Patent Application
20230040993
This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “SWIP-002PUS_ST25.txt” created on Oct. 11, 2022 and is 3,474 bytes in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
The invention relates to a fiber-optic wave guide sensor of aptamers, and more particularly, to a fiber-optic wave guide sensor of aptamers having functions of in situ target enrichment and purification (SPME-OWS), which can achieve the ultrasensitive and ultrahigh specific quick detection for all types of small molecule targets regardless of water solubility, belonging to analytical chemistry technical field.
Small organic molecules are one large classification of food and environmental contaminants, having features of numerous varieties, different water solubility, and less specific antibody, etc. Therefore, their analytical test mainly use the ways based on the Large-scale Instruments, such as Vapour phase chromatography, High performance liquid chromatography (HPLC), Gas chromatography mass spectrometry, liquid chromatography mass spectrometry, etc., which require expensive equipment, high work environment, and equipment maintenance, and which are not suitable for field testing. In recent years, various biosensors are developing at flying speed for satisfying quick detection for small molecule targets.
Aptamers are the single-stranded or the double-stranded DNA or RNA obtained by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology (Nature, 1990, 346, 818-822; Nature, 1992, 355, 564-566). Aptamers can specifically recognize the varied target molecule ranging from proteins, small molecules, cells, to tissue, which have high chemical stability, are easy to synthetize and modify, have low cost, and have broad application prospects in biosensor field. Aptamers for detection of small molecules especially are very attractive as the high specificity antibodies of small molecules are not easy to be obtained. However, affinity of aptamers of small molecules is generally much lower than antibody. In general, signal is enlarged based on enzyme and Nanomaterial for improving detection sensitivity, but detection sensitivity cannot achieve the actual required level.
The fiber-optic wave guide sensor is one kind of portable fluorescent sensor. It is mainly based on that, total reflection of light is generated when laser enters into the light sparing substance from the light dense substance at a certain angle of incidence, a portion of laser will transmit in vertical direction of optical fiber, and the strength of this portion of laser will decrease exponentially with distance left from the optical fiber, which is called the evanescent wave. The evanescent wave can excite fluorescent group within the range of the evanescent wave transmission (the evanescent wave field). Therefore, target's antibody, receptor, and complementary chains of aptamers can be specifically recognized, or target molecules are attached on the surface of the optical fiber and fluorescence is labeled on target molecules, aptamers or antibody so as to realize quantitative fluorescence detection of target. The fiber-optic wave guide sensor is very suitable for cheap detection of environmental and food contaminants because it is simply and quickly operated, industrialization has been realized, regeneration of hundred times can be made on the sensing interface. However, the present detection limit is mostly the level of nM, which cannot achieve the limit standard of small molecules contaminants in the complex medium.
Extraction technology is one common sample preparation method in the analysis method based on the instrument, so as to remove substrate and enrich target to realize quantitative or qualitative detection of target. Solid-phase microextraction (SPME) is one new type of extraction technology in rapid development in recent years, which uses various enrichment materials attached to solid-phase to enrich and purify all kinds of targets (Trac-Trends in Analytical Chemistry 2018, 108, 154-166. Trac-Trends in Analytical Chemistry 2019. 110, 66-80).
In order to overcome defection in the prior art, we combine SPME and aptamers for the first time in the invention, via simultaneous target enrichment, purification, and detection taking the fiber-optic wave guide sensor as an example so as to achieve the ultrasensitive and high specific detection for small molecule targets. The invention realized quantitative detection of small molecules in various complex substrates samples and all the detection limits are lower over 20 times than the State Limit Standard. Further, liquid samples only need to be diluted and solid samples only need to be extracted without the complicated sample pretreatment. The methods of the invention have enormous development prospects.
The object of the invention is to provide a fiber-optic wave guide sensor of aptamers having functions of in situ target enrichment and purification (SPME-OWS) and a detection method of its application to achieve the high sensitive and high specific detection for small molecule targets. The methods of the invention synchronously assembly the extraction layer SPME having high efficiency target extraction capability, for example, bare fiber, Tween 80, and aptamers having target specificity on the fiber-optic sensing interface to realize synchronous target enrichment, purification and the specific detection, which can achieve the ultrasensitive and ultrahigh specific detection. The invention can realize the quantitative detection of small molecules targets based on that small molecules targets and the aptamers complementary short strand DNA (cDNA) competitively bind with aptamers tethered on the fiber surface. The SPME on the fiber surface high effectively enriched small molecules in the solution nearby the fiber surface, which substantially bind small molecules with aptamers tethered on the fiber surface, substantially decreasing hybridization of the fluorophore labeled aptamers complementary DNA (cDNA) with aptamers, enabling the ultrasensitive and highly specific detection of targets. The method of the invention is a general way, taking detections of four representative environmental and food small molecules contaminants as examples, respectively comprising kanamycin A (Kana) of hydrophilic small molecule antibiotic, sulfadimethoxine (SDM) of hydrophobic small molecule antibiotic, Alternariol (AOH) of small molecule mycotoxin, and Di-(2-ethylhexyl) phthalate (DEHP) of high hydrophobic small molecule. Especially, the method of the invention can be used for the direct detection of targets in the complicated samples (milk, lake water, Wine, wheat), only requiring diluting the liquid sample, without any time consuming and complicated sample pretreatment. All detection sensitivity satisfies the limit standard of each target in the food and environment.
The method has the following advantages:
1) The method of the invention realizes synchronous target enrichment, purification and the specific detection, which is realized in the prior art for the first time. And the operation is very convenient and fast.
2) The method of the invention can achieve the ultrasensitive and ultrahigh specific detection, the detection of limits of which is lower 625-2000, 000 times than that of the conventional fiber-optic wave guide sensor, even still lower 325-20, 000 times than that of the electrochemical detection method.
3) The method of the invention has ultrahigh specificity (the selectivity>1000) and anti-matrix interference ability, which can be used for the direct detection of targets in the complicated samples (milk, lake water, Wine, wheat), only requiring to dilute the liquid sample, without any time consuming and complicated sample pretreatment. All detection sensitivity satisfies the limit standard of each target in the food and environment.
4) The sensor of the invention has superior target generality, which can used for high hydrophobic, hydrophobic and hydrophobic small molecules.
5) One of unique advantages of the invention is conveniently controlling of the detection sensitivity and dynamic interval. For example, the efficiency of in situ target enrichment and purification can be controlled by simply changing constitution of SPME or adding other ingredients which affects target enrichment in the buffers, so as to control the dynamic range. The detection sensitivity and dynamic interval of the sensor are adjusted by changing the surface density of the probe or using aptamers with the different affinity commonly in the prior art. However, it is very difficult for the accurate control of the probe density on the surface without good repeatability. Further, aptamers probes with the different affinity require the complicated engineering design. The method of the invention is more concise without these limitations.
6) The test cost is lower, and the stability is better among batches by using aptamers to realize the specific recognition of targets compared to the sensor based on antibody.
7) The sensor of the invention can recycle and reproduce for many times (>100 times) and has stability (Fluorescence signal change is ±6%).
8) The sensor of the invention can achieve quick detection within several minutes.
9) The sensor of the invention does not limit to be used for the small molecules, which can be used for other types of targets, for example, protein, heavy metal ion, etc., by changing extractant.
The specific experimental procedures of the invention are as follows:
1) Hydroxylation of the optic fiber surface: Firstly, The optical fiber with clean surface was dipped into a 3:1 v/v concentrated sulfuric acid and a 30% hydrogen peroxide mixing solution at 100-120° C. for 1 h, then, the fiber was taken from the mixing solution and washed to neutral with the ultrapure water, followed by blowing dry with nitrogen and drying in an oven at 70-90° C. for 4-6 h, taking the fiber in the dryer and cooling to room temperature;
2) Silylanization of the optic fiber surface: the above fiber was immersed APTS anhydrous toluene solution at room temperature for 1-2 h, followed by rinsing with Anhydrous toluene, toluene-ethyl alcohol (v/v=1:1) and ethyl alcohol wash (three time), blowing dry with nitrogen and drying in an oven at 180° C. for 4-6 h, taking the fiber in the dryer and cooling to room temperature;
3) Aptamers coupling of the optic fiber surface: the optical fiber of silylanization was immersed in 10 mM phosphate buffered solution (PB) containing glutaraldehyde for 4 h at room temperature, after the reaction being finished, washed with the ultrapure water three times, blowing dry with nitrogen. The fiber was then immersed in the amino modified aptamers solution 6-8 h at room temperature, washed then with the ultrapure water three times;
4) Restoring and sealing: the above fiber was immersed in sodium borohydride (NaBH4) solution for 30 minutes, sealing the fiber interface with a certain concentration of extractant, for example, Tween 80 solution (When fabricating SPME-OWS of the bare fiber, the fiber interface was sealed without the extractant), washed then with the ultrapure water three times and stored in the refrigerator of 4° C.
5) The optic fiber was assembled into the reaction chamber of the waveguide sensor, after the baseline being stabled, pumping the mixed solution containing a certain concentration of small molecule target and complementary chains of fluorescent modified aptamers in the reaction chamber, measuring the change of fluorescence signal in real time;
6) The fiber was flushed with solution of sodium dodecyl sulfate (SDS) to regenerate the sensor interface; repeating 5);
7) Drawing the working curves of optical waveguide sensor detecting different targets;
8) Selectivity test: targets of 5) was changed to substances of Selectivity test.
The detailed descriptions of embodiments in the invention for further understanding of the invention are as follows. The following embodiments are used to illustrate the invention but are not used to limit the scope.
In general, the technical scheme of the invention can realize the quantitative detection of small molecules targets based on that small molecules targets and the aptamers complementary short strand DNA (cDNA) competitively bind with aptamers tethered on the fiber surface. The SPME on the fiber surface high effectively enriched small molecules in the solution nearby the fiber surface, which substantially bind small molecules with aptamers tethered on the fiber surface, substantially decreasing hybridation of the fluorophore labeled aptamers complementary DNA (cDNA) with aptamers, enabling the ultrasensitive and highly specific detection of targets. It comprises the following detailed experiment procedure:
1) Hydroxylation of the optic fiber surface: Firstly, The optical fiber with clean surface was dipped into a 3:1 v/v concentrated sulfuric acid and a 30% hydrogen peroxide mixing solution at 100-120° C. for 1 h, then, the fiber was taken from the mixing solution and washed to neutral with the ultrapure water, followed by blowing dry with nitrogen and drying in an oven at 70-90° C. for 4-6 h, taking the fiber in the dryer and cooling to room temperature;
2) Silylanization of the optic fiber surface: the above fiber was immersed APTS anhydrous toluene solution at room temperature for 1-2 h, followed by rinsing with Anhydrous toluene, toluene-ethyl alcohol (v/v=1:1) and ethyl alcohol wash (three time), blowing dry with nitrogen and drying in an oven at 180° C. for 4-6 h, taking the fiber in the dryer and cooling to room temperature;
3) Aptamers coupling of the optic fiber surface: the optical fiber of silylanization was immersed in 10 mM phosphate buffered solution (PB) containing glutaraldehyde for 4 h at room temperature, after the reaction being finished, washed with the ultrapure water three times, blowing dry with nitrogen. The fiber was then immersed in the amino modified aptamers solution 6-8 h at room temperature, washed then with the ultrapure water three times;
4) Restoring and sealing: the above fiber was immersed in sodium borohydride (NaBH4) solution for 30 minutes, sealing the fiber interface with a certain concentration of extractant, for example, Tween 80 solution (When fabricating SPME-OWS of the bare fiber, the fiber interface was sealed without the extractant), washed then with the ultrapure water three times and stored in the refrigerator of 4° C.
5) The optic fiber was assembled into the reaction chamber of the waveguide sensor, after the baseline being stabled, pumping the mixed solution containing a certain concentration of small molecule target and complementary chains of fluorescent modified aptamers in the reaction chamber, measuring the change of fluorescence signal in real time;
6) The fiber was flushed with solution of sodium dodecyl sulfate (SDS) to regenerate the sensor interface; repeating 5);
7) Drawing the working curves of optical waveguide sensor detecting different targets;
8) Selectivity test: targets of 5) was changed to substances of Selectivity test.
The invention provides a fiber-optic wave guide sensor of aptamers having functions of in situ target enrichment and purification (SPME-OWS) and a detection method of its application to achieve the high sensitive and high specific detection for small molecule targets. The principle of the invention is as shown in Part A of
According to the invention, the fiber modification process of SPME-OWS is as shown in
1) Hydroxylation of the optic fiber surface: Firstly, The optical fiber with clean surface was dipped into a 3:1 v/v concentrated sulfuric acid and a 30% hydrogen peroxide mixing solution at 100-120° C. for 1 h, then, the fiber was taken from the mixing solution and washed to neutral with the ultrapure water, followed by blowing dry with nitrogen and drying in an oven at 70-90° C. for 4-6 h, taking the fiber in the dryer and cooling to room temperature;
2) Silylanization of the optic fiber surface: the above fiber was immersed APTS anhydrous toluene solution at room temperature for 1-2 h, followed by rinsing with Anhydrous toluene, toluene-ethyl alcohol (v/v=1:1) and ethyl alcohol wash (three time), blowing dry with nitrogen and drying in an oven at 180° C. for 4-6 h, taking the fiber in the dryer and cooling to room temperature;
3) Aptamers coupling of the optic fiber surface: the optical fiber of silylanization was immersed in 10 mM phosphate buffered solution (PB) containing glutaraldehyde for 4 h at room temperature, after the reaction being finished, washed with the ultrapure water three times, blowing dry with nitrogen. The fiber was then immersed in the amino modified aptamers solution 6-8 h at room temperature, washed then with the ultrapure water three times;
4) Restoring and sealing: the above fiber was immersed in sodium borohydride (NaBH4) solution for 30 minutes, sealing the fiber interface with a certain concentration of extractant, for example, Tween 80 solution (When fabricating SPME-OWS of the bare fiber, the fiber interface was sealed without the extractant), washed then with the ultrapure water three times and stored in the refrigerator of 4° C.
The above fabricated optic fiber was assembled into the reaction chamber of the waveguide sensor to begin test of target. The fluorescent detector online installed on the sensor recorded changes of the fluorescent signals in real time for quantitative analysis of target concentration. After each test, washed the fiber with 0.5% SDS (pH=1.9) for 60 seconds to regenerate the sensing interface. After washed the fiber with the corresponding detection buffer again to begin the next test.
The fiber modification process of classic-OWS is as shown in
1) Hydroxylation of the optic fiber surface: Firstly, The optical fiber with clean surface was dipped into a 3:1 v/v concentrated sulfuric acid and a 30% hydrogen peroxide mixing solution at 100-120° C. for 1 h, then, the fiber was taken from the mixing solution and washed to neutral with the ultrapure water, followed by blowing dry with nitrogen and drying in an oven at 70-90° C. for 4-6 h, taking the fiber in the dryer and cooling to room temperature;
2) Silylanization of the optic fiber surface: the above fiber was immersed APTS anhydrous toluene solution at room temperature for 1-2 h, followed by rinsing with Anhydrous toluene, toluene-ethyl alcohol (v/v=1:1) and ethyl alcohol wash (three time), blowing dry with nitrogen and drying in an oven at 180° C. for 4-6 h, taking the fiber in the dryer and cooling to room temperature;
3) Kana Aor SDM coupling of the optic fiber surface: the optical fiber of silylanization was immersed in 10 mM phosphate buffered solution (PB) containing glutaraldehyde for 4 h at room temperature, after the reaction being finished, washed with the ultrapure water three times, blowing dry with nitrogen. The fiber was then immersed in the amino modified aptamers solution 6-8 h at room temperature, washed then with the ultrapure water three times;
4) Restoring: the above fiber was immersed in sodium borohydride (NaBH4) solution for 30 minutes, washed then with the ultrapure water three times and stored in the refrigerator of 4° C.
The above fabricated optic fiber was assembled into the reaction chamber of the waveguide sensor to begin test of target. The fluorescent detector online installed on the sensor recorded changes of the fluorescent signals in real time for quantitative analysis of target concentration. After each test, washed the fiber with 0.5% SDS (pH=1.9) for 60 seconds to regenerate the sensing interface. After washed the fiber with the corresponding detection buffer again to begin the next test.
Preparing Kana A and SDM standard solutions having the different final concentration (0, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 200 nM, 500 nM, 800 nM, 1 μM, 10 μM) in buffer 2 (trihydroxymethylaminomethane, 20 mM; NaCl, 50 mM; KCl, 5 mM; MgCl 5 mM; pH 7.0), respectively mixing with 100 nM fluorescent modified aptamers (Cy5.5-Kana or Cy5.5-SDM, Table 1), in proper order putting in the optic-fiber sensor from the low concentration to the high concentration, after finishing each test, regenerating the interface and cleaning up the pipe to drop the fluorescent signal to the baseline, recording the fluorescent changes with time in the different concentrations, drawing the working curves with relative fluorescence signal reduction percentage value at different target concentrations being vertical coordinates.
The result is as shown in
Hydroxylation, silylanization, coupling, sealing and restoring process of the optic fiber surface are same as Embodiment 1, without sealing after the fiber being restored, in which the coupling aptamer is NH2-Kana (Table 1). During experiment of the working curves of for Kana A, 0.5% SDS should be added to destroy the G-quadruplex structure formed by aptamer of Kana on the fiber surface.
Preparing Kana standard solutions having the different final concentration (0,100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM) in buffer 1 ((phosphate, 10 Mm; NaCl, 50 mM; KCl, 5 mM; MgCl 5 mM; pH 7.0), respectively mixing with 100 nM fluorescent modified complementary chains of aptamers (Cy5.5-Kana or Cy5.5-SDM, Table 1), in proper order putting in the optic-fiber sensor from the low concentration to the high concentration, after finishing each test, regenerating the interface and cleaning up the pipe to drop the fluorescent signal to the baseline, recording the fluorescent changes with time in the different concentrations, drawing the working curves with relative fluorescence signal reduction percentage value at different target concentrations being vertical coordinates.
Respectively preparing TET, AMP, SDM and DEHP standard solutions having the final concentration of 10 nM to test target selectivity.
Respectively dissolving Kana (0 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM) in the lake water, using buffer 1 for 1000 times dilution, to detect Kana in the lake water. Respectively dissolving Kana (0 pM, 10 nM, 100 nM, 1 μM, 10 μM) in the skim milk, using buffer 1 for 10000 times dilution, to detect Kana in the milk.
The result is as shown in
Hydroxylation, silylanization, coupling, sealing and restoring process of the optic fiber surface are same as Embodiment 1, without sealing after the fiber being restored, in which the coupling aptamer is NH2-SDM (Table 1). Preparing SDM standard solutions having the different final concentration (0, 10 aM, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM) in buffer 2 (trihydroxymethylaminomethane, 20 mM; NaCl, 50 mM; KCl, 5 mM; MgCl 5 mM; pH 7.0), respectively mixing with 100 nM fluorescent modified complementary chains of aptamers (c-SDM-Cy 5.5), in proper order putting in the optic-fiber sensor from the low concentration to the high concentration, after finishing each test, regenerating the interface and cleaning up the pipe to drop the fluorescent signal to the baseline, recording the fluorescent changes with time in the different concentrations, drawing the working curves with relative fluorescence signal reduction percentage value at different target concentrations being vertical coordinates.
Respectively preparing TET, AMP, SDM and DEHP standard solutions having the final concentration of 10 nM to test target selectivity.
Respectively dissolving SDM (0 pM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM) in the lake water, using buffer 2 for 1000 times dilution, to detect SDM in the lake water. Respectively dissolving SDM (0 pM, 3 nM, 30 nM, 50 nM, 300 nM, 3 μM, 5 μM) in the skim milk, using buffer 1 for 10000 times dilution, to detect SDM in the milk.
The result is as shown in
Hydroxylation, silylanization, coupling, sealing and restoring process of the optic fiber surface are same as Embodiment 1, in which the coupling aptamer is NH2-DEHP (Table 1). Preparing DEHP standard solutions having the different final concentration (0, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 200 nM) in buffer 3 (NaCl, 100 mM; trihydroxymethylaminomethane, 20 Mm; MgCl 2 mM; KCl, 5 mM; CaCl, 1 mM; 0.03% Triton X-100, 2% Dimethyl Sulfoxide, pH 7.9), respectively mixing with 100 nM fluorescent modified aptamers (c-DEHP-Cy5.5), in proper order putting in the optic-fiber sensor from the low concentration to the high concentration, after finishing each test, regenerating the interface and cleaning up the pipe to drop the fluorescent signal to the baseline, recording the fluorescent changes with time in the different concentrations, drawing the working curves with relative fluorescence signal reduction percentage value at different target concentrations being vertical coordinates.
Respectively preparing SDM, Kana, BA, PA, Hg2+, Pb2+ having the final concentration of 100 nM and fluorescent modified complementary chains of aptamers (c-DEHP-Cy 5.5) having the final concentration of 100 nM in buffer 3. Respectively mixing the every target solution with the complementary chain solution to test target selectivity according to the following procedure. The first step is putting buffer 3 into the reaction chamber for 60 seconds, the second step is putting the mixing solutions of target and complementary chain into the reaction chamber for 20 seconds and then retaining in the reaction chamber for 200 seconds, the third step is putting 0.5% SDS (pH 1.9) into the reaction chamber for 60 seconds, and finally putting buffer 3 into the reaction chamber for 50 seconds till the baseline returned to original position.
Respectively dissolving DEHP (0 pM, 100 pM, 1 nM, 10 nM, 100 nM) in the lake water, using buffer 3 for 1000 times dilution, to detect DEHP in the lake water. Respectively dissolving DEHP (0 pM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM) in the Wine, using buffer 3 for 1000 times dilution, to detect DEHP in the Wine.
The result is as shown in
Hydroxylation, silylanization, coupling, sealing and restoring process of the optic fiber surface are same as Embodiment 1, in which the coupling aptamer is NH2-AOH (Table 1). Preparing AOH standard solutions having the different final concentration (0, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM) in buffer 4 (CaCl, 0.9 mM; KCl, 2.685 mM; KDP 1.47 mM; MgCl 0.49 mM; NaCl, 137 mM; DSP, 8.1 mM; pH 7.4), respectively mixing with 50 nM fluorescent modified aptamers (c-AOH-Cy 5.5, Table 1), in proper order putting in the optic-fiber sensor from the low concentration to the high concentration, after finishing each test, regenerating the interface and cleaning up the pipe to drop the fluorescent signal to the baseline, recording the fluorescent changes with time in the different concentrations, drawing the working curves with relative fluorescence signal reduction percentage value at different target concentrations being vertical coordinates.
Respectively preparing standard solutions of other toxin small molecules such as AME, Patulin, ZEA, OTA and DON having the final concentration of 100 pM to test target selectivity.
Respectively dissolving AOH (0, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM) in the wheat extract (taking wheat flour, 1 g, adding pure acetonitrile, 6 mL, mediating the mixing solution for 3 minutes, then centrifugating for 10 minutes at 9000 r/min, taking the supernate, 1 mL, no membrane), using buffer 4 for 100 times dilution, to labelly detect AOH in the wheat extract.
The result is as shown in
SPMES-OWS of the invention realized conveniently controlling of the detection dynamic interval. Hydroxylation, silylanization, coupling, sealing and restoring process of the optic fiber surface are same as Embodiment 1. Taking the detection of Kana A as one example, the method of the invention can conveniently control of the detection dynamic interval by change components of the buffer. Taking the detection of SDM as one example, the method of the invention can conveniently control of the detection dynamic interval by change SPME layers. All experiment processes are same as Embodiments 4-7, only requiring changes of the buffer and SPME layers.
The result is as shown in
As shown in
The fiber surface of SPMES-OWS of the invention has the superior interface regeneration performance. As shown in
It should be understood that the foregoing discussion, embodiments and examples merely present a detailed description of certain preferred embodiments. It will be apparent to those of ordinary skill in the art that various modifications and equivalents can be made without departing from the spirit and scope of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201910509959.0 | Jun 2019 | CN | national |
This is a U.S. national stage of application No. PCT/CN2020/079442, filed on Mar. 16, 2020. Priority is claimed on the following application: Country: China, Application No.: 201910509959.0, Filed: Jun. 13, 2019, the entire content of which is incorporated here by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/079442 | 3/16/2020 | WO |
