Method and scanning device for the quantitative detection of fluorescence

Abstract

The application provides a fluorescence quantitative detection method and a scanning device, which comprises the following steps: projecting laser with constant power to the surface of a micro-mirror array of a digital micro-mirror device, so that the micro-mirror array reflects the laser to a measured point on a biochip; receiving fluorescence photons emitted by the measured point through a single photon avalanche diode, adjusting the number of micro-mirrors in an open state in the digital micro-mirror device until the single photon avalanche diode only detects one fluorescence photon, and recording the number of micro-mirrors in the open state at this time to obtain an effective micro-mirror number; and comparing the effective micro-mirror number with a preset standard curve to obtain the concentration of a fluorescence marker of the measured point.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a method and scanning device for quantitative fluorescence detection. Background Technology

[0002] Fluorescent detection technology combines biological DNA fragments with fluorescent labels to form fluorescently labeled biochip probes. After the gene sequence of the sample to be tested completes a biochemical hybridization reaction with the detection probe, followed by cleaning and drying, quantitative information about the sample is obtained by scanning the number of fluorescent molecules immobilized on the biochip.

[0003] Due to the low excitation efficiency and weak response signal of fluorescent markers, fluorescence detection instruments place extremely high demands on sensor gain. Currently, mainstream laser confocal scanners use photomultiplier tubes as sensing elements. Although they have the advantage of high gain, photomultiplier tube components are expensive, the assembly process of the two-dimensional scanning system is complex, and miniaturization of the entire machine is difficult, making it difficult to apply on a large scale in grassroots units. Summary of the Invention

[0004] The technical problem to be solved by this invention is: how to achieve high-sensitivity scanning of fluorescent biochips at a lower cost.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A method for quantitative fluorescence detection, using a single-photon avalanche diode as a photoelectric sensor, enables high-sensitivity reception and detection of fluorescent photons. The method includes: A constant power laser is projected onto the surface of a micromirror array of a digital micromirror device, causing the micromirror array to reflect the laser onto the measurement point on the biochip. The fluorescent photon emitted from the measured point is received by a single-photon avalanche diode, and the number of micromirrors in the digital micromirror device in the on state is adjusted until the single-photon avalanche diode detects only one fluorescent photon. The number of micromirrors in the on state at this time is recorded to obtain the effective number of micromirrors. The effective number of micromirrors is compared with a preset standard curve to obtain the concentration of the fluorescent marker at the test site. To solve the above technical problem, another technical solution adopted by the present invention is as follows: A scanning device for quantitative fluorescence detection, comprising: Laser generator; A digital micromirror device is deployed at a preset angle in the output light path of the laser generator; the intensity of the reflected light is adjusted by changing the number of micromirrors in the open state. A dichroic beam splitter is deployed in the output light path of the digital micromirror device and is parallel to the digital micromirror device; A motion platform, deployed on the first optical path of the dichroic beam splitter, is used to place the biochip; A single-photon avalanche diode is deployed in the second optical path of the dichroic beam splitter to receive fluorescent photons from the second optical path; the fluorescent photons are excited by light from the first optical path received at the test point on the biochip; the wavelength of the fluorescent photons is different from the wavelength of the light emitted by the laser generator.

[0006] The beneficial effects of this invention are as follows: Single-photon excitation of fluorescent markers is achieved using a digital micromirror device as the quantitative adjustment mechanism for incident light. A single-photon avalanche diode replaces a photomultiplier tube as the sensing element. Based on the single-photon level detection capability of the single-photon avalanche diode, the excitation light intensity is changed by adjusting the number of open-state micromirrors until the single-photon avalanche diode detects exactly one fluorescent photon. Since the single-photon avalanche diode generates only one avalanche electrical pulse when a single photon is incident, a precise inverse quantitative relationship is formed between the number of effective micromirrors recorded and the concentration of the fluorescent marker at the measured site. This achieves accurate quantitative results without the need for a photomultiplier tube, simplifying the mechanical structure, reducing manufacturing costs, and obtaining accurate measurement results. Attached Figure Description

[0007] Figure 1 This is a flowchart illustrating the steps of a fluorescence quantitative detection method according to an embodiment of the present invention; Figure 2 This is a flowchart illustrating the steps of the binary search algorithm in an embodiment of the present invention; Figure 3 This is a schematic diagram of the structure of a scanning device for quantitative fluorescence detection in an embodiment of the present invention; Figure 4 This is a schematic diagram of the optical path of a scanning device for quantitative fluorescence detection in an embodiment of the present invention; Label Explanation: 1. Laser generator; 2. Digital micromirror device; 3. Dichroic beam splitter; 4. Motion platform; 5. Single-photon avalanche diode; 6. Beam expander; 7. First filter; 8. Second filter; 9. First focusing lens; 10. Second focusing lens. Detailed Implementation

[0008] Definitions:

[0009] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments and accompanying drawings.

[0010] In existing technologies, biochip imaging techniques are mainly divided into laser confocal scanning technology and CCD wide-field imaging technology. However, although laser confocal scanning technology has high detection accuracy, it is complex in structure and expensive. CCD wide-field imaging technology is limited by insufficient intensity of the illumination source, especially the attenuation and high energy consumption of high-pressure gas lamps, as well as the disadvantages of large chip area and low power of high-power LED lighting, resulting in low detection sensitivity of CCD-based fluorescence scanners.

[0011] To at least address the aforementioned problems, this invention provides a method and scanning device for quantitative fluorescence detection. A constant-power laser is projected onto the surface of a micromirror array of a digital micromirror device (DMicromirror Device), causing the micromirror array to reflect the laser onto the measurement site on a biochip. A single-photon avalanche diode (SPDB) receives the fluorescent photons emitted from the measurement site. The number of open micromirrors in the DMicromirror Device is adjusted, and the effective number of micromirrors is recorded when the SPDB detects exactly one fluorescent photon. Finally, the concentration of the fluorescent marker at the measurement site is obtained based on the effective number of micromirrors. This method achieves accurate quantitative results without the need for a photomultiplier tube, simplifying the mechanical structure, reducing manufacturing costs, and providing precise measurement results.

[0012] The following describes a method for quantitative fluorescence detection according to the present invention. Please refer to [link / reference]. Figure 1 The method 100 includes steps 101 to 103: Step 101: Project a constant power laser onto the surface of the micromirror array of the digital micromirror device 2, so that the micromirror array reflects the laser onto the test site on the biochip; Step 102: Receive the fluorescent photon emitted from the measured point through the single-photon avalanche diode 5, and adjust the number of micromirrors in the on state in the digital micromirror device 2 until the single-photon avalanche diode 5 detects only one fluorescent photon, and record the number of micromirrors in the on state at this time to obtain the effective number of micromirrors.

[0013] In this system, because the single-photon avalanche diode 5 operates in Geiger mode, a single incident photon will undergo an internal photoelectric effect in the SPAD, generating an electron-hole pair and instantaneously forming a macroscopic current pulse in avalanche multiplication mode. At this point, the component voltage can be quickly reduced by an external quenching circuit to terminate the avalanche and reset, thus forming a detection. When the fluorescent label on the biochip is irradiated by external excitation light, it will emit corresponding fluorescent excitation photons. Typically, in biochip detection systems, the irradiation power of the excitation source is constant, so the intensity of the illumination source remains unchanged. Therefore, the fluorescence intensity generated on the biochip under illumination excitation is directly proportional to the concentration of the fluorescent label on the biochip. Furthermore, the higher the concentration of the fluorescent label on the biochip, the stronger the excited fluorescence. Therefore, the detection instrument can collect this data using a photomultiplier tube or CCD sensor, thus obtaining a one-to-one correspondence between the excited fluorescence intensity and the concentration of the fluorescent label on the biochip.

[0014] Considering that SPADs can only detect single photons, this embodiment modifies the intensity of the external excitation light irradiating the biochip. When the illumination intensity is adjusted to precisely excite a single photon from the fluorescent marker on the biochip, the SPAD will complete an accurate avalanche detection. At this point, the illumination intensity and the concentration of the fluorescent marker are in one-to-one correspondence; the higher the concentration of the fluorescent marker on the biochip, the weaker the required illumination intensity, thus forming a new quantitative detection approach. If there is no fluorescent marker at that location, the SPAD will not experience an avalanche effect, meaning no macroscopic current pulse will be generated.

[0015] Considering the differences in conversion efficiency and the stabilization time of light power during the electro-optical energy conversion process, this invention adjusts the reflection ratio of the DMD micromirror array (i.e., the number of micromirrors in the "on" state in the digital micromirror device 2) in real time. The DMD is used to digitally control the output of excitation light with constant irradiation power, forming an excitation light illumination source with adjustable output intensity. Because a constant illumination source projected onto the DMD surface can produce light reflection, the microprocessor can precisely control the reflection ratio of the DMD micromirror array. This allows for accurate detection of the reflection ratio coefficient of the DMD micromirror array at the moment when the fluorescent label is excited by a single photon. This coefficient (the number of micromirrors in the "on" state at this time) corresponds one-to-one with the concentration of the fluorescent label on the biochip.

[0016] Step 103: Compare the effective number of micromirrors with the preset standard curve to obtain the concentration of the fluorescent marker at the test site.

[0017] As can be seen from the above description, the beneficial effects of the present invention are as follows: the digital micromirror device 2 realizes the quantitative light intensity output adjustment function in numerical control mode; the single-photon avalanche diode 5 replaces the photomultiplier tube as the sensing element; based on the single-photon level detection capability of the single-photon avalanche diode 5, the excitation light intensity is changed by adjusting the number of open-state micromirrors until the single-photon avalanche diode 5 detects exactly one fluorescent photon; since the single-photon avalanche diode 5 generates only one avalanche electrical pulse when a single photon is incident, a precise inverse quantitative relationship is formed between the number of effective micromirrors recorded and the concentration of fluorescent markers at the measured point, thereby realizing accurate quantitative results without the need for a photomultiplier tube, which simplifies the mechanical structure, reduces manufacturing costs, and obtains accurate measurement results.

[0018] In one embodiment of the present invention, step 102 involves adjusting the number of micromirrors in the on state of the digital micromirror device 2 until the single-photon avalanche diode 5 detects only one fluorescent photon. This includes steps 201 to 203. Please refer to [link / reference needed]. Figure 2 : Step 201: Obtain the total number of micromirrors of the digital micromirror device 2. For the first calculation, the total number of micromirrors is used as the upper bound of the search, and zero is used as the lower bound of the search; that is, perform initialization, low (lower bound of the search) = 0, high (upper bound of the search) = N (total number of micromirrors).

[0019] Step 202: Determine whether there is an optional intermediate value between the current upper search bound and the lower search bound. If so, use the intermediate value as the number of open-state micromirrors and determine whether the fluorescent photons are read. If read, use the intermediate value as the new upper search bound; if not read, use the intermediate value as the new lower search bound, until there is no optional intermediate value between the upper and lower search bounds. For example, during the first search, mid (intermediate value) = (low + high) / 2; when fluorescence is detected, use the intermediate value as the new upper search bound (i.e., high = mid); otherwise, use the intermediate value as the new lower search bound (i.e., low = mid). The options for "selectable middle value" and "unselectable middle value" are as follows: The middle value is an integer, and when it is not an integer, both sides can be selected. For example, if low=1 and high=6, then mid=3.5, and the selectable middle value includes 3 or 4. The options for "unselectable middle value" are as follows: When the middle value is not an integer, both sides cannot be selected. For example, if low=5 and high=6, then mid=5.5, and only the upper or lower bound of the search can be selected, which is considered unselectable. When the number of open micromirrors is expressed as a ratio, the search can be stopped when the middle value reaches a preset ratio precision.

[0020] Step 203: If there is no selectable intermediate value between the upper search bound and the lower search bound, then the upper search bound or the lower search bound is taken as the effective number of micromirrors. For example, if low=4, high=6, mid=5, and no fluorescence is detected, then low is updated to low=5; at this time, there is no selectable intermediate value between the upper search bound and the lower search bound, and no fluorescence is detected when low=5, therefore the upper search bound (high=6) is taken as the effective number of micromirrors. If fluorescence is detected, high is updated to high=5. At this point, there is no selectable intermediate value between the upper and lower search bounds, and fluorescence is detected at high=5. Therefore, the upper search bound (high=5) is taken as the effective number of micromirrors.

[0021] As described above, the binary search algorithm for closed-loop search adjustment of the number of open-state micromirrors in DMD significantly shortens the detection time per point compared to the sequential search method that increments from zero. This effectively improves the throughput and efficiency of scanning the entire biochip and ensures that each search accurately locates the critical illumination intensity that "just triggers the SPAD single-photon avalanche" (if a fluorescent marker is present at that point), thus ensuring the consistency and repeatability of the detection results.

[0022] In other optional implementations, the search can be performed by sequentially increasing the number of open-state micromirrors from 0, sequentially decreasing the number of open-state micromirrors from the maximum value, or by using other search algorithms.

[0023] In one embodiment of the present invention, before step 102 adjusts the number of micromirrors in the digital micromirror device 2 that are in the open state, the method includes: setting all micromirrors in the digital micromirror device 2 to the open state; the incident light scans and irradiates each test point on the biochip point by point; for each target test point traversed, it is determined whether the fluorescent photon is read; if not, the target test point is marked as an empty point; if so, the target test point is marked as a valid point.

[0024] As described above, by setting all DMD micromirrors to the open state through pre-scanning and traversing all test sites with maximum excitation light intensity, it is possible to quickly distinguish between valid sites (containing fluorescent markers) and empty sites (not containing fluorescent markers). The pre-screening mechanism avoids the need to perform a complete open-state micromirror number search process for empty sites, thus saving time spent on ineffective searches. In typical detection scenarios with high density of biochip arrays and sparse positive sites, it can significantly reduce the total scan time, increase detection throughput, and provide site validity labels for subsequent data analysis.

[0025] Please refer to Figure 3The present invention also provides a scanning device for quantitative fluorescence detection, comprising a laser generator 1, a digital micromirror device 2, a dichroic beam splitter 3, a motion platform 4, and a single-photon avalanche diode 5. The digital micromirror device 2 is deployed at a preset angle in the output light path of the laser generator 1, and the intensity of reflected light is adjusted by changing the number of micromirrors in the open state. The dichroic beam splitter 3 is deployed in the output light path of the digital micromirror device 2 and is parallel to the digital micromirror device 2. The motion platform 4 is deployed in the first light path of the dichroic beam splitter 3 for placing a biochip. The single-photon avalanche diode 5 is deployed in the second light path of the dichroic beam splitter 3 to receive fluorescent photons from the second light path. The fluorescent photons are excited by the light received from the first light path at the test point on the biochip. The wavelength of the fluorescent photons is different from the wavelength of the light emitted by the laser generator 1.

[0026] As can be seen from the above description, the beneficial effects of the present invention are as follows: by replacing the traditional PMT-based optoelectronic detection link with DMD+SPAD, the advantages of high energy density and good imaging quality of laser confocal excitation light are fully utilized. At the same time, by utilizing the small size, all-solid-state nature and low operating voltage of SPAD, the overall structure is simpler and more robust than the traditional PMT solution, which can significantly reduce the instrument manufacturing and maintenance costs, and is conducive to the large-scale popularization of high-end biochip detection equipment in primary medical institutions.

[0027] In one embodiment of the present invention, a Cy3 fluorescent marker is used. The first optical path of the dichroic beam splitter 3 is a reflected optical path, and the second optical path is a transmitted optical path; the wavelength of the fluorescent photons is greater than the wavelength of the light emitted by the laser generator 1. Figure 3 As shown, if the wavelength of the light emitted by laser generator 1 is 532 nm and the wavelength of the fluorescent photon is 550 nm, the light emitted by laser generator 1 is reflected downwards by the dichroic beam splitter 3, while the fluorescent photon is transmitted from the dichroic beam splitter 3 to the single-photon avalanche diode 5. In other optional embodiments, the dichroic beam splitter can be changed from low-reflection, high-transmission to high-reflection, low-transmission, thereby detecting upconversion fluorescence with a shorter fluorescence wavelength.

[0028] As described above, the wavelength selectivity of the dichroic beam splitter 3 enables the co-focusing design of the excitation light path and the fluorescence acquisition light path. The excitation light (short wavelength, such as 532nm) is reflected by the dichroic beam splitter 3 and enters the objective lens to be focused onto the biochip, while the fluorescent photons emitted by the fluorescent markers are longer in wavelength and penetrate the dichroic beam splitter 3 to enter the SPAD detection channel.

[0029] In one embodiment of the present invention, a beam expander 6 is further included, which is deployed between the laser generator 1 and the digital micromirror device 2 and is parallel to the laser generator 1.

[0030] As described above, deploying a beam expander 6 between the laser generator 1 and the DMD shapes the point-like or small-aperture beam emitted by the laser generator 1 into a large-aperture parallel beam, enabling it to uniformly cover the entire surface of the DMD micromirror array. This ensures that each micromirror pixel of the DMD receives a uniform incident light power density, avoiding errors in reflected light intensity control caused by beam inhomogeneity. This provides a precise physical basis for subsequent quantitative concentration calculations based on the number of open states.

[0031] In one embodiment of the present invention, a first filter 7 is further included, which is deployed in the output light path of the laser generator 1 and parallel to the laser generator 1. For example, a 532nm filter is set as the first filter 7.

[0032] As described above, a first filter 7 is placed between the laser generator 1 and the beam expander 6 to limit the narrowband spectrum of the laser output, filter out stray light and other non-target wavelength stray light components in the spontaneous emission background of the laser generator 1, effectively avoid the potential risk of non-target band stray light entering the fluorescence acquisition channel through DMD reflection and dichroic beam splitter 3, thereby reducing the detection background noise and improving the signal-to-noise ratio and detection specificity of SPAD single-photon detection.

[0033] In one embodiment of the present invention, a second filter 8 is further included, disposed between the single-photon avalanche diode 5 and the dichroic beam splitter 3, and parallel to the single-photon avalanche diode 5. For example, a 550nm filter is used as the second filter 8.

[0034] As described above, a second filter 8 is placed between the dichroic spectrometer 3 and the SPAD to perform secondary spectral purification of fluorescent photons before they enter the SPAD photosensitive window. This filter allows only narrow-band fluorescent photons of the target wavelength (such as the 550nm green fluorescence of Cy3) to pass through, further blocking excitation light and other stray light from potentially penetrating the dichroic spectrometer 3 and entering the detection channel. This ensures that every pulse detected by the SPAD is generated by single-photon excitation of the target fluorescent label, improving the accuracy and repeatability of quantitative detection.

[0035] In one embodiment of the present invention, a first focusing lens 9 and a second focusing lens 10 are further included. The first focusing lens 9 is deployed between the dichroic beam splitter 3 and the motion platform 4, and is parallel to the motion platform 4; the second focusing lens 10 is deployed between the dichroic beam splitter 3 and the single-photon avalanche diode 5, and is parallel to the single-photon avalanche diode 5.

[0036] As described above, a first focusing lens 9 and a second focusing lens 10 are deployed between the dichroic beam splitter 3 and the biochip, and between the dichroic beam splitter 3 and the SPAD, respectively, forming a complete optical path structure for laser confocalization. The first focusing lens 9 focuses the parallel excitation light reflected by the DMD and the dichroic beam splitter 3, realizing the precise excitation of a single detection point on the biochip. The second focusing lens 10 refocuses the parallel fluorescent beam transmitted by the dichroic beam splitter 3 after the biochip is excited and emitted onto the micron-scale photosensitive window of the SPAD, avoiding the loss of fluorescent photons by scattering. The cooperation of the two focusing lenses ensures the maximum utilization of excitation light energy and the maximum efficiency of fluorescent photon collection, enabling the SPAD to stably detect single photons.

[0037] This embodiment uses the 550nm green fluorescence of a Cy3 biochip as an example to illustrate the above-mentioned fluorescence quantitative detection method and scanning device: Please refer to Figure 4 The light source emitted by laser generator 1 is shaped into a parallel light source by beam expander 6, reflected by DMD, and enters the low-reflection, high-transmission dichroic beam splitter 3 arranged at 45 degrees. Because the excitation light source has higher energy and therefore a higher frequency, it can be reflected by the low-reflection, high-transmission dichroic beam splitter 3 and focused onto the DNA biochip by the first focusing lens 9. Simultaneously, the fluorescent marker at that point on the microarray chip is excited by laser irradiation to emit fluorescence with a longer wavelength. This fluorescence is converted into parallel light by the first focusing lens 9, forming a confocal excitation light path and a collection light path. At this time, the collected fluorescence passes through the low-reflection, high-transmission dichroic beam splitter 3 and is focused onto the SPAD photosensitive window by the second focusing lens 10, obtaining laser confocal fluorescence photon collection. The specific steps are as follows: S1, 532nm laser generator 1 emits 532nm laser light to provide a light source.

[0038] S2. Place a 532nm first filter 7 in front of the 532nm laser generator 1 to filter stray light.

[0039] S3. Place a beam expander 6 parallel to the 532nm excitation filter in front of it to form a 532nm parallel excitation beam.

[0040] S4. Place the DMD chip at a 45º angle in front of the beam expander 6.

[0041] S5. A 540nm low-reflection, high-transmittance dichroic beam splitter 3 is placed parallel to the light output port of the DMD chip. The 532nm excitation light is reflected by the 540nm dichroic beam splitter 3.

[0042] S6. Place a first focusing lens 9 at a 45º angle to the 540nm low-reflection, high-transmission dichroic beam splitter 3 below it.

[0043] S7. Place an XY two-dimensional motion platform 4 (5µm precision) parallel to the first focusing lens 9, and place the DNA biochip treated with Cy3 fluorescent dye on the XY two-dimensional motion platform 4. The 532nm excitation light is focused onto the confocal point on the DNA biochip through the beam expander 6-DMD reflection-dichroic mirror reflection 2-first focusing lens 9. The fluorescent dye on the DNA biochip excited by the 532nm focusing light will emit fluorescent photons. The spectrum of these fluorescent photons is greater than that of the 540nm band and can penetrate the 540nm low-reflection, high-transmission dichroic beam splitter 3.

[0044] S8. Fluorescent photons with a wavelength greater than 540nm transmitted upward will be focused by the upper second focusing lens 10.

[0045] S9. A second 550nm filter 8 is placed above the second focusing lens 10 and parallel to it to form narrow-band fluorescence excitation light after filtering.

[0046] S10. Deploy a single-photon avalanche diode 5 at the upward focal point of the fluorescent light path.

[0047] S11. The single-photon avalanche diode 5 receives the single photon. The charge carriers excited by the single photon induce avalanche multiplication in the strong electric field, forming a detectable electric pulse.

[0048] S12. The number of micromirrors in the "on state" when the detected point on the DNA biochip emits only one fluorescent photon is calculated using a binary search algorithm. This yields the number of fluorescent probes. The smaller the number of "on state" micromirrors on the DMD chip, the lower the illumination intensity of the 532nm excitation light, indicating a higher concentration of Cy3 on the biochip. In other words, the Cy3 concentration is inversely proportional to the number of "on state" micromirrors on the DMD chip.

[0049] S13 and XY two-dimensional motion platform 4 move to measure the next measurement point of the DNA biochip.

[0050] S14. Repeat the above steps until all measurements on the DNA biochip are completed.

[0051] In summary, the present invention provides a method and scanning device for quantitative fluorescence detection, which differs from the traditional forward mode of fluorescence detection, which uses constant excitation light and infers concentration by collecting fluorescence intensity. This invention establishes a one-to-one correspondence between "excitation light intensity and label concentration" by fixing the fluorescence response end (SPAD detects only one photon) and adjusting the excitation light intensity in reverse (the number of open-state micromirrors in the DMD), thus realizing a new paradigm of digital and highly sensitive quantitative fluorescence detection. Simultaneously, it combines the precision of DMD digital light control with the single-photon-level detection sensitivity of SPAD and integrates them into a laser confocal optical path, forming a conjugate focal structure of "reflection excitation—transmission acquisition." This combination replaces the traditional PMT scheme, resulting in a simpler structure and significantly reduced cost.

[0052] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for quantitative fluorescence detection, characterized in that, Using a single-photon avalanche diode as a photoelectric sensor, the method for highly sensitive reception and detection of fluorescent photons includes: A constant power laser is projected onto the surface of a micromirror array of a digital micromirror device, causing the micromirror array to reflect the laser onto the measurement point on the biochip. The fluorescent photon emitted from the measured point is received by a single-photon avalanche diode, and the number of micromirrors in the digital micromirror device in the on state is adjusted until the single-photon avalanche diode detects only one fluorescent photon. The number of micromirrors in the on state at this time is recorded to obtain the effective number of micromirrors. The effective number of micromirrors is compared with a preset standard curve to obtain the concentration of the fluorescent marker at the test site.

2. The method for quantitative fluorescence detection according to claim 1, characterized in that, Adjusting the number of micromirrors in the on state in the digital micromirror device until the single-photon avalanche diode detects only one fluorescent photon includes: Obtain the total number of micromirrors in the digital micromirror device. For the first calculation, the total number of micromirrors is used as the upper bound of the search, and zero is used as the lower bound of the search. Determine whether there is an optional intermediate value between the current upper search bound and the lower search bound. If so, use the intermediate value as the number of open-state micromirrors and determine whether the fluorescent photons are read. If they are read, use the intermediate value as the new upper search bound. If they are not read, use the intermediate value as the new lower search bound until there is no optional intermediate value between the upper search bound and the lower search bound. If there is no selectable intermediate value between the upper search bound and the lower search bound, then the upper search bound or the lower search bound is taken as the effective number of micromirrors.

3. The method for quantitative fluorescence detection according to claim 1, characterized in that, Before adjusting the number of micromirrors in the on state in the digital micromirror device, the following steps are included: Set all micromirrors in the digital micromirror device to the on state; The biochip is irradiated at each test point. For each target test point, it is determined whether the fluorescent photon is read. If not, the target test point is marked as an empty point; if so, the target test point is marked as a valid point.

4. A scanning device for quantitative fluorescence detection, characterized in that, include: Laser generator; A digital micromirror device is deployed at a preset angle in the output light path of the laser generator; The intensity of reflected light can be adjusted by changing the number of micromirrors in the open state; A dichroic beam splitter is deployed in the output light path of the digital micromirror device and is parallel to the digital micromirror device; A motion platform, deployed on the first optical path of the dichroic beam splitter, is used to place the biochip; A single-photon avalanche diode is deployed in the second optical path of the dichroic beam splitter to receive fluorescent photons from the second optical path; the fluorescent photons are excited by light from the first optical path received at the test point on the biochip; the wavelength of the fluorescent photons is different from the wavelength of the light emitted by the laser generator.

5. The scanning device for quantitative fluorescence detection according to claim 4, characterized in that, The first optical path of the dichroic beam splitter is a reflected optical path, and the second optical path is a transmitted optical path; The wavelength of the fluorescent photon is greater than the wavelength of the light emitted by the laser generator.

6. The scanning device for quantitative fluorescence detection according to claim 4, characterized in that, Also includes: A beam expander is positioned between the laser generator and the digital micromirror device, and is parallel to the laser generator.

7. The scanning device for quantitative fluorescence detection according to claim 4, characterized in that, Also includes: The first filter is deployed in the output light path of the laser generator and is parallel to the laser generator.

8. The scanning device for quantitative fluorescence detection according to claim 4, characterized in that, Also includes: The second filter is deployed between the single-photon avalanche diode and the dichroic beam splitter, and is parallel to the single-photon avalanche diode.

9. The scanning device for quantitative fluorescence detection according to claim 4, characterized in that, Also includes: A first focusing lens is positioned between the dichroic beam splitter and the motion platform, and is parallel to the motion platform. The second focusing lens is positioned between the dichroic beam splitter and the single-photon avalanche diode, and is parallel to the single-photon avalanche diode.

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