Flow fluorescence detection method and detection system based on microwave field on-off state

Abstract

The invention discloses a flow-type fluorescence detection method and detection system based on a microwave field on-off state, and the method comprises the following steps: when a target material specifically bound with a nano-diamond mark flows through the first half detection area of a sample flow channel, the NV color center in the nano-diamond generates a dark-state fluorescence signal; when the target material specifically bound with the nano-diamond mark flows through the rear half detection area of the sample flow channel, the NV color center in the nano-diamond generates a bright-state fluorescence signal; wherein the exciting light module is used for exciting an NV color center of a detection area of the sample flow channel, so that electrons of the NV color center are transitioned to generate fluorescence, and the microwave radiation module is used for providing a microwave field for the detection area of the sample flow channel; the light detection module collects a fluorescence signal at a preset collection frequency; and the data processing module judges whether the target object contains the to-be-detected biomolecules or not according to the magnitude relationship between the intensity of the dark-state fluorescence signal and the intensity of the bright-state fluorescence signal. According to the technical scheme provided by the embodiment of the invention, the detection sensitivity of the fluorescence detection method is improved.

Description

Technical Field

[0001] The present invention relates to the field of detection technologies, and in particular, to a flow cytometry fluorescence detection method and a detection system based on the switching state of a microwave field. Background Art

[0002] Flow cytometry fluorescence technology can achieve high-throughput, high-speed, and multiplex biomarker joint detection. However, due to the too low intensity of existing fluorescent dyes, the fluorescence signal recognition of single molecules cannot be achieved, and the lower limit of detection sensitivity is limited. It cannot meet the requirements of special application scenarios, such as the detection of low-abundance biomarkers of diseases, etc.

[0003] Therefore, there is an urgent need for a fluorescence detection method with high detection sensitivity. Summary of the Invention

[0004] The present invention provides a flow cytometry fluorescence detection method and a detection system based on the switching state of a microwave field to improve the detection sensitivity of the fluorescence detection system.

[0005] According to one aspect of the present invention, there is provided a flow cytometry fluorescence detection method based on the switching state of a microwave field, including:

[0006] When the target substance specifically bound to the nanodiamond flows through the first half of the detection area of the sample flow channel, a modulated fluorescence signal is generated in the NV centers in the nanodiamond, and the modulated fluorescence signal includes a dark-state fluorescence signal; the dark-state fluorescence signal is the fluorescence signal generated in the NV centers in the nanodiamond when some or all of the NV centers resonate with the microwave field.

[0007] When the target substance specifically bound to the nanodiamond flows through the second half of the detection area of the sample flow channel, a modulated fluorescence signal is generated in the NV centers in the nanodiamond, and the modulated fluorescence signal includes a bright-state fluorescence signal; the bright-state fluorescence signal is the fluorescence signal generated in the NV centers in the nanodiamond when no NV center resonates with the microwave field.

[0008] Wherein, an excitation light module is used to emit excitation light, the excitation light is used to excite and cause the electrons of the NV centers in the detection area of the sample flow channel to undergo electronic transitions, a microwave radiation module is used to provide a microwave field for the detection area of the sample flow channel, the microwave field resonates with all or some of the NV centers in the nanodiamond, and the microwave radiation module is in an on state or an off state under the control of a control signal; when the microwave radiation module is in the on state, the NV centers in the nanodiamond generate the dark-state fluorescence signal, and when the microwave radiation module is in the off state, the NV centers in the nanodiamond generate the bright-state fluorescence signal;

[0009] A light detection module collects the modulated fluorescence signal at a preset collection frequency;

[0010] The data processing module determines whether the target contains the biomolecule to be detected according to the magnitude relationship between the intensity of the dark-state fluorescence signal and the intensity of the bright-state fluorescence signal.

[0011] "The microwave field resonates with all or part of the NV color centers in the nanodiamond", specifically referring to: the microwave frequency of the microwave field is equal to the energy level difference of the electron spin transition of the NV color center, which enables the microwave field to resonate with the NV color center.

[0012] Optionally, when the target specifically bound to the nanodiamond marker flows through the first half of the detection area of the sample flow channel, the NV color centers in the nanodiamond generate a modulated fluorescence signal, and the modulated fluorescence signal includes a dark-state fluorescence signal including:

[0013] The control module issues a control signal, and the control signal includes a microwave field on signal and a microwave field off signal;

[0014] According to the microwave field on signal, the microwave radiation module is in the on state, and the microwave radiation module provides a microwave field for the detection area of the sample flow channel. When the NV color centers in the nanodiamond flow through the first half of the detection area of the sample flow channel, they emit a dark-state fluorescence signal under the irradiation of the excitation light.

[0015] Optionally, when the target specifically bound to the nanodiamond marker flows through the second half of the detection area of the sample flow channel, the NV color centers in the nanodiamond generate a modulated fluorescence signal, and the modulated fluorescence signal includes a bright-state fluorescence signal including:

[0016] According to the microwave field off signal, the microwave radiation module is in the off state, and the microwave radiation module stops providing a microwave field for the detection area of the sample flow channel. When the NV color centers in the nanodiamond flow through the second half of the detection area of the sample flow channel, they emit a bright-state fluorescence signal under the irradiation of the excitation light.

[0017] Optionally, the data processing module determines whether the target contains the biomolecule to be detected according to the magnitude relationship between the intensity of the dark-state fluorescence signal and the intensity of the bright-state fluorescence signal, including:

[0018] If the intensity of the dark-state fluorescence signal is less than the intensity of the bright-state fluorescence signal, and the absolute value of the difference between the intensity of the dark-state fluorescence signal and the intensity of the bright-state fluorescence signal is greater than the second preset threshold, it is determined that the target contains the biomolecule to be detected.

[0019] Optionally, after the NV color centers in the nanodiamond generate a modulated fluorescence signal when the target specifically bound to the nanodiamond marker flows through the second half of the detection area of the sample flow channel, it further includes:

[0020] After each detection window of the optical detection module, a detection window without microwave radiation signal is added as a reference signal.

[0021] Optionally, the magnetic field strength in the detection area satisfies the zero-field condition, and the microwave radiation signal provided by the microwave field has a fixed frequency.

[0022] Optionally, when the magnetic field strength near the detection area of the sample flow channel monitored by the magnetic field monitoring module satisfies the zero-field condition, the energy level splitting of the NV color centers in the nanodiamond occurs, and the offset between the resonance frequency of the wave valley of the split energy level and the frequency of the microwave signal of the microwave field is less than half of the full width at half maximum of the wave valley of this energy level.

[0023] Optionally, the fixed frequency of the microwave radiation signal provided by the microwave field is greater than or equal to D - 50 MHz and less than or equal to D + 50 MHz, where D is the zero-field splitting parameter of the NV color center.

[0024] Optionally, the particle size range of the nanodiamond is greater than or equal to 40 nm and less than or equal to 1 μm;

[0025] When the target fluid flows through the detection area, within a single period of the modulated fluorescence signal, the number of detection windows collected by the optical detection module is at least one, and the fluorescence counting rate of the nanodiamond, the contrast of the modulated fluorescence signal, and the exposure time of a single detection window satisfy the following relationship:

[0026]

[0027] where C is the contrast of the modulated fluorescence signal, R is the fluorescence counting rate of the nanodiamond, and t is the exposure time of a single detection window.

[0028] Optionally, the excitation light module includes an optical pump, and the optical pump is used to emit laser light;

[0029] The contrast of the modulated fluorescence signal generated by the NV color centers in the nanodiamond satisfies the following relationship:

[0030]

[0031] where Θ is the normalization constant, Γ p is the laser polarization rate, Γ c is the coherent relaxation rate caused by the optical pump, Ω R is the frequency of the Rabi oscillation of the NV color center driven by the microwave field, Γ p 、Γ c the two terms are related to the laser power, and the Ω R term is positively correlated with the microwave field strength felt by the NV color centers in the nanodiamond.

[0032] Optionally, the microwave field intensity sensed by the NV color centers in the nanodiamond satisfies the following relationship:

[0033]

[0034] where B is the microwave field intensity sensed by the NV color centers in the nanodiamond, and B1 is the microwave field intensity of the microwave field.

[0035] Optionally, for a single detection window, the excitation light remains on all the time, the microwave radiation module provides a microwave radiation signal, and the light detection module uses the entire detection window length as the exposure time to obtain the fluorescence signal intensity of the nanodiamond within a single sampling point.

[0036] Optionally, the delay time between the excitation light initialization pulse and the modulation fluorescence signal collection pulse window is greater than a preset delay time.

[0037] According to another aspect of the present invention, there is provided a flow fluorescence detection system based on the microwave field switching state, and the flow fluorescence detection system based on the microwave field switching state is applicable to any of the flow fluorescence detection methods based on the microwave field switching state in the first aspect of the present invention.

[0038] The embodiment of the present invention provides a flow fluorescence detection method based on the microwave field switching state. The flow fluorescence detection method is different from the traditional light detection method based on NV color center in that the nanodiamond particles to be detected are not stably laid on the substrate, but are placed inside the sample flow channel and are in a moving state. The target flow specifically bound to the nanodiamond label flows through the detection area of ​​the sample flow channel. The nanodiamond includes an NV color center. The NV color center in the nanodiamond is irradiated by the excitation light emitted by the excitation light module. The excitation light is used to excite and cause the electrons of the NV color center in the detection area of ​​the sample flow channel to transition, and the NV color center in the nanodiamond can emit a fluorescence signal. When the target flow specifically bound to the nanodiamond label flows through the first half of the detection area of ​​the sample flow channel, the microwave radiation module is in the open state, and there is a microwave radiation signal in the detection area. The NV color center in the nanodiamond is irradiated by the excitation light, and the electrons of the NV color center transition to produce a dark state fluorescence signal. When the target flow specifically bound to the nano-diamond marker flows through the latter half of the detection area of ​​the sample flow channel, the microwave radiation module is in the off state, and there is no microwave radiation signal in the detection area. The NV color center in the nano-diamond is irradiated by the excitation light, and the electrons of the NV color center transition to produce a bright state fluorescence signal. The light detection module collects the modulated fluorescence signal generated by the NV color center in the nano-diamond when the target flow specifically bound to the nano-diamond marker flows through the detection area of ​​the sample flow channel at a preset collection frequency. Since the modulated fluorescence signal is associated with whether the target contains the biological molecule to be detected, the data processing module determines whether the target contains the biological molecule to be detected based on the magnitude relationship between the intensity of the dark state fluorescence signal and the intensity of the bright state fluorescence signal. Due to the NV color center system due to its stability, room temperature atmospheric environment compatibility and biocompatibility, and nano-diamond as a new type of biological fluorescence marker, low-frequency noise interference can be eliminated through signal modulation and demodulation technology, so it can achieve a detection sensitivity far higher than other fluorescent labeling detection methods. In summary, the technical solution provided in the embodiment of the present invention improves the detection sensitivity of the fluorescence detection method, and can realize the fluorescence signal recognition of a single molecule.

[0039] It should be understood that the contents described in this section are not intended to identify the key or important features of the embodiments of the present invention, nor are they intended to limit the scope of the present invention. Other features of the present invention will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0041] Figure 1It is a schematic structural diagram of a flow cytometry fluorescence detection system provided according to an embodiment of the present invention;

[0042] Figure 2 It is a block diagram of the structure of a flow cytometry fluorescence detection system provided according to an embodiment of the present invention;

[0043] Figure 3 It is a schematic structural diagram of another flow cytometry fluorescence detection system provided according to an embodiment of the present invention;

[0044] Figure 4 It is a schematic diagram of the optical path system of a flow cytometry fluorescence detection system provided according to an embodiment of the present invention;

[0045] Figure 5 It is a schematic structural diagram of yet another flow cytometry fluorescence detection system provided according to an embodiment of the present invention;

[0046] Figure 6 It is Figure 5 A cross-sectional view of a simulation diagram of the microwave field provided by the microwave radiation structure in the structure of the flow cytometry fluorescence detection system;

[0047] Figure 7 It is Figure 5 A three-dimensional view of a simulation diagram of the microwave field provided by the microwave radiation structure in the structure of the flow cytometry fluorescence detection system;

[0048] Figure 8 It is a schematic structural diagram of yet another flow cytometry fluorescence detection system provided according to an embodiment of the present invention;

[0049] Figure 9 It is Figure 8 A cross-sectional view of a simulation diagram of the microwave field provided by the microwave radiation structure in the structure of the flow cytometry fluorescence detection system;

[0050] Figure 10 It is a schematic diagram showing the relationship between the fluorescence intensity of the NV color center and the frequency of the microwave radiation signal provided according to an embodiment of the present invention;

[0051] Figure 11 It is a schematic diagram of the optical path structure of an optical detection module provided according to an embodiment of the present invention;

[0052] Figure 12 It is a schematic flowchart of a flow cytometry fluorescence detection method based on zero field provided according to an embodiment of the present invention;

[0053] Figure 13 It is Figure 12 The flowchart included in S110;

[0054] Figure 14 It is Figure 12 The flowchart included in S130;

[0055] Figure 15 It is a schematic diagram showing the relationship between the frequency of a microwave radiation signal and the flow rate of nanodiamonds according to an embodiment of the present invention;

[0056] Figure 16 It is a schematic diagram for collecting a modulated fluorescence signal according to an embodiment of the present invention;

[0057] Figure 17 It is a schematic flow diagram of a flow cytometry fluorescence detection method based on the switching state of a microwave field according to an embodiment of the present invention;

[0058] Figure 18 is Figure 17 the schematic flow diagram included in S210 in Detailed implementation manners

[0059] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0060] It should be noted that the terms "first", "second", etc. in the description and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged under appropriate circumstances so that the embodiments of the present invention described herein can be implemented in an order different from those illustrated or described herein. In addition, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device that includes a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0061] In order to improve the detection sensitivity of the fluorescence detection method, an embodiment of the present invention provides a flow cytometry fluorescence detection method based on the switching state of a microwave field. Before introducing the flow cytometry fluorescence detection method based on the switching state of a microwave field provided by the embodiment of the present invention, the flow cytometry fluorescence detection system applicable to the flow cytometry fluorescence detection method provided by the embodiment of the present invention will be introduced first.

[0062] As Figure 1 shown, Figure 1 it is a schematic structural diagram of a flow cytometry fluorescence detection system according to an embodiment of the present invention, Figure 2It is a structural block diagram of a flow cytometry fluorescence detection system provided according to an embodiment of the present invention. The flow cytometry fluorescence detection system includes:

[0063] A sample flow channel 100 for transporting a target specifically bound to a nanodiamond label. The nanodiamond includes an NV color center, and a detection area S1 is provided in the sample flow channel 100.

[0064] An excitation light module 200 for emitting excitation light to excite the NV color centers in the detection area S1 of the sample flow channel 100, causing electron transitions to generate fluorescence.

[0065] A microwave radiation module 300 for providing a microwave field to the detection area S1 of the sample flow channel 100. The microwave field can resonate with all or part of the NV color centers in the nanodiamond. The frequency of the microwave radiation signal provided by the microwave field is equal to the energy level difference of the electron spin transition of the NV color center, enabling resonance between the microwave field and the NV color center. The microwave radiation module 300 includes at least two microwave radiation structures 301 distributed along the transport direction of the target and arranged around the detection area S1 of the sample flow channel 100.

[0066] A light detection module 400 for collecting, at a preset collection frequency, fluorescence signals with periodically varying intensities emitted by the NV color centers in the nanodiamond when a target specifically bound to the nanodiamond label flows through the detection area S1 of the sample flow channel 100 by controlling at least one of the intensity, frequency, and on / off of the microwave field; the fluorescence signals are associated with whether the target contains a biomolecule to be detected.

[0067] It is known that quantum sensing systems include Rydberg atoms, atomic magnetometers, superconducting quantum interference devices, diamond NV color centers, etc. Among them, the NV color center system is one of the most promising solid-state quantum sensors in the fields of life science and medical detection due to its stability, compatibility with room temperature atmospheric environment, and biocompatibility. As a new type of bio-fluorescent label, nanodiamonds can eliminate low-frequency noise interference through signal modulation and demodulation techniques, thus achieving detection sensitivity far higher than other fluorescence label detection methods.

[0068] Under the irradiation of the excitation light emitted by the excitation light module 200, the NV color centers in the nanodiamond are used to excite the NV color centers in the detection region S1 of the sample channel 100, causing the electrons to transition and generate fluorescence. Exemplarily, when irradiated with a laser of 532 nm wavelength emitted by the excitation light module 200, the NV color centers will emit fluorescence signals in the wavelength range of 630 - 800 nm. In this embodiment, the excitation light generation module 500 generates the excitation light, and the excitation light module 200 emits the excitation light as a flat-top beam, which can uniformly converge the excitation light on the detection system of the sample channel 100.

[0069] The microwave radiation module 300 includes at least two microwave radiation structures 301, which are distributed along the conveying direction of the target, and the microwave radiation structures 301 are arranged around the detection region S1 of the sample channel 100. As Figure 1 shown, the conveying direction of the target is from top to bottom. The at least two microwave radiation structures are distributed along the conveying direction of the target, which can ensure that during the process of the target flowing through the detection region S1, the microwave field provided by the microwave radiation module 300 can cover the entire detection region S1. Then, when the microwave radiation signal provided by the microwave field changes, the fluorescence signal generated by the NV color centers in the nanodiamond will change accordingly.

[0070] In this embodiment, when the microwave radiation module 300 provides a microwave field for the detection region S1 of the sample channel 100, the microwave field can provide a microwave radiation signal. Due to the presence and absence of the microwave radiation signal, the fluorescence signals generated by the NV color centers in the nanodiamond are different; or when the frequency or power of the microwave radiation signal changes, the fluorescence signal generated by the NV color centers in the nanodiamond will change accordingly. The specific changes are as follows:

[0071] When the microwave radiation module 300 is in the on state and there is a microwave radiation signal in the detection region S1, under the irradiation of the excitation light, the electrons of the NV color centers in the nanodiamond transition to generate dark-state fluorescence signals. The dark-state fluorescence signals are the fluorescence signals generated by the NV color centers in the nanodiamond when some or all of the NV color centers resonate with the microwave field.

[0072] When the microwave radiation module 300 is in the off state and there is no microwave radiation signal in the detection region S1, under the irradiation of the excitation light, the electrons of the NV color centers in the nanodiamond transition to generate bright-state fluorescence signals. The bright-state fluorescence signals are the fluorescence signals generated by the NV color centers in the nanodiamond when no NV color centers resonate with the microwave field. The intensity of the dark-state fluorescence signals is less than the intensity of the bright-state fluorescence signals.

[0073] When the frequency or radiation power of the microwave radiation signal provided by the microwave field changes, a fluorescence signal that changes accordingly is generated by the NV color centers in the nanodiamond. In this embodiment, since the frequency or power of the microwave radiation signal provided by the microwave field changes periodically, when the nanodiamond is irradiated with excitation light, the electrons of the NV color centers undergo transitions, the number of NV color centers resonating with the microwave field changes periodically, and / or the degree of resonance of the NV color centers changes periodically, and the NV color centers generate a fluorescence signal with a periodically changing intensity.

[0074] Specifically, when the modulation mode of the microwave field is amplitude modulation, the power of the microwave radiation signal provided by the microwave field changes. For example, in the simplest square wave modulation, sinusoidal wave modulation and triangular wave modulation achieved by changing the microwave power. When the modulation mode of the microwave field is frequency modulation, the frequency of the microwave radiation signal provided by the microwave field changes. For example, the microwave resonance-non-resonance mode. When the frequency of the microwave radiation signal provided by the microwave field is equal to the energy level difference of the electron spin transition of the NV color centers, the microwave field can be made to resonate with the NV color centers.

[0075] In a flow cytometry fluorescence detection system implemented based on a nanodiamond fluorescence marker provided by an embodiment of the present invention, the difference between the flow cytometry fluorescence detection system and the traditional optical detection system based on NV centers is that the to-be-detected nanodiamond particles are not stably laid flat on the substrate, but are placed inside the sample flow channel 100 and are in a moving state. The target substance specifically bound to the nanodiamond flows through the detection area S1 of the sample flow channel 100. The nanodiamond includes NV centers. Under the irradiation of the excitation light emitted by the excitation light module 200, the excitation light is used to excite the NV centers in the detection area S1 of the sample flow channel 100, causing the electrons to transition and generate fluorescence. When the microwave radiation module 300 provides a microwave field for the detection area S1 of the sample flow channel 100, the microwave field can provide a microwave radiation signal. When the microwave radiation signal exists and does not exist, the fluorescence signals generated by the NV centers in the nanodiamond are different; alternatively, when the frequency or radiation power of the microwave radiation signal changes, the fluorescence signals generated by the NV centers in the nanodiamond change accordingly, that is, by controlling at least one of the intensity, frequency, and on / off of the microwave field, fluorescence signals with periodically changing intensities are emitted by the NV centers in the nanodiamond. The light detection module 400 collects the fluorescence signals generated by the NV centers in the nanodiamond when the target substance specifically bound to the nanodiamond flows through the detection area S1 of the sample flow channel 100 at a preset collection frequency. Since the fluorescence signals are related to whether the target substance contains the biomolecule to be detected, it is possible to determine whether the target substance contains the biomolecule to be detected based on the fluorescence signals. Due to the stability, room-temperature atmospheric environment compatibility, and biocompatibility of the NV center system, and the fact that nanodiamonds are a new type of biological fluorescence marker, low-frequency noise interference can be excluded through signal modulation and demodulation techniques, so the detection sensitivity far higher than other fluorescence marker detection methods can be achieved. In summary, the flow cytometry fluorescence detection system provided by the embodiment of the present invention improves the detection sensitivity of the fluorescence detection system and can achieve the recognition of single-molecule fluorescence signals.

[0076] Optionally, on the basis of the above technical solution, as Figure 2 shown, it further includes a control module 600. The control module 600 is connected to the microwave radiation module 300, and the control module 600 is used to emit control signals.

[0077] The control signals include at least one of a microwave field turn-on signal, a microwave field turn-off signal, a microwave field power periodically changing signal, and a microwave field frequency periodically changing signal.

[0078] Optionally, based on the above technical solution, the microwave radiation module 300 is configured to provide a modulated microwave field for the detection region S1 of the sample flow channel 100 according to the microwave field turn-on signal. The NV centers in the nanodiamonds emit a dark-state fluorescence signal under the excitation light irradiation. The dark-state fluorescence signal is the fluorescence signal generated by the NV centers in the nanodiamonds when some or all of the NV centers resonate with the microwave field. The microwave radiation module 300 is configured to stop providing the modulated microwave field for the detection region S1 of the sample flow channel 100 according to the microwave field turn-off signal. The NV centers in the nanodiamonds emit a bright-state fluorescence signal under the excitation light irradiation. The bright-state fluorescence signal is the fluorescence signal generated by the NV centers in the nanodiamonds when no NV center resonates with the microwave field.

[0079] Optionally, based on the above technical solution, the microwave radiation module 300 is configured to emit a microwave modulation field with a fixed frequency and a periodically varying power according to the periodically varying signal of the microwave field power, so that the NV centers in the nanodiamonds emit a fluorescence signal with a periodically varying intensity under the excitation light irradiation.

[0080] Optionally, based on the above technical solution, the microwave radiation module 300 is configured to emit a microwave modulation field with a fixed power and a periodically varying frequency according to the periodically varying signal of the microwave field frequency, so that the NV centers in the nanodiamonds emit a fluorescence signal with a periodically varying intensity under the excitation light irradiation.

[0081] Specifically, the microwave radiation module 300 is in the on state under the control of the microwave field turn-on signal, and there is a microwave radiation signal in the detection region S1. When the NV centers in the nanodiamonds are irradiated with the excitation light, the electrons of the NV centers undergo transitions to generate a dark-state fluorescence signal. The dark-state fluorescence signal is the fluorescence signal generated by the NV centers in the nanodiamonds when some or all of the NV centers resonate with the microwave field.

[0082] The microwave radiation module 300 is in the off state under the control of the microwave field turn-off signal, and there is no microwave radiation signal in the detection region S1. When the NV centers in the nanodiamonds are irradiated with the excitation light, the electrons of the NV centers undergo transitions to generate a bright-state fluorescence signal. The bright-state fluorescence signal is the fluorescence signal generated by the NV centers in the nanodiamonds when no NV center resonates with the microwave field. The intensity of the dark-state fluorescence signal is less than the intensity of the bright-state fluorescence signal.

[0083] Alternatively, the microwave radiation module 300 is controlled by the periodically varying signal of the microwave field power to emit a microwave modulation field with a fixed frequency and a periodically varying power, so that when the NV centers in the nanodiamonds are irradiated with the excitation light, the electrons of the NV centers undergo transitions, the number of NV centers resonating with the microwave field varies periodically, and / or the degree of resonance of the NV centers varies periodically, and the NV centers generate a fluorescence signal with a periodically varying intensity.

[0084] Alternatively, under the control of a microwave field frequency periodic change signal, the microwave radiation module 300 emits a microwave modulation field with a fixed power and a periodically changing frequency, so that under the excitation light irradiation of the NV centers in the nanodiamond, the electrons of the NV centers undergo transitions, and the number of NV centers resonating with the microwave field changes periodically, and / or the degree of resonance of the NV centers changes periodically, and the NV centers generate a fluorescence signal with a periodically changing intensity.

[0085] Optionally, on the basis of the above technical solution, a data processing module 700 is further included. The data processing module 700 is connected to the optical detection module 400, and the data processing module 700 determines whether the target contains the biomolecule to be detected according to the fluorescence signal.

[0086] Specifically, the frequency or power of the microwave radiation signal provided by the microwave field changes periodically, the number of NV centers resonating with the microwave field changes periodically, and / or the degree of resonance of the NV centers changes periodically, and the NV centers generate a fluorescence signal with a periodically changing intensity. The first judgment method is adopted: the data processing module 700 performs Fourier transform on the fluorescence signal to obtain a frequency-domain signal, and the part with the same frequency as the control signal is the demodulation signal; if the intensity of the demodulation signal is greater than the first preset threshold, it is determined that the target contains the biomolecule to be detected.

[0087] The microwave radiation module 300 is in an on state under the control of a microwave field turn-on signal, and the electrons of the NV centers undergo transitions to generate a dark-state fluorescence signal. The dark-state fluorescence signal is the fluorescence signal generated by the NV centers in the nanodiamond when some or all of the NV centers resonate with the microwave field. The microwave radiation module 300 is in an off state under the control of a microwave field turn-off signal, and the electrons of the NV centers undergo transitions to generate a bright-state fluorescence signal. The bright-state fluorescence signal is the fluorescence signal generated by the NV centers in the nanodiamond when no NV centers resonate with the microwave field. The intensity of the dark-state fluorescence signal is less than the intensity of the bright-state fluorescence signal. The second judgment method is adopted. The intensity of the dark-state fluorescence signal is less than the intensity of the bright-state fluorescence signal, and the absolute value of the difference between the intensity of the dark-state fluorescence signal and the intensity of the bright-state fluorescence signal is greater than the second preset threshold. The data processing module 700 determines that the target contains the biomolecule to be detected, and the second preset threshold is at least 1 times the noise level when the bright-state fluorescence signal is generated.

[0088] Optionally, on the basis of the above technical solution, as Figure 1 and Figure 3 shown, the microwave radiation module 300 includes two microwave radiation structures 301. One microwave radiation structure 301 is located at the starting position of the detection area S1, and the other microwave radiation structure 301 is located at the ending position of the detection area S1. The direction from the starting position of the detection area S1 to the ending position of the detection area S1 is parallel to the transmission direction of the target.

[0089] As Figure 4 shown, in a flow cytometry detection system, a sheath fluid is arranged around a sample flow channel 100, and a target substance specifically bound to a nanodiamond marker flows through a detection area S1 of the sample flow channel 100. Under the irradiation of excitation light emitted by an excitation light module 200, the excitation light is used to excite the NV centers in the detection area S1 of the sample flow channel 100, causing the electrons to undergo transitions and generate fluorescence. When a microwave radiation module 300 provides a microwave field for the detection area S1 of the sample flow channel 100, the microwave field can provide a microwave radiation signal. When the microwave radiation signal changes, the NV centers in the nanodiamond generate corresponding fluorescence signals that change accordingly. As Figure 1 and 3 shown, the transmission direction of the target substance in the figure is from top to bottom. A microwave radiation structure 301 is located at the starting position of the detection area S1, and another microwave radiation structure 301 is located at the ending position of the detection area S1, which can ensure that during the process of the target substance flowing through the detection area S1, the microwave radiation signal emitted by the microwave radiation module 300 covers the entire detection area S1. Then, when the microwave radiation signal changes, the NV centers in the nanodiamond generate corresponding fluorescence signals that change accordingly. When the microwave radiation module 300 provides a microwave field for the detection area S1 of the sample flow channel 100, the microwave field can provide a microwave radiation signal. When the microwave radiation signal is present or absent, the fluorescence signals generated by the NV centers in the nanodiamond are different; or when the frequency or radiation power of the microwave radiation signal changes, the NV centers in the nanodiamond generate corresponding fluorescence signals that change accordingly.

[0090] Optionally, on the basis of the above technical solution, as Figure 5 shown, the microwave radiation structure 301 includes a microstrip line L1 and a microwave resonant cavity structure L2; the microwave resonant cavity structure L2 is arranged around the detection area S1 of the sample flow channel 100, and the microstrip line L1 is located on one side of the microwave resonant cavity structure L2.

[0091] Specifically, Figure 6 and Figure 7 shown, the gray level of the detection area S1 of the sample flow channel 100 is uniform. The microstrip line L1 generates a microwave radiation signal under the action of an electrical signal, and the microwave resonant cavity structure L2 provides a microwave field with a very high degree of uniformity for the detection area S1 of the sample flow channel 100. The microwave field can provide a microwave radiation signal, and the microwave radiation signal irradiates the entire detection area S1.

[0092] Optionally, on the basis of the above technical solution, as Figure 5 shown, the microwave resonant cavity structure L2 includes two coaxial radiation coils.

[0093] Specifically, two coaxial radiation coils form a microwave resonance cavity structure L2, and the microwave radiation signal generated by the microstrip line L1 under the action of an electrical signal irradiates the entire detection area S1.

[0094] Optionally, on the basis of the above technical solution, as Figure 8 shown, the microwave radiation structure 301 includes a radiation antenna of an "Ω"-shaped coil.

[0095] Specifically, as Figure 9 shown, the gray level of the detection area S1 of the sample flow channel 100 is uniform.

[0096] The radiation antenna of the "Ω"-shaped coil provides a microwave field with a very high degree of uniformity for the detection area S1 of the sample flow channel 100 under the action of an electrical signal. The microwave field can provide a microwave radiation signal, and the microwave radiation signal irradiates the entire detection area S1. The "Ω"-shaped coil can be a single-turn or multi-turn copper wire. Among them, as Figure 9 shown, the detection area S1 includes a detection point 100a of the optical detection module 400.

[0097] Optionally, on the basis of the above technical solution, as Figure 1 and Figure 2 shown, a magnetic field monitoring module 800 is further included. The magnetic field monitoring module 800 is used to monitor the magnetic field intensity near the detection area S1 of the sample flow channel 100. When the magnetic field intensity monitored by the magnetic field monitoring module 800 near the detection area S1 of the sample flow channel 100 meets the zero-field condition, the microwave field is used to provide a microwave radiation signal with a fixed frequency.

[0098] In an external magnetic field, due to the Zeeman effect, the electrons of the NV color centers in the nanodiamond cause the energy levels between the |m s = 0> state and the |m s = ±1> state to shift in opposite directions, and the frequency difference is proportional to the magnitude of the projection of the external magnetic field in the NV color center crystal axis direction. A single nanodiamond contains multiple NV color centers, and the crystal axis directions of different NV color centers are different; moreover, the spatial directions of different nanodiamond particles when flowing through the sample flow channel 100 are random. Therefore, for the NV color centers in each axial direction within different nanodiamond particles, the projection components of the external magnetic field are different, resulting in different transition frequencies between the |m s = 0> state and the |m s = ±1> state. Under the control of a microwave with a single frequency, the quantum states of the NV color centers in some nanodiamond particles do not change, and the overall fluorescence brightness change is not obvious, thus affecting the signal-to-noise ratio of the measurement scheme, and further affecting the accuracy and reliability of the detection of the analyte.

[0099] Therefore, when the magnetic field intensity near the detection region S1 of the sample flow channel 100 monitored by the magnetic field monitoring module 800 satisfies the zero-field condition, the resonance frequency of some or all of the NV color centers with the microwave field is the natural frequency. The microwave field is used to provide a microwave radiation signal with this fixed frequency, which can improve the signal-to-noise ratio of the fluorescence signal, and thus improve the detection sensitivity of the flow cytometry fluorescence detection system.

[0100] Optionally, on the basis of the above technical solution, as Figure 10 shown, when the magnetic field intensity near the detection region S1 of the sample flow channel 100 monitored by the magnetic field monitoring module 800 satisfies the zero-field condition, the energy level splitting of the NV color centers in the nanodiamond caused by the magnetic field is less than half of the linewidth of its optically detected magnetic resonance spectrum.

[0101] "The energy level splitting of the NV color centers in the nanodiamond" refers to the shift in the resonance peak position of the optically detected magnetic resonance spectrum caused by the Zeeman shift of the NV energy levels due to an external magnetic field in the optically detected magnetic resonance spectrum of the NV color centers - that is, half of the difference between the two microwave resonance frequencies corresponding to the energy level splitting of the same NV color center; "the linewidth of its optically detected magnetic resonance spectrum" refers to the full width at half maximum of the resonance peak of the optically detected magnetic resonance spectrum of the NV color centers.

[0102] Among them, when the magnetic field intensity near the detection region S1 of the sample flow channel 100 monitored by the magnetic field monitoring module 800 satisfies the zero-field condition, the fixed frequency of the microwave radiation signal provided by the microwave field is greater than or equal to 2820 MHz and less than or equal to 2920 MHz, so that the NV color centers in all crystal axis directions in the nanodiamond particles can be quantum state controlled by microwaves with a frequency located in the zero-field splitting greater than or equal to 2820 MHz and less than or equal to 2920 MHz. Therefore, the system can use a frequency greater than or equal to 2820 MHz and less than or equal to 2920 MHz as the fixed operating frequency of the microwave radiation signal provided by the microwave radiation module 300. As is well known to those skilled in the art, the zero-field splitting D of the NV color centers at room temperature has a value of 2870 MHz, but the exact value of the zero-field splitting D has a linear relationship with temperature; it is not difficult to understand that the fixed frequency of the above microwave radiation signal is set between 2820 MHz and 2920 MHz on the premise that the zero-field splitting D = 2870 MHz is default.

[0103] In order to make the embodiments of the present invention more concise and clear, it is default that the experimental environment temperature is room temperature in the following text, that is, the value of the zero-field splitting D is 2870 MHz; however, it is not difficult for those skilled in the art to understand that the zero-field splitting D may show other values due to the influence of different experimental environment temperatures, and the fixed frequency of the microwave radiation signal can usually be set between D - 50 MHz and D + 50 MHz.

[0104] The technical solution provided by the embodiments of the present invention is to improve the signal-to-noise ratio of the fluorescence signal and enhance the recognition accuracy of the analyte. The system introduces a microwave field to modulate the fluorescence signal of the nanodiamond particles: when the microwave radiation module 300 turns on microwaves with a frequency greater than or equal to 2820 MHz and less than or equal to 2920 MHz and enters the radiation coil, the NV color centers in the nanodiamond particles in the sample flow channel 100 transition from the |m s = 0> state to the |m s = ±1> state, and the fluorescence intensity of the nanodiamond particles decreases; when the microwave in the radiation coil is turned off, the excitation light in the detection area S1 initializes the NV color centers to the |m s = 0> state, and the fluorescence intensity recovers. When the radiation power of the microwave radiation signal provided by the microwave radiation module 300 changes, a fluorescence signal that changes accordingly is generated in the NV color centers in the nanodiamonds. The microwave radiation module 300 modulates the fluorescence signal in the time domain, and then the optical detection module 400 collects it at a certain frequency. Finally, the algorithm identifies the analyte signal at a specific frequency and excludes the interference of the static noise signal.

[0105] Optionally, on the basis of the above technical solution, as Figure 11 shown, the flow-through fluorescence detection system includes at least two optical detection modules 400, which can ensure that the fluorescence signals at all angles in the circumferential direction of the detection area S1 can be collected by the optical detection module 400.

[0106] Exemplarily, Figure 1 and Figure 11 show two optical detection modules 400. The optical detection modules 400 are arranged around the detection area of the sample flow channel and are symmetrically arranged with respect to the detection area S1. When the number of the optical detection modules 400 is greater than two, multiple optical detection modules 400 are arranged around the detection area S1 of the sample flow channel 100, and it is only necessary to ensure that the fluorescence signals at all angles in the circumferential direction of the detection area S1 can be collected by the optical detection module 400. On the one hand, the above technical solution can increase the collection amount of the fluorescence signal, thereby providing a lower detection limit; on the other hand, it can overcome the problem that the target object blocks the fluorescence of the NV color centers.

[0107] Since in the single-molecule detection application scenario, the fluorescence intensity of a single nanodiamond particle is weak, the optical detection module 400 preferably uses an avalanche photodiode photodetector (APD), secondly, a scientific camera can be selected, and thirdly, a photomultiplier tube sensor (PMT) can be selected.

[0108] In addition, the interference of the excitation light and the stray fluorescence signal can be filtered out by a notch filter, a long-pass filter, and a short-pass filter.

[0109] As Figure 12 shown, Figure 12It is a schematic flow chart of a flow cytometry fluorescence detection method based on the switching state of a microwave field provided by the present invention. The flow cytometry fluorescence detection method based on the switching state of a microwave field includes the following steps:

[0110] S110. When the target substance labeled with nanodiamond flows through the detection area of the sample flow channel, by controlling at least one of the intensity, frequency, and on / off of the microwave field, a fluorescence signal with a periodically changing intensity is emitted from the NV color centers in the nanodiamond.

[0111] Among them, as Figure 1 shown, the magnetic field intensity in the detection area S1 satisfies the zero-field condition. The excitation light module 200 emits excitation light, which is used to excite the NV color centers in the detection area S1 of the sample flow channel 100, causing the electrons to undergo transitions and generate fluorescence. The microwave radiation module 300 is used to provide a microwave field for the detection area S1 of the sample flow channel 100. The microwave field resonates with all or part of the NV color centers in the nanodiamond. The frequency of the microwave radiation signal provided by the microwave field is equal to the energy level difference of the electron spin transition of the NV color center, enabling the microwave field to resonate with the NV color center. The microwave radiation signal provided by the microwave field has a fixed frequency.

[0112] In this embodiment, when the microwave radiation module 300 provides a microwave field for the detection area S1 of the sample flow channel 100, the microwave field can provide a microwave radiation signal. When the microwave radiation signal is present or absent, the fluorescence signals generated by the NV color centers in the nanodiamond are different; or when the frequency or power of the microwave radiation signal changes, the fluorescence signals generated by the NV color centers in the nanodiamond change accordingly.

[0113] When the microwave radiation module 300 is in the on state and there is a microwave radiation signal in the detection area S1, when the NV color centers in the nanodiamond are irradiated with excitation light, the electrons of the NV color centers undergo transitions to generate a dark-state fluorescence signal. The dark-state fluorescence signal is the fluorescence signal generated by the NV color centers in the nanodiamond when all or part of the NV color centers resonate with the microwave field.

[0114] When the microwave radiation module 300 is in the off state and there is no microwave radiation signal in the detection area S1, when the NV color centers in the nanodiamond are irradiated with excitation light, the electrons of the NV color centers undergo transitions to generate a bright-state fluorescence signal. The bright-state fluorescence signal is the fluorescence signal generated by the NV color centers in the nanodiamond when no NV color centers resonate with the microwave field. The intensity of the dark-state fluorescence signal is less than the intensity of the bright-state fluorescence signal.

[0115] When the frequency or power of the microwave radiation signal provided by the microwave field changes, the NV color centers in the nanodiamond generate fluorescence signals that change accordingly. In this embodiment, since the frequency or radiation power of the microwave radiation signal provided by the microwave field changes periodically, when the NV color centers in the nanodiamond are irradiated with excitation light, the electrons of the NV color centers undergo transitions, and the number of NV color centers resonating with the microwave field changes periodically, and / or the degree of resonance of the NV color centers changes periodically, and the NV color centers generate fluorescence signals with periodically changing intensities.

[0116] Specifically, when the modulation mode of the microwave field is amplitude modulation, the radiation power of the microwave radiation signal provided by the microwave field changes. For example, the simplest square wave modulation, sine wave modulation, and triangular wave modulation achieved by changing the microwave power. When the modulation mode of the microwave field is frequency modulation, the frequency of the microwave radiation signal provided by the microwave field changes. For example, the microwave resonance-non-resonance mode. When the frequency of the microwave radiation signal provided by the microwave field is equal to the energy level difference of the electron spin transition of the NV color center, the microwave field can be made to resonate with the NV color center.

[0117] Optionally, on the basis of the above technical solution, as Figure 1 and Figure 2 shown, the magnetic field monitoring module 800 monitors the magnetic field intensity near the detection area S1 of the sample flow channel 100; when the magnetic field intensity near the detection area S1 of the sample flow channel 100 monitored by the magnetic field monitoring module 800 satisfies the zero-field condition, the microwave field is used to provide a microwave radiation signal with a fixed frequency.

[0118] In an external magnetic field, due to the Zeeman effect, the electrons of the NV color centers in the nanodiamond cause the energy levels between the |m s = 0> state and the |m s = ±1> state to shift in opposite directions, and the frequency difference is proportional to the magnitude of the projection of the external magnetic field in the crystal axis direction of the NV color center. A single nanodiamond contains multiple NV color centers, and the crystal axis directions of different NV color centers are different; and the spatial directions of different nanodiamond particles when flowing through the sample flow channel 100 are random. Therefore, for the NV color centers in each axial direction in different nanodiamond particles, the projection components of the external magnetic field are different, resulting in different transition frequencies between the |m s = 0> state and the |m s = ±1> state. Under the control of microwaves with a single frequency, the quantum states of the NV color centers in some nanodiamond particles do not change, and the overall fluorescence brightness change is not obvious, thus affecting the signal-to-noise ratio of the measurement scheme, and further affecting the accuracy and reliability of the detection of the analyte.

[0119] Therefore, when the magnetic field intensity near the detection area S1 of the sample flow channel 100 monitored by the magnetic field monitoring module 800 satisfies the zero-field condition, the resonance frequency of some or all of the NV color centers with the microwave field is the natural frequency. The microwave field is used to provide a microwave radiation signal with this fixed frequency, which can improve the signal-to-noise ratio of the fluorescence signal, and thus improve the detection sensitivity of the flow cytometry fluorescence detection system.

[0120] Optionally, on the basis of the above technical solution, as Figure 10 shown, when the magnetic field intensity near the detection area S1 of the sample flow channel 100 monitored by the magnetic field monitoring module 800 satisfies the zero-field condition, the energy level splitting of the NV color centers in the nanodiamond caused by the magnetic field is less than half of the linewidth broadening of its optically detected magnetic resonance spectrum.

[0121] The "energy level splitting of the NV color centers in the nanodiamond" refers to the shift in the position of the resonance peak of the optically detected magnetic resonance spectrum of the NV color centers due to the Zeeman shift of the NV energy levels caused by the external magnetic field - that is, half of the difference between the two microwave resonance frequencies corresponding to the energy level splitting of the same NV color center; the "linewidth broadening of its optically detected magnetic resonance spectrum" refers to the full width at half maximum of the resonance peak of the optically detected magnetic resonance spectrum of the NV color centers.

[0122] Among them, when the magnetic field intensity near the detection area S1 of the sample flow channel 100 monitored by the magnetic field monitoring module 800 satisfies the zero-field condition, the fixed frequency of the microwave radiation signal provided by the microwave field is greater than or equal to 2820 MHz and less than or equal to 2920 MHz, so that the NV color centers in all crystal axis directions in the nanodiamond can be quantum state controlled by microwaves with a frequency located in the zero-field splitting greater than or equal to 2820 MHz and less than or equal to 2920 MHz. Therefore, the system can use a frequency greater than or equal to 2820 MHz and less than or equal to 2920 MHz as the fixed operating frequency of the microwave radiation signal provided by the microwave radiation structure 300.

[0123] It should be noted that in the embodiments of the present invention, it is required that the spectral peak shift caused by the magnetic field does not exceed the intrinsic broadening of the resonance (CW) spectrum, and the intrinsic broadening of the CW spectrum is determined by its dephasing time. However, in nanodiamonds with different manufacturing processes and diameters, the noise environments of the NV color centers are different, corresponding to different dephasing times, and the zero-field limit needs to be determined according to the selected particles. Taking HPHT-processed nanodiamonds with a diameter of 40 nm and an NV concentration of 1.5 ppm as an example, the typical value of the intrinsic broadening of its CW spectrum is 20 MHz, corresponding to a requirement that the external magnetic field is below 3.56 Gs. The resonance (CW) spectrum is Figure 10 the spectral line in.

[0124] S120. The optical detection module collects the fluorescence signal at a preset collection frequency.

[0125] As Figure 11As shown in the figure, the flow cytometry system includes at least two light detection modules 400, which can ensure that the fluorescence signals at all angles in the circumferential direction of the detection area S1 can be collected by the light detection modules 400.

[0126] Exemplarily, Figure 1 and Figure 11 Two light detection modules 400 are shown. The light detection modules 400 are arranged around the detection area S1 of the sample flow channel 100 and are symmetrically arranged with respect to the detection area S1.

[0127] When the number of the light detection modules 400 is greater than two, the multiple light detection modules 400 are arranged around the detection area S1 of the sample flow channel 100, and it is only necessary to ensure that the fluorescence signals at all angles in the circumferential direction of the detection area S1 can be collected by the light detection modules 400.

[0128] On the one hand, the above technical solution can increase the collection amount of fluorescence signals, thereby providing a lower detection limit; on the other hand, it can overcome the problem of the target object blocking the fluorescence of the NV center.

[0129] Since in the application scenario of single molecule detection, the fluorescence intensity of a single nanodiamond particle is weak, the light detection module 400 preferably uses an avalanche photodiode photodetector (APD), secondly, a scientific camera can be selected, and thirdly, a photomultiplier tube sensor (PMT) can be selected.

[0130] In addition, the interference of the excitation light and the stray fluorescence signal can be filtered out by a notch filter, a long-pass filter, and a short-pass filter.

[0131] S130. The data processing module determines whether the target object contains the biomolecule to be detected according to the fluorescence signal.

[0132] Specifically, the frequency or power of the microwave radiation signal provided by the microwave field changes periodically, the number of NV centers resonating with the microwave field changes periodically, and / or the degree of resonance of the NV centers changes periodically. The NV centers generate fluorescence signals with periodically changing intensities. The first judgment method is adopted: the data processing module 700 performs Fourier transform on the fluorescence signal to obtain a frequency domain signal, and the part with the same frequency as the control signal is the demodulation signal; if the intensity of the demodulation signal is greater than the first preset threshold, it is determined that the target object contains the biomolecule to be detected.

[0133] The microwave radiation module 300 is in an on state under the control of a microwave field on signal. The electrons of the NV color centers undergo transitions to generate a dark state fluorescence signal. The dark state fluorescence signal is the fluorescence signal generated by the NV color centers in the nanodiamond when some or all of the NV color centers resonate with the microwave field. The microwave radiation module 300 is in an off state under the control of a microwave field off signal. The electrons of the NV color centers undergo transitions to generate a bright state fluorescence signal. The bright state fluorescence signal is the fluorescence signal generated by the NV color centers in the nanodiamond when no NV color centers resonate with the microwave field. The intensity of the dark state fluorescence signal is less than the intensity of the bright state fluorescence signal. Using the second determination method, the intensity of the dark state fluorescence signal is less than the intensity of the bright state fluorescence signal, and the absolute value of the difference between the intensity of the dark state fluorescence signal and the intensity of the bright state fluorescence signal is greater than a second preset threshold. The data processing module 700 determines that the target contains the biomolecule to be detected. The second preset threshold is at least 1 times the noise level when generating the bright state fluorescence signal.

[0134] An embodiment of the present invention provides a flow cytometry fluorescence detection method based on the switching state of a microwave field. The difference between the flow cytometry fluorescence detection method and the traditional optical detection method based on NV centers is that the nanodiamond particles to be measured are not stably laid on the substrate, but are placed inside the sample flow channel 100 and are in a moving state. The target substance specifically bound to the nanodiamond flows through the detection area S1 of the sample flow channel 100. The nanodiamond includes NV centers. Under the irradiation of the excitation light emitted by the excitation light module 200, the excitation light is used to excite the NV centers in the detection area S1 of the sample flow channel 100, causing the electrons to transition and generate fluorescence. When the microwave radiation module 300 provides a microwave field for the detection area S1 of the sample flow channel 100, the microwave field can provide a microwave radiation signal. When the microwave radiation signal exists and does not exist, the fluorescence signals generated by the NV centers in the nanodiamond are different; or, when the frequency or power of the microwave radiation signal changes, the fluorescence signals generated by the NV centers in the nanodiamond change accordingly, that is, by controlling at least one of the intensity, frequency, and on / off of the microwave field, a fluorescence signal with a periodically changing intensity is emitted by the nanodiamond. The optical detection module 400 collects the fluorescence signals generated by the NV centers in the nanodiamond when the target substance specifically bound to the nanodiamond flows through the detection area S1 of the sample flow channel 100 at a preset collection frequency. Since the fluorescence signal is related to whether the target substance contains the biomolecule to be detected, it is possible to determine whether the target substance contains the biomolecule to be detected based on the fluorescence signal. Due to the stability, room-temperature atmospheric environment compatibility, and biocompatibility of the NV center system, and the nanodiamond as a new type of biofluorescent label, low-frequency noise interference can be excluded through signal modulation and demodulation techniques, so the detection sensitivity far higher than other fluorescence label detection methods can be achieved. In summary, the technical solution provided by the embodiment of the present invention improves the detection sensitivity of the fluorescence detection method and can realize the recognition of single-molecule fluorescence signals. In the embodiment of the present invention, the magnetic field intensity near the detection area S1 of the sample flow channel 100 satisfies the zero-field condition, and the resonance frequency of some or all of the NV centers with the microwave field is the natural frequency. The microwave field is used to provide a microwave radiation signal with this fixed frequency, which can improve the signal-to-noise ratio of the fluorescence signal and further improve the detection sensitivity of the flow cytometry fluorescence detection method.

[0135] Optionally, on the basis of the above technical solution, as Figure 13 shown, Figure 13 is Figure 12 the flow schematic diagram included in 110. When the target substance specifically bound to the nanodiamond flows through the detection area of the sample flow channel, the generation of fluorescence signals by the NV centers in the nanodiamond includes:

[0136] S1101. The control module issues a control signal, which includes at least one of a microwave field turn-on signal, a microwave field turn-off signal, a periodically varying microwave field power signal, and a periodically varying microwave field frequency signal.

[0137] S1102. According to the microwave field turn-on signal, the microwave radiation module is in an on state. The microwave radiation module provides a microwave field for the detection area of the sample flow channel. When the NV centers in the nanodiamond are irradiated by the excitation light, a dark-state fluorescence signal is emitted. The dark-state fluorescence signal is the fluorescence signal generated by the NV centers in the nanodiamond when some or all of the NV centers resonate with the microwave field.

[0138] Specifically, under the control of the microwave field turn-on signal, the microwave radiation module 300 is in an on state, and there is a microwave radiation signal in the detection area S1. When the NV centers in the nanodiamond are irradiated by the excitation light, the electrons of the NV centers undergo transitions to generate a dark-state fluorescence signal. The dark-state fluorescence signal is the fluorescence signal generated by the NV centers in the nanodiamond when some or all of the NV centers resonate with the microwave field.

[0139] S1103. According to the microwave field turn-off signal, the microwave radiation module is in an off state. The microwave radiation module stops providing a microwave field for the detection area of the sample flow channel. When the NV centers in the nanodiamond are irradiated by the excitation light, a bright-state fluorescence signal is emitted. The bright-state fluorescence signal is the fluorescence signal generated by the NV centers in the nanodiamond when no NV centers resonate with the microwave field.

[0140] Under the control of the microwave field turn-off signal, the microwave radiation module 300 is in an off state, and there is no microwave radiation signal in the detection area S1. When the NV centers in the nanodiamond are irradiated by the excitation light, the electrons of the NV centers undergo transitions to generate a bright-state fluorescence signal. The bright-state fluorescence signal is the fluorescence signal generated by the NV centers in the nanodiamond when no NV centers resonate with the microwave field. The intensity of the dark-state fluorescence signal is less than the intensity of the bright-state fluorescence signal.

[0141] Alternatively, S1104. According to the periodically varying microwave field power signal, the microwave radiation module emits a microwave modulation field with a fixed frequency and a periodically varying power, so that the NV centers in the nanodiamond emit a fluorescence signal with a periodically varying intensity under the irradiation of the excitation light.

[0142] Under the control of the periodically varying microwave field power signal, the microwave radiation module 300 emits a microwave modulation field with a fixed frequency and a periodically varying power, so that when the NV centers in the nanodiamond are irradiated by the excitation light, the electrons of the NV centers undergo transitions, the number of NV centers resonating with the microwave field varies periodically, and / or the degree of resonance of the NV centers varies periodically, and the NV centers generate a fluorescence signal with a periodically varying intensity.

[0143] Alternatively, S1105: The microwave radiation module emits a microwave modulation field with a fixed power and a periodically varying frequency according to the periodically varying signal of the microwave field frequency, so that the NV color centers in the nanodiamond emit fluorescence signals with periodically varying intensities under the illumination of the excitation light.

[0144] Under the control of the control signal of the periodically varying signal of the microwave field frequency, the microwave radiation module 300 emits a microwave modulation field with a fixed power and a periodically varying frequency, so that under the illumination of the excitation light, the electrons of the NV color centers in the nanodiamond undergo transitions, and the number of NV color centers resonating with the microwave field varies periodically, and / or the degree of resonance of the NV color centers varies periodically, and the NV color centers generate fluorescence signals with periodically varying intensities.

[0145] The above technical solution provides a solution in which when the microwave radiation module 300 is in an on state and an off state or the power or frequency of the emitted microwave radiation signal with a fixed frequency varies periodically under the control of the control signal, the NV color centers in the nanodiamond emit fluorescence signals that change accordingly under the illumination of the excitation light.

[0146] Optionally, on the basis of the above technical solution, as Figure 14 shown, Figure 14 is Figure 12 the schematic flowchart included in S130. The data processing module in S130 determines whether the target contains the biomolecule to be detected according to the fluorescence signal, including:

[0147] S1301: The data processing module performs Fourier transform on the fluorescence signal to obtain a frequency-domain signal, and the part with the same frequency as the control signal is the demodulated signal.

[0148] S1302: If the intensity of the demodulated signal is greater than the first preset threshold, it is determined that the target contains the biomolecule to be detected.

[0149] The above technical solution is directed to a method for determining whether the target contains the biomolecule to be detected, in which the frequency or power of the microwave radiation signal provided by the microwave field varies periodically, the number of NV color centers resonating with the microwave field varies periodically, and / or the degree of resonance of the NV color centers varies periodically, and the NV color centers generate fluorescence signals with periodically varying intensities, and the data processing module 700 determines whether the target contains the biomolecule to be detected according to the fluorescence signal.

[0150] Optionally, on the basis of the above technical solution, the data processing module in S130 determines whether the target contains the biomolecule to be detected according to the fluorescence signal, including:

[0151] The intensity of the dark-state fluorescence signal is less than that of the bright-state fluorescence signal, and the absolute value of the difference between the intensity of the dark-state fluorescence signal and that of the bright-state fluorescence signal is greater than a second preset threshold. The data processing module determines that the target contains the biomolecule to be detected. The second preset threshold is at least 1 times the noise level when the bright-state fluorescence signal is generated.

[0152] The above technical solution is directed to a determination method in which the microwave radiation module 300 is in an on state or an off state under the control of a control signal, and the data processing module 700 determines whether the target contains the biomolecule to be detected according to the fluorescence signal. Among them, the microwave radiation module 300 is in an on state under the control of a microwave field turn-on signal, and the electrons of the NV color center undergo a transition to generate a dark-state fluorescence signal. The dark-state fluorescence signal is the fluorescence signal generated by the NV color center in the nanodiamond when some or all of the NV color centers resonate with the microwave field. The microwave radiation module 300 is in an off state under the control of a microwave field turn-off signal, and the electrons of the NV color center undergo a transition to generate a bright-state fluorescence signal. The bright-state fluorescence signal is the fluorescence signal generated by the NV color center in the nanodiamond when no NV color center resonates with the microwave field.

[0153] Optionally, on the basis of the above technical solution, the number of periodically changing fluorescence signals presented by a single target in the detection area is pieces;

[0154] wherein, N is the number of targets flowing through the detection area of the sample flow channel per second, and the value of N is an integer greater than or equal to 1; F M is the frequency of the control signal.

[0155] As Figure 15 shown, if the number of targets detected by the flow cytometry system per unit time is N per second, the time required for a single target to flow through the detection area S1 is 1 / N seconds. Taking F M as the frequency of the control signal, a single target presents cycles of fluorescence signals in the detection area S1. The above technical solution gives the relationship between the frequency of the control signal and the flow rate of the target.

[0156] Here, when the microwave field modulation mode is amplitude modulation, the radiation power of the microwave radiation signal provided by the microwave field changes, such as the simplest square wave modulation, sine wave modulation and triangular wave modulation realized by changing the microwave power. When the microwave field modulation mode is frequency modulation, the frequency of the microwave radiation signal provided by the microwave field changes, such as the microwave resonance-non-resonance mode. When the frequency of the microwave radiation signal provided by the microwave field is equal to the energy level difference of the electron spin transition of the NV color center, the microwave field can resonate with the NV color center.

[0157] Optionally, on the basis of the above technical solution, when the target fluid passes through the detection area, within the period of a single fluorescence signal, the number of detection windows collected by the optical detection module satisfies the following relationship:

[0158] M = F s / F M

[0159] where F s is the sampling frequency of the optical detection module, and F M is the frequency of the control signal.

[0160] The above technical solution gives the relationship between the sampling frequency of the optical detection module 400 and the frequency of the control signal. As Figure 16 shown, it also gives the number of detection windows collected by the optical detection module 400 within the period of a single fluorescence signal when the target fluid passes through the detection area S1.

[0161] Optionally, on the basis of the above technical solution, when the target fluid specifically bound by S110 and labeled with nanodiamonds passes through the detection area of the sample flow channel, after the NV color centers in the nanodiamonds generate fluorescence signals, it includes:

[0162] After each detection window of the optical detection module, a detection window without microwave radiation signal is added as a reference signal.

[0163] Within each detection window, the optical detection module 400 simultaneously collects the fluorescence signals of the entire detection area; a pinhole is added in front of the fluorescence collector for spatial filtering to weaken the interference of background stray signals; after a single detection window, a detection window without modulated microwave manipulation can be added as a reference signal to be used for calibrating the time jitter and spatial inhomogeneity of the excitation light.

[0164] Optionally, on the basis of the above technical solution, several scientific cameras are used as the optical detection module 400. Here, an sCMOS high-speed scientific camera can be selected, or an intensified charge-coupled device (ICCD) can be selected; within each detection window, the detector performs fluorescence imaging on the entire detection area; a pinhole can be added in front of the fluorescence collector for spatial filtering to weaken the interference of background stray signals; for the fluorescence images of each detection window, the position trajectories of the nanodiamond fluorescence particles in the images are found through algorithm recognition, and several points are selected outward from the trajectory as the fluorescence counts of the nanodiamond particles within this window, and the background fluorescence signals of the rest are discarded to improve the signal-to-noise ratio of the fluorescence signal to be measured.

[0165] Optionally, on the basis of the above technical solution, the particle size range of the nanodiamonds is greater than or equal to 40 nm and less than or equal to 1 μm; preferably, the particle size range of the nanodiamonds is greater than or equal to 100 nm and less than or equal to 300 nm.

[0166] The fluorescence counting rate of nanodiamond, the contrast of the fluorescence signal, and the exposure time of a single detection window satisfy the following relationship:

[0167]

[0168] Among them, C is the contrast of the fluorescence signal, R is the fluorescence counting rate of nanodiamond, t is the exposure time of a single detection window; NV color center |m s = 0> bright state and |m s = ±1> the relative counting difference between the dark states is the contrast.

[0169] The particle size range of the nanodiamond satisfies the above range, the contrast of the fluorescence signal, the fluorescence counting rate of the nanodiamond, and the exposure time of a single detection window satisfy the above formula. At this time, within each detection window, the amplitude of the fluorescence signal of the nanodiamond particles is greater than the amplitude of the noise. Here, the main consideration is the photon shot noise, which can ensure that the signal-to-noise ratio of a single measurement is greater than 1:1, thereby improving the signal-to-noise ratio of the fluorescence signal. Optionally, on the basis of the above technical solution, the excitation light module 200 includes an optical pump, and the optical pump is used to emit laser light;

[0170] The contrast of the fluorescence signal generated by the NV color center in the nanodiamond satisfies the following relationship:

[0171]

[0172] Among them, Θ is the normalization constant, Γ p is the laser polarization rate, Γ c is the coherent relaxation rate caused by the optical pump, Ω R is the frequency of the Rabi oscillation of the NV color center driven by the microwave field, Γ p 、Γ c The two terms are related to the laser power, and the Ω R term is positively correlated with the intensity of the microwave field felt by the NV color center in the nanodiamond.

[0173] The above technical solution gives the calculation formula for the contrast of the fluorescence signal generated by the NV color center in the nanodiamond. In order to ensure that the amplitude of the fluorescence signal of a single nanodiamond particle is always consistent in the detection area, the laser power density and the microwave field intensity distribution in the detection area should be as uniform as possible. Optionally, on the basis of the above technical solution, the intensity of the microwave field felt by the NV color center in the nanodiamond satisfies the following relationship:

[0174]

[0175] Among them, B is the intensity of the microwave field felt by the NV color center in the nanodiamond, and B1 is the intensity of the microwave field provided by the microwave radiation module 300.

[0176] Different from the traditional NV - center - based optical detection method, in the flow - through fluorescence detection method, the nanodiamond particles to be measured are not stably laid on the substrate, but are placed inside the sample flow channel 100 and are in a moving state. The nanodiamonds selected in the embodiments of the present invention contain hundreds of NV centers with different crystal axis directions. Therefore, it can be considered that the angles between the main axes of the NV centers and the microwave field are completely averaged, and the microwave field intensity sensed by all NV centers in a single nanodiamond particle is:

[0177]

[0178] It can be seen from the above formula that the microwave field intensity sensed by all NV centers in a single nanodiamond particle is proportional to the microwave field intensity provided by the microwave radiation module 300.

[0179] Optionally, on the basis of the above - mentioned technical solution, for a single detection window, the excitation light remains on all the time, the microwave radiation module 300 provides a microwave radiation signal, and the optical detection module 400 uses the entire length of the detection window as the exposure time to obtain the fluorescence signal intensity of the nanodiamonds within a single sampling point.

[0180] Specifically, for a single detection window, the laser remains on all the time, the microwave radiation module 300 provides a microwave radiation signal, and the fluorescence detector uses the entire length of the detection window as the exposure time to obtain the fluorescence signal intensity of the nanodiamond particles within a single sampling point. In this way, the duty cycle of the excitation light is high, and the number of effective fluorescence photons emitted by the nanodiamonds collected by the optical detection module 400 is relatively high.

[0181] Optionally, on the basis of the above - mentioned technical solution, the delay time between the excitation light initialization pulse and the fluorescence signal collection pulse window is greater than a preset delay time.

[0182] Specifically, since the fluorescence lifetime of the NV center is relatively long, the detector can be used to select an appropriate pulsed delayed fluorescence window, that is, the delay time between the excitation light initialization pulse and the fluorescence signal collection pulse window is greater than a preset delay time. The preset delay time can be, for example, 20 ns; then the excitation - waiting - fluorescence collection sequence is repeated multiple times to obtain the fluorescence signal intensity of a single sampling point. At this time, the background fluorescence signal caused by non - target substances has a short lifetime and has been greatly attenuated; while the fluorescence of the NV centers in the nanodiamond particles still exists, thereby filtering the interference of the background fluorescence signal in terms of time and improving the signal - to - noise ratio of the detection.

[0183] In the technical solution provided by the embodiment of the present invention, in order to improve the signal-to-noise ratio of the fluorescence signal and enhance the recognition accuracy of the analyte, the system introduces a microwave field to modulate the fluorescence signal of the nanodiamond particles: when the microwave radiation module 300 emits microwaves greater than or equal to 2820 MHz and less than or equal to 2920 MHz into the radiation coil, the NV color centers in the nanodiamond particles in the sample flow channel 100 transition from the |m s =0> state to the |m s =±1> state, and the fluorescence intensity of the nanodiamond particles decreases; when the microwave in the radiation coil is turned off, the excitation light in the detection area S1 initializes the NV color centers to the |m s =0> state, and the fluorescence intensity is restored. When the radiation power of the microwave radiation signal provided by the microwave radiation module 300 changes, a corresponding fluorescence signal is generated in the NV color centers in the nanodiamond. The microwave radiation module 300 modulates the fluorescence signal in the time domain, and then the optical detection module 400 collects it at a certain frequency. Finally, the algorithm identifies the analyte signal at a specific frequency and excludes the interference of static noise signals.

[0184] The embodiment of the present invention also provides a zero-field flow fluorescence detection system, and this zero-field flow fluorescence detection system is applicable to any of the zero-field flow fluorescence detection methods described in the embodiments of the present invention. The beneficial effects of the zero-field flow fluorescence detection system include those of any of the zero-field flow fluorescence detection methods described in the embodiments of the present invention, which will not be elaborated here.

[0185] As Figure 17 shown, Figure 17 is a flowchart of another zero-field flow fluorescence detection method based on the switching state of the microwave field provided by the embodiment of the present invention. This zero-field flow fluorescence detection method based on the switching state of the microwave field includes the following steps:

[0186] S210. When the target substance labeled with nanodiamonds specifically binds and flows through the first half of the detection area of the sample flow channel, fluorescence signals are generated in the NV color centers in the nanodiamonds, and the fluorescence signals include dark-state fluorescence signals; the dark-state fluorescence signals are the fluorescence signals generated in the NV color centers in the nanodiamonds when some or all of the NV color centers resonate with the microwave field.

[0187] In this embodiment, when the microwave radiation module 300 provides a microwave field for the detection area S1 of the sample flow channel 100, the microwave field can provide a microwave radiation signal. When the microwave radiation signal exists and does not exist, the fluorescence signals generated in the NV color centers in the nanodiamonds are different.

[0188] When the target substance specifically binding to the nanodiamond label flows through the first half of the detection region S1 of the sample flow channel 100, the microwave radiation module 300 is in the on state, and there is a microwave radiation signal in the detection region S1. When the NV centers in the nanodiamonds are irradiated with excitation light, the electrons of the NV centers undergo transitions to generate dark-state fluorescence signals. The dark-state fluorescence signals are the fluorescence signals generated by the NV centers in the nanodiamonds when some or all of the NV centers resonate with the microwave field.

[0189] S220. When the target substance specifically binding to the nanodiamond label flows through the second half of the detection region of the sample flow channel, the NV centers in the nanodiamonds generate fluorescence signals, and the fluorescence signals include bright-state fluorescence signals. The bright-state fluorescence signals are the fluorescence signals generated by the NV centers in the nanodiamonds when no NV centers resonate with the microwave field.

[0190] Among them, the excitation light module is used to emit excitation light, and the excitation light is used to excite the NV centers in the detection region of the sample flow channel to cause the electrons to undergo transitions to generate fluorescence. The microwave radiation module is used to provide a microwave field for the detection region of the sample flow channel. The microwave field resonates with all or some of the NV centers in the nanodiamonds. The frequency of the microwave radiation signal provided by the microwave field is equal to the energy level difference of the electron spin transition of the NV centers, which can enable the microwave field to resonate with the NV centers. The microwave radiation module is in the on state or off state under the control of a control signal. When the microwave radiation module is in the on state, the NV centers in the nanodiamonds generate dark-state fluorescence signals. When the microwave radiation module is in the off state, the NV centers in the nanodiamonds generate bright-state fluorescence signals.

[0191] When the target substance specifically binding to the nanodiamond label flows through the second half of the detection region S1 of the sample flow channel 100, the microwave radiation module 300 is in the off state, and there is no microwave radiation signal in the detection region S1. When the NV centers in the nanodiamonds are irradiated with excitation light, the electrons of the NV centers undergo transitions to generate bright-state fluorescence signals. The bright-state fluorescence signals are the fluorescence signals generated by the NV centers in the nanodiamonds when no NV centers resonate with the microwave field. The intensity of the dark-state fluorescence signals is less than the intensity of the bright-state fluorescence signals.

[0192] S230. The light detection module collects the fluorescence signals at a preset collection frequency.

[0193] As Figure 11 shown, the flow-through fluorescence detection system includes at least two light detection modules 400. The at least two light detection modules 400 are arranged around the detection region S1 of the sample flow channel, which can ensure that the fluorescence signals at all angles in the circumferential direction of the detection region S1 can be collected by the light detection modules 400.

[0194] Exemplarily, Figure 1 and Figure 11Two photodetection modules 400 are shown. The photodetection modules 400 are arranged around the detection area S1 of the sample flow channel 100 and are symmetrically arranged with respect to the detection area S1.

[0195] When the number of photodetection modules 400 is greater than two, multiple photodetection modules 400 are arranged around the detection area S1 of the sample flow channel 100. It is only necessary to ensure that the fluorescence signals at all angles in the circumferential direction of the detection area S1 can be collected by the photodetection modules 400.

[0196] On the one hand, the above technical solution can increase the collection amount of fluorescence signals, thereby providing a lower detection limit; on the other hand, it can overcome the problem of the target object blocking the fluorescence of the NV center.

[0197] Since in the application scenario of single-molecule detection, the fluorescence intensity of a single nanodiamond particle is weak, the photodetection module 400 preferably uses an avalanche photodiode photodetector (APD), followed by a scientific research camera, and then a photomultiplier tube sensor (PMT).

[0198] In addition, the excitation light and the interference of stray fluorescence signals can be filtered out by a notch filter, a long-pass filter, and a short-pass filter.

[0199] S240. The data processing module determines whether the target object contains the biomolecule to be detected according to the magnitude relationship between the intensity of the dark-state fluorescence signal and the intensity of the bright-state fluorescence signal.

[0200] The microwave radiation module 300 is in an on state under the control of the microwave field on signal. The electrons of the NV center undergo transitions to generate dark-state fluorescence signals. The dark-state fluorescence signals are the fluorescence signals generated by the NV centers in the nanodiamond when some or all of the NV centers resonate with the microwave field. The microwave radiation module 300 is in an off state under the control of the microwave field off signal. The electrons of the NV center undergo transitions to generate bright-state fluorescence signals. The bright-state fluorescence signals are the fluorescence signals generated by the NV centers in the nanodiamond when no NV center resonates with the microwave field. The intensity of the dark-state fluorescence signal is less than the intensity of the bright-state fluorescence signal. Therefore, if the intensity of the dark-state fluorescence signal is less than the intensity of the bright-state fluorescence signal, the data processing module 700 determines that the target object contains the biomolecule to be detected.

[0201] An embodiment of the present invention provides a flow cytometry fluorescence detection method based on the switching state of a microwave field. The difference between the flow cytometry fluorescence detection method and the traditional optical detection method based on NV centers is that the to-be-detected nanodiamond particles are not stably laid on the substrate, but are placed inside the sample flow channel 100 and are in a moving state. The target substance specifically bound to the nanodiamond flows through the detection area S1 of the sample flow channel 100. The nanodiamond includes an NV center. Under the irradiation of the excitation light emitted by the excitation light module 200, the excitation light is used to excite the NV centers in the detection area S1 of the sample flow channel 100, causing the electrons to undergo transitions and generate fluorescence. When the target substance specifically bound to the nanodiamond flows through the first half of the detection area S1 of the sample flow channel 100, the microwave radiation module 300 is in the on state, and there is a microwave radiation signal in the detection area S1. Under the irradiation of the excitation light, the electrons of the NV centers in the nanodiamond undergo transitions to generate dark-state fluorescence signals. When the target substance specifically bound to the nanodiamond flows through the second half of the detection area S1 of the sample flow channel 100, the microwave radiation module 300 is in the off state, and there is no microwave radiation signal in the detection area S1. Under the irradiation of the excitation light, the electrons of the NV centers in the nanodiamond undergo transitions to generate bright-state fluorescence signals. The light detection module 400 collects the fluorescence signals generated by the NV centers in the nanodiamond when the target substance specifically bound to the nanodiamond flows through the detection area S1 of the sample flow channel 100 at a preset collection frequency. Since the fluorescence signals are related to whether the target substance contains the biomolecule to be detected, the data processing module 700 determines whether the target substance contains the biomolecule to be detected based on the magnitude relationship between the intensities of the dark-state fluorescence signals and the bright-state fluorescence signals. Due to the stability, room-temperature atmospheric environment compatibility, and biocompatibility of the NV center system, and the fact that nanodiamonds, as a new type of biological fluorescence marker, can exclude low-frequency noise interference through signal modulation and demodulation techniques, the detection sensitivity far higher than other fluorescence marker detection methods can be achieved. In summary, the technical solution provided by the embodiment of the present invention improves the detection sensitivity of the fluorescence detection method and can achieve the recognition of single-molecule fluorescence signals.

[0202] Optionally, on the basis of the above technical solution, as Figure 18 shown, when the target substance specifically bound to the nanodiamond flows through the first half of the detection area of the sample flow channel, the NV centers in the nanodiamond generate fluorescence signals, and the fluorescence signals include dark-state fluorescence signals, including:

[0203] S2101. The control module issues a control signal, and the control signal includes a microwave field turn-on signal and a microwave field turn-off signal.

[0204] S2102. When the microwave radiation module receives the microwave field turn-on signal, it is in the on state. The microwave radiation module provides a microwave field for the detection area of the sample flow channel. When the NV centers in the nanodiamonds flow through the first half of the detection area of the sample flow channel, they emit dark-state fluorescence signals under the excitation light irradiation.

[0205] Specifically, the microwave radiation module 300 is in the on state under the control of the microwave field turn-on signal. When the NV centers in the nanodiamonds flow through the first half of the detection area S1 of the sample flow channel 100, there is a microwave radiation signal in the detection area S1. Under the excitation light irradiation, the electrons of the NV centers in the nanodiamonds undergo transitions to generate dark-state fluorescence signals. The dark-state fluorescence signals are the fluorescence signals generated by the NV centers in the nanodiamonds when some or all of the NV centers resonate with the microwave field.

[0206] The above technical solution provides a scheme for the fluorescence signals emitted by the NV centers in the nanodiamonds under the excitation light irradiation when the microwave radiation module 300 is in the on state under the control of the control signal.

[0207] Optionally, on the basis of the above technical solution, when the target substance labeled with nanodiamonds that specifically binds to S220 flows through the second half of the detection area of the sample flow channel, the NV centers in the nanodiamonds generate fluorescence signals, and the fluorescence signals include bright-state fluorescence signals, including:

[0208] When the microwave radiation module receives the microwave field turn-off signal, it is in the off state. The microwave radiation module stops providing a microwave field for the detection area of the sample flow channel. When the NV centers in the nanodiamonds flow through the second half of the detection area of the sample flow channel, they emit bright-state fluorescence signals under the excitation light irradiation.

[0209] The microwave radiation module 300 is in the off state under the control of the microwave field turn-off signal. When the NV centers in the nanodiamonds flow through the second half of the detection area S1 of the sample flow channel 100, there is no microwave radiation signal in the detection area S1. Under the excitation light irradiation, the electrons of the NV centers in the nanodiamonds undergo transitions to generate bright-state fluorescence signals. The bright-state fluorescence signals are the fluorescence signals generated by the NV centers in the nanodiamonds when there is no NV center resonating with the microwave field. The intensity of the dark-state fluorescence signals is less than the intensity of the bright-state fluorescence signals.

[0210] The above technical solution provides a scheme for the fluorescence signals emitted by the NV centers in the nanodiamonds under the excitation light irradiation when the microwave radiation module 300 is in the off state under the control of the control signal.

[0211] Optionally, on the basis of the above technical solution, the data processing module S240 determines whether the target substance contains the biomolecule to be detected according to the magnitude relationship between the intensity of the dark-state fluorescence signals and the intensity of the bright-state fluorescence signals, including:

[0212] The intensity of the dark-state fluorescence signal is less than that of the bright-state fluorescence signal, and the absolute value of the difference between the intensity of the dark-state fluorescence signal and the intensity of the bright-state fluorescence signal is greater than a second preset threshold. It is determined that the target contains the biomolecule to be detected, and the second preset threshold is at least 1 times the noise level when the bright-state fluorescence signal is generated.

[0213] The above technical solution is directed to a determination method in which the microwave radiation module 300 is in an on state or an off state under the control of a control signal, and the data processing module 700 determines whether the target contains the biomolecule to be detected according to the fluorescence signal.

[0214] Optionally, on the basis of the above technical solution, after the specifically bound nanodiamond-labeled target flows through the latter half of the detection area of the sample flow channel and the NV color centers in the nanodiamonds generate fluorescence signals, it further includes:

[0215] After each detection window of the optical detection module, a detection window without a microwave radiation signal is added as a reference signal.

[0216] Within each detection window, the detector simultaneously collects the fluorescence signals of the entire detection area; a pinhole is added in front of the fluorescence collector for spatial filtering to weaken the interference of background stray signals; after a single detection window, a detection window without modulated microwave manipulation can be added as a reference signal to be used for calibrating the time jitter and spatial inhomogeneity of the excitation light.

[0217] Optionally, on the basis of the above technical solution, several scientific research cameras are used as the optical detection module 400. Here, an sCMOS high-speed scientific research camera can be selected, or an intensified charge-coupled device (ICCD) can be selected; within each detection window, the detector performs fluorescence imaging on the entire detection area; a pinhole can be added in front of the fluorescence collector for spatial filtering to weaken the interference of background stray signals; for the fluorescence images of each detection window, the position trajectories of the nanodiamond fluorescence particles in the figure are found by algorithm recognition, and several points are selected outward from the trajectory as the fluorescence count of the nanodiamond particles in this window, and the background fluorescence signals of the rest are discarded to improve the signal-to-noise ratio of the fluorescence signal to be measured.

[0218] Optionally, on the basis of the above technical solution, the magnetic field intensity of the detection area satisfies the zero-field condition, and the microwave radiation signal provided by the microwave field has a fixed frequency.

[0219] Optionally, on the basis of the above technical solution, such as Figure 1 and Figure 2As shown, the magnetic field monitoring module 800 monitors the magnetic field intensity near the detection area S1 of the sample flow channel 100; when the magnetic field intensity near the detection area S1 of the sample flow channel 100 monitored by the magnetic field monitoring module 800 satisfies the zero-field condition, the microwave field is used to provide a microwave radiation signal with a fixed frequency.

[0220] In an external magnetic field, due to the Zeeman effect, the electrons of the NV color centers in the nanodiamond cause the energy levels between the |m s = 0> state and the |m s = ±1> state to shift in opposite directions, and the frequency difference is proportional to the magnitude of the projection of the external magnetic field in the crystal axis direction of the NV color center. A single nanodiamond contains multiple NV color centers, and the crystal axis directions of different NV color centers are different; moreover, the spatial directions of different nanodiamond particles when flowing through the sample flow channel 100 are random. Therefore, for the NV color centers in each axial direction within different nanodiamond particles, the projection components of the external magnetic field are different, resulting in different transition frequencies between the |m s = 0> state and the |m s = ±1> state. Under the control of microwaves with a single frequency, the quantum states of the NV color centers in some nanodiamond particles do not change, and the overall fluorescence bright-dark change is not obvious, thus affecting the signal-to-noise ratio of the measurement scheme, and further affecting the accuracy and reliability of the detection of the analyte.

[0221] Therefore, when the magnetic field intensity near the detection area S1 of the sample flow channel 100 monitored by the magnetic field monitoring module 800 satisfies the zero-field condition, the resonance frequencies of some or all of the NV color centers with the microwave field are the natural frequencies, and the microwave field is used to provide a microwave radiation signal with this fixed frequency, which can improve the signal-to-noise ratio of the fluorescence signal, and further improve the detection sensitivity of the flow cytometry fluorescence detection system.

[0222] Optionally, on the basis of the above technical solution, as Figure 10 shown, when the magnetic field intensity near the detection area S1 of the sample flow channel 100 monitored by the magnetic field monitoring module 800 satisfies the zero-field condition, the energy level splitting of the NV color centers in the nanodiamond caused by the magnetic field is less than half of the linewidth of its optically detected magnetic resonance spectrum.

[0223] The "energy level splitting of the NV color centers in the nanodiamond" refers to the shift amount of the resonance peak position of the optically detected magnetic resonance spectrum due to the Zeeman shift of the NV energy level caused by the external magnetic field in the optically detected magnetic resonance spectrum of the NV color center - that is, half of the difference between the two microwave resonance frequencies corresponding to the energy level splitting of the same NV color center; the "linewidth of its optically detected magnetic resonance spectrum" refers to the full width at half maximum of the resonance peak of the optically detected magnetic resonance spectrum of the NV color center.

[0224] When the magnetic field intensity near the detection area S1 of the sample flow channel 100 monitored by the magnetic field monitoring module 800 satisfies the zero-field condition, the fixed frequency of the microwave radiation signal provided by the microwave field is greater than or equal to 2820 MHz and less than or equal to 2920 MHz, so that all the NV color centers in all crystal axis directions in the nanodiamond can be subjected to quantum state control by microwaves with a frequency located at a zero-field splitting greater than or equal to 2820 MHz and less than or equal to 2920 MHz. Therefore, the system can use a frequency greater than or equal to 2820 MHz and less than or equal to 2920 MHz as the fixed operating frequency of the microwave radiation signal provided by the microwave radiation module 300.

[0225] It should be noted that in the embodiments of the present invention, it is required that the spectral peak shift caused by the magnetic field does not exceed the intrinsic broadening of the resonance (CW) spectrum, and the intrinsic broadening of the CW spectrum is determined by its dephasing time. However, in nanodiamonds with different manufacturing processes and diameters, the noise environments of the NV color centers are different, corresponding to different dephasing times, and the zero-field limit needs to be determined according to the selected particles. Taking HPHT-processed nanodiamonds with a diameter of 40 nm and an NV concentration of 1.5 ppm as an example, the typical value of the intrinsic broadening of its CW spectrum is 20 MHz, corresponding to a requirement that the external magnetic field is below 3.56 Gs. The resonance (CW) spectrum is Figure 10 the spectral line in.

[0226] Optionally, on the basis of the above technical solution, the particle size range of the nanodiamond is greater than or equal to 40 nm and less than or equal to 1 μm; preferably, the particle size range of the nanodiamond is greater than or equal to 100 nm and less than or equal to 300 nm.

[0227] When the target fluid flows through the detection area, within the period of a single fluorescence signal, the number of detection windows collected by the optical detection module is at least one, and the fluorescence counting rate of the nanodiamond, the contrast of the fluorescence signal, and the exposure time of a single detection window satisfy the following relationship:

[0228]

[0229] where C is the contrast of the fluorescence signal, R is the fluorescence counting rate of the nanodiamond, and t is the exposure time of a single detection window; the relative counting difference between the bright state of the NV color center |m s =0> and the dark state of |m s =±1> is the contrast.

[0230] The particle size range of the nanodiamonds meets the above range, and the contrast of the fluorescence signal, the fluorescence counting rate of the nanodiamonds, and the exposure time of a single detection window satisfy the above formula. At this time, within each detection window, the amplitude of the fluorescence modulation signal of the nanodiamond particles is greater than the amplitude of the noise. Here, the main consideration is the photon shot noise, which can ensure that the signal-to-noise ratio of a single measurement is greater than 1:1, thereby improving the signal-to-noise ratio of the fluorescence signal.

[0231] Optionally, on the basis of the above technical solution, the excitation light module 200 includes an optical pump, and the optical pump is used to emit laser light;

[0232] The contrast of the fluorescence signal generated by the NV color centers in the nanodiamonds satisfies the following relationship:

[0233]

[0234] where Θ is the normalization constant, Γ p is the laser polarization rate, Γ c is the coherent relaxation rate caused by the optical pump, Ω R is the frequency of the Rabi oscillation of the NV color centers driven by the microwave field, Γ p and Γ c are related to the laser power, and the Ω R term is positively correlated with the intensity of the microwave field sensed by the NV color centers in the nanodiamonds.

[0235] The above technical solution gives the calculation formula for the contrast of the fluorescence signal generated by the NV color centers in the nanodiamonds. To ensure that the fluorescence signal amplitude of a single nanodiamond particle is always consistent within the detection area, the laser power density and the microwave field strength distribution within the detection area should be as uniform as possible.

[0236] Optionally, on the basis of the above technical solution, the intensity of the microwave field sensed by the NV color centers in the nanodiamonds satisfies the following relationship:

[0237]

[0238] where B is the intensity of the microwave field sensed by the NV color centers in the nanodiamonds, and B1 is the intensity of the microwave field provided by the microwave radiation module.

[0239] What is different between the flow detection method and the traditional optical detection method based on NV color centers is that the nanodiamond particles to be measured are not stably spread on the substrate, but are placed inside the flow channel and are in a moving state. The nanodiamonds we selected contain hundreds of NV color centers with different crystal axis directions. Therefore, it can be considered that the angle between the main axis direction of the NV color centers and the microwave field is completely averaged, and the intensity of the microwave field sensed by all the NV color centers in a single nanodiamond particle is:

[0240]

[0241] As can be seen from the above formula, the microwave field intensity felt by all NV color centers in a single nanodiamond particle is proportional to the microwave field intensity.

[0242] Optionally, based on the above technical solution, for a single detection window, the excitation light remains on all the time, the microwave radiation module provides a microwave radiation signal, and the light detection module uses the entire length of the detection window as the exposure time to obtain the fluorescence signal intensity of the nanodiamonds within a single sampling point.

[0243] Specifically, for a single detection window, the laser remains on all the time, the microwave radiation module provides a microwave radiation signal, and the fluorescence detector uses the entire length of the detection window as the exposure time to obtain the fluorescence signal intensity of the nanodiamond particles within a single sampling point. In this way, the duty cycle of the excitation light is high, and the number of effective fluorescence photons emitted by the nanodiamonds collected by the light detection module is relatively high.

[0244] Optionally, based on the above technical solution, the delay time between the excitation light initialization pulse and the fluorescence signal collection pulse window is greater than a preset delay time.

[0245] Specifically, since the fluorescence lifetime of the NV color center is relatively long, a detector can be used to select an appropriate pulsed delayed fluorescence window, that is, the delay time between the excitation light initialization pulse and the fluorescence signal collection pulse window is greater than the preset delay time. The preset delay time can be, for example, 20 ns; then the excitation-wait-fluorescence collection sequence is repeated multiple times to obtain the fluorescence signal intensity of a single sampling point. At this time, the background fluorescence signal caused by non-target substances has a short lifetime and has been greatly attenuated; while the fluorescence of the NV color centers in the nanodiamond particles still exists, thereby filtering the interference of the background fluorescence signal in terms of time and improving the signal-to-noise ratio of the detection.

[0246] For the technical solution provided by the embodiments of the present invention, in order to improve the signal-to-noise ratio of the fluorescence signal and enhance the recognition accuracy of the analyte, the system introduces a microwave field to modulate the fluorescence signal of the nanodiamond particles: when the microwave radiation module 300 turns on microwaves with a frequency greater than or equal to 2820 MHz and less than or equal to 2920 MHz and enters the radiation coil, the NV color centers in the nanodiamond particles in the sample flow channel 100 transition from the |m s =0> state to the |m s =±1> state, and the fluorescence intensity of the nanodiamond particles decreases; when the microwave in the radiation coil is turned off, the excitation light in the detection area S1 initializes the NV color centers to |m s= 0> state, the fluorescence intensity recovers. When the radiation power of the microwave radiation signal provided by the microwave radiation module 300 changes, a corresponding fluorescence signal is generated in the NV color centers within the nanodiamonds. By modulating the fluorescence signal in the time domain through the microwave radiation module 300, and then collecting it at a certain frequency through the optical detection module 400, and finally identifying the signal to be measured at a specific frequency through an algorithm to exclude the interference of static noise signals. Specifically, when the target object passes through the detection area S1, the microwave radiation module 300 is turned on to modulate the microwave in the first half of the detection area S1, and the NV color centers in the nanodiamond particles within the detection area S1 are manipulated to change from the |m s = 0> state to the |m s = ±1> state; in the second half of the detection area S1, the microwave field is turned off, and the NV color centers are initialized to the |m s = 0> state by laser; by comparing the fluorescence signal intensities of the nanodiamonds in the first and second halves of the detection area S1, it can be determined whether the detected object contains nanodiamond labels and whether the target object contains the biomolecule to be detected.

[0247] The embodiments of the present invention also provide a flow cytometry fluorescence detection system based on the switching state of the microwave field. This flow cytometry fluorescence detection system based on the switching state of the microwave field is applicable to any of the flow cytometry fluorescence detection methods based on the switching state of the microwave field described in the embodiments of the present invention. The beneficial effects of the flow cytometry fluorescence detection system based on the switching state of the microwave field include those of any of the flow cytometry fluorescence detection methods based on the switching state of the microwave field described in the embodiments of the present invention, which will not be elaborated here.

[0248] It should be understood that various forms of the processes shown above can be used, with steps reordered, added, or deleted. For example, the steps described in the present invention can be executed in parallel, sequentially, or in a different order, as long as the desired results of the technical solution of the present invention can be achieved, and no limitations are imposed herein.

[0249] The above specific embodiments do not constitute a limitation to the protection scope of the present invention. Those skilled in the art should understand that various modifications, combinations, sub - combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A flow cytometry fluorescence detection method based on the switching state of a microwave field, characterized in that, it includes: When the target substance specifically bound to the nanodiamond flows through the first half of the detection area of the sample flow channel, the NV color centers in the nanodiamond generate fluorescence signals, and the fluorescence signals include dark-state fluorescence signals; the dark-state fluorescence signals are the fluorescence signals generated by the NV color centers in the nanodiamond when some or all of the NV color centers resonate with the microwave field; When the target substance specifically bound to the nanodiamond flows through the second half of the detection area of the sample flow channel, the NV color centers in the nanodiamond generate fluorescence signals, and the fluorescence signals include bright-state fluorescence signals; the bright-state fluorescence signals are the fluorescence signals generated by the NV color centers in the nanodiamond when no NV color centers resonate with the microwave field; Among them, the excitation light module is used to emit excitation light, and the excitation light is used to excite the NV color centers in the detection area of the sample flow channel, so that the electrons undergo transitions to generate fluorescence. The microwave radiation module is used to provide a microwave field for the detection area of the sample flow channel, and the microwave field resonates with all or some of the NV color centers in the nanodiamond. The microwave radiation module is in an on state or an off state under the control of a control signal; when the microwave radiation module is in the on state, the NV color centers in the nanodiamond generate the dark-state fluorescence signals, and when the microwave radiation module is in the off state, the NV color centers in the nanodiamond generate the bright-state fluorescence signals; The light detection module collects the fluorescence signals at a preset collection frequency; The data processing module judges whether the target substance contains the biomolecule to be detected according to the magnitude relationship between the intensity of the dark-state fluorescence signal and the intensity of the bright-state fluorescence signal.

2. The flow cytometry fluorescence detection method based on the switching state of a microwave field according to claim 1, characterized in that, When the target substance specifically bound to the nanodiamond flows through the first half of the detection area of the sample flow channel, the NV color centers in the nanodiamond generate fluorescence signals, and the fluorescence signals including the dark-state fluorescence signals include: The control module issues a control signal, and the control signal includes a microwave field opening signal and a microwave field closing signal; The microwave radiation module is in the on state according to the microwave field opening signal. The microwave radiation module provides a microwave field for the detection area of the sample flow channel. When the NV color centers in the nanodiamond flow through the first half of the detection area of the sample flow channel, they emit dark-state fluorescence signals under the irradiation of the excitation light.

3. The flow cytometry fluorescence detection method based on the switching state of a microwave field according to claim 2, characterized in that, When the target substance specifically bound to the nanodiamond flows through the second half of the detection area of the sample flow channel, the NV color centers in the nanodiamond generate fluorescence signals, and the fluorescence signals including the bright-state fluorescence signals include: The microwave radiation module is in the off state according to the microwave field closing signal. The microwave radiation module stops providing a microwave field for the detection area of the sample flow channel. When the NV color centers in the nanodiamond flow through the second half of the detection area of the sample flow channel, they emit bright-state fluorescence signals under the irradiation of the excitation light.

4. The flow cytometry fluorescence detection method based on the switching state of the microwave field according to claim 1, characterized in that, the data processing module determines whether the target contains the biomolecule to be detected according to the magnitude relationship between the intensity of the dark-state fluorescence signal and the intensity of the bright-state fluorescence signal, including: if the intensity of the dark-state fluorescence signal is less than the intensity of the bright-state fluorescence signal, and the absolute value of the difference between the intensity of the dark-state fluorescence signal and the intensity of the bright-state fluorescence signal is greater than a second preset threshold, it is determined that the target contains the biomolecule to be detected, and the second preset threshold is at least 1 time the noise level when the bright-state fluorescence signal is generated.

5. The flow cytometry fluorescence detection method based on the switching state of the microwave field according to claim 1, characterized in that, when the target specifically bound to the nanodiamond flows through the latter half of the detection area of the sample flow channel, after the NV color center in the nanodiamond generates a fluorescence signal, it further includes: after each detection window of the light detection module, a detection window without microwave radiation signal is added as a reference signal.

6. The flow cytometry fluorescence detection method based on the switching state of the microwave field according to any one of claims 1-5, characterized in that, the magnetic field intensity in the detection area satisfies the zero-field condition, and the microwave radiation signal provided by the microwave field has a fixed frequency.

7. The flow cytometry fluorescence detection method based on the switching state of the microwave field according to claim 6, characterized in that, when the magnetic field monitoring module monitors that the magnetic field intensity near the detection area of the sample flow channel satisfies the zero-field condition, the energy level splitting of the NV color center in the nanodiamond caused by the magnetic field is less than one-half of the linewidth of its optically detected magnetic resonance spectrum.

8. The flow cytometry fluorescence detection method based on the switching state of the microwave field according to claim 6, characterized in that, the fixed frequency of the microwave radiation signal provided by the microwave field is greater than or equal to D - 50 MHz and less than or equal to D + 50 MHz, where D is the zero-field splitting parameter of the NV color center.

9. The flow cytometry fluorescence detection method based on the switching state of the microwave field according to claim 6, characterized in that, the particle size range of the nanodiamond is greater than or equal to 40 nm and less than or equal to 1 μm; when the target flows through the detection area, within the period of a single fluorescence signal, the number of detection windows collected by the light detection module is at least one, and for the fluorescence counting rate of the nanodiamond, the contrast of the fluorescence signal, and the exposure time of a single detection window, the following relationship is satisfied: where C is the contrast of the fluorescence signal, R is the fluorescence counting rate of the nanodiamond, and t is the exposure time of a single detection window.

10. The flow cytometry fluorescence detection method based on the switching state of the microwave field according to claim 9, characterized in that, the excitation light module includes an optical pump, and the optical pump is used to emit laser light; the contrast of the fluorescence signal generated by the NV color center in the nanodiamond satisfies the following relationship: where Θ is the normalization constant, Γ p is the laser polarization rate, Γ c is the coherence relaxation rate caused by optical pumping, Ω R is the frequency of the Rabi oscillation of the NV center driven by the microwave field, Γ p and Γ c are related to the laser power, and Ω R is positively correlated with the microwave field intensity felt by the NV centers in the nanodiamond.

11. The flow cytometry fluorescence detection method based on the switching state of the microwave field according to claim 10, characterized in that, the microwave field intensity sensed by the NV color center in the nanodiamond satisfies the following relationship: Wherein, B is the intensity of the microwave field sensed by the NV color centers in the nanodiamond, and B1 is the intensity of the microwave field.

12. The flow cytometry fluorescence detection method based on the switching state of the microwave field according to claim 6, characterized in that for a single detection window, the excitation light remains constantly on, the microwave radiation module provides a microwave radiation signal, and the light detection module uses the entire length of the detection window as the exposure time to obtain the fluorescence signal intensity of the nanodiamond within a single sampling point.

13. The flow cytometry fluorescence detection method based on the switching state of the microwave field according to claim 6, characterized in that the delay time between the excitation light initialization pulse and the fluorescence signal collection pulse window is greater than a preset delay time.

14. A flow cytometry fluorescence detection system based on the switching state of the microwave field, characterized in that the flow cytometry fluorescence detection system based on the switching state of the microwave field is applicable to the flow cytometry fluorescence detection method according to any one of claims 1-13.

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