Near-infrared fluorescence sensor, preparation method, traditional Chinese medicine optical nose based on near-infrared fluorescence sensor, application of traditional Chinese medicine optical nose and detection method for volatile components of traditional Chinese medicine

Abstract

The invention discloses a near-infrared fluorescence sensor, a preparation method, a traditional Chinese medicine optical nose based on the near-infrared fluorescence sensor, application and a method for detecting volatile components of traditional Chinese medicine, and belongs to the technical field of traditional Chinese medicine. The near-infrared fluorescence sensor comprises a single-walled carbon nanotube, a single-stranded DNA (deoxyribonucleic acid) and a paper base, the single-stranded DNA is fixed on the surface of the single-walled carbon nanotube, and the single-walled carbon nanotube with the surface modified with the single-stranded DNA is fixed on the paper base. The 11 near-infrared fluorescence sensors form the traditional Chinese medicine optical nose. In traditional Chinese medicine volatile component detection, the traditional Chinese medicine optical nose can rapidly capture changes of various components in a to-be-detected object and make a response to fluorescence signal changes. Whether the components are changed or not can be visualized by means of the method. The types of the traditional Chinese medicine volatile components or the change conditions of the traditional Chinese medicine volatile components are judged through response information of the multiple sensors, and therefore rapid identification of the traditional Chinese medicine volatile components and whether the traditional Chinese medicine is processed or not is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and specifically relates to a near-infrared fluorescence sensor, a preparation method, a traditional Chinese medicine optical nose based on the near-infrared fluorescence sensor, an application, and a method for detecting volatile components of traditional Chinese medicine. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Volatile oil is a general term for volatile oily components that are widely present in plants and can be obtained by steam distillation. They are widely distributed in Chinese medicinal materials, such as Bupleurum, Citrus aurantium, and Paeonia suffruticosa. The content and composition of volatile components play an important therapeutic role in many Chinese medicines and are indispensable effective ingredients. The distribution of volatile components in plants varies, and is affected by factors such as different medicinal parts, collection time, storage conditions, and processing technology. The content and composition of volatile components vary greatly, and the amount of volatile components directly affects its efficacy, safety, and quality stability. The characteristics of high heat sensitivity and easy volatilization and loss make its quality control difficult. Only 38 kinds of Chinese medicinal materials in the "Chinese Pharmacopoeia" stipulate the limit standards for volatile components, and Chinese patent medicines rich in volatile components also lack quality control of volatile components.

[0004] In order to detect the composition and content of volatile components, analytical techniques such as gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC) are usually used, but these conventional techniques require complex pretreatment processes and cannot be easily applied to online monitoring. At present, there are new methods for the detection of volatile components of traditional Chinese medicine in the gas phase, such as headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS), which achieves the enrichment of volatile components through the adsorption / desorption process, thereby achieving the purpose of volatile component detection. However, this method has a long analysis time, expensive equipment and high cost. Electronic nose (odor scanner) is an analytical technology for determining the flavor of volatile compounds. Slight changes in the flavor of the sample will cause differences in the sensor response value. It has a fast response and high sensitivity. However, due to the complexity and high specialization of electronic nose technology, its equipment and R&D costs are relatively high, and it is greatly affected by environmental factors and has limited recognition capabilities. Therefore, it is very meaningful to develop new technologies for rapid characterization of volatile components of traditional Chinese medicine. Summary of the invention

[0005] In order to solve the shortcomings of the prior art, the purpose of the present invention is to provide a near-infrared fluorescence sensor, a preparation method and a traditional Chinese medicine optical nose based on the near-infrared fluorescence sensor, and an application and a detection method for volatile components of traditional Chinese medicine. The present invention utilizes the difference in fluorescence response of single-walled carbon nanotubes (SWCNTs) modified with single-stranded DNA (ssDNA) to different volatile components of traditional Chinese medicine to distinguish different components, and has the advantages of high sensitivity, fast response, and sample non-destructiveness, and has good application value.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] The first aspect of the present invention provides a near-infrared fluorescence sensor, comprising: single-walled carbon nanotubes, single-stranded DNA and a paper substrate;

[0008] The single-stranded DNA is fixed on the surface of the single-walled carbon nanotube, and the single-walled carbon nanotube with the single-stranded DNA modified on the surface is fixed on the paper base.

[0009] A second aspect of the present invention provides a method for preparing the above-mentioned near-infrared fluorescence sensor, comprising:

[0010] The single-stranded DNA and the single-walled carbon nanotubes are mixed and dissolved in a solvent to be evenly dispersed, undispersed single-walled carbon nanotubes and unconnected single-stranded DNA are removed, and a near-infrared fluorescent sensor dispersion is obtained after resuspending and diluting;

[0011] The near-infrared fluorescent sensor dispersion is added to a substrate and dried to obtain a near-infrared fluorescent sensor.

[0012] The third aspect of the present invention provides a traditional Chinese medicine optical nose based on a near-infrared fluorescence sensor, comprising 11 near-infrared fluorescence sensors as described in any one of claims 1-3, the nucleic acid sequence of the single-stranded DNA in each near-infrared fluorescence sensor is different, and the nucleic acid sequences of the single-stranded DNA in the 11 near-infrared fluorescence sensors are respectively shown in SEQ ID NOs: 1-11.

[0013] The fourth aspect of the present invention provides the above-mentioned near-infrared fluorescence sensor, or the near-infrared fluorescence sensor prepared by the above-mentioned preparation method, or the above-mentioned traditional Chinese medicine optical nose based on the near-infrared fluorescence sensor for use in the detection of volatile components of traditional Chinese medicine.

[0014] A fifth aspect of the present invention provides a method for detecting volatile components of traditional Chinese medicine, comprising:

[0015] The above-mentioned Chinese medicine optical nose is fixed in a closed device, and the fluorescence intensity of the Chinese medicine optical nose is measured, which is recorded as the initial fluorescence intensity I0; different volatile components of Chinese medicine are introduced into the closed device, and the test substances are allowed to volatilize naturally. After the fluorescence signal of the Chinese medicine optical nose is stable, the fluorescence intensity is recorded again, which is recorded as I1;

[0016] The relative value of fluorescence intensity change was used for result analysis, i.e. (I1-I0) / I0; principal component cluster analysis was used to evaluate the differentiation of volatile components of different traditional Chinese medicines.

[0017] A sixth aspect of the present invention provides an application of the above-mentioned method for detecting volatile components of traditional Chinese medicine in distinguishing traditional Chinese medicine before and after processing.

[0018] The beneficial effects of the present invention are:

[0019] The present invention uses SWCNT modified by ssDNA non-covalently as a basis to construct a near-infrared fluorescence sensor, and fixes the sensor on a paper substrate to prepare a sensor array, i.e., a traditional Chinese medicine optical nose. The sensor array preparation method is simple, low-cost, good in response, and good in stability. After the volatile components of traditional Chinese medicine gas are in contact with the sensor array, the difference in the fluorescence response of the sensor is used to quantitatively and qualitatively analyze the volatile components of traditional Chinese medicine. The method analyzes the volatile components of traditional Chinese medicine in the gas phase, is simple to operate, has a short response time, and is highly sensitive. In addition, the method does not require pretreatment before detection, and is suitable for online monitoring of volatile components of traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0021] Figure 1 The 11 near-infrared fluorescent sensor dispersions prepared in Example 1 of the present invention are (GT)5-SWCNT, (GT) 10 -SWCNT, (GT) 15 -SWCNT, (GT) 20 -SWCNT, (GT) 30 -SWCNT, (AT) 15 -SWCNT, (AAT) 10 -SWCNT, (AAAT)7-SWCNT, (T) 30 -SWCNT, (GC) 15 -SWCNT, (C) 30 -SWCNT near-infrared fluorescence sensor dispersion;

[0022] Figure 2 The UV-visible-near-infrared absorption spectra of 11 kinds of near-infrared fluorescence sensor dispersions prepared in Example 1 of the present invention;

[0023] Figure 3Atomic force microscope (AFM) images of 11 near-infrared fluorescent sensor dispersions and single-walled carbon nanotubes prepared in Example 1 of the present invention;

[0024] Figure 4 The fluorescence spectra of 11 kinds of near-infrared fluorescence sensor dispersions prepared in Example 1 of the present invention;

[0025] Figure 5 The paper-based near-infrared fluorescence sensor array (a) prepared in Example 2 of the present invention and its wide-field fluorescence imaging image (b);

[0026] Figure 6 Schematic diagram of a static response device for volatile components of traditional Chinese medicine constructed in Example 3 of the present invention;

[0027] Figure 7 (GT) prepared in Example 3 of the present invention 15 -Linear relationship between the fluorescence response of SWCNT sensor array to paeonol and the mass of paeonol;

[0028] Figure 8 The fluorescence response of the traditional Chinese medicine optical nose to different amounts of the volatile component limonene standard of the traditional Chinese medicine in Example 4 of the present invention, wherein a is 1 μL, b is 2 μL, and c is 3 μL;

[0029] Fig. 9 The fluorescence response of the Chinese medicine optical nose to different amounts of the Chinese medicine volatile component geraniol standard in Example 5 of the present invention, where a is 1 μL, b is 2 μL, and c is 3 μL;

[0030] Fig.10 The fluorescence response of the Chinese medicine optical nose to different amounts of the volatile component cinnamaldehyde standard of Chinese medicine in Example 6 of the present invention, where a is 1 μL, b is 2 μL, and c is 3 μL;

[0031] Fig.11 The fluorescence response of the Chinese medicine optical nose to different amounts of the Chinese medicine volatile component pelargonium alcohol standard in Example 7 of the present invention, where a is 1 μL, b is 2 μL, and c is 3 μL;

[0032] Fig.12 The figure shows how the optical nose for Chinese medicine in Example 8 of the present invention distinguishes different amounts of volatile components of Chinese medicine, namely, limonene, geraniol, cinnamaldehyde, and geranyl alcohol; wherein a to c are heat maps of the response of the near-infrared fluorescence sensor array to different volatile components in the presence of 1 μL, 2 μL, and 3 μL of the test substance, respectively; d to f are cluster analyses of the response of the near-infrared fluorescence sensor array to different volatile components in the presence of 1 μL, 2 μL, and 3 μL of the test substance, respectively;

[0033] Fig.13The fluorescence response of the optical nose of traditional Chinese medicine to the volatile components of Fructus Aurantii Immaturus before (a) and after (b) processing and the cluster analysis (c) in Example 9 of the present invention. DETAILED DESCRIPTION

[0034] In view of the problems of long detection time, expensive equipment and high cost of existing volatile components of traditional Chinese medicine, the present invention proposes a near-infrared fluorescence sensor, a preparation method and a traditional Chinese medicine optical nose based on the near-infrared fluorescence sensor, and an application and a method for detecting volatile components of traditional Chinese medicine.

[0035] A typical embodiment of the present invention provides a near-infrared fluorescence sensor, comprising: single-walled carbon nanotubes (SWCNTs), single-stranded DNA (ssDNA) and a paper substrate;

[0036] The single-stranded DNA is fixed on the surface of the single-walled carbon nanotube, and the single-walled carbon nanotube with the single-stranded DNA modified on the surface is fixed on the paper base.

[0037] SWCNTs can emit photoluminescence (PL) in the near-infrared region (NIR), and the required excitation wavelength and the resulting emission wavelength depend on the chirality of the SWCNTs. The photoluminescence intensity and the precise emission wavelength are affected by the local environment created by the solvent and the absorbing molecules and their aggregation state. SWCNTs are extremely hydrophobic and form insoluble aggregates in water, which quenches their fluorescence. Therefore, it is necessary to functionalize the surface of carbon nanotubes by covalent or non-covalent modification. ssDNA is a commonly used modifier to improve the water solubility of SWCNTs. SWCNTs modified with ssDNA have good stability and can be stored for a long time. The four types of aromatic nitrogenous bases interact with the side walls of SWCNTs, exposing the hydrophilic sugar phosphate backbone to the aqueous environment, which can improve the water solubility of the DNA-SWCNT complex.

[0038] In some examples of this embodiment, the nucleic acid sequence of the single-stranded DNA is any one of the sequences shown in SEQ ID NOs: 1-11. The specific information of the sequences shown in SEQ ID NOs: 1-11 is as follows.

[0039] Table 1 Sequences shown in SEQ ID NOs: 1-11

[0040]

[0041]

[0042] The present invention fixes single-walled carbon nanotubes modified with specific ssDNA on a paper substrate, which can improve the stability of the sensor and make it easier to store for a long time. In addition, the sensor can be directly in contact with volatile gases, reducing the response time.

[0043] In some examples of this embodiment, the paper base is mixed cellulose paper, and the pore size of the mixed cellulose paper is 1-1.5 μm, preferably 1.2 μm.

[0044] It should be noted that, compared with mixed cellulose papers with pore sizes of 0.2 μm, 0.22 μm, 0.45 μm, etc., the near-infrared fluorescence sensor prepared from mixed cellulose paper with the above pore sizes has the best sensing effect.

[0045] Another typical embodiment of the present invention provides a method for preparing the above-mentioned near-infrared fluorescence sensor, comprising:

[0046] The single-stranded DNA and the single-walled carbon nanotubes are mixed and dissolved in a solvent to be evenly dispersed, undispersed single-walled carbon nanotubes and unconnected single-stranded DNA are removed, and a near-infrared fluorescent sensor dispersion is obtained after resuspending and diluting;

[0047] The near-infrared fluorescent sensor dispersion is added to a substrate and dried to obtain a near-infrared fluorescent sensor.

[0048] In some examples of this embodiment, the mass ratio of the single-stranded DNA to the single-walled carbon nanotube is (0.9-1.1):(0.9-1.1).

[0049] In some examples of this embodiment, the solvent is ultrapure water;

[0050] In some examples of this embodiment, the concentration of the near-infrared fluorescence sensor dispersion is 10-20 mg / L, specifically 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, 20 mg / L, and the like.

[0051] It should be noted that the concentration of the near-infrared fluorescent sensor dispersion is calculated based on the absorbance value at 632 nm using the Lambert-Beer law under ultraviolet-visible near-infrared spectrophotometer. If the concentration is higher than 10-20 mg / L, ultrapure water can be used to dilute until the target concentration is obtained.

[0052] In some examples of this embodiment, the single-stranded DNA and the single-walled carbon nanotubes are mixed and dissolved in a solvent, and dispersed uniformly at a low temperature, which is -5°C to 5°C, preferably in an ice bath environment.

[0053] Disperse evenly at low temperature. To improve the dispersion effect, the dispersion can be carried out with the help of tip ultrasound. For example, a handheld 3mm probe tip ultrasound can be used to connect ssDNA and SWCNT under the condition of 15% power, and the probe is about 5mm below the liquid surface. The ultrasound process is carried out in a way of working for 2s and stopping for 1s, with a total working time of 60min. The entire ultrasound dispersion process is carried out in an ice bath.

[0054] In some examples of this embodiment, undispersed single-walled carbon nanotubes are removed by centrifugation. For example, the obtained dispersed solution can be centrifuged at a centrifugal force of 16,100×g for three times, each time for 60 minutes, and 80% of the supernatant is aspirated each time, and the precipitate is discarded to remove the undispersed SWCNTs.

[0055] In some examples of this embodiment, unconnected single-stranded DNA can be removed by ultrafiltration and centrifugation. For example, 80% of the supernatant after removing undispersed SWCNTs can be added to an ultrafiltration tube with a cutoff molecular weight of 100 kDa, centrifuged at a centrifugal force of 12,000 × g for 10 min, and then 200 μL of ultrapure water is added to resuspend the retentate, and centrifuged at 12,000 × g for 10 min, and the washing is repeated 3 times to remove unconnected ssDNA. After elution, the near-infrared fluorescent sensor is retained in the ultrafiltration tube.

[0056] It should be noted that the near-infrared fluorescent sensor precipitates that are aggregated and difficult to disperse due to ultrafiltration also need to be removed. The near-infrared fluorescent sensor after ultrafiltration can be resuspended in 400 μL PBS, and ultrasonic dispersion is performed for 10 minutes using a handheld 3 mm probe tip ultrasound at 15% power to disperse the aggregated sensors. The sensor precipitates that are aggregated and difficult to disperse due to ultrafiltration are removed by centrifugation at 16,100 × g centrifugal force for 30 minutes, and the first 80% of the supernatant is taken to obtain a near-infrared fluorescent sensor dispersion.

[0057] It should be noted that the preparation methods of near-infrared fluorescent sensors prepared from different ssDNAs are the same, and only the type of ssDNA needs to be changed during the preparation.

[0058] A third typical embodiment of the present invention provides a traditional Chinese medicine optical nose based on a near-infrared fluorescence sensor, comprising 11 of the above-mentioned near-infrared fluorescence sensors, the nucleic acid sequence of the single-stranded DNA in each near-infrared fluorescence sensor is different, and the nucleic acid sequences of the single-stranded DNA in the 11 near-infrared fluorescence sensors are respectively shown in SEQ ID NOs: 1-11.

[0059] According to the different structures of volatile components of different traditional Chinese medicines, the present invention uses single-walled carbon nanotubes (SWCNTs) as the central material, modifies the surface of the SWCNTs with different ssDNAs to prepare fluorescent sensors with different response properties, fixes them with the help of paper substrates to prepare near-infrared fluorescent sensors, and then prepares a traditional Chinese medicine optical nose based on near-infrared fluorescent sensors, that is, a near-infrared fluorescent sensor array.

[0060] In the detection of volatile components of traditional Chinese medicine, an array composed of multiple near-infrared fluorescence sensors can quickly capture the changes in multiple components in the test object and respond to the changes in fluorescence signals. Relying on this array, the slight changes in the medium components can be converted into different degrees of fluorescence signal changes, that is, whether the components have changed can be "visualized". The array is composed of multiple different universal response sensors, so even for weak material changes, they will be reflected because there are multiple different sensors to capture different fluorescence signal changes. The response information of multiple sensors is used to determine the type of volatile components of traditional Chinese medicine or the changes in volatile components in traditional Chinese medicine, thereby realizing the rapid identification of volatile components of traditional Chinese medicine and whether the traditional Chinese medicine has been processed. The array has the characteristics of high sensitivity, short response time, small sample size, and low preparation and operation costs.

[0061] It should be noted that the traditional Chinese medicine optical nose based on near-infrared fluorescence sensor of the present invention is used for qualitative detection of volatile components of traditional Chinese medicine. It can be prepared into a traditional Chinese medicine volatile component detection kit to achieve rapid detection of volatile components of traditional Chinese medicine with short detection time and high sensitivity.

[0062] A fourth typical embodiment of the present invention provides the above-mentioned near-infrared fluorescence sensor, or the near-infrared fluorescence sensor prepared by the above-mentioned preparation method, or the application of the above-mentioned traditional Chinese medicine optical nose based on the near-infrared fluorescence sensor in the detection of volatile components of traditional Chinese medicine.

[0063] The traditional Chinese medicine optical nose based on near-infrared fluorescence sensor is composed of multiple different universal response sensors. Even for weak material changes, they will be reflected because multiple different sensors are used to capture different fluorescence signal changes. The response information of multiple sensors is used to determine the type of volatile components of traditional Chinese medicine or the changes in volatile components in traditional Chinese medicine, thereby realizing rapid identification of volatile components of traditional Chinese medicine and whether the traditional Chinese medicine has been processed. The array has the characteristics of high sensitivity, short response time, small sample size, and low preparation and operation costs.

[0064] In some examples of this embodiment, the volatile components of the traditional Chinese medicine include but are not limited to at least one of paeonol, limonene, geraniol, cinnamaldehyde and geranyl alcohol.

[0065] A fifth typical embodiment of the present invention provides a method for detecting volatile components of traditional Chinese medicine, comprising:

[0066] The above-mentioned Chinese medicine optical nose is fixed in a closed device, and the fluorescence intensity of the Chinese medicine optical nose is measured, which is recorded as the initial fluorescence intensity I0; different volatile components of Chinese medicine are introduced into the closed device, and the test substances are allowed to volatilize naturally. After the fluorescence signal of the Chinese medicine optical nose is stable, the fluorescence intensity is recorded again, which is recorded as I1;

[0067] The relative value of fluorescence intensity change was used for result analysis, i.e. (I1-I0) / I0; principal component cluster analysis was used to evaluate the differentiation of volatile components of different traditional Chinese medicines.

[0068] The present invention successfully prepared a fluorescent sensor array for rapid detection of volatile components in traditional Chinese medicine, which can qualitatively detect the changes in the content of volatile components of a single traditional Chinese medicine, and can detect the changes in the content of different volatile components in traditional Chinese medicine. Using this traditional Chinese medicine optical nose, the detection process of volatile components in traditional Chinese medicine can be accelerated in actual production.

[0069] A sixth typical embodiment of the present invention provides an application of the above-mentioned method for detecting volatile components of traditional Chinese medicine in distinguishing traditional Chinese medicine before and after processing.

[0070] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0071] The reagents used in the following examples are all commercially available products. The 11 kinds of ssDNA in the following examples are respectively marked as follows:

[0072] GTGTGTGTGT is marked as (GT)5;

[0073] GTGTGTGTGTGTGTGTGTGTGT marked as (GT) 10 ;

[0074] GTGTGTGTGTGTGTGTGTGTGTGTGT is marked (GT) 15 ;

[0075] GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT is marked with (GT) 20 ;

[0076] GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT GTGTGTGT marked with (GT) 30 ;

[0077] ATATATATATATATATATATATATATATATATATATATATMarked as (AT) 15 ;

[0078] AATAATAATAATAATAATAATAATAATAAT is marked (AAT) 10 ;

[0079] AAATAAATAAATAAATAAATAAATAAAT is marked as (AAAT)7;

[0080] TTTTTTTTTTTTTTTTTTTTTTTTTTTT marked with (T) 30 ;

[0081] GCGCGCGCGCGCGCGCGCGCGCGCGCGCGC is marked with (GC) 15 ;

[0082] CCCCCCCCCCCCCCCCCCCCCCCCCCCCCC marked with (C) 30 .

[0083] (GT)5, (GT) 10 , (GT) 15 , (GT) 20 , (GT) 30 , (AT) 15 、(AAT) 10 、(AAAT)7、(T) 30 , (GC) 15 (C) 30 All are ssDNA sequences and are artificially synthesized.

[0084] Example 1

[0085] This example is to prepare 11 kinds of ssDNA modified near-infrared fluorescent sensor dispersions, and the specific steps are as follows:

[0086] 0.2 mg (GT) 5 was weighed and dissolved in 1 mL ultrapure water, to which 0.2 mg single-walled carbon nanotube powder was added, and ssDNA was connected to SWCNT using a handheld 3 mm probe tip ultrasound at 15% power, with the probe penetrating about 5 mm below the liquid surface. The ultrasound process was carried out in a 2 s on, 1 s off mode, with a total working time of 60 min, and the entire ultrasound dispersion process was carried out in an ice bath.

[0087] The obtained dispersed solution was centrifuged at 16,100×g for three times, each time for 60 min, and 80% of the supernatant was aspirated each time, and the precipitate was discarded to remove the undispersed SWCNT. After the three centrifugations, 80% of the supernatant obtained from the last centrifugation was taken into an ultrafiltration tube with a cutoff molecular weight of 100 kDa, and after centrifugation at 12,000×g for 10 min, 200 μL of ultrapure water was added to resuspend the retentate, and centrifuged at 12,000×g for 10 min, and the washing was repeated 3 times to remove the unattached ssDNA. After centrifugation, 400 μL of ultrapure water was added to resuspend the retentate, and ultrasonic dispersion was performed for 10 min using a handheld 3mm probe tip ultrasound at 15% power to disperse the aggregated sensors, and centrifuged at 16,100×g for 30 min to remove the sensor precipitate that was aggregated and difficult to disperse due to ultrafiltration. The first 80% of the supernatant was taken to obtain a near-infrared fluorescent sensor dispersion, which was named (GT)5-SWCNT near-infrared fluorescent sensor dispersion.

[0088] The preparation methods of the remaining 10 ssDNA modified near-infrared fluorescent sensor dispersions were the same as above, and (GT) 10 -SWCNT, (GT) 15 -SWCNT, (GT) 20 -SWCNT, (GT) 30 -SWCNT, (AT) 15 -SWCNT, (AAT) 10 -SWCNT, (AAAT)7-SWCNT, (T) 30 -SWCNT, (GC) 15 -SWCNT, (C) 30 -SWCNT near-infrared fluorescence sensor dispersion.

[0089] The 11 kinds of near-infrared fluorescent sensor dispersions prepared in Example 1 are shown in Figure 1 , from left to right: (GT)5-SWCNT, (GT) 10 -SWCNT, (GT) 15 -SWCNT, (GT) 20 -SWCNT, (GT) 30 -SWCNT, (AT) 15 -SWCNT, (AAT) 10 -SWCNT, (AAAT)7-SWCNT, (T) 30 -SWCNT, (GC) 15 -SWCNT, (C) 30-SWCNT sensor dispersion The near-infrared fluorescence sensor dispersion prepared in Example 1 has high dispersibility or stability, and no visible precipitation is generated during the solution preparation process.

[0090] Performance characterization of 11 near-infrared fluorescence sensor dispersions prepared in Example 1

[0091] 1. UV-visible near-infrared absorption spectroscopy to characterize whether SWCNT is successfully dispersed

[0092] 1 mL of each near-infrared fluorescence sensor dispersion prepared in Example 1 was transferred to a quartz cuvette, and the absorption spectrum of the optical sensor dispersion solution in the wavelength range of 400 nm to 1100 nm was measured using an ultraviolet-visible near-infrared spectrophotometer.

[0093] The UV-visible near-infrared absorption spectra of the 11 near-infrared fluorescence sensor dispersions prepared in Example 1 are shown in Figure 2 .Depend on Figure 2 It was observed that each dispersion had characteristic absorption peaks of SWCNT near 560 nm and 990 nm, indicating that SWCNT in the 11 near-infrared fluorescence sensor dispersions prepared in Example 1 could be uniformly dispersed in ultrapure water.

[0094] 2. Atomic force microscopy (AFM) imaging to characterize whether ssDNA is successfully modified on the SWCNT surface

[0095] (1) AFM sample preparation

[0096] 1 μL of each near-infrared fluorescent sensor dispersion was taken and diluted to 500 μL with ultrapure water to obtain the SWCNTAFM sample stock solution of each near-infrared fluorescent sensor dispersion.

[0097] Weigh about 0.2 mg of SWCNT, add 1 mL of 1% (m / v) sodium deoxycholate (DOC) aqueous solution, use tip ultrasound to disperse at 15% power for 60 min, centrifuge at 16,100 × g centrifugal force for 60 min, remove the precipitate, repeat three times, and take 80% of the supernatant as the DOC-dispersed SWCNT sample solution. Take 1 μL of the dispersion and dilute it to 500 μL with ultrapure water to obtain the DOC-dispersed SWCNT sample stock solution.

[0098] Fix the clean mica sheet on the AFM iron sheet, and remove the surface mica sheet with transparent tape before use. Take 3 μL of each of the above 11 near-infrared fluorescent sensor dispersions and DOC-dispersed SWCNT AFM sample stock solution, drop them on the clean mica sheet, and place them in a clean space to fully dry.

[0099] (2) AFM imaging

[0100] The samples on the mica sheet were measured in the gas phase environment using an atomic force microscope equipped with an RTESP-300 probe with a curvature radius of 10 nm to obtain the height of each sample. The scanning area was 1 μm and the scanning rate was 0.5 Hz. AFM images of SWCNT and each near-infrared fluorescent sensor dispersion are shown in Figure 3 .

[0101] According to the imaging results ( Figure 3 ) shows that the diameter of SWCNT is <1nm, and the diameters of the 11 optical sensors prepared are all significantly larger than 1nm. The height difference between the prepared complex and the SWCNT treated with DOC indicates that ssDNA is wrapped around SWCNT to increase its diameter, that is, all 11 ssDNAs can be successfully connected and modified to the SWCNT surface.

[0102] 3. Characterization of sensor response performance by steady-state transient fluorescence spectroscopy

[0103] Under the detection of UV-visible near-infrared spectrophotometer, the sensor concentration was calculated according to the absorbance value at 632 nm using the Lambert-Beer law. After the 11 near-infrared fluorescent sensor dispersions prepared in Example 1 were diluted with ultrapure water to a concentration of about 15 mg / L, the original fluorescence spectra of each near-infrared fluorescent sensor dispersion were obtained by scanning on an FLS1000 steady-state transient fluorescence spectrometer. The scanning starting wavelength was 900 nm, the cutoff wavelength was 1100 nm, the step size was 1 nm, and the excitation wavelength was 570 nm.

[0104] The fluorescence spectra of the 11 optical sensors prepared in Example 1 are shown in Figure 4 All sensor solutions have characteristic fluorescence signals at 995 nm.

[0105] Example 2

[0106] This embodiment uses 11 kinds of near-infrared fluorescence sensor dispersions prepared in Example 1 to prepare near-infrared fluorescence sensors and a traditional Chinese medicine optical nose based on near-infrared fluorescence sensors, that is, a near-infrared fluorescence sensor array. The specific steps are as follows:

[0107] Under the detection of UV-visible near-infrared spectrophotometer, the sensor concentration was calculated according to the absorbance value at 632 nm using the Lambert-Beer law, and the 11 near-infrared fluorescent sensor dispersions prepared in Example 1 were diluted with ultrapure water to a concentration of about 15 mg / L. 1 μL of each of the 11 prepared near-infrared fluorescent sensor dispersions was respectively drawn and dropped onto a mixed cellulose (MCE) membrane with a pore size of 1.2 μm, and heated and dried at 90°C to obtain a paper-based near-infrared fluorescent sensor array, i.e., a traditional Chinese medicine optical nose, such as Figure 5 (a) shown.

[0108] The prepared Chinese medicine optical nose was placed in a wide-field imaging system, and an LED light source with a 561nm bandpass filter was used to excite the Chinese medicine optical nose. The excited fluorescence signal was monitored using a XEVA near-infrared optimized InGaAs camera with a 990nm long-pass filter. The fluorescence imaging results are shown in Figure 2. Figure 5 (b) as shown.

[0109] Example 3

[0110] This example is (GT) prepared using Example 1 15 -SWCNT near-infrared fluorescence sensor dispersion was used to prepare a near-infrared fluorescence sensor and detect its response to paeonol. The specific steps are as follows:

[0111] Under ultraviolet-visible-near-infrared spectrophotometer detection, the sensor concentration was calculated according to the absorbance value at 632 nm using the Lambert-Beer law, and the (GT) prepared in Example 1 was diluted with ultrapure water. 15 -SWCNT near-infrared fluorescence sensor dispersion to a concentration of about 15 mg / L. Absorb and configure (GT) 15 -1 μL of SWCNT near-infrared fluorescent sensor dispersion was dripped onto a mixed cellulose (MCE) membrane with a pore size of 1.2 μm. This was repeated 4 times to prepare four identical sensors for parallel testing. The sensors were then heated and dried at 90°C to obtain a near-infrared fluorescent sensor array, named (GT) 15 -SWCNT sensor array.

[0112] Build as Figure 6 The static response device shown in the figure is used to prepare (GT) 15 -The SWCNT sensor array is fixed on the bottom of the sealed box and uses an LED light source with a 561nm bandpass filter (GT) 15 -SWCNT sensor array was excited, and the excited fluorescence signal was monitored using a XEVA near-infrared optimized InGaAs camera with a 990nm long-pass filter. Six groups of paeonol standards were accurately weighed, with masses of 9.71 mg, 15.91 mg, 5.24 mg, 2.65 mg, 20.29 mg, and 24.45 mg, respectively. Before the experiment began, record (GT) 15 -The initial fluorescence signal of the SWCNT sensor array is recorded as I0. The paeonol standard is introduced into the sealed small box, left to evaporate naturally, and the entire evaporation process is recorded (GT) 15 -Changes in fluorescence signal of SWCNT sensor array after a period of time, (GT) 15 -The fluorescence signal of the SWCNT sensor array remains stable, and the stabilized fluorescence signal is recorded as I. Remove the analyte in the small box to make (GT) 15-The fluorescence signal of the SWCNT sensor array returned to the baseline level. Repeat the above experimental operation to detect 6 groups of paeonol, and record the fluorescence signal before each experiment and the fluorescence signal after stabilization to obtain two groups of fluorescence values, I0 and I. The relative value of the change in fluorescence intensity was used for result analysis, i.e. (I-I0) / I0. The analysis results are shown in the table. Linear regression was performed on the obtained data, and the results are shown in Table 2 and Figure 7 As shown, the linear equation is Y = 0.002328X + 0.01163, r 2 =0.9397.

[0113] Table 2 Statistics of relative values ​​of paeonol mass and fluorescence intensity changes

[0114]

[0115] Example 4

[0116] This example uses the traditional Chinese medicine optical nose prepared in Example 2 to detect its response to the volatile component of traditional Chinese medicine, limonene. The specific steps are as follows:

[0117] Using the static response device built in Example 3, the Chinese medicine optical nose prepared in Example 2 is fixed to the bottom of the sealed small box, and the Chinese medicine optical nose is excited using an LED light source with a 561nm bandpass filter, and the excited fluorescence signal is monitored using a XEVA near-infrared optimized InGaAs camera with a 990nm long-pass filter. Before the experiment begins, the initial fluorescence signal of the Chinese medicine optical nose is recorded, recorded as I0. 1 μL of limonene standard is introduced into the sealed small box, and it is allowed to evaporate naturally, and the changes in the fluorescence signal of the Chinese medicine optical nose during the entire volatilization process are recorded, and the fluorescence signal after the introduction of the analyte for 20 minutes is recorded as I. After the analyte is introduced for 30 minutes, the analyte in the small box is removed to restore the fluorescence signal of the sensor to the baseline level. Repeat the above operation four times, and record the fluorescence signal before the start of each experiment and the fluorescence signal 20 minutes after the introduction of the analyte, and obtain two groups of fluorescence values ​​of I0 and I.

[0118] 2 μL and 3 μL of limonene standard were introduced into the small box respectively. The experimental steps were the same as above, and two groups of fluorescence values, I0 and I, were obtained under different limonene content conditions. The relative value of the fluorescence intensity change, i.e. (I-I0) / I0, was used for result analysis.

[0119] The experimental results are as follows Figure 8 As shown, analysis of the experimental results shows that different sensors have different fluorescence responses to limonene gas, and the fluorescence intensity increases with the increase in the content of limonene standard added.

[0120] Example 5

[0121] This example uses the traditional Chinese medicine optical nose prepared in Example 2 to detect its response to the volatile component geraniol of traditional Chinese medicine. The specific steps are as follows:

[0122] Using the static response device built in Example 3, the Chinese medicine optical nose prepared in Example 2 is fixed to the bottom of the sealed small box, and the Chinese medicine optical nose is excited using an LED light source with a 561nm bandpass filter, and the excited fluorescence signal is monitored using a XEVA near-infrared optimized InGaAs camera with a 990nm long-pass filter. Before the experiment begins, the initial fluorescence signal of the Chinese medicine optical nose is recorded, recorded as I0. 1 μL of geraniol standard is introduced into the sealed small box, and it is allowed to evaporate naturally, and the changes in the fluorescence signal of the Chinese medicine optical nose during the entire volatilization process are recorded, and the fluorescence signal after the introduction of the analyte for 20 minutes is recorded as I. After the analyte is introduced for 30 minutes, the analyte in the small box is removed to restore the fluorescence signal of the sensor to the baseline level. Repeat the above operation four times, and record the fluorescence signal before the start of each experiment and the fluorescence signal after the introduction of the analyte for 20 minutes, and obtain two groups of fluorescence values ​​of I0 and I.

[0123] 2 μL and 3 μL of geraniol standard were introduced into the small box respectively. The experimental steps were the same as above, and two groups of fluorescence values, I0 and I, were obtained under different geraniol content conditions. The relative value of the fluorescence intensity change, i.e. (I-I0) / I0, was used for result analysis.

[0124] The experimental results are as follows Fig. 9 As shown, analysis of the experimental results shows that different sensors have different fluorescence responses to geraniol gas, and as the content of geraniol standard increases, the fluorescence intensity also increases.

[0125] Example 6

[0126] This example uses the Chinese medicine optical nose prepared in Example 2 to detect its response to cinnamaldehyde, a volatile component of Chinese medicine. The specific steps are as follows:

[0127] Using the static response device built in Example 3, the Chinese medicine optical nose prepared in Example 2 is fixed to the bottom of the sealed small box, and the Chinese medicine optical nose is excited using an LED light source with a 561nm bandpass filter, and the excited fluorescence signal is monitored using a XEVA near-infrared optimized InGaAs camera with a 990nm long-pass filter. Before the experiment begins, the initial fluorescence signal of the Chinese medicine optical nose is recorded, recorded as I0. 1 μL of cinnamaldehyde standard is introduced into the sealed small box, and it is allowed to evaporate naturally, and the changes in the fluorescence signal of the Chinese medicine optical nose during the entire volatilization process are recorded, and the fluorescence signal after the introduction of the analyte for 20 minutes is recorded as I. After the analyte is introduced for 30 minutes, the analyte in the small box is removed to restore the fluorescence signal of the sensor to the baseline level. Repeat the above operation four times, and record the fluorescence signal before the start of each experiment and the fluorescence signal 20 minutes after the introduction of the analyte, and obtain two groups of fluorescence values ​​of I0 and I.

[0128] 2 μL and 3 μL of cinnamaldehyde standard were introduced into the small box respectively. The experimental steps were the same as above, and two groups of fluorescence values, I0 and I, were obtained under different conditions of cinnamaldehyde content. The relative value of fluorescence intensity change, i.e. (I-I0) / I0, was used for result analysis.

[0129] The experimental results are as follows Fig.10 As shown, analysis of the experimental results shows that there are differences in the fluorescence responses of different sensors to cinnamaldehyde gas, and as the content of cinnamaldehyde standard increases, the fluorescence intensity also increases.

[0130] Example 7

[0131] This example uses the traditional Chinese medicine optical nose prepared in Example 2 to detect its response to the volatile component of traditional Chinese medicine, pelargonium alcohol. The specific steps are as follows:

[0132] Using the static response device built in Example 3, the Chinese medicine optical nose prepared in Example 2 is fixed to the bottom of the closed small box, and the Chinese medicine optical nose is excited using an LED light source with a 561nm bandpass filter, and the excited fluorescence signal is monitored using a XEVA near-infrared optimized InGaAs camera with a 990nm long-pass filter. Before the experiment begins, the initial fluorescence signal of the Chinese medicine optical nose is recorded, denoted as I0. 1 μL of geranium alcohol standard is introduced into the closed small box, and it is allowed to evaporate naturally, and the change of the fluorescence signal of the Chinese medicine optical nose during the entire volatilization process is recorded, and the fluorescence signal after the introduction of the analyte for 20 minutes is recorded as I. After the analyte is introduced for 30 minutes, the analyte in the small box is removed to restore the fluorescence signal of the sensor to the baseline level. Repeat the above operation four times, and record the fluorescence signal before the start of each experiment and the fluorescence signal after the introduction of the analyte for 20 minutes, and obtain two groups of fluorescence values ​​of I0 and I.

[0133] 2 μL and 3 μL of pelargonium alcohol standard were introduced into the small box respectively. The experimental steps were the same as above, and two groups of fluorescence values, I0 and I, were obtained under different conditions of pelargonium alcohol content. The relative value of the change in fluorescence intensity, i.e. (I-I0) / I0, was used for result analysis.

[0134] The experimental results are as follows Fig.11 As shown, analysis of the experimental results shows that different sensors have different fluorescence responses to geranium alcohol gas, and as the content of geranium alcohol standard increases, the fluorescence intensity also increases.

[0135] Example 8

[0136] This example is the identification of volatile components of traditional Chinese medicine by the traditional Chinese medicine optical nose prepared in Example 2, and the specific steps are as follows:

[0137] The data collected in Examples 4, 5, 6, and 7 were analyzed and processed, and principal component cluster analysis was performed to evaluate the paper-based sensor array's ability to distinguish the volatile components of traditional Chinese medicine. Fig.12 As shown, each group of volatile components of different traditional Chinese medicines can be distinguished.

[0138] Example 9

[0139] This example is the identification of Fructus Aurantii Immaturus before and after processing by the traditional Chinese medicine optical nose prepared in Example 2, and the specific steps are as follows:

[0140] A static response device was built based on the static response device built in Example 3. The difference from the static response device in Example 3 is that a heating device is placed under the sealed small box of the sample. An LED light source with a 561nm bandpass filter is used to excite the Chinese medicine optical nose, and the excited fluorescence signal is monitored using a XEVA near-infrared optimized InGaAs camera with a 990nm longpass filter. The pre-processed Citrus aurantium was crushed and sieved, and 50mg of the pre-processed Citrus aurantium coarse powder was weighed. Before the experiment began, the initial fluorescence signal of the Chinese medicine optical nose was recorded and recorded as I0. The weighed pre-processed Citrus aurantium sample was introduced into the sealed small box, heated at 90°C to volatilize the volatile components in the medicinal material, and the changes in the fluorescence signal of the Chinese medicine optical nose during the entire volatilization process were recorded, and the fluorescence signal after the introduction of the analyte for 8min was recorded as I1. The analyte in the small box was removed 10min after the introduction of the analyte to restore the fluorescence signal of the sensor to the baseline level. The above operation was repeated four times, and the fluorescence signal before each experiment and the fluorescence signal 20 minutes after the introduction of the analyte were recorded to obtain two groups of fluorescence values, I0 and I1.

[0141] The processed Citrus aurantium was crushed and sieved, and 50 mg of processed Citrus aurantium coarse powder was weighed. Before the experiment, the initial fluorescence signal of the Chinese medicine optical nose was recorded and recorded as I0. The weighed processed Citrus aurantium sample was introduced into a sealed small box, heated at 90°C to volatilize the volatile components in the medicinal material, and the changes in the fluorescence signal of the Chinese medicine optical nose during the entire volatilization process were recorded. The fluorescence signal 8 minutes after the introduction of the analyte was recorded as I2. After 10 minutes of the introduction of the analyte, the analyte in the small box was removed to restore the fluorescence signal of the sensor to the baseline level. Repeat the above operation four times, and record the fluorescence signal before the start of each experiment and the fluorescence signal 20 minutes after the introduction of the analyte to obtain two sets of fluorescence values, I0 and I2.

[0142] The relative value of fluorescence intensity change was used for result analysis, i.e. (I1-I0) / I0, (I2-I0) / I0; principal component cluster analysis was used to evaluate the distinction between the processed and unprocessed Citrus aurantium. Fig.13 As shown, the Citrus aurantium before processing can be distinguished from the Citrus aurantium after processing.

[0143] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A near-infrared fluorescence sensor, characterized in that: include: single-walled carbon nanotubes, single-stranded DNA, and paper substrates; The single-stranded DNA is fixed on the surface of the single-walled carbon nanotube, and the single-walled carbon nanotube with the single-stranded DNA modified on the surface is fixed on the paper base. 2 . The near-infrared fluorescence sensor according to claim 1 , wherein the nucleic acid sequence of the single-stranded DNA is any one of the sequences shown in SEQ ID NOs: 1-11.

3. The near-infrared fluorescence sensor according to claim 1, characterized in that: The paper base is mixed cellulose paper, and the pore size of the mixed cellulose paper is 1-1.5 μm.

4. A method for preparing a near-infrared fluorescence sensor according to any one of claims 1 to 3, characterized in that: include: The single-stranded DNA and the single-walled carbon nanotubes are mixed and dissolved in a solvent to be evenly dispersed, undispersed single-walled carbon nanotubes and unconnected single-stranded DNA are removed, and a near-infrared fluorescent sensor dispersion is obtained after resuspending and diluting; The near-infrared fluorescent sensor dispersion is added to a substrate and dried to obtain a near-infrared fluorescent sensor.

5. The preparation method according to claim 4, characterized in that: The mass ratio of the single-stranded DNA to the single-walled carbon nanotube is (0.9-1.1):(0.9-1.1); The solvent is ultrapure water; The concentration of the near-infrared fluorescent sensor dispersion is 10-20 mg / L.

6. A Chinese medicine optical nose based on near-infrared fluorescence sensor, characterized in that: It comprises 11 near-infrared fluorescent sensors according to any one of claims 1-3, the nucleic acid sequence of the single-stranded DNA in each near-infrared fluorescent sensor is different, and the nucleic acid sequences of the single-stranded DNA in the 11 near-infrared fluorescent sensors are respectively shown as SEQ ID NOs: 1-11.

7. Use of a near-infrared fluorescence sensor as described in any one of claims 1 to 3, or a near-infrared fluorescence sensor prepared by the preparation method as described in any one of claims 4 to 5, or a traditional Chinese medicine optical nose based on a near-infrared fluorescence sensor as described in claim 6 in the detection of volatile components of traditional Chinese medicine.

8. The use according to claim 7, characterized in that The volatile components of traditional Chinese medicine include at least one of paeonol, limonene, geraniol, cinnamaldehyde and pelargonium alcohol.

9. A method for detecting volatile components of traditional Chinese medicine, characterized in that: include: Fix the Chinese medicine optical nose as claimed in claim 6 in a closed device, measure the fluorescence intensity of the Chinese medicine optical nose, and record it as the initial fluorescence intensity I0; introduce different volatile components of Chinese medicine into the closed device, let it stand to allow the test substance to volatilize naturally, and after the fluorescence signal of the Chinese medicine optical nose is stable, record the fluorescence intensity again, and record it as I1; The relative value of fluorescence intensity change was used for result analysis, i.e. (I1-I0) / I0; principal component cluster analysis was used to evaluate the differentiation of volatile components of different traditional Chinese medicines.

10. Use of the method for detecting volatile components of traditional Chinese medicine according to claim 9 in distinguishing traditional Chinese medicine before and after processing.

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