CN114778589B - Longitudinal relaxation rate measuring system and measuring method using same - Google Patents
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Abstract
The invention provides a longitudinal relaxation rate measuring system and a measuring method using the system. The measuring method using the system comprises the following steps: s1, connecting a first biomolecule to hydrogel in a biochemical reaction mode; s2, cleaning the hydrogel after the operation of S1, dripping second and third biomolecules with different spin labels onto the hydrogel for reaction, and then fully cleaning to obtain a sample to be detected, wherein the interaction of the first biomolecule with the second and third biomolecules is realized; and S3, measuring the longitudinal relaxation rate of the sample to be measured. The system can realize the measurement of the interaction of the biomolecules, and has the advantages of stable signal, high signal-to-noise ratio, capability of detecting multi-channel biological samples and the like.
Description
Technical Field
The invention relates to the field of biological sample detection, in particular to a longitudinal relaxation rate measuring system and a measuring method using the system.
Background
The conformational changes of the biomolecules themselves and the biomolecular interactions affected by them are fundamental and important processes of biological life activities. Therefore, the detection of the above process is a very important research direction in life science.
Among the many methods of detecting molecular interactions, fluorescent biomarker detection is a widely used method. The method is characterized in that fluorescent molecules capable of being excited by light with a specific wave band are marked on target protein molecules, and then final result detection is carried out by means of a specific optical excitation and collection imaging system. However, one important drawback of fluorescent biomarkers is poor stability. Despite improvements over multiple upgrade iterations, it is still difficult for fluorescent molecules to maintain stable luminescence on the order of minutes in the face of continued illumination with excitation light. This means that fluorescent biomarker detection does not provide a continuous observation measurement for a sufficiently long time for normal life response activity (typically on the order of minutes to hours).
Faced with the above problems, S. Steinert et al (S. Steinert)et al."Magnetic spin imaging under atmospheric conditions with sub-cellular resolution," Nat Commun, Vol 4, number 1, p 1607, Jun 2013, doi: 10.1038/ncomms 2588) is linked to a target protein by using metal ions as biomarkers (hereinafter referred to as ion markers), and then Magnetic noise disturbance is generated on nitrogen-vacancy color centers (NV color centers) in diamond by using the ion markers, so as to detect the reduction of the longitudinal relaxation decay characteristic time (T1). In this way, they also performed signal detection and imaging of interacting proteins. However, the method still faces two problems, namely, the signal to noise ratio is low due to the limited protein connection density, and the subsequent measurement and imaging are influenced; secondly, the method can not carry out differentiated multi-channel detection on the mixed reaction of a plurality of target proteins, thereby distinguishing which protein plays a role in the process.
There is therefore a need for a system and method for measuring biomolecular interactions that can perform multi-channel measurements.
Disclosure of Invention
To solve the problems in the prior art, in one aspect of the present disclosure, there is provided a longitudinal relaxation rate measuring system, which is characterized by comprising a pump light unit 1, a confocal microscopic collection unit 2, a displacement unit 6, a microwave radiation unit, a static magnetic field unit 4, a diamond probe 3 containing a nitrogen-vacancy color center, and a computer manipulation processing unit 5, wherein:
the pumping light unit 1 is used for generating laser to initialize the quantum state of a nitrogen-vacancy color center in the diamond to a first quantum state, reading the quantum state of the current nitrogen-vacancy color center and performing fluorescence scanning imaging on a sample 20 to be detected, wherein the first quantum state is a nitrogen-vacancy color center |0> state;
the confocal microscope collecting unit 2 is used for collecting fluorescence emitted by a nitrogen-vacancy color center which is positioned at the focus of the microscope lens 10 at present and transmitting data to the computer control processing unit 5;
the displacement unit 6 is used for accurately controlling the relative position between the diamond probe 3 containing the nitrogen-vacancy color center and the microscope lens 10 in a nanoscale, and ensuring that a nitrogen-vacancy color center spinning signal from a target region to be detected is collected;
the diamond probe 3 containing the nitrogen-vacancy color center is used as a detector, and the whole system obtains the interaction information of the biomolecules with spin labels by measuring the longitudinal relaxation rate of the nitrogen-vacancy color center in the diamond;
the microwave radiation unit is used for applying pulse radiation of a microwave band to the nitrogen-vacancy color center, so that the quantum state of the nitrogen-vacancy color center is regulated;
the static magnetic field unit 4 is used for changing the transition frequency of the nitrogen-vacancy color center so as to influence the longitudinal relaxation disturbance intensity of the spin label on the nitrogen-vacancy color center, thereby distinguishing different spin labels and different corresponding biomolecules;
and the computer operation processing unit 5 is used for controlling the trigger time and the application length of the pump light and the trigger time and the application length of the microwave, adjusting the position of the displacement unit 6 and processing the signal returned by the confocal microscope collecting unit 2.
In some embodiments, the depth of the color center in the diamond should not be too deep, and the concentration of the color center is determined according to the requirements; generally, NV colour centres in diamond are responsive to magnetic signals in the surrounding range of about 10nm, beyond which little is affected, i.e. the target signal cannot be measured, thus meaning that it needs to be at a shallow depth from the surface (preferably 3-5 nm); for a lower NV centre concentration (generally defined as the concentration at which a single colour centre can be distinguished), the measured result generally has a higher signal contrast due to the fact that the collected signal is almost always from that colour centre compared to a high NV centre concentration with multiple colour centres in the collection range, whereas a low concentration at the same time poses the problem of high noise, and therefore the required concentration needs to be specifically chosen according to the needs of the implementer.
In some embodiments, the pump light unit 1 comprises:
a light source 7 for emitting a free space beam;
an acousto-optic modulator 8 for emitting a free space beam of a predetermined duration at a predetermined time under the manipulation of the computer manipulation processing unit 5;
a lens group for converging and diverging the free space beam to pass through the acousto-optic modulator 8;
and the optical fiber coupling system is used for coupling the free space light beam into an optical fiber and taking emergent light of the free space light beam as a light source of the diamond probe 3.
In some embodiments, the pump light unit 1 further comprises a circular continuously variable reflective Neutral Density (ND) filter for adjusting the power of the free space beam.
In some embodiments, the pump light unit 1 further comprises a mirror for changing the optical path direction; a quarter wave plate corresponding to the free space beam for switching the beam between linear and circular polarization; and the polarization beam splitter is used for guiding the free space light beam modulated by the acousto-optic modulator 8 out to the optical fiber coupling system.
In some embodiments, the displacement unit 6 comprises:
the first three-dimensional displacement table is used for bearing a microwave radiation structure 15, the diamond probe 3 containing the nitrogen-vacancy color center and a sample 20 to be detected, and controlling the nitrogen-vacancy color center of the diamond to be locked at the focus of the microscope lens 10 of the confocal microscope collection unit 2 at the nanoscale precision so as to collect a fluorescence signal from the nitrogen-vacancy color center of the diamond; the microwave radiation structure 15 is used for applying pulse radiation of a microwave band to the diamond nitrogen-vacancy color center;
and the second three-dimensional displacement table is used for adjusting the relative position of the diamond probe 3 containing the nitrogen-vacancy color center and the microscope lens focus 10 of the confocal microscope collection unit 2 on a micrometer scale.
In some embodiments, the confocal microscopy collection unit 2 comprises:
a micro lens 10 for focusing the free space beam generated by the light source 7 onto the nitrogen-vacancy color center of the diamond probe and collecting the fluorescence emitted by the nitrogen-vacancy color center of the diamond probe;
a pinhole 11 for spatially filtering the collected fluorescence;
a single photon detector 12 for measuring the collected fluorescence counts and returning the data to the computer manipulation processing unit 5;
a dichroic mirror 9 for separating incident light generated by the light source 7 from the collected fluorescence;
a plurality of filters for filtering noise fluorescence of non-target wavelength bands;
and the achromatic lens group 13 is used for converging the collected fluorescence through the pinhole 11 and finally converging the fluorescence onto the single-photon detector 12.
In some embodiments, the confocal microscopy collection unit 2 further comprises: and the third three-dimensional displacement table is used for fixing and adjusting the spatial positions of the achromatic lens group 13 and the single-photon detector 12.
In some embodiments, the microwave radiation unit includes:
a wave source 14 for generating a microwave signal of a predetermined frequency for manipulating the target nitrogen-vacancy color center quantum state;
the microwave switch is used for controlling the on-off of the microwave signal generated by the wave source 14 on the nanosecond precision;
and the radiation structure 15 is fixedly arranged between the first three-dimensional displacement table and the diamond probe 3 containing the nitrogen-vacancy color center and is used for radiating the microwave signal to the nitrogen-vacancy color center of the diamond probe.
In some embodiments, the microwave radiation unit further comprises: and the power amplifier is used for increasing the amplitude of the microwave signal to a required value.
In some embodiments, the static magnetic field unit 4 includes:
a magnet 16 for providing a desired magnetic field strength for the spin label on the sample 20 to be measured;
a fourth three-dimensional displacement stage 17 for fixing the magnet 16 and changing its position in space relative to the sample 20 to be measured to provide different magnetic field strengths.
In some embodiments, the magnet 16 is a permanent magnet.
In some embodiments, the permanent magnet is a cubic permanent magnet.
In some embodiments, the computer-operated processing unit 5 comprises:
the multifunctional I/O equipment card 19 is used for carrying out analog signal operation on the first three-dimensional displacement platform and reading data returned by the single-photon detector 12;
a pulse sequence generator for controlling respective trigger times of the pump light unit 1, the microwave radiation unit, and the multifunctional I/O device card 19, thereby completing a measurement sequence;
and the computer host 18 is used for storing an interaction program of the hardware and the computer and a LabVIEW-based program and is used for overall planning and controlling the normal work of the hardware.
In another aspect of the present disclosure, there is provided a method of measuring biomolecular interactions, using the above system, comprising:
s1, connecting the first biological molecule to the hydrogel through a biochemical reaction mode;
s2, washing the hydrogel after the operation of S1, dripping second and third biomolecules with different spin labels on the hydrogel for reaction, and fully washing to obtain a sample to be detected through interaction of the first biomolecule with the second and third biomolecules;
s3, measuring the longitudinal relaxation rate of the sample to be measured, if the longitudinal relaxation rate is obviously improved under the magnetic field intensity suitable for the spin label carried by the second biological molecule and not suitable for the spin label carried by the third biological molecule, indicating that the second biological molecule and the first biological molecule have stronger interaction, and the obvious improvement means that the original longitudinal relaxation rate is the measured longitudinal relaxation rate
The color center of (1), wherein
Is a result of a longitudinal relaxation rate measurement,
to measure the error in the result, if the sample is loaded, its increased relaxation rate
The improvement is remarkable, wherein gamma is a longitudinal relaxation rate result measured after sample loading; if the longitudinal relaxation rate is obviously improved under the magnetic field intensity suitable for the spin label carried by the third biomolecule and the spin label carried by the second biomolecule, the third biomolecule and the first biomolecule have stronger interaction.
Spin labeling refers to a substance that accelerates the longitudinal relaxation rate of NV by means of magnetic noise at the NV color center |0> state to ± |1> state transition frequency (typically 2.87GHz without external static magnetic field), and is mostly present in the form of metal ions, including but not limited to trivalent gadolinium ions, divalent manganese ions, and divalent copper ions.
In some embodiments, the spin label is selected from trivalent gadolinium ions, divalent manganese ions, and divalent copper ions.
A magnetic field strength suitable for a spin label means that the spin label has a magnetic noise distribution at said magnetic field strength that is above 70% of the peak of the noise spectrum, and a magnetic field strength unsuitable for a spin label means that the spin label has a magnetic noise distribution at said magnetic field strength that is below 30% of the peak of the noise spectrum.
Biochemical reaction modes include, but are not limited to, chemical ligation, biomolecule binding.
Chemical linkage refers to a linkage established by chemical reaction between molecules to form a covalent bond, such as the reaction of an amino group with a carboxyl group. Biomolecule binding refers to the establishment of highly specific physical contact between biomolecules due to electrostatic forces, hydrogen bonding, hydrophobic effects, etc., such as the binding of streptavidin and biotin.
The hydrogel is a crosslinked hydrophilic polymer, insoluble in water, and can be prepared from various biochemical monomers (such as four-arm-polyethylene glycol-succinimidyl glutarate (4-arm PEG-SG) and four-arm-polyethylene glycol-amino (4-arm PEG-NH) 2 ) Are obtained) are crosslinked, and the surface thereof will generally have a correspondingly high density of groups serving as modification sites due to the tight crosslinking.
In some embodiments, the hydrogel is an amino-rich hydrogel composed of 4-arm PEG-SG (four-arm-polyethylene glycol-succinimidyl glutarate ) and 4-arm PEG-NH 2 (four-arm-polyethylene glycol-amino) crosslinking reaction.
The first biomolecule and the second biomolecule may be selected according to a particular subject of investigation.
In the embodiment of the invention, the first biomolecule is streptavidin, and the second biomolecule is biotin with a trivalent gadolinium ion label; the third biomolecule is biotin with a bivalent copper ion label.
Definition of
Pump light: the pump light is a light having high coherence and directivity generated by exciting atoms or molecules with light.
A displacement unit: the device is provided with a first displacement platform (a nanometer displacement platform) and a second displacement platform (a micrometer displacement platform), and can enable the spatial position of a target NV color center in a diamond probe to be just positioned at the focus of a microscope lens of a confocal microscope collection unit through adjustment, so that the collection of signals is completed.
Static magnetic field unit: a device for generating static magnetic field, and the intensity of the magnetic field can be adjusted by the device.
Longitudinal relaxation of NV: under the excitation of laser with wavelength of 532nm, the quantum state |0 is utilized>NV color center ratio of state at | + -1>The color centers of the states emit about 30% more fluorescence within the same time, so that the spin state of the NV color center can be judged by reading the fluorescence intensity, and the NV color center is polarized to a certain quantum state (| 0)>Or + - |1>NV color center of state) the quantum state also slowly returns to the thermal equilibrium state over time in the absence of excitation light
The characteristic time from full polarization to return to equilibrium is called NV longitudinal relaxation time (T1), and the reciprocal of the characteristic time is called NV longitudinal relaxation rate, denoted as Γ, i.e., Γ = 1/T1.
NV degree of population: the ratio of NV in a quantum state to the total number of NVs (e.g., NV color center |0> state population of 0.7, where 70% of the measured results are all at |0 >).
Free space beam: refers to a light beam propagating in free space, the propagation medium of which is air.
Advantageous effects
Compared with the prior art, the invention at least has the following advantages:
the invention provides a longitudinal relaxation rate measuring system, which can obviously improve a signal of a sample to be measured, which really generates biomolecule interaction, and can detect a biomolecule to be measured with a spin label; in addition, the invention provides a method for measuring the interaction of biological molecules by using the system, the method uses hydrogel as a reaction substrate of the interaction of the biological molecules, and then carries out longitudinal relaxation rate measurement on the prepared sample to be measured, compared with the surface modification of common diamond, the surface of the hydrogel has a plurality of biochemical reaction binding sites, compared with the traditional technology, the method can detect the interaction of the biological molecules of the sample to be measured more efficiently (about 16 times of signal enhancement), and simultaneously compared with the traditional technology, the method can realize the simultaneous detection of multi-channel biological samples.
Drawings
In order to more clearly illustrate the embodiments or technical solutions of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
Fig. 1 is a schematic diagram of a longitudinal relaxation rate measurement system provided by an embodiment of the present disclosure, where: 1 is a pump light unit, 2 is a confocal microscopic collection unit, 3 is a diamond probe containing a nitrogen-vacancy color center, 4 is a static magnetic field unit, 5 is a computer control processing unit, 6 is a displacement unit, 7 is a light source, 8 is an acousto-optic modulator, 9 is a dichroic mirror, 10 is a microscope lens, 11 is a pinhole, 12 is a single-photon detector, 13 is an achromatic lens group, 14 is a wave source, 15 is a radiation structure, 16 is a magnet, 17 is a fourth three-dimensional displacement table, 18 is a computer host, and 19 is a multifunctional I/O equipment card;
FIG. 2 is a schematic diagram of the placement of a diamond probe and a sample to be tested in an embodiment of the present disclosure, where 16 is a magnet, 20 is the sample to be tested, 3 is the diamond probe containing a nitrogen-vacancy color center, 15 is a radiation structure, and 10 is a microscope lens;
FIG. 3 is a schematic diagram of a longitudinal relaxation rate measurement sequence of an embodiment of the present disclosure;
FIG. 4 is a graphical illustration of longitudinal relaxation rate measurements in accordance with an embodiment of the disclosure;
FIG. 5 is a schematic diagram illustrating the principle of measuring the biomolecular interaction in an embodiment of the present disclosure;
FIG. 6 is a schematic flow chart of a method for preparing a sample to be tested for biomolecular interaction according to an embodiment of the present disclosure;
fig. 7 is a graph illustrating comparative results of measurements provided by embodiments of the present disclosure.
Detailed Description
In order to make the technical solutions of the present invention better understood, the present invention will be described in further detail with reference to the accompanying drawings and the detailed description below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The technical solution of the present disclosure will be described in detail below with reference to specific examples. It should be noted that the following specific examples are only for illustration and are not intended to limit the disclosure.
Fig. 1 is a schematic diagram of a longitudinal relaxation rate measurement system provided by an embodiment of the present disclosure.
As shown in fig. 1, the system for measuring a longitudinal relaxation rate provided by this embodiment is implemented based on the optical property of the NV color center of diamond, and is used for measuring the longitudinal relaxation rate of NV, and specifically includes: a pump light unit 1, a confocal microscopic collection unit 2, a displacement unit 6, a microwave radiation unit, a static magnetic field unit 4, a diamond probe 3 containing a nitrogen-vacancy color center, and a computer manipulation processing unit 5.
The diamond used in the application has an NV color center which has good optical properties, and is in a quantum state |0 under excitation of laser with a wavelength of 532nm>NV color center ratio of state at | + -1>The color centers of the states emit about 30% more fluorescence within the same time, so that the spin state of the NV color center can be judged by reading the fluorescence intensity, and the NV color center is polarized to a certain quantum state (| 0)>Or + - |1>NV color center of state) the quantum state also slowly returns to the thermal equilibrium state over time in the absence of excitation light
The characteristic time from full polarization to return to equilibrium is called NV longitudinal relaxation time (T1), and the reciprocal of the characteristic time is called NV longitudinal relaxation rate, denoted as Γ, i.e., Γ = 1/T1. The longitudinal relaxation time of the NV color center is reduced by the noise spectrum of said spin label within about 10nm of its vicinity at the transition frequency, the higher the noise spectrum distribution at the transition frequency, upon longitudinal relaxationThe more pronounced the inter-reduction effect, the faster the corresponding longitudinal relaxation rate. Therefore, the longitudinal relaxation rates of NV before and after the sample suspected to be provided with the metal spin label is added are measured, and the difference before and after the sample is compared, so that whether the sample really has a certain biomolecule marked with the spin label can be judged. In the invention, the depth of the color center in the diamond is not suitable to be too deep, and the concentration of the color center and the surface treatment mode can be determined according to the requirements.
The pump light unit 1 is used for generating laser with a wavelength of 532nm to initialize the quantum state of a nitrogen-vacancy color center in the diamond to a first quantum state, reading out the quantum state where the current nitrogen-vacancy color center is located, and performing fluorescence scanning imaging on a sample 20 to be detected, wherein the first quantum state is a nitrogen-vacancy color center |0> state in the invention.
The confocal microscopic collection unit 2 is used for collecting fluorescence emitted by a nitrogen-vacancy color center which is currently located in a micron-scale range of a focus of the microscope 10, ensuring high signal-to-noise ratio and high spatial resolution, transmitting data to the computer control processing unit 5, and converting optical signals into electric signals.
The displacement unit 6 is used to precisely control the relative position between the diamond probe 3 containing the nitrogen-vacancy colour centre and the microscope lens 10 in the confocal microscopy collection unit 2, ensuring that a relaxation rate signal from the target nitrogen-vacancy colour centre spin is collected.
The microwave radiation unit generates a microwave signal by a wave source 14, a power amplifier amplifies the signal, a switch of the wave source 14 controls the on-off of the microwave signal, and a radiation structure 15 radiates the microwave signal to an NV color center in the diamond; the unit is used for applying pulse radiation corresponding to a microwave band to the diamond nitrogen-vacancy color center, so that the quantum state of the color center is regulated.
The static magnetic field unit 4 changes the position of the magnet 16 relative to the NV color center through the three-dimensional displacement table, so as to adjust the magnitude and the direction of the magnetic field strength, change the transition frequency of the NV color center (the position is changed, the magnetic field strength is changed, so that the transition frequency is changed), further influence the longitudinal relaxation disturbance intensity of the metal spin mark on the NV color center, and further distinguish different spin marks and corresponding different biomolecules; when a static magnetic field is applied along the direction of the NV color center main axis, the splitting of the NV color center ground state non-magnetic field energy level (2.87 GHz) is changed, the relation between the transition frequency and the applied static magnetic field size is omega =2.87 +/-gamma B (GHz), gamma is the NV electron spin magnetic ratio, and B is the static magnetic field intensity.
The computer operation processing unit 5 is used for controlling the triggering time and the application length of the pump light, the microwave and the multifunctional I/O equipment card, adjusting the position of the displacement unit 6 and processing the signal returned by the confocal microscopic collection unit 2.
The computer operation processing unit 5 is connected with the pump light unit 1, the confocal micro-collection unit 2, the displacement unit 6, the microwave radiation unit and the static magnetic field unit 4 through wired or wireless connection, wherein the wired connection comprises but is not limited to a data bus and a USB interface, and the wireless connection comprises but is not limited to Bluetooth and Wi-Fi.
When the device works, exciting light generated by the pumping light unit 1 is reflected by the dichroic mirror 9 and converged into the position of the microscope lens 10 of the confocal microscope collection unit 2, the radiation structure 15 is fixedly connected above the displacement unit 6 by a screw, the diamond probe 3 containing a nitrogen-vacancy color center is fixedly connected on the displacement unit by transparent glue, the magnet 16 is fixed at the suspended position above the displacement structure by the fourth three-dimensional displacement table 17, and a fluorescent signal collected by the microscope lens 10 is transmitted into the confocal microscope collection unit 2 by the dichroic mirror 9.
Fig. 2 is a schematic diagram illustrating a placement manner of a diamond probe and a sample to be tested in the embodiment of the present disclosure.
As shown in fig. 2, 532nm laser with wavelength from the pump light unit 1 enters through the confocal microscopic collection unit 2 and is converged by the microscope 10, and then passes through the radiation structure 15 fixed on the displacement unit 6 and is focused on the diamond fixed on the radiation structure 15. The sample to be tested is closely attached to the nitrogen-vacancy color center surface above the diamond and can have a spin-labeled biomolecule interaction to be tested. And a static magnetic field unit 4 is disposed over the entire sample area for adjusting the static magnetic field applied to NV in the diamond.
Fig. 3 is a schematic diagram of a longitudinal relaxation rate measurement sequence of an embodiment of the disclosure.
As shown in FIG. 3, for a single NV color centerLongitudinal relaxation Rate measurement NV color center quantum states are first initialized to a first quantum state (i.e., | 0) by excitation with a laser at 532nm wavelength for a longer period of time (about 10 μ s)>State), then laser irradiation is stopped, and the quantum state of the NV color center begins to gradually revert from the first quantum state to the thermal equilibrium state (i.e., the quantum state is in the thermal equilibrium state)
) After the evolution of a given time tau, 532nm laser is applied again to read the count of the quantum state at the time to obtain a tau time evolution state (as shown by tau time in fig. 3), and then the current standard count can be obtained as a reference value of the counting disturbance after the initialization and the instant reading. As can be seen from the above, the above operation yields |0 corresponding to time τ in FIG. 4>Degree of state settlement
A single measurement. If τ time |1 is to be obtained>And the measurement value of the state population degree at one time only needs to apply a section of pi-pulse to the NV color center of the diamond by using the microwave radiation unit after the initialization and before tau time evolution so as to turn the population degree. Will be |0 of the above-mentioned tau time>State, |1>The state measurement is repeated a plurality of times (typically about 10) 4 Magnitude), the quantum state of NV color center at τ time with lower error can be obtained. By changing τ and repeating the above measurement, the relationship between the population of the quantum state where the NV color center is located and the evolution time can be drawn (fig. 4), so as to calculate the longitudinal relaxation rate of the NV color center in the current environment.
FIG. 5 is a schematic diagram of the principle of biomolecular interaction measurement in the disclosed embodiment.
As shown in fig. 5, for different metal spin labels 1 and 2, their magnetic noise spectra at different frequencies may be different, mainly due to the difference in the magnitude of their noise broadening. The spin label 1 has a greater spread of magnetic noise as in FIG. 5
Has stronger magnetic noise distribution at the lower part, and the spin mark 2 has smaller magnetic noise broadening at
There is a strong magnetic noise distribution when the magnetic field strength becomes
The magnetic noise distribution is sharply reduced. Thus, when the transition frequency of the NV color center is at
When the metal mark is used, the longitudinal relaxation rate of the metal mark is strongly disturbed by the two metal marks; when the NV color center changes to the transition frequency after changing the applied static magnetic field
The influence of the spin label 2 on its longitudinal relaxation rate is greatly reduced, mainly due to the contribution of the spin label 1. Therefore, by changing the static magnetic field applied to the NV color center, the detection channel of the metal marker can be changed, and the sample detection of the multi-channel metal marker biomolecule interaction can be completed even under the condition that a plurality of metal markers exist simultaneously.
Fig. 6 is a schematic flow chart of a method for preparing a sample to be tested for biomolecular interaction according to an embodiment of the present disclosure.
As shown in FIG. 6, the procedure for preparing the sample 20 to be tested for biomolecular interaction using hydrogel is as follows:
s1, connecting the first biological molecule to the hydrogel through a biochemical reaction mode;
s2, cleaning the hydrogel after the operation of S1, dripping second and third biomolecules with different spin labels on the hydrogel for reaction, and then fully cleaning to obtain a sample to be detected, wherein the interaction of the first biomolecule with the second and third biomolecules is realized;
s3, the reaction surface of the sample to be tested is tightly attached to a diamond probe 3 containing an NV color center in a longitudinal relaxation rate measuring system, and NV color center longitudinal relaxation rate measurement is carried out on the diamond probe 3.
The biochemical reaction mode comprises various modes such as chemical connection, biomolecule combination and the like.
The hydrogel is a cross-linked hydrophilic polymer, is insoluble in water, can be obtained by cross-linking a plurality of biochemical monomers, and has compact cross-linking, so that the surface of the hydrogel can be modified by corresponding high-density groups.
In the present examples, the hydrogel is an amino-rich hydrogel composed of 4-ARM PEG-SG (JenKem Technology USA, 4 ARM-SG) and 4-ARM PEG-NH 2 (Sigma, 672130) was formed by a crosslinking reaction at room temperature.
The first biomolecule and the second biomolecule may be selected according to a particular subject of investigation.
In an embodiment of the present invention, the first biomolecule is streptavidin, and the second biomolecule is biotin labeled with trivalent gadolinium ions.
Fig. 7 is a schematic diagram of measurement comparison results, and in the present invention, biotin with a trivalent gadolinium metal spin label is used as a target biomolecule to be detected, and through a conventional method of directly connecting the biomolecule to a diamond probe 3 and a comparison experiment based on a hydrogel reaction and measurement scheme provided by the scheme of the present invention, we observe a spin signal increase of about 16 times.
The specific operations of the conventional method sequentially comprise:
1. performing carboxylation treatment on the surface of the diamond (using concentrated sulfuric acid with the mass fraction of 98%, perchloric acid with the mass fraction of 70% and concentrated nitric acid with the mass fraction of 63% to prepare triacid and boiling the diamond for 4 hours at 180 ℃ according to the ratio of 1: 1: 1;
2. then sequentially treating with 1 mol/L (hereinafter, mol/L is abbreviated as M, and millimole/L is abbreviated as mM) hydrochloric acid, 1M sodium hydroxide and 1M hydrochloric acid (heating at 90 ℃ for 1 hour);
3. streptavidin ligation (addition of 10mM streptavidin to the previously treated diamond surface in 20mM EDC-NHS for 40 min);
4. the biotin with trivalent gadolinium metal spin labeling is dissolved in 0.1M phosphate buffer normal saline to prepare a solution with the concentration of 50mM, and the solution is dripped on the surface of the diamond for reaction.
Measuring the change of the longitudinal relaxation rate of the nitrogen-vacancy color center in the diamond before and after the biotin is connected, wherein the change is accelerated by 0.211 kHz; by utilizing the hydrogel-based reaction and measurement scheme provided by the scheme of the invention, the acceleration of 3.392kHz before and after the connection of biotin with the same concentration is obtained, and the comparison of the two methods shows that the signal intensity of the invention is improved by 16 times when the invention is used for detecting a biological sample with a spin label.
After the preparation of the hydrogel sample with different spin-labeled biomolecule interactions is completed, the NV longitudinal relaxation rate measurement system (fig. 1) based on the NV color center of diamond according to the embodiment of the present invention is used, the sample and the diamond are placed as shown in fig. 2, the measurement sequence shown in fig. 3 is adopted, the static magnetic field applied to the NV of diamond is changed to make the NV longitudinal relaxation rate measurement system suitable for the spin label of the second biomolecule and not suitable for the spin label of the third biomolecule, if the measured longitudinal relaxation rate is significantly improved, it indicates that the second biomolecule and the first biomolecule have stronger interactions, and the significantly improved longitudinal relaxation rate is the original measured longitudinal relaxation rate
The color center of (1), wherein
Is a result of a longitudinal relaxation rate measurement,
to measure the error in the result, if the sample is loaded, its increased relaxation rate
The improvement is remarkable, wherein gamma is a longitudinal relaxation rate result measured after sample loading; similarly, the static magnetic field applied to diamond NV is modified to suit the third generationThe spin label carried by the biomolecule is not suitable for the spin label carried by the second biomolecule, and if the longitudinal relaxation rate obtained by measurement is remarkably improved, the third biomolecule has stronger interaction with the first biomolecule.
The magnetic field strength suitable for the spin label means that the spin label has a strong magnetic noise distribution at the transition frequency, and the magnetic field strength unsuitable for the spin label means that the spin label has a weak magnetic noise distribution at the transition frequency.
Therefore, the measurement system, the sample preparation mode and the measurement mode provided by the invention can be used by those skilled in the art for carrying out multi-channel biological signal detection on the sample to be detected through the relevant biomolecule interaction.
The terms "first," "second," "third," "fourth," and the like in the description and claims of this application and in the above-described drawings are used for distinguishing between different objects and not for describing a particular order. Furthermore, the terms "comprising" and "having," as well as any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, system, article, or apparatus that comprises a list of steps or elements is not limited to only those steps or elements but may include other steps or elements not expressly listed.
And the shapes and sizes of the respective components in the drawings do not reflect actual sizes and proportions, but merely illustrate the contents of the embodiments of the present disclosure. Furthermore, the word "comprising" does not exclude the presence of elements or steps not listed in a claim. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.
Similarly, it should be appreciated that in the foregoing description of exemplary embodiments of the disclosure, various features of the disclosure are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. However, the method of the invention should not be construed to reflect the intent: that is, the claimed disclosure requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing inventive embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this disclosure.
Claims (9)
1. A longitudinal relaxation rate measurement system, comprising a pump light unit, a confocal microscopy collection unit, a displacement unit, a microwave radiation unit, a static magnetic field unit, a diamond probe containing a nitrogen-vacancy color center, and a computer manipulation processing unit, wherein:
the pumping light unit is used for generating laser so as to initialize the quantum state of a nitrogen-vacancy color center in the diamond to a first quantum state, reading the quantum state of the current nitrogen-vacancy color center and carrying out fluorescence scanning imaging on a sample to be detected, wherein the first quantum state is a nitrogen-vacancy color center |0> state;
the confocal microscopic collection unit is used for collecting fluorescence emitted by a nitrogen-vacancy color center which is currently located in the range of the focus of the microscope lens and transmitting data to the computer control processing unit;
the displacement unit is used for accurately controlling the relative position between the diamond probe containing the nitrogen-vacancy color center and the microscope lens in a nanoscale, and ensuring that a nitrogen-vacancy color center spinning signal from a target region to be detected is collected;
the diamond probe containing the nitrogen-vacancy color center is used as a detector, and the whole system obtains biomolecular interaction information with metal spin labels by measuring the longitudinal relaxation rate of the nitrogen-vacancy color center in the diamond;
the microwave radiation unit is used for applying pulse radiation of a microwave band to the nitrogen-vacancy color center, so that the quantum state of the nitrogen-vacancy color center is regulated;
the static magnetic field unit changes the position of a magnet relative to a nitrogen-vacancy color center through a three-dimensional displacement table, further adjusts the intensity and the direction of a magnetic field, changes the transition frequency of the nitrogen-vacancy color center, further influences the longitudinal relaxation disturbance intensity of a metal spin label on the nitrogen-vacancy color center, and distinguishes different metal spin labels and different corresponding biomolecules, and the static magnetic field unit also changes a detection channel of the metal spin label by changing a static magnetic field applied to the nitrogen-vacancy color center, so that the detection of the interaction of the biomolecules of the multichannel metal spin label is completed under the condition that a plurality of metal spin labels exist simultaneously;
and the computer control processing unit is used for controlling the triggering time and the application length of the pump light and the triggering time and the application length of the microwave, adjusting the position of the displacement unit and processing the signal returned by the confocal microscopic collection unit.
2. The system of claim 1, wherein the pump light unit comprises:
a light source for emitting a free space beam;
an acousto-optic modulator for emitting a free space beam of light for a predetermined length of time at a predetermined time under manipulation of the computer manipulation processing unit;
the lens group is used for converging and diverging the free space light beam to pass through the acousto-optic modulator;
and the optical fiber coupling system is used for coupling the free space light beam into an optical fiber and taking emergent light of the free space light beam as a light source of the diamond probe.
3. The system of claim 2, wherein the pump light unit further comprises a circular continuously variable reflection neutral density filter for adjusting the power of the free space beam.
4. The system of claim 3, wherein the pump light unit further comprises a mirror for changing the optical path direction; a quarter wave plate corresponding to the free space beam for switching the free space beam between linear polarization and circular polarization; and the polarization beam splitter is used for guiding the free space light beam modulated by the acousto-optic modulator out to the optical fiber coupling system.
5. The system of claim 1, wherein the displacement unit comprises:
the first three-dimensional displacement platform is used for bearing a microwave radiation structure of the microwave radiation unit, the diamond probe containing the nitrogen-vacancy color center and a sample to be detected, and controlling the nitrogen-vacancy color center of the diamond probe to be locked at the focus of the microscope lens of the confocal microscopic collection unit at the nanoscale precision so as to collect a fluorescence signal from the diamond nitrogen-vacancy color center; the microwave radiation structure is used for applying pulse radiation of a microwave band to the diamond nitrogen-vacancy color center;
and the second three-dimensional displacement table is used for adjusting the relative positions of the diamond probe containing the nitrogen-vacancy color center and the focus of the microscope lens of the confocal microscopy collection unit on a micrometer scale.
6. The system of claim 2, wherein the confocal microscopy collection unit comprises:
the microscope lens is used for focusing the free space light beam generated by the light source on the nitrogen-vacancy color center of the diamond probe and collecting fluorescence emitted by the nitrogen-vacancy color center of the diamond probe;
a pinhole for spatially filtering the collected fluorescence;
a single photon detector for measuring the collected fluorescence counts and returning data to the computer manipulation processing unit;
a dichroic mirror for separating incident light generated by the light source from the collected fluorescent light;
a plurality of filters for filtering noise fluorescence of non-target wavelength bands;
and the achromatic lens group is used for converging the collected fluorescence to pass through the pinhole and finally to the single-photon detector.
7. The system of claim 6, wherein the confocal microscopy collection unit further comprises: and the third three-dimensional displacement platform is used for fixing and adjusting the spatial positions of the achromatic lens group and the single photon detector.
8. The system of claim 5, wherein the microwave radiation unit comprises:
a wave source for generating a microwave signal of a predetermined frequency for manipulating the target nitrogen-vacancy color center quantum state;
the microwave switch is used for controlling the on-off of a microwave signal generated by the wave source on the nanosecond precision;
and the radiation structure is fixedly arranged between the first three-dimensional displacement table and the diamond probe containing the nitrogen-vacancy color center and is used for radiating the microwave signal to the nitrogen-vacancy color center of the diamond probe.
9. A method of measuring biomolecular interactions using a system according to any of claims 1-8, comprising:
s1, connecting the first biological molecule to the hydrogel through a biochemical reaction mode;
s2, washing the hydrogel after the operation of S1, dripping second and third biomolecules with different metal spin labels on the hydrogel for reaction, and fully washing to obtain a sample to be detected, wherein the interaction of the first biomolecule with the second and third biomolecules is realized;
s3, measuring the longitudinal relaxation rate of the sample to be measured, if the longitudinal relaxation rate is obviously improved under the magnetic field intensity suitable for the metal spin label carried by the second biological molecule and not suitable for the metal spin label carried by the third biological molecule, indicating that the second biological molecule and the first biological molecule have stronger interaction, wherein the obvious improvement means that the original longitudinal relaxation rate is the measured longitudinal relaxation rate
A color center of wherein
Is a result of a longitudinal relaxation rate measurement,
to measure the error in the result, if the sample is loaded, its increased relaxation rate
The improvement is remarkable, wherein gamma is a longitudinal relaxation rate result measured after sample loading; if the longitudinal relaxation rate is obviously improved under the magnetic field intensity suitable for the metal spin label carried by the third biomolecule and the metal spin label carried by the second biomolecule, the third biomolecule and the first biomolecule have stronger interaction.
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