CN115308132A - Optical rotation biological sensing system and detection method of biological molecule interaction - Google Patents
Optical rotation biological sensing system and detection method of biological molecule interaction Download PDFInfo
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Abstract
The invention discloses an optical rotation biological sensing system based on optical weak measurement, which comprises a light source, a first lens, a second lens, a spectrometer, a processing unit, a first polaroid, a sample cell made of a light-transmitting material, an optical rotation dispersion element and a second polaroid, wherein light emitted by the light source sequentially enters the sample cell through the first lens and the first polaroid, light emitted from the sample cell sequentially passes through the optical rotation dispersion element and then sequentially reaches the spectrometer through the second polaroid and the second lens, the spectrometer is used for recording the change of the central wavelength of emergent light, and the processing unit is connected with the spectrometer and used for obtaining parameters of biomolecule interaction according to the change of the central wavelength of the emergent light. The invention also discloses a detection method of the interaction of the biomolecules based on the weak optical measurement. The invention does not need pretreatment, has lower detection cost of the optical rotation biological sensing system and single detection, and can provide quantitative parameters of reaction interaction.
Description
Technical Field
The invention relates to the technical field of molecular detection, in particular to an optical rotation biosensing system based on optical weak measurement and a detection method of biomolecule interaction based on optical weak measurement.
Background
The detection of biomolecular interactions, which includes the characterization of biomolecular interactions and the accurate measurement of parameters related to biomolecular interactions, is one of the fundamental research subjects of life science research. At present, researchers have developed various biomolecule interaction detection methods, which are widely used in the establishment of biomolecule interaction detection methods due to the unique combined advantages exhibited by optical detection methods, and from the viewpoint of biomolecules to be detected, these methods can be classified into methods for detecting immobilized biomolecules, including surface plasmon resonance techniques, etc., and methods for detecting non-immobilized biomolecules, including fluorescence quenching techniques, ultraviolet-visible absorption spectroscopy, infrared spectroscopy, etc. The result and related parameters obtained by detecting the interaction of immobilized biomolecules are apparent parameters, that is, the detection result may be different from the detection result corresponding to the interaction in the natural state because the molecules interact in an immobilized manner, and the result obtained by detecting the non-immobilized biomolecules is often closer to the corresponding result in the natural state. In the conventional detection method for detecting non-immobilized biomolecules, the ultraviolet-visible absorption spectroscopy and infrared spectroscopy are less applied to quantitative high-precision detection due to low sensitivity, and the accuracy and sensitivity of the fluorescence quenching technique commonly used for quantitative detection are often influenced by the fluorescence intensity of the biomolecules to be detected, i.e., when the autofluorescence intensity is weak, the result of the biomolecule interaction detection is poor, and at the moment, the biomolecules to be detected need to be subjected to fluorescence modification by a pretreatment method, so that the experiment difficulty and the experiment cost are increased.
The above background disclosure is only provided to assist understanding of the concept and technical solution of the present invention, which does not necessarily belong to the prior art of the present patent application, and should not be used to evaluate the novelty and inventive step of the present application in the case that there is no clear evidence that the above content is disclosed at the filing date of the present patent application.
Disclosure of Invention
In order to solve the technical problems, the invention provides an optical rotation biosensing system and a method for detecting the interaction of biomolecules, which do not need pretreatment, have lower detection cost and single detection cost of the optical rotation biosensing system and can provide quantitative parameters of reaction interaction.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention discloses an optical rotation biosensing system based on optical weak measurement, which comprises a detection assembly, a front selection state, a weak interaction assembly and a rear selection state, wherein the detection assembly comprises a light source, a first lens, a second lens, a spectrometer and a processing unit, the front selection state comprises a first polaroid and a sample cell made of a light-transmitting material, the weak interaction assembly comprises an optical rotation dispersion element, the rear selection state comprises a second polaroid, light emitted by the light source sequentially passes through the first lens and the first polaroid and enters the sample cell, light emitted from the sample cell sequentially passes through the optical rotation dispersion element and then sequentially passes through the second polaroid and the second lens to reach the spectrometer, the spectrometer is used for recording the change of the central wavelength of emergent light, and the processing unit is connected with the spectrometer and is used for obtaining the parameters of biomolecular interaction according to the change of the central wavelength of the emergent light.
Preferably, the optically dispersive element is a quartz optically active plate.
Preferably, the first lens is a collimating lens and the second lens is a coupling lens.
Preferably, an included angle between the first polarizing plate and the second polarizing plate is 89 ° to 91 °.
The invention also discloses a detection method of the interaction of the biomolecules based on the weak optical measurement, which adopts the optical rotation biological sensing system to detect the parameters of the interaction of the biomolecules and comprises the following steps:
s1: adjusting the included angle between the first polaroid and the second polaroid to be 89-91 degrees;
s2: adjusting the optical dispersion element to enable the optical rotation biosensing system to work in a weak measurement state;
s3: introducing a solvent into the sample cell, and measuring the central wavelength offset delta lambda of the emergent light at the moment 0 To perform calibration;
s4: extracting the solvent, introducing optically active biomolecule A solution into the sample cell, and measuring the central wavelength ratio of the emergent light at the moment 0 Is offset amount of
S5: dropwise adding a solution of molecule B into the sample pool, and measuring the central wavelength ratio delta lambda of the emergent light after the jth titration after each titration 0 Offset Δ λ of (2) j ；
S6: multiple titrations to obtain the central wavelength ratio Delta lambda of the emergent light 0 Offset Δ λ of (2) j Fitting to obtain the binding equilibrium constant K of the biomolecule A and the molecule B A And a value combining the number of sites n.
Preferably, the step S1 further includes adjusting the first polarizer to have an angle pi/4 with the vertical direction.
Preferably, the solvent in step S3 is deionized water.
Preferably, step S6 specifically includes:
s61: determination of product B n Whether the A has optical rotation or not, if so, executing step S62, and if not, executing step S63;
s62: titrating the solution of the molecule B until the central wavelength of the emergent light is compared with delta lambda 0 Offset amount of (a) Δ λ j The central wavelength of the emergent light reaches the minimum value of rapid change, and the central wavelength of the emergent light at the time is recorded to be compared with delta lambda 0 Offset amount of (a) Δ λ V And then obtaining the central wavelength ratio delta lambda of the emergent light according to multiple titrations 0 Fitting to obtain the binding equilibrium constant K of the biomolecule A and the molecule B A And a value of the number of binding sites n;
s63: titrating a solution of molecule B at least 10 times and obtaining from multiple titrationsCentral wavelength ratio of emergent light Delta lambda 0 Fitting to obtain the binding equilibrium constant K of the biomolecule A and the molecule B A And a value combining the number of sites n.
Preferably, in step S62, the following formula is adopted to perform fitting according to the central wavelength shift of the outgoing light obtained by multiple titrations:
wherein, the first and the second end of the pipe are connected with each other,
is the concentration of biomolecule a when only biomolecule a is in solution,
the total concentration of the biomolecule A solution after the v-th titration of the molecule B solution, v being the ratio of the central wavelength of the emitted light to the wavelength of Delta lambda 0 The number of titrations of the solution of molecule B when the offset of (a) reaches a rapidly changing minimum,
the central wavelength of emergent light corresponding to the condition that only the biomolecule A exists in the solution is compared with delta lambda 0 The amount of the offset of (a) is,
for the total concentration of biomolecule a solution after the jth titration of molecule B solution,
is the total concentration of the molecule B solution after the jth titration of the molecule B solution.
Preferably, in step S63, the following formula is adopted to perform fitting according to the central wavelength shift of the emergent light obtained by multiple titrations:
wherein the content of the first and second substances,
for the total concentration of the molecule B solution after the jth titration of the molecule B solution,
for the total concentration of biomolecule a solution after the jth titration of molecule B solution,
the central wavelength of emergent light corresponding to the condition that only the biomolecule A exists in the solution is compared with delta lambda 0 The amount of the offset of (a) is,
is the concentration of biomolecule a when only biomolecule a is in solution.
Compared with the prior art, the invention has the beneficial effects that: the method realizes real-time monitoring of the interaction of the optical rotation biomolecules by high-sensitivity calculation of the change of optical rotation signals generated during the interaction of the biomolecules based on the optical weak measurement theory, and uses the combination equilibrium constant K of the spectrum center wavelength signals output by the optical rotation biosensor system to the interaction of the biomolecules A And calculating in conjunction with the number of sites n. In the prior art, the most similar optical methods for detecting interaction of non-immobilized biomolecules in a solution can be provided, the most common method is a fluorescence quenching technology, but the accuracy and sensitivity of the fluorescence quenching technology are often influenced by the fluorescence intensity of the biomolecules to be detected, that is, when the autofluorescence intensity is weak, the result of the interaction detection of the biomolecules is poor, and at this time, the biomolecules to be detected need to be subjected to fluorescence modification by a pretreatment method, but the experiment difficulty and the experiment cost are increased. Compared with the prior art, the optical rotation biosensing system based on optical weak measurement and the biomolecule interaction detection method based on optical weak measurement optical rotation detection both provided by the invention are based on high-sensitivity calculation of optical rotation signals, and the system and the method have good optical rotation capability due to ubiquitous optical rotation capability of biomoleculesThe method is universal and does not need complex pretreatment, and the method is based on a simple optical system to realize a complex detection principle, so that the detection cost of the optical rotation biosensor system and the single detection cost are both low, quantitative reaction interaction parameters can be provided, and a novel simple method can be provided for the detection of the interaction between the biomolecules with low autofluorescence and the optical rotation effect.
Drawings
FIG. 1 is a schematic diagram of an optically active biosensor system for biomolecule interaction detection in accordance with a preferred embodiment of the present invention;
FIG. 2 is a schematic diagram of the detection principle of the biomolecule interaction detection according to the preferred embodiment of the present invention;
FIG. 3 is a flow chart of the detection process for detecting biomolecular interactions according to the preferred embodiment of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the embodiments of the present invention more clearly apparent, the present invention is further described in detail below with reference to the accompanying drawings and the embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
It will be understood that when an element is referred to as being "secured to" or "disposed on" another element, it can be directly on the other element or be indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or be indirectly connected to the other element. In addition, the connection may be for either a fixing function or a circuit connection function.
It is to be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like are used in an orientation or positional relationship indicated in the drawings for convenience in describing the embodiments of the present invention and to simplify the description, and are not intended to indicate or imply that the referenced device or element must have a particular orientation, be constructed in a particular orientation, and be in any way limiting of the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the embodiments of the present invention, "a plurality" means two or more unless specifically limited otherwise.
The invention provides a universal optical weak measurement optical rotation biosensing system, which comprises a front selection state, a detection device and a weak interaction and a rear selection state of a physical system to be detected, wherein in the optical weak measurement-based optical rotation biosensing system, a front polaroid and a sample pool containing an optical rotation biomolecular solution are selected as the front selection state, a rear polaroid which is approximately orthogonal to the front selection state is used for providing the rear selection state, a quartz optical rotation sheet is used for modulating the proper weak interaction, and when the optical weak measurement optical rotation biosensing system is properly modulated, the central wavelength of system output light can amplify and detect the optical rotation of the solution. On the whole, the optical rotation biological sensing system based on the optical weak measurement comprises a laser with high bandwidth and high intensity, an optical weak measurement linear detection system, a sample cell in which an optical rotation biological solution to be measured is located, a spectrometer for receiving an output light beam of the optical weak measurement linear detection system, and a computer for analyzing spectral data received by the spectrometer, calculating and displaying the central wavelength of output light in real time, and dynamically monitoring the interaction of biomolecules in real time through the deviation of the central wavelength. Furthermore, the laser can be a superluminescent diode, the linear detection system for optical weak measurement and the sample cell are placed in the order of a collimating lens, a front polarizing film, the sample cell, an optical rotation film, a rear polarizing film and a coupling lens, after light beams are emitted from the superluminescent diode, collimated parallel light is formed after passing through the collimating lens, and the collimated parallel light vertically passes through the front polarizing film, the sample cell, the optical rotation film and the rear polarizing film in sequence and enters the spectrometer through optical fibers after passing through the coupling lens.
As shown in fig. 1, the optical rotation biosensing system based on optical weak measurement according to the preferred embodiment of the present invention includes a superluminescent light emitting diode 1, a collimating lens 2, a first linear polarizer 3, a glass sample cell 4, a quartz optical rotation sheet 5, a second linear polarizer 6, a coupling lens 7, a spectrometer 8 and a computer 9, wherein the glass sample cell 4 is a sample cell portion where a biomolecule interaction occurs, and the superluminescent light emitting diode 1, the collimating lens 2, the first linear polarizer 3, the quartz optical rotation sheet 5, the second linear polarizer 6, the coupling lens 7, the spectrometer 8 and the computer 9 are optical sensor portions.
Incident light is emitted by the superluminescent light-emitting diode 1 and enters the glass sample cell 4 through the collimating lens 2 and the first linear polaroid 3, light rays are subjected to optical angle rotation under the action of a light rotating substance in the glass sample cell 4, and the optical angle rotation and the first linear polaroid 3 work to provide a front choice in a weak measurement system. Light rays emitted from the glass sample cell 4 are subjected to optical rotation dispersion action of the quartz optical rotation sheet 5 to generate coupling between optical frequency and polarization state, weak interaction between a generating system and a pointer (Gaussian laser beams emitted by a laser) occurs, and finally the light rays reach a spectrometer 8 through a second linear polarization sheet 6 and a coupling lens 7 which are provided and selected to be approximately orthogonal to the first linear polarization sheet 3, wherein the light beams in the whole optical path are perpendicular to optical devices and parallel to an optical platform. In the detection method of weak measurement, a high-sensitivity linear correlation relationship between the central wavelength of output light and the optical rotation of the solution can be constructed, so that spectral signals collected by a spectrometer can be further analyzed and calculated through a self-made program, a real-time curve of the central wavelength offset of the output light is obtained in a computer 9, the optical rotation of the solution in a sample cell can be quantitatively calculated through the curve, the concentration of biomolecules in the solution is obtained through calculation, the purpose of representing the reaction of a biological sample in real time is achieved, and parameters required by analysis of interaction of the biomolecules such as a binding constant and the like are obtained through calculation.
Adjusting the angle of the first linear polarizer 3 to be pi/4 of the included angle with the vertical direction, recording the optical rotation angle of the solution in the glass sample cell 4 as alpha, wherein the front selection state is the initial light quiltThe first linear polarizer 3 and the optical rotation of the solution in the glass sample cell 4 jointly select the formed polarization state
Wherein, | H > represents horizontal polarization and | V > represents vertical polarization. The angle of the second linear polarizer 6 is adjusted to form an angle with the vertical direction
The rear selected state is the polarization state of the emergent light selectively formed by the second linear polarizer 6
Also, operators of the system can be written as
Next, a quartz polarising plate 5 is used to induce an interaction between the system and the pointer (gaussian laser beam from the laser), with an interaction intensity τ = β d/P 0 Wherein beta is the central frequency P of the selected quartz optical rotation sheet 5 0 The specific optical rotation at this time, d, is the thickness of the quartz optical rotation piece 5. When the interaction strength tau is very small, the system satisfies weak measurement conditions, introducing
Weak value of (2)
According to weak measurement theory, the weak value introduces a magnification factor in the measurement result, and the momentum shift deltaP can be obtained through weak value calculation.
When χ < P 0 Time (χ is the spectral width, P, of the selected light source, i.e., superluminescent light emitting diode 1 0 Is the center frequency of a gaussian laser beam), the center wavelength of the output light is λ =2 pi/P, where P is the momentum of photons, and thus, the output light resulting from a solution with an optical rotation of α can be obtainedCentral wavelength shift of emitted light
Further, when the approximation condition | τ P is satisfied 0 When-epsilon < tau x, the linear relation between the central wavelength offset of the emergent light and the optical rotation of the solution to be measured can be obtained
Therefore, the amount of shift Δ λ of the center wavelength of outgoing light due to the solution having the optical rotation α α Shift amount Δ λ of central wavelength of emitted light with respect to optical rotation of 0 0 Is changed into
Thus, a linear corresponding relation between the central wavelength offset of the emergent light of the system and the optical rotation of the solution to be measured is established.
The relation between the optical rotation of the solution and the concentration of the solution to be measured is alpha = [ alpha ]]l.C, wherein, [ alpha ]]Is the characteristic specific optical rotation of the sample to be measured, the unit is angle, C is the concentration of the solution to be measured, the unit is g/mL, and l is the distance of the light beam passing through the sample solution, and the unit is dm. When more than one optically active solute molecule is contained in the solution to be tested,
wherein, [ alpha ] is i ]Is the characteristic specific optical rotation, C, of each solute molecule i The remaining parameters and all units are the same as in the previous formula for the concentration of each solute. According to the method, the optical rotation degree of the solution in the sample cell can be quantitatively calculated by the optical rotation biosensor system based on optical weak measurement, and the concentration of the biomolecules in the solution can be obtained through calculation, so that the optical rotation biosensor system based on optical weak measurement can be used for quantitatively calculating the optical rotation degree of the solution in the sample cell, and further obtaining the concentration of the biomolecules in the solutionThe purpose of real-time characterization of the biological sample reaction is achieved, and parameters required by biomolecule interaction analysis such as binding constant and the like are calculated and obtained. The system realizes a complex detection principle through a simpler device, and the biological reaction to be detected is generated in a common glass sample cell, so that the expansibility is better.
The invention also provides a general method for calculating the biomolecule interaction parameters based on the central wavelength signals output by the optical weak measurement optical rotation biosensing system, which can provide a combination equilibrium constant K A And determination of the number of binding sites n. Here, considering the combination of a biomolecule A having optical rotation ability with a molecule B, the reaction between them can be expressed as a reaction assuming that there are n binding sites for the molecule B on the molecule A
Wherein, B n A is a composite molecule generated after the A and the B are combined.
When the product B is formed n When A has optical activity, the detection method comprises the following steps: a small amount (not excessive) of a solution containing B molecules is titrated into a solution containing optically active biomolecules A, assuming that there are n nearly identical and independent binding sites on molecules A, according to the binding constant K A Can be given by
Wherein the content of the first and second substances,
and
respectively the concentration of the optically active biomolecule A and the concentration of the optically active biomolecule B after the reaction,
is a compound molecule B generated after the molecule A is combined with the molecule B n The concentration of A. Let the total concentration of molecule A after the jth titration reaction be
The concentration of the remaining molecules A which have not reacted with molecules B is
The total concentration of the molecule B is
The concentration of the remaining molecules B which have not reacted with the molecules A is
A compound molecule B generated after the molecule A is combined with the molecule B n A has a concentration of
Then
This is always true. By measuring the corresponding relation between the central wavelength of the output light of the optical rotation biosensor system and the optical rotation of the solution through optical weak measurement, the method can obtain
In the formula, l is the length of the sample cell, k is the corresponding proportion between the central wavelength offset of the emergent light output by the optical weak measurement optical rotation biosensor and the optical rotation of the solution, j is the titration frequency,
the offset of the corresponding center wavelength when only the molecule A exists in the solution compared with the offset when only the water exists in the sample cell,
the concentration of molecule A in the solution, Δ λ j The shift amount of the center wavelength of the emergent light after the jth titration to the center wavelength of the emergent light at the optical rotation of 0, alpha A The optical rotation of the solution is the optical rotation of the solution with only molecule A in the solution. When the solution of the molecule B is dropped into the solution of the molecule A for v times, the central wavelength shift amount reaches the minimum value of rapid change after the molecule A which is not combined with the molecule B does not exist, at this time,
the shift amount of the center wavelength at this time was recorded as Δ λ V Then, then
To this end, it can be obtained by calculation
To facilitate the calculation and verify the reliability of the data, the formula is processed into
Therefore, when titrating the solution of the molecule B to the solution of the optically active biomolecule A step by step, the binding equilibrium constant K can be obtained by fitting the stable central wavelength value after multiple titrations A And a value combining the number of sites n.
When the optical rotation of the product is not available (or the optical rotation of the product is extremely small and can be ignored), the detection method comprises the following steps: detection by titration of a solution containing the B molecule into a solution containing the optically active biomolecule A is also carried out when the resulting complex molecule B is known n Specific rotation of A
At 0 or negligibly small, the optical rotation of the solution after the reaction is only produced by the remaining molecules a. The optical rotation of the solution before the reaction (i.e., when only the molecule A is present in the solution) is recorded as α A And the optical rotation of the solution after the jth titration is recorded as alpha j With water (or otherwise)Solvent) as the center wavelength calibration zero point, and the offset of the center wavelength caused by the original molecule A in the sample cell is set as
The shift amount of the center wavelength after the jth titration is Delta lambda j Then, then
After operation, can obtain
To obtain the binding constant K of the reaction of the molecule A and the molecule B by convenient calculation A And combining the number n of the sites to obtain
Thus, in the stepwise titration of the solution of molecule B into the solution of optically active biomolecule A, the binding equilibrium constant K can be obtained by fitting the stable shift of the center wavelength after multiple titrations A And a value combining the number of sites n.
Specifically, as shown in fig. 3, in the detection process, the light source and the computer are turned on, the front and rear polarizers (the first linear polarizer 3 and the second linear polarizer 6) are adjusted to be nearly orthogonal (for example, between 89 ° and 91 °), and then the quartz rotation plate 5 is adjusted to operate the optical rotation biosensing system in the "weak measurement" state.
Deionized water is injected into the glass sample cell 4, and when the central wavelength of the output light of the optical rotation biological sensing system calculated and displayed by the computer is stable, the central wavelength delta lambda of the output light is recorded 0 As a blank set scale for the system, i.e., a "0" point scale.
Then, a solution of the optical rotation biomolecule A in the interaction of the biomolecule to be detected is introduced, and the solution is countedWhen the central wavelength of the output light of the optical rotation biological sensing system calculated and displayed by the computer is stable, the offset of the central wavelength at the moment compared with the blank calibration is recorded
A solution of another reactant molecule B with which biomolecules interact is dropped dropwise, the volume and concentration of the solution of molecule B dropped each time are the same, and the reaction of molecule B is always kept not excessive. As shown in FIG. 2, after each titration, a portion of molecule A was bound to molecule B due to the interaction to form molecule B n A, resulting in a change in the optical rotation of the solution (from. Alpha.) 1 Change to alpha 2 In which α is 1 The optical rotation of the solution, alpha, before dropping of the molecule B 2 The optical rotation of the solution after the molecule B is dropped), thereby causing the central wavelength of the output light of the optically active biosensing system to change. After titration, the central wavelength of the output light of the optical rotation biological sensing system is calculated and displayed by the computer to be stable, and the central wavelength offset Delta lambda of the output light of the optical rotation biological sensing system obtained after titration is recorded j Central wavelength offset DeltaLambda obtained after multiple titrations j The curve characterizes the interaction between biomolecules A and B.
When the product B is formed n When the A has optical activity and the optical activity is larger and cannot be ignored, the detection of the biomolecule interaction parameters needs to be titrated for many times until the central wavelength offset reaches the minimum value (or the maximum value) of rapid change, and the stable central wavelength value delta lambda is recorded at the moment V The central wavelength offset value after each titration is divided into delta lambda i And Δ λ V And inputting the corresponding parameters into a computer by using a formula
Fitting to obtain a combined equilibrium constant K A And the number of binding sites n.
When the product has no optical activity or the optical activity of the product is negligible, after a plurality of titrations (generally up to 10 times), the central wavelength shift value Δ λ is determined for each titration i And inputting the corresponding parameters into a computer by using a formula
Fitting to obtain a combined equilibrium constant K A And the number of binding sites n.
The invention aims to provide a novel optical device and a method for detecting interaction of non-fixed biomolecules, and enriches detection methods in the field. The existing method provides a detection method with similar detection function, which is a fluorescence quenching method, but the precision, the sensitivity and the like of the method are related to the fluorescence of biomolecules, so when the autofluorescence intensity of the biomolecules is low, the fluorescence modification of the biomolecules is needed to perform the biomolecule interaction detection by using the fluorescence quenching method, and the complexity and the cost of the detection are increased. The invention can introduce the analysis of optical rotation signals into the field of non-fixed biomolecule interaction detection, does not need pretreatment, provides a new method for optical rotation biomolecule interaction detection, and particularly provides a better scheme for optical rotation biomolecule interaction detection with low fluorescence intensity. The method achieves the purpose of calculating the change of the optical rotation of the solution in which the biomolecular interaction occurs by the high sensitivity of the central wavelength signal of the output spectrum of the optical weak measurement optical rotation biological sensing system through the high-magnification detection of the phase difference change of the polarized light caused by the optical rotation by the weak measurement principle, analyzes the contribution condition of each biomolecule in the solution to the whole optical rotation of the solution, completes the representation of the biomolecular interaction condition and completes the combination equilibrium constant K of the biomolecular interaction A And determination of the number of binding sites n.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. It will be apparent to those skilled in the art that various equivalent substitutions and obvious modifications can be made without departing from the spirit of the invention, and all changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope.
Claims (10)
1. The utility model provides an optical rotation biosensing system based on optical weak measurement, its characterized in that, includes detection components, preceding option state, weak interaction subassembly and back option state, detection components includes light source, first lens, second lens, spectrum appearance and processing unit, preceding option state includes the sample cell that first polaroid and printing opacity material were made, weak interaction subassembly includes optical rotation dispersion element, the back option state includes the second polaroid, the light of light source outgoing passes through in proper order first lens with first polaroid gets into the sample cell, follows the light process of sample cell outgoing passes through in proper order behind the optical rotation dispersion element the second polaroid with the second lens reachs the spectrum appearance, the spectrum appearance is used for recording the change of emergent light center wavelength, processing unit connects the spectrum appearance is in order to be used for obtaining the parameter of biomolecular interaction according to the change of emergent light center wavelength.
2. The optical biosensing system of claim 1, wherein said optically dispersive element is a quartz polarimeter.
3. The system of claim 1, wherein the first lens is a collimating lens and the second lens is a coupling lens.
4. The system according to any of claims 1 to 3, wherein the angle between the first polarizer and the second polarizer is 89 ° to 91 °.
5. A method for detecting biomolecular interactions based on weak optical measurements, characterized in that the optically active biosensing system according to any of claims 1 to 3 is used to detect parameters of biomolecular interactions, comprising the steps of:
s1: adjusting the included angle between the first polaroid and the second polaroid to be 89-91 degrees;
s2: adjusting the optical dispersion element to make the optical biosensor system work in a weak measurement state;
s3: introducing a solvent into the sample cell, and measuring the central wavelength offset delta lambda of the emergent light at the moment 0 To perform calibration;
s4: extracting the solvent, introducing optically active biomolecule A solution into the sample cell, and measuring the central wavelength ratio of the emergent light at the moment 0 Is offset amount of
S5: dropwise adding a solution of molecule B into the sample pool, and measuring the central wavelength ratio delta lambda of emergent light obtained after the jth titration after each titration 0 Offset amount of (a) Δ λ j ；
S6: multiple titrations to obtain the central wavelength ratio Delta lambda of the emergent light 0 Offset amount of (a) Δ λ j Fitting to obtain the binding equilibrium constant K of the biomolecule A and the molecule B A And a value combining the number of sites n.
6. The detecting method according to claim 5, wherein the step S1 further comprises adjusting the first polarizer to have an angle of π/4 with respect to the vertical direction.
7. The detection method according to claim 5, wherein the solvent in step S3 is deionized water.
8. The detection method according to any one of claims 5 to 7, characterized in that step S6 specifically comprises:
s61: determination of product B n Whether the A has optical rotation or not, if so, executing step S62, and if not, executing step S63;
s62: titrating the solution of the molecule B until the central wavelength of the emergent light is compared with delta lambda 0 Offset amount of (a) Δ λ j The central wavelength of the emergent light reaches the minimum value of rapid change, and the central wavelength ratio Delta lambda of the emergent light at the moment is recorded 0 Offset amount of (a) Δ λ V And then obtaining the central wavelength ratio delta lambda of the emergent light according to multiple titrations 0 Fitting to obtain the binding equilibrium constant K of the biomolecule A and the molecule B A And a value of the number of binding sites n;
s63: titrating the solution of the molecule B for at least 10 times, and comparing the central wavelength of the emergent light with the central wavelength of the emergent light obtained by multiple titrations 0 Fitting to obtain the binding equilibrium constant K of the biomolecule A and the molecule B A And a value combining the number of sites n.
9. The detection method according to claim 8, wherein the step S62 is performed by fitting the shift amount of the central wavelength of the outgoing light obtained by the multiple titrations according to the following formula:
wherein the content of the first and second substances,
the concentration of biomolecule a when only biomolecule a is in solution,
the total concentration of the biomolecule A solution after the v-th titration of the molecule B solution, v being the ratio of the central wavelength of the emitted light to the wavelength of Delta lambda 0 The number of titrations of the solution of molecule B when the offset of (a) reaches a rapidly changing minimum,
the central wavelength of emergent light corresponding to the condition that only the biomolecule A exists in the solution is compared with delta lambda 0 The amount of the offset of (a) is,
for the total concentration of biomolecule a solution after the jth titration of molecule B solution,
is the total concentration of the molecule B solution after the jth titration of the molecule B solution.
10. The detection method according to claim 8, wherein the step S63 is performed by fitting the shift amount of the central wavelength of the outgoing light obtained by the multiple titrations according to the following formula:
wherein the content of the first and second substances,
for the total concentration of the molecule B solution after the jth titration of the molecule B solution,
for the total concentration of biomolecule a solution after the jth titration of molecule B solution,
the central wavelength of emergent light corresponding to the condition that only the biomolecule A exists in the solution is compared with delta lambda 0 The amount of the offset of (a) is,
is the concentration of biomolecule a when only biomolecule a is in solution.
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