A method for reducing saliva viscosity and its application

Abstract

The invention belongs to the field of biochemical technology, and provides a method for reducing saliva viscosity and an application thereof. A disulfide bond reducing agent or an aluminum salt and an alkaline solution are mixed with saliva to quickly reduce the saliva viscosity. The flow rate of the treated saliva is not less than 2.1 mm / min. The method of the invention can be applied to paper-based microfluidic detection to detect the components of saliva, so that the saliva component detection speed is fast, the efficiency is high, and the result is accurate. The defect of the existing saliva component detection method that relies on the detection equipment is overcome, and the detection efficiency and accuracy are avoided to be affected by the excessively long detection time and the large individual differences. After being treated with the method of the invention, the relative standard deviation of the flow length of different individual samples in the same time is reduced to 45%-85% of the untreated ones.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry, and more specifically, to a method for reducing saliva viscosity and application thereof. Background Art

[0002] Saliva is a mixture of multiple components including DNA, RNA, fatty acids, and microorganisms. Changes in the protein level in the blood will be reflected in the protein content of saliva. Therefore, saliva contains a variety of biomarkers that can reflect the health level of an individual. At the same time, the advantages of non-invasive and easy access to saliva sampling make saliva a highly potential and application-valued instant detection sample. At present, there are many methods for biomolecular analysis and monitoring of saliva, such as high-performance liquid chromatography, surface-enhanced Raman spectroscopy, gas chromatography-mass spectrometry, enzyme colorimetry, etc., but these methods have more or less some defects, such as: high-performance liquid chromatography and gas chromatography-mass spectrometry require complex sample pretreatment processes, surface-enhanced Raman spectroscopy and gas chromatography-mass spectrometry require precision measuring instruments, and are difficult to apply in real-time scenarios outside the laboratory. Enzyme colorimetry is expensive and time-consuming. Therefore, the detection of saliva components requires the development of an instant detection tool that is simpler to operate, more portable, and less expensive.

[0003] Paper-based microfluidic detection refers to the use of different methods to form patterned hydrophobic walls in paper, so that microchannels can be made on paper, which can then be used for the analysis and detection of various biochemical reactions. This new microfluidic analysis method is called paper-based microfluidic detection. Since the chip used in paper-based microfluidics has low processing cost, simple operation, and portable device, it is particularly suitable for instant diagnosis, environmental monitoring and other scenarios, and has attracted widespread attention in the field of microfluidics in recent years.

[0004] At present, paper-based microfluidic chips have been proven to be applicable to saliva detection. The concentrations of glucose, nitrite, uric acid, viruses, banned drugs, harmful ions and other components in saliva have been proven to be detectable by paper-based microfluidic chips. Based on the colorimetric principle, de Castro et al. developed a wearable paper-based microfluidic device (colorimetric method) that can simultaneously detect glucose and nitrite in saliva; Huang et al. integrated composite material electrodes into paper-based microfluidic chips to achieve the detection of uric acid in saliva; Zangmo et al. developed a paper-based adsorption device wrapped with selenium nanoparticles to capture and analyze mercury in saliva samples. The color of the adsorbent changes with the increase of mercury ion concentration, and the mercury ion concentration can be accurately determined by inductively coupled plasma mass spectrometry (ICP-MS) (electrochemical method). The existing methods for detecting saliva components on paper-based microfluidic chips mostly use colorimetric methods, electrochemical methods, etc. Although the above methods can realize the detection of saliva components on paper-based microfluidic chips, most of them require additional equipment for auxiliary concentration detection, and the operation steps are relatively cumbersome.

[0005] Recently, a method for detecting the concentration of substances on a paper-based microfluidic chip based on the difference in flow length has emerged. The principle is that after the detection substrate reacts chemically with a specific substance in the reaction area of ​​the paper-based microfluidic chip, the reaction product flows into the microfluidic channel with the mixed solution and reacts with the color developer on the channel. After the solution flows, a color development segment of a certain length can be observed in the channel, and then the content of the reaction product and the detection object can be determined. The device is simple and easy to operate. However, the paper-based microfluidic chip is a porous medium. Since there is a negative correlation between the flow velocity and viscosity of the fluid on the porous medium, saliva is a high-viscosity fluid. The time required to flow the same length on the paper-based microfluidic chip increases significantly, resulting in a long detection time. The long detection time will cause the liquid to evaporate, the object to be detected to react with external components, etc. In addition, due to the large individual differences in saliva viscosity, the flow velocity varies greatly. The above uncontrollable factors affect the detection efficiency and may also interfere with the detection results. Anirudh et al. measured that the average time required for five saliva samples to flow 5 mm on a paper-based microfluidic chip at room temperature was about 2-3 times the flow time of pure water under the same conditions, and there were problems of large differences in flow speed and slow flow speed. Therefore, it is urgent to solve the problem that saliva flows slowly in porous media, is greatly affected by individual differences, and has large differences in flow speed. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a method for reducing saliva viscosity and its application, which can be applied to increase the flow rate of saliva in porous media by mixing saliva with a disulfide bond reducing agent or an aluminum salt and an alkaline solution. The method of the present invention is applied to paper-based microfluidics to detect saliva components, which can improve the detection efficiency, realize rapid detection of saliva, and avoid being affected by individual differences. The flow rate of the sample treated by the method of the present invention is not less than 2.1 mm / min and can reach 15 mm / min. The relative standard deviation of the flow length of the sample treated by the method of the present invention is reduced to 45%-85% of that before treatment.

[0007] A first aspect of the present invention provides a method for reducing saliva viscosity.

[0008] Specifically, a method for reducing saliva viscosity comprises the following steps:

[0009] A chemical is mixed with the saliva, wherein the chemical is selected from at least one of a disulfide bond reducing agent, an aluminum salt, or an alkaline solution.

[0010] Mucin is the main source of saliva viscosity. The present invention utilizes chemicals to react with saliva, and adopts a disulfide bond reducing agent as a chemical, or adopts aluminum salt and alkaline solution as chemicals. The sulfhydryl group or other effective ingredients in the disulfide bond reducing agent can reduce the polypeptide or protein in the saliva to denature it, and the mixture of aluminum salt and alkaline solution will produce Al(OH)3 precipitation. During the precipitation, aluminum ions will settle together with mucin in the saliva. The above two chemicals can denature or settle mucin in the saliva, thereby reducing the viscosity of the saliva. The method for reducing the viscosity of the saliva of the present invention can be applied to increasing the flow rate of saliva in a porous medium, for example, in the paper-based microfluidic detection of saliva components, the flow rate of saliva on the porous medium is significantly improved, thereby improving the efficiency of paper-based microfluidic detection.

[0011] Preferably, the disulfide bond reducing agent is one or more of mercaptoethanol, dithiothreitol (DTE), dithioerythritol, and tris(hydroxymethylaminomethane)phosphine. The disulfide bond reducing agent can undergo redox reaction with the disulfide bonds in the polypeptide or protein, thereby reducing the disulfide bonds and denaturing the polypeptide or protein.

[0012] More preferably, the disulfide bond reducing agent is dithioerythritol.

[0013] Preferably, the dithioerythritol is mixed with saliva in the form of solid powder or in the form of dithioerythritol solution.

[0014] Preferably, the concentration of the dithioerythritol solution is 10-210 mg / mL.

[0015] More preferably, the concentration of the dithioerythritol solution is 50-200 mg / mL.

[0016] More preferably, the concentration of dithioerythritol is 200 mg / mL.

[0017] Preferably, the aluminum salt is aluminum sulfate, and the alkaline solution is sodium hydroxide solution.

[0018] Preferably, the aluminum salt is mixed with the saliva simultaneously with the alkaline solution in the form of an aluminum salt solution, and the concentration of the aluminum salt solution is 10-110 mg / mL.

[0019] More preferably, the concentration of the aluminum salt solution is 50-100 mg / mL.

[0020] Further preferably, the concentration of the aluminum salt solution is 50 mg / mL.

[0021] Preferably, the molar concentration of aluminum ions in the aluminum salt is three times that of hydroxide ions in the alkaline solution.

[0022] A second aspect of the present invention provides an application of the method for reducing saliva viscosity.

[0023] The present invention protects the application of the above method for reducing saliva viscosity in increasing the flow rate of saliva in a porous medium.

[0024] Preferably, when the flow region is a straight channel with a width of 1 mm and the flow driving force is the hydrostatic pressure, the flow velocity of the saliva in the porous medium is not less than 2.1 mm / min.

[0025] The present invention protects the application of the above method for reducing saliva viscosity in paper-based microfluidic detection of saliva components.

[0026] Preferably, the components of the saliva include one or more of water, drugs, hormones, proteins, nucleic acids, antibodies, glucose, nitrites, uric acid, and harmful ions. Since DTE can reduce disulfide bonds in proteins, the detection method using DTE as a chemical to reduce saliva viscosity is not suitable for detecting protein components in saliva or when protein reagents such as enzymes are required in the detection method.

[0027] Preferably, the disulfide bond reducing agent is pre-placed on the reaction area of ​​the paper-based microfluidic chip, and then the saliva sample is added to the reaction area for detection.

[0028] Preferably, the disulfide bond reducing agent is mixed with the saliva sample and then dripped into the reaction area of ​​the paper-based microfluidic chip for detection.

[0029] Preferably, the aluminum salt and alkaline solution are mixed with the saliva sample and then dripped into the reaction area of ​​the paper-based microfluidic chip for detection.

[0030] Preferably, the method for preparing the paper-based microfluidic chip comprises the following steps:

[0031] A paper-based microfluidic chip is produced by drawing a channel pattern on a porous medium material using a hydrophobic material.

[0032] Preferably, the porous medium material is a paper-based material.

[0033] More preferably, the porous medium material is filter paper.

[0034] Preferably, the hydrophobic material is a patternable hydrophobic material.

[0035] More preferably, the hydrophobic material is wax and / or polydimethylsiloxane (PDMS).

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] (1) The present invention uses a disulfide bond reducing agent or an aluminum salt and an alkaline solution to mix with saliva to denature or precipitate the mucin in the saliva, thereby quickly reducing the viscosity of the saliva, greatly accelerating the flow rate of the saliva on the porous medium, and the flow rate is not less than 2.1 mm / min, and can reach 15 mm / min. The method of reducing the viscosity of saliva of the present invention can be applied to paper-based microfluidic detection. By reducing the viscosity of saliva, the flow rate of saliva in the porous medium material is improved, and the flow length of saliva in the paper-based microfluidic chip channel per unit time is significantly increased, thereby shortening the detection time, improving the length-based saliva detection efficiency, and minimizing the amount of saliva. The influence of external factors on the test results is reduced. For samples with large differences in saliva viscosity collected from different individuals at different times, the relative standard deviation of the flow length within the same time is reduced to reduce the measurement error caused by the difference in flow speed, so that the length-based saliva component detection speed is faster, more efficient, and the results are more accurate. The defect of existing saliva component detection methods (such as colorimetry, etc.) that they rely on detection equipment is overcome, and the detection efficiency and accuracy are avoided to be affected by long collection time and individual differences. After being treated with the method of the present invention, the relative standard deviation of the flow length of samples from different individuals is reduced to 45%-85% of that before treatment.

[0038] (2) The present invention applies the method of reducing saliva viscosity to the detection of saliva components by paper-based microfluidics. The added form of chemical drugs can be selected according to the specific components to be detected and the detection principle to meet the requirements of different paper-based microfluidic chip detection methods. The disulfide bond reducing agent can be directly pre-set in the reaction area of ​​the paper-based microfluidic chip, or the saliva sample can be pre-treated with the disulfide bond reducing agent or aluminum salt and alkaline solution before detection. The sample processing time in the early stage of detection does not exceed three minutes, and the operation is simple and fast. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A schematic diagram of a paper-based microfluidic chip used in Application Example 1 of the present invention;

[0040] Figure 2 It is the technical roadmap of Application Example 1 and Application Example 2 of the present invention;

[0041] Figure 3 This is the technical roadmap of Application Example 3 of the present invention;

[0042] Figure 4 The flow velocity diagram of pure water on the microfluidic chip is shown in Application Example 8 of the present invention, Comparative Example 1, and FIG.

[0043] Figure 5 The flow velocity diagram of pure water on the microfluidic chip is shown in Application Example 2 of the present invention, Comparative Example 1, and FIG.

[0044] Figure 6Flow diagrams of Application Example 4, Application Example 5, and Application Example 1 of the present invention on a paper-based microfluidic chip;

[0045] Figure 7 Flow diagrams of Application Example 6, Application Example 2, and Application Example 7 of the present invention on a paper-based microfluidic chip;

[0046] Figure 8 A schematic diagram of the improvement effect of the method for reducing saliva viscosity of the present invention on the difference in flow speed of saliva of different individuals on a paper-based microfluidic chip;

[0047] Fig. 9 This is a diagram showing the processing of saliva samples from different individuals using the method of Application Example 2 of the present invention;

[0048] Fig.10 This is a relative standard deviation diagram of saliva samples from different individuals processed by the method of Application Example 2 of the present invention. DETAILED DESCRIPTION

[0049] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0050] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0051] Example 1

[0052] A method for reducing saliva viscosity comprises the following steps: mixing 200 mg / mL dithioerythritol solution with saliva.

[0053] Example 2

[0054] A method for reducing saliva viscosity comprises the following steps: mixing an aluminum sulfate solution and a sodium hydroxide solution with saliva, wherein the concentration of aluminum ions in the aluminum sulfate solution is 50 mg / mL.

[0055] Application Example 1

[0056] The method for reducing saliva viscosity in Example 1 is applied to paper-based microfluidic detection, comprising the following steps:

[0057] A 200 mg / mL dithioerythritol solution was mixed with saliva and dripped onto a paper-based microfluidic chip to observe its flow. The paper-based microfluidic chip used in the present invention is as follows: Figure 1 As shown, the paper-based microfluidic chip includes a sample reaction area (100), a hydrophobic channel (200), and a color development area (300).

[0058] Application Example 2

[0059] The method for reducing saliva viscosity of Example 2 is applied to paper-based microfluidic detection, comprising the following steps:

[0060] Prepare Al2(SO4)3 and NaOH solutions respectively, and ensure that Al 3+ The molar concentration of OH - Take an appropriate amount of saliva sample to be tested, add equal volumes of Al2(SO4)3 and NaOH solution to produce Al(OH)3 precipitation in the mixed solution, and centrifuge the mixed solution. 3+ It settles together with mucin, and the solution after centrifugation is taken and then subjected to paper-based microfluidic detection. Its flow is observed on the paper chip and a color reaction occurs.

[0061] The technical roadmap of the method for pre-treating saliva to reduce saliva viscosity in the above application examples 1 and 2 is as follows: Figure 2 As shown, a saliva collector is first used to collect saliva samples. The specific collection method is as follows: rinse the mouth with drinking water until there is no food residue or other debris in the mouth, and use the tip of the tongue to press against the upper or lower jaw tooth root to enrich saliva; gently spit saliva into the collection funnel until the liquid height reaches the scale line; hold the saliva collection tube upright, remove the collection funnel and tighten the tube cover. The saliva source is healthy people with no previous medical history. The sample collection time of each experiment is consistent, and it is fresh saliva collected on the same day. Then, the saliva sample collected by the above method is added to the DTE solution or the alkaline solution containing aluminum ions to obtain a treated saliva sample, and the treated saliva sample is dripped on the paper-based microfluidic chip, and finally a color reaction occurs on the paper-based microfluidic chip.

[0062] The time for the preliminary sample processing in Application Example 1 and Application Example 2 does not exceed 3 minutes. The method of the present invention can simply and quickly process the sample and then perform subsequent detection.

[0063] Application Example 3

[0064] The method for reducing saliva viscosity in Example 1 is applied to paper-based microfluidic detection, comprising the following steps:

[0065] DTE is prepared into a 200 mg / mL solution, and the supernatant is added dropwise to the sample reaction area of ​​the paper-based microfluidic chip so that the reaction area is filled with the solution. After air drying, DTE is pre-set on the paper-based microfluidic chip. Then, the saliva sample is directly added dropwise to the sample reaction area of ​​the paper-based microfluidic chip. DTE can denature the mucin in the saliva, thereby reducing the viscosity of the saliva.

[0066] Application Example 3: Pre-setting DTE on a paper-based microfluidic chip to reduce saliva viscosity Figure 3As shown, a DTE solution is first pre-placed on the prepared paper-based microfluidic chip, and then a saliva sample is added, and a color reaction will occur.

[0067] Application Example 4

[0068] The difference between Application Example 4 and Application Example 1 is that the concentration of the DTE solution is replaced with 50 mg / mL.

[0069] Application Example 5

[0070] The difference between Application Example 5 and Application Example 1 is that the concentration of the DTE solution is replaced with 100 mg / mL.

[0071] Application Example 6

[0072] The difference between Application Example 6 and Application Example 2 is that the concentration of aluminum ions is replaced with 20 mg / mL.

[0073] Application Example 7

[0074] The difference between Application Example 7 and Application Example 2 is that the concentration of aluminum ions is replaced with 100 mg / mL.

[0075] Application Example 8

[0076] The difference between Application Example 8 and Application Example 1 is that the concentration of the DTE solution is replaced with 10 mg / mL.

[0077] Comparative Example 1

[0078] Comparative Example 1 is untreated saliva, which is different from Application Example 1 in that the saliva is not treated with the dithioerythritol solution.

[0079] Product effect testing

[0080] 1. Flow velocity on paper-based microfluidic chips

[0081] The untreated saliva and pure water of Comparative Example 1 were added to the paper-based microfluidic chip to observe their flow conditions, and then compared with the test results of Application Example 8. The results are as follows: Figure 4 As shown, the results show that after adding DTE in Application Example 8, the average flow rate of saliva in the first five minutes reaches 2.1 mm / min, which is greatly improved compared with the flow rate of 1.3 mm / min of the untreated saliva in Comparative Example 1.

[0082] The untreated saliva and pure water of Comparative Example 1 were added to the paper-based microfluidic chip to observe their flow conditions and test their flow speeds, and then compared with the test results of Application Example 2. The results are as follows: Figure 5As shown, the results show that after adding aluminum salt and alkaline solution in Application Example 2, the flow rate can reach 15 mm / min, which is greatly improved compared with the untreated saliva in Comparative Example 1 and is close to the flow rate of pure water.

[0083] The flow conditions of Application Examples 1, 4 and 5 on paper-based microfluidic chips are shown in Figure 2. Figure 6 As shown, the results show that the effect of reducing saliva viscosity by using DTE with a concentration of 200 mg / mL in Application Example 1 is the best.

[0084] The flow conditions of Application Examples 2, 6 and 7 on paper-based microfluidic chips are shown in Figure 2. Figure 7 As shown, the results show that the effect of reducing saliva viscosity using aluminum salt and alkaline solution with a concentration of 50 mg / mL in Application Example 2 is the best.

[0085] 2. Differences in the speed at which saliva from different individuals flows on the paper chip

[0086] like Figure 8 As shown, by using the method of the present invention, after saliva samples A, B, and C of different individuals are treated by the method of the present invention, not only can the saliva viscosity be reduced, thereby lengthening the flow length, but also the difference in flow speed of saliva of different individuals on the paper-based microfluidic chip can be reduced, and the difference in flow length within the same time is reduced.

[0087] To verify the results of the method of the present invention on reducing individual viscosity differences, three saliva samples A, B, and C were collected from different individuals, and treated with aluminum salt and alkaline solution using the method of Application Example 2. The differences in flow lengths within the same time were observed on a paper-based microfluidic chip. The results are as follows: Fig. 9 As shown in Figure 2, after treatment, not only did the saliva flow speed become faster, but the relative difference in flow length between samples also decreased. Fig.10 It can be seen that after treatment with aluminum salt and alkaline solution, the relative standard deviation of the flow length of the three saliva samples in the same time period is reduced to 45%-85% of that of the untreated samples, wherein the relative standard deviation of the sample flow length = standard deviation of the flow length of each sample / average value.

Claims

1. The method for reducing saliva viscosity is applied in paper-based microfluidic detection of saliva components, characterized in that: The method for reducing saliva viscosity comprises the following steps: mixing a chemical with the saliva, the chemical being a disulfide bond reducing agent, or the chemical being a combination of an aluminum salt and an alkaline solution; The saliva components include one or more of water, medicine, hormone, protein, nucleic acid, antibody, glucose, nitrite, uric acid, and harmful ions.

2. The application according to claim 1, characterized in that: The disulfide bond reducing agent is one or more of mercaptoethanol, dithiothreitol, dithioerythritol, and tris(hydroxymethyl)aminomethanephosphine.

3. The application according to claim 1, characterized in that: The aluminum salt is aluminum sulfate, and the alkaline solution is sodium hydroxide solution.

4. The use according to claim 1, characterized in that: The flow rate of the saliva in the porous medium is not less than 2.1 mm / min.

5. The use according to claim 1, characterized in that: The disulfide bond reducing agent is pre-placed on the reaction area of ​​the paper-based microfluidic chip, and then the saliva sample is added to the reaction area for in-situ detection.

6. The use according to claim 1, characterized in that: The disulfide bond reducing agent is mixed with the saliva sample and then dripped into the reaction area of ​​the paper-based microfluidic chip for detection.

7. The use according to claim 1, characterized in that: The aluminum salt and alkali solution are mixed with the saliva sample and then dripped into the reaction area of ​​the paper-based microfluidic chip for detection.

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