Microwave field switching state-based flow fluorescence detection method and detection system

Abstract

The application discloses a flow fluorescence detection method and a detection system based on a microwave field switching state, and the method comprises the following steps: when specific binding nanodiamond labeled target objects flow through a first half of a detection area of a sample flow channel, NV color centers in the nanodiamonds generate dark state fluorescence signals; when the specific binding nanodiamond labeled target objects flow through a second half of the detection area of the sample flow channel, the NV color centers in the nanodiamonds generate bright state fluorescence signals; wherein, an excitation light module is used for exciting 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; a microwave radiation module is used for providing a microwave field for the detection area of the sample flow channel; a light detection module collects the fluorescence signals at a preset collection frequency; and a data processing module judges whether the target objects contain biological molecules to be detected according to the size relationship between the intensity of the dark state fluorescence signals and the intensity of the bright state fluorescence signals. The technical scheme provided in the application embodiment improves the detection sensitivity of the fluorescence detection method.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection, in particular to a flow fluorescence detection method based on microwave field switching state and a detection system. BACKGROUND

[0002] Flow fluorescence technology can realize high-throughput, high-speed and multiple marker joint detection. However, due to the low intensity of existing fluorescent dyes, it is difficult to realize single molecule fluorescence signal recognition, 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 method with high detection sensitivity. SUMMARY

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

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

[0006] When the target material 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 modulated fluorescence signal, and the modulated fluorescence signal includes 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 center resonates with the microwave field;

[0007] When the target material specifically combined with the nanodiamond label flows through the second half of the detection area of the sample flow channel, the NV color center in the nanodiamond generates a modulated fluorescence signal, and the modulated fluorescence signal includes 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;

[0008] Wherein, the excitation light module is used to emit excitation light, the excitation light is used to excite and make the electrons of the NV color center in the detection area of the sample flow channel jump, the microwave radiation module is used to provide a microwave field for the detection area of the sample flow channel, the microwave field resonates with all or part of the NV color center in the nanodiamond, and the microwave radiation module is in an on state or an off state under the control of a control signal; when the microwave radiation module is in the on state, the NV color center in the nanodiamond generates the dark state fluorescence signal, and when the microwave radiation module is in the off state, the NV color center in the nanodiamond generates the bright state fluorescence signal;

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

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

[0011] The microwave field resonates with all or part of the NV centers in the nanodiamond, specifically, the microwave frequency of the microwave field is equal to the energy level difference of the electron spin transition of the NV center, so that the microwave field resonates with the NV center.

[0012] Optionally, when the nanodiamond-labeled target object flows through the first half of the detection area of the sample flow channel, the NV centers in the nanodiamond generate a modulated fluorescence signal, and the modulated fluorescence signal includes a dark-state fluorescence signal, which includes:

[0013] The control module sends a control signal, which includes a microwave field opening signal and a microwave field closing signal.

[0014] The microwave radiation module is in an 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, and the NV centers in the nanodiamond emit a dark-state fluorescence signal under excitation light irradiation when flowing through the first half of the detection area of the sample flow channel.

[0015] Optionally, when the nanodiamond-labeled target object flows through the second half of the detection area of the sample flow channel, the NV centers in the nanodiamond generate a modulated fluorescence signal, and the modulated fluorescence signal includes a bright-state fluorescence signal, which includes:

[0016] The microwave radiation module is in a closed state according to the microwave field closing signal, the microwave radiation module stops providing a microwave field for the detection area of the sample flow channel, and the NV centers in the nanodiamond emit a bright-state fluorescence signal under excitation light irradiation when flowing through the second half of the detection area of the sample flow channel.

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

[0018] 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, indicating that the target object contains the biological molecule to be detected.

[0019] Optionally, when the nanodiamond-labeled target object flows through the second half of the detection area of the sample flow channel, the NV centers in the nanodiamond generate a modulated fluorescence signal, which further includes:

[0020] A detection window without microwave radiation signal is added as a reference signal after each detection window of the light detection module.

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

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

[0023] Optionally, 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, and D is the NV center zero field splitting parameter.

[0024] Optionally, the particle size of the nanodiamond ranges from greater than or equal to 40nm to less than or equal to 1μm.

[0025] When the target flow passes through the detection area, the number of detection windows collected by the light detection module within a single modulation fluorescence signal cycle is at least one, and the fluorescence counting rate of the nanodiamond, the contrast of the modulation fluorescence signal and the single detection window exposure time satisfy the following relationship:

[0026]

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

[0028] Optionally, the excitation light module includes an optical pump for emitting laser light.

[0029] The contrast of the modulation fluorescence signal generated by the NV center in the nanodiamond satisfies the following relationship:

[0030]

[0031] 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 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 strength experienced by the NV center in the nanodiamond.

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

[0033]

[0034] wherein B is the microwave field intensity sensed by the NV center in the nanodiamond, and B1 is the microwave field intensity of the microwave field.

[0035] 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.

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

[0037] According to another aspect of the present application, there is provided a microwave field switch state-based flow fluorescence detection system, which is applicable to any of the microwave field switch state-based flow fluorescence detection methods according to the first aspect of the present application.

[0038] 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 a substrate, but are placed in a sample flow channel and are in a moving state, specific binding nanodiamond labeled target objects flow through a detection area of the sample flow channel, the nanodiamonds include NV color centers, the NV color centers in the nanodiamonds emit fluorescence signals under irradiation of excitation light emitted by an excitation light module, the excitation light is used for exciting and causing the electrons of the NV color centers in the detection area of the sample flow channel to jump, and the NV color centers in the nanodiamonds can emit fluorescence signals. When the specific binding nanodiamond labeled target objects flow through the first half of the detection area of the sample flow channel, a microwave radiation module is in an open state, the detection area has a microwave radiation signal, the electrons of the NV color centers in the nanodiamonds jump to generate dark-state fluorescence signals under irradiation of the excitation light. When the specific binding nanodiamond labeled target objects flow through the second half of the detection area of the sample flow channel, the microwave radiation module is in a closed state, the detection area does not have a microwave radiation signal, the electrons of the NV color centers in the nanodiamonds jump to generate bright-state fluorescence signals under irradiation of the excitation light. A light detection module collects the modulated fluorescence signals generated by the NV color centers in the nanodiamonds at a preset collection frequency when the specific binding nanodiamond labeled target objects flow through the detection area of the sample flow channel, and the modulated fluorescence signals are related to whether the target objects contain the biological molecules to be detected. Therefore, a data processing module determines whether the target objects contain the biological molecules to be detected according to the size relationship between the intensity of the dark-state fluorescence signals and the intensity of the bright-state fluorescence signals. 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 marker, low-frequency noise interference can be excluded through signal modulation and demodulation technology, and therefore the detection sensitivity is much higher than that of other fluorescence marker 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 single-molecule fluorescence signal recognition can be realized.

[0039] 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 present application, nor is it used to limit the scope of the present application. Other features of the present application will become apparent from the following description. BRIEF DESCRIPTION OF DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present 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 present application, and other drawings can also be obtained by those skilled in the art without creative labor.

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

[0042] Figure 2 It is a structural block diagram of a flow fluorescence detection system according to an embodiment of the present application;

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

[0044] Figure 4 It is a light path system schematic diagram of a flow fluorescence detection system according to an embodiment of the present application;

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

[0046] Figure 6 It is Figure 5 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;

[0047] Figure 7 It is Figure 5 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;

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

[0049] Figure 9 It is Figure 8 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;

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

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

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

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

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

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

[0056] Figure 16 is a schematic diagram of the acquisition of a modulated fluorescent signal according to an embodiment of the present application;

[0057] Figure 17 is a flowchart of a flow-based fluorescent detection method based on microwave field switching states according to an embodiment of the present application;

[0058] Figure 18 is Figure 17 a flowchart included in S210. DETAILED DESCRIPTION

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

[0060] It should be noted that the terms "first", "second", and the like in the specification and claims of the present application and the above-described drawings are used to distinguish similar objects, and do not necessarily have to be used to describe 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 that includes a series of steps or units does not have to be limited to only those steps or units clearly listed, but can include other steps or units that are not clearly listed or inherent to the process, method, product, or device.

[0061] In order to improve the detection sensitivity of the fluorescent detection method, the present application provides a flow-based fluorescent detection method based on microwave field switching states. Before introducing the flow-based fluorescent detection method based on microwave field switching states provided by the embodiments of the present application, the flow-based fluorescent detection system to which the flow-based fluorescent detection method based on microwave field switching states provided by the embodiments of the present application is introduced.

[0062] As shown in Figure 1 , Figure 1 is a structural schematic diagram of a flow-based fluorescent detection system according to an embodiment of the present application, Figure 2is a structural block diagram of a flow fluorescence detection system according to an embodiment of the present application, the flow fluorescence detection system comprising:

[0063] A sample flow channel 100 is configured to transport target objects specifically combined with nanodiamond markers, the nanodiamonds including NV color centers.

[0064] An excitation light module 200 is configured to emit excitation light, which 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.

[0065] A microwave radiation module 300 is configured to provide a microwave field for the detection area S1 of the sample flow channel 100, the microwave field can resonate with all or part of the NV color centers in the nanodiamonds, 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 color centers, so that the microwave field resonates with the NV color centers, and the microwave radiation module 300 includes at least two microwave radiation structures 301, the microwave radiation structures 301 are distributed along the transport direction of the target objects, and the microwave radiation structures 301 are arranged around the detection area S1 of the sample flow channel 100.

[0066] A light detection module 400 is configured to collect 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 S1 of the sample flow channel 100, the light detection module 400 is configured to control at least one of the intensity, frequency and on-off of the microwave field to realize that the fluorescence signals emitted by the NV color centers in the nanodiamonds present periodic changes in intensity, and the fluorescence signals are associated with whether the target objects contain biological molecules to be detected.

[0067] As known, quantum sensing systems include Rydberg atoms, atomic magnetometers, superconducting quantum interference devices, diamond NV color centers, etc., among which the NV color center system is one of the most promising solid-state quantum sensors in the fields of life science and medical detection due to its stability, 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, so that the detection sensitivity can be much higher than that of other fluorescent marker detection methods.

[0068] The NV centers in the nanodiamonds are excited by the excitation light emitted by the excitation light module 200, which is used to excite the NV centers in the detection area S1 of the sample flow channel 100, so that the electrons of the NV centers are excited to produce fluorescence. For example, the NV centers emit fluorescence signals in the wavelength range of 630-800 nm when excited by the 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.

[0069] The microwave radiation module 300 includes at least two microwave radiation structures 301, which are distributed along the conveying direction of the target objects and are arranged around the detection area S1 of the sample flow channel 100. As shown in the figure, the conveying direction of the target objects is from top to bottom, and the at least two microwave radiation structures are distributed along the conveying direction of the target objects, which 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 objects flow through the detection area S1, and then when the microwave radiation signal provided by the microwave field changes, the NV centers in the nanodiamonds produce fluorescence signals that change accordingly. Figure 1

[0070] 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, and the fluorescence signals produced by the NV centers in the nanodiamonds are different due to the presence and absence of the microwave radiation signal; or the frequency or power of the microwave radiation signal changes, and the NV centers in the nanodiamonds produce fluorescence signals that change accordingly, which are as follows:

[0071] When the microwave radiation module 300 is in the open state, the detection area S1 has a microwave radiation signal, and the electrons of the NV centers in the nanodiamonds are excited to produce a dark-state fluorescence signal under the irradiation of the excitation light. The dark-state fluorescence signal is the fluorescence signal produced by the NV centers in the nanodiamonds when some or all of the NV centers resonate with the microwave field.

[0072] When the microwave radiation module 300 is in the closed state, the detection area S1 does not have a microwave radiation signal, and the electrons of the NV centers in the nanodiamonds are excited to produce a bright-state fluorescence signal under the irradiation of the excitation light. The bright-state fluorescence signal is the fluorescence signal produced 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] ​The microwave field provides microwave radiation signal of frequency or radiation power change, and the NV color center in the nanodiamond produces the fluorescent signal which changes accordingly. In this embodiment, since the microwave field provides microwave radiation signal of frequency or power change periodically, the NV color center in the nanodiamond under the excitation light irradiation, the electron of the NV color center jumps, the number of the NV color center and the microwave field resonance changes periodically, and / or the degree of the NV color center resonance changes periodically, and the NV color center produces the fluorescent signal with the intensity changing periodically.

[0074] Specifically, when the microwave field modulation mode is amplitude modulation, the microwave field provides microwave radiation signal of power change, for example, the simplest square wave modulation, the sinusoidal wave modulation and the triangular wave modulation realized by changing the microwave power. When the microwave field modulation mode is frequency modulation, the microwave field provides microwave radiation signal of frequency change, for example, the microwave resonance-non resonance mode. When the microwave field provides microwave radiation signal of frequency equal to the energy level difference of the electron spin transition of the NV color center, the microwave field can resonate with the NV color center.

[0075] 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 the irradiation 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, the frequency, and the on-off of the microwave field. A light detection module 400 collects the fluorescence signals generated by the NV color centers in the nanodiamonds when the target objects specifically combined with the nanodiamond labels flow through the detection area S1 of the sample flow channel 100 at a preset collection frequency. Since the fluorescence signals are related to whether the target 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 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 fluorescence detection system and can realize single-molecule fluorescence signal recognition.

[0076] Optionally, based on the technical scheme above, as shown in the following Figure 2 The control module 600 is further connected with the microwave irradiation module 300, and the control module 600 is configured to emit a control signal.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] Specifically, 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.

[0092] Optionally, based on the above technical solutions, such as Figure 5 As shown, the microwave resonant cavity structure L2 includes two coaxial radiating coils.

[0093] 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 the electrical signal radiates the entire detection area S1.

[0094] 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.

[0095] Specifically, as shown in Figure 9 The detection area S1 of the sample flow channel 100 has uniform gray scale,

[0096] The radiation antenna of the "Ω" shaped coil provides a microwave field with high uniformity for the detection area S1 of the sample flow channel 100 under the action of the electrical signal. The microwave field can provide a microwave radiation signal, and the microwave radiation signal radiates 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.

[0097] Optionally, based on the technical scheme above, as shown in Figure 1 and Figure 2 It further includes a magnetic field monitoring module 800, which is used to monitor the magnetic field strength near the detection area S1 of the sample flow channel 100. When the magnetic field strength near the detection area S1 of the sample flow channel 100 monitored by the magnetic field monitoring module 800 satisfies the zero field condition, the microwave field is used to provide a microwave radiation signal with a fixed frequency.

[0098] 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. Moreover, the spatial directions of different nanodiamond particles flowing through the sample flow channel 100 are 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 |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 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.

[0099] 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.

[0100] 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 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 center.

[0101] 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.

[0102] 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.

[0103] 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 exhibit other values affected by different experimental environment temperatures, and the fixed frequency of the microwave radiation signal can generally be set to be between D-50 MHz and D+50 MHz.

[0104] 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.

[0105] 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.

[0106] 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, and can overcome the problem of shielding the NV center fluorescence by the target object.

[0107] 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).

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

[0109] As shown in Figure 12 , the light detection module 400 is preferably an avalanche photodiode photodetector (APD), followed by a research camera, and then a photomultiplier tube sensor (PMT). Figure 12This is a schematic flowchart of a flow cytometry fluorescence detection method based on microwave field switching states according to the present invention. The flow cytometry fluorescence detection method based on microwave field switching states includes the following steps:

[0110] S110. When the target material specifically bound to nanodiamond passes through the detection area of ​​the sample channel, by controlling at least one of the intensity, frequency, and on / off state of the microwave field, the NV color center within the nanodiamond emits a fluorescence signal with periodically changing intensity.

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

[0112] In this embodiment, when the microwave radiation module 300 provides a microwave field 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; or the frequency or power of the microwave radiation signal changes, causing the NV color centers within the nanodiamond to generate correspondingly changing fluorescence signals.

[0113] When the microwave radiation module 300 is in the open state, there is a microwave radiation signal in the detection area S1. When the NV color center in the nanodiamond is irradiated by the excitation light, the electrons of the NV color center 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.

[0114] When the microwave radiation module 300 is off, there is no microwave radiation signal in the detection area S1. Under excitation light, the electrons of the NV color centers within the nanodiamond undergo transitions, generating a bright-state fluorescence signal. This 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 that of the bright-state fluorescence signal.

[0115] As the frequency or power of the microwave radiation signal provided by the microwave field changes, the NV centers within the nanodiamond generate correspondingly varying fluorescence signals. In this embodiment, because the frequency or power of the microwave radiation signal provided by the microwave field varies periodically, the electrons in the NV centers within the nanodiamond undergo transitions under excitation light irradiation. The number of NV centers resonating with the microwave field varies periodically, and / or the degree of NV center resonance varies periodically, resulting in the NV centers generating fluorescence signals with periodically varying intensity.

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

[0117] Optionally, based on the above technical solutions, such as Figure 1 and Figure 2 As shown, 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 strength near the detection area S1 of the sample flow channel 100 monitored by the magnetic field monitoring module 800 meets the zero field condition, the microwave field is used to provide a microwave radiation signal with a fixed frequency.

[0118] In an applied magnetic field, the electrons of the NV centers within the nanodiamond are affected by the Zeeman effect, causing them to move from |m s =0> state and |m s The energy levels between the ±1> states shift in opposite directions, and the frequency difference is proportional to the magnitude of the projection of the applied magnetic field onto the crystal axis of the NV color center. A single nanodiamond contains multiple NV color centers, each with a different crystal axis; furthermore, the spatial orientation of different nanodiamond particles flowing through the sample channel 100 is random. Therefore, the projection components of the applied magnetic field onto the NV color centers in different axes within different nanodiamond particles differ, leading to |m s =0> state and |m s The transition frequencies between the ±1 states are different. Under microwave manipulation at a single frequency, the NV color center quantum states within some nanodiamond particles do not change, and the overall fluorescence brightness does not change significantly, thus affecting the signal-to-noise ratio of the measurement scheme, and consequently affecting the accuracy and reliability of the analyte detection.

[0119] 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 intrinsic 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.

[0120] Optionally, based on the technical scheme, 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 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.

[0121] 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, 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 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.

[0122] 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 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 structure 300.

[0123] 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, and the dephasing time is different, and the zero field limit needs to be determined according to the selected particles. Taking a 40nm diameter, 1.5ppm NV concentration HPHT method nanodiamond as an example, the typical value of the CW spectrum intrinsic broadening is 20MHz, and the external magnetic field is required to be below 3.56Gs. The resonance (CW) spectrum is the spectral line in Figure 10

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

[0125] As shown in Figure 11 ​As shown, the flow fluorescence detection system includes at least two light detection modules 400, which can ensure that the fluorescence signals at each angle around the detection area S1 can be collected by the light detection modules 400.

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

[0127] 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 fluorescence signals at each angle around the detection area S1 can be collected by the light detection modules 400.

[0128] 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.

[0129] 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).

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

[0131] S130, the data processing module determines whether the target contains the biological molecule to be detected according to the fluorescence signal.

[0132] Specifically, the frequency or power of the microwave radiation signal provided by the microwave field changes periodically, the number of NV 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.

[0133] 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 the 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.

[0134] 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 in the detection area S1 of the sample flow channel 100 jump to generate fluorescence. When the microwave radiation module 300 provides a microwave field for the detection area S1 of the sample flow channel 100, the microwave field can provide a microwave radiation signal, and the fluorescence signals generated by the NV color center in the nanodiamond are different when the microwave radiation signal exists and does not exist; or the frequency or power of the microwave radiation signal changes, and the fluorescence signal generated by the NV color center in the nanodiamond changes accordingly, that is, at least one of the intensity, frequency and on-off of the microwave field is controlled to realize that the nanodiamond emits a fluorescence signal with periodic variation of intensity. 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, whether the target object contains the biological molecule to be detected can be judged according to the 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 of the flow fluorescence detection method is much higher than that of other fluorescence label detection methods. In summary, the technical scheme provided by the embodiment of the present application improves the detection sensitivity of the fluorescence detection method, and the single molecule fluorescence signal recognition can be realized. In the embodiment of the present application, the magnetic field strength 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 intrinsic 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 method is further improved.

[0135] Optionally, on the basis of the above technical scheme, as shown in Figure 13 , Figure 13 is Figure 12 the flowchart included in 110, when the target object specifically combined with the nanodiamond label flows through the detection area of the sample flow channel, the fluorescence signal generated by the NV color center in the nanodiamond includes:

[0136] 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.

[0137] 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.

[0138] 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 undergoes transition under excitation light irradiation to generate 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.

[0139] 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, the microwave radiation module is in a closed state, 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.

[0140] 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 undergoes transition under excitation light irradiation to generate 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.

[0141] 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.

[0142] 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 undergoes transition under excitation light irradiation. The number of NV centers resonating with the microwave field and / or the degree of NV center resonance varies periodically, and the NV center generates a fluorescence signal with a periodically varying intensity.

[0143] 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.

[0144] The microwave radiation module 300 emits a microwave modulation field with fixed power and periodically changed frequency under the control of the control signal 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, the number of NV center resonance with the microwave field is periodically changed, and / or the degree of NV center resonance is periodically changed, and the NV center produces a fluorescence signal with periodically changed intensity.

[0145] The above technical solution provides a scheme for the NV center in the nanodiamond to emit a fluorescence signal that changes accordingly under excitation light irradiation when the microwave radiation module 300 is used to periodically change the power or frequency of the microwave radiation signal emitted in the on state and the off state or the fixed frequency of the control signal.

[0146] Alternatively, on the basis of the above technical solution, as shown in Figure 14 , Figure 14 is Figure 12 S130 includes a flowchart, and the data processing module determines whether the target object contains the biological molecule to be detected according to the fluorescence signal.

[0147] 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.

[0148] 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.

[0149] 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 resonance with the microwave field is periodically changed, and / or the degree of NV center resonance is periodically changed, the NV center produces a fluorescence signal with periodically changed intensity, and the data processing module 700 determines whether the target object contains the biological molecule to be detected according to the fluorescence signal.

[0150] Alternatively, on the basis of the above technical solution, the data processing module determines whether the target object contains the biological molecule to be detected according to the fluorescence signal.

[0151] 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.

[0152] 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 fluorescence signal. When the microwave radiation module 300 is in the closed state under the control of a microwave field closing signal, the electrons of the NV color center jump to generate a bright-state fluorescence signal, and the bright-state fluorescence signal is generated by the NV color center in the nanodiamond when there is no NV color center resonating with the microwave field.

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

[0154] 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.

[0155] As shown in Figure 15 , the number of target objects detected per unit time by the flow fluorescence system is N per second, and the time required for a single target object to flow through the detection area S1 is 1 / N second. With F M as the frequency of the control signal, a single target object presents periodic 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.

[0156] Here, when the microwave field modulation mode is amplitude modulation, the radiation power of the microwave radiation signal provided by the microwave field changes, 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.

[0157] 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 within the period of a single fluorescent signal satisfies the following relationship:

[0158] M=F s / F M

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

[0160] 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 , and also give the number of detection windows collected by the light detection module 400 within the period of a single fluorescent signal when the target stream passes through the detection area S1.

[0161] 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:

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

[0163] 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.

[0164] Optionally, on the basis of the above technical solutions, a number of scientific cameras are used as the light detection module 400, which can be sCMOS high-speed scientific cameras or intensified electron-coupled devices (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.

[0165] 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.

[0166] 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:

[0167]

[0168] 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 =0>and the dark state of |m p =±1>is the contrast.

[0169] When 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, which mainly considers the photon shot noise, and the signal-to-noise ratio of a single measurement can be greater than 1:1, thereby improving the signal-to-noise ratio of the fluorescent signal. Optionally, on the basis of the above technical solution, the excitation light module 200 includes an optical pump for emitting laser light;

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

[0171]

[0172] Wherein, Θ is a normalized 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 and Γ c 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.

[0173] The above technical solution 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 solution, the microwave field intensity experienced by the NV color center in the nanodiamond satisfies the following relationship:

[0174]

[0175] 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 300.

[0176] The flow fluorescence detection method is different from the traditional NV color center-based light detection method in that the nanodiamond particles to be detected are not stably laid on the substrate, but are placed inside the sample flow channel 100 and are in a moving state. The nanodiamonds selected in the embodiment of the present application contain hundreds of NV color centers with different crystal axis directions, so it can be considered that the angle between the NV color center main axis direction and the microwave field is completely averaged, and the microwave field strength sensed by all NV color centers in a single nanodiamond particle is:

[0177]

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

[0179] 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 strength of the nanodiamond in a single sampling point.

[0180] 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 strength of the nanodiamond particle in a single sampling point. This way, the excitation light duty cycle is high, and the number of effective fluorescence photons emitted by the nanodiamond collected by the light detection module 400 is higher.

[0181] 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.

[0182] Specifically, since the NV color 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; then the excitation-waiting-fluorescence collection sequence is repeated multiple times to obtain the fluorescence signal strength 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 color 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.

[0183] The technical scheme provided by the embodiment of the application is to 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 particles; when the microwave radiation module 300 emits microwave 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 particles of 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 particles decreases; when the microwave in the radiation coil is turned off, the excitation light in the detection area S1 initializes the NV center 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 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 of a specific frequency is identified by an algorithm to exclude the interference of static noise signals.

[0184] The embodiment of the application also provides a zero-field flow fluorescent detection system, which is suitable for the zero-field flow fluorescent detection method according to any of the embodiments of the application. The zero-field flow fluorescent detection system has the beneficial effects of the zero-field flow fluorescent detection method according to any of the embodiments of the application, which will not be repeated here.

[0185] As shown in FIG. 1, Figure 17 Figure 17 is a flowchart of another zero-field flow fluorescent detection method based on a microwave field switch state according to an embodiment of the application. The zero-field flow fluorescent detection method based on a microwave field switch state includes the following steps:

[0186] S210, when the nanodiamond-labeled target object flows through the first half of the detection area of the sample flow channel, the NV center in the nanodiamond generates a fluorescent signal, and the fluorescent signal includes a dark-state fluorescent signal; the dark-state fluorescent signal is the fluorescent signal generated by the NV center in the nanodiamond when part or all of the NV center resonates with the microwave field.

[0187] In this embodiment, when the microwave radiation module 300 provides a microwave field for the detection area S1 of the sample flow channel 100, the microwave field can provide a microwave radiation signal, and the fluorescent signal generated by the NV center in the nanodiamond is different when the microwave radiation signal exists and does not exist.

[0188] ​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.

[0189] 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.

[0190] The excitation light module is configured to emit excitation light, and 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. 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 field provides a microwave radiation signal with 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.

[0191] 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.

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

[0193] 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 modules 400.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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).

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

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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:

[0203] S2101, the control module sends a control signal, and the control signal includes a microwave field opening signal and a microwave field closing signal.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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:

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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:

[0212] 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.

[0213] The technical solution is directed to the microwave radiation module 300 being in an open state or a closed state under the control of the control signal, and the data processing module 700 judging whether the target object contains the to-be-detected biomolecule according to the fluorescence signal.

[0214] Optionally, on the basis of the above 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 color center generates a fluorescence signal, and then the following is included:

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

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

[0217] Optionally, on the basis of the above technical solution, a plurality of scientific research cameras are used as the light detection module 400, and sCMOS high-speed scientific research cameras or enhanced electron-coupled devices (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 remaining background fluorescence signals are discarded to improve the signal-to-noise ratio of the to-be-detected fluorescence signal.

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

[0219] Optionally, on the basis of the above technical solution, 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.

[0220] In an applied magnetic field, NV centers cause the electron in the nanodiamond NV center 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 of the applied magnetic field on 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 applied magnetic field on the different axial NV 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 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.

[0221] 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 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.

[0222] 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 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 center.

[0223] 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 caused by the Zeeman shift of the NV energy level due to the external magnetic field, i.e., one-half of the two microwave resonance frequency difference corresponding to the energy level splitting of the same NV center; and the "spectral line broadening of the optical detection magnetic resonance spectrum of the NV center" refers to the half-peak width of the optical detection magnetic resonance spectrum resonance peak of the NV center.

[0224] 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.

[0225] 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, in nanodiamonds with different diameters and different noise environments of NV centers, the dephasing time is different, and 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 made 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 the formula. Figure 10

[0226] 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.

[0227] When the target flow passes through the detection area, the number of detection windows collected by the light detection module within the cycle of a single fluorescent signal is at least one, and the fluorescent counting rate of the nanodiamond, the contrast of the fluorescent signal and the single detection window exposure time satisfy the following relationship:

[0228]

[0229] Where C is the contrast of the fluorescent signal, R is the fluorescent 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.

[0230] ​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.

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

[0232] The contrast of the fluorescence signal generated by the NV color center in the nanodiamond meets the following relationship:

[0233]

[0234] 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.

[0235] 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.

[0236] 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:

[0237]

[0238] 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.

[0239] 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:

[0240]

[0241] 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.

[0242] Optionally, on the basis of the above technical solution, 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.

[0243] 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.

[0244] 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.

[0245] 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 for example; 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.

[0246] The technical solution provided by the embodiment of the present application introduces a microwave field to modulate the fluorescence signal of the nanodiamond particle in order to improve the signal-to-noise ratio of the fluorescence signal and improve the recognition accuracy of the measured object: when the microwave radiation module 300 opens the microwave of 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 |m s =0> state to |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 |m s =0> state by 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.

[0247] The embodiment of the present application also provides a flow fluorescence detection system based on microwave field switching state, which is suitable for the flow fluorescence detection method based on microwave field switching state of any of the embodiments of the present application. The flow fluorescence detection system based on microwave field switching state has the beneficial effects of the flow fluorescence detection method based on microwave field switching state of any of the embodiments of the present application, which will not be repeated here.

[0248] It should be understood that the various forms of 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 order, as long as the desired results of the technical solutions of the present application can be achieved, which is not limited herein.

[0249] 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 within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A method of flow fluorescence detection based on microwave field switching state, characterized in that, The NV color center in the nanodiamond emits a fluorescent signal when the target object specifically combined with the nanodiamond flows through the first half of the detection area of the sample flow channel, and the fluorescent signal is a dark-state fluorescent signal; the NV color center in the nanodiamond emits a fluorescent signal when the target object specifically combined with the nanodiamond flows through the second half of the detection area of the sample flow channel, and the fluorescent signal is a bright-state fluorescent signal; wherein the excitation light module is used to emit excitation light, the excitation light is used to excite the NV color center in the detection area of the sample flow channel, so that the electron of the NV color center jumps to produce fluorescence, the microwave radiation module is used to provide a microwave field for the detection area of the sample flow channel, the microwave field is resonant with all or part of the NV color center in the nanodiamond, and the microwave radiation module is in an open state or a closed state under the control of a control signal; when the microwave radiation module is in the open state, the NV color center in the nanodiamond emits the dark-state fluorescent signal, and when the microwave radiation module is in the closed state, the NV color center in the nanodiamond emits the bright-state fluorescent signal; the light detection module collects the fluorescent signal at a preset collection frequency; the data processing module determines whether the target object contains the biological molecule to be detected according to the size relationship between the intensity of the dark-state fluorescent signal and the intensity of the bright-state fluorescent signal. The NV color center in the nanodiamond emits a fluorescent signal when the target object specifically combined with the nanodiamond flows through the first half of the detection area of the sample flow channel, and the fluorescent signal is a dark-state fluorescent signal; the NV color center in the nanodiamond emits a fluorescent signal when the target object specifically combined with the nanodiamond flows through the second half of the detection area of the sample flow channel, and the fluorescent signal is a bright-state fluorescent signal; wherein the excitation light module is used to emit excitation light, the excitation light is used to excite the NV color center in the detection area of the sample flow channel, so that the electron of the NV color center jumps to produce fluorescence, the microwave radiation module is used to provide a microwave field for the detection area of the sample flow channel, the microwave field is resonant with all or part of the NV color center in the nanodiamond, and the microwave radiation module is in an open state or a closed state under the control of a control signal; when the microwave radiation module is in the open state, the NV color center in the nanodiamond emits the dark-state fluorescent signal, and when the microwave radiation module is in the closed state, the NV color center in the nanodiamond emits the bright-state fluorescent signal; the light detection module collects the fluorescent signal at a preset collection frequency; the data processing module determines whether the target object contains the biological molecule to be detected according to the size relationship between the intensity of the dark-state fluorescent signal and the intensity of the bright-state fluorescent signal. The NV color center in the nanodiamond emits a fluorescent signal when the target object specifically combined with the nanodiamond flows through the first half of the detection area of the sample flow channel, and the fluorescent signal is a dark-state fluorescent signal; the NV color center in the nanodiamond emits a fluorescent signal when the target object specifically combined with the nanodiamond flows through the second half of the detection area of the sample flow channel, and the fluorescent signal is a bright-state fluorescent signal; wherein the excitation light module is used to emit excitation light, the excitation light is used to excite the NV color center in the detection area of the sample flow channel, so that the electron of the NV color center jumps to produce fluorescence, the microwave radiation module is used to provide a microwave field for the detection area of the sample flow channel, the microwave field is resonant with all or part of the NV color center in the nanodiamond, and the microwave radiation module is in an open state or a closed state under the control of a control signal; when the microwave radiation module is in the open state, the NV color center in the nanodiamond emits the dark-state fluorescent signal, and when the microwave radiation module is in the closed state, the NV color center in the nanodiamond emits the bright-state fluorescent signal; the light detection module collects the fluorescent signal at a preset collection frequency; the data processing module determines whether the target object contains the biological molecule to be detected according to the size relationship between the intensity of the dark-state fluorescent signal and the intensity of the bright-state fluorescent signal. The NV color center in the nanodiamond emits a fluorescent signal when the target object specifically combined with the nanodiamond flows through the first half of the detection area of the sample flow channel, and the fluorescent signal is a dark-state fluorescent signal; the NV color center in the nanodiamond emits a fluorescent signal when the target object specifically combined with the nanodiamond flows through the second half of the detection area of the sample flow channel, and the fluorescent signal is a bright-state fluorescent signal; wherein the excitation light module is used to emit excitation light, the excitation light is used to excite the NV color center in the detection area of the sample flow channel, so that the electron of the NV color center jumps to produce fluorescence, the microwave radiation module is used to provide a microwave field for the detection area of the sample flow channel, the microwave field is resonant with all or part of the NV color center in the nanodiamond, and the microwave radiation module is in an open state or a closed state under the control of a control signal; when the microwave radiation module is in the open state, the NV color center in the nanodiamond emits the dark-state fluorescent signal, and when the microwave radiation module is in the closed state, the NV color center in the nanodiamond emits the bright-state fluorescent signal; the light detection module collects the fluorescent signal at a preset collection frequency; the data processing module determines whether the target object contains the biological molecule to be detected according to the size relationship between the intensity of the dark-state fluorescent signal and the intensity of the bright-state fluorescent signal. ​ ​ 2. The microwave field-switched regime based flow- fluorescence detection method according to claim 1, characterized in that, ​ ​ ​ 3. The microwave field-switched state-based flow fluorescence detection method according to claim 2, characterized in that, ​ ​ 4. The microwave field-switched regime based flow- fluorescence detection method according to claim 1, wherein, ​ 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, which is at least 1 times the noise level when the bright-state fluorescence signal is generated.

5. The microwave field-switched regime based flow- fluorescence detection method according to claim 1, wherein, When the target object specifically combined with the nanodiamond-labeled target object flows through the second half of the sample flow channel detection area, the NV color center in the nanodiamond generates a fluorescence signal, and then further includes: After each detection window of the light detection module, a detection window without microwave radiation signal is added as a reference signal.

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

7. The microwave field-switched regime based flow- fluorescence detection method according to claim 6, characterized in that, When the magnetic field monitoring module monitors that the magnetic field strength 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.

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

9. The microwave field-switched regime based flow- fluorescence detection method according to claim 6, wherein, The particle size of the nanodiamond ranges from greater than or equal to 40 nm to less than or equal to 1 μm. When the target object flows through the detection area, the number of detection windows collected by the light detection module within the cycle of a single fluorescence signal is at least one, and the fluorescence counting rate of the nanodiamond, the contrast of the fluorescence signal, and the single detection window exposure time satisfy the following relationship: ； Where C is the contrast of the fluorescence signal, R is the fluorescence counting rate of the nanodiamond, and t is the exposure time of a single detection window.

10. The microwave field-switched regime based flow- fluorescence detection method according to claim 9, characterized in that, The excitation light module includes an optical pump for emitting laser light. The contrast of the fluorescence signal generated by the NV color center in the nanodiamond satisfies the following relationship: ； where Θ is a normalization constant, is the rate of laser polarization, is the rate of coherent relaxation due to optical pumping, is the frequency of the NV color center Rabi oscillation driven by the microwave field, , Two terms are related to the laser power, The term is positively related to the microwave field strength experienced by the NV color centers in the nanodiamond.

11. The microwave field-switched regime based flow- fluorescence detection method according to claim 10, wherein, The microwave field strength sensed by the NV color center in the nanodiamond satisfies the following relationship: ； Where B is the microwave field strength sensed by the NV color center in the nanodiamond, and B1 is the strength of the microwave field.

12. The microwave field-switched regime based flow- fluorescence detection method according to claim 6, wherein, For a single detection window, the excitation light remains on, the microwave radiation module provides a microwave radiation signal, and the light detection module uses the entire detection window length as the exposure time to obtain the fluorescence signal intensity of the nanodiamond in a single sampling point.

13. The microwave field-switched regime based flow- fluorescence detection method according to claim 6, wherein, The delay time between the excitation light initialization pulse and the fluorescence signal collection pulse window is greater than a preset delay time.

14. A microwave field-switched state based flow fluorescence detection system, characterized in that, The microwave field switch state-based flow fluorescence detection system is used to implement the microwave field switch state-based flow fluorescence detection method of any one of claims 1-13, comprising: A sample flow channel for transporting a target object specifically combined with a nanodiamond-labeled target object, the sample flow channel being provided with a detection area; An excitation light module for emitting excitation light to the detection area to excite the NV color center in the detection area to emit a fluorescence signal; A control module for issuing a control signal, the control signal including a microwave field opening signal and a microwave field closing signal; The control module for issuing a control signal, the control signal including a microwave field opening signal and a microwave field closing signal; a microwave radiation module, configured to provide a microwave field to the detection area, the microwave field being in an on state or an off state under control of the control signal; a light detection module, configured to collect the fluorescent signal at a preset collection frequency; a data processing module, configured to determine whether the target object contains the biological molecule to be detected according to a size relationship between intensities of the dark-state fluorescent signal and the bright-state fluorescent signal in the fluorescent signal.

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