Colorimetric array sensor constructed by combining hydroxylamine hydrochloride with pH indicator and application of colorimetric array sensor

Abstract

The invention discloses a colorimetric array sensor constructed by combining hydroxylamine hydrochloride with a pH indicator and application of the colorimetric array sensor, a detection material in a sensing array does not need to be subjected to a complex synthesis process, each sensing unit is composed of one hydroxylamine hydrochloride and one pH indicator, immediate response to aldehyde and ketone compounds can be realized within 4 minutes, and the detection sensitivity is high. And the effect is obvious. Compared with the existing array sensor, the sensor disclosed by the invention does not need to be synthesized, is low in cost, convenient to implement and rapid in response, and can be used for rapid identification of aldehyde and ketone compounds and visual detection of complex samples rich in the compounds.

Description

Technical Field

[0001] The present invention belongs to the field of bionic array sensors, and particularly relates to a colorimetric array sensor constructed by combining hydroxylamine hydrochloride with a pH indicator and its application, which can be used for the rapid identification of aldehyde and ketone compounds and complex samples rich in such compounds. Background Art

[0002] Optical sensing arrays have been widely used due to their advantages such as low cost, simple operation, fast response speed, and rich information. Optical sensing arrays are usually measured by fluorescence and colorimetric methods, which convert the interaction between the sensing unit and the analyte into a fluorescence signal or a color signal output. The types of such interactions include van der Waals forces, hydrogen bonds, charge transfer, ionic bonds, covalent bonds, coordination bonds, acid-base interactions, and so on. This multi-type of interaction provides more selectivity for the design and construction of sensing unit materials. Compared with electronic noses / electronic tongues that only rely on simple physical adsorption or van der Waals forces, they can provide stronger sensitivity for the analysis of analytes and better discrimination ability for complex matrices, and have become a powerful tool for analyzing and detecting food quality. They identify different target analytes by obtaining unique color fingerprints through the color difference value before and after the array reaction.

[0003] Currently, the detection materials in most sensing arrays are mainly optical nanomaterials such as quantum dots, nano-gold and silver, etc. They often have defects such as relatively high toxicity, difficulty in ensuring the stability of multi-batch synthesis, and easy aggregation and morphological changes during storage, which greatly limit the development of actual detection applications. In addition, another part of the sensing materials often involves complex synthesis methods, such as organic light-emitting molecules, etc., which have disadvantages such as cumbersome production processes and high costs.

[0004] Currently, the design of fluorescent probes or dyes for aldehyde and ketone compounds mostly relies on the interaction between amino groups and aldehydes and ketones. Among them, hydroxylamine, as an inexpensive and easily available reagent, can act as a nucleophile to undergo a nucleophilic addition reaction with aldehydes or ketones to form oximes. Therefore, the re-development, design and utilization of hydroxylamine reagents have good application prospects in the visual detection of aldehyde and ketone compounds and the identification of complex food matrices. The specific reaction between aldehydes and ketones and hydroxylamine hydrochloride releases hydrochloric acid after the reaction, which changes the pH value in the system and thus induces a change in the color of the indicator. This reaction is stable, simple, fast and can be used for real-time detection without complex synthesis methods. However, this analysis method has not achieved the detection of aldehyde and ketone compounds and their identification in complex systems. Summary of the Invention

[0005] In order to solve the defects existing in the above-mentioned array sensors, the present invention provides a colorimetric array sensor constructed by combining hydroxylamine hydrochloride with a pH indicator. The detection materials in the sensing array do not need to go through a complex synthesis process. Each sensing unit is composed of a hydroxylamine hydrochloride and a pH indicator, and can achieve an instant response to aldehyde and ketone compounds within 3 minutes, and the effect is obvious.

[0006] The technical solution adopted by the present invention to solve the above-mentioned problems is as follows:

[0007] As a first aspect, a colorimetric array sensor constructed by combining hydroxylamine hydrochloride with a pH indicator is provided. The array sensor is composed of M×N sensing units, where M is the number of hydroxylamine hydrochlorides and N is the number of pH indicators. Each sensing unit is sequentially added with hydroxylamine hydrochloride, the analyte, and a pH indicator.

[0008] Furthermore, the concentration of the hydroxylamine hydrochloride solution is 0.0005 - 0.25 mmol / L, and the concentration of the pH indicator is 0.05 - 5 mmol / L. The concentrations of both can be appropriately adjusted according to the concentration of the analyte compound; the volumes of the hydroxylamine hydrochloride solution and the pH indicator added to each sensing unit are the same.

[0009] Preferably, the array sensor is composed of 12 sensing units. Each sensing unit is respectively hydroxylamine hydrochloride (HA-1) + congo red (CR), hydroxylamine hydrochloride (HA-1) + methyl orange (MO), hydroxylamine hydrochloride (HA-1) + brilliant yellow (BY), hydroxylamine hydrochloride (HA-1) + solvent yellow 2 (SY), N-benzylhydroxylamine hydrochloride (HA-2) + congo red (CR), N-benzylhydroxylamine hydrochloride (HA-2) + methyl orange (MO), N-benzylhydroxylamine hydrochloride (HA-2) + brilliant yellow (BY), N-benzylhydroxylamine hydrochloride (HA-2) + solvent yellow 2 (SY), O-butylhydroxylamine hydrochloride (HA-3) + congo red (CR), O-butylhydroxylamine hydrochloride (HA-3) + methyl orange (MO), O-butylhydroxylamine hydrochloride (HA-3) + brilliant yellow (BY), O-butylhydroxylamine hydrochloride (HA-3) + solvent yellow 2 (SY). Each sensing point is a mixed solution of the two.

[0010] As a second aspect, a method for identifying compounds based on the above-mentioned colorimetric array sensor constructed by combining hydroxylamine hydrochloride with a pH indicator is provided, including the following steps: extracting the R, G, B differences of the sensor units added with the analyte and the control, as features, and analyzing by chemometric methods for the identification of different compounds.

[0011] As a third aspect, there is provided an application of a colorimetric array sensor constructed based on the above-mentioned hydroxylamine hydrochloride combined with a pH indicator in the identification of aldehydes and ketones compounds. Specifically, it is used for identifying aldehyde and ketone organic small molecules, aldehyde and ketone mixed compounds, samples rich in such small molecules, etc.

[0012] Preferably, the application method is as follows: First, add hydroxylamine hydrochloride solution, ethanol solution, and pH indicator as a control to each well in the first row of a 96-well plate in sequence. Replace the ethanol solution in each row below the first row with different concentrations of aldehyde and ketone compound solutions prepared with the same volume of ethanol. The order of adding samples is the same as the order of the solutions in the control. Among them, equal volumes, equal concentrations, and the same type of hydroxylamine hydrochloride and pH indicator are added to each column. The final concentrations of the aldehyde and ketone compounds prepared in each row in the system are the same. After adding the samples, mix and react at room temperature for 2 - 10 min; by obtaining the R, G, B values of each analyte (aldehyde and ketone compounds) and its control (in the same column in the first row), the corresponding R, G, B differences are obtained. As can be seen from the above solution, the sensing array with M*N channels can form M*N*3 characteristic values, and each concentration and each organic small molecule has its corresponding M*N*3 characteristic values. Thus, the compounds are discriminated by chemometric methods such as hierarchical cluster analysis, so as to achieve the distinction of different aldehyde and ketone compounds and further identify the types, origins, and brands of complex samples rich in such compounds.

[0013] According to the above solution, the concentrations of all aldehyde and ketone compounds need to be diluted with ethanol to 0.25 - 25 mmol / L (final concentration in the system).

[0014] According to the above solution, place the 96-well plate on a shadowless bottom lamp, fix the camera on a tripod and align the lens with the 96-well plate. When the reaction time is reached, use the camera to record the image for later processing of the image in Photoshop and extraction of the R, G, B values. The above-mentioned photographing methods are all carried out in a darkroom to minimize the influence of ambient light on the acquisition of the fingerprint spectrum.

[0015] As a fourth aspect, there is provided an application of an array sensor constructed based on the above-mentioned hydroxylamine hydrochloride combined with a pH indicator in the identification of Chinese Baijiu.

[0016] Preferably, the application method is as follows: Take a 96-well plate. Add hydroxylamine hydrochloride solution, ethanol solution and pH indicator into each well in the first row in sequence as a control. Replace the ethanol solution in each row below the first row with different types of Chinese liquor samples of the same volume. The adding order of the samples is the same as that of the solutions in the control. Among them, the volume, concentration and type of hydroxylamine hydrochloride and pH indicator added in each column are the same, and the types of Chinese liquor samples in each row are the same. After adding the samples, mix and react at room temperature for 2 - 10 min; By obtaining the R, G, B values between the reaction wells where Chinese liquor is located and the controls in the same column in the first row, the corresponding R, G, B differences are obtained; In the M*N channel sensing array, each type of liquor has its corresponding M*N*3 characteristic values. Analyze different types of Chinese liquor through principal component analysis or linear discriminant analysis to achieve the identification of liquor samples; Among them, the Chinese liquor samples do not need to be further diluted, and the original liquor samples are used.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The present invention constructs an array sensor constructed by combining hydroxylamine hydrochloride with a pH indicator. Compared with traditional sensors, this sensor does not need to synthesize sensing materials or molecules in advance, and only realizes an instant color reaction at room temperature. It has many advantages such as low cost, simple operation, fast and efficient, and easy to realize on-line detection. It can be used for the rapid identification of various aldehydes and ketones and complex samples rich in such small molecules. Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the method for constructing a novel array sensor by combining hydroxylamine hydrochloride with a pH indicator according to the present invention;

[0020] Figure 2 It is a color difference diagram of the array sensor for 20 aldehyde and ketone compounds;

[0021] Figure 3 It is a HCA dendrogram of 20 aldehydes and ketones;

[0022] Figure 4 It is a fingerprint map of the array sensor for 27 Chinese strong-flavor liquors;

[0023] Figure 5 It is the linear discriminant analysis result of the array sensor for classifying 27 strong-flavor liquors by grade. Detailed Embodiments

[0024] The following embodiments facilitate a better understanding of the present invention, but do not limit the present invention. The following takes aldehyde and ketone compounds and Chinese strong-flavor liquors from different origins and brands as examples, and further elaborates on the technical solutions of the present invention in conjunction with the drawings.

[0025] The array sensor constructed in the present invention is used to detect aldehyde and ketone compounds. Each individual sensing unit does not need to have a high selectivity for the target analyte, but has different response capabilities to different analytes. By utilizing the differences in the overall responses of the sensing array to various aldehyde and ketone compounds, the differentiation of different aldehyde and ketone compounds can be achieved. The detection mechanism lies in that due to the specific nucleophilic addition reaction between aldehydes and ketones and hydroxylamine hydrochloride, the hydrochloric acid released after the reaction changes the pH value in the system, thereby inducing a change in the color of the indicator. Since the reaction capabilities of each aldehyde and ketone with hydroxylamine hydrochloride are different and the amounts of hydrochloric acid released are different, differential color fingerprints can be achieved. By analyzing the differential color fingerprints through chemometrics, more accurate differentiation and identification of multiple compounds can be realized.

[0026] As Figure 1 shown, first, a 3×4 colorimetric sensing array is constructed by using 3 kinds of hydroxylamine hydrochloride and 4 kinds of pH indicators. Secondly, after adding hydroxylamine hydrochloride, aldehyde and ketone compounds, and pH indicators into a 96-well plate and reacting for 4 min, the fingerprint pattern corresponding to each compound is recorded with a camera. The image is imported into an image processing software such as Photoshop to extract the R, G, B values of each reaction well point. Then, hierarchical cluster analysis (HCA) can be carried out by using the R, G, B differences between the control and the addition of aldehyde and ketone organic compounds. Introducing chemometric methods helps to obtain more accurate analysis results. Finally, the array sensor is used to identify actual complex samples rich in such compounds, and the operation method is the same as that shown in the previous steps.

[0027] Example 1:

[0028] The array sensor constructed by the present invention can be used to distinguish 20 different concentrations of aldehyde and ketone compounds. The sensing array constructed by the present invention consists of 12 sensing units, and each sensing unit is HA-1 (0.05 mol / L, 45 μL) + CR (2.5 mmol / L, 50 μL), HA-1 (0.05 mol / L, 45 μL) + MO (1 mmol / L, 50 μL), HA-1 (0.05 mol / L, 45 μL) + BY (5 mmol / L, 50 μL), HA-1 (0.05 mol / L, 45 μL) + SY (1 mmol / L, 50 μL), HA-2 (0.05 mol / L, 45 μL) + CR (2.5 mmol / L, 50 μL), HA-2 (0.25 mol / L, 45 μL) + MO (2.5 mmol / L, 50 μL), HA-2 (0.05 mol / L, 45 μL, 45 μL) + BY (5 mmol / L, 50 μL), HA-2 (0.05 mol / L, 45 μL) + SY (1 mmol / L, 50 μL), HA-3 (0.05 mol / L, 45 μL) + CR (2.5 mmol / L, 50 μL), HA-3 (0.05 mol / L, 45 μL) + MO (0.5 mmol / L, 50 μL), HA-3 (0.05 mol / L, 45 μL) + BY (5 mmol / L, 50 μL), HA-3 (0.05 mol / L, 45 μL) + SY (1 mmol / L, 50 μL). 5 μL of absolute ethanol is added to the control well, and 5 μL of aldehyde and ketone compounds with different concentrations or types are added to the remaining reaction wells to ensure that the total volume in each reaction well spot is 100 μL. The concentrations of hydroxylamine hydrochloride and pH indicator at this place are the concentrations before addition.

[0029] The aldehyde and ketone compounds identified in this example include: acetaldehyde (AH1), valeraldehyde (AH2), heptaldehyde (AH3), isobutyraldehyde (AH4), furfural (AH5), 5-methylfurfural (AH6), cinnamaldehyde (AH7), benzaldehyde (AH8), phenylpropionaldehyde (AH9), vanillin (AH10), acetal (AH11), 2-pentanone (KT1), 2,3-butanedione (KT2), 2,3-pentanedione (KT3), 3-methyl-2-butanone (KT4), 3-hydroxy-2-butanone (KT5), acetylacetone (KT6), cyclohexanone (KT7), acetophenone (KT8), 4-(4-hydroxyphenyl)-2-butanone (KT9). Each aldehyde and ketone has 9 concentrations, and the final concentration range in the detection system is 0.25 mmol / L - 25 mmol / L. The specific concentrations are as Figure 2 shown (0.25, 1.25, 2.5, 4, 6, 7.5, 10, 12.5, 17.5, 25 mmol / L respectively).

[0030] The order of adding samples into each reaction well is hydroxylamine hydrochloride, ethanol or aldehyde / ketone compound, and pH indicator. To control the influence of sample addition time on the reaction, all samples of the same type are added using an Eppendorf multi-channel pipette. After adding the pH indicator, mix it evenly at room temperature and react for 4 min. Then, place the 96-well plate on a shadowless bottom lamp and take pictures with a single-lens reflex camera in a darkroom, that is, record the fingerprint spectra of each aldehyde / ketone, as Figure 2 shown. Each compound is repeated with 3 parallel experiments. It can be seen from the figure that the array sensor has different responses to different aldehyde / ketone compounds only by the method of visual discrimination. The pictures taken are used to extract the R, G, B values through Photoshop to obtain the R, G, B differences under each aldehyde / ketone, and then hierarchical clustering analysis (HCA) is performed through Matlab R2018b.

[0031] The results of hierarchical clustering analysis for each concentration of 20 aldehyde / ketone compounds are as Figure 3 shown. Each individual aldehyde or ketone in each small category is well classified. Secondly, multiple aldehydes and multiple ketones are well clustered together. This may be due to the smaller steric hindrance of the aldehyde carbonyl group and the greater electron cloud density on the carbon of the ketone carbonyl group, which jointly result in the higher reactivity of aldehydes than ketones. It is worth mentioning that acetaldehyde is classified into the large category of ketones, while 2,3-butanedione and 2,3-pentanedione are classified into the large category of aldehydes. This may be because the 3 hydrogen atoms on the methyl group in acetaldehyde reduce the reactivity of the carbonyl group with hydroxylamine through the σ-π hyperconjugation effect with the carbonyl group, thus making it have similar properties to ketone compounds. While 2,3-butanedione and 2,3-pentanedione have stronger reaction efficiency due to having multiple reaction sites, and thus show more similar properties to aldehyde compounds.

[0032] Example 2:

[0033] The array sensor constructed by the present invention can, on the basis of identifying aldehyde / ketone compounds, also realize the identification of different types of Chinese strong-flavor liquor samples. Liquor contains rich flavor substances, including aldehyde / ketone flavor substances. The content and proportion of aldehyde / ketone compounds contained in each liquor are different. Aldehydes and ketones play a very important role in the aroma coordination of liquor, especially in strong-flavor liquor. And strong-flavor liquor is a kind of liquor loved by the majority of consumers with a market share of more than 70%. Therefore, 27 different brands and grades of strong-flavor liquors from three production areas of Sichuan, Jiangsu, and Inner Mongolia are selected as the detection objects, including 6 kinds of Luzhou Laojiao, 7 kinds of Wuliangye, 8 kinds of Yanghe, and 6 kinds of Yili liquor. Their specific information is shown in Table 1.

[0034] Table 1 Detailed information of 27 kinds of strong-flavor liquors

[0035]

[0036]

[0037] The method and steps for identifying liquor by the array are the same as those for identifying aldehyde and ketone compounds in Example 1. It is only necessary to replace the liquor of the same volume with organic compounds (the liquor does not need to be diluted, and the original liquor sample can be directly detected). The fingerprint spectrum of liquor identified by the array sensor is as follows Figure 4 As shown in the color difference spectrum, it can be found that the array has a similar color rendering effect on each brand of liquor, and the color rendering effect on Wuliangye liquor is the most obvious, followed by Yanghe, Yili, and Luzhou Laojiao is the weakest, indicating that the aldehyde and ketone compound content of different brands of Luzhou-flavor liquor can be preliminarily evaluated by naked eye observation alone, and the overall content is ranked as follows: Wuliangye>Yanghe>Yili>Luzhou Laojiao.

[0038] In order to obtain a more accurate quality distinction between different grades of liquor, the LDA model was introduced for analysis. The LDA discrimination results showed that when Luzhou Laojiao, Wuliangye, Yanghe and Yili were divided into 6, 7, 8 and 6 categories respectively, there was no overlap between each type of liquor, and the discrimination accuracy was higher than 96% ( Figure 5 ). It shows that the colorimetric sensor array based on aldehyde and ketone specificity can realize preliminary visual evaluation of the aldehyde and ketone content in different brands of Luzhou-flavor liquor, and accurately distinguish different grades of Luzhou-flavor liquor of the same brand under the chemometric mode, which proves its excellent performance in the quality classification of Luzhou-flavor liquor.

[0039] The embodiments of the present invention are described in detail above, but the present invention is not limited to the above embodiments. In addition, all equivalent changes and modifications made without departing from the method and scope of the present invention should be included in the scope of the present invention.

Claims

1. A colorimetric array sensor constructed by combining hydroxylamine hydrochloride with a pH indicator, characterized in that: The array sensor is composed of M*N sensing units, where M is the number of hydroxylamine hydrochloride and N is the number of pH indicators. Hydroxylamine hydrochloride, an object to be detected and a pH indicator are sequentially added to each sensing unit.

2. The colorimetric array sensor constructed by combining hydroxylamine hydrochloride with a pH indicator according to claim 1, characterized in that: The concentration of the hydroxylamine hydrochloride solution is 0.0005-0.25 mmol / L, and the concentration of the pH indicator is 0.05-5 mmol / L; the volumes of the hydroxylamine hydrochloride solution and the pH indicator added to each sensing unit are the same.

3. The colorimetric array sensor constructed by combining hydroxylamine hydrochloride with a pH indicator according to claim 2, characterized in that: The array sensor is composed of 12 sensing units, each of which is hydroxylamine hydrochloride + Congo red, hydroxylamine hydrochloride + methyl orange, hydroxylamine hydrochloride + brilliant yellow, hydroxylamine hydrochloride + solvent yellow 2, N-benzylhydroxylamine hydrochloride + Congo red, N-benzylhydroxylamine hydrochloride + methyl orange, N-benzylhydroxylamine hydrochloride + brilliant yellow, N-benzylhydroxylamine hydrochloride + solvent yellow 2, O-butylhydroxylamine hydrochloride + Congo red, O-butylhydroxylamine hydrochloride + methyl orange, O-butylhydroxylamine hydrochloride + brilliant yellow, O-butylhydroxylamine hydrochloride + solvent yellow 2, and each sensing point is a mixed solution of the two.

4. A compound identification method based on a colorimetric array sensor constructed by combining hydroxylamine hydrochloride with a pH indicator according to any one of claims 1 to 3, characterized in that: The R, G, and B differences of the sensor units with the test substance added and the control substance added are extracted as features and analyzed by chemometric methods to identify different compounds.

5. Use of the array sensor according to any one of claims 1 to 3 in identifying aldehyde and ketone compounds.

6. The use according to claim 5, characterized in that: The specific application method is as follows: take a 96-well plate, add hydroxylamine hydrochloride solution, ethanol solution and pH indicator to each well in the first row as a control, replace the ethanol solution in each row below the first row with aldehyde and ketone compound solutions of different concentrations prepared with the same volume of ethanol, and the order of adding samples is the same as the order of solutions in the control, wherein the volume, concentration and type of hydroxylamine hydrochloride and pH indicator added to each column are the same, and the final concentration of aldehyde and ketone compounds prepared in each row is the same in the system, and after the addition is completed, mix and react for 2-10 minutes at room temperature; obtain the R, G, B values ​​of the control of the reaction wells where the aldehyde and ketone compounds are located and the same column as the first row, and obtain the corresponding R, G, B difference values. In the sensor array of the M*N channel, each concentration and each aldehyde and ketone compound has its corresponding M*N*3 eigenvalues; distinguish different aldehyde and ketone compounds by hierarchical cluster analysis to achieve qualitative detection of different aldehyde and ketone compounds.

7. The use according to claim 5, characterized in that: The final concentration of all aldehyde and ketone compounds in the system is 0.25-25 mmol / L.

8. Use of the array sensor according to claims 1-3 in identifying Chinese liquor.

9. The use according to claim 8, characterized in that: The application method is as follows: a 96-well plate is taken, and hydroxylamine hydrochloride solution, ethanol solution and pH indicator are added to each well in the first row in sequence as a control, and the ethanol solution in each row below the first row is replaced by Chinese liquor samples of the same volume but different types, and the order of adding samples is the same as the order of solutions in the control, wherein the volume, concentration and type of hydroxylamine hydrochloride and pH indicator added to each column are the same, and the type of Chinese liquor samples in each row is the same. After the addition is completed, the mixture is reacted for 2-10 minutes at room temperature; by obtaining the R, G, B values ​​between the reaction well where the Chinese liquor is located and the control in the same column of the first row, the corresponding R, G, B difference values ​​are obtained; in the sensor array of the M*N channels, each type of liquor has its corresponding M*N*3 eigenvalues, and Chinese liquors from different types are analyzed by principal component analysis or linear discriminant analysis to achieve identification of liquor samples; wherein the Chinese liquor samples do not need to be further diluted, and the original liquor samples are used.

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