A flow-through fluorescence detection system

Abstract

The application discloses a kind of flow fluorescence detection systems, comprising: sample flow channel, sample flow channel is used to transport specific binding nanodiamond mark target, nanodiamond includes NV color center, sample flow channel is provided with detection area;Excitation light module, excitation light module is used to excite the NV color center of the detection area of sample flow channel, make its electron transition produce fluorescence;Microwave radiation module, microwave radiation module is used to provide microwave field for the detection area of sample flow channel;Light detection module, light detection module is with preset collection frequency is used to collect specific binding nanodiamond mark target when flowing through the detection area of sample flow channel, by controlling at least one of the intensity, frequency and on-off of microwave field realizes that NV color center in nanodiamond emits fluorescence signal with intensity presenting periodic change;Fluorescence signal is associated with whether target contains biological molecule to be detected.The technical scheme provided in the embodiment of the application improves the detection sensitivity of the fluorescence detection system.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection, in particular to a flow fluorescence detection system. BACKGROUND

[0002] The flow fluorescence technology can realize high-throughput, high-speed and multiple marker joint detection. However, due to the low intensity of the existing fluorescent dye, it is difficult to realize the fluorescence signal recognition of a single molecule, and the lower limit of detection sensitivity is limited. It cannot meet the requirements of special application scenarios, such as detection of low-abundance markers of diseases.

[0003] Therefore, there is an urgent need for a fluorescence detection system with high detection sensitivity. SUMMARY

[0004] The present application provides a flow fluorescence detection system to improve the detection sensitivity of the fluorescence detection system.

[0005] According to an aspect of the present application, a flow fluorescence detection system is provided, comprising:

[0006] A sample flow channel for transporting target objects specifically combined with nanodiamond markers, the nanodiamonds comprising NV color centers, the sample flow channel being provided with a detection area;

[0007] An excitation light module for emitting excitation light for exciting the NV color centers in the detection area of the sample flow channel to make the electrons thereof transition to produce fluorescence;

[0008] A microwave radiation module for providing a microwave field for the detection area of the sample flow channel, the microwave field being resonant with all or part of the NV color centers in the nanodiamonds, the microwave radiation signal provided by the microwave field having a frequency equal to the energy level difference of the electron spin transition of the NV color centers, so that the microwave field is resonant with the NV color centers, the microwave radiation module comprising at least two microwave radiation structures, the microwave radiation structures being distributed along the transport direction of the target objects and being arranged around the detection area of the sample flow channel;

[0009] An optical detection module for collecting fluorescence signals emitted by the NV color centers in the nanodiamonds when the target objects specifically combined with the nanodiamond markers flow through the detection area of the sample flow channel, the fluorescence signals having a periodic variation in intensity, the fluorescence signals being associated with whether the target objects contain the biological molecules to be detected, the optical detection module being configured to control at least one of the intensity, frequency and on-off of the microwave field.

[0010] As a person skilled in the art, it is not difficult to understand that "the sample flow channel is used for transporting the target specifically combined with the nanodiamond marker", but it does not mean that it is only used for transporting the target specifically combined with the nanodiamond marker; in actual detection, the sample flow channel may transport the target without the target, or the target not specifically combined with the nanodiamond marker, and the like.

[0011] Optionally, a control module is further included, which is connected with the microwave radiation module, and the control module is used for emitting a control signal.

[0012] The control signal includes at least one of a microwave field opening signal and a microwave field closing signal, a microwave field power periodic change signal, and a microwave field frequency periodic change signal.

[0013] Optionally, the microwave radiation module is used for providing a modulated microwave field for a detection area of the sample flow channel according to the microwave field opening signal, and the NV center in the nanodiamond emits a dark state fluorescence signal under irradiation of excitation light, the dark state fluorescence signal being a fluorescence signal generated by the NV center in the nanodiamond when part or all of the NV centers are in resonance with the microwave field.

[0014] The microwave radiation module is used for stopping providing a modulated microwave field for the detection area of the sample flow channel according to the microwave field closing signal, and the NV center in the nanodiamond emits a bright state fluorescence signal under irradiation of excitation light, the bright state fluorescence signal being a fluorescence signal generated by the NV center in the nanodiamond when no NV center is in resonance with the microwave field.

[0015] Optionally, the microwave radiation module is used for emitting a microwave modulation field with a fixed frequency and a periodically changed power according to the microwave field power periodic change signal, so that the NV center in the nanodiamond emits a fluorescence signal with a periodically changed intensity under irradiation of excitation light.

[0016] Optionally, the microwave radiation module is used for emitting a microwave modulation field with a fixed power and a periodically changed frequency according to the microwave field frequency periodic change signal, so that the NV center in the nanodiamond emits a fluorescence signal with a periodically changed intensity under irradiation of excitation light.

[0017] Optionally, a data processing module is further included, which is connected with the light detection module, and the data processing module is used for judging whether the target contains a biological molecule to be detected according to the fluorescence signal.

[0018] Optionally, the microwave radiation module comprises two microwave radiation structures, one microwave radiation structure is located at the start position of the detection area, and the other microwave radiation structure is located at the end position of the detection area, and the start position of the detection area is parallel to the direction of the target object and the direction of the end position of the detection area.

[0019] Optionally, the microwave radiation structure comprises a microstrip line and a microwave resonant cavity structure.

[0020] The microwave resonant cavity structure is arranged around the detection area of the sample flow channel, and the microstrip line is located on one side of the microwave resonant cavity structure.

[0021] Optionally, the microwave resonant cavity structure comprises two coaxial radiation coils.

[0022] Optionally, the microwave radiation structure comprises an "Ω" shaped coil radiation antenna.

[0023] Optionally, further comprising a magnetic field monitoring module, the magnetic field monitoring module is used for monitoring the magnetic field intensity near the detection area of the sample flow channel.

[0024] 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 microwave field is used for providing a microwave radiation signal with a fixed frequency.

[0025] Optionally, 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 spectral line broadening of the optical detection magnetic resonance spectrum.

[0026] Optionally, 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 fixed frequency of the microwave radiation signal provided by the microwave field is greater than or equal to D-50MHz and less than or equal to D+50MHz, D is the zero field splitting parameter of the NV color center.

[0027] Optionally, the flow cytometry fluorescence detection system comprises at least two light detection modules.

[0028] The flow fluorescence detection system based on the nanodiamond fluorescent marker provided by the embodiment of the application is different from the traditional NV color center-based light detection system in that the nanodiamond particles to be detected are not stably laid on a substrate but are placed in a sample flow channel and are in a moving state, and the target objects specifically combined with the nanodiamond markers flow through a detection area of the sample flow channel. The nanodiamonds include NV color centers. The NV color centers in the nanodiamonds are excited by excitation light emitted by an excitation light module, the excitation light is used to excite the NV color centers in the detection area of the sample flow channel, so that the electrons of the NV color centers jump to generate fluorescence. When a microwave radiation module provides a microwave field for the detection area of the sample flow channel, the microwave field can provide a microwave radiation signal. The fluorescence signals generated by the NV color centers in the nanodiamonds are different when the microwave radiation signal exists and does not exist. Alternatively, the fluorescence signals generated by the NV color centers in the nanodiamonds change with the change of the frequency or power of the microwave radiation signal, that is, the intensity of the fluorescence signals generated by the NV color centers in the nanodiamonds presents periodic changes by controlling at least one of the intensity, frequency and on-off of the microwave field. When the light detection module collects the fluorescence signals generated by the NV color centers in the nanodiamonds at a preset collection frequency, the target objects specifically combined with the nanodiamond markers flow through the detection area of the sample flow channel, and the fluorescence signals generated by the NV color centers in the nanodiamonds. Since the fluorescence signals are related to whether the target objects contain the biological molecules to be detected, whether the target objects contain the biological molecules to be detected can be determined according to the fluorescence signals. Since the NV color center system has stability, room temperature atmospheric environment compatibility and biological compatibility, and the nanodiamonds are used as new biological fluorescent markers, the low-frequency noise interference can be eliminated by using signal modulation and demodulation technology, so that the detection sensitivity is much higher than that of other fluorescent marker detection methods. In conclusion, the flow fluorescence detection system provided by the embodiment of the application improves the detection sensitivity of the fluorescent detection system and can realize the recognition of single-molecule fluorescence signals.

[0029] It should be understood that the content described in this part is not intended to identify the key or important features of the embodiments of the application, nor is it used to limit the scope of the application. Other features of the application will become apparent from the following description. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the application, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0031] Figure 1 It is a structural schematic diagram of a flow fluorescence detection system provided by the embodiment of the application;

[0032] Figure 2is a structural block diagram of a flow fluorescence detection system according to an embodiment of the present application;

[0033] Figure 3 is another structural schematic diagram of a flow fluorescence detection system according to an embodiment of the present application;

[0034] Figure 4 is a light path system schematic diagram of a flow fluorescence detection system according to an embodiment of the present application;

[0035] Figure 5 is still another structural schematic diagram of a flow fluorescence detection system according to an embodiment of the present application;

[0036] Figure 6 is Figure 5 is a cross-sectional view of a simulation diagram of a microwave field provided by a microwave radiation structure in a structure of a flow fluorescence detection system;

[0037] Figure 7 is Figure 5 is a perspective view of a simulation diagram of a microwave field provided by a microwave radiation structure in a structure of a flow fluorescence detection system;

[0038] Figure 8 is still another structural schematic diagram of a flow fluorescence detection system according to an embodiment of the present application;

[0039] Figure 9 is Figure 8 is a cross-sectional view of a simulation diagram of a microwave field provided by a microwave radiation structure in a structure of a flow fluorescence detection system;

[0040] Figure 10 is a diagram of a relationship between a frequency of a microwave radiation signal and a NV color center fluorescence intensity according to an embodiment of the present application;

[0041] Figure 11 is a light path structure schematic diagram of an optical detection module according to an embodiment of the present application;

[0042] Figure 12 is a flow schematic diagram of a flow fluorescence detection method based on a zero field according to an embodiment of the present application;

[0043] Figure 13 is Figure 12 is a flow schematic diagram included in S110;

[0044] Figure 14 is Figure 12 is a flow schematic diagram included in S130;

[0045] Figure 15 is a diagram of a relationship between a frequency of a microwave radiation signal and a flow rate of nanodiamonds according to an embodiment of the present application;

[0046] Figure 16 is a collection schematic diagram of a modulated fluorescent signal according to an embodiment of the present application;

[0047] Figure 17 is a flowchart of a flow cytometric fluorescent detection method based on microwave field switching state according to an embodiment of the present application;

[0048] Figure 18 is Figure 17 comprises a flowchart. DETAILED DESCRIPTION

[0049] In order to make the personnel in the technical field better understand the present application scheme, the technical scheme in the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should belong to the scope of protection of the present application.

[0050] It should be noted that the terms "first", "second" and the like in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and do not necessarily indicate a specific order or sequence. It should be understood that the data thus used can be interchanged under appropriate circumstances, so that the embodiments of the present application described herein can be implemented in an order other than that illustrated or described herein. In addition, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusion, for example, a process, method, system, product or device including a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but can include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0051] In order to improve the detection sensitivity of the fluorescent detection system, the embodiments of the present application provide a flow cytometric fluorescent detection system.

[0052] As shown in Figure 1 , Figure 1 is a structural schematic diagram of a flow cytometric fluorescent detection system according to an embodiment of the present application, Figure 2 is a structural block diagram of a flow cytometric fluorescent detection system according to an embodiment of the present application, the flow cytometric fluorescent detection system comprising:

[0053] The sample flow channel 100 is used to transport the target object specifically combined with nanodiamond markers, the nanodiamonds including NV color centers, and the sample flow channel 100 is provided with a detection area S1.

[0054] The excitation light module 200 is configured to emit excitation light for exciting NV centers in the detection region S1 of the sample flow channel 100 to make the electrons of the NV centers transition to generate fluorescence.

[0055] The microwave radiation module 300 is configured to provide a microwave field for the detection region S1 of the sample flow channel 100. The microwave field can resonate with all or part of the NV centers in the nanodiamond. The microwave radiation signal provided by the microwave field has a frequency equal to the energy level difference of the electron spin transition of the NV center, so that the microwave field can resonate with the NV center. The microwave radiation module 300 includes at least two microwave radiation structures 301. The microwave radiation structures 301 are distributed along the conveying direction of the target object, and the microwave radiation structures 301 are arranged around the detection region S1 of the sample flow channel 100.

[0056] The light detection module 400 is configured to collect fluorescence signals emitted by the NV centers in the nanodiamond at a preset collection frequency. The fluorescence signals have a periodic variation in intensity. The fluorescence signals are associated with whether the target object contains the biological molecule to be detected. The light detection module 400 is configured to control at least one of the intensity, frequency, and on-off of the microwave field to achieve the periodic variation in intensity of the fluorescence signals emitted by the NV centers in the nanodiamond.

[0057] It is known that quantum sensing systems include Rydberg atoms, atomic magnetometers, superconducting quantum interference devices, and diamond NV centers. Among them, the NV 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, room temperature atmospheric environment compatibility, and biological compatibility. Nanodiamonds as a new type of biological fluorescent marker can eliminate low-frequency noise interference through signal modulation and demodulation technology, thereby achieving a detection sensitivity much higher than other fluorescent marker detection methods.

[0058] The NV centers in the nanodiamond emit fluorescence under the irradiation of excitation light emitted by the excitation light module 200. The excitation light is used to excite the NV centers in the detection region S1 of the sample flow channel 100 to make the electrons of the NV centers transition to generate fluorescence. For example, the NV centers emit fluorescence signals in the wavelength range of 630-800 nm under the irradiation of 532 nm laser emitted by the excitation light module 200. In this embodiment, the excitation light is generated by the excitation light generation module 500, and the excitation light module 200 emits the excitation light in a flat-top beam, which can uniformly converge the excitation light on the detection system of the sample flow channel 100.

[0059] The microwave radiation module 300 includes at least two microwave radiation structures 301. The microwave radiation structures 301 are distributed along the conveying direction of the target object, and the microwave radiation structures 301 are arranged around the detection region S1 of the sample flow channel 100. Figure 1As shown, the transmission direction of the target is from top to bottom, and the at least two microwave radiation structures are distributed along the transmission direction of the target. This can ensure that the microwave field provided by the microwave radiation module 300 can cover the entire detection area S1 during the process that the target flows through the detection area S1, and then when the microwave radiation signal provided by the microwave field changes, the NV center in the nanodiamond generates a fluorescent signal that changes accordingly.

[0060] 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. Due to the presence and absence of the microwave radiation signal, the fluorescent signal generated by the NV center in the nanodiamond is different. Or the change of the frequency or power of the microwave radiation signal, the fluorescent signal generated by the NV center in the nanodiamond changes accordingly, and the specific changes are as follows:

[0061] The microwave radiation module 300 is in an open state, and the detection area S1 has a microwave radiation signal. Under the irradiation of the excitation light, the electrons of the NV center in the nanodiamond jump to generate a dark-state fluorescent signal. The fluorescent signal generated by the NV center in the nanodiamond when part or all of the NV centers resonate with the microwave field is the dark-state fluorescent signal.

[0062] The microwave radiation module 300 is in a closed state, and the detection area S1 does not have a microwave radiation signal. Under the irradiation of the excitation light, the electrons of the NV center in the nanodiamond jump to generate a bright-state fluorescent signal. The fluorescent signal generated by the NV center in the nanodiamond when no NV center resonates with the microwave field is the bright-state fluorescent signal. The intensity of the dark-state fluorescent signal is less than that of the bright-state fluorescent signal.

[0063] The change of the frequency or power of the microwave radiation signal provided by the microwave field causes the fluorescent signal generated by the NV center in the nanodiamond to change accordingly. In this embodiment, due to the periodic change of the frequency or power of the microwave radiation signal provided by the microwave field, under the irradiation of the excitation light, the electrons of the NV center in the nanodiamond jump, the number of NV centers that resonate with the microwave field changes periodically, and / or the degree of NV center resonance changes periodically, and the NV center generates a fluorescent signal with a periodically changing intensity.

[0064] Specifically, when the microwave field modulation mode is amplitude modulation, the power of the microwave radiation signal provided by the microwave field changes, for example, the simplest square wave modulation, sinusoidal wave modulation and triangular wave modulation achieved 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, 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 center, the microwave field can resonate with the NV center.

[0065] The flow fluorescence detection system based on the nanodiamond fluorescent label provided by the embodiment of the application is different from the traditional NV color center-based light detection system in that the nanodiamond particles to be detected are not stably laid on a substrate but are placed in a sample flow channel 100 and are in a moving state, and the target objects specifically combined with the nanodiamond labels flow through a detection area S1 of the sample flow channel 100. The nanodiamonds include NV color centers. The NV color centers in the nanodiamonds are excited by excitation light emitted by an excitation light module 200, the excitation light is used to excite the NV color centers in the detection area S1 of the sample flow channel 100, so that the electrons of the NV color centers are transitioned to generate fluorescence. When a microwave irradiation module 300 provides a microwave field for the detection area S1 of the sample flow channel 100, the microwave field can provide a microwave irradiation signal. The fluorescence signals generated by the NV color centers in the nanodiamonds are different when the microwave irradiation signal exists and does not exist. Alternatively, the fluorescence signals generated by the NV color centers in the nanodiamonds change when the frequency or power of the microwave irradiation signal changes, that is, the intensity of the fluorescence signals generated by the NV color centers in the nanodiamonds presents periodic changes by controlling at least one of the intensity, frequency and on-off of the microwave field. When the light detection module 400 collects the fluorescence signals generated by the NV color centers in the nanodiamonds at a preset collection frequency when the target objects specifically combined with the nanodiamond labels flow through the detection area S1 of the sample flow channel 100, the fluorescence signals generated by the NV color centers in the nanodiamonds are associated with whether the target objects contain the biological molecules to be detected. Therefore, whether the target objects contain the biological molecules to be detected can be determined according to the fluorescence signals. Because the NV color center system has stability, room temperature atmospheric environment compatibility and biological compatibility, and the nanodiamonds are used as a new type of biological fluorescent label, the signal modulation and demodulation technology can be used to eliminate low-frequency noise interference, so that the detection sensitivity is much higher than that of other fluorescent label detection methods. In summary, the flow fluorescence detection system provided by the embodiment of the application improves the detection sensitivity of the fluorescent detection system and can realize single-molecule fluorescence signal recognition.

[0066] Optionally, based on the above technical solution, as shown in Figure 2 The control module 600 is connected with the microwave irradiation module 300, and the control module 600 is used to emit a control signal.

[0067] The control signal includes at least one of a microwave field opening signal and a microwave field closing signal, a microwave field power periodic change signal and a microwave field frequency periodic change signal.

[0068] Optionally, on the basis of the above technical solutions, the microwave radiation module 300 is configured to provide a modulated microwave field for the detection area S1 of the sample flow channel 100 according to a microwave field opening signal, and the NV centers in the nanodiamonds emit a dark-state fluorescence signal under irradiation of the excitation light; the dark-state fluorescence signal is a fluorescence signal generated by the NV centers in the nanodiamonds when part or all of the NV centers resonate with the microwave field.

[0069] Optionally, on the basis of the above technical solutions, the microwave radiation module 300 is configured to emit a microwave modulation field with a fixed frequency and a periodically varying power according to a microwave field power periodic variation signal, so that the NV centers in the nanodiamonds emit a fluorescence signal with a periodically varying intensity under irradiation of the excitation light.

[0070] Optionally, on the basis of the above technical solutions, the microwave radiation module 300 is configured to emit a microwave modulation field with a fixed power and a periodically varying frequency according to a microwave field frequency periodic variation signal, so that the NV centers in the nanodiamonds emit a fluorescence signal with a periodically varying intensity under irradiation of the excitation light.

[0071] Specifically, the microwave radiation module 300 is in an open state under the control of a microwave field opening signal, and the detection area S1 has a microwave radiation signal; under irradiation of the excitation light, the electrons of the NV centers in the nanodiamonds undergo transition to generate a dark-state fluorescence signal, which is a fluorescence signal generated by the NV centers in the nanodiamonds when part or all of the NV centers resonate with the microwave field.

[0072] The microwave radiation module 300 is in a closed state under the control of a microwave field closing signal, and the detection area S1 has no microwave radiation signal; under irradiation of the excitation light, the electrons of the NV centers in the nanodiamonds undergo transition to generate a bright-state fluorescence signal, which is a fluorescence signal generated by the NV centers in the nanodiamonds when none of the NV centers resonate with the microwave field. The intensity of the dark-state fluorescence signal is less than that of the bright-state fluorescence signal.

[0073] Alternatively, the microwave radiation module 300 emits a microwave modulation field with a fixed frequency and a periodically varying power under the control of a microwave field power periodic variation signal, so that, under irradiation of the excitation light, the electrons of the NV centers in the nanodiamonds undergo transition, 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.

[0074] Alternatively, the microwave radiation module 300 emits a microwave modulation field with fixed power and periodically changing frequency under the control of a microwave field frequency periodic change signal, so that the NV centers in the nanodiamonds produce a fluorescent signal with intensity periodically changing when the electrons of the NV centers jump under the irradiation of the excitation light, the number of NV centers resonating with the microwave field periodically changes, and / or the degree of NV center resonance periodically changes.

[0075] Optionally, on the basis of the above technical solutions, the data processing module 700 is further included, the data processing module 700 is connected with the light detection module 400, and the data processing module 700 judges whether the target object contains the biological molecule to be detected according to the fluorescent signal.

[0076] Specifically, the frequency or power of the microwave radiation signal provided by the microwave field periodically changes, the number of NV centers resonating with the microwave field periodically changes, and / or the degree of NV center resonance periodically changes, the NV centers produce a fluorescent signal with intensity periodically changing, and a first judgment mode is adopted: the data processing module 700 performs Fourier transform on the fluorescent signal to obtain a frequency domain signal, wherein the part with the same frequency as the control signal is a demodulation signal; if the intensity of the demodulation signal is greater than a first preset threshold, it is judged that the target object contains the biological molecule to be detected.

[0077] The microwave radiation module 300 is in an open state under the control of the microwave field opening signal, the electrons of the NV centers jump to produce a dark state fluorescent signal, and the dark state fluorescent signal is the fluorescent signal produced by the NV centers in the nanodiamonds when part or all of the NV centers resonate with the microwave field. The microwave radiation module 300 is in a closed state under the control of the microwave field closing signal, the electrons of the NV centers jump to produce a bright state fluorescent signal, and the bright state fluorescent signal is the fluorescent signal produced by the NV centers in the nanodiamonds when no NV center resonates with the microwave field. The intensity of the dark state fluorescent signal is less than the intensity of the bright state fluorescent signal. A second judgment mode is adopted, the intensity of the dark state fluorescent signal is less than the intensity of the bright state fluorescent signal, and the absolute value of the difference between the intensity of the dark state fluorescent signal and the intensity of the bright state fluorescent signal is greater than a second preset threshold, the data processing module 700 judges that the target object contains the biological molecule to be detected, and the second preset threshold is at least 1 times the noise level when the bright state fluorescent signal is produced.

[0078] Optionally, on the basis of the above technical solutions, as shown in Figure 1 and Figure 3 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 end position of the detection area S1, and the starting position of the detection area S1 is parallel to the direction of the target object in the direction pointing to the end position of the detection area S1.

[0079] like Figure 4 As shown, in the flow cytometry detection system, a sheath fluid is placed around the sample channel 100. Target material specifically bound to nanodiamonds flows through the detection region S1 of the sample channel 100. The NV centers within the nanodiamonds are irradiated by excitation light emitted from the excitation light module 200. The excitation light excites the NV centers in the detection region S1 of the sample channel 100, causing electron transitions and generating fluorescence. When the microwave radiation module 300 provides a microwave field to the detection region S1 of the sample channel 100, the microwave field provides a microwave radiation signal. Changes in the microwave radiation signal cause corresponding changes in the fluorescence signal generated by the NV centers within the nanodiamonds. Figure 1 and 3 As shown, the transmission direction of the target object is from top to bottom in the figure. One microwave radiation structure 301 is located at the beginning of the detection area S1, and another microwave radiation structure 301 is located at the end of the detection area S1. This ensures that the microwave radiation signal emitted by the microwave radiation module 300 covers the entire detection area S1 as the target object flows through it. Consequently, when the microwave radiation signal changes, the NV color centers within the nanodiamond generate corresponding fluorescence signals. When the microwave radiation module 300 provides a microwave field to the detection area S1 of the sample flow channel 100, the microwave field provides a microwave radiation signal. The presence or absence of the microwave radiation signal results in different fluorescence signals generated by the NV color centers within the nanodiamond; or, changes in the frequency or power of the microwave radiation signal cause corresponding changes in the fluorescence signals generated by the NV color centers within the nanodiamond.

[0080] Optionally, based on the above technical solutions, such as Figure 5 As 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.

[0081] Specifically, such as Figure 6 and Figure 7 As shown, the grayscale 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 the electrical signal. The microwave resonant cavity structure L2 provides a microwave field with high 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 radiates the entire detection area S1.

[0082] Optionally, based on the above technical solutions, such as Figure 5 As shown, the microwave resonant cavity structure L2 includes two coaxial radiating coils.

[0083] Specifically, the two coaxial radiation coils constitute a microwave resonance cavity structure L2, and the microwave radiation signal generated by the microstrip line L1 under the action of an electrical signal is radiated to the entire detection area S1.

[0084] Optionally, based on the technical scheme above, as shown in Figure 8 The microwave radiation structure 301 includes a radiation antenna of an "Ω" shaped coil.

[0085] Specifically, as shown in Figure 9 The detection area S1 of the sample flow channel 100 has uniform gray scale, and the radiation antenna of the "Ω" shaped coil provides a microwave field with high uniformity to 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 is radiated to the entire detection area S1. The "Ω" shaped coil can be a single-turn or multi-turn copper wire. As shown in Figure 9 The detection area S1 includes a detection point 100a of the light detection module 400.

[0086] Optionally, based on the technical scheme above, as shown in Figure 1 and Figure 2 The technical scheme further includes a magnetic field monitoring module 800, which is configured to monitor the magnetic field strength near the detection area S1 of the sample flow channel 100. When the magnetic field monitoring module 800 monitors that the magnetic field strength near the detection area S1 of the sample flow channel 100 satisfies a zero field condition, the microwave field is configured to provide a microwave radiation signal with a fixed frequency.

[0087] In an external magnetic field, NV centers cause the energy levels of NV centers in nanodiamonds to move in opposite directions between |m s = 0> and |m s = ±1> states, and the frequency difference is proportional to the projection size of the external magnetic field in the NV center crystal axis direction. A single nanodiamond contains multiple NV centers, and the crystal axis directions of different NV centers are different. In addition, the spatial direction of different nanodiamond particles flowing through the sample flow channel 100 is random. Therefore, the projection components of the external magnetic field on the NV centers in different axial directions in different nanodiamond particles are different, resulting in different transition frequencies between |m s = 0> and |m s = ±1> states. Under the control of a single frequency microwave, the quantum state of the NV center in part of the nanodiamond particles does not change, and the overall fluorescence bright-dark change is not obvious, thereby affecting the signal-to-noise ratio of the measurement scheme and further affecting the accuracy and reliability of the detection of the measured object.

[0088] Therefore, when the magnetic field monitoring module 800 monitors that the magnetic field intensity near the detection area S1 of the sample flow channel 100 satisfies the zero field condition, the frequency at which part or all of the NV centers resonate with the microwave field is the intrinsic frequency, the microwave field is used to provide a microwave radiation signal with the fixed frequency, which can improve the signal-to-noise ratio of the fluorescence signal, and further improve the detection sensitivity of the flow fluorescence detection system.

[0089] Optionally, on the basis of the above technical solutions, as shown in Figure 10 When the magnetic field monitoring module 800 monitors that the magnetic field intensity near the detection area S1 of the sample flow channel 100 satisfies the zero field condition, the energy level splitting of the NV center in the nanodiamond is less than one-half of the spectral line broadening of the optical detection magnetic resonance spectrum of the NV center.

[0090] The "energy level splitting of the NV center in the nanodiamond" refers to the amount of movement of the optical detection magnetic resonance spectrum resonance peak position of the NV center caused by the Zeeman shift of the NV energy level due to the external magnetic field, that is, one-half of the difference between the two microwave resonance frequencies corresponding to the energy level splitting of the same NV center; and the "spectral line broadening of the optical detection magnetic resonance spectrum" refers to the half-peak width of the optical detection magnetic resonance spectrum resonance peak of the NV center.

[0091] 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 NV centers in the nanodiamond particles in all crystal axis directions can be quantum state regulated by the microwave with a frequency greater than or equal to 2820 MHz and less than or equal to 2920 MHz, so that the system can use a frequency greater than or equal to 2820 MHz and less than or equal to 2920 MHz as the fixed working frequency of the microwave radiation signal provided by the microwave radiation module 300. It is well known to those skilled in the art that the zero field splitting D of the NV center at room temperature is 2870 MHz, but the accurate value of the zero field splitting D has a linear relationship with the temperature. It is not difficult to understand that the fixed frequency of the above-mentioned microwave radiation signal is set between 2820 MHz and 2920 MHz, which is based on the premise that the zero field splitting D is 2870 MHz.

[0092] In order to make the embodiments of the present application more concise and clear, it is assumed in the following that the experimental environment temperature is room temperature, that is, the value of the zero field splitting D is 2870 MHz; but it is not difficult for those skilled in the art to understand that the zero field splitting D may be affected by different experimental environment temperatures and show other values, and the fixed frequency of the microwave radiation signal can usually be set between D-50 MHz and D+50 MHz.

[0093] The technical scheme provided by the embodiment of the application can improve the signal-to-noise ratio of the fluorescent signal and improve the identification accuracy of the to-be-detected object, and the system introduces a microwave field to modulate the fluorescent signal of the nanodiamond particle; when the microwave radiation module 300 opens the microwave with a frequency greater than or equal to 2820 MHz and less than or equal to 2920 MHz into the radiation coil, the NV center in the nanodiamond particle in the sample flow channel 100 transitions from the |m s = 0> state to the |m s = ± 1> state, and the fluorescent intensity of the nanodiamond particle decreases; when the microwave in the radiation coil is turned off, the excitation light initializes the NV center in the detection area S1 to the |m s = 0> state, and the fluorescent intensity is restored. When the radiation power of the microwave radiation signal provided by the microwave radiation module 300 changes, the NV center in the nanodiamond particle generates a fluorescent signal that changes accordingly. The fluorescent signal is modulated in the time domain by the microwave radiation module 300, then collected at a certain frequency by the light detection module 400, and finally the to-be-detected signal at a specific frequency is identified by an algorithm to exclude the interference of static noise signals.

[0094] Optionally, based on the technical scheme described above, as shown in Figure 11 , the flow fluorescence detection system includes at least two light detection modules 400, which can ensure that the fluorescent signals at all angles around the detection area S1 can be collected by the light detection module 400.

[0095] For example, Figure 1 and Figure 11 two light detection modules 400 are shown, which 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 light detection modules 400 is greater than two, the plurality of 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 fluorescent signals at all angles around the detection area S1 can be collected by the light detection module 400. The above technical scheme can improve the collection amount of the fluorescent signal, thereby providing a lower detection limit; on the other hand, the problem of the target object shielding the NV center fluorescent light can be overcome.

[0096] Since the fluorescent intensity of a single nanodiamond particle is weak in a single molecule detection application scenario, the light detection module 400 is preferably an avalanche photodiode photodetector (APD), followed by a research camera, and then a photomultiplier tube sensor (PMT).

[0097] In addition, the interference of the excitation light and stray fluorescent signals can be filtered out by using wave-trap filters, long-wave-pass filters and short-wave-pass filters.

[0098] As shown in Figure 12 , Figure 12is a flowchart of a zero-field-based flow cytometry fluorescence detection method provided by the present application, which comprises the following steps:

[0099] In S110, when the target labeled by the nanodiamonds flows through the detection region of the sample flow channel, the intensity, frequency and on-off of the microwave field are controlled to realize that the NV centers in the nanodiamonds emit a fluorescence signal with periodically changing intensity.

[0100] As shown in Figure 1 The magnetic field intensity of the detection region S1 satisfies the zero-field condition, the excitation light module 200 emits excitation light, the excitation light is used to excite the NV centers in the detection region S1 of the sample flow channel 100 to make the electrons thereof transition to produce fluorescence, the microwave radiation module 300 is used to provide a microwave field for the detection region S1 of the sample flow channel 100, the microwave field resonates with all or part of the NV centers in the nanodiamonds, the microwave radiation signal provided by the microwave field has a fixed frequency and is equal to the energy level difference of the electron spin transition of the NV centers, so that the microwave field resonates with the NV centers.

[0101] In this embodiment, when the microwave radiation module 300 provides a microwave field for the detection region S1 of the sample flow channel 100, the microwave field can provide a microwave radiation signal, the fluorescence signal produced by the NV centers in the nanodiamonds is different when the microwave radiation signal exists and does not exist; or the frequency or radiation power of the microwave radiation signal changes, and the fluorescence signal produced by the NV centers in the nanodiamonds changes accordingly.

[0102] The microwave radiation module 300 is in the open state, and the detection region S1 has the microwave radiation signal. Under the irradiation of the excitation light, the electrons of the NV centers in the nanodiamonds transition to produce a dark-state fluorescence signal. The dark-state fluorescence signal is the fluorescence signal produced by the NV centers in the nanodiamonds when part or all of the NV centers resonate with the microwave field.

[0103] The microwave radiation module 300 is in the closed state, and the detection region S1 does not have the microwave radiation signal. Under the irradiation of the excitation light, the electrons of the NV centers in the nanodiamonds transition to produce a bright-state fluorescence signal. The bright-state fluorescence signal is the fluorescence signal produced 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 that of the bright-state fluorescence signal.

[0104] The microwave field provides microwave radiation signals with varying frequency or power, and the NV centers in the nanodiamonds produce fluorescent signals that vary accordingly. In this embodiment, because the microwave field provides microwave radiation signals with varying frequency or power, the NV centers in the nanodiamonds, under excitation light irradiation, the electrons of the NV centers transition, the number of NV centers resonating with the microwave field varies periodically, and / or the degree of NV center resonance varies periodically, and the NV centers produce fluorescent signals with varying intensity.

[0105] Specifically, when the microwave field modulation mode is amplitude modulation, the microwave field provides microwave radiation signals with varying radiation power, for example, the simplest square wave modulation, sinusoidal wave modulation achieved by changing the microwave power, and triangular wave modulation. When the microwave field modulation mode is frequency modulation, the microwave field provides microwave radiation signals with varying frequency, for example, microwave resonance-non-resonance mode. When the frequency of the microwave field provided by the microwave radiation signal is equal to the energy level difference of the electron spin transition of the NV center, the microwave field can resonate with the NV center.

[0106] Optionally, on the basis of the above technical solutions, as shown in Figure 1 and Figure 2 The magnetic field monitoring module 800 monitors the magnetic field strength near the detection area S1 of the sample flow channel 100; when the magnetic field monitoring module 800 monitors the magnetic field strength near the detection area S1 of the sample flow channel 100 satisfies the zero field condition, the microwave field is used to provide microwave radiation signals with a fixed frequency.

[0107] In an external magnetic field, the NV center causes the electron of the NV center in the nanodiamond to move in the opposite direction between the |m s =0> state and the |m s =±1> state, and the frequency difference is proportional to the projection size of the external magnetic field in the NV center crystal axis direction. A single nanodiamond contains multiple NV centers, and the crystal axis directions of different NV centers are different; and the spatial direction of different nanodiamond particles flowing through the sample flow channel 100 is random. Therefore, the projection components of the external magnetic field are different for the NV centers in different axial directions in different nanodiamond particles, resulting in different transition frequencies between the |m s =0> state and the |m s =±1> state. Under the control of a single frequency microwave, the quantum state of the NV center in part of the nanodiamond particles does not change, and the overall fluorescence bright-dark change is not obvious, thereby affecting the signal-to-noise ratio of the measurement scheme and further affecting the accuracy and reliability of the detection of the measured object.

[0108] Therefore, when the magnetic field monitoring module 800 monitors that the magnetic field intensity near the detection area S1 of the sample flow channel 100 satisfies the zero field condition, the frequency at which part or all of the NV color centers resonate with the microwave field is the fixed frequency, the microwave field is used to provide a microwave radiation signal with the fixed frequency, the signal-to-noise ratio of the fluorescence signal can be improved, and the detection sensitivity of the flow fluorescence detection system is further improved.

[0109] Optionally, based on the technical scheme above, as shown in the figure, Figure 10 When the magnetic field monitoring module 800 monitors that the magnetic field intensity near the detection area S1 of the sample flow channel 100 satisfies the zero field condition, the energy level splitting of the NV color center in the nanodiamond is less than one-half of the spectral line broadening of the optical detection magnetic resonance spectrum of the NV color center caused by the magnetic field.

[0110] The "energy level splitting of the NV color center in the nanodiamond" refers to the amount of movement of the optical detection magnetic resonance spectrum resonance peak position of the NV color center caused by the Zeeman shift of the NV energy level due to the external magnetic field, that is, one-half of the difference between the two microwave resonance frequencies corresponding to the energy level splitting of the same NV color center; and the "spectral line broadening of the optical detection magnetic resonance spectrum" refers to the half-peak width of the optical detection magnetic resonance spectrum resonance peak of the NV color center.

[0111] In the above technical scheme, when the magnetic field monitoring module 800 monitors that the magnetic field intensity near the detection area S1 of the sample flow channel 100 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 of the nanodiamond can be quantum state controlled by the microwave with a frequency greater than or equal to 2820 MHz and less than or equal to 2920 MHz, and thus the system can use a frequency greater than or equal to 2820 MHz and less than or equal to 2920 MHz as the fixed working frequency of the microwave radiation signal provided by the microwave radiation structure 300.

[0112] It should be noted that in the embodiment of the present application, the spectral peak displacement caused by the magnetic field is required to be within the intrinsic broadening of the resonance (CW) spectrum, and the intrinsic broadening of the CW spectrum is determined by the dephasing time. However, in nanodiamonds with different diameters and different manufacturing processes, the noise environment of the NV color center is different, corresponding to different dephasing times, and the zero field limit needs to be determined according to the selected particles. For example, for a 40 nm diameter nanodiamond made by HPHT method with an NV concentration of 1.5 ppm, the typical value of the CW spectrum intrinsic broadening is 20 MHz, and the external magnetic field is required to be below 3.56 Gs. The resonance (CW) spectrum is the spectral line in Figure 10 .

[0113] S120, the optical detection module collects the modulated fluorescence signal at a preset collection frequency.

[0114] As shown in the figure, Figure 11As shown, the flow fluorescence detection system includes at least two light detection modules 400, which can ensure that the modulated fluorescence signals at each angle around the detection area S1 can be collected by the light detection modules 400.

[0115] Exemplarily, Figure 1 and Figure 11 Two light detection modules 400 are shown, which are arranged around the detection area S1 of the sample flow channel 100 and symmetrically arranged with respect to the detection area S1.

[0116] When the number of light detection modules 400 is greater than two, the plurality of 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 modulated fluorescence signals at each angle around the detection area S1 can be collected by the light detection modules 400.

[0117] The above technical solution can improve the collection amount of fluorescence signals on the one hand, thereby providing a lower detection limit; on the other hand, it can overcome the problem of target shielding NV color center fluorescence.

[0118] Since the fluorescence intensity of a single nanodiamond particle is weak in single molecule detection application scenarios, the light detection module 400 is preferably an avalanche photodiode photodetector (APD), followed by a research camera, and then a photomultiplier tube sensor (PMT).

[0119] In addition, the interference of excitation light and stray fluorescence signals can be filtered out by notch filters, long-wave pass filters, and short-wave pass filters.

[0120] S130, the data processing module determines whether the target contains the biological molecule to be detected according to the fluorescence signal.

[0121] Specifically, the frequency or power of the microwave radiation signal provided by the microwave field changes periodically, the number of NV color centers and microwave field resonances changes periodically, and / or the degree of NV color center resonance changes periodically, and the intensity of the fluorescence signal generated by the NV color center changes periodically. The first judgment method is adopted: the data processing module 700 performs Fourier transform on the fluorescence signal to obtain a frequency domain signal, wherein 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 biological molecule to be detected.

[0122] The microwave radiation module 300 is in an open state under the control of the microwave field opening signal, and the electrons of the NV color center jump to generate a dark state fluorescence signal. The dark state fluorescence signal is the fluorescence signal generated by the NV color center in the nanometer diamond when part or all of the NV color centers resonate with the microwave field. The microwave radiation module 300 is in a closed state under the control of the microwave field closing signal, and the electrons of the NV color center jump to generate a bright state fluorescence signal. The bright state fluorescence signal is the fluorescence signal generated by the NV color center in the nanometer diamond when no NV color 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. In the second judgment mode, 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 value. The data processing module 700 judges that the target object contains the biological molecule to be detected, and the second preset threshold value is at least 1 times the noise level when the bright state fluorescence signal is generated.

[0123] The embodiment of the present application provides a kind of based on zero field's flow fluorescence detection method, and flow fluorescence detection method is different from traditional based on NV color center's light detection method, the nanodiamond particles to be measured is not stably laid on substrate, but is placed in sample flow channel 100 inside, in motion state, specific binding nanodiamond labeled target flows through the detection area S1 of sample flow channel 100, nanodiamond includes NV color center, NV color center in nanodiamond under the irradiation of excitation light emitted by excitation light module 200, excitation light is used to excite the NV color center of the detection area S1 of sample flow channel 100, make its electron transition produce fluorescence.When microwave radiation module 300 provides microwave field for the detection area S1 of sample flow channel 100, microwave field can provide microwave radiation signal, and the fluorescence signal produced by NV color center in nanodiamond is different when microwave radiation signal exists and does not exist;Or, the frequency or power of microwave radiation signal changes, and the fluorescence signal produced by NV color center in nanodiamond changes accordingly, i.e. by controlling at least one of the intensity, frequency and on-off of microwave field, nanodiamond emits fluorescence signal with periodic variation of intensity.Light detection module 400 collects the fluorescence signal produced by NV color center in nanodiamond when specific binding nanodiamond labeled target flows through the detection area S1 of sample flow channel 100 at preset collection frequency, because fluorescence signal is associated with whether target contains biological molecule to be detected, therefore, whether target contains biological molecule to be detected can be judged according to fluorescence signal.Because of the stability of NV color center system, room temperature atmospheric environment compatibility and biological compatibility, and nanodiamond as a new type of biological fluorescent marker, low-frequency noise interference can be excluded by signal modulation and demodulation technology, so that the detection sensitivity of the method is much higher than that of other fluorescent marker detection methods.Summarized above, the technical scheme provided by the embodiment of the present application improves the detection sensitivity of fluorescence detection method, and single molecule fluorescence signal recognition can be realized.In the embodiment of the present application, the magnetic field intensity near the detection area S1 of sample flow channel 100 satisfies the zero field condition, and the frequency of partial or all NV color centers resonating with microwave field is inherent frequency, and microwave field is used to provide microwave radiation signal with the fixed frequency, which can improve the signal-to-noise ratio of modulated fluorescence signal, and further improve the detection sensitivity of flow fluorescence detection method.

[0124] Optionally, on the basis of the above technical scheme, as shown in Figure 13 , Figure 13 is Figure 12 S110 includes flow chart, when specific binding nanodiamond labeled target flows through the detection area of sample flow channel, the fluorescence signal produced by NV color center in nanodiamond includes:

[0125] S1101, the control module sends a control signal, the control signal including at least one of a microwave field opening signal and a microwave field closing signal, a microwave field power periodic variation signal, and a microwave field frequency periodic variation signal.

[0126] S1102, the microwave radiation module provides a microwave field for the detection area of the sample flow channel according to the microwave field opening signal, the microwave radiation module is in an open state, and the NV center in the nanodiamond emits a dark state fluorescence signal under excitation light irradiation. The dark state fluorescence signal is a fluorescence signal generated by the NV center in the nanodiamond when part or all of the NV centers resonate with the microwave field.

[0127] Specifically, the microwave radiation module 300 is in an open state under the control of the microwave field opening signal, and the detection area S1 has a microwave radiation signal. The NV center in the nanodiamond emits a dark state fluorescence signal under excitation light irradiation, and the electron of the NV center jumps to produce a dark state fluorescence signal. The dark state fluorescence signal is a fluorescence signal generated by the NV center in the nanodiamond when part or all of the NV centers resonate with the microwave field.

[0128] S1103, the microwave radiation module stops providing a microwave field for the detection area of the sample flow channel according to the microwave field closing signal, and the NV center in the nanodiamond emits a bright state fluorescence signal under excitation light irradiation. The bright state fluorescence signal is a fluorescence signal generated by the NV center in the nanodiamond when no NV center resonates with the microwave field.

[0129] The microwave radiation module 300 is in a closed state under the control of the microwave field closing signal, and the detection area S1 has no microwave radiation signal. The NV center in the nanodiamond emits a bright state fluorescence signal under excitation light irradiation, and the electron of the NV center jumps to produce a bright state fluorescence signal. The bright state fluorescence signal is a fluorescence signal generated by the NV center 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.

[0130] Alternatively, S1104, the microwave radiation module emits a microwave modulation field with a fixed frequency and a periodically varying power according to the microwave field power periodic variation signal, so that the NV center in the nanodiamond emits a fluorescence signal with a periodically varying intensity under excitation light irradiation.

[0131] The microwave radiation module 300 emits a microwave modulation field with a fixed frequency and a periodically varying power under the control of the microwave field power periodic variation signal, so that the NV center in the nanodiamond emits a fluorescence signal with a periodically varying intensity under excitation light irradiation. The electron of the NV center jumps, the number of NV centers resonating with the microwave field periodically varies, and / or the degree of NV center resonance periodically varies, and the NV center generates a fluorescence signal with a periodically varying intensity.

[0132] Alternatively, the microwave radiation module emits a microwave modulation field with fixed power and periodically changed frequency according to the microwave field frequency periodic change signal, so that the NV center in the nanodiamond emits a fluorescence signal with periodically changed intensity under excitation light irradiation.

[0133] The microwave radiation module 300 emits a microwave modulation field with fixed power and periodically changed frequency under the control of the microwave field frequency periodic change signal, so that the NV center in the nanodiamond emits a fluorescence signal with periodically changed intensity under excitation light irradiation.

[0134] The above technical solution provides a scheme for the microwave radiation module 300 to emit a modulated fluorescence signal that changes accordingly when the microwave radiation signal is in the on state and the off state or the power or frequency of the microwave radiation signal emitted by the microwave radiation module 300 changes periodically under the control of the control signal.

[0135] Alternatively, on the basis of the above technical solution, as shown in Figure 14 Figure 14 is Figure 12 S130 includes a flowchart as shown in the figure, and the data processing module determines whether the target object contains the biological molecule to be detected according to the modulated fluorescence signal.

[0136] S1301, the data processing module performs Fourier transform on the fluorescence signal to obtain a frequency domain signal, wherein the part with the same frequency as the control signal is a demodulation signal.

[0137] S1302, if the intensity of the demodulation signal is greater than a first preset threshold, it is determined that the target object contains the biological molecule to be detected.

[0138] The above technical solution is aimed at the frequency or power of the microwave radiation signal provided by the microwave field, the number of NV center and microwave field resonance changes periodically, and / or the degree of NV center resonance changes periodically, and the NV center generates a fluorescence signal with periodically changed intensity. The data processing module 700 determines whether the target object contains the biological molecule to be detected according to the modulated fluorescence signal.

[0139] Alternatively, on the basis of the above technical solution, the data processing module 700 determines whether the target object contains the biological molecule to be detected according to the modulated fluorescence signal.

[0140] ​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 value, and the data processing module determines that the target object contains the to-be-detected biological molecule, and the second preset threshold value is at least 1 times the noise level when the bright-state fluorescence signal is generated.

[0141] The technical solution is directed to a judgment method for determining whether a target object contains a to-be-detected biological molecule by a data processing module 700 according to a fluorescence signal, wherein the microwave radiation module 300 is in an open state or a closed state under the control of a control signal, when the microwave radiation module 300 is in the open state under the control of a microwave field opening signal, the electrons of the NV color center jump to generate a dark-state fluorescence signal, and when the dark-state fluorescence signal is generated by the NV color center in the nanodiamond under the resonance of part or all of the NV color center and the microwave field, the bright-state fluorescence signal is generated by the NV color center in the nanodiamond when the microwave radiation module 300 is in the closed state under the control of a microwave field closing signal, and the electrons of the NV color center jump to generate a bright-state fluorescence signal.

[0142] Optionally, based on the technical solution, the number of periodically changed fluorescence signals presented by a single target object in the detection area is ;

[0143] wherein N is the number of target objects flowing through the detection area of the sample flow channel per second, and N is an integer greater than or equal to 1; F M is the frequency of the control signal.

[0144] As shown in Figure 15 , the number of target objects detected by the flow fluorescence system per unit time is N per second, and the time required for a single target object to flow through the detection area S1 is 1 / N seconds. With F M as the frequency of the control signal, a single target object presents periodic modulation 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 object.

[0145] Here, when the microwave field modulation mode is amplitude modulation, the microwave radiation signal provided by the microwave field changes in radiation power, for example, the simplest square wave modulation, the sinusoidal modulation and the 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, 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 and the NV color center can resonate.

[0146] Optionally, on the basis of the above technical solutions, when the target stream passes through the detection area, the number of detection windows collected by the light detection module in the period of a single fluorescent signal satisfies the following relationship:

[0147] M=F s / F M

[0148] Wherein, F s is the sampling frequency of the light detection module, and F M is the frequency of the control signal.

[0149] The above technical solutions give the relationship between the sampling frequency of the light detection module 400 and the frequency of the control signal, as shown in Figure 16 , also give the number of detection windows collected by the light detection module 400 in the period of a single modulated fluorescent signal when the target stream passes through the detection area S1.

[0150] Optionally, on the basis of the above technical solutions, when the target stream labeled with nanodiamonds specifically binds to S110 passes through the detection area of the sample flow channel, the following is included after the nanodiamonds produce a fluorescent signal:

[0151] After each detection window of the light detection module, a detection window without microwave radiation signal is added as a reference signal.

[0152] In each detection window, the light detection module 400 collects the fluorescent signal of the entire detection area at the same time; a pinhole is added in front of the fluorescent collector for spatial filtering to reduce the interference of background stray signals; after a single detection window, a detection window without modulated microwave control can be added as a reference signal for calibrating the time jitter and spatial non-uniformity of the excitation light.

[0153] Optionally, on the basis of the above technical solutions, a number of scientific cameras are used as the light detection module 400, which can choose sCMOS high-speed scientific cameras or enhanced ICCD; in each detection window, the detector performs fluorescent imaging on the entire detection area; a pinhole can be added in front of the fluorescent collector for spatial filtering to reduce the interference of background stray signals; for the fluorescent images of each detection window, the position trajectory of the nanodiamond fluorescent particles in the image is found by algorithm recognition, and a number of points are selected as the fluorescent count of the nanodiamond particles in the window, and the rest of the background fluorescent signal is discarded to improve the signal-to-noise ratio of the measured fluorescent signal.

[0154] Optionally, on the basis of the above technical solutions, 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.

[0155] The fluorescent counting rate of the nanodiamond, the contrast of the fluorescent signal and the exposure time of the single detection window satisfy the following relationship:

[0156]

[0157] Wherein, C is the contrast of the fluorescent signal, R is the fluorescent counting rate of the nanodiamond, and t is the exposure time of the single detection window. s The relative counting difference between the bright state of |m s = ±1> dark state is the contrast.

[0158] The particle size range of the nanodiamond satisfies the above range, the contrast of the fluorescent signal, the fluorescent counting rate of the nanodiamond, and the exposure time of the single detection window satisfy the above formula, at this time, the fluorescent signal amplitude of the nanodiamond particles in each detection window is greater than the amplitude of the noise, here mainly considering the photon shot noise, which can ensure that the signal-to-noise ratio of single measurement is greater than 1:1, thereby improving the signal-to-noise ratio of the fluorescent signal.

[0159] Optionally, on the basis of the above technical scheme, the excitation light module 200 includes an optical pump, and the optical pump is used to emit laser light.

[0160] The contrast of the fluorescent signal generated by the NV color center in the nanodiamond satisfies the following relationship:

[0161]

[0162] Wherein, Θ is a 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 NV color center Rabi oscillation under the driving of the microwave field, Γ p , Γ c two terms are related to the laser power, and the Ω R term is positively related to the microwave field intensity experienced by the NV color center in the nanodiamond.

[0163] The above technical scheme gives the calculation formula of the contrast of the fluorescent signal generated by the NV color center in the nanodiamond. In order to ensure that the fluorescent signal amplitude of a single nanodiamond particle in the detection area is always consistent, 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 scheme, the microwave field intensity experienced by the NV color center in the nanodiamond satisfies the following relationship:

[0164]

[0165] Wherein, B is the microwave field intensity sensed by NV centers in the nanodiamond, and B1 is the intensity of the microwave field provided by the microwave radiation module 300.

[0166] The flow fluorescence detection method is different from the traditional NV center-based light detection method in that the nanodiamond particles to be detected are not stably laid on the substrate but are placed in the sample flow channel 100 and are in a moving state. The nanodiamonds selected in the embodiment of the present application contain hundreds of NV centers with different crystal axis directions, so it can be considered that the angle between the NV center main axis direction and the microwave field is completely averaged, and the microwave field intensity sensed by all NV centers in a single nanodiamond particle is:

[0167]

[0168] As can be seen from the above formula, 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.

[0169] Optionally, on the basis of the above technical solution, for a single detection window, the excitation light is kept on, the microwave radiation module 300 provides a microwave radiation signal, and the light detection module 400 takes the entire detection window length as the exposure time to obtain the fluorescence signal intensity of the nanodiamond in a single sampling point.

[0170] Specifically, for a single detection window, the laser is kept on, the microwave radiation module 300 provides a microwave radiation signal, and the fluorescence detector takes the entire detection window length as the exposure time to obtain the fluorescence signal intensity of the nanodiamond particle in a single sampling point. This mode has a high excitation light duty cycle, and the light detection module 400 collects a higher number of effective fluorescence photons emitted by the nanodiamond.

[0171] Optionally, on the basis of the above technical solution, the delay time between the excitation light initialization pulse and the fluorescence signal collection pulse window is greater than the preset delay time.

[0172] Specifically, since the NV center fluorescence lifetime is long, the detector can be used to select an appropriate pulse delay 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, which can be 20 ns for example; and 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 objects has a short lifetime and has been greatly attenuated; while the NV center fluorescence in the nanodiamond particle still exists, thereby filtering the interference of the background fluorescence signal in time and improving the signal-to-noise ratio of the detection.

[0173] The technical solution provided in this invention, in order to improve the signal-to-noise ratio of the fluorescence signal and enhance the accuracy of analyte identification, 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 2820MHz and less than or equal to 2920MHz into the radiation coil, the NV color centers within the nanodiamond particles in the sample flow channel 100 change from |m s =0> State transition to |m s =±1> state, the fluorescence intensity of 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 center to |m s =0> state, fluorescence intensity recovers. When the radiation power of the microwave radiation signal provided by the microwave radiation module 300 changes, the NV color centers within the nanodiamond generate a correspondingly changing fluorescence signal. The fluorescence signal is modulated in the time domain by the microwave radiation module 300, then collected at a certain frequency by the photodetector module 400, and finally the specific frequency of the test signal is identified by an algorithm to eliminate interference from static noise signals.

[0174] This invention also provides a zero-field flow cytometry fluorescence detection system, which is applicable to any of the zero-field flow cytometry fluorescence detection methods described in this invention. This zero-field flow cytometry fluorescence detection system incorporates the beneficial effects of any of the zero-field flow cytometry fluorescence detection methods described in this invention, which will not be elaborated further here.

[0175] like Figure 17 As shown, Figure 17 This is a schematic flowchart of a flow cytometry fluorescence detection method based on microwave field switching states according to an embodiment of the present invention. The flow cytometry fluorescence detection method based on microwave field switching states includes the following steps:

[0176] S210. When the target material specifically bound to nanodiamond passes through the first half of the detection area of ​​the sample channel, the NV color centers within the nanodiamond generate a fluorescence signal, including a dark-state fluorescence signal. The dark-state fluorescence signal is the fluorescence signal generated by the NV color centers within the nanodiamond when some or all of the NV color centers resonate with the microwave field.

[0177] In this embodiment, when the microwave radiation module 300 provides a microwave field to the detection area S1 of the sample flow channel 100, the microwave field can provide a microwave radiation signal. The presence or absence of the microwave radiation signal results in different fluorescence signals generated by the NV color centers within the nanodiamond.

[0178] When the target substance specifically combined with the nanodiamond marker flows through the first half of the detection region S1 of the sample flow channel 100, the microwave radiation module 300 is in the open state, and the microwave radiation signal exists in the detection region S1. Under the irradiation of the excitation light, the NV center in the nanodiamond produces a dark-state fluorescence signal by the transition of the electron of the NV center. When the dark-state fluorescence signal is partially or entirely in resonance with the microwave field, the NV center in the nanodiamond produces a fluorescence signal.

[0179] S220, when the target substance specifically combined with the nanodiamond marker flows through the second half of the detection region of the sample flow channel, the NV center in the nanodiamond produces a fluorescence signal, and the fluorescence signal includes a bright-state fluorescence signal. When the bright-state fluorescence signal is not in resonance with the microwave field, the NV center in the nanodiamond produces a fluorescence signal.

[0180] The excitation light module is configured to emit excitation light, the excitation light is configured to excite the NV center in the detection region of the sample flow channel to make the electron of the NV center transition to produce fluorescence, and the microwave radiation module is configured to provide a microwave field for the detection region of the sample flow channel. The microwave field is in resonance with all or part of the NV center in the nanodiamond. The microwave radiation signal provided by the microwave field has a frequency equal to the energy level difference of the electron spin transition of the NV center, so that the microwave field is in resonance with the NV center. The microwave radiation module is in the open state or the closed state under the control of the control signal. When the microwave radiation module is in the open state, the NV center in the nanodiamond produces a dark-state fluorescence signal. When the microwave radiation module is in the closed state, the NV center in the nanodiamond produces a bright-state fluorescence signal.

[0181] When the target substance specifically combined with the nanodiamond marker flows through the second half of the detection region S1 of the sample flow channel 100, the microwave radiation module 300 is in the closed state, and the microwave radiation signal does not exist in the detection region S1. Under the irradiation of the excitation light, the NV center in the nanodiamond produces a bright-state fluorescence signal by the transition of the electron of the NV center. When the bright-state fluorescence signal is not in resonance with the microwave field, the NV center in the nanodiamond produces a fluorescence signal. The intensity of the dark-state fluorescence signal is less than the intensity of the bright-state fluorescence signal.

[0182] S230, the light detection module collects the fluorescence signal at a preset collection frequency.

[0183] As shown in Figure 11 The flow 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, so that the fluorescence signals at all angles around the detection region S1 can be collected by the light detection module 400.

[0184] For example, Figure 1 and Figure 11Two light detection modules 400 are shown, which are arranged around the detection area S1 of the sample flow channel 100 and symmetrically arranged with respect to the detection area S1.

[0185] When the number of light detection modules 400 is greater than two, the plurality of light detection modules 400 are arranged around the detection area S1 of the sample flow channel 100, and it is necessary to ensure that the fluorescence signals at each angle around the detection area S1 can be collected by the light detection module 400.

[0186] The above technical solution can improve the collection amount of fluorescence signals, thereby providing a lower detection limit, and can overcome the problem of target shielding NV color center fluorescence.

[0187] Since the fluorescence intensity of a single nanodiamond particle is weak in single molecule detection application scenarios, the light detection module 400 is preferably an avalanche photodiode photodetector (APD), followed by a scientific camera, and then a photomultiplier tube sensor (PMT).

[0188] In addition, the interference of excitation light and stray fluorescence signals can be filtered out by notch filters, long-wave pass filters, and short-wave pass filters.

[0189] S240, the data processing module determines whether the target contains the biological molecule to be detected according to the size relationship between the intensity of the dark-state fluorescence signal and the intensity of the bright-state fluorescence signal.

[0190] The microwave radiation module 300 is in an open state under the control of the microwave field opening signal, and the electrons of the NV color center jump 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 part or all of the NV color centers resonate with the microwave field. The microwave radiation module 300 is in a closed state under the control of the microwave field closing signal, and the electrons of the NV color center jump 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. 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 contains the biological molecule to be detected.

[0191] The embodiment of the present application provides a flow fluorescence detection method based on microwave field switching state, which is different from the traditional NV color center-based light detection method, wherein the nanodiamond particles to be detected are not stably laid on the substrate, but are placed in the sample flow channel 100 and are in a moving state, the target object specifically combined with the nanodiamond label flows through the detection area S1 of the sample flow channel 100, the nanodiamond includes the NV color center, and the NV color center in the nanodiamond is excited by the excitation light emitted by the excitation light module 200 to make the electrons of the NV color center jump to generate fluorescence. When the target object specifically combined with the nanodiamond label flows through the first half of the detection area S1 of the sample flow channel 100, the microwave radiation module 300 is in an open state, the microwave radiation signal exists in the detection area S1, the electrons of the NV color center in the nanodiamond jump to generate a dark-state fluorescence signal under the irradiation of the excitation light. When the target object specifically combined with the nanodiamond label flows through the second half of the detection area S1 of the sample flow channel 100, the microwave radiation module 300 is in a closed state, the microwave radiation signal does not exist in the detection area S1, and the electrons of the NV color center in the nanodiamond jump to generate a bright-state fluorescence signal under the irradiation of the excitation light. The light detection module 400 collects the fluorescence signal generated by the NV color center in the nanodiamond when the target object specifically combined with the nanodiamond label 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 object contains the biological molecule to be detected, the data processing module 700 judges whether the target object contains the biological molecule to be detected according to the size relationship between the intensity of the dark-state fluorescence signal and the intensity of the bright-state fluorescence signal. Since the NV color center system has stability, room temperature atmospheric environment compatibility and biological compatibility, and the nanodiamond is a new type of biological fluorescence label, the signal modulation and demodulation technology can be used to eliminate low-frequency noise interference, so that the detection sensitivity is much higher than that of other fluorescence label detection methods. In conclusion, the technical scheme provided by the embodiment of the present application improves the detection sensitivity of the fluorescence detection method, and can realize the fluorescence signal recognition of a single molecule.

[0192] Optionally, on the basis of the above technical scheme, as shown in Figure 18 S210, when the target object specifically combined with the nanodiamond label flows through the first half of the detection area of the sample flow channel, the NV color center in the nanodiamond generates a fluorescence signal, and the fluorescence signal includes a dark-state fluorescence signal, which includes:

[0193] S2101, the control module sends a control signal, and the control signal includes a microwave field opening signal and a microwave field closing signal.

[0194] S2102. The microwave radiation module is in the open 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 center in the nanodiamond flows through the first half of the detection area of ​​the sample flow channel, it emits a dark state fluorescence signal under the excitation light.

[0195] Specifically, the microwave radiation module 300 is in the open state under the control of the microwave field opening signal. When the NV color center in the nanodiamond flows 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 irradiation of the excitation light, the electrons of the NV color center in the nanodiamond undergo transition and 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.

[0196] The above technical solution provides a scheme for the microwave radiation module 300 to emit fluorescence signals from the NV color centers in nanodiamonds under excitation light when the module is in the open state under the control of a control signal.

[0197] Optionally, based on the above technical solution, when the target material labeled with nanodiamond specifically binds to S220 and passes through the latter half of the detection area of ​​the sample channel, the NV color center within the nanodiamond generates a fluorescence signal. The fluorescence signal includes bright-state fluorescence signals, including:

[0198] According to the microwave field shutdown signal, the microwave radiation module is in the off state and stops providing microwave field to the detection area of ​​the sample flow channel. When the NV color center in the nanodiamond flows through the second half of the detection area of ​​the sample flow channel, it emits a bright fluorescence signal under the excitation light.

[0199] The microwave radiation module 300 is in the off state under the control of the microwave field shutdown signal. When the NV color centers within the nanodiamond flow through the latter 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 color centers within the nanodiamond undergo transitions, generating a bright-state fluorescence signal. The bright-state fluorescence signal is the fluorescence signal generated by the NV color centers within the nanodiamond when there is no resonance between the NV color centers and the microwave field. The intensity of the dark-state fluorescence signal is less than the intensity of the bright-state fluorescence signal.

[0200] The above technical solution provides a scheme for the microwave radiation module 300 to emit fluorescence signals from the NV color centers in nanodiamonds under excitation light when the module is in the off state under the control of the control signal.

[0201] Optionally, based on the above technical solution, the S240 data processing module determines whether the target analyte contains the biomolecule to be detected based on the relationship between the intensity of the dark-state fluorescence signal and the intensity of the bright-state fluorescence signal, including:

[0202] 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 value, the target object contains the to-be-detected biomolecule, and the second preset threshold value is at least 1 times the noise level when the bright-state fluorescence signal is generated.

[0203] The technical solution is directed to a judgment method for determining whether a target object contains a to-be-detected biomolecule by using a microwave radiation module 300 in an open state or a closed state under the control of a control signal and a data processing module 700 to determine whether the target object contains the to-be-detected biomolecule according to a fluorescence signal.

[0204] Optionally, based on the technical solution, after the S220 specific binding nanodiamond labeled target flows through the second half of the sample flow channel detection area, the nanodiamond NV center generates a fluorescence signal, and then the following steps are further included:

[0205] After each detection window of the light detection module, a detection window without a microwave radiation signal is added as a reference signal.

[0206] In each detection window, the detector simultaneously collects 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; and after a single detection window, a detection window without a modulated microwave control can be added as a reference signal for calibrating the time jitter and spatial non-uniformity of the excitation light.

[0207] Optionally, based on the technical solution, a plurality of scientific cameras are used as the light detection module 400, and an sCMOS high-speed scientific camera or an intensified electron-coupled device (ICCD) can be selected; in 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 trajectory of the nanodiamond fluorescence particles in the image is found through algorithm recognition, and a plurality of points are selected as the fluorescence count of the nanodiamond particles in the window, and the background fluorescence signals of the remaining part are discarded to improve the signal-to-noise ratio of the to-be-detected fluorescence signal.

[0208] Optionally, based on the technical solution, the magnetic field strength of the detection area satisfies a zero-field condition, and the microwave radiation signal provided by the microwave field has a fixed frequency.

[0209] Optionally, based on the technical solution, 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 monitoring module 800 monitors that the magnetic field intensity 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.

[0210] In an external magnetic field, NV color centers cause the electrons in the nanodiamond NV color centers to move in opposite directions between the |m s =0> state and the |m s =±1> state, and the frequency difference is proportional to the projection of the external magnetic field on 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; and the spatial direction of different nanodiamond particles flowing through the sample flow channel 100 is random. Therefore, the projection components of the external magnetic field on the different axial NV color centers in different nanodiamond particles are different, resulting in different transition frequencies between the |m s =0> state and the |m s =±1> state. Under the control of a single frequency microwave, the quantum state of the NV color center in part of the nanodiamond particles does not change, and the bright and dark changes of the overall fluorescence are not obvious, thereby affecting the signal-to-noise ratio of the measurement scheme and further affecting the accuracy and reliability of the detection of the measured object.

[0211] Therefore, when the magnetic field monitoring module 800 monitors that the magnetic field intensity near the detection area S1 of the sample flow channel 100 meets the zero field condition, the frequency at which part or all of the NV color centers resonate with the microwave field is the intrinsic frequency, and the microwave field is used to provide a microwave radiation signal with the fixed frequency, which can improve the signal-to-noise ratio of the fluorescence signal and further improve the detection sensitivity of the flow cytometry detection system.

[0212] Optionally, based on the above technical solutions, as shown, Figure 10 When the magnetic field monitoring module 800 monitors that the magnetic field intensity near the detection area S1 of the sample flow channel 100 meets 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 spectral line broadening of the optical detection magnetic resonance spectrum of the NV color center.

[0213] The energy level splitting of the NV color center in the nanodiamond refers to the amount of movement of the optical detection magnetic resonance spectrum resonance peak position caused by the Zeeman shift of the NV energy level due to the external magnetic field, i.e., one-half of the frequency difference between the two microwave resonance frequencies corresponding to the energy level splitting of the same NV color center; and the spectral line broadening of the optical detection magnetic resonance spectrum of the NV color center refers to the half-peak width of the optical detection magnetic resonance spectrum resonance peak of the NV color center.

[0214] The magnetic field monitoring module 800 monitors the magnetic field intensity near the detection area S1 of the sample flow channel 100 to satisfy the zero field condition. When 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, the NV centers in the nanodiamonds can be controlled by the microwave with a frequency 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 working frequency of the microwave radiation signal provided by the microwave radiation module 300.

[0215] It should be noted that in the embodiment of the present application, the spectral peak shift caused by the magnetic field does not exceed the intrinsic broadening of the CW spectrum, and the intrinsic broadening of the CW spectrum is determined by the dephasing time. However, the noise environment of the NV center in nanodiamonds with different diameters and different production processes is different, and the dephasing time is different. Therefore, the zero field limit needs to be determined according to the selected particles. For example, for a 40 nm diameter nanodiamond with an NV concentration of 1.5 ppm produced by HPHT method, the typical value of the CW spectrum intrinsic broadening is 20 MHz, and the requirement for the external magnetic field is below 3.56 Gs. The resonance (CW) spectrum is the spectral line in Figure 10

[0216] Alternatively, on the basis of the above technical solutions, the particle size of the nanodiamond is greater than or equal to 40 nm and less than or equal to 1 μm; preferably, the particle size of the nanodiamond is greater than or equal to 100 nm and less than or equal to 300 nm.

[0217] When the target flow passes through the detection area, the number of detection windows collected by the light detection module within the period of a single fluorescence signal is at least one. The fluorescence counting rate of the nanodiamond, the contrast of the fluorescence signal, and the single detection window exposure time satisfy the following relationship:

[0218]

[0219] 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 |m s = 0> and the dark state |m s = ±1> is the contrast.

[0220] ​The particle size range of the nanodiamond meets 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 meet the above formula, at this time, in each detection window, the fluorescence modulation signal amplitude of the nanodiamond particles is greater than the amplitude of the noise, mainly considering the photon shot noise here, 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.

[0221] Optionally, on the basis of the above technical solution, the excitation light module 200 comprises an optical pump, and the optical pump is used for emitting laser light.

[0222] The contrast of the fluorescence signal generated by the NV color center in the nanodiamond meets the following relationship:

[0223]

[0224] Wherein, Θ is a normalized constant, Γ p is a laser polarization rate, Γ c is a coherent relaxation rate caused by the optical pump, Ω R is the frequency of the NV color center Rabi oscillation driven by the microwave field, Γ p , Γ c Two terms are related to the laser power, Ω R The term is positively related to the microwave field intensity experienced by the NV color center in the nanodiamond.

[0225] The above technical solution gives the calculation formula of the contrast of the fluorescence signal generated by the NV color center in the nanodiamond, in order to ensure that the fluorescence signal amplitude of a single nanodiamond particle in the detection area is always consistent, the laser power density and the microwave field intensity distribution in the detection area should be as uniform as possible.

[0226] Optionally, on the basis of the above technical solution, the microwave field intensity experienced by the NV color center in the nanodiamond meets the following relationship:

[0227]

[0228] Wherein, B is the microwave field intensity experienced by the NV color center in the nanodiamond, and B1 is the intensity of the microwave field provided by the microwave radiation module.

[0229] The flow detection method is different from the traditional NV color center-based optical detection method, the nanodiamond particles to be measured are not stably laid on the substrate, but are placed in the flow channel and are in a moving state. The nanodiamonds we selected contain hundreds of NV color centers with different crystal axis directions, so it can be considered that the angle between the NV color center principal axis direction and the microwave field is completely averaged, and the microwave field intensity experienced by all NV color centers in a single nanodiamond particle is:

[0230]

[0231] As can be seen from the above formula, the microwave field intensity sensed by all NV centers in a single nanodiamond particle is proportional to the microwave field intensity.

[0232] Optionally, for a single detection window, the excitation light is kept on, the microwave radiation module provides a microwave radiation signal, and the light detection module takes the entire detection window length as the exposure time to obtain the fluorescence signal intensity of the nanodiamond in a single sampling point.

[0233] Specifically, for a single detection window, the laser is kept on, the microwave radiation module provides a microwave radiation signal, and the fluorescence detector takes the entire detection window length as the exposure time to obtain the fluorescence signal intensity of the nanodiamond particle in a single sampling point. This way, the duty cycle of the excitation light is high, and the number of effective fluorescence photons emitted by the nanodiamond collected by the light detection module is high.

[0234] Optionally, on the basis of the above technical solution, the delay time between the excitation light initialization pulse and the fluorescence signal collection pulse window is greater than the preset delay time.

[0235] Specifically, since the NV center fluorescence lifetime is long, an appropriate pulse delay fluorescence window can be selected by the detector, 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, which can be 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 objects has a short lifetime and has been greatly attenuated; while the NV center fluorescence in the nanodiamond particle still exists, thereby filtering the interference of the background fluorescence signal in time and improving the signal-to-noise ratio of the detection.

[0236] The technical solution provided by the embodiment of the application improves the signal-to-noise ratio of the fluorescence signal and improves the recognition accuracy of the to-be-detected object. The system introduces a microwave field to modulate the fluorescence signal of the nanodiamond particle: when the microwave radiation module 300 opens the microwave with a frequency greater than or equal to 2820 MHz and less than or equal to 2920 MHz into the radiation coil, the NV center in the nanodiamond particle of the sample flow channel 100 transitions from the |m s =0> state to the |m s =±1> state, and the fluorescence intensity of the nanodiamond particle decreases; when the microwave in the radiation coil is turned off, the excitation light initializes the NV center in the detection area S1 to the |m sThe fluorescence intensity is recovered when the NV center in the nanodiamond changes from the |m s =0> state to the |m s =±1> state; in the second half of the detection region S1, the microwave field is turned off, and the NV center is initialized to the |m s =0> state by the laser; by comparing the fluorescence signal intensity of the nanodiamond in the two detection regions S1, it can be determined whether the target object contains nanodiamond markers and whether the target object contains the biological molecules to be detected.

[0237] The embodiment of the present application also provides a flow fluorescence detection system based on a microwave field switching state, which is suitable for the flow fluorescence detection method based on the microwave field switching state of any of the embodiments of the present application. The flow fluorescence detection system based on the microwave field switching state has the beneficial effects of the flow fluorescence detection method based on the microwave field switching state of any of the embodiments of the present application, which will not be repeated here.

[0238] It should be understood that the various forms of the flow shown above can be reordered, added or deleted steps. For example, the steps described in the present application can be executed in parallel, sequentially or in different orders, as long as the desired results of the technical solutions of the present application can be achieved, which are not limited herein.

[0239] The above specific embodiments do not constitute a limitation on the scope of protection of the present application. 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 replacements and improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A flow cytometry fluorescence detection system, characterized in that, include: A sample flow channel is provided for delivering a target object specifically bound to a nanodiamond label, the nanodiamond including NV color centers, and the sample flow channel is provided with a detection area. An excitation light module is used to emit excitation light, which is used to excite the NV color center in the detection area of ​​the sample flow channel, causing its electrons to transition and generate fluorescence. A microwave radiation module is provided to provide a microwave field to the detection area of ​​the sample flow channel. The microwave field can resonate with all or part of the NV 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 centers, which enables the microwave field to resonate with the NV centers. The microwave radiation module includes at least two microwave radiation structures, which are distributed along the transport direction of the target object and are arranged around the detection area of ​​the sample flow channel. The optical detection module is used to collect the target material specifically bound to nanodiamond labels through the detection area of ​​the sample channel at a preset collection frequency. By controlling at least one of the intensity, frequency, and on / off state of the microwave field, the NV color centers within the nanodiamond emit fluorescence signals with periodically changing intensity. The fluorescence signal is correlated with whether the target material contains the biomolecule to be detected.

2. The flow cytometry fluorescence detection system according to claim 1, characterized in that, It also includes a control module, which is connected to the microwave radiation module and is used to transmit control signals; The control signals include at least one of the following: microwave field on signal and microwave field off signal, microwave field power periodic change signal, and microwave field frequency periodic change signal.

3. The flow cytometry fluorescence detection system according to claim 2, characterized in that, The microwave radiation module is used to provide a modulated microwave field to the detection area of ​​the sample flow channel according to the microwave field opening signal. The NV color center in the nanodiamond emits a dark state fluorescence signal under the excitation light irradiation. 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 is used to stop providing a modulated microwave field to the detection area of ​​the sample flow channel according to the microwave field shutdown signal. The NV color center in the nanodiamond emits a bright fluorescence signal under excitation light irradiation. The bright fluorescence signal is the fluorescence signal generated by the NV color center in the nanodiamond when there is no resonance between the NV color center and the microwave field.

4. The flow cytometry fluorescence detection system according to claim 2, characterized in that, The microwave radiation module is used to emit a microwave modulation field with a fixed frequency and periodically varying power according to the periodically varying power signal of the microwave field, so that the NV color centers in the nanodiamond emit a fluorescence signal with periodically varying intensity under the excitation light.

5. The flow cytometry fluorescence detection system according to claim 2, characterized in that, The microwave radiation module is used to emit a microwave modulation field with fixed power and periodically changing frequency according to the periodically changing frequency signal of the microwave field, so that the NV color centers in the nanodiamond emit a fluorescence signal with periodically changing intensity under the excitation light.

6. The flow cytometry fluorescence detection system according to claim 1, characterized in that, It also includes a data processing module, which is connected to the photodetector module. The data processing module determines whether the target contains the biomolecule to be detected based on the fluorescence signal.

7. The flow cytometry fluorescence detection system according to claim 1, characterized in that, The microwave radiation module includes two microwave radiation structures. One microwave radiation structure is located at the beginning of the detection area, and the other microwave radiation structure is located at the end of the detection area. The direction from the beginning of the detection area to the end of the detection area is parallel to the transmission direction of the target object.

8. The flow cytometry fluorescence detection system according to claim 1 or 7, characterized in that, The microwave radiation structure includes a microstrip line and a microwave resonant cavity structure. The microwave resonant cavity structure is arranged around the detection area of ​​the sample flow channel, and the microstrip line is located on one side of the microwave resonant cavity structure.

9. The flow cytometry fluorescence detection system according to claim 8, characterized in that, The microwave resonant cavity structure includes two coaxial radiating coils.

10. The flow cytometry fluorescence detection system according to claim 1 or 7, characterized in that, The microwave radiating structure includes an Ω-shaped coil radiating antenna.

11. The flow cytometry fluorescence detection system according to claim 1, characterized in that, It also includes a magnetic field monitoring module, which is used to monitor the magnetic field strength near the detection area of ​​the sample flow channel; When the magnetic field monitoring module monitors the magnetic field strength near the detection area of ​​the sample flow channel and the condition of zero field is met, the microwave field is used to provide a microwave radiation signal with a fixed frequency.

12. The flow cytometry fluorescence detection system according to claim 11, characterized in that, When the magnetic field monitoring module monitors the magnetic field strength near the detection area of ​​the sample flow channel and the condition of zero field is met, the energy level splitting of the NV color center in the nanodiamond caused by the magnetic field is less than half of the broadening of its photodetector magnetic resonance spectrum.

13. The flow cytometry fluorescence detection system according to claim 11, characterized in that, When the magnetic field monitoring module monitors the magnetic field strength near the detection area of ​​the sample flow channel and it meets the zero-field condition, the fixed frequency of the microwave radiation signal provided by the microwave field is greater than or equal to D-50MHz and less than or equal to D+50MHz, where D is the zero-field splitting parameter of the NV color center.

14. The flow cytometry fluorescence detection system according to claim 1, characterized in that, The flow cytometry fluorescence detection system includes at least two of the aforementioned photodetector modules.

Images