A zero-field-based flow fluorescence detection method and detection system

Abstract

This invention discloses a zero-field-based flow cytometry fluorescence detection method and system. The method includes: when a target material specifically bound to nanodiamonds flows through the detection region of a sample channel, controlling at least one of the intensity, frequency, and on / off state of a microwave field to induce periodically varying fluorescence signals emitted by the NV centers within the nanodiamonds; the magnetic field strength in the detection region satisfies the zero-field condition; an excitation light module excites the NV centers in the detection region of the sample channel; a microwave radiation module provides a microwave field to the detection region of the sample channel, and the microwave radiation signal provided by the microwave field has a fixed frequency; a photodetector module collects the fluorescence signal at a preset collection frequency; and a data processing module determines whether the target analyte contains the biomolecule to be detected based on the fluorescence signal. The technical solution provided by this invention improves the detection sensitivity of the fluorescence detection method.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and in particular to a flow cytometry fluorescence detection method and system based on zero field. Background Technology

[0002] Flow cytometry enables high-throughput, high-speed, and multi-marker detection. However, due to the low intensity of existing fluorescent dyes, single-molecule fluorescence signal recognition is not possible, limiting the lower limit of detection sensitivity. This prevents it from meeting the requirements of specific applications, such as the detection of low-abundance biomarkers for diseases.

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

[0004] This invention provides a zero-field-based flow cytometry fluorescence detection method and system to improve the detection sensitivity of fluorescence detection systems.

[0005] According to one aspect of the present invention, a zero-field-based flow cytometry fluorescence detection method is provided, comprising: when a target material specifically bound to nanodiamond passes through the detection area of ​​a sample channel, the nanodiamond emits a fluorescence signal with periodically changing intensity by controlling at least one of the intensity, frequency, and on / off state of a microwave field;

[0006] The magnetic field strength of the detection area satisfies the zero-field condition. The excitation light module emits excitation light, which is used to excite the NV color centers in the detection area of ​​the sample flow channel, causing their electrons to undergo transitions and generate fluorescence. The microwave radiation module provides a microwave field for the detection area of ​​the sample flow channel. The microwave field can resonate with all or part of the NV color centers in the nanodiamond. The frequency of the microwave radiation signal provided by the microwave field is equal to the energy level difference of the electron spin transition of the NV color 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.

[0007] The optical detection module collects the fluorescence signal at a preset collection frequency;

[0008] The data processing module determines whether the target analyte contains the biomolecule to be detected based on the fluorescence signal.

[0009] Optionally, when the magnetic field monitoring module monitors the magnetic field strength near the detection area of ​​the sample flow channel and the condition of zero field is met, the energy level splitting of the NV color center in the nanodiamond caused by the magnetic field is less than half of the broadening of its photodetector magnetic resonance spectrum.

[0010] 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, where D is the zero-field splitting parameter of the NV color center.

[0011] Optionally, when the target material specifically bound to nanodiamond passes through the detection area of ​​the sample channel, the NV color centers within the nanodiamond generate a fluorescence signal, including:

[0012] The control module sends control signals, which include at least one of the following: microwave field on signal and microwave field off signal, microwave field power periodic change signal and microwave field frequency periodic change signal;

[0013] According to the microwave field activation signal, the microwave radiation module is in the open state and provides a microwave field for the detection area of ​​the sample flow channel. The NV color centers in the nanodiamond emit dark-state fluorescence signals under excitation light irradiation. The dark-state fluorescence signals are the fluorescence signals generated by the NV color centers in the nanodiamond when some or all of the NV color centers resonate with the microwave field.

[0014] According to the microwave field shutdown signal, the microwave radiation module is in the off state, and the microwave radiation module stops providing microwave field to the detection area of ​​the sample flow channel. The NV color center in the nanodiamond emits a bright fluorescence signal under the excitation light. The bright fluorescence signal is the fluorescence signal generated by the NV color center in the nanodiamond when there is no resonance between the NV color center and the microwave field.

[0015] Alternatively, the microwave radiation module emits a microwave modulation field with a fixed frequency and periodically varying power based on the periodically varying power signal of the microwave field power, so that the NV color centers in the nanodiamond emit a fluorescence signal with periodically varying intensity under excitation light irradiation.

[0016] Alternatively, the microwave radiation module emits a microwave modulation field with fixed power and periodically varying frequency according to the periodically changing frequency signal of the microwave field, causing the NV color centers in the nanodiamond to emit a fluorescence signal with periodically varying intensity under excitation light irradiation.

[0017] Optionally, the data processing module determines whether the target analyte contains the biomolecule to be detected based on the fluorescence signal, including:

[0018] The data processing module performs a 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 demodulated signal;

[0019] If the intensity of the demodulated signal is greater than a first preset threshold, it is determined that the target object contains the biomolecule to be detected.

[0020] Optionally, the data processing module determines whether the target analyte contains the biomolecule to be detected based on the fluorescence signal, including:

[0021] If the intensity of the dark-state fluorescence signal is less than the intensity of the bright-state fluorescence signal, and the absolute value of the difference between the intensity of the dark-state fluorescence signal and the intensity of the bright-state fluorescence signal is greater than a second preset threshold, the data processing module determines that the target analyte contains the biomolecule to be detected. The second preset threshold is at least 1 times the noise level when the bright-state fluorescence signal is generated.

[0022] Optionally, the number of periodically changing fluorescence signals exhibited by a single target within the detection area is... indivual;

[0023] Where N is the number of target objects flowing through the detection area of ​​the sample flow channel per second, and the value of N is an integer greater than or equal to 1; F M The frequency of the control signal is denoted as .

[0024] Optionally, when the target material passes through the detection area, the number of detection windows acquired by the photodetector module within a single fluorescence signal period satisfies the following relationship:

[0025] M = F s / F M

[0026] Among them, F s F is the sampling frequency of the optical detection module. M The frequency of the control signal is denoted as .

[0027] Optionally, when the target material specifically bound to nanodiamond passes through the detection area of ​​the sample channel, after the NV color center within the nanodiamond generates a fluorescence signal, the process includes:

[0028] After each detection window of the optical detection module, a detection window without microwave radiation signal is added as a reference signal.

[0029] Optionally, the nanodiamond has a particle size range greater than or equal to 40 nm and less than or equal to 1 μm;

[0030] The fluorescence counting rate of the nanodiamond, the contrast of the fluorescence signal, and the exposure time of a single detection window satisfy the following relationship:

[0031]

[0032] Where C is the contrast of the fluorescence signal, R is the fluorescence count rate of the nanodiamond, and t is the exposure time of a single detection window.

[0033] Optionally, the excitation optical module includes an optical pump for emitting laser light;

[0034] The contrast of the fluorescence signal generated by the NV color centers within the nanodiamond satisfies the following relationship:

[0035]

[0036] Where Θ is the normalization constant, Γ p Γ represents the laser polarization rate. c For the coherent relaxation rate caused by optical pumping, Ω R Γ is the frequency of the Rabi oscillation of the NV color center driven by the microwave field. p ,Γ c Two items are related to laser power, Ω R The term is positively correlated with the microwave field intensity sensed by the NV color center within the nanodiamond.

[0037] Optionally, the microwave field intensity sensed by the NV color center within the nanodiamond satisfies the following relationship:

[0038]

[0039] Wherein, B is the intensity of the microwave field sensed by the NV color center within the nanodiamond, and B1 is the intensity of the microwave field provided by the microwave radiation module.

[0040] Optionally, for a single detection window, the excitation light remains constantly on, the microwave radiation module provides a microwave radiation signal, and the photodetector module uses the entire length of the detection window as the exposure time to obtain the fluorescence signal intensity of the nanodiamond within a single sampling point.

[0041] Optionally, the delay time between the excitation light initialization pulse and the fluorescence signal collection pulse window is greater than a preset delay time.

[0042] According to another aspect of the present invention, a zero-field-based flow cytometry fluorescence detection system is provided, which is applicable to any of the zero-field-based flow cytometry fluorescence detection methods described in the first aspect of the present invention.

[0043] This invention provides a zero-field-based flow cytometry fluorescence detection method. Unlike traditional NV center-based optical detection methods, the nanodiamond particles to be tested are not stably laid flat on a substrate, but rather placed inside the sample channel, in a state of motion. The target material, specifically bound to nanodiamond-labeled particles, flows through the detection area of ​​the sample channel. The nanodiamond particles include NV centers. When these NV centers are irradiated by excitation light emitted from an excitation light module, the excitation light excites the NV centers in the detection area of ​​the sample channel, causing electron transitions and generating fluorescence. When a microwave radiation module provides a microwave field to the detection area of ​​the sample channel, 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 centers within the nanodiamond particles; alternatively, changes in the frequency or power of the microwave radiation signal cause corresponding changes in the fluorescence signal generated by the NV centers within the nanodiamond particles. In other words, by controlling at least one of the intensity, frequency, and on / off states of the microwave field, the NV centers within the nanodiamond particles emit fluorescence signals with periodically varying intensity. The optical detection module collects fluorescence signals generated by the NV centers within the nanodiamonds as the target material, specifically bound to nanodiamonds, flows through the detection area of ​​the sample channel at a preset collection frequency. Since the fluorescence signal is correlated with whether the target analyte contains the biomolecule to be detected, it is possible to determine whether the target analyte contains the biomolecule based on the fluorescence signal. Due to the stability, room temperature and atmospheric environment compatibility, and biocompatibility of the NV center system, and because nanodiamonds, as a novel biofluorescent label, can eliminate low-frequency noise interference through signal modulation and demodulation techniques, a detection sensitivity far exceeding that of other fluorescence labeling detection methods can be achieved. In summary, the technical solution provided by this invention improves the detection sensitivity of the fluorescence detection method, enabling the identification of single-molecule fluorescence signals. Furthermore, in this invention, the magnetic field strength near the detection area of ​​the sample channel satisfies the zero-field condition, and the resonant frequency of some or all NV centers with the microwave field is their natural frequency. The microwave field is used to provide a microwave radiation signal with this fixed frequency, which can improve the signal-to-noise ratio of the fluorescence signal, thereby improving the detection sensitivity of the flow cytometry fluorescence detection method.

[0044] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of the present invention, nor is it intended to limit the scope of the invention. Other features of the invention will become readily apparent from the following description. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0046] Figure 1 This is a schematic diagram of a flow cytometry fluorescence detection system according to an embodiment of the present invention;

[0047] Figure 2 This is a structural block diagram of a flow cytometry fluorescence detection system provided according to an embodiment of the present invention;

[0048] Figure 3 This is a schematic diagram of another flow cytometry fluorescence detection system provided according to an embodiment of the present invention;

[0049] Figure 4 This is a schematic diagram of the optical path system of a flow cytometry fluorescence detection system according to an embodiment of the present invention;

[0050] Figure 5 This is a schematic diagram of the structure of another flow cytometry fluorescence detection system provided according to an embodiment of the present invention;

[0051] Figure 6 yes Figure 5 A cross-sectional view of the simulated microwave field provided by the microwave radiation structure in the mid-flow fluorescence detection system.

[0052] Figure 7 yes Figure 5 A three-dimensional diagram of the microwave field provided by the microwave radiation structure in the structure of a mid-flow fluorescence detection system;

[0053] Figure 8 This is a schematic diagram of another flow cytometry fluorescence detection system provided according to an embodiment of the present invention;

[0054] Figure 9 yes Figure 8 A cross-sectional view of the simulated microwave field provided by the microwave radiation structure in the mid-flow fluorescence detection system.

[0055] Figure 10 This is a schematic diagram illustrating the relationship between the fluorescence intensity of an NV color center and the frequency of a microwave radiation signal, according to an embodiment of the present invention.

[0056] Figure 11 This is a schematic diagram of the optical path structure of a photodetector module according to an embodiment of the present invention;

[0057] Figure 12 This is a schematic flowchart of a zero-field-based flow cytometry fluorescence detection method provided according to an embodiment of the present invention;

[0058] Figure 13 yes Figure 12 A schematic diagram of the process included in S110;

[0059] Figure 14 yes Figure 12A schematic diagram of the process included in S130;

[0060] Figure 15 This is a schematic diagram illustrating the relationship between the frequency of a microwave radiation signal and the flow velocity of nanodiamonds according to an embodiment of the present invention.

[0061] Figure 16 This is a schematic diagram of a modulated fluorescence signal acquisition method according to an embodiment of the present invention;

[0062] Figure 17 This is a schematic flowchart of a flow cytometry fluorescence detection method based on microwave field switching state according to an embodiment of the present invention;

[0063] Figure 18 yes Figure 17 The process diagram included in S210. Detailed Implementation

[0064] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0065] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0066] To improve the detection sensitivity of fluorescence detection methods, this invention provides a zero-field-based flow cytometry fluorescence detection method. Before introducing the zero-field-based flow cytometry fluorescence detection method provided by this invention, we will first introduce the flow cytometry fluorescence detection system to which the flow cytometry fluorescence detection method provided by this invention is applicable.

[0067] like Figure 1 As shown, Figure 1 This is a schematic diagram of a flow cytometry fluorescence detection system according to an embodiment of the present invention.Figure 2 This is a structural block diagram of a flow cytometry fluorescence detection system according to an embodiment of the present invention. The flow cytometry fluorescence detection system includes:

[0068] The sample channel 100 is used to deliver a target object specifically bound to nanodiamond markers, the nanodiamonds including NV color centers, and the sample channel 100 is provided with a detection area S1.

[0069] The excitation light module 200 is used to emit excitation light, which is used to excite the NV color center of the detection area S1 of the sample flow channel 100, causing its electrons to transition and generate fluorescence.

[0070] The microwave radiation module 300 provides a microwave field to the detection area S1 of the sample flow channel 100. The microwave field can resonate with all or part of the NV color centers in the nanodiamond. The frequency of the microwave radiation signal provided by the microwave field is equal to the energy level difference of the electron spin transition of the NV color center, which enables the microwave field to resonate with the NV color center. The microwave radiation module 300 includes at least two microwave radiation structures 301, which are distributed along the transport direction of the target object and are arranged around the detection area S1 of the sample flow channel 100.

[0071] The optical detection module 400 collects the target material specifically bound to nanodiamond labels through the detection area S1 of the sample flow channel 100 at a preset collection frequency. By controlling at least one of the intensity, frequency, and on / off state of the microwave field, the NV color center within the nanodiamond emits a fluorescence signal with periodic intensity changes. The fluorescence signal is correlated with whether the target material contains the biomolecule to be detected.

[0072] Known quantum sensing systems include Rydberg atoms, atomic magnetometers, superconducting quantum interference devices (SQIs), and diamond NV centers, among which the NV center system is one of the most promising solid-state quantum sensors in life sciences and medical detection due to its stability, room temperature and atmospheric environment compatibility, and biocompatibility. Nanodiamond, as a novel biofluorescent label, can eliminate low-frequency noise interference through signal modulation and demodulation techniques, thus achieving detection sensitivity far exceeding that of other fluorescent labeling detection methods.

[0073] When the NV color centers within the nanodiamond are irradiated by the excitation light emitted by the excitation light module 200, the excitation light excites the NV color centers in the detection region S1 of the sample flow channel 100, causing their electrons to transition and generate fluorescence. For example, a 532nm wavelength laser emitted by the excitation light module 200 will cause the NV color centers to radiate fluorescence signals in the 630-800nm ​​wavelength range. In this embodiment, the excitation light generation module 500 generates the excitation light, and the excitation light module 200 emits the excitation light as a flat-top beam, which can uniformly focus the excitation light onto the detection system of the sample flow channel 100.

[0074] The microwave radiation module 300 includes at least two microwave radiation structures 301, which are distributed along the transport direction of the target object and are arranged around the detection area S1 of the sample flow channel 100. Figure 1 As shown, the transmission direction of the target object is from top to bottom, and at least two microwave radiation structures are distributed along the transmission direction of the target object. This ensures that during the process of the target object flowing through the detection area S1, the microwave field provided by the microwave radiation module 300 can cover the entire detection area S1. Consequently, when the microwave radiation signal provided by the microwave field changes, the NV color center in the nanodiamond generates a correspondingly changing fluorescence signal.

[0075] 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. Due to the presence or absence of the microwave radiation signal, the fluorescence signal generated by the NV color centers in the nanodiamond is different; or, the change in the frequency or power of the microwave radiation signal will cause the NV color centers in the nanodiamond to generate a correspondingly changing fluorescence signal, as follows:

[0076] 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.

[0077] 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.

[0078] As the frequency or power of the microwave radiation signal provided by the microwave field changes, the NV color 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 changes periodically, the electrons in the NV color centers within the nanodiamond undergo transitions under excitation light irradiation. The number of NV color centers resonating with the microwave field changes periodically, and / or the degree of NV color center resonance changes periodically, resulting in the NV color centers generating fluorescence signals with periodically varying intensity.

[0079] Specifically, when the microwave field modulation mode is amplitude modulation, the 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.

[0080] This invention provides a flow cytometry fluorescence detection system based on nanodiamond fluorescent labels. Unlike traditional optical detection systems based on NV centers, the nanodiamond particles to be tested are not stably laid flat on the substrate, but are placed inside the sample channel 100 and are in motion. The target material specifically bound to the nanodiamond labels flows through the detection area S1 of the sample channel 100. The nanodiamonds include NV centers. Under the irradiation of the excitation light emitted by the excitation light module 200, the NV centers within the nanodiamonds are excited by the excitation light to excite the NV centers in the detection area S1 of the sample channel 100, causing their electrons to transition and generate fluorescence. 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 centers within the nanodiamond. Alternatively, changes in the frequency or power of the microwave radiation signal cause the NV centers within the nanodiamond to generate modulated fluorescence signals that change accordingly. That is, by controlling at least one of the intensity, frequency, and on / off state of the microwave field, the NV centers within the nanodiamond emit fluorescence signals with periodically changing intensity. The photodetector module 400 collects the modulated fluorescence signals generated by the NV centers within the nanodiamond as the target material specifically bound to the nanodiamond passes through the detection area S1 of the sample flow channel 100 at a preset collection frequency. Since the fluorescence signal is associated with whether the target material contains the biomolecule to be detected, the presence of the biomolecule to be detected can be determined based on the fluorescence signal. Due to the stability, room temperature and atmospheric environment compatibility, and biocompatibility of the NV center system, and because nanodiamond, as a novel biofluorescent label, can eliminate low-frequency noise interference through signal modulation and demodulation technology, a detection sensitivity far exceeding that of other fluorescent labeling detection methods can be achieved. In summary, the flow cytometry fluorescence detection system provided in this embodiment of the invention improves the detection sensitivity of the fluorescence detection system and can realize the identification of single-molecule fluorescence signals.

[0081] Optionally, based on the above technical solutions, such as Figure 2 As shown, it also includes a control module 600, which is connected to the microwave radiation module 300 and is used to transmit control signals.

[0082] The control signals include at least one of the following: microwave field turn-on signal and microwave field turn-off signal, microwave field power periodic change signal, and microwave field frequency periodic change signal.

[0083] Optionally, based on the above technical solution, the microwave radiation module 300 is used to provide a modulated microwave field to the detection area S1 of the sample flow channel 100 according to the microwave field opening signal. The NV color centers in the nanodiamond emit dark-state fluorescence signals under excitation light irradiation. The dark-state fluorescence signals are the fluorescence signals generated by the NV color centers in the nanodiamond when some or all of the NV color centers resonate with the microwave field. The microwave radiation module 300 is used to stop providing a modulated microwave field to the detection area S1 of the sample flow channel 100 according to the microwave field closing signal. The NV color centers in the nanodiamond emit bright-state fluorescence signals under excitation light irradiation. The bright-state fluorescence signals are the fluorescence signals generated by the NV color centers in the nanodiamond when no NV color centers resonate with the microwave field.

[0084] Optionally, based on the above technical solution, the microwave radiation module 300 is used to transmit a microwave modulation field with a fixed frequency and periodically varying power according to the periodically varying power signal of the microwave field, so that the NV color centers in the nanodiamond emit a fluorescence signal with periodically varying intensity under the excitation light irradiation.

[0085] Optionally, based on the above technical solution, the microwave radiation module 300 is used to emit a microwave modulation field with fixed power and periodically changing frequency according to the periodically changing frequency signal of the microwave field, so that the NV color center in the nanodiamond emits a fluorescence signal with periodically changing intensity under the excitation light irradiation.

[0086] Specifically, the microwave radiation module 300 is in the open state under the control of the microwave field open signal, and there is a microwave radiation signal in the detection area S1. Under the irradiation of the excitation light, the electrons of the NV color centers in the nanodiamond undergo transitions to generate dark-state fluorescence signals. The dark-state fluorescence signals are the fluorescence signals generated by the NV color centers in the nanodiamond when some or all of the NV color centers resonate with the microwave field.

[0087] The microwave radiation module 300 is in the off state under the control of the microwave field shutdown signal, and there is no microwave radiation signal in the detection area S1. Under the irradiation of the excitation light, the electrons of the NV color centers in 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 in 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.

[0088] Alternatively, under the control of a periodically changing microwave field power signal, the microwave radiation module 300 emits a microwave modulation field with a fixed frequency and periodically changing power. This causes the electrons of the NV color centers in the nanodiamond to undergo transitions under excitation light irradiation. The number of NV color centers resonating with the microwave field changes periodically, and / or the degree of NV color center resonance changes periodically. The NV color centers then generate fluorescence signals with periodically changing intensity.

[0089] Alternatively, under the control of a periodically changing microwave field frequency signal, the microwave radiation module 300 emits a microwave modulation field with a fixed power and a periodically changing frequency, causing the electrons of the NV color centers in the nanodiamond to undergo transitions under excitation light irradiation. The number of NV color centers resonating with the microwave field changes periodically, and / or the degree of NV color center resonance changes periodically, resulting in the NV color centers generating fluorescence signals with periodically changing intensity.

[0090] Optionally, based on the above technical solution, a data processing module 700 is also included. The data processing module 700 is connected to the photodetector module 400. The data processing module 700 determines whether the target object contains the biomolecule to be detected based on the fluorescence signal.

[0091] Specifically, the frequency or power of the microwave radiation signal provided by the microwave field varies periodically, the number of NV color centers and microwave field resonances varies periodically, and / or the degree of NV color center resonance varies periodically. The NV color centers generate fluorescence signals with periodically varying intensity. The first judgment method is adopted: the data processing module 700 performs Fourier transform on the fluorescence signal to obtain the frequency domain signal, where the part with the same frequency as the control signal is the demodulated signal; if the intensity of the demodulated signal is greater than the first preset threshold, it is determined that the target object contains the biomolecule to be detected.

[0092] The microwave radiation module 300 is in the "on" state under the control of the microwave field activation signal. Electrons in the NV color centers undergo transitions, generating a dark-state fluorescence signal. This dark-state fluorescence signal is the fluorescence signal generated by the NV color centers within the nanodiamond when some or all of the NV color centers resonate with the microwave field. The microwave radiation module 300 is in the "off" state under the control of the microwave field deactivation signal. Electrons in the NV color centers undergo transitions, generating a second fluorescence signal. This second fluorescence signal is the fluorescence signal generated by the NV color centers within the nanodiamond when no NV color centers resonate with the microwave field. The intensity of the dark-state fluorescence signal is less than the intensity of the bright-state fluorescence signal. Using a second judgment method, if the intensity of the dark-state fluorescence signal is less than the intensity of the bright-state fluorescence signal, and the absolute value of the difference between the intensity of the dark-state fluorescence signal and the intensity of the bright-state fluorescence signal is greater than a second preset threshold, the data processing module 700 determines that the target analyte contains the biomolecule to be detected. The second preset threshold is at least one times the noise level when the bright-state fluorescence signal is generated.

[0093] Optionally, based on the above technical solutions, such as Figure 1 and Figure 3 As shown, the microwave radiation module 300 includes two microwave radiation structures 301. One microwave radiation structure 301 is located at the beginning of the detection area S1, and the other microwave radiation structure 301 is located at the end of the detection area S1. The direction from the beginning of the detection area S1 to the end of the detection area S1 is parallel to the transmission direction of the target object.

[0094] 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 area S1 of the sample channel 100. Under the irradiation of excitation light emitted by the excitation light module 200, the NV centers within the nanodiamonds are excited and cause electron transitions in the detection area S1 of the sample channel 100, generating fluorescence. When the microwave radiation module 300 provides a microwave field to the detection area 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.

[0095] 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.

[0096] Specifically, such as Figure 6 and Figure 7 As shown, the grayscale of the detection area S1 of the sample flow channel 100 is uniform. The microstrip line L1 generates a microwave radiation signal under the action of the electrical signal. The microwave resonant cavity structure L2 provides a microwave field with high uniformity for the detection area S1 of the sample flow channel 100. The microwave field can provide a microwave radiation signal, and the microwave radiation signal radiates the entire detection area S1.

[0097] Optionally, based on the above technical solutions, such as Figure 5 As shown, the microwave resonant cavity structure L2 includes two coaxial radiating coils.

[0098] Specifically, two coaxial radiation coils form a microwave resonant cavity structure L2, which radiates the microwave radiation signal generated by the microstrip line L1 under the action of electrical signals to the entire detection area S1.

[0099] Optionally, based on the above technical solutions, such as Figure 8 As shown, the microwave radiating structure 301 includes a radiating antenna with an "Ω"-shaped coil.

[0100] Specifically, such as Figure 9 As shown, the detection area S1 of the sample flow channel 100 has uniform grayscale. The radiating antenna of the "Ω"-shaped coil, under the influence of an electrical signal, provides a highly uniform microwave field to the detection area S1 of the sample flow channel 100. This microwave field provides a microwave radiation signal, radiating the entire detection area S1. The "Ω"-shaped coil can be a single-turn or multi-turn copper wire. For example... Figure 9 As shown, the detection area S1 includes the detection point 100a of the photodetector module 400.

[0101] Optionally, based on the above technical solutions, such as Figure 1 and Figure 2 As shown, it also 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 meets the zero field condition, the microwave field is used to provide a microwave radiation signal with a fixed frequency.

[0102] 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.

[0103] Therefore, 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 frequency of resonance between some or all NV color centers and the microwave field is the natural frequency. The microwave field is used to provide a microwave radiation signal with this fixed frequency, which can improve the signal-to-noise ratio of the modulated fluorescence signal, thereby improving the detection sensitivity of the flow cytometry fluorescence detection system.

[0104] Optionally, based on the above technical solutions, such as Figure 10 As shown, 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 energy level splitting of the NV color center in the nanodiamond caused by the magnetic field is less than half of the broadening of its photodetector magnetic resonance spectrum.

[0105] "Energy level splitting of NV color centers in nanodiamonds" refers to the shift in the position of the resonance peak in the photodetector magnetic resonance spectrum caused by the Zeeman shift of the NV energy level due to the external magnetic field—that is, half of the difference between the two microwave resonance frequencies corresponding to the energy level splitting of the same NV color center; "spectral broadening of its photodetector magnetic resonance spectrum" refers to the half-width at half-maximum (WHM) of the resonance peak in the photodetector magnetic resonance spectrum of the NV color center.

[0106] Specifically, 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 fixed frequency of the microwave radiation signal provided by the microwave field is greater than or equal to 2820MHz and less than or equal to 2920MHz. This allows all NV color centers in all crystal axis directions within the nanodiamond particles to be quantum-state-controlled by microwaves with a frequency at the zero-field split greater than or equal to 2820MHz and less than or equal to 2920MHz. Therefore, the system can use a frequency greater than or equal to 2820MHz and less than or equal to 2920MHz as the fixed operating frequency of the microwave radiation signal provided by the microwave radiation module 300. It is well known to those skilled in the art that the zero-field split D of the NV color center at room temperature is 2870MHz, but the accurate value of the zero-field split D has a linear relationship with temperature. It is easy to understand that the fixed frequency setting of the microwave radiation signal between 2820MHz and 2920MHz is based on the premise that the zero-field split D = 2870MHz.

[0107] To make the embodiments of the present invention more concise and clear, the experimental environment temperature is assumed to be room temperature, i.e., the value of zero field splitting D is 2870MHz. However, those skilled in the art will understand that the zero field splitting D may be affected by different experimental environment temperatures and exhibit other values. Therefore, the fixed frequency of the microwave radiation signal can usually be set between D-50MHz and D+50MHz.

[0108] The technical solution provided in this invention aims to improve the signal-to-noise ratio of fluorescence signals and enhance the accuracy of analyte identification. The system introduces a microwave field to modulate the fluorescence signal of nanodiamond particles: when the microwave radiation module 300 is activated and microwaves with a frequency greater than or equal to 2820 MHz and less than or equal to 2920 MHz enter the radiation coil, the NV color centers within the nanodiamond particles in the sample flow channel 100 change from |m s =0> State transition to |m s =±1> state, the fluorescence intensity of nanodiamond particles decreases; when the microwave in the radiation coil is turned off, the excitation light in the detection area S1 initializes the NV color center to |m s =0> state, fluorescence intensity recovers. When the radiation power of the microwave radiation signal provided by the microwave radiation module 300 changes, the NV color centers within the nanodiamond generate a correspondingly changing fluorescence signal. The fluorescence signal is modulated in the time domain by the microwave radiation module 300, then collected at a certain frequency by the photodetector module 400, and finally the specific frequency of the test signal is identified by an algorithm to eliminate interference from static noise signals.

[0109] Optionally, based on the above technical solutions, such as Figure 11 As shown, the flow cytometry fluorescence detection system includes at least two photodetector modules 400, which can ensure that fluorescence signals from all angles around the detection area S1 can be collected by the photodetector modules 400.

[0110] For example, Figure 1 and Figure 11 Two photodetector modules 400 are shown, arranged symmetrically around the detection area S1 of the sample flow channel 100. When the number of photodetector modules 400 is greater than two, multiple photodetector modules 400 are arranged around the detection area S1 of the sample flow channel 100, ensuring that fluorescence signals from all angles circumferentially around the detection area S1 can be collected by the photodetector modules 400. This technical solution can, on the one hand, increase the amount of fluorescence signal collected, thereby providing a detection limit; on the other hand, it can overcome the problem of the target object obscuring the NV color center fluorescence.

[0111] Since the fluorescence intensity of a single nanodiamond particle is weak in single-molecule detection applications, the photodetector module 400 preferably uses an avalanche photodiode photodetector (APD), followed by a scientific camera, and then a photomultiplier tube sensor (PMT).

[0112] In addition, interference from excitation light and stray fluorescence signals can be filtered out by using notch filters, long-pass filters, and short-pass filters.

[0113] like Figure 12 As shown, Figure 12This is a schematic flowchart of a zero-field-based flow cytometry fluorescence detection method according to the present invention, which includes the following steps:

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] Therefore, 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 frequency of resonance between some or all NV color centers and the microwave field is the natural frequency. The microwave field is used to provide a microwave radiation signal with this fixed frequency, which can improve the signal-to-noise ratio of the fluorescence signal and thus improve the detection sensitivity of the flow cytometry fluorescence detection system.

[0124] Optionally, based on the above technical solutions, such as Figure 10 As shown, 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 energy level splitting of the NV color center in the nanodiamond caused by the magnetic field is less than half of the broadening of its photodetector magnetic resonance spectrum.

[0125] "Energy level splitting of NV color centers in nanodiamonds" refers to the shift in the position of the resonance peak in the photodetector magnetic resonance spectrum caused by the Zeeman shift of the NV energy level due to the external magnetic field—that is, half of the difference between the two microwave resonance frequencies corresponding to the energy level splitting of the same NV color center; "spectral broadening of its photodetector magnetic resonance spectrum" refers to the half-width at half-maximum (WHM) of the resonance peak in the photodetector magnetic resonance spectrum of the NV color center.

[0126] When the magnetic field monitoring module 800 monitors the magnetic field strength near the detection area S1 of the sample flow channel 100 and meets the zero-field condition, the fixed frequency of the microwave radiation signal provided by the microwave field is greater than or equal to 2820MHz and less than or equal to 2920MHz. This allows all NV color centers in the crystal axis direction within the nanodiamond to be quantum-state regulated by microwaves with a frequency greater than or equal to 2820MHz and less than or equal to 2920MHz at the zero-field split. Thus, the system can use a frequency greater than or equal to 2820MHz and less than or equal to 2920MHz as the fixed operating frequency of the microwave radiation signal provided by the microwave radiation structure 300.

[0127] It should be noted that, in this embodiment of the invention, the spectral peak shift caused by the magnetic field is required not to exceed the intrinsic broadening of the resonance (CW) spectrum, which is determined by its dephase time. However, in nanodiamonds with different fabrication processes and diameters, the noise environment of the NV color centers varies, corresponding to different dephase times, requiring the zero-field constraint to be determined based on the selected particle. Taking HPHT-fabricated nanodiamonds with a diameter of 40 nm and an NV concentration of 1.5 ppm as an example, its typical intrinsic broadening of the CW spectrum is 20 MHz, corresponding to a required external magnetic field below 3.56 Gs. The resonance (CW) spectrum is... Figure 10 Spectral lines in.

[0128] S120, the photodetector module collects fluorescence signals at a preset collection frequency.

[0129] like Figure 11As shown, the flow cytometry fluorescence detection system includes at least two photodetector modules 400, which can ensure that the modulated fluorescence signals at all angles around the detection area S1 can be collected by the photodetector modules 400.

[0130] For example, Figure 1 and Figure 11 Two photodetector modules 400 are shown, arranged symmetrically around the detection area S1 of the sample flow channel 100. When the number of photodetector modules 400 is greater than two, multiple photodetector modules 400 are arranged around the detection area S1 of the sample flow channel 100, ensuring that fluorescence signals from all angles circumferentially around the detection area S1 can be collected by the photodetector modules 400.

[0131] The above technical solution can, on the one hand, increase the amount of fluorescence signal collected, thereby providing a detection limit; on the other hand, it can overcome the problem of the target object obscuring the fluorescence of the NV color center.

[0132] Since the fluorescence intensity of a single nanodiamond particle is weak in single-molecule detection applications, the photodetector module 400 preferably uses an avalanche photodiode photodetector (APD), followed by a scientific camera, and then a photomultiplier tube sensor (PMT).

[0133] In addition, interference from excitation light and stray fluorescence signals can be filtered out by using notch filters, long-pass filters, and short-pass filters.

[0134] S130 The data processing module determines whether the target analyte contains the biomolecule to be detected based on the fluorescence signal.

[0135] Specifically, the frequency or power of the microwave radiation signal provided by the microwave field varies periodically, the number of NV color centers and microwave field resonances varies periodically, and / or the degree of NV color center resonance varies periodically. The NV color centers generate fluorescence signals with periodically varying intensity. The first judgment method is adopted: the data processing module 700 performs Fourier transform on the fluorescence signal to obtain the frequency domain signal, where the part with the same frequency as the control signal is the demodulated signal; if the intensity of the demodulated signal is greater than the first preset threshold, it is determined that the target object contains the biomolecule to be detected.

[0136] The microwave radiation module 300 is in the "on" state under the control of the microwave field activation signal. Electrons in the NV color centers undergo transitions, generating a dark-state fluorescence signal. This dark-state fluorescence signal is the fluorescence signal generated by the NV color centers within the nanodiamond when some or all of the NV color centers resonate with the microwave field. The microwave radiation module 300 is in the "off" state under the control of the microwave field deactivation signal. Electrons in the NV color centers 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 no NV color centers resonate with the microwave field. The intensity of the dark-state fluorescence signal is less than the intensity of the bright-state fluorescence signal. Using a second judgment method, if the intensity of the dark-state fluorescence signal is less than the intensity of the bright-state fluorescence signal, and the absolute value of the difference between the intensities of the dark-state and bright-state fluorescence signals is greater than a second preset threshold, the data processing module 700 determines that the target analyte contains the biomolecule to be detected. The second preset threshold is at least one times the noise level when the bright-state fluorescence signal is generated.

[0137] This invention provides a zero-field-based flow cytometry fluorescence detection method. Unlike traditional photodetectors based on NV centers, the nanodiamond particles to be tested are not stably laid flat on a substrate, but are placed inside the sample channel 100, in a state of motion. Target materials specifically bound to nanodiamonds flow through the detection area S1 of the sample channel 100. The nanodiamonds include NV centers. When 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 area 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 area S1 of the sample 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 centers within the nanodiamonds; or, changes in the frequency or power of the microwave radiation signal cause corresponding changes in the fluorescence signal generated by the NV centers within the nanodiamonds. In other words, by controlling at least one of the intensity, frequency, and on / off states of the microwave field, the nanodiamonds emit fluorescence signals with periodically varying intensity. The optical detection module 400 collects the fluorescence signal generated by the NV centers within the nanodiamond as the target material specifically bound to nanodiamond passes through the detection region S1 of the sample channel 100 at a preset collection frequency. Since the fluorescence signal is correlated with whether the target analyte contains the biomolecule to be detected, it can be used to determine whether the target analyte contains the biomolecule to be detected. Due to the stability, room temperature and atmospheric environment compatibility, and biocompatibility of the NV center system, and because nanodiamond, as a novel biofluorescent label, can eliminate low-frequency noise interference through signal modulation and demodulation techniques, it can achieve a detection sensitivity far exceeding that of other fluorescence labeling detection methods. In summary, the technical solution provided by this embodiment improves the detection sensitivity of the fluorescence detection method, enabling the identification of single-molecule fluorescence signals. Furthermore, in this embodiment, the magnetic field strength near the detection region S1 of the sample channel 100 satisfies the zero-field condition, and the frequency at which some or all of the NV centers resonate with the microwave field is their natural frequency. The microwave field is used to provide a microwave radiation signal with this fixed frequency, which can improve the signal-to-noise ratio of the fluorescence signal, thereby improving the detection sensitivity of the flow cytometry fluorescence detection method.

[0138] Optionally, based on the above technical solutions, such as Figure 13 As shown, Figure 13 yes Figure 12 The flowchart of S110 includes the following: When the target material labeled with nanodiamonds specifically binds to S110 and passes through the detection area of ​​the sample channel, the NV color centers within the nanodiamonds generate fluorescence signals, including:

[0139] S1101, The control module sends a control signal, which includes at least one of the following: microwave field on signal, microwave field off signal, microwave field power periodic change signal, and microwave field frequency periodic change signal.

[0140] S1102. The microwave radiation module is in the open state according to the microwave field opening signal. The microwave radiation module provides a microwave field to the detection area of ​​the sample flow channel. The NV color centers in the nanodiamond emit dark-state fluorescence signals under the excitation light. The dark-state fluorescence signal is the fluorescence signal generated by the NV color centers in the nanodiamond when some or all of the NV color centers resonate with the microwave field.

[0141] Specifically, the microwave radiation module 300 is in the open state under the control of the microwave field open signal, and there is a microwave radiation signal in the detection area S1. Under the irradiation of the excitation light, the electrons of the NV color centers in the nanodiamond undergo transitions to generate dark-state fluorescence signals. The dark-state fluorescence signals are the fluorescence signals generated by the NV color centers in the nanodiamond when some or all of the NV color centers resonate with the microwave field.

[0142] S1103. According to the microwave field shut-off signal, the microwave radiation module is in the off state. The microwave radiation module stops providing microwave field to the detection area of ​​the sample flow channel. The NV color center in the nanodiamond emits a bright fluorescence signal under the excitation light. The bright fluorescence signal is the fluorescence signal generated by the NV color center in the nanodiamond when there is no NV color center resonating with the microwave field.

[0143] The microwave radiation module 300 is in the off state under the control of the microwave field shutdown signal, and there is no microwave radiation signal in the detection area S1. Under the irradiation of the excitation light, the electrons of the NV color centers in 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 in 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.

[0144] Alternatively, S1104, the microwave radiation module, emits a microwave modulation field with a fixed frequency and periodically varying power based on the periodically changing power signal of the microwave field, causing the NV color centers in the nanodiamond to emit a fluorescence signal with periodically varying intensity under excitation light irradiation.

[0145] Under the control of a periodically changing microwave field power signal, the microwave radiation module 300 emits a microwave modulation field with a fixed frequency and periodically changing power. This causes the electrons in the NV color centers within the nanodiamond to undergo transitions under excitation light irradiation. The number of NV color centers resonating with the microwave field changes periodically, and / or the degree of NV color center resonance changes periodically. As a result, the NV color centers generate fluorescence signals with periodically changing intensity.

[0146] Alternatively, S1105, the microwave radiation module, emits a microwave modulation field with fixed power and periodically changing frequency based on the periodically changing frequency signal of the microwave field, causing the NV color centers in the nanodiamond to emit a fluorescence signal with periodically changing intensity under excitation light irradiation.

[0147] Under the control of a periodically changing microwave field frequency signal, the microwave radiation module 300 emits a microwave modulation field with a fixed power and a periodically changing frequency. This causes the electrons in the NV color centers within the nanodiamond to undergo transitions under excitation light irradiation. The number of NV color centers resonating with the microwave field changes periodically, and / or the degree of NV color center resonance changes periodically. As a result, the NV color centers generate fluorescence signals with periodically changing intensity.

[0148] The above technical solution provides a scheme for the microwave radiation module 300 to emit a fluorescent signal that changes accordingly when the power or frequency of the microwave radiation signal emitted at a fixed frequency changes periodically under the control of the control signal, so that the NV color center in the nanodiamond emits a fluorescent signal that changes accordingly under the excitation light.

[0149] Optionally, based on the above technical solutions, such as Figure 14 As shown, Figure 14 yes Figure 12 The flowchart of S130 shows that the S130 data processing module determines whether the target analyte contains the biomolecule to be detected based on the fluorescence signal, including:

[0150] S1301 The data processing module performs a Fourier transform on the fluorescence signal to obtain a frequency domain signal, where the part with the same frequency as the control signal is the demodulated signal.

[0151] S1302. If the intensity of the demodulated signal is greater than the first preset threshold, it is determined that the target object contains the biomolecule to be detected.

[0152] The above technical solution addresses a method for determining whether a target analyte contains a biomolecule to be detected based on the fluorescence signal generated by the NV color center, where 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.

[0153] Optionally, based on the above technical solution, the S130 data processing module determines whether the target analyte contains the biomolecule to be detected based on the modulated fluorescence signal, including:

[0154] If the intensity of the dark fluorescence signal is less than that of the bright fluorescence signal, and the absolute value of the difference between the intensity of the dark fluorescence signal and the intensity of the bright fluorescence signal is greater than the second preset threshold, the data processing module determines that the target analyte contains the biomolecule to be detected. The second preset threshold is at least 1 times the noise level when the bright fluorescence signal is generated.

[0155] The above technical solution addresses a method for determining whether a target analyte contains the biomolecule to be detected, based on fluorescence signals, when the microwave radiation module 300 is in an on or off state under the control of a control signal. Specifically, when the microwave radiation module 300 is in an on state under the control of a microwave field on signal, electrons in the NV color centers undergo transitions, generating a dark-state fluorescence signal. This dark-state fluorescence signal is the fluorescence signal generated by the NV color centers within the nanodiamond when some or all of the NV color centers resonate with the microwave field. When the microwave radiation module 300 is in an off state under the control of a microwave field off signal, electrons in the NV color centers 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 no NV color centers resonate with the microwave field.

[0156] Optionally, based on the above technical solution, the number of periodically changing fluorescence signals exhibited by a single target within the detection area is: indivual;

[0157] Where N is the number of target objects flowing through the detection area of ​​the sample flow channel per second, and the value of N is an integer greater than or equal to 1; F M To control the frequency of the signal.

[0158] like Figure 15 As shown, if the flow cytometry system detects N targets per second, then the time required for a single target to flow through the detection area S1 is 1 / N seconds. Let F... M To control the frequency of the signal, a single target object appears within the detection area S1. The above technical solution provides the relationship between the frequency of the control signal and the flow rate of the target object.

[0159] Here, 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.

[0160] Optionally, based on the above technical solution, when the target material passes through the detection area, the number of detection windows acquired by the photodetector module within the period of a single fluorescence signal satisfies the following relationship:

[0161] M = F s / F M

[0162] Among them, F s F is the sampling frequency of the optical detection module. M To control the frequency of the signal.

[0163] The above technical solution provides the relationship between the sampling frequency of the optical detection module 400 and the frequency of the control signal, such as... Figure 16 As shown, the number of detection windows collected by the photodetector module 400 within a single fluorescence signal period is also given when the target material passes through the detection area S1.

[0164] Optionally, based on the above technical solution, when the target material specifically bound to nanodiamond by S110 passes through the detection area of ​​the sample channel, after the NV color center within the nanodiamond generates a fluorescence signal, the process includes:

[0165] After each detection window of the optical detection module, a detection window without microwave radiation signal is added as a reference signal.

[0166] Within each detection window, the photodetector module 400 simultaneously collects fluorescence signals from the entire detection area; a pinhole is added before the fluorescence collector for spatial filtering to reduce interference from background stray signals; after a single detection window, an unmodulated microwave-controlled detection window can be added as a reference signal to calibrate the temporal jitter and spatial inhomogeneity of the excitation light.

[0167] Optionally, based on the above technical solution, several scientific cameras are used as the photodetector module 400. Here, sCMOS high-speed scientific cameras or enhanced electronically coupled devices (ICCDs) can be selected. Within each detection window, the detector performs fluorescence imaging of the entire detection area. A pinhole can be added in front of the fluorescence collector for spatial filtering to reduce the interference of background stray signals. For the fluorescence image of each detection window, the position trajectory of the nanodiamond fluorescent particles in the image is identified by an algorithm. Several points are selected outward from the trajectory as the center as the fluorescence count of the nanodiamond particles in that window, and the background fluorescence signal of the rest is discarded to improve the signal-to-noise ratio of the fluorescence signal to be measured.

[0168] Optionally, based on the above technical solution, the particle size range of nanodiamond is greater than or equal to 40 nm and less than or equal to 1 μm; preferably, the particle size range of nanodiamond is greater than or equal to 100 nm and less than or equal to 300 nm.

[0169] The fluorescence counting rate, fluorescence signal contrast, and exposure time of a single detection window for nanodiamonds satisfy the following relationship:

[0170]

[0171] Where C is the contrast of the fluorescence signal, R is the fluorescence count rate of the nanodiamond, and t is the exposure time of a single detection window; NV color center |m s =0> Bright state and |m s =±1> The relative count difference between dark states is the contrast.

[0172] The particle size range of nanodiamonds meets the above range, and the contrast of the fluorescence signal, the fluorescence count rate of nanodiamonds, and the exposure time of a single detection window meet the above formula. At this time, within each detection window, the fluorescence signal amplitude of the nanodiamond particles is greater than the noise amplitude. Here, the main consideration is photon shot noise, which can ensure that the signal-to-noise ratio of a single measurement is greater than 1:1, thereby improving the signal-to-noise ratio of the fluorescence signal.

[0173] Optionally, based on the above technical solution, the excitation optical module 200 includes an optical pump, which is used to emit laser light;

[0174] The contrast of the fluorescence signal generated by the NV color centers within nanodiamond satisfies the following relationship:

[0175]

[0176] Where Θ is the normalization constant, Γ p Γ represents the laser polarization rate. c For the coherent relaxation rate caused by optical pumping, Ω R Γ is the frequency of the Rabi oscillation of the NV color center driven by the microwave field. p ,Γ c Two items are related to laser power, Ω R The term is positively correlated with the microwave field intensity sensed by the NV color center within the nanodiamond.

[0177] The above technical solution provides a formula for calculating the contrast of the fluorescence signal generated by the NV color center in nanodiamond. In order to ensure that the fluorescence signal amplitude of a single nanodiamond particle remains consistent within the detection area, the laser power density and microwave field strength distribution within the detection area should be as uniform as possible.

[0178] Optionally, based on the above technical solution, the microwave field intensity sensed by the NV color center within the nanodiamond satisfies the following relationship:

[0179]

[0180] Where B is the intensity of the microwave field sensed by the NV color center within the nanodiamond, and B1 is the intensity of the microwave field provided by the microwave radiation module 300.

[0181] Unlike traditional photodetectors based on NV centers, the flow cytometry fluorescence detection method involves a moving nanodiamond particle, which is not stably laid flat on the substrate but rather placed inside the sample channel 100. The nanodiamond selected in this embodiment contains hundreds of NV centers with different crystal axis orientations. Therefore, it can be assumed that the angle between the principal axis of the NV center and the microwave field is completely averaged. The microwave field intensity sensed by all NV centers within a single nanodiamond particle is:

[0182]

[0183] As can be seen from the above formula, the microwave field intensity sensed by all NV color centers within a single nanodiamond particle is proportional to the microwave field intensity provided by the microwave radiation module 300.

[0184] Optionally, based on the above technical solution, for a single detection window, the excitation light remains constantly on, the microwave radiation module 300 provides a microwave radiation signal, and the photodetector module 400 uses the entire length of the detection window as the exposure time to obtain the fluorescence signal intensity of the nanodiamond within a single sampling point.

[0185] Specifically, for a single detection window, the laser remains constantly on, the microwave radiation module 300 provides a microwave radiation signal, and the fluorescence detector uses the entire length of the detection window as the exposure time to obtain the fluorescence signal intensity of the nanodiamond particles within a single sampling point. This method results in a high excitation light duty cycle and a high number of effective fluorescent photons emitted by the nanodiamond particles collected by the photodetector module 400.

[0186] Optionally, based on the above technical solution, the delay time between the excitation light initialization pulse and the fluorescence signal collection pulse window is greater than a preset delay time.

[0187] Specifically, due to the long fluorescence lifetime of NV centers, an appropriate pulse-delayed fluorescence window can be selected using a detector. This means the delay time between the excitation initialization pulse and the fluorescence signal collection pulse window is greater than a preset delay time, such as 20 ns. Then, the excitation-wait-fluorescence collection sequence is repeated multiple times to obtain the fluorescence signal intensity of a single sampling point. At this point, the background fluorescence signal caused by non-target substances has a short lifetime and has significantly attenuated; however, the NV center fluorescence in the nanodiamond particles still exists, thus filtering out background fluorescence interference over time and improving the signal-to-noise ratio of the detection.

[0188] The technical solution provided in this invention, in order to improve the signal-to-noise ratio of the fluorescence signal and enhance the accuracy of analyte identification, introduces a microwave field to modulate the fluorescence signal of the nanodiamond particles: when the microwave radiation module 300 emits microwaves greater than or equal to 2820MHz and less than or equal to 2920MHz into the radiation coil, the NV color centers within the nanodiamond particles in the sample flow channel 100 change from |m s =0> State transition to |m s =±1> state, the fluorescence intensity of nanodiamond particles decreases; when the microwave in the radiation coil is turned off, the excitation light in the detection area S1 initializes the NV color center to |m s =0> state, fluorescence intensity recovers. When the radiation power of the microwave radiation signal provided by the microwave radiation module 300 changes, the NV color centers within the nanodiamond generate a correspondingly changing fluorescence signal. The fluorescence signal is modulated in the time domain by the microwave radiation module 300, then collected at a certain frequency by the photodetector module 400, and finally the specific frequency of the test signal is identified by an algorithm to eliminate interference from static noise signals.

[0189] This invention also provides a zero-field flow cytometry fluorescence detection system, which is applicable to any of the zero-field flow cytometry fluorescence detection methods described in this invention. This zero-field flow cytometry fluorescence detection system incorporates the beneficial effects of any of the zero-field flow cytometry fluorescence detection methods described in this invention, which will not be elaborated further here.

[0190] like Figure 17 As shown, Figure 17 This is a schematic flowchart of another zero-field-based flow cytometry fluorescence detection method according to an embodiment of the present invention. The zero-field-based flow cytometry fluorescence detection method includes the following steps:

[0191] S210. When the target material specifically bound to nanodiamond passes through the first half of the detection area of ​​the sample channel, the NV color centers within the nanodiamond generate a fluorescence signal, including a dark-state fluorescence signal. The dark-state fluorescence signal is the fluorescence signal generated by the NV color centers within the nanodiamond when some or all of the NV color centers resonate with the microwave field.

[0192] 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.

[0193] When the target material specifically bound to nanodiamond passes through the first half of the detection area S1 of the sample flow channel 100, the microwave radiation module 300 is in the open state, and there is a microwave radiation signal in the detection area S1. Under the irradiation of the excitation light, the electrons of the NV color centers in the nanodiamond undergo transitions to generate dark-state fluorescence signals. The dark-state fluorescence signals are the fluorescence signals generated by the NV color centers in the nanodiamond when some or all of the NV color centers resonate with the microwave field.

[0194] S220. When the target material specifically bound to nanodiamond passes through the latter half of the detection area of ​​the sample channel, the NV color center within the nanodiamond generates a fluorescence signal, including a bright fluorescence signal. The bright fluorescence signal is the fluorescence signal generated by the NV color center within the nanodiamond when there is no NV color center and it resonates with the microwave field.

[0195] The excitation light module emits excitation light, which excites the NV centers in the detection area of ​​the sample flow channel, causing their electrons to transition and generate fluorescence. The microwave radiation module provides a microwave field to the detection area of ​​the sample flow channel. The microwave field resonates with all or part of the NV centers in the nanodiamond. The frequency of the microwave radiation signal provided by the microwave field is equal to the energy level difference of the electron spin transition of the NV centers, which enables the microwave field to resonate with the NV centers. The microwave radiation module is in an on or off state under the control of the control signal. When the microwave radiation module is in the on state, the NV centers in the nanodiamond generate a dark fluorescence signal. When the microwave radiation module is in the off state, the NV centers in the nanodiamond generate a bright fluorescence signal.

[0196] When the target material specifically bound to nanodiamond passes through the latter half of the detection area S1 of the sample flow channel 100, the microwave radiation module 300 is off, and 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.

[0197] S230, the photodetector module collects fluorescence signals at a preset collection frequency.

[0198] like Figure 11 As shown, the flow cytometry fluorescence detection system includes at least two photodetector modules 400; the at least two photodetector modules 400 are arranged around the detection area S1 of the sample flow channel, which can ensure that the fluorescence signals at all angles in the circumference of the detection area S1 can be collected by the photodetector modules 400.

[0199] For example, Figure 1 and Figure 11Two optical detection modules 400 are shown, which are arranged around the detection area S1 of the sample flow channel 100 and are symmetrical about the detection area S1.

[0200] When the number of photodetector modules 400 is greater than two, multiple photodetector modules 400 are arranged around the detection area S1 of the sample flow channel 100. It is necessary to ensure that the fluorescence signal at each angle in the circumferential direction of the detection area S1 can be collected by the photodetector modules 400.

[0201] The above technical solution can, on the one hand, increase the amount of fluorescence signal collected, thereby providing a detection limit; on the other hand, it can overcome the problem of the target object obscuring the fluorescence of the NV color center.

[0202] Since the fluorescence intensity of a single nanodiamond particle is weak in single-molecule detection applications, the photodetector module 400 preferably uses an avalanche photodiode photodetector (APD), followed by a scientific camera, and then a photomultiplier tube sensor (PMT).

[0203] In addition, interference from excitation light and stray fluorescence signals can be filtered out by using notch filters, long-pass filters, and short-pass filters.

[0204] S240 The 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 second fluorescence signal.

[0205] The microwave radiation module 300 is in the "on" state under the control of the microwave field activation signal. Electrons in the NV color centers undergo transitions, generating a dark-state fluorescence signal. This dark-state fluorescence signal is the fluorescence signal generated by the NV color centers within the nanodiamond when some or all of the NV color centers resonate with the microwave field. The microwave radiation module 300 is in the "off" state under the control of the microwave field deactivation signal. Electrons in the NV color centers undergo transitions, generating a second fluorescence signal. This second fluorescence signal is the fluorescence signal generated by the NV color centers within the nanodiamond when no NV color centers resonate with the microwave field. The intensity of the dark-state fluorescence signal is less than the intensity of the second fluorescence signal. Therefore, if the intensity of the dark-state fluorescence signal is less than the intensity of the second fluorescence signal, the data processing module 700 determines that the target analyte contains the biomolecule to be detected.

[0206] This invention provides a zero-field-based flow cytometry fluorescence detection method. Unlike traditional photodetectors based on NV centers, the nanodiamond particles to be tested are not stably laid flat on a substrate, but are placed inside the sample channel 100 and are in motion. The target material specifically bound to nanodiamond particles flows through the detection region S1 of the sample channel 100. The nanodiamond particles include NV centers. When the NV centers within the nanodiamond particles 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 their electrons to transition and generate fluorescence. When the target material specifically bound to nanodiamond particles flows through the first half of the detection region S1 of the sample channel 100, the microwave radiation module 300 is in the open state, and a microwave radiation signal exists in the detection region S1. Under the irradiation of the excitation light, the electrons of the NV centers within the nanodiamond particles undergo transitions, generating a dark-state fluorescence signal. When the target material specifically bound to nanodiamond passes through the latter half of the detection area S1 of the sample flow channel 100, the microwave radiation module 300 is off, and there is no microwave radiation signal in the detection area S1. Under excitation light irradiation, the electrons of the NV centers within the nanodiamond undergo transitions, generating a bright-state fluorescence signal. The photodetector module 400 collects the fluorescence signal generated by the NV centers within the nanodiamond as the target material specifically bound to nanodiamond passes through the detection area S1 of the sample flow channel 100 at a preset collection frequency. Since the fluorescence signal is correlated with whether the target contains the biomolecule to be detected, the data processing module 700 determines whether the target 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. Due to the stability, room temperature and atmospheric environment compatibility, and biocompatibility of the NV center system, and because nanodiamond, as a novel biofluorescent label, can eliminate low-frequency noise interference through signal modulation and demodulation technology, a detection sensitivity far exceeding that of other fluorescence labeling detection methods can be achieved. In summary, the technical solution provided by this embodiment of the invention improves the detection sensitivity of the fluorescence detection method and can achieve single-molecule fluorescence signal recognition.

[0207] Optionally, based on the above technical solutions, such as Figure 18 As shown, when the S210-specifically bound nanodiamond-labeled target stream passes through the first half of the detection area of ​​the sample channel, the NV color centers within the nanodiamond generate fluorescence signals. These fluorescence signals include dark-state fluorescence signals.

[0208] S2101, The control module sends control signals, including microwave field turn-on signals and microwave field turn-off signals.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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:

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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:

[0217] If the intensity of the dark-state fluorescence signal is less than the intensity of the bright-state fluorescence signal, and the absolute value of the difference between the intensity of the dark-state fluorescence signal and the intensity of the bright-state fluorescence signal is greater than a second preset threshold, it is determined that the target analyte contains the biomolecule to be detected. The second preset threshold is at least 1 times the noise level when the bright-state fluorescence signal is generated.

[0218] The above technical solution addresses a method for determining whether a target object contains the biomolecule to be detected by a data processing module 700 when the microwave radiation module 300 is in an on or off state under the control of a control signal, based on the fluorescence signal.

[0219] Optionally, based on the above technical solution, after the NV color center within the nanodiamond generates a fluorescence signal when the target material specifically bound to the S220 passes through the latter half of the detection area of ​​the sample flow channel, the process further includes:

[0220] After each detection window of the optical detection module, a detection window without microwave radiation signal is added as a reference signal.

[0221] Within each detection window, the detector simultaneously collects fluorescence signals from the entire detection area; a pinhole is added before the fluorescence collector for spatial filtering to reduce interference from background stray signals; after each detection window, an unmodulated microwave-controlled detection window can be added as a reference signal to calibrate the temporal jitter and spatial inhomogeneity of the excitation light.

[0222] Optionally, based on the above technical solution, several scientific cameras are used as the photodetector module 400. Here, sCMOS high-speed scientific cameras or enhanced electronically coupled devices (ICCDs) can be selected. Within each detection window, the detector performs fluorescence imaging of the entire detection area. A pinhole can be added in front of the fluorescence collector for spatial filtering to reduce the interference of background stray signals. For the fluorescence image of each detection window, the position trajectory of the nanodiamond fluorescent particles in the image is identified by an algorithm. Several points are selected outward from the trajectory as the center as the fluorescence count of the nanodiamond particles in that window, and the background fluorescence signal of the rest is discarded to improve the signal-to-noise ratio of the fluorescence signal to be measured.

[0223] Optionally, based on the above technical solution, the magnetic field strength in the detection area meets the zero-field condition, and the microwave radiation signal provided by the microwave field has a fixed frequency.

[0224] Optionally, based on the above technical solutions, such as Figure 1 and Figure 2As 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.

[0225] 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.

[0226] Therefore, 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 frequency of resonance between some or all NV color centers and the microwave field is the natural frequency. The microwave field is used to provide a microwave radiation signal with this fixed frequency, which can improve the signal-to-noise ratio of the fluorescence signal and thus improve the detection sensitivity of the flow cytometry fluorescence detection system.

[0227] Optionally, based on the above technical solutions, such as Figure 10 As shown, 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 energy level splitting of the NV color center in the nanodiamond caused by the magnetic field is less than half of the broadening of its photodetector magnetic resonance spectrum.

[0228] "Energy level splitting of NV color centers in nanodiamonds" refers to the shift in the position of the resonance peak in the photodetector magnetic resonance spectrum caused by the Zeeman shift of the NV energy level due to the external magnetic field—that is, half of the difference between the two microwave resonance frequencies corresponding to the energy level splitting of the same NV color center; "its photodetector magnetic resonance spectrum line broadening" refers to the half-width at half-maximum (WHM) of the resonance peak in the photodetector magnetic resonance spectrum of the NV color center.

[0229] When the magnetic field monitoring module 800 monitors the magnetic field strength near the detection area S1 of the sample flow channel 100 and meets the zero-field condition, the fixed frequency of the microwave radiation signal provided by the microwave field is greater than or equal to 2820MHz and less than or equal to 2920MHz. This allows all NV color centers in the crystal axis direction within the nanodiamond to be quantum-state-controlled by microwaves with a frequency greater than or equal to 2820MHz and less than or equal to 2920MHz at the zero-field split. Thus, the system can use a frequency greater than or equal to 2820MHz and less than or equal to 2920MHz as the fixed operating frequency of the microwave radiation signal provided by the microwave radiation module 300.

[0230] It should be noted that, in this embodiment of the invention, the spectral peak shift caused by the magnetic field is required not to exceed the intrinsic broadening of the resonance (CW) spectrum, which is determined by its dephase time. However, in nanodiamonds with different fabrication processes and diameters, the noise environment of the NV color centers varies, corresponding to different dephase times, requiring the zero-field constraint to be determined based on the selected particle. Taking HPHT-fabricated nanodiamonds with a diameter of 40 nm and an NV concentration of 1.5 ppm as an example, its typical intrinsic broadening of the CW spectrum is 20 MHz, corresponding to a required external magnetic field below 3.56 Gs. The resonance (CW) spectrum is... Figure 10 Spectral lines in.

[0231] Optionally, based on the above technical solution, the particle size range of nanodiamond is greater than or equal to 40 nm and less than or equal to 1 μm; preferably, the particle size range of nanodiamond is greater than or equal to 100 nm and less than or equal to 300 nm.

[0232] When the target material passes through the detection area, within the period of a single fluorescence signal, the number of detection windows acquired by the photodetector module is at least one. The fluorescence count rate of the nanodiamond, the contrast of the fluorescence signal, and the exposure time of a single detection window satisfy the following relationship:

[0233]

[0234] Where C is the contrast of the fluorescence signal, R is the fluorescence count rate of the nanodiamond, and t is the exposure time of a single detection window; NV color center |m s =0> Bright state and |m s =±1> The relative count difference between dark states is the contrast.

[0235] The particle size range of nanodiamonds meets the above range, and the contrast of the fluorescence signal, the fluorescence count rate of nanodiamonds, and the exposure time of a single detection window meet the above formula. At this time, within each detection window, the amplitude of the fluorescence modulation signal of the nanodiamond particles is greater than the amplitude of the noise. Here, the main consideration is photon shot noise, which can ensure that the signal-to-noise ratio of a single measurement is greater than 1:1, thereby improving the signal-to-noise ratio of the fluorescence signal.

[0236] Optionally, based on the above technical solution, the excitation optical module 200 includes an optical pump, which is used to emit laser light;

[0237] The contrast of the fluorescence signal generated by the NV color centers within nanodiamond satisfies the following relationship:

[0238]

[0239] Where Θ is the normalization constant, Γ p Γ represents the laser polarization rate. c For the coherent relaxation rate caused by optical pumping, Ω R Γ is the frequency of the Rabi oscillation of the NV color center driven by the microwave field. p ,Γ c Two items are related to laser power, Ω R The term is positively correlated with the microwave field intensity sensed by the NV color center within the nanodiamond.

[0240] The above technical solution provides a formula for calculating the contrast of the fluorescence signal generated by the NV color center in nanodiamond. In order to ensure that the fluorescence signal amplitude of a single nanodiamond particle remains consistent within the detection area, the laser power density and microwave field strength distribution within the detection area should be as uniform as possible.

[0241] Optionally, based on the above technical solution, the microwave field intensity sensed by the NV color center within the nanodiamond satisfies the following relationship:

[0242]

[0243] Where B is the intensity of the microwave field sensed by the NV color center within the nanodiamond, and B1 is the intensity of the microwave field provided by the microwave radiation module.

[0244] Unlike traditional optical detection methods based on NV centers, the flow cytometry method involves the nanodiamond particles being tested not being stably laid flat on the substrate, but rather placed inside the flow channel and in motion. The nanodiamond particles we selected contain hundreds of NV centers with different crystal axis orientations. Therefore, it can be assumed that the angle between the principal axes of the NV centers and the microwave field is completely averaged. The microwave field intensity sensed by all NV centers within a single nanodiamond particle is:

[0245]

[0246] As can be seen from the above formula, the microwave field intensity sensed by all NV color centers within a single nanodiamond particle is directly proportional to the microwave field intensity.

[0247] Optionally, based on 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 photodetector module uses the entire length of the detection window as the exposure time to obtain the fluorescence signal intensity of the nanodiamond within a single sampling point.

[0248] Specifically, for a single detection window, the laser remains constantly on, the microwave radiation module provides the microwave radiation signal, and the fluorescence detector uses the entire length of the detection window as the exposure time to obtain the fluorescence signal intensity of the nanodiamond particles within a single sampling point. This method results in a high excitation light duty cycle and a high number of effective fluorescent photons emitted by the nanodiamond particles collected by the photodetector module.

[0249] Optionally, based on the above technical solution, the delay time between the excitation light initialization pulse and the fluorescence signal collection pulse window is greater than a preset delay time.

[0250] Specifically, due to the long fluorescence lifetime of NV centers, an appropriate pulse-delayed fluorescence window can be selected using a detector. This means the delay time between the excitation initialization pulse and the fluorescence signal collection pulse window is greater than a preset delay time, such as 20 ns. Then, the excitation-wait-fluorescence collection sequence is repeated multiple times to obtain the fluorescence signal intensity of a single sampling point. At this point, the background fluorescence signal caused by non-target substances has a short lifetime and has significantly attenuated; however, the NV center fluorescence in the nanodiamond particles still exists, thus filtering out background fluorescence interference over time and improving the signal-to-noise ratio of the detection.

[0251] The technical solution provided in this invention aims to improve the signal-to-noise ratio of fluorescence signals and enhance the accuracy of analyte identification. The system introduces a microwave field to modulate the fluorescence signal of nanodiamond particles: when the microwave radiation module 300 is activated and microwaves with a frequency greater than or equal to 2820 MHz and less than or equal to 2920 MHz enter the radiation coil, the NV color centers within the nanodiamond particles in the sample flow channel 100 change from |m s =0> State transition to |m s =±1> state, the fluorescence intensity of nanodiamond particles decreases; when the microwave in the radiation coil is turned off, the excitation light in the detection area S1 initializes the NV color center to |m s=0> state, fluorescence intensity recovers. When the radiation power of the microwave radiation signal provided by the microwave radiation module 300 changes, the NV color centers in the nanodiamond generate a correspondingly changing fluorescence signal. The fluorescence signal is modulated in the time domain by the microwave radiation module 300, then collected at a certain frequency by the photodetector module 400, and finally the target signal at a specific frequency is identified by an algorithm to eliminate the interference of static noise signals. Specifically, when the target object passes through the detection area S1, the microwave radiation module 300 is turned on to modulate microwaves in the first half of the detection area S1, manipulating the NV color centers in the nanodiamond particles in the detection area S1 from |m s =0> State transition to |m s =±1> state; the microwave field is turned off in the second half of the detection area S1, and the NV color center is initialized to |m by laser. s =0> state; by comparing the fluorescence signal intensity of nanodiamond in the two detection areas S1 before and after, it can be determined whether the analyte contains nanodiamond label and whether the target analyte contains the biomolecule to be detected.

[0252] This invention also provides a flow cytometry fluorescence detection system based on microwave field switching states. This system is applicable to any of the flow cytometry fluorescence detection methods based on microwave field switching states described in this invention. The beneficial effects of this system on microwave field switching states, as described in any of the flow cytometry fluorescence detection methods based on microwave field switching states, are not repeated here.

[0253] It should be understood that the various forms of processes shown above can be used, with steps reordered, added, or deleted. For example, the steps described in this invention can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution of this invention can be achieved, and this is not limited herein.

[0254] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A flow cytometry fluorescence detection method based on zero field, characterized in that, include: When the target material specifically bound to nanodiamond passes through the detection area of ​​the sample channel, the intensity of the NV color center inside the nanodiamond is periodically changed by controlling at least one of the intensity, frequency and on / off of the microwave field. The detection area's magnetic field strength satisfies the zero-field condition. An excitation light module emits excitation light to excite the NV centers in the detection area of ​​the sample flow channel, causing electrons to transition and generate fluorescence. A microwave radiation module provides a microwave field to the detection area of ​​the sample flow channel. This microwave field can resonate with all or part of the NV centers within the nanodiamond. The frequency of the microwave radiation signal provided by the microwave field is equal to the energy level difference of the electron spin transition of the NV centers, allowing the microwave field to resonate with the NV centers. The microwave radiation signal provided by the microwave field has a fixed frequency. The zero-field condition is the condition that the frequency at which some or all NV centers resonate with the microwave field is their natural frequency. The optical detection module collects the fluorescence signal at a preset collection frequency; The data processing module determines whether the target analyte contains the biomolecule to be detected based on the fluorescence signal.

2. The flow cytometry fluorescence detection method based on zero field according to claim 1, characterized in that, When the magnetic field monitoring module monitors the magnetic field strength near the detection area of ​​the sample flow channel and the condition of zero field is met, the energy level splitting of the NV color center in the nanodiamond caused by the magnetic field is less than half of the broadening of its photodetector magnetic resonance spectrum.

3. The flow cytometry fluorescence detection method based on zero field according to claim 1, characterized in that, The fixed frequency of the microwave radiation signal provided by the microwave field is greater than or equal to D-50 MHz and less than or equal to D+50 MHz, where D is the zero-field splitting parameter of the NV color center.

4. The flow cytometry fluorescence detection method based on zero field according to claim 1, characterized in that, When the target material specifically bound to nanodiamond passes through the detection area of ​​the sample channel, the NV color centers within the nanodiamond generate fluorescence signals, including: The control module sends control signals, which include at least one of the following: microwave field on signal and microwave field off signal, microwave field power periodic change signal and microwave field frequency periodic change signal; According to the microwave field activation signal, the microwave radiation module is in the open state and provides a microwave field for the detection area of ​​the sample flow channel. The NV color centers in the nanodiamond emit dark-state fluorescence signals under excitation light irradiation. The dark-state fluorescence signals are the fluorescence signals generated by the NV color centers in the nanodiamond when some or all of the NV color centers resonate with the microwave field. According to the microwave field shutdown signal, the microwave radiation module is in the off state, and the microwave radiation module stops providing microwave field to the detection area of ​​the sample flow channel. The NV color center in the nanodiamond emits a bright fluorescence signal under the excitation light. The bright fluorescence signal is the fluorescence signal generated by the NV color center in the nanodiamond when there is no resonance between the NV color center and the microwave field. Alternatively, the microwave radiation module emits a microwave modulation field with a fixed frequency and periodically varying power based on the periodically varying power signal of the microwave field power, so that the NV color centers in the nanodiamond emit a fluorescence signal with periodically varying intensity under excitation light irradiation. Alternatively, the microwave radiation module emits a microwave modulation field with fixed power and periodically varying frequency according to the periodically changing frequency signal of the microwave field, causing the NV color centers in the nanodiamond to emit a fluorescence signal with periodically varying intensity under excitation light irradiation.

5. The flow cytometry fluorescence detection method based on zero field according to claim 4, characterized in that, The data processing module determines whether the target analyte contains the biomolecule to be detected based on the fluorescence signal, including: The data processing module performs a 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 demodulated signal; If the intensity of the demodulated signal is greater than a first preset threshold, it is determined that the target object contains the biomolecule to be detected.

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

7. The flow cytometry fluorescence detection method based on zero field according to claim 4, characterized in that, The number of periodically changing fluorescence signals exhibited by a single target within the detection area is indivual; Wherein, N is the number of target objects flowing through the detection area of ​​the sample flow channel per second, and the value of N is an integer greater than or equal to 1; The frequency of the control signal is denoted as .

8. The flow cytometry fluorescence detection method based on zero field according to claim 7, characterized in that, When the target material passes through the detection area, the number of detection windows acquired by the photodetector module within a single fluorescence signal period satisfies the following relationship: ； in, This refers to the sampling frequency of the optical detection module. The frequency of the control signal is denoted as .

9. The flow cytometry fluorescence detection method based on zero field according to claim 8, characterized in that, When the target material specifically bound to nanodiamond passes through the detection area of ​​the sample channel, the NV color center within the nanodiamond generates a fluorescence signal, followed by: After each detection window of the optical detection module, a detection window without microwave radiation signal is added as a reference signal.

10. The zero-field-based flow cytometry fluorescence detection method according to claim 8, characterized in that, The nanodiamonds have a particle size range greater than or equal to 40 nm and less than or equal to 1 μm. The fluorescence counting rate of the nanodiamond, the contrast of the fluorescence signal, and the exposure time of a single detection window satisfy the following relationship: ； Where C is the contrast of the fluorescence signal, R is the fluorescence count rate of the nanodiamond, and t is the exposure time of a single detection window.

11. The flow cytometry fluorescence detection method based on zero field according to claim 10, characterized in that, The excitation optical module includes an optical pump, which is used to emit laser light; The contrast of the fluorescence signal generated by the NV color centers within the nanodiamond satisfies the following relationship: ； Where Θ is the normalization constant, For laser polarization rate, The coherent relaxation rate caused by optical pumping. The frequency of the Rabi oscillation of the NV color center driven by the microwave field. , Both are related to laser power. The term is positively correlated with the microwave field intensity sensed by the NV color center within the nanodiamond.

12. The flow cytometry fluorescence detection method based on zero field according to claim 11, characterized in that, The microwave field intensity sensed by the NV color center within the nanodiamond satisfies the following relationship: ； Wherein, B is the intensity of the microwave field sensed by the NV color center within the nanodiamond, and B1 is the intensity of the microwave field provided by the microwave radiation module.

13. The flow cytometry fluorescence detection method based on zero field according to claim 8, characterized in that, For a single detection window, the excitation light remains constantly on, the microwave radiation module provides a microwave radiation signal, and the photodetector module uses the entire length of the detection window as the exposure time to obtain the fluorescence signal intensity of the nanodiamond within a single sampling point.

14. The flow cytometry fluorescence detection method based on zero field according to claim 8, characterized in that, The delay time between the excitation light initialization pulse and the fluorescence signal collection pulse window is greater than the preset delay time.

15. A zero-field-based flow cytometry fluorescence detection system, characterized in that, The zero-field-based flow cytometry fluorescence detection system is applicable to the zero-field-based flow cytometry fluorescence detection method according to any one of claims 1-14; The zero-field-based flow cytometry fluorescence detection system includes: A sample flow channel is provided for delivering a target object specifically bound to a nanodiamond marker, the nanodiamond including NV color centers, and the sample flow channel is provided with a detection area. An excitation light module is used to emit excitation light, which is used to excite the NV color center in the detection area of ​​the sample flow channel, causing the electrons of the NV color center to undergo transition and generate fluorescence; A microwave radiation module is provided to provide a microwave field to the detection area of ​​the sample flow channel. The microwave field resonates with all or part of the NV centers within the nanodiamond. The frequency of the microwave radiation signal provided by the microwave field is equal to the energy level difference of the electron spin transition of the NV centers, thus causing the microwave field to resonate with the NV centers. The microwave radiation module includes at least two microwave radiation structures distributed along the transport direction of the target object and arranged around the detection area of ​​the sample flow channel. The optical detection module collects the target material specifically bound to nanodiamond labels through the detection area of ​​the sample channel at a preset collection frequency. By controlling at least one of the intensity, frequency, and on / off state of the microwave field, the NV color centers within the nanodiamond emit a fluorescence signal with periodically changing intensity. The fluorescence signal is correlated with whether the target material contains the biomolecule to be detected. A data processing module is used to determine whether the target analyte contains the biomolecule to be detected based on the fluorescence signal. A magnetic field monitoring module is used to monitor the magnetic field strength near the detection area of ​​the sample flow channel; when the magnetic field strength near the detection area of ​​the sample flow channel meets the zero field condition, the microwave field is used to provide a microwave radiation signal with a fixed frequency.

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