CN110794070B - Taylor diffusion analysis device and method for determining size of substance in solution - Google Patents

Taylor diffusion analysis device and method for determining size of substance in solution Download PDF

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CN110794070B
CN110794070B CN201911117652.2A CN201911117652A CN110794070B CN 110794070 B CN110794070 B CN 110794070B CN 201911117652 A CN201911117652 A CN 201911117652A CN 110794070 B CN110794070 B CN 110794070B
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不公告发明人
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Abstract

The invention provides a Taylor diffusion analysis device and a method for measuring the size of a substance in a solution, and relates to the technical field of substance analysis in a liquid phase medium.

Description

Taylor diffusion analysis device and method for determining size of substance in solution
Technical Field
The invention relates to the technical field of substance analysis in a liquid-phase medium, in particular to a Taylor diffusion analysis device and a method for determining the size of a substance in a solution.
Background
Taylor Diffusion Analysis (TDA) is an analysis method for measuring the hydrodynamic radius of a single substance in a liquid phase environment based on substance diffusion characteristics by using a capillary as a channel in the liquid phase environment, and is difficult to realize wide application due to the lack of multi-component structural analysis capability of a complex system and complex correction process.
In taylor diffusion analysis experiments, constant pressure/flow rate fluid is used to drive analytes in laminar flow, allowing analysis of the extent of analyte diffusion by a detector, which is readily available on commercial Capillary Electrophoresis (CE) instruments. The traditional taylor diffusion analysis is generally used for analyzing the diffusion degree of substances with single-component structures, but the analysis efficiency is low due to the method.
Disclosure of Invention
The invention aims to provide a device and a method for measuring the size of a liquid phase substance based on Taylor diffusion mass spectrometry, so as to solve the problems in the prior art.
In a first aspect, an embodiment of the present invention provides a taylor diffusion analysis apparatus, including: the microfluidic circuit comprises at least two microfluidic circuit branches with different lengths, and is used for dividing a sample to be detected into at least two parts, respectively flowing through the at least two microfluidic circuit branches, and then carrying out mass spectrometry.
In an alternative embodiment, the microfluidic circuit further comprises: the liquid inlet shunt assembly comprises a first liquid inlet and at least two first liquid outlets; the liquid outlet converging assembly comprises at least two second liquid inlets and a second liquid outlet; two ends of each of the at least two micro-flow passage branches are respectively connected with the first liquid outlet and the second liquid inlet;
the liquid inlet shunt assembly is used for receiving the sample to be detected through the first liquid inlet, dividing the sample to be detected into at least two parts through at least two first liquid outlets and respectively introducing the two parts into each microflow passage branch; the liquid outlet convergence assembly is used for receiving part of samples to be detected in each microflow passage branch through at least two second liquid inlets and respectively enabling the part of samples to be detected to flow out through the second liquid outlets so as to carry out mass spectrum detection respectively.
In an alternative embodiment, the device further comprises a thermostatic assembly for controlling the temperature within the at least two microfluidic circuit branches to be constant; a pump assembly for providing a constant carrier current to the microfluidic pathway required for analysis of the sample to be tested.
In an alternative embodiment, the microfluidic circuit is implemented by a capillary or microfluidic chip.
In an optional embodiment, the sample feeding device further comprises a sample feeding switching assembly, wherein the sample feeding switching assembly is connected with the microfluidic channel, and the sample feeding switching assembly is used for switching between constant current carrying and a sample to be detected.
In a second aspect, an embodiment of the present invention provides a multidimensional joint liquid phase structure analysis apparatus, including: the taylor diffusion analysis apparatus and the mass spectrometry detection apparatus as in any one of the preceding embodiments, wherein the mass spectrometry detection apparatus comprises an ionization part and a mass spectrometry detector, the ionization part is connected to the microfluidic channel, and the ionization part is used for ionizing a sample to be detected from the microfluidic channel and then analyzing the sample by the mass spectrometry detector.
In an alternative embodiment, the apparatus further comprises: and the front stage separation equipment is connected with the microfluidic channel and is used for providing the sample to be detected for the microfluidic channel.
According to the Taylor diffusion analysis device and the multidimensional combined liquid phase structure analysis device, at least two micro-flow passage branches with different lengths in the micro-flow passage are used for dividing a sample to be detected into at least two parts and respectively flowing through the at least two micro-flow passage branches to perform mass spectrum detection, so that Taylor diffusion analysis and mass spectrum detection of a multi-component sample to be detected are simultaneously completed, and the requirement of high-throughput analysis is met.
In a third aspect, an embodiment of the present invention provides a method for determining a size of a substance in a solution based on taylor diffusion analysis, the method including: determining target mass spectrum data detected after a target section sample to be detected is divided into two parts of samples to be detected and respectively passes through two microflow passages with different path lengths; determining the diffusion degree and the peak-off time of the target substance corresponding to the two parts of samples to be detected based on the target mass spectrum data; and determining the size of the target substance according to the diffusion degree and the peak-off time corresponding to the target substance.
In an alternative embodiment, the step of determining the size of the target substance based on the corresponding degree of diffusion and the time-to-peak of the target substance comprises: determining the size of the target substance according to the diffusion degree and the peak-off time of the target substance corresponding to the two parts of samples to be detected; determining two chromatographic peaks of a target substance corresponding to the two parts of samples to be detected based on the target mass spectrum data; and fitting through a Gaussian function based on the spectral peak center position parameter and the peak width parameter of each chromatographic peak to determine the diffusion degree and the peak emergence time of the target substance corresponding to the two parts of samples to be detected.
In a fourth aspect, an embodiment of the present invention provides a computer device, including a memory and a processor, where the memory stores a computer program executable on the processor, and the processor implements the steps of the method according to any one of the above embodiments when executing the computer program.
According to the Taylor diffusion analysis device and the method for measuring the size of the substance in the solution, the sample to be measured is divided into at least two parts by the at least two micro-flow passage branches with different lengths in the micro-flow passage, and the at least two parts of the sample to be measured are subjected to mass spectrometry detection after respectively flowing through the at least two micro-flow passage branches, so that Taylor diffusion analysis and mass spectrometry detection of the multi-component sample to be measured are simultaneously completed, the components separated by various liquid phase analysis methods can be further subjected to size measurement, the continuous analysis capability of the system is improved, and the requirement of high-throughput analysis is met.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, 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 some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a structural diagram of a Taylor diffusion analyzer according to an embodiment of the present invention;
FIG. 2 is a structural diagram of a Taylor diffusion analyzer according to an embodiment of the present invention;
fig. 3 is a structural diagram of a multidimensional joint liquid phase structure analysis device according to an embodiment of the present invention;
FIG. 4 is a flow chart of a method for determining the size of a substance in a solution based on Taylor diffusion analysis according to an embodiment of the present invention;
FIG. 5 is a flow chart of another method for determining the size of a substance in a solution based on Taylor diffusion analysis according to an embodiment of the present invention;
FIG. 6 is a schematic diagram of a Taylor diffusion mass spectrometry method according to an embodiment of the present invention;
fig. 7 is a diagram illustrating a result of analyzing an influence of a condition on a characteristic ion flow graph according to a method provided in an embodiment of the present invention;
FIG. 8 is a mass spectrum of a mixture of phenylalanine and angiotensin II according to the present invention;
FIG. 9 is a graph of fitting results for bimodal parameters provided by an embodiment of the present invention;
fig. 10 is a flow chart of an extracted ion beam according to an embodiment of the present invention;
FIG. 11 is a graph showing the results of measuring the size of the liquid phase of a mixed sample (phenylalanine and angiotensin II) according to an embodiment of the present invention;
fig. 12 is a schematic structural diagram of a computer device according to an embodiment of the present invention.
Icon: 100-Taylor diffusion analysis device; 11-microfluidic pathway; 111-microfluidic pathway branch; 112-microfluidic pathway branch; 210-an inlet liquid shunt assembly; 220-liquid outlet convergence component; 211-a first liquid inlet; 212-a first outlet port; 221-a second liquid inlet; 222-a second exit port; 230-a capillary tube; 231-a sleeve; 240-a thermostatic component; 250-sample introduction switching component; 300-mass spectrometric detection means; 302-an ionizing component; 303-mass spectrometric detector.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments 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.
It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.
Analysis of the molecular structure is helpful for understanding information such as physicochemical properties and biological activity. X-ray crystallography, nuclear magnetic resonance spectroscopy, and cryoelectron microscopy are among the most commonly used techniques for structural analysis. When the three technologies are used for analyzing the structure, a large amount of samples with high purity need to be obtained firstly, the preliminary treatment steps are complicated, and the reconstruction after data collection also faces the problems of large data volume and the like. Therefore, a method with high sensitivity and complex component structure analysis capability is needed, and moreover, the method for firstly determining the basic size and conformation of a substance by using auxiliary means can be helpful for the subsequent prediction and characterization of a high-resolution structure.
The ion mobility mass spectrometry is a novel two-dimensional mass spectrometry technology combining an ion mobility separation technology and a mass spectrometry in a gas phase environment. Measurement of ion mobility rate utilizes an electric field intensity gradient and a laterally flowing buffer gas, and the mobility rate depends on the collision cross section and the charged state of ions. The rapid separation and detection of complex samples in the gas phase can be realized, and the structural information of substances under the gas phase condition can also be obtained. However, the gas phase structure information obtained by the method often cannot accurately reflect the liquid phase structure of the molecule and the properties of the molecule under the solution condition, and the structure function information of the biomacromolecule under the physiological condition is difficult to obtain.
The conventional taylor diffusion analysis method does not have a mixture separation/analysis capability, and thus cannot simultaneously analyze a plurality of components. To solve this problem, Billy a. williams and gyulavirh propose a time-sharing technique, in which the analytes are first separated in the conventional CE mode, the instrument is then adjusted to the TDA mode for structural analysis, and the hydrodynamic radii of the different molecules in the mixture are measured in two steps. However, this method still has difficulty in satisfying the requirement of high-throughput analysis.
Based on the above, the embodiment of the invention provides a Taylor diffusion analysis device, a multi-dimensional combined liquid phase structure analysis device and a method for determining the size of a substance in a solution based on Taylor diffusion analysis, which can simultaneously complete Taylor diffusion analysis and mass spectrum detection of a multi-component sample to be determined, realize further size determination of components separated by various liquid phase analysis methods, and meet the requirement of high-throughput analysis. Some embodiments of the invention are described in detail below with reference to the accompanying drawings. The embodiments described below and the features of the embodiments can be combined with each other without conflict.
The embodiment of the invention provides a Taylor diffusion analysis device, which comprises the following structures shown in figure 1:
the microfluidic circuit 11 includes at least two microfluidic circuit branches with different lengths, and is used for dividing a sample to be detected into at least two parts, and performing mass spectrometry after the sample flows through the at least two microfluidic circuit branches respectively. For example, a microfluidic channel branch 111 and a microfluidic channel branch 112 are shown in fig. 1.
The microfluidic circuit is mainly used for dividing a sample to be detected into at least two parts, and each part reaches the next-stage equipment at a preset interval, wherein the next-stage equipment can be a mass spectrometry detection device or other liquid phase analysis devices.
It should be noted that the microfluidic channel may also be referred to as a microfluidic channel, a microfluidic circuit, a microfluidic line, or the like.
According to the embodiment of the application, Taylor diffusion analysis and mass spectrum detection of a multi-component sample to be detected are completed simultaneously, components separated by various liquid phase analysis methods can be subjected to further size measurement, the continuous analysis capability of a system is improved, and the requirement of high-throughput analysis is met. In some embodiments, the microfluidic circuit may include various implementations, for example, implemented by a capillary or a microfluidic chip.
As an example, as shown in fig. 2, the microfluidic circuit may include an inlet branching assembly 210, an outlet converging assembly 220, and at least two microfluidic circuit branches of unequal lengths.
The inlet shunt assembly 210 includes a first inlet 211 and at least two first outlet 212.
The liquid outlet converging assembly 220 comprises at least two second liquid inlets 221 and a second liquid outlet 222;
at least two microflow passages with different lengths are branched. For example, the microfluidic circuit may be a capillary 230. Two ends of each micro-flow passage branch are respectively connected with a first liquid outlet 212 and a second liquid inlet 221.
The liquid inlet shunt assembly 210 is configured to receive a sample to be tested through a first liquid inlet 211, divide the sample to be tested into at least two parts through at least two first liquid outlets 212, and respectively introduce the two parts into each branch of the microfluidic channel; the liquid outlet converging assembly 220 is configured to receive a portion of the sample to be detected in each branch of the microfluidic channel through at least two second liquid inlets 221, and to separately flow out a portion of the sample to be detected through second liquid outlets 222 so as to perform mass spectrometry.
As an example, the microfluidic circuit may be implemented by a microfluidic chip. The microfluidic circuit may include an inlet circuit, an outlet circuit, and at least two microfluidic circuit branches of unequal lengths. The liquid inlet passage receives a sample to be detected, divides the sample to be detected into at least two parts and respectively introduces the parts into the microflow passage branches; the liquid outlet channel receives each part of samples to be detected in each channel of the microfluidic channel branches, and the samples to be detected flow out of each part of samples to be detected so as to carry out mass spectrum detection respectively.
It should be noted that the implementation manner of the microfluidic path through the capillary or the microfluidic chip is only an example, and the embodiment of the present application may further include other manners that the sample to be detected can be divided into multiple portions in the flow-through process and reach at fixed intervals, which is not described herein again.
In some embodiments, the taylor diffusion analysis device may further include a thermostatic assembly. The constant temperature component is used for controlling the temperature in the at least two microflow passage branches to be constant. By providing a constant temperature, environmental disturbances during the analysis can be reduced, making the analysis results more accurate.
In some embodiments, the taylor diffusion analysis device may further comprise at least one pump assembly.
As an example, the taylor diffusion analysis device may comprise a pump assembly for providing a constant flow rate of buffer solution for providing a constant carrier flow to the microfluidic channel required for analysis of the sample to be tested. The pump assembly may be a micro-fluid syringe pump or a liquid chromatography pump, or the like.
As another example, the taylor diffusion analysis device can include a sample switching assembly including a pump assembly coupled to the microfluidic circuit, the sample switching assembly configured to switch between a constant current carrying (e.g., provided by a microfluidic syringe pump, a liquid chromatography pump, etc.) and a sample to be tested. It should be noted that the constant current may also be referred to as a constant current or a constant voltage.
The taylor diffusion analytical equipment that this embodiment provided divides into at least two parts through the inlet liquid shunt circuit subassembly with the sample that awaits measuring, and at least two parts samples that await measuring flow out through two at least ways capillary and play liquid convergence subassemblies that length is unequal, have realized accomplishing the taylor diffusion analysis of multicomponent sample that awaits measuring simultaneously, have satisfied high throughput analysis's demand.
Embodiments of the present application will be described in detail below with reference to specific examples, which take the case of implementing a microfluidic circuit by a capillary tube.
The sample to be tested may be a discrete or continuous sample, typically a mixed sample comprising a plurality of solutions of different concentrations, for example, a 0.5ppm solution of phenylalanine and a 2ppm solution of angiotensin II. When Taylor diffusion analysis is carried out, the mixed sample to be measured flows along with the buffer liquid, namely, the constant current carrying required by analysis. For example, a constant fluid injection pump can be used to provide a constant pressure of 0-10000mbar or equivalent flow rate for providing a constant carrier flow required for Taylor diffusion analysis.
The inlet shunt assembly 210 may be configured to receive a sample to be tested, divide the sample into at least two portions, and further introduce the sample to be tested into the capillary. The liquid inlet branching assembly may include a first liquid inlet 211 and at least two first liquid outlets 212, the first liquid inlet 211 may be configured to introduce a sample to be tested into the liquid inlet branching assembly 210, and the at least two first liquid outlets 212 may be configured to divide the sample to be tested in the liquid inlet branching assembly 210 into at least two parts, and introduce the two parts into each capillary respectively, so as to split the sample to be tested. For example, the liquid inlet shunt assembly 210 may be a liquid phase three-way valve, and the valve body of the three-way valve has three outlets. One liquid inlet can be used for introducing a sample to be detected into the liquid phase three-way valve, and the two liquid outlets can be used for dividing the sample to be detected in the liquid phase three-way valve into at least two parts and respectively introducing the two parts into each path of capillary tube to realize the shunting of the sample to be detected.
The liquid outlet convergence component 220 can be used for receiving a part of the sample to be detected and discharging a part of the sample to be detected so as to perform mass spectrometry detection respectively. The liquid outlet converging assembly 220 may include at least two second liquid inlets 221 and a second liquid outlet 222, where the second liquid inlet 221 may be configured to receive a portion of the sample to be detected in each capillary, and the second liquid outlet 222 may be configured to flow out a portion of the sample to be detected so as to perform mass spectrometry detection, respectively. For example, the liquid outlet converging component 220 may be a liquid phase three-way valve, and the valve body of the three-way valve has three outlets. Two liquid inlets can be used for receiving part of samples to be detected in each capillary, and one liquid outlet can be used for flowing out part of samples to be detected so as to carry out mass spectrum detection respectively.
Capillaries are typically thin tubes or microchannels having an internal diameter of no more than 1 mm, which can be used to provide a channel for taylor diffusion analysis of a sample to be tested. In this embodiment, the lengths of the at least two capillaries 230 are different, that is, the lengths of the branch paths of the samples to be measured are different. The two ends of each capillary 230 are connected with a sleeve 231, and are respectively connected with a first liquid outlet 212 and a second liquid inlet 221 through the sleeves 231, so that the connection of the liquid inlet branching assembly and the liquid outlet converging assembly is realized.
In some embodiments, the taylor diffusion analysis device further comprises a thermostat assembly 240 that can be used to control temperature constancy in the analysis pathway. For example, the temperature control assembly can be adjusted by cooling air, cooling fluid and the like, so that the temperature in at least two capillary tubes is maintained to be a constant value, and the analysis of a sample to be detected is facilitated.
In some embodiments, the taylor diffusion analyzer further comprises a sample introduction switching assembly 250, which can be used for introducing a sample to be tested, controlling the introduced sample to be tested during introduction, and switching between a constant current carrying and the sample to be tested, wherein the introduced sample to be tested can be a discrete or continuous sample. For example, sample injection control may be achieved using a six-way valve, a liquid chromatography autosampler, or the like. The sample switching assembly may also be used to provide a buffer, such as a constant carrier current, that flows with the mixed sample to be measured.
The taylor diffusion analysis device provided by the above embodiment comprises a liquid inlet shunt component, a liquid outlet convergence component and at least two paths of capillaries with different lengths, and is used for realizing taylor diffusion analysis with correction; a thermostatic assembly may also be included, using adjustments of cooling air, coolant flow, etc., for maintaining the temperature of the tube analysis passage constant.
The embodiment of the invention also provides a multidimensional joint liquid phase structure analysis device, which comprises the following structures shown in figure 3:
the device comprises the Taylor diffusion analysis device 100 and the mass spectrum detection device 300 in any one of the above embodiments, wherein the mass spectrum detection device 300 comprises an ionization part 302 and a mass spectrum detector 303, the ionization part 302 is connected with the Taylor diffusion analysis device 100, and the ionization part 302 is used for ionizing part of samples to be detected from the micro-flow passage and then analyzing the part by the mass spectrum detector 303.
The Ionization module is configured to ionize a sample to be measured, and may use an electrospray Ionization (ESI), an Atmospheric Pressure Chemical Ionization (APCI), an Atmospheric Pressure Photoionization (APPI) or an ion source associated with a mass spectrometer. The mass spectrometer is used for detecting the substance to be detected and acquiring data.
In some embodiments, the multidimensional joint use liquid phase structure analysis device further comprises a pre-stage separation device connected with the liquid inlet shunt assembly for providing a sample to be tested. As an example, the pre-stage separation apparatus may also be used to provide a constant carrier flow, for example a binary constant flow pump in liquid chromatography.
The embodiment of the invention also provides a method for determining the size of a substance in a solution based on Taylor diffusion analysis, which comprises the following steps as shown in figure 4:
s401, determining that a target section sample to be detected is divided into two parts of samples to be detected, and respectively passing through the detected target mass spectrum data after the two parts of samples to be detected are branched by microfluidic channels with different path lengths;
s402, determining the diffusion degree and the peak-off time of the target substance corresponding to the two parts of samples to be detected based on the target mass spectrum data;
and S403, determining the size of the target substance according to the diffusion degree and the peak-off time corresponding to the target substance.
The microfluidic channel branches with different path lengths are also provided with constant current carriers, and the constant current carriers are used for pushing a sample to be detected to flow at a constant speed in the microfluidic channel branches. The target mass spectral data may be a target ion flow chromatogram.
For the above S402, the following steps may be specifically implemented:
determining the size of the target substance according to the diffusion degree and the peak-off time of the target substance corresponding to the two parts of samples to be detected;
determining two chromatographic peaks of a target substance corresponding to two parts of samples to be detected based on target mass spectrum data; and fitting through a Gaussian function based on the spectral peak center position parameter and the peak width parameter of each chromatographic peak to determine the diffusion degree and the peak emergence time of the target substance corresponding to the two parts of samples to be detected.
Specifically, the diffusion degree and the peak-off time of the target substance corresponding to the two parts of the sample to be measured can be determined by the following steps:
step 1), determining two chromatographic peaks of a target substance corresponding to two parts of samples to be detected based on target mass spectrum data;
and 2) fitting a Gaussian function to each chromatographic peak to determine the diffusion degree and the peak-off time of the target substance. Wherein, the chromatographic peak corresponds to a central position parameter and a peak width parameter of the chromatographic peak.
For example, the peak fitting can be performed using the following Exponential Modified Gaussian (EMG) formula:
Figure BDA0002273621180000111
wherein, a0Is the peak area, a1Is the center of the peak, a2Is the peak width, a3Is the degree of torsion.
It should be noted that other gaussian functions may be used for fitting. For example, an intensity form (amplitude) gaussian function or an area form (area) gaussian function, etc.
For S403 described above, the size of the target substance may be determined according to the diffusion coefficient of the target substance.
For the diffusion coefficient of the target substance, the diffusion coefficient D of the target substance can be calculated by the following formulad：
Figure BDA0002273621180000121
Wherein Rc is the inner diameter of the capillary channel, and the diffusion degrees of the target substance corresponding to the two parts of the samples to be measured are respectively1And2the peak-off time of the target substance corresponding to the two parts of the sample to be tested is t1 and t2 respectively.
Based on the diffusion coefficient of the target substance, the size R of the target substance can be calculated by the following formulah：
Figure BDA0002273621180000122
Wherein, k isbIs the boltzmann constant, T is the temperature, and η is the solution viscosity coefficient.
Based on the above equations (1-2) and (1-3), the following equations can be obtained in order to achieve the calculation of the size of the target substance:
Figure BDA0002273621180000123
the measurement of the size of a target substance (which may be a single component or a complex having an interaction, for example) in a measurement solution can be achieved based on the above formulas (1 to 4).
The following describes an embodiment of a method for measuring the size of a substance in a solution based on taylor diffusion analysis provided by the present application in detail with reference to specific examples.
The application discloses another method for determining the size of a substance in a solution based on Taylor diffusion analysis, which can be divided into the following steps as shown in figure 5:
s501, collecting Taylor diffusion analysis device and mass spectrum data;
s502, extracting target ion current and fitting a peak shape;
and S503, calculating the hydrodynamic radius of the sample.
For the above S501, one embodiment is to use the apparatus shown in FIGS. 1 to 2, and provide a constant flow rate (10 μ L/min) of the ionized buffer solution by a constant flow syringe pump; the buffer solution is injected into the TDA analysis capillary 230 through the liquid inlet shunt assembly 210 and washed for 5 min; the mass spectrum signal is detected by the mass spectrum detector 303, and the six-way valve is switched after the signal baseline is stable. Another constant flow injection pump is used for providing a constant flow rate (2mL/h) to sample a certain amount of samples, the sample injection is suspended, and the sample injection switching component 250 is switched.
Wherein the sample to be tested can be diffused in the analysis capillary by injecting the buffer solution at a constant flow rate. And (3) ionizing the sample by adopting an electrospray ion source, detecting a signal by using a time-of-flight mass spectrum and acquiring data, and finishing data acquisition after two sample peaks appear. The buffer used here was a volume ratio of acetonitrile in water (containing 0.1% formic acid).
For the above S502, the extracted ion chromatogram of the target substance is obtained by extracting the target ion current, as shown in fig. 6, the analysis sample includes three mass spectrum characteristic peaks (part (a) of fig. 6), the substance ion flow data with the charge-to-mass ratio is extracted, two gaussian chromatographic peaks (part (b) of fig. 6) are obtained, and the corresponding information contained in the chromatographic peaks is obtained by peak shape fitting. The fitted functional form should contain two key pieces of information, namely the spectral peak center (time to peak) and the peak width (degree of spread). Fitting may be done using a Gaussian (area form) function, an Exponentially Modified Gaussian (EMG) function, etc. In this embodiment, an EMG function is used, which is in the form of:
Figure BDA0002273621180000131
wherein a is0Is the peak area, a1Is the center of the peak, a2Is the peak width, a3Is the degree of torsion.
EMG fitting is carried out on the first peak to obtain a parameter a1 which is the peak-appearing time (recorded as t1), and obtain a parameter a2 which is the diffusion degree (recorded as t1)1). Performing EMG fitting on the second peak to obtain a peak-appearing time related parameter (denoted as t2) and a diffusion degree related parameter (denoted as t2)2)。
For the above S503, the hydrodynamic radius of the sample is calculated, the measured parameter (t,) and the sample diffusion coefficient (D,) are calculatedd) The relationship of (a) to (b) is as follows:
Figure BDA0002273621180000132
where Rc is the capillary passage inner diameter.
Hydrodynamic radius (R) of sampleh) The relationship to the diffusion coefficient of the sample is as follows:
Figure BDA0002273621180000141
in the above formula, kbIs the boltzmann constant, T is the temperature, and η is the solution viscosity coefficient. The hydrodynamic radius R of the sample can be solved by substituting experimental datah。
After passing through the above steps S501 to S503, the hydrodynamic radius of the sample under the test conditions can be obtained.
In order to embody the features and advantages of the present invention, a taylor diffusion analysis apparatus, a multi-dimensional combined liquid phase structure analysis apparatus, and a method for measuring a size of a substance in a solution based on taylor diffusion analysis, which are provided in connection with the above embodiments, will be described in detail in the following description. It is to be understood that the invention is capable of modification in various experimental examples without departing from the scope of the invention, and that the description and drawings are to be regarded as illustrative in nature, and not as restrictive.
Experiment one: the influence of the sample introduction time on the peak shape of the sample is considered.
The experimental conditions are as follows: the sample was a 0.5ppm phenylalanine solution, the buffer was a 70% acetonitrile aqueous solution (w: w contains 0.1% formic acid), and the viscosity coefficient was 0.59 mPas. The length of the L1 tube in the capillary tube was 50cm, the length of the L2 tube in the capillary tube was 25cm, and the inner diameter of the tube was 50 μm. The constant assay flow rate was 0.4. mu.L/min and the sampling rate was 1 graph/s. The operation mode is as follows: the samples were injected at a flow rate of 2mL/h for 0.15, 0.1, 0.05, 0.03 minutes, and the results of the experiment were observed as shown in FIG. 7 (a). Therefore, the signal intensity of the sample is improved along with the increase of the sample introduction time, and the signal acquisition is facilitated. However, too high a signal intensity will change the peak shape, affecting the EMG fitting accuracy.
Experiment two: consider the effect of sampling rate on the peak shape of a sample.
The experimental conditions are as follows: the sample was 300ppm of cytochrome C solution, the buffer was 70% acetonitrile aqueous solution (w: w contains 0.1% formic acid), and the viscosity coefficient was 0.59 mPas. The L1 tube length of the capillary tube was 50cm, the L2 tube length of the capillary tube was 25cm, and the tube inner diameter was 50 μm. The constant analysis flow rate is 0.4 mu L/min, and the sample injection adopts 2mL/h flow rate for 0.03 min. The operation mode is as follows: the sampling rates were selected to be 1, 2, and 3 plots/s, respectively, and the experimental results were observed, as shown in fig. 7 (b). It can be seen that as the sampling rate increases, the sample signal intensity decreases. However, too low a sampling rate results in less fittable data and decreases EMG fitting accuracy.
Experiment three: the effect of constant flow rate on the peak shape of the sample was considered.
The experimental conditions are as follows: the sample was a 5ppm phenylalanine solution, the buffer was a 70% acetonitrile aqueous solution (w: w contains 0.1% formic acid), and the viscosity coefficient was 0.59 mPas. The L1 tube length of the capillary tube was 50cm, the L2 tube length of the capillary tube was 25cm, and the tube inner diameter was 50 μm. The sampling rate was 1 picture/s. The sample introduction is carried out at a flow rate of 2mL/h for 0.1 min. The operation mode is as follows: the constant analysis flow rates were set at 0.2. mu.L/min, 0.4. mu.L/min, 0.6. mu.L/min, and 0.8. mu.L/min for observation, as shown in (c) of FIG. 7. It can be seen that with increasing flow rate, the separation of the sample doublets increases and the signal intensity increases. According to the method, two chromatographic peaks with good separation degree need to be obtained for correcting TDA analysis, the separation degree of double peaks and signal intensity need to be considered, and the EMG fitting precision is guaranteed.
Experiment four: the effect of separation tube length ratio on the peak shape of the sample was considered.
The experimental conditions are as follows: the sample was a 5ppm phenylalanine solution, the buffer was a 70% acetonitrile aqueous solution (w: w contains 0.1% formic acid), and the viscosity coefficient was 0.59 mPas. The capillary tube had an L2 tube length of 15cm and a tube inner diameter of 50 μm. The constant analysis flow rate is 0.4 mu L/min, and the sample injection adopts 2mL/h flow rate for 0.1 min. The operation mode is as follows: the results of observation of L1 tubes of capillaries respectively having lengths of 50cm, 25cm and 10cm were obtained as shown in FIG. 7 (d). It can be seen that as the separation tube length ratio increases, the separation of the sample doublets increases, but the signal intensity of the first peak increases and the signal intensity of the second peak decreases. The bimodal separation degree and the signal intensity are considered, and the EMG fitting precision is guaranteed.
Experiment five: the structure of the mixed sample was determined.
The experimental conditions are as follows: the samples were 0.5ppm phenylalanine and 2ppm angiotensin II solutions, the buffer was 70% acetonitrile in water (w: w contains 0.1% formic acid), and the viscosity coefficient was 0.59 mPas. The L1 tube length of the capillary tube was 50cm, the L2 tube length of the capillary tube was 25cm, and the tube inner diameter was 50 μm. The constant analysis flow rate is 0.4 mu L/min, and the sample injection adopts 2mL/h flow rate for 0.1 min. The mass spectrum of the sample is shown in fig. 8, wherein the m/z 166 mass peak is a characteristic peak of phenylalanine, and an m/z 166 ion flow diagram is extracted. The chromatographic peaks were fitted with EMG function with a goodness of fit R2 of 0.9994, as shown in fig. 9. Five parallel experiments were performed, and the extracted ion flow diagrams are shown in fig. 10. The hydrodynamic radii of the two materials are calculated by the formula: phenylalanine with a radius of 0.441nm and angiotensin II with a radius of 1.025nm, the results of the analysis are shown in FIG. 11. The experimental result shows that the EMG fitting goodness is high, the sizes of the two mixtures can be measured at one time, the method is stable in measurement, high in precision and good in reproducibility. As shown in fig. 12, an embodiment of the present application provides a computer device 1200, including: a processor 1201, a memory 1202 and a bus, the memory 1202 storing machine readable instructions executable by the processor 1201, the processor 1201 and the memory 1202 communicating via the bus when the electronic device is operating, the processor 1201 executing the machine readable instructions to perform the steps of the method for sizing a substance in a solution based on taylor diffusion analysis as described above.
Specifically, the memory 1202 and the processor 1201 can be general-purpose memories and processors, and are not specifically limited herein, and the size determination method for a substance in a solution based on taylor diffusion analysis can be performed when the processor 1201 runs a computer program stored in the memory 1202.
In accordance with the taylor diffusion analysis-based method for determining the size of a substance in a solution, a computer-readable storage medium is provided, the computer-readable storage medium storing machine-executable instructions that, when invoked and executed by a processor, cause the processor to perform the steps of the taylor diffusion analysis-based method for determining the size of a substance in a solution.
Corresponding to the method for measuring the size of the substance in the solution based on the taylor diffusion analysis, the embodiment of the application also provides a device for measuring the size of the substance in the solution based on the taylor diffusion analysis, and the device can comprise modules required for realizing the steps of the method for measuring the size of the substance in the solution based on the taylor diffusion analysis.
The device for determining the size of the substance in the solution based on the taylor diffusion analysis provided by the embodiment of the application can be specific hardware on equipment or software or firmware installed on the equipment, and the like. The device provided by the embodiment of the present application has the same implementation principle and technical effect as the foregoing method embodiments, and for the sake of brief description, reference may be made to the corresponding contents in the foregoing method embodiments where no part of the device embodiments is mentioned. It is clear to those skilled in the art that, for convenience and brevity of description, the specific working processes of the foregoing systems, apparatuses and units may refer to the corresponding processes in the foregoing method embodiments, and are not described herein again.
In the embodiments provided in the present application, it should be understood that the disclosed apparatus and method may be implemented in other ways. The above-described embodiments of the apparatus are merely illustrative, and for example, the division of the units is only one logical division, and there may be other divisions when actually implemented, and for example, a plurality of units or components may be combined or integrated into another system, or some features may be omitted, or not executed. In addition, the shown or discussed mutual coupling or direct coupling or communication connection may be an indirect coupling or communication connection of devices or units through some communication interfaces, and may be in an electrical, mechanical or other form.
The units described as separate parts may or may not be physically separate, and parts displayed as units may or may not be physical units, may be located in one place, or may be distributed on a plurality of network units. Some or all of the units can be selected according to actual needs to achieve the purpose of the solution of the embodiment.
In addition, functional units in the embodiments provided in the present application may be integrated into one processing unit, or each unit may exist alone physically, or two or more units are integrated into one unit.
The functions, if implemented in the form of software functional units and sold or used as a stand-alone product, may be stored in a computer readable storage medium. Based on such understanding, the technical solution of the present application or portions thereof that substantially contribute to the prior art may be embodied in the form of a software product stored in a storage medium and including instructions for causing a computer device (which may be a personal computer, a server, or a network device) to execute all or part of the steps of the mobile control method according to the embodiments of the present application. And the aforementioned storage medium includes: a U-disk, a removable hard disk, a Read-Only Memory (ROM), a Random Access Memory (RAM), a magnetic disk or an optical disk, and other various media capable of storing program codes.
It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus once an item is defined in one figure, it need not be further defined and explained in subsequent figures, and moreover, the terms "first", "second", "third", etc. are used merely to distinguish one description from another and are not to be construed as indicating or implying relative importance.
In the description of the present invention, it should also be noted that, unless otherwise explicitly specified or limited, the terms "disposed," "mounted," "connected," and "connected" are to be construed broadly and may, for example, be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
Finally, it should be noted that: the above-mentioned embodiments are only specific embodiments of the present application, and are used for illustrating the technical solutions of the present application, but not limiting the same, and the scope of the present application is not limited thereto, and although the present application is described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: any person skilled in the art can modify or easily conceive the technical solutions described in the foregoing embodiments or equivalent substitutes for some technical features within the technical scope disclosed in the present application; such modifications, changes or substitutions do not depart from the scope of the embodiments of the present application. Are intended to be covered by the scope of the present application.

Claims (7)

1. A multidimensional joint liquid phase structure analysis device is characterized by comprising: the mass spectrometer comprises a Taylor diffusion analysis device, a mass spectrum detection device and a preceding stage separation device, wherein the Taylor diffusion analysis device comprises a microfluidic channel;
the front stage separation equipment is connected with the microfluidic channel and is used for providing a sample to be detected for the microfluidic channel;
the microfluidic channel comprises a liquid inlet channel, a liquid outlet channel and at least two microfluidic channel branches with different lengths, wherein the liquid inlet channel is used for receiving the sample to be detected, dividing the sample to be detected into at least two parts and respectively introducing the parts into each microfluidic channel branch; the liquid outlet passage is used for receiving part of samples to be detected in each microfluidic passage branch and respectively discharging the part of samples to be detected so as to respectively carry out mass spectrum detection;
the mass spectrum detection device comprises an ionization part and a mass spectrum detector, wherein the ionization part is connected with the microfluidic channel, and the ionization part is used for analyzing a sample to be detected from the microfluidic channel after ionizing the sample to be detected by the mass spectrum detector.
2. The device of claim 1, wherein the microfluidic circuit further comprises:
the liquid inlet channel is a liquid inlet shunt assembly and comprises a first liquid inlet and at least two first liquid outlets;
the liquid outlet passage is a liquid outlet convergence assembly and comprises at least two second liquid inlets and a second liquid outlet;
two ends of each of the at least two micro-flow passage branches are respectively connected with the first liquid outlet and the second liquid inlet;
the liquid inlet shunt assembly is used for receiving the sample to be detected through the first liquid inlet, dividing the sample to be detected into at least two parts through at least two first liquid outlets and respectively introducing the two parts into each microflow passage branch; the liquid outlet convergence assembly is used for receiving part of samples to be detected in each microflow passage branch through at least two second liquid inlets and respectively enabling the part of samples to be detected to flow out through the second liquid outlets so as to carry out mass spectrum detection respectively.
3. The apparatus of claim 1, further comprising:
a thermostatic assembly for controlling a temperature within the at least two microfluidic pathway branches to be constant;
a pump assembly for providing a constant carrier current to the microfluidic pathway required for analysis of the sample to be tested.
4. The device of claim 1, wherein the microfluidic circuit is implemented by a capillary or a microfluidic chip.
5. The device of claim 3, further comprising a sample switching assembly, the sample switching assembly being connected to the microfluidic channel, the sample switching assembly being configured to switch between a constant current carrying and a sample to be measured.
6. A method for determining the size of a substance in a solution based on taylor diffusion analysis, which is applied to the multi-dimensional liquid phase structure analysis device according to any one of claims 1 to 5, comprising:
determining target mass spectrum data detected after a target section sample to be detected is divided into two parts of samples to be detected and respectively passes through two microflow passages with different path lengths;
determining the diffusion degree and the peak-off time of the target substance corresponding to the two parts of samples to be detected based on the target mass spectrum data;
determining the size of the target substance according to the diffusion degree and the peak-off time corresponding to the target substance; the step of determining the size of the target substance according to the corresponding diffusion degree and the peak-off time of the target substance comprises:
determining the size of the target substance according to the diffusion degree and the peak-off time of the target substance corresponding to the two parts of samples to be detected;
determining two chromatographic peaks of a target substance corresponding to the two parts of samples to be detected based on the target mass spectrum data; and fitting through a Gaussian function based on the spectral peak center position parameter and the peak width parameter of each chromatographic peak to determine the diffusion degree and the peak emergence time of the target substance corresponding to the two parts of samples to be detected.
7. A computer device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor implements the steps of the method of claim 6 when executing the computer program.
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