Single-molecule detection method based on raster-scan recognition

Abstract

A single molecule detection method based on raster scan recognition, in which a large number of capture beads are arranged on a bead fixing plate in a dispersed manner, at least some of the capture beads are specifically bound to an analyte, and each analyte is bound to a luminescent substance, and the grid openings of a grid detection device are used to start scanning the capture beads row by row starting from the capture beads arranged at one end, the grid detection device synchronously counts the number of capture beads bound to markers, and the capture beads on the entire bead fixing plate are scanned by the relative lateral movement of the grid openings and the capture beads, and the total number of analytes is obtained when the scan is completed. The present invention significantly improves the detection speed by counting the number of capture beads bound to markers while scanning the capture beads, and obtaining the number of detected objects in real time.

Description

[Technical Field]

[0001] The present invention relates to a detection method, in particular to a single molecule detection method based on raster scan recognition, which belongs to the technical field of single molecule detection. [Background technology]

[0002] Single Molecule Detection (SMD) is an ultra-sensitive detection technology that has developed rapidly in recent years. It refers to measuring and analyzing targets at the single-molecule level. It is a new detection method and has opened up a new field of detection. This detection method can detect very low concentrations of analytes, and requires very little reagent and space. For example, in "CN202080000774.8 - Single Molecule Quantitative Detection Method and Detection System" disclosed by Suzhou Yushou Biotechnology Co., Ltd., in situ signal-enhancing nanoparticles with optical effects are introduced during the immunological recognition process to mark the target molecules, thereby enhancing the signal generated by the original luminescent material and improving recognition. Then, an optical imaging device is used to obtain the number of target molecules with spots. Also, for example, in "CN201180019462.2 - Ultrasensitive detection of molecules or particles using beads or other captures" disclosed by Quanterix, USA, the captures are spatially divided into certain sections, then imaged, and the concentration is determined by counting the number of analytes that emit light in the image. Both of these methods can significantly improve detection sensitivity. However, since both of these methods involve counting the number of analytes that emit light in the image after imaging, the requirements for imaging equipment are high and the detection speed is slow. Summary of the Invention [Problem to be solved by the invention]

[0003] In response to the problems of current single-molecule detection methods using imaging methods to calculate the concentration of analytes, which results in high requirements for imaging equipment and slow detection speeds, the present invention proposes a single-molecule detection method based on raster scan recognition, which uses the raster scan method to obtain the number of captured analytes in real time, resulting in simple equipment and fast detection speeds. [Means for solving the problem]

[0004] The technical means by which the present invention solves the above-mentioned problems is a single-molecule detection method based on raster scan recognition, in which a large number of capture beads are arranged dispersedly on a bead fixing plate (or detection chip), at least some of the capture beads are specifically bound to one analyte, and each analyte is bound to a luminescent substance, and the grid openings of the grid detection device are used to scan the capture beads row by row, starting from the capture beads arranged at one end, and the grid detection device synchronously counts the number of capture beads bound to markers, and the capture beads are scanned across the entire bead fixing plate by moving the grid openings and the capture beads laterally relative to each other, and the total number of analytes is obtained when the scan is completed.

[0005] Furthermore, one set of grating detection devices includes a grating head, an optical sensor, and a signal processor, wherein the grating head includes a plurality of independent grating openings arranged in a row, and the optical sensor includes a plurality of independent sensing elements, and during scanning, one grating opening is aligned with one capture bead or not aligned with the capture bead, one sensing element corresponds to one grating opening, and can sense the light incident on this grating opening, and when the sensing element senses the incident light, it feeds back to the signal processor, and the signal processor calculates the number of analytes according to the feedback result.

[0006] Furthermore, the sensing element includes photosensitive tubes that output pulse signals to a signal processor upon sensing the incident light, and the signal processor calculates the number of analytes according to the number of photosensitive tubes that output pulse signals.

[0007] Furthermore, the grid openings of one set of grid detection devices are arranged in one column, the number of grid openings in the whole column is equal to or greater than the number of rows of capture beads arranged on the bead fixing plate, and the center distance between two adjacent grid openings is equal to the center distance between two adjacent rows of capture beads in the same column.

[0008] Furthermore, the grid openings of one set of grid detection devices are arranged in a staggered pattern in two columns, the total number of grid openings in the two columns is equal to or greater than the number of rows of capture beads on the bead fixing plate, the center distance between adjacent grid openings in each column is equal to the center distance between two rows of capture beads every other row in the same column, and the grid openings in the two columns work together to scan one column of capture beads.

[0009] Being arranged in a staggered pattern means that the positions between adjacent grid openings are V-shaped, and when one column of grid openings is aligned with capture beads in several rows on the bead fixing plate, another column of grid openings is aligned with capture beads in the row adjacent to the capture beads; that is, two adjacent rows of capture beads arranged in the same column are not aligned with one column of grid openings at the same time, but are aligned with the two columns of grid openings in turn during lateral movement along the rows of the capture beads and grid openings.

[0010] Furthermore, the center-to-center distance between two adjacent capture beads is at least twice the outer diameter of the capture beads.

[0011] Furthermore, the center distance between two adjacent capture beads is at least three times the outer diameter of the capture beads.

[0012] Furthermore, the distance between the lattice opening and the surface of the capture bead is no more than twice the outer diameter of the capture bead.

[0013] Furthermore, the distance between the grid opening and the surface of the capture bead is equal to 1.5, 1, or 0.5 times the outer diameter of the capture bead. Theoretically, the shorter the distance between the grid opening and the surface of the capture bead, the better, provided that the grid opening does not come into contact with the material on the surface of the capture bead. This allows as much light emitted by the marker as possible to enter the grid opening, and avoids light loss that would otherwise occur and cause inaccurate detection results.

[0014] Furthermore, the inner diameter of one lattice opening is 0.5 to 1.5 times the outer diameter of the capture bead.

[0015] Furthermore, the inner diameter of one grid opening is equal to the outer diameter of the capture bead. Preferably, the area of ​​the light-transmitting region of one grid opening is close to the area of ​​the light-emitting region of the capture bead projected onto the grid opening, and the distance between the grid opening and the surface of the capture bead is controlled, so that the synergistic effect of these factors ensures that the maximum amount of light emitted from the light-emitting substance on the surface of the capture bead can be incident into the grid opening, while preventing light emitted from the light-emitting substance on the surface of another adjacent capture bead from incident into this grid opening and affecting the detection results.

[0016] Preferably, the capture beads are arranged in an array on a bead fixing plate to minimize the size of the device and control the amount of light that can enter the grid opening, not only to turn on the sensing element and transmit a signal, but also to prevent light from the surface of adjacent capture beads from entering the grid opening and causing the sensing element to transmit an erroneous signal.

[0017] Additionally, luminescent materials include fluorescent or chemiluminescent reagents.

[0018] Furthermore, when the luminescent substance is fluorescent, an excitation light source that irradiates the capture beads is installed near the bead fixing plate.

[0019] Furthermore, there are two or more types of analytes, two or more types of luminescent substances, one type of analyte is bound to one type of luminescent substance, there are two or more sets of lattice detection devices, and one set of lattice detection devices counts one type of luminescent substance.

[0020] The beneficial effects of the present invention are as follows:

[0021] 1. The present invention significantly improves the detection speed by counting the number of capture beads to which markers are bound while scanning the capture beads and obtaining the number of detected objects in real time.

[0022] 2. The present invention can realize real-time statistics of the number of detected objects by converting the optical signal of the luminous object into an electrical signal during scanning.

[0023] 3. In the present invention, the arrangement of the grating openings in a staggered two-row configuration corresponds to increasing the distance between adjacent grating openings, which avoids optical interference between adjacent capture beads during synchronous detection and improves the accuracy of the detection results. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 is a schematic diagram showing magnetic beads arranged on a microwell array plate in Example 1. [Figure 2] FIG. 2 is a schematic diagram of automatic scan recognition in the first embodiment. [Figure 3] FIG. 2 is a schematic diagram illustrating an arrangement of grating openings of a grating head in Example 1. [Figure 4] FIG. 10 is a schematic diagram illustrating the arrangement of grating openings of a grating head in Example 2. [Figure 5] FIG. 10 is a schematic diagram showing the correspondence between a top view of a microwell array plate and a bottom view of a grid opening. [Figure 6A] 1A-1C are schematic diagrams of signals corresponding to the detection of luminescent magnetic beads arranged in different positions. [Figure 6B] 1A-1C are schematic diagrams of signals corresponding to the detection of luminescent magnetic beads arranged in different positions. [Figure 6C] 1A-1C are schematic diagrams of signals corresponding to the detection of luminescent magnetic beads arranged in different positions. [Figure 6D] 1A-1C are schematic diagrams of signals corresponding to the detection of luminescent magnetic beads arranged in different positions. [Figure 6E] 1A-1C are schematic diagrams of signals corresponding to the detection of luminescent magnetic beads arranged in different positions. [Figure 6F] 1A-1C are schematic diagrams of signals corresponding to the detection of luminescent magnetic beads arranged in different positions. [Figure 7] FIG. 10 is a schematic diagram showing the positional relationship between an excitation light source, capture beads, and a lattice detection device in Example 3. [Figure 8] 10A to 10C are schematic diagrams illustrating two types of installation forms of the lattice detection device in Example 4. [Figure 9] 10A to 10C are schematic diagrams illustrating two types of installation forms of the lattice detection device in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention will now be further described with reference to the drawings. [Example]

[0026] In this example, the markers are fluorescent substances, the capture beads are magnetic beads, the bead immobilization plate is a long microwell array plate with a microwell array, some microwells contain one magnetic bead and some microwells contain no magnetic beads, at most one analyte is specifically bound to the surface of each magnetic bead, and each analyte is bound to a fluorescent marker. The magnetic beads (capture beads) in adjacent rows or columns described herein refer to magnetic beads (capture beads) in microwells in adjacent rows or columns in the array. Even if there are no magnetic beads in the microwells in these adjacent rows or columns, the description of the magnetic beads (capture beads) in adjacent rows or columns in this specification still refers to the positions of the microwells in the adjacent rows or columns in the array.

[0027] This is a single molecule detection method based on raster scan recognition, in which a certain number of magnetic beads 12 are arranged on a microwell array plate 10, as shown in Figure 1, some of the magnetic beads 12 have no analyte bound to their surface, some have an analyte bound to their surface, i.e., a fluorescent marker 13 bound to them, and at most one magnetic bead 12 is arranged in each microwell 11, and some microwells 11 have no magnetic beads 12.

[0028] Next, raster scan recognition and detection are performed. As shown in FIG. 2, an excitation light source 24 is installed above the microwell array plate 10. The emitted excitation light is reflected by a reflecting mirror 25 and irradiated onto the surface of the magnetic beads 12 to be scanned. After excitation is complete, the excitation light is turned off. The grid head 21 of the grid detection device 20 is placed above the microwell array plate 10 (although it can also be installed below), and the grid openings 211 of the grid head 21 are aligned with the magnetic beads 12 so that the fluorescence emitted after excitation of the fluorescent markers 13 can be maximized and enter the grid openings 211. As shown in FIG. 3, the number of grid openings 211 must be equal to or greater than the number of microwells 11 in one row of the microwell array plate 10, and the center-to-center distance between two adjacent grid openings 211 must be equal to the center-to-center distance between two adjacent microwells 11, allowing all rows of magnetic beads 12 in the microwell array plate 10 to be scanned and recognized simultaneously. In addition, in order to accurately count the number of grating openings 211 into which fluorescence is incident, the optical sensor 22 of the grating detection device 20 is installed at the other end opposite the grating openings 211 of the grating head 21, and the sensing elements such as photosensitive tubes of the optical sensor 22 correspond one-to-one to the grating openings 211. When fluorescence is incident on a specific grating opening 211, the corresponding sensing element receives the incident light and transmits a signal to the signal processor 23 of the grating detection device 20. For example, when the photosensitive tube senses the light, it can generate a pulse signal as a signal. The signal processor 23 receives this pulse signal and performs further processing. By counting the number of signals transmitted by the optical sensor 22, the number of fluorescent magnetic beads 12 can be counted, and the number of specific analytes can be obtained. Of course, the method of converting this optical signal into an electrical signal for counting can be easily realized by adopting conventional technology.

[0029] During the scanning recognition process, one row of magnetic beads 12 is detected each time, so to complete the detection of all the magnetic beads 12 in the microwell array, relative movement between the microwell array plate 10 and the grid detection device 20 is required, so that the grid head 21 passes above (or below) each row of microwells 12 in turn for detection. As shown in Figure 2, this relative movement can be achieved by horizontal movement of the microwell array plate 10 to the right, or of course, by horizontal movement of the grid head 21 to the left.

[0030] In this detection method, the number of all magnetic beads 12 is determined when the magnetic beads 12 are arranged, and then the number of luminescent magnetic beads 12, i.e., the number of analytes, is scanned and recognized to obtain the proportion of the analyte, and the concentration of the analyte in the detection solution can be obtained by comparing it with a standard concentration curve. [Example]

[0031] The principle of this embodiment is the same as that of embodiment 1, except that the arrangement of the lattice openings 211 of the lattice head 21 is different. As shown in FIG. 4, the lattice openings 211 are arranged in a staggered pattern in two rows, that is, the positional relationship between adjacent lattice openings 211 is V-shaped, which increases the distance between two adjacent lattice openings 211. In this case, the total number of lattice openings 211 in the two rows is greater than the number of microwells 11 in one row. As shown in FIG. 5, during the scanning recognition process, after the lattice head 21 is aligned with the microwell array plate 10 from above, the lattice opening 211 marked A' is located directly above the magnetic bead 12 marked A and is close to the magnetic bead 12, and the lattice opening 211 marked B' is located directly above the magnetic bead 12 marked B and is close to the magnetic bead 12. During the detection process, if the microwell array plate 10 moves laterally from left to right to achieve relative movement between the microwell array plate 10 and the grid detection device 20, when scanning recognition begins, the grid head 21 is positioned above the microwells 12 in the rightmost row of the microwell array plate 10, and the grid opening 211 marked B' is aligned with and detects the magnetic bead 12 marked B, while the grid opening 211 marked A' does not face the microwell 11, and the magnetic bead 12 marked A is not detected in this case. After the grid opening 211 marked B' detects the magnetic bead 12 marked B, the microwell array plate 10 moves laterally from left to right to complete the relative movement between the microwell array plate 10 and the grid detection device 20. In this case, the grid opening 211 marked B' is aligned with the magnetic bead 12 in the next adjacent column in the same row as the magnetic bead marked B, and the grid opening 211 marked A' is aligned with the magnetic bead 12 marked A, completing the synchronous detection of the magnetic beads 12 at the offset locations in the two adjacent columns. This process is repeated until detection of all magnetic beads 12 in the microwell array plate 10 is completed. In this way, increasing the distance between adjacent grid openings 211 reduces optical interference between adjacent magnetic beads 12, resulting in more accurate detection results.

[0032] Detection results According to the experimental results, in the following, the detection results are illustrated by way of example, and in order to simplify the experimental operation and facilitate understanding, only one grating opening 211 and one photosensitive tube are used for the operation.

[0033] As shown in Figure 6A, when one luminescent capture bead passes under the grating opening, it is processed by the signal processor to generate one normal signal wave. As shown in Figure 6B, when two luminescent capture beads, spaced apart, pass under the grating opening in sequence, it is processed by the signal processor to generate two normal signal waves, with clear and uniform peak-to-valley intervals. As shown in Figure 6C, when two luminescent capture beads are too close together and partially overlap at the edges, the signal waves generated by the signal processor have peaks that are too close and the valleys are not clear. As shown in Figure 6D, when three luminescent capture beads that are too close to each other pass under the grating opening in sequence, the signal waves generated by the signal processor have peaks and valleys that are not clearly distinguishable. As shown in Figures 6E and 6F, when three luminescent capture beads are not collinear, with one capture bead aligned directly under the grating opening and the other two positioned near the grating opening, both of these conditions affect detection and prevent the generation of normal signal waves.

[0034] Therefore, to ensure accurate detection results and achieve a compact detection system, it is important to select capture beads with appropriate specifications, such as beads with a diameter of 2.7 μm. When arranging the capture beads, the center-to-center distance between adjacent capture beads should be controlled to 2.5–3 times the diameter. Furthermore, the size of the lattice openings should be minimized, such as by controlling the light transmission area within the cross-sectional area of ​​the capture beads, so that the transmitted light can turn on the sensing element and transmit a signal that can be easily recognized by the signal processor. Furthermore, the distance between the lattice openings and the surface of the capture beads should be minimized to prevent light from neighboring emitting capture beads from entering the lattice openings, thereby reducing optical interference at adjacent positions.

[0035] Although the above examples use magnetic beads as capture beads, in actual use, other shapes of magnetic or non-magnetic particles may be used, the analyte may be a protein, a nucleic acid molecule, etc., and the bead fixing plate may have a hole or a slightly recessed pit structure, or may even be flat. If the bead fixing plate has holes or pits, these holes or pits are pre-fabricated and can be processed into a regular array shape, allowing the capture beads to be regularly arranged in the holes or pits, making it easier for the row-arranged lattice openings to cooperate with them. If the bead fixing plate is flat, some auxiliary structures may be used to arrange the capture beads in a regular array on the surface of the bead fixing plate, or the capture beads may be randomly dispersed on the surface of the bead fixing plate. In the case of randomly distributing and arranging capture beads on a plane, the distance between the capture beads needs to be as large as possible to avoid the capture beads being too close and therefore gathering into clusters. Therefore, for the same number of capture beads, when dispersed on a plane, the surface area of ​​the bead fixing plate is larger than the surface area of ​​the bead fixing plate with holes or pits, and the positional relationship between the capture beads is highly irregular. However, generally speaking, the capture beads can still be considered to be arranged in a multi-row and multi-column array, although there are no capture beads at many positions in this array, and the capture beads in the same row or column are not strictly collinear. Therefore, the column-by-column scanning detection method in Example 1 or Example 2 above can still be adopted. [Example]

[0036] 7, in this embodiment, the bead fixing plate and the lattice detection device 20 are installed one above the other, and two or more excitation light sources 24 and reflective mirrors 25 are evenly arranged around the periphery of the bead fixing plate, and during the detection process, the excitation light sources 24 and reflective mirrors 25 remain stationary, while the bead fixing plate or the lattice detection device 20 moves within the space between the excitation light sources 24. Of course, the excitation light sources 24 may be installed in other ways as long as they can ensure that the surface of each capture bead is illuminated and can excite the markers on its surface to emit fluorescence. [Example]

[0037] In this embodiment, two or more analytes can be detected on one bead-immobilized plate. In this case, different luminescent materials are bound to different analytes, and the excitation light source 24 must also be capable of emitting light of two or more wavelengths. Two or more sets of lattice detectors 20 are installed, and each set of lattice detectors 20 detects and counts one analyte. The two or more sets of lattice detectors 20 may be installed in various forms.

[0038] (1) As shown in Figure 8, each set of grid detection devices 20 includes a grid head 21, an optical sensor 22, and a signal processor 23 that are connected to each other. Two or more sets of grid detection devices 20 are installed in parallel in a row, and scanning begins with the captured beads at the same end of the bead fixing plate, and scanning detection of all captured beads is completed by relative movement between the bead fixing plate and the grid detection devices 20.

[0039] (2) As shown in Figure 9, two or more sets of lattice detectors 20 are symmetrically mounted on a bracket 26, which supports the bead, via a single rotation center 27. The rotation center 27 can rotate around the bracket 26. If there are two sets of lattice detectors 20, they are mounted on both ends of the rotation center 27. If there are three sets of lattice detectors 20, the angle between two adjacent sets of lattice detectors is 120°. If there are more sets of lattice detectors 20, they are also symmetrically mounted according to this rule. During scanning, one set of lattice detectors 20 starts scanning one analyte from one end of the bead fixing plate, and after scanning to the other end and completing the scanning of that analyte, the rotation center 27 is rotated, and the other set of lattice detectors 20 is moved to the end of the bead fixing plate. The bead fixing plate or bracket 26 moves in the opposite direction to the previous detection process, completing the scanning of the second analyte. This process is repeated in sequence until all analytes have been scanned.

[0040] (3) In the embodiment of two or more sets of grating detection devices, a set of grating head 21, optical sensor 22 and signal processor 23 is installed, and the filter at the grating opening of grating head 21 is replaceable. In step 1, the required filter for the grid opening is selected, and the grid detection device 20 starts scanning from the capture bead (start end) at one end of the bead fixing plate, and through the relative movement between the bead fixing plate and the grid detection device 20, this set of grid detection device 20 scans to the capture bead (end end) at the other end of the bead fixing plate, completing the scanning detection of one kind of marker; In step 2, the filter at the lattice opening is replaced, and the lattice detection device 20 scans from the capture bead at the end of the bead fixing plate to the starting end of the scan in step 1 by relative movement between the bead fixing plate and the lattice detection device 20 in the opposite direction to the movement direction in step 1, completing the scan detection of another type of marker; Repeat until all markers have been scanned.

[0041] The above examples are only used to illustrate the present invention, and do not limit the present invention. Those skilled in the art can make various modifications and variations without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions should also fall within the protection scope of the present invention, and the protection scope of the present invention should be defined in each claim. [Explanation of symbols]

[0042] 10 Microwell Array Plates 11 microwells 12 Magnetic beads 13 Fluorescent Markers 20 Grating detector 21 Lattice Head 211 Lattice opening 22 Optical Sensor 23 Signal Processor 24 Excitation light source 25 Reflective mirror 26 Bracket 27 Center of rotation

Claims

1. A single molecule detection method based on raster scan recognition, comprising: distributing a large number of capture beads on a bead fixing plate or a detection chip; specifically binding one analyte to at least some of the capture beads; and binding a luminescent substance to each analyte; and using an array of apertures in a detection device, scanning the capture beads row by row, starting from the capture beads located at one end; the detection device synchronously counting the number of capture beads bound to markers; scanning the capture beads across the entire bead fixing plate by moving the apertures and the capture beads laterally relative to each other; and obtaining the total number of analytes upon completion of the scan. The set of detection devices includes a grid head, an optical sensor, and a signal processor, the grid head includes a plurality of independent openings arranged in a row, the optical sensor includes a plurality of independent sensing elements, and during scanning, one opening is aligned with one capture bead or not aligned with the capture bead, one sensing element corresponds to one of the openings and can sense the light incident on the opening, the sensing element senses the incident light and feeds it back to the signal processor, and the signal processor calculates the number of analytes according to the feedback result; A single molecule detection method based on raster scan recognition, characterized in that the openings of the detection device are arranged in a single row, the number of openings in the total row is equal to or greater than the number of rows of capture beads arranged on the bead fixing plate, and the center distance between two adjacent openings is equal to the center distance between two adjacent rows of capture beads in the same column.

2. A single molecule detection method based on raster scan recognition, comprising: distributing a large number of capture beads on a bead fixing plate or a detection chip; specifically binding one analyte to at least some of the capture beads; and binding a luminescent substance to each analyte; and using an array of apertures in a detection device, scanning the capture beads row by row, starting from the capture beads located at one end; the detection device synchronously counting the number of capture beads bound to markers; scanning the capture beads across the entire bead fixing plate by moving the apertures and the capture beads laterally relative to each other; and obtaining the total number of analytes upon completion of the scan. The set of detection devices includes a grid head, an optical sensor, and a signal processor, the grid head includes a plurality of independent openings arranged in a row, the optical sensor includes a plurality of independent sensing elements, and during scanning, one opening is aligned with one capture bead or not aligned with the capture bead, one sensing element corresponds to one of the openings and can sense the light incident on the opening, the sensing element senses the incident light and feeds it back to the signal processor, and the signal processor calculates the number of analytes according to the feedback result; A single molecule detection method based on raster scan recognition, characterized in that the openings of the detection device are arranged in a staggered pattern in two rows, the total number of openings in the two rows is equal to or greater than the number of rows of capture beads on the bead fixing plate, the center distance between adjacent openings in each row is equal to the center distance between two rows of capture beads every other row in the same column, and the openings in the two rows cooperate to scan one row of capture beads.

3. The method for single molecule detection based on raster scan recognition according to claim 1 or 2, characterized in that the sensing element includes a photomultiplier tube, which outputs a pulse signal to a signal processor when it senses incident light, and the signal processor calculates the number of analytes according to the number of photomultiplier tubes that output pulse signals.

4. 3. The single molecule detection method based on raster scan recognition according to claim 1, wherein the center distance between two adjacent capture beads is at least twice the outer diameter of the capture beads.

5. A single molecule detection method based on raster scan recognition as described in claim 1 or 2, characterized in that the distance between the opening and the surface of the capture bead is less than twice the outer diameter of the capture bead.

6. A single-molecule detection method based on raster scan recognition as described in claim 5, characterized in that the distance between the opening and the surface of the capture bead is less than one time the outer diameter of the capture bead.

7. 3. The single molecule detection method based on raster scan recognition according to claim 1, wherein the inner diameter of one of the openings is 0.5 to 1.5 times the outer diameter of the capture bead.

8. The single molecule detection method based on raster scan recognition according to claim 1 or 2, characterized in that there are two or more types of analytes, two or more types of luminescent substances, one type of analyte is bound to one type of luminescent substance, there are two or more sets of detection devices, and one set of detection devices counts one type of luminescent substance.

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