaning steps of the assay and reduce the time required for these steps.

[0016] In some instances, the particles may comprise mainly superparamagnetic materials. In other words, the particles may comprise magnetic particles having a size that allows only a single magnetic domain to be formed in the particles. As is well known in the art, superparamagnetic materials generally exhibit relatively high magnetic susceptibility, thereby allowing them to be relatively strongly magnetized in an external field. Therefore, for example, during cleaning and separation, the particles can be relatively effectively controlled. In addition, it is also known that when the external magnetic field is removed, superparamagnetic materials exhibit a residual magnetization that is substantially zero. Advantageously, this can reduce their tendency to form clumps on flat surfaces.

[0017] However, by applying an alternating magnetic field as described above, aggregation can be further reduced. This may be caused by the alternating magnetic field removing or at least reducing the residual magnetization in the particles, also for predominantly superparamagnetic particles. The alternating magnetic field can help reduce the alignment of the magnetic moments of the magnetic domains of the particles, thereby increasing the degree of randomization of the magnetic domains.

[0018] The particle can have a shape that conforms to a sphere, and can also be referred to as a microsphere, a bead, or a microbead. However, other shapes and forms are also possible, and it should be understood that the concepts of the present invention as described herein may not necessarily be limited to spherical particles. Other suitable shapes include rods, stars, tubes, plates, wires, and shells (spherical particles with hollow cores). However, for purposes disclosed herein, the terms "particle" and "microsphere" are used interchangeably to illustrate some examples of suitable particles.

[0019] The detection molecule may contain or be conjugated with a fluorescent marker, which is also known as a fluorophore or fluorescent dye, which is configured to absorb light of a specific wavelength and re-emit light typically at a longer wavelength. Different fluorescent markers may be used to distinguish different detection molecules that bind to different analytes. Each fluorescent marker may have a unique emission spectrum, allowing them to emit light at different wavelengths when excited by a light source. The use of different fluorescent markers allows for the simultaneous detection of multiple analytes in a single sample, as each analyte can be associated with a unique fluorescent signal. Examples of fluorescent markers range from small organic dyes to large quantum dots and nanoparticles.

[0020] Detection molecules may include antibodies, which may be understood as proteins that can bind to a specific target analyte such as an antigen with high specificity and affinity. Other examples include aptamers, which may be understood as short chains of DNA or RNA that are engineered to fold into a specific three-dimensional shape that can bind to a specific target molecule. Still other examples include peptides and various enzymes.

[0021] In some instances, the detection molecule can be configured to bind to the analyte, which in turn can bind to the particle via the capture molecule. The capture molecule can be configured to selectively bind to a specific analyte and separate the specific analyte from the sample. The capture molecule is typically bound to a solid support such as a particle to facilitate separation of the analyte from the rest of the sample. Typical examples of capture molecules include antibodies, aptamers, peptides, and lectins.

[0022] In some instances, each particle can be dyed with a fluorophore, which can be configured similarly to the fluorophores discussed above for the detection molecules. The light emitted from the dye can then be used to determine the position of the particle on the flat surface. Therefore, a position that can represent the center of the particle can be determined without using any dedicated structure on the flat surface, such as a groove or indentation that is commonly used to locate the particle in the prior art. Information about the position of the particle can be used to improve the efficiency and accuracy of the analysis of the light signal emitted by the detection molecule labeled with the fluorophore. For example, the determined position can be used to delimit an area of ​​interest in an image of a flat surface, in which a signal can be expected to be found. The position of the particle can also make it easier to identify and distinguish signals from two or more particles that are arranged relatively close to each other.

[0023] The particles can also be dyed to achieve multiplexing. In some instances, the particles are dyed with different fluorescent dyes. As mentioned, each dye can have a unique emission spectrum that allows the dye to be distinguished from other dyes when excited with light of a specific wavelength. By using different dye combinations, a relatively large number of particle subsets of different colors can be produced. By allowing each subset to capture a specific analyte, the multiplexing capability of the assay can be improved.

[0024] In one example, each subset of particles can be dyed with one or more fluorescent dyes. The concentration of each dye used can be changed to produce particles with different color intensities. By coupling each uniquely dyed subset of particles with a capture molecule specific for a particular analyte, the dyed subsets can be mixed, and different colors are used to distinguish between the subsets and therefore different analytes. For multiplexed detection, reducing the degree of aggregation of the particles can be particularly beneficial because this can make it easier to distinguish between different subsets of particles and their co-located analyte signals.

[0025] Thus, in one example, a first subset of particles may be stained with a first concentration of a first type of fluorophore and a second concentration of a second type of fluorophore. Additionally, a second subset of particles may be stained with a third concentration of the first type of fluorophore and a fourth concentration of the second type of fluorophore. The ratio between the first concentration and the second concentration may be greater than the ratio between the third concentration and the fourth concentration. Then, for each of the plurality of particles, it may be determined that the particle forms part of the first subset or the second subset based, at least in part, on a difference in the intensity of detected photons emitted from the first type of fluorophore and the second type of fluorophore, respectively.

[0026] The concepts of the present invention may be applied to any research technique involving the analysis of a plurality of particles, such as microspheres or microbeads, arranged on a flat surface.The confocal scanning microscopy discussed below is to be understood as an illustrative example only.

[0027] Other features and advantages of the present invention will become apparent from the following description of preferred embodiments of the invention, given by way of example only, and made with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A system according to an embodiment of the invention is schematically shown.

[0029] Figure 2 A fluorophore-labeled detection molecule bound to a particle according to an embodiment of the present invention is schematically shown.

[0030] Figure 3a-3d This is a photograph of particles settling on a flat surface.

[0031] Figure 4 is a flow chart showing a method according to an embodiment of the present invention. DETAILED DESCRIPTION

[0032] Figure 1 A system 100 for determining the presence of a plurality of fluorophore-labeled detection molecules bound to a plurality of particles 110 is schematically shown. The system 100 includes an illumination tool 140 configured to illuminate the particles 110 when the particles 110 are deposited on a flat surface 122 of a container 120, and a detector 150 configured to generate a signal indicating photons emitted from the fluorophore-labeled detection molecules when illuminated. In addition, a processor 160 is provided to determine the presence of the detection molecules based on the signal from the detector 150. The illumination tool 140, the detector 150, and the processor 160 may form part of a reader 180, such as a confocal laser scanning microscope. However, the inventive concept that the aggregation of particles 110 may be reduced by exposure to an alternating magnetic field may also be used in other types of assays using other types of imaging techniques other than confocal laser scanning microscopy.

[0033] The system 100 may further include a stirrer 170 configured to stir the sample and a magnetic field generator 130 configured to expose the sample to an alternating magnetic field B.

[0034] It should be noted that the multiple components of the above-mentioned system 100, i.e., the irradiation tool 140, the detector 150, the processor 160, the stirrer 170 and the magnetic field generator 130 can be arranged as structurally separated components, or integrated in one or more common platforms. Therefore, in one example, the irradiation tool 140, the detector 150 and the processor 160 can be arranged in the reader 180, and the stirrer 170 and the magnetic field generator 130 can be arranged as separate independent units. In some examples, the magnetic field generator 130 and the stirrer 170 can be integrated into a single unit. In some examples, the magnetic field generator 130 and / or the stirrer 170 can be integrated into the reader 180 to help the automation of the smooth processing and / or analysis process. In other examples, the processor 160 or at least a part of its functionality can be arranged in a separate location, such as in a separate server forming a part of a local area network or in a cloud-based server.

[0035] The particles 110 may be provided in a liquid medium 115, such as distilled water or a buffer solution, such as phosphate buffered saline (PBS), contained in a container 120. The container 120 may be, for example, a well of a multi-well plate (not shown). Figure 1 The sample is shown in three different situations - when exposed to an alternating magnetic field B from a magnetic field generator 130, when stirred by a stirrer 170, and when analyzed in a reader 180. The stirrer 170 can be used to help distribute the particles 110 throughout the liquid medium 115, thereby creating a particle suspension. The particles 110 can then be allowed to settle on the flat surface 122 forming the bottom of the container 120 before the sample is analyzed in the reader 180. It will be appreciated that when the particles 110 are suspended in the liquid medium 115 (e.g., Figure 1 ), the particles 110 may be exposed to an alternating magnetic field B while the particles 110 are deposited on the flat surface 122, or while the particles 110 are arranged in any state therebetween.

[0036] The illumination tool 140 and the detector 150 can be configured to illuminate the sample and detect photons emitted or scattered from the sample. The light from the illumination tool 140 or the light source can be directed to the sample by a first optical system (e.g., including a lens 144, a beam splitter 142, and an objective lens 148). The first optical system can be configured to direct the light in a scanning motion on the sample so that the fluorescent marker of the dye of the detection molecule or particle 110 emits fluorescence. Therefore, the sample can be scanned by at least two different types of light-the wavelength of the first light excites the fluorescent marker of the detection molecule, and the wavelength of the second light excites the dye of the particle 110. In one example, light of a wavelength of 532nm can be used to generate a signal indicating an analyte, while 638nm can be used to generate a signal indicating the particle 110.

[0037] For example, scanning can be achieved by a galvanometer mirror that moves rapidly to scan light across the sample. Fluorescence can be collected by an objective lens 148, collimated and passed through a beam splitter 142. A second optical system 146 can direct the fluorescence to a detector 150, which can preferably be a light detector configured to generate a signal that can be processed by a processor 160. In addition, a sample stage (not shown) can be provided that is configured to move the sample in various directions to allow scanning and focusing on different portions of the sample.

[0038] However, it should be understood that the specific components and exact assembly of the system 100 disclosed herein are exemplary only. For example, the reader 180 may well use multiple light sources that emit light with different properties (such as wavelengths), and / or filters for providing light of the desired color, as well as enabling multiplexed detection. In addition, the configuration of the optical system including, for example, mirrors, beam splitters, apertures, pinholes, and filters may vary.

[0039] like Figure 1 As shown, the sample can be analyzed from below through the bottom of the container. Therefore, the flat surface 122 on which the particles 110 can rest when they settle can be formed by a light-transmitting material that allows light to be transmitted from the irradiation tool 140 to the sample and allows the fluorescence generated by the sample to escape to the detector 150 through the flat surface 122. In some instances, the bottom of the container can be formed by a light-transmitting or transparent plastic material or glass. However, in alternative instances, the sample can be irradiated from above through a liquid medium. The detector 150 can be arranged below the sample or above the sample.

[0040] The agitator 170 can be used to generate motion of the particles 110 in the liquid medium 115, typically by rotation or vibration, to keep the particles 110 substantially evenly distributed in the liquid medium 115 and to reduce the degree of aggregation. By placing a container 120 (such as a multi-well plate) in mechanical contact with the rotating portion of the agitator 170, the vibration can be transmitted to the sample.

[0041] The magnetic field generator 130 is an electrically operated device including one or more electromagnetic coils that can be combined with a capacitor to produce one or more oscillating LC circuits. When connected to a power source, the LC circuit begins to generate an oscillating current, which in turn generates an oscillating magnetic field B. The particles 110 can be placed in the magnetic field B, for example, by placing the container 120 on a dedicated surface of the magnetic field generator 130 to increase the randomization of the magnetic domains of the magnetic material of the particles 110. The sample can be moved or rotated to allow the magnetic field B to pass through the sample from different directions or angles. According to some observations, the magnetic field B can reduce or neutralize the magnetic field in each particle 110 by changing the arrangement of the magnetic domains within the magnetizable material of the particles 110. In magnetized particles, the magnetic domains are usually arranged in the same direction, which produces the overall magnetic field of the particles. However, when exposed to the alternating field B generated by the magnetic field generator 130, the magnetic domains can begin to change direction and follow the external field pattern. By designing the magnetic field generator 130 to repeatedly change the direction of the generated magnetic field B, the magnetic domains in the particles 110 can be flipped back and forth. As the magnetic field strength is reduced, for example, by increasing the distance between the magnetic field generator 130 and the particle 110, these flips can become less complete, ultimately leaving most of the magnetic domains in random orientations. The randomization of the magnetic domains means that their respective magnetic fields largely cancel each other out, resulting in the particle having little to no overall magnetization. Preferably, the particle 110 can be slowly removed from the magnetic field B while the magnetic field is still effective to prevent the magnetic domains from rearranging themselves to any residual magnetic field. The power, frequency, and duration of the demagnetization process can depend on factors such as the type of magnetic material of the particle 110, the size and shape of the particle 110, and the strength and orientation of any initial magnetization of the particle 110. The same is true for the direction or orientation of the alternating field, which can vary depending on the type of material of the particle 110 and any initial or residual magnetization. Therefore, it should be understood that the magnetic field B can be alternating in one, two, or three dimensions.

[0042] In an exemplary sequence, a container 120 including a sample is placed on a magnetic field generator 130 to expose the sample to an alternating magnetic field B, and then moved to an agitator 170, where the sample is agitated to more evenly distribute the particles 110 in a liquid medium 115. By moving the container 120 to the agitator 170, the sample can experience a gradual decrease in magnetic field strength as the sample leaves the magnetic field generator 130. The sample can then be placed aside to rest until at least some of the particles 110 settle on a flat surface 122 at the bottom of the container 120. This process can typically take 10-20 minutes, depending on the type of particles and the type of liquid medium. Thereafter, the sample can be inserted into a sample stage of a reader 180. In the case where the sample is arranged in a multi-well plate, the sample stage can be operated to move each well into the field of view of the detector 150, and the reading of the fluorescence signal can be initiated by scanning the sample with light from the irradiation means 140. The imaging of the sample can be substantially automatic, requiring little or no operator intervention.

[0043] Figure 2 is a cross-section of a particle 110 that is combined with a plurality of fluorophore-labeled detection molecules 114. Figure 1 The particles 110 and detection molecules 114 are configured in a similar manner to those discussed above. In this example, the detection molecules 114 are bound to the particles 110 via the analyte 113, which in turn is bound to the capture molecules 112 attached to the surface of the particles 110. Each of these components will be discussed in more detail below. It should be noted that Figure 2 is a schematic diagram of the functional elements of particle 110 and related molecules / substances, and sizes and measurements may be exaggerated for illustrative purposes.

[0044] The particles 110 can be magnetic microspheres, also referred to as beads or microbeads. The microspheres provide a solid carrier for the capture molecules 112, which are bound to the surface of the microspheres. The microspheres can typically be formed by a polymer matrix in which magnetic particles 116 are dispersed. The polymer matrix can provide structural stability and a surface for the microspheres, which can be modified to attach the capture molecules 112, while the magnetic particles 116 can provide the microspheres with their magnetic properties. As will be known to those skilled in the art, various types of polymers can be used for the matrix, including polystyrene and poly (lactic acid-co-glycolic acid) (PLGA).

[0045] Depending on the specific requirements of the assay, such as signal intensity and the need for a particular surface chemistry, particles 110 can be used in a variety of shapes and sizes, typically ranging in size from nanometers to micrometers. Relatively small microspheres can have a beneficial surface-to-volume ratio, which provides a relatively large surface for a given volume, on which capture molecules 112 can be attached. However, in absolute numbers, larger microspheres have a larger surface, which can bind more capture molecules 112. This in turn allows more analytes 113 and detection molecules 114 to bind to each particle 110, resulting in an increase in the intensity of the fluorescent signal emitted from the particle 110.

[0046] Larger particles are also more easily captured in a magnetic field and are therefore less likely to be lost during, for example, cleaning and separation processes. However, a disadvantage is that it has been observed that larger particles are more prone to aggregation. By exposing particles 110 to an alternating magnetic field, the size of particles 110 can be increased without increasing their tendency to aggregate. In some examples, the microspheres have an average diameter in the range of 2-10 μm, such as 4-8 μm. In the present example, the microspheres have an average diameter of about 8 μm.

[0047] The magnetic particles 116 may be formed of a ferromagnetic material, such as magnetite (Fe 2 O 4 ) or hematite (γ-Fe 2 O 3 ), and may have a size that allows the magnetic particles 116 to primarily exhibit superparamagnetism.

[0048] In some examples, the particles 110 may be coated with a layer that protects the magnetic particles 116 from exposure to the surrounding environment. The coating may further assist in the attachment of the capture molecules 112.

[0049] In addition, the microspheres can be dyed with one or more types of fluorophores or dyes (not shown) to enable identification of microspheres on a flat surface. Two or more subgroups or populations of microspheres can be dyed with two or more types of dyes, each dye having a different emission spectrum. By varying the relative concentrations of the dyes between different populations, the populations can be identified based on the intensity composition of the fluorescence emitted by the dyes.

[0050] Capture molecules 112 are provided to selectively bind to target analytes 113 and immobilize analytes 113 on a solid support provided by microspheres. In one example, capture molecules 112 are antibodies, such as monoclonal antibodies, which are specific to a particular epitope. Therefore, capture molecules 112 may also be referred to as capture antibodies.

[0051] Analyte 113 is a specific substance or molecular target that the assay is designed to detect. The nature of analyte 113 can vary widely based on the purpose of the assay. In one example, analyte 113 is a protein, such as a protein present in blood. For example, the protein can be prostate specific antigen or PSA, a specific antibody (such as an antibody produced in response to a viral infection, such as HIV), or a signal transduction protein, such as a cytokine (such as interleukin-6 or IL-6 in an inflammatory response).

[0052] The detection molecule 114 is typically designed to bind to a portion of the analyte 113 that is different from the portion that binds to the capture molecule 112. The detection molecule 114 can be labeled with a fluorophore that emits light of a specific wavelength when excited, and in some instances can be referred to as a fluorophore-conjugated antibody. By providing different detection molecules 114 with different fluorophores, each fluorophore has a unique excitation and emission spectrum, it can allow for the simultaneous detection of multiple analytes in a single sample. This can also be referred to as multiplexed detection.

[0053] Figure 3a-3d As mentioned above, Figure 1 The photographs are taken from a computer program product using a method such as that for Figure 1 The stirrer in question stirs two identical samples containing microspheres suspended in a liquid medium. The particles are then allowed to settle on a flat surface. Figure 3a and Figure 3b A first sample of particles 110 is shown in FIG. Figure 3a The particles are shown 10 minutes after stirring, and Figure 3b The particles are shown 40 minutes after stirring.

[0054] exist Figure 3c and Figure 3d A second sample of particles 110 is shown in FIG. Figure 3c The particles are shown 10 minutes after stirring, and Figure 3d The particles are shown 40 minutes after stirring. However, the second sample differs from the first sample in that, before stirring, the particles were stirred as described for Figure 1 The magnetic field generator in question exposes the particles 110 to an alternating magnetic field. Therefore, the goal of the experiment is to investigate whether exposure to an alternating magnetic field has any effect on the degree of aggregation in the short term (10 minutes) and in the long term (40 minutes). In this experimental procedure, 30 minutes is the time it takes for the reader to scan the entire multi-well plate. Therefore, the photo taken after 10 minutes can be considered to represent the first well scanned, and the photo taken after 40 minutes represents the last well scanned.

[0055] Will Figure 3a and Figure 3bIn comparison, the sample not exposed to the alternating magnetic field shows that the initial degree of aggregation increases with time. After 10 minutes, a considerable number of relatively small agglomerates are formed, which merge into larger agglomerates after 40 minutes. This trend of agglomerate formation and merging over time is shown in Figure 3c and Figure 3d are not visible in the figure, these figures show the sample that has been exposed to an alternating magnetic field. In addition, when comparing Figure 3a (10 minutes, not demagnetized) and Figure 3c (10 minutes, demagnetization), Figure 3c The initial aggregation degree of the samples in was significantly lower.

[0056] In summary, the extent of aggregation can be significantly reduced by exposing the particles to an alternating magnetic field before allowing them to settle onto a flat surface. This effect appears to last for at least over the 40 minutes during which the particles were observed in the experiment, and has been observed to persist for several hours thereafter, as long as the particles remain in solution.

[0057] Figure 4 is a flow chart outlining a method 10 for determining the presence of a plurality of detection molecules according to some embodiments of the present invention. Figure 1 A system similar to the one shown in the figure is configured to bind the detection molecule to the particle, such as the one described above for Figure 2 The magnetic microspheres in question.

[0058] according to Figure 4 In the example shown in FIG. 1 , method 10 includes providing 12 a sample including particles suspended in a liquid medium. It should be noted that the particles may have been subjected to some treatment in previous steps, which is not described in the present invention. Figure 4 In the previous steps, for example, the particles can be dyed with one or more fluorescent dyes to allow detection and identification of the particles, and the particles can be functionalized, that is, the particles are treated to introduce reactive groups on their surface. These groups can be chemically reactive and allow covalent attachment to capture the molecules to be formed. The functionalized particles can be mixed with capture molecules to allow the capture molecules to be covalently attached to the reactive groups on the surface of the particles. After this process, excess capture molecules can be washed away, leaving particles with capture molecules bound to their surfaces. The capture molecules can then be exposed to target analytes bound to the capture molecules, and unbound substances can be washed away after a specific incubation period. Finally, the particles can be exposed to fluorophore-labeled detection molecules, which can bind to the analyte, and the excess detection molecules can then be washed away.

[0059] The resulting particles may be a complex of particle-capture molecule-analyte-detection molecule provided 12 in step. In a next step, the particles may be exposed 14 to an alternating magnetic field to reduce the degree of aggregation of the particles. Additionally, the particles may be stirred 15 to further reduce the degree of aggregation. Thereafter, the particles may be allowed to settle 16 on a flat surface.

[0060] When at least some of the particles, such as a majority of the particles, have settled on the flat surface, the particles may be illuminated 18 by a light source, such as a laser from a confocal laser scanning microscope. The resulting fluorescence emitted from the fluorescent label of the detection molecule may then be detected 20, and the presence of the detection molecule determined 22 based at least in part on the detected photons. In some examples, the method 10 may also include a step of determining 21 that the particles form part of a particular subgroup or population of particles based at least in part on a difference in intensities of photons emitted from the first type of dye and the second type of dye of the detected particles.

[0061] The above embodiments should be understood as illustrative examples of the present invention. Other embodiments of the present invention are contemplated. For example, the particles may not necessarily be limited to microspheres. Other particles, such as rods or ellipsoids, are also contemplated. In addition, other types of imaging techniques may also be possible, including fluorescence microscopy. It should be understood that any feature described in conjunction with any one embodiment may be used alone, or in combination with other described features, and may also be used in combination with one or more features of any other embodiment or any combination of any other embodiments. In addition, equivalent forms and modifications not described above may also be used without departing from the scope of the present invention, and the scope of the present invention is defined in the appended claims.

Claims

1. A method for determining the presence of a plurality of fluorophore-labeled detection molecules bound to a plurality of particles arranged on a planar surface, the method comprising: providing a sample comprising the particles suspended in a liquid medium; exposing the particles to an alternating magnetic field to reduce the tendency of the particles to aggregate on the planar surface; allowing the particles to settle on the planar surface; irradiating the particles; detecting photons emitted from the illuminated detection molecules; and The presence of the detection molecule is determined based at least in part on the detected photons.

2. The method of claim 1, wherein each detection molecule is bound to an analyte, wherein the analyte is bound to a capture molecule, and wherein the capture molecule is bound to the particle.

3. The method of claim 2, wherein the capture molecule is a capture antibody, and wherein the analyte is a protein present in blood.

4. The method of claim 1, wherein each particle is dyed with a fluorophore, and wherein the method further comprises detecting photons emitted from the fluorophore to determine the position of the particle on the planar surface.

5. The method according to claim 4, wherein: staining a first subset of the particles with a first concentration of a first type of fluorophore and a second concentration of a second type of fluorophore; staining a second subset of the particles with a third concentration of the first type of fluorophore and a fourth concentration of the second type of fluorophore; and A ratio of the first concentration to the second concentration is greater than a ratio of the third concentration to the fourth concentration.

6. The method according to claim 5 also includes, for each of the plurality of particles, determining that the particle forms part of the first subgroup or the second subgroup based at least in part on the difference in intensity detected from the first type of fluorophore and the second type of fluorophore, respectively.

7. The method of claim 1, wherein each of the plurality of particles comprises a ferromagnetic material, and wherein exposing the ferromagnetic material to the alternating magnetic field comprises at least partially randomizing magnetic domains of the ferromagnetic material.

8. A method according to claim 7, wherein the ferromagnetic material is provided as a plurality of magnetic particles having a size allowing them to be predominantly superparamagnetic.

9. The method of claim 1, wherein each of the plurality of particles is substantially spherical, and wherein an average diameter of the plurality of particles is in the range of 2-10 μm.

10. The method of claim 1, further comprising agitating the sample to further reduce the tendency of the particles to aggregate.

11. The method of claim 1, wherein exposing the particles to an alternating magnetic field comprises gradually decreasing the strength of the magnetic field.

12. A system for determining the presence of a plurality of fluorophore-labeled detection molecules bound to a plurality of particles arranged on a planar surface, the system comprising: a magnetic field generator configured to expose a sample comprising said particles suspended in a liquid medium to an alternating magnetic field to reduce a tendency of said particles to aggregate on said planar surface; irradiation means for irradiating said particles deposited on said flat surface; a detector configured to generate a signal indicative of photons emitted from the illuminated detection molecules; and A processor is configured to determine the presence of the detection molecule based at least in part on the signal from the detector.

13. The system of claim 12, further comprising a stirrer configured to stir the sample to further reduce the tendency of the particles to aggregate, wherein the stirrer is structurally integrated with the magnetic field generator.

14. A system according to claim 12 or 13, wherein each particle is dyed with a fluorophore, wherein the detector is configured to generate a signal indicative of photons emitted from the fluorophore, and wherein the processor is configured to determine the position of the particle based at least in part on the signal from the detector.

15. A system according to claim 12 or 13, wherein the illumination means and the detector form part of a confocal laser scanning microscope.

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