CN117517427A - Quantitative rapid detection method for purity of annular RNA vaccine based on nano holes - Google Patents
Quantitative rapid detection method for purity of annular RNA vaccine based on nano holes Download PDFInfo
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Abstract
The invention discloses a quantitative rapid detection method for purity of a circular RNA vaccine based on a nanopore, and relates to the technical field of biological detection. The invention adopts the nanopore chip for detecting the annular RNA or the linear RNA, and because the annular RNA and the linear RNA have different two-dimensional structures, two molecules can generate different via hole electric signals when passing through the nanopore, the current change amplitude value caused by the annular RNA when passing through the nanopore is larger and the via hole time is longer, the current change amplitude value caused by the linear RNA passing through the nanopore is smaller, and the annular RNA and the linear RNA can be identified by carrying out statistical analysis on the signals of via hole events within a certain time range, so that the invention is applied to quantitative detection of the purity of the annular RNA vaccine. The detection method is simple to operate, quantitative detection of the purity of the annular RNA can be effectively realized without sample pretreatment steps such as PCR amplification, the sample demand is small, and a result can be obtained after sample addition for a few minutes.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a quantitative rapid detection method for purity of a circular RNA vaccine based on nanopores.
Background
Artificially synthesized RNA vaccines are usually circular RNA molecules of about one kilobase in length, however, due to the purity of the synthesis, the vaccine sample contains non-synthesized linear RNA, which affects the synthesis yield and purity of the RNA vaccine.
However, the purity of the vaccine has a great influence on the effect, so that quantitative detection of the purity of the circular RNA molecules in the synthesized vaccine sample has extremely important significance in the field of developing RNA vaccines. Currently, the commonly used method for analyzing the purity of RNA vaccines is chromatography or genetic sequencing, however, the above two methods have complicated detection process, high use cost and long detection time.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a quantitative rapid detection method for the purity of a nanopore-based annular RNA vaccine.
The invention is realized in the following way:
in a first aspect, embodiments of the present invention provide a method for detecting purity of a circular RNA vaccine, comprising the steps of: detecting a sample to be detected by adopting a nanopore chip, applying a set voltage on one side of the nanopore to drive annular RNA molecules or linear RNA molecule vias in the sample, and extracting the number of via events of the sample to be detected within a set time and the current change amplitude value caused by each via event; maximum current change amplitude delta I of via hole under the set voltage condition based on linear RNA molecule Line Judging a via event of the sample to be tested in a set time range, and enabling the current change amplitude value in the via event of the sample to be tested to be more than delta I Line As a via event for a circular RNA molecule; the amplitude value of the current change in the via event of the sample to be tested is less than or equal to the delta I Line The via event is used as a via event of a linear RNA molecule.
In a second aspect, embodiments of the present invention provide the use of a detection reagent for the detection or identification of the purity of a circular RNA vaccine, the detection reagent comprising: the nanopore chip described in the previous embodiments.
In a third aspect, embodiments of the present invention provide the use of a detection reagent for the preparation of a product for the detection or identification of a circular RNA molecule or a linear RNA molecule for the purity of a circular RNA vaccine, the detection reagent comprising: the nanopore chip described in the previous embodiments.
The invention has the following beneficial effects:
according to the invention, the nanopore chip is used for detecting the annular RNA or the linear RNA, as the annular RNA and the linear RNA have different two-dimensional structures, two molecules can generate different via hole electric signals when passing through the nanopore, the current change amplitude value caused by the annular RNA when passing through the nanopore is larger and the via hole time is longer, the current change amplitude value caused by the linear RNA passing through the nanopore is smaller, the annular RNA and the linear RNA can be identified by carrying out statistical analysis on the signals of via hole events within a certain time range, and the quantitative detection of the purity of the annular RNA vaccine is further realized. The detection method is simple to operate, quantitative detection of the purity of the annular RNA can be effectively realized without sample pretreatment steps such as PCR amplification, the sample demand is small, and a result can be obtained after sample addition for a few minutes.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a quantitative determination method of purity of a circular RNA vaccine according to the present invention;
FIG. 2 is a graph showing current traces of the low noise 10nm diameter nanopores of the present invention for opening at different voltages;
FIG. 3 is a current trace of a 10nm diameter nanopore of the present invention for detection of linear RNA at 200mV voltage;
FIG. 4 is a plot of linear RNA via events at 200mV voltage in accordance with the present invention;
FIG. 5 is a current trace of a sample of the detection RNA vaccine of the present invention at a voltage of 200mV for a 10nm diameter nanopore;
FIG. 6 is a plot of via events for RNA vaccine samples at 200mV voltage according to the present invention;
FIG. 7 is a plot of via events for RNA vaccine samples at 250mV voltage according to the present invention;
FIG. 8 is a plot of via events for RNA vaccine samples at 300mV voltage according to the present invention;
FIG. 9 is a current trace of a sample of the detection RNA vaccine of the present invention at a voltage of 200mV for a 15nm diameter nanopore;
FIG. 10 is a plot of via events for a sample of RNA vaccine of the present invention at a 15nm diameter 200mV voltage;
FIG. 11 is a plot of current trace at 300mV voltage at 1M LiCl,10mM Tris-HCl electrolyte at 10nm diameter in accordance with the present invention;
FIG. 12 is a plot of via events at 300mV for a 10nm diameter RNA vaccine sample under 1M LiCl,10mM Tris-HCl electrolyte in accordance with the present invention;
FIG. 13 is a graph comparing current power spectral density of low noise nanopores of the present invention with conventional nanopores.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In order to solve the problem of low detection efficiency of annular RNA molecules in an RNA vaccine sample, the inventor of the application provides an annular RNA vaccine purity quantitative detection method based on solid nano holes. By applying bias voltages to both sides of the nanopore chip, RNA molecules in the sample solution will pass through the nanopore under the action of an electric field force, thereby causing a change in the pore opening current of the nanopore, and generating a downward current pulse signal. Since the two types of molecular circular RNAs in solution have different two-dimensional structures from the non-circular linear RNAs, different shaped via electrical signals are generated when the two molecules pass through the nanopore. The circular RNA has a larger space structure, so that the current change amplitude (delta I) caused when passing through the nanopore is larger and the via time (delta t) is longer; whereas the current change amplitude of the via signal of the linear RNA molecule is small. The ratio of the number of each type of event can be obtained by counting the number of the via events with different current change amplitudes in a period of time under different voltages, so that the concentration ratio of the corresponding type of via RNA molecules can be obtained. Therefore, the method can realize rapid quantitative detection of the purity of the circular RNA vaccine.
In one aspect, the embodiment of the invention provides a method for detecting the purity of a circular RNA vaccine, which comprises the following steps:
detecting a sample to be detected by adopting a nanopore chip, adding the sample to one side of a nanopore, applying a set voltage to the other side of the nanopore to drive annular RNA molecules or linear RNA molecule through holes (penetrating the nanopore) in the sample, and extracting the number of through hole events and the current change amplitude value caused by each through hole event of the sample to be detected within a set time;
maximum current change amplitude delta I of via hole based on linear RNA molecule under set voltage Line Judging a via event of the sample to be tested in a set time range, and enabling the current change amplitude value in the via event of the sample to be tested to be more than delta I Line As a via event for a circular RNA molecule; the amplitude value of the current change in the via event of the sample to be tested is less than or equal to the delta I Line The via event is used as a via event of a linear RNA molecule.
In some embodiments, the set voltage is 50-300 mV. The voltage used in the detection is determined by the type of sample and the diameter of the nanopore. The nucleic acid (RNA) has a large negative charge, and is subjected to a large electrophoretic force under the same voltage, so that the nucleic acid (RNA) is easy to pass through holes. Typically, signals occur at voltages in excess of 50mV. The range of 50 to 300mV is suitable for the diameter (5 to 50 nm) of the nanopore employed in the present invention, and if voltage is increased on this basis, the pore blocking may be increased, so that it is usually less than 300mV. Specifically, the set voltage may be in a range between any one or any two of 50, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300mV.
The set time may be determined according to experimental conditions, and theoretically, the longer the time is, the more the event amount is recorded. However, the unlimited time also affects the performance of the chip, which decreases with the time of use. In some embodiments, the set time is 1s to 30min. Specifically, the set time may be any one or a range between any two of 1s, 2s, 4s, 6s, 8s, 10s, 15s, 20s, 25s, 30s, 35s, 40s, 45s, 50s, 55s, 60s, 1min, 2min, 4min, 6min, 8min, 10min, 15min, 20min, 30min.
In some embodiments, the detection method further comprises: detecting the maximum current change amplitude delta I of the linear RNA molecule through holes under the set voltage Line Is carried out by a method comprising the steps of. The steps may be: detecting a sample containing only linear RNA molecules by adopting the nanopore chip to obtain the current change amplitude of the sample within the set voltage and the set time range, wherein the maximum value is taken as the delta I Line 。
In some embodiments, the nanopore chip contains a dielectric film with a nanopore open in a silicon nitride film.
In some embodiments, the dielectric film may be selected from any one of a silicon nitride film, an aluminum oxide film, a hafnium oxide film, and a graphene film.
In some embodiments, the nanopore has a pore size of 5 to 50nm. Unlike conventional RNA molecules, circular RNA has a larger spatial structure size and cannot pass through nanopores of smaller diameter, and therefore, the minimum nanopore diameter proposed by the present invention is 5nm. In addition, the nanopore diameter cannot be too large, and linear RNA molecules with smaller spatial dimensions cannot generate significant current change signals when detected using larger diameter nanopores. Thus, the maximum diameter of the nanopores proposed by the present invention is 50nm. Specifically, the pore diameter may be in a range between any one or any two of 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50nm.
In some embodiments, the dielectric film has a thickness of 10 to 50nm. Films below 10nm are difficult to process and too thin films have poor mechanical stability, resulting in elevated baseline noise of the current. However, too thick (greater than 50 nm) a thin film can result in an increase in the volume of the entire nanopore, reducing the resolution of the assay. Specifically, the thickness may be in a range between any one or any two of 10, 15, 20, 25, 30, 35, 40, 45, 50nm.
The invention adopts the chip of low-noise dielectric film material, obtains the optimal solid state nano pore sensor chip through the optimal specific diameter and the thickness of the dielectric film, and can more effectively distinguish the pore event of the annular RNA molecule and the linear RNA molecule which is not annular. The concentration ratio of the RNA molecules in two shapes in the RNA vaccine can be obtained by simply calculating the signal quantity of different current change amplitude values in a period of time. The method is simple to operate, sample pretreatment steps such as PCR amplification are not needed, the sample demand is small, and the result can be obtained a few minutes after sample addition.
In some embodiments, the nanopore chip further comprises a support substrate having an opening, the support substrate being coupled to the lower surface of the dielectric membrane, the opening being in communication with the nanopore without affecting passage of RNA molecules from the nanopore. During detection, the nanopore chip is immersed in the electrolyte solution, and the nanopore on the chip is the only channel for ions in the electrolyte solution to pass through.
In some embodiments, the material of the support substrate comprises silicon.
In some embodiments, the support substrate has a thickness of 100 to 300 μm. The thickness of the support substrate is typically less than 300 μm because an excessively thick base may result in prolonged window etching time in the preparation of the chip and the overall thickness of the chip may also become thicker. While too thin (less than 100 μm) substrates tend to degrade the protection of the silicon nitride film and the chip is prone to breakage. Specifically, the thickness may be in a range between any one or any two of 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300 μm.
In some embodiments, the outer diameter of the open pores is greater than or equal to the pore diameter of the nanopores (5-50 nm).
In some embodiments, the openings have an outer diameter of 10 to 100nm. The outer diameter may specifically be in a range between any one or any two of 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100nm.
The present invention is not particularly limited to the shape of the openings, and in some embodiments, the cross-sectional shape of the openings includes, but is not limited to, circular, oval, rectangular, diamond, etc., preferably rectangular. The outer diameter of the foregoing embodiments is understood to be the largest diameter of the opening.
In some embodiments, when the sample to be detected is detected by using the nanopore chip, electrolyte solution is poured into two sides of a nanopore of the nanopore chip, and positive and negative electrodes are applied to two sides of the nanopore.
In some embodiments, the electrolyte solution includes: any one or more of lithium chloride, sodium chloride and potassium chloride.
In some embodiments, the electrolyte solution contains a buffer solution of NaCl, specifically Tris-HCl solution containing NaCl, wherein the final concentration of NaCl is 0.5-1.5M, and the final concentration of Tris-HCl is 1-20 mM. The final concentration of NaCl may specifically be in the range between any one or any two of 0.5, 0.6, 0.8, 1, 1.2, 1.4, 1.5M. The final concentration of Tris-HCl may be in the range between any one or any two of 1, 5, 10, 15, 20mM.
In some embodiments, the electrolyte solution contains a buffer solution of LiCl, specifically, may be a Tris-HCl solution containing LiCl, wherein the final concentration of LiCl is 0.5-1.5M, and the final concentration of Tris-HCl is 1-20 mM. The final concentration of LiCl may specifically be in the range between any one or any two of 0.5, 0.6, 0.8, 1, 1.2, 1.4, 1.5M. The final concentration of Tris-HCl may be in the range between any one or any two of 1, 5, 10, 15, 20mM.
The detection method of the present application is not particularly limited in terms of the length of the circular RNA molecule and the linear RNA molecule. The length of the circular RNA molecule and the linear RNA molecule may be 3nt to 2000nt, and specifically may be any one or any two or more of 3, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, and 2000 nt.
The sample to be tested may be a circular RNA vaccine. The sample volume may be 1 to 10. Mu.L. Specifically, the concentration may be in a range of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Mu.L or any two or more thereof.
In some embodiments, the detection method further comprises: calculating the ratio of the number of the annular RNA molecule via events to the number of the via events of the sample to be detected within a set time to obtain the purity of the annular RNA molecule.
In another aspect, the embodiment of the present invention further provides an application of a detection reagent in detecting or identifying a cyclic RNA molecule or a linear RNA molecule in the purity of a cyclic RNA vaccine, where the detection reagent includes: the nanopore chip of any of the preceding embodiments.
In some embodiments, the detection reagent further comprises: the electrolyte solution of any of the preceding embodiments.
In addition, the embodiment of the invention also provides application of the detection reagent in preparing a product for detecting or identifying the purity of the circular RNA vaccine, wherein the detection reagent comprises the following components: the nanopore chip of any of the preceding embodiments.
In some embodiments, the detection reagent further comprises: the electrolyte solution of any of the preceding embodiments.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The embodiment provides a quantitative detection method for purity of a circular RNA vaccine, which is used for detecting a sample to be detected (the sample to be detected is a vaccine sample with the purity of 30% identified by mass spectrometry), and the steps of the detection method are as follows, and a schematic diagram can be referred to in FIG. 1.
(1) Obtaining a nanopore chip; in this embodiment, the nanopore chip includes a silicon nitride film and a silicon substrate; the thickness of the silicon nitride film is 30nm, and the film is provided with a nano hole with the diameter of 10 nm; the silicon nitride film is deposited on a silicon substrate by a chemical vapor deposition method, the thickness of the silicon substrate is 200 mu m, rectangular open pores are also arranged on the silicon substrate and are communicated with the nano-pores, and the side length of the rectangular open pores is larger than the diameter of the nano-pores.
(2) Detection preparation: mounting the nanopore chip in a fluid pool, and dividing the fluid pool into 2 chambers; an electrolyte solution (1M NaCl,10mM Tris-HCl) was added to the 2-side chamber. Two Ag/AgCl electrode wires are respectively inserted into two chambers of the fluid pool to form positive and negative electrodes, and the other two ends of the electrodes are respectively connected with a patch clamp current amplifier.
The pore currents at different voltages (-200 mV) were recorded to confirm that no false positive signal was present before addition of RNA samples, and the results are shown in FIG. 2. And adding 2 mu L of linear RNA samples with the same number of bases as the number of bases of the annular RNA to be detected into a cavity at one side of the fluid pool, applying positive voltage at the other side of the fluid pool, and observing and recording the change condition of the current base line. Under the action of electric field force, the linear RNA can pass through the nanopore, so that a current change pulse signal is generated. As shown in fig. 3.
(3) Obtaining the maximum current variation amplitude (delta I) caused by the through hole of the linear RNA molecule under the set voltage (200 mV in the embodiment) Line )：
The maximum current change amplitude (delta I) caused by linear RNA molecular via hole is determined by extracting the via hole event of linear RNA molecules under the voltage of 200mV and carrying out data analysis Line ) 1nA, as shown in the scatter diagram of FIG. 4.
(4) Detecting and analyzing a via hole signal of a sample to be detected:
and taking the nanopore chip out of the fluid pool, fully flushing and drying by using ethanol and deionized water, then reinstalling the chip into the fluid pool, adding electrolyte solution (1M NaCl,10mM Tris-HCl), adding 2 mu L of synthesized vaccine sample into one side of the fluid pool after confirming that the open-pore current is stable, applying positive voltage on the other side of the fluid pool, and observing and recording the change condition of the current base line under different voltages.
Under the action of electric field force, circular RNA and linear RNA in vaccine sample can pass through the nano hole, so that current pulse signals with different current change amplitudes are caused.
As shown in fig. 5, I 1 The current level is the current change amplitude generated by the linear RNA via hole, I 2 The magnitude of the current change generated for the circular RNA via.
The current change amplitude (Δi) and the via time (Δt) of the vaccine sample via event at the set voltage (200 mV) for 30s were extracted and a two-dimensional scattergram was made, as shown in fig. 6. The total number of events within 30s was determined to be 203 (see Table 1), wherein the number of events in which the current change amplitude was greater than 1nA was 43, and thus the event amount of the circular RNA was 43/203.
TABLE 1 detection results
Example 2
The present example provides a quantitative determination method for purity of circular RNA vaccine, which is substantially the same as that of example 1, except that the set voltage at the time of detection is 250mV. The current baseline change at this voltage was recorded, and the current change amplitude (Δi) and the via time (Δt) of the via event signal were extracted, and a two-dimensional scattergram was made, as shown in fig. 7, with an event ratio of 48/168 for the annular RNA.
Example 3
The present example provides a quantitative determination method for purity of circular RNA vaccine, which is substantially the same as that of example 1, except that the set voltage at the time of detection is 300mV. The current baseline change condition under the voltage is recorded, the current change amplitude (delta I) and the via time (delta t) of the via event signal are extracted, and a two-dimensional scatter diagram is made, and the event ratio of the annular RNA is 160/594 as shown in FIG. 8.
Example 4
The present example provides a quantitative determination of purity of circular RNA vaccine, which is substantially the same as example 1, except that the diameter of the nanopore is 15nm. The change in current baseline at this diameter was recorded as shown in fig. 9. The current change amplitude (Δi) and the via time (Δt) of the via signal event are extracted and a two-dimensional scattergram is made, as shown in fig. 10.
Example 5
The present example provides a quantitative determination method for purity of circular RNA vaccine, which is substantially the same as example 1, except that:
(1) The electrolyte solution is a mixed solution of LiCl and Tris-HCl (1M LiCl,10mM Tris-HCl);
(2) The set voltage at the time of detection was 300mV.
The change in current baseline at this voltage was recorded as shown in fig. 11. The current change amplitude (Δi) and the via time (Δt) of the via signal event are extracted and a two-dimensional scattergram is made, as shown in fig. 12.
Example 6
The nanopore chip provided in comparative example 1 was compared to conventional nanopore chips for detection of purity of circular RNA vaccine.
The conventional nanopore chip is an in-situ chip, and the product number is G20L08P004. The process of the detection method is approximately the same as that of example 1 (the pore opening voltage is-150 mV), the current baseline at 100mV voltage for a period of time is taken to calculate the power average density, and the power average density is compared with the current power spectrum density at the same voltage of the low-noise nanopore chip (example 1 of the application), as shown in fig. 13. From the graph, the power spectral density of the current baseline of the conventional nanopore is obviously higher than that of the nanopore used in the invention, which indicates that the nanopore chip used in the invention has lower noise level.
The test samples of examples 2 to 6 were the same as example 1.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The method for detecting the purity of the circular RNA vaccine is characterized by comprising the following steps of:
detecting a sample to be detected by adopting a nanopore chip, applying a set voltage on one side of the nanopore to drive annular RNA molecules or linear RNA molecule vias in the sample, and extracting the number of via events of the sample to be detected within a set time and the current change amplitude value caused by each via event;
maximum current change amplitude delta I of via hole based on linear RNA molecule under set voltage Line Judging a via event of the sample to be tested in a set time range, and enabling the current change amplitude value in the via event of the sample to be tested to be more than delta I Line As a via event for a circular RNA molecule; the amplitude value of the current change in the via event of the sample to be tested is less than or equal to the delta I Line The via event is used as a via event of a linear RNA molecule.
2. The method of claim 1, wherein the nanopore chip comprises a dielectric film having a nanopore open therein;
optionally, the dielectric film is selected from any one of a silicon nitride film, an aluminum oxide film, a hafnium oxide film and a graphene film;
optionally, the aperture of the nano hole is 5-50 nm;
optionally, the thickness of the dielectric film is 10-50 nm.
3. The method of claim 2, wherein the nanopore chip further comprises a support substrate having an opening, the support substrate being coupled to a lower surface of the dielectric membrane, the opening being in communication with the nanopore;
optionally, the material of the support substrate comprises silicon;
alternatively, the thickness of the support substrate is 100 to 300 μm.
4. The method according to claim 3, wherein the outer diameter of the open pores is not less than the diameter of the nanopores;
optionally, the outer diameter of the opening is 10-100 nm.
5. The method according to claim 1, wherein the set voltage is 50 to 300mV.
6. The method according to claim 1, wherein the set time is 1s to 30min.
7. The method according to any one of claims 1 to 6, wherein when a sample to be detected is detected by using a nanopore chip, electrolyte solution is poured into both sides of a nanopore of the nanopore chip, and positive and negative electrodes are applied to both sides of the nanopore;
optionally, the electrolyte solution includes: any one or more of lithium chloride, sodium chloride and potassium chloride.
8. The method according to any one of claims 1 to 6, further comprising: and obtaining the ratio of the number of the via events of the annular RNA molecules in the set time range to the number of the samples to be tested in the set time range so as to obtain the purity of the annular RNA.
9. Use of a detection reagent for the detection or identification of the purity of a circular RNA vaccine, or a linear RNA molecule, characterized in that the detection reagent comprises: a nanopore chip as claimed in any one of claims 1 to 8;
optionally, the detection reagent further comprises: the electrolyte solution as recited in claim 7.
10. Use of a detection reagent for the preparation of a product for the detection or identification of the purity of a circular RNA vaccine, a circular RNA molecule or a linear RNA molecule, characterized in that the detection reagent comprises: a nanopore chip as claimed in any one of claims 1 to 8;
optionally, the detection reagent further comprises: the electrolyte solution as recited in claim 7.
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