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 been rapidly developed in recent years. It refers to measuring and analyzing targets at the single molecule level. It is a new detection method and opens up a new detection field. This detection method can detect very low concentrations of analytes, and the reagents and space required for detection are both very small. For example, in "CN202080000774.8-Single Molecule Quantitative Detection Method and Detection System" disclosed by Suzhou Yusue Biological Technology Co., Ltd., in situ signal enhancing nanoparticles with optical effect are introduced in the immunological recognition process to mark the molecules to be detected, thereby enhancing the signal generated by the original luminescent material and increasing the recognition degree, and then an optical imaging instrument is used to obtain the number of molecules to be detected that have spots. Also, for example, in "CN201180019462.2-Ultrasensitive detection of molecules or particles using beads or other captures" disclosed by Quanterix, the captures are spatially divided into certain sections, and then the captures are imaged, and the concentration is determined by counting the number of analytes that emit light in the image. All of these methods can greatly improve the sensitivity of detection. However, all of the above methods require high requirements for imaging equipment and slow detection speed because they all involve counting the number of analytes that emit light in the image after imaging. 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 have 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, with simple equipment and fast detection speed. [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 on a bead fixing plate (or detection chip) 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 lattice openings of a lattice detection device are used to start scanning the capture beads row by row starting from the capture beads arranged at one end, the lattice detection device synchronously collects statistics on 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 lattice openings and the capture beads, and the total number of analytes is obtained when the scan is completed.

[0005] Further, the set of grating detection devices includes a grating head, an optical sensor and a signal processor, the grating head includes a plurality of independent grating openings arranged in a row, 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, the sensing element feeds back to the signal processor when it senses the incident light, and the signal processor statistics the number of analytes according to the feedback result.

[0006] Furthermore, the sensing element includes photosensitive tubes, which output a pulse signal to the 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 the pulse signal.

[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-to-center distance between two adjacent grid openings is equal to the center-to-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 in 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] Staggered arrangement means that the positions between adjacent grid openings are V-shaped, and when the grid openings in one column are aligned with the capture beads in several rows on the bead fixing plate, the grid openings in another column are aligned with the capture beads in the row adjacent to the capture beads, i.e., the capture beads in two adjacent rows among the capture beads arranged in the same column are not aligned with the grid openings in one column at the same time, but are aligned with the grid openings in two columns in sequence during lateral movement along the rows of the capture beads and the 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-to-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 openings and the surface of the capture bead is less than or equal to twice the outer diameter of the capture bead.

[0013] In addition, the distance between the lattice opening and the surface of the capture bead is equal to 1.5 times, 1 times or 0.5 times the outer diameter of the capture bead. Theoretically, on the premise that the lattice opening does not contact the material on the surface of the capture bead, the smaller the distance between the lattice opening and the surface of the capture bead, the better, so as to make the light emitted by the marker enter the lattice opening as much as possible, and to avoid the light loss caused by the distance being too large, which will cause inaccurate detection results.

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

[0015] In addition, the inner diameter of one lattice opening is equal to the outer diameter of the capture bead. Preferably, the area of ​​the light-transmitting region in one lattice opening is close to the area of ​​the projection of the light-emitting region of the capture bead on the lattice opening, and the distance between the lattice opening and the surface of the capture bead is controlled, so that the light emitted by the light-emitting substance on the surface of the capture bead can be maximally incident into the lattice opening by the synergistic effect, and the light emitted by the light-emitting substance on the surface of another capture bead at an adjacent position is prevented from incident into the lattice opening and affecting the detection result.

[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 an adjacent capture bead from entering the grid opening and causing the sensing element to transmit an erroneous signal.

[0017] Further, luminescent materials include fluorescent or chemiluminescent agents.

[0018] Furthermore, when the luminescent material is fluorescent, an excitation light source for irradiating the capture beads is placed 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 collecting statistics on 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 configuration in which the grating openings are arranged in a staggered pattern in two rows 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 description of the drawings]

[0024] [Figure 1] FIG. 2 is a schematic diagram showing magnetic beads in Example 1 arranged on a microwell array plate. [Diagram 2] FIG. 2 is a schematic diagram of automatic scan recognition in the first embodiment. [Diagram 3] FIG. 4 is a schematic diagram showing an arrangement of grating openings of a grating head in the first embodiment. [Figure 4] FIG. 11 is a schematic diagram showing an arrangement of grating openings of a grating head in Example 2. [Diagram 5] FIG. 1 is a schematic diagram showing the correspondence between a top view of a microwell array plate and a bottom view of a lattice opening. [Figure 6A] 1A-1D are signal schematics corresponding to the detection of luminescent magnetic beads arranged in different orientations. [Figure 6B] 1A-1D are signal schematics corresponding to the detection of luminescent magnetic beads arranged in different orientations. [Figure 6C] 1A-1D are signal schematics corresponding to the detection of luminescent magnetic beads arranged in different orientations. [Figure 6D] 1A-1D are signal schematics corresponding to the detection of luminescent magnetic beads arranged in different orientations. [Figure 6E] 1A-1D are signal schematics corresponding to the detection of luminescent magnetic beads arranged in different orientations. [Figure 6F] 1A-1D are signal schematics corresponding to the detection of luminescent magnetic beads arranged in different orientations. [Figure 7] FIG. 11 is a schematic diagram showing the positional relationship between an excitation light source, a trapping bead, and a lattice detection device in Example 3. [Figure 8] 11A to 11C are schematic diagrams showing two types of installation forms of the lattice detection device in the fourth embodiment. [Figure 9] 11A to 11C are schematic diagrams showing two types of installation forms of the lattice detection device in the fourth embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0026] In this embodiment, the marker is a fluorescent substance, the capture beads are magnetic beads, a long microwell array plate with a microwell array is a bead immobilization plate, some microwells have one magnetic bead, some microwells do not have 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) of adjacent rows or adjacent columns described herein refer to magnetic beads (capture beads) in the microwells of adjacent rows or adjacent columns in the array, and even if there are no magnetic beads in the microwells of the adjacent rows or adjacent columns, the description of the magnetic beads (capture beads) of adjacent rows or adjacent columns in this specification still refers to the position of the microwells of adjacent rows or adjacent columns in the array.

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

[0028] Then, raster scan recognition and detection is performed. As shown in FIG. 2, an excitation light source 24 is installed above the microwell array plate 10, and the emitted excitation light is reflected by a reflecting mirror 25 to irradiate the surface of the magnetic beads 12 to be scanned, and the excitation light is turned off after the excitation is completed. The lattice head 21 of the lattice detection device 20 is placed above the microwell array plate 10 (of course, it may be installed below), and the lattice openings 211 of the lattice head 21 are aligned with the magnetic beads 12, so that the fluorescence emitted after the fluorescent markers 13 are excited can be incident on the lattice openings 211 to the maximum extent. As shown in FIG. 3, the number of lattice openings 211 must be equal to or greater than the number of microwells 11 in one row in the microwell array plate 10, and the center distance between two adjacent lattice openings 211 must be equal to the center distance between two adjacent microwells 11, so that all rows of magnetic beads 12 in the microwell array plate 10 are scanned and recognized at once. In addition, in order to accurately count the number of lattice openings 211 into which fluorescence is incident, the optical sensor 22 of the lattice detection device 20 is installed at the other end opposite to the lattice openings 211 of the lattice head 21, and the sensing elements such as photosensitive tubes of the optical sensor 22 correspond one-to-one to the lattice openings 211. When fluorescence is incident on a specific lattice opening 211, the corresponding sensing element receives the incident light and transmits a signal to the signal processor 23 of the lattice detection device 20. For example, when the photosensitive tube senses light, it can generate a pulse signal as a signal. The signal processor 23 receives this pulse signal and performs subsequent processing, and the number of signals transmitted by the optical sensor 22 can be counted to count the magnetic beads 12 that emit fluorescence, 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] In 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, a 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 realized by a lateral movement of the microwell array plate 10 to the right, or of course, by a lateral 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 with a standard concentration curve. EXAMPLES

[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 manner in two rows, that is, the positional relationship between adjacent lattice openings 211 is V-shaped, thereby making the distance between two adjacent lattice openings 211 large. In this case, the total number of lattice openings 211 in the two rows is greater than or equal to the number of microwells 11 in one row. As shown in FIG. 5, in 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 realize relative movement between the microwell array plate 10 and the grid detection device 20, when the scan recognition begins, the grid head 21 is located above the microwells 12 in the rightmost row of the microwell array plate 10, the grid opening 211 marked B' is aligned with and detects the magnetic bead 12 marked B, 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 lattice opening 211 marked with B' detects the magnetic bead 12 marked with 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 lattice detection device 20, in which the lattice opening 211 marked with B' is aligned with the magnetic bead 12 in the next adjacent row in the same row as the magnetic bead marked with B, and the lattice opening 211 marked with A' is aligned with the magnetic bead 12 marked with A, and the detection of the magnetic beads 12 at the offset portion of the two adjacent rows is completed synchronously. This is repeated until the detection of all the magnetic beads 12 in the microwell array plate 10 is completed. In this way, by increasing the distance between the adjacent lattice openings 211, the optical interference between the adjacent magnetic beads 12 can be reduced, and the detection result can be made more accurate.

[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 FIG. 6A, when one luminescent capture bead passes under the lattice opening, it is processed by the signal processor to generate one normal signal wave. As shown in FIG. 6B, when two luminescent capture beads with a suitable distance pass under the lattice opening in sequence, it is processed by the signal processor to generate two normal signal waves, the interval between the peaks and valleys of which is clear and uniform. As shown in FIG. 6C, when the distance between the two luminescent capture beads is too close and evenly overlaps at the edge, the peaks of the two waves of the signal waves processed and generated by the signal processor are too close and the valleys are not obvious. As shown in FIG. 6D, when three luminescent capture beads that are too close to each other pass under the lattice opening in sequence, the peaks and valleys of the signal waves processed and generated by the signal processor are not clearly distinguishable. As shown in FIG. 6E and 6F, when the three luminescent capture beads are not collinear, one capture bead is aligned directly under the lattice opening and the other two capture beads are located near the lattice opening, both of them affect the detection and cannot form normal signal waves.

[0034] Therefore, in order to ensure the accuracy of the detection results and to realize a compact detection system, capture beads with appropriate specifications, such as beads with a diameter of 2.7 um, should be selected as much as possible, and when arranging the capture beads, the center distance between two adjacent capture beads should be controlled to 2.5 to 3 times the diameter as much as possible. In addition, it is necessary to make the size of the lattice opening as small as possible, such as by controlling the light transmission area within the range of the cross-sectional area of ​​the capture beads, so that it is possible to ensure that the transmitted light can turn on the sensing element and transmit a signal that can be easily recognized by the signal processor. In addition, the distance between the lattice opening and the surface of the capture bead is made as close as possible to prevent light from adjacent emitting capture beads from entering the lattice opening as much as possible, that is, to reduce the interference of light at adjacent positions.

[0035] The above embodiment uses magnetic beads as the capture beads, but in actual use, other shapes of particles with or without magnetism 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 concave pit structure, or may even be flat. If the bead fixing plate has holes or pits, the holes or pits are pre-processed, so that they can be processed into a regular array shape, and the capture beads can be regularly arranged in the holes or pits, and the lattice openings arranged in rows are easy 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 distributed and arranged on the surface of the bead fixing plate. In the case where the capture beads are randomly distributed and arranged on a plane, the distance between the capture beads needs to be as large as possible to avoid the capture beads being too close to each other and thus gathering into clusters. Therefore, for the same number of capture beads, when distributed 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 an array of multiple rows and columns, 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 in the same line. Therefore, the method of scanning and detecting each column in the above Example 1 or Example 2 can still be adopted. EXAMPLES

[0036] 7, in this embodiment, the bead fixing plate and the lattice detection device 20 are installed vertically, and two or more excitation light sources 24 and reflecting mirrors 25 are evenly installed around the bead fixing plate, and during the detection process, the excitation light sources 24 and reflecting mirrors 25 remain stationary, and 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 it can ensure that the surface of each captured bead is illuminated and the markers on the surface can be excited to emit fluorescence. EXAMPLES

[0037] In this embodiment, two or more analytes can be detected on one bead immobilization plate, in which 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, and two or more sets of lattice detection devices 20 are installed, and each set of lattice detection devices 20 detects and counts one analyte. The two or more sets of lattice detection devices 20 may be installed in various forms.

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

[0039] (2) As shown in Fig. 9, two or more sets of lattice detection devices 20 are symmetrically installed on a bracket 26 that plays a supporting role by one rotation center 27, and the rotation center 27 can rotate around the bracket 26. When there are two sets of lattice detection devices 20, they are installed on both ends of the rotation center 27, and when there are three sets of lattice detection devices 20, the angle between two adjacent sets of lattice detection devices is 120°. When there are more sets of lattice detection devices 20, they are also installed symmetrically according to this rule. During scanning detection, one set of lattice detection devices 20 starts scanning detection of one analyte from one end of the bead fixing plate, and after detecting to the other end and completing the scanning detection of this analyte, the rotation center 27 is rotated, and the other set of lattice detection devices 20 is moved to the end of the bead fixing plate, and the bead fixing plate or bracket 26 moves in the opposite direction to the previous detection process to complete the scanning detection of the second analyte. This is repeated in sequence until the scanning detection of all analytes is completed.

[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. During scanning, In step 1, the filter required for the lattice opening is selected, and the lattice 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 lattice detection device 20, this set of lattice detection devices 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 in 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 embodiments are only used to explain the present invention, and are not intended to limit the present invention. Those skilled in the art may 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 scope of protection of the present invention, and the scope of protection of the present invention should be defined in the claims. [Explanation of symbols]

[0042] 10 Microwell Array Plate 11 Microwell 12 Magnetic beads 13 Highlighter 20 Lattice detector 21 Lattice Head 211 Lattice opening 22 Optical Sensor 23 Signal Processor 24 Excitation Light Source 25 Reflective Mirror 26 Bracket 27 Rotation Center

Claims

1. A single molecule detection method based on raster scan recognition, comprising: arranging a large number of capture beads on a bead fixing plate (or detection chip) in a dispersed manner; at least some of the capture beads are specifically bound to one analyte, and each analyte is bound to a luminescent substance; using a grid opening of a grid detection device, scanning the capture beads row by row, starting from the capture beads arranged at one end; the grid detection device synchronously collects statistics on the number of capture beads bound to markers; scanning the capture beads across the entire bead fixing plate by lateral relative movement between the grid opening and the capture beads; and obtaining the total number of analytes when the scan is completed.

2. The method for detecting single molecules based on raster scanning recognition according to claim 1, characterized in that the set of lattice detection devices includes a lattice head, an optical sensor and a signal processor, the lattice head includes a plurality of independent lattice openings arranged in a row, the optical sensor includes a plurality of independent sensing elements, and during scanning, one lattice opening is aligned with one capture bead or not aligned with the capture bead, one sensing element corresponds to one lattice opening and can sense the light incident on this lattice opening, the sensing element feeds back to the signal processor when sensing the incident light, and the signal processor calculates the number of analytes according to the feedback result.

3. The method for single molecule detection based on raster scan recognition as described in claim 2, characterized in that the sensing element includes a photosensitive 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 photosensitive tubes that output the pulse signal.

4. The method for single molecule detection based on raster scan recognition as described in claim 2, characterized in that the grid openings of the grid detection device are arranged in a row, the number of grid 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-to-center distance between two adjacent grid openings is equal to the center-to-center distance between two adjacent rows of capture beads in the same column.

5. The method for single molecule detection based on raster scan recognition as described in claim 2, characterized in that the grid openings of the grid detection device 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-to-center distance between adjacent grid openings in each column is equal to the center-to-center distance between two rows of capture beads in every other row in the same column, and the two columns of grid openings work together to scan one column of capture beads.

6. 2. The method for single molecule detection 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.

7. 2. The method for single molecule detection based on raster scan recognition according to claim 1, wherein the distance between the lattice opening and the surface of the capture bead is less than or equal to twice the outer diameter of the capture bead.

8. 8. The method for single molecule detection based on raster scan recognition according to claim 7, wherein the distance between the lattice opening and the surface of the capture bead is equal to or less than one time the outer diameter of the capture bead.

9. 2. The method for single molecule detection based on raster scan recognition according to claim 1, wherein the inner diameter of one lattice opening is 0.5 times to 1.5 times the outer diameter of the capture bead.

10. The method for single molecule detection based on raster scan recognition as described in claim 1, 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 grid detection devices, and one set of grid detection devices counts one type of luminescent substance.

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