A paper-based microfluidic chip for dental plaque detection, a kit, a method for detecting dental plaque and application thereof

Abstract

The application provides a paper-based microfluidic chip, a kit and a method for detecting dental plaque. The paper-based microfluidic chip for detecting dental plaque comprises a paper-based material, a closed hydrophobic isolation belt made of a hydrophobic material is arranged on the paper-based material, and a detection area is formed in the hydrophobic isolation belt; the kit for detecting dental plaque based on the paper-based microfluidic chip comprises the paper-based microfluidic chip for detecting dental plaque, a dyeing agent and a flushing agent. The paper-based microfluidic chip for detecting dental plaque, the kit and the method for detecting dental plaque use paper as a basic material, build an in-vitro paper-based microfluidic dental plaque detection chip, utilize the filtering performance of the 3D porous structure of the paper fiber and the preferential selectivity of the dyeing agent to the paper fiber and dental plaque, determine the content of the dental plaque to be detected through the color area of the reactant, and can overcome the disadvantages of conventional in-vivo detection methods and in-vitro detection methods, and the operation process is simple and convenient, so that the detection method is a low-cost, rapid and environmentally-friendly detection method.

Description

Technical Field

[0001] This invention belongs to the field of paper-based microfluidic chip technology, specifically relating to a paper-based microfluidic chip, reagent kit, and method and application for dental plaque detection. Background Technology

[0002] Dental plaque is a typical bacterial biofilm, composed of microbial cells and extracellular polysaccharide matrix of Streptococcus, Lactobacillus, Actinomycetes and other genera. Various bacteria exist in the three-dimensional structure of the host and bacterial extracellular polymer matrix, adhering to or attaching to each other, and colonizing the tooth surface, between teeth or on the surface of restorations.

[0003] Dental plaque biofilm is the initiating factor for most oral diseases (such as periodontitis, gingivitis, tartar, and dental caries). Studies have shown that the occurrence of dental caries is determined by the balance of the overall microbial community within the dental plaque biofilm. When the microbial community is placed in a fermentable carbohydrate environment, acid-producing bacteria produce large amounts of acidic substances, lowering the pH of the oral environment. Once the pH falls below 5.5, it leads to enamel demineralization, ultimately resulting in dental caries. Furthermore, numerous studies have shown that bacteria and their metabolites within dental plaque can directly enter adjacent tissues or organs, or spread to distant organs throughout the body via damaged epithelial tissue, causing systemic infections of multiple organs or systems, such as cardiovascular disease, lung and bronchial infections, chronic gastritis, peptic ulcers, and obstetric infections in premature and low-birth-weight infants. Therefore, rapid detection of dental plaque is not only crucial for the prevention and treatment of oral diseases but also of great significance for maintaining overall health and reducing serious illnesses.

[0004] Currently, the commonly used method for detecting dental plaque involves staining the oral cavity with a plaque display solution, and then performing visual quantitative analysis based on the displayed area and distribution of the stained plaque biofilm. The plaque display solution is prepared into solutions or tablets using dyes such as iodine, mercurochrome, basic brown, erythrosine, and fast green, or other colored preparations. This in vivo testing method has several drawbacks. For example, some individuals may experience allergic reactions to certain components of the display solution; the test subject may experience taste discomfort during the test; and because the staining agent also stains the tongue and oral cavity walls, staining residues may remain on these areas after testing, causing aesthetic concerns. Furthermore, the visualization of dental plaque inside the human oral cavity is relatively low. Existing in vitro testing methods all require laboratory equipment and are not suitable for daily personal use. Summary of the Invention

[0005] The technical problem solved by this invention is to provide a paper-based microfluidic chip, reagent kit, and method for detecting dental plaque, as well as its application. Using paper as the base material, an in vitro paper-based microfluidic dental plaque detection chip is constructed. By transferring in vivo dental plaque detection onto paper, the filtration performance of the 3D porous structure of paper fibers and the selective staining of paper fibers and dental plaque by dyes are utilized. The content of the dental plaque to be tested is determined by the color region of the reactants. The operation process is simple and convenient, and it is a low-cost, rapid, and environmentally friendly detection method.

[0006] To address the aforementioned problems, this invention provides a paper-based microfluidic chip for dental plaque detection, comprising a paper-based material, on which a closed hydrophobic isolation band made of a hydrophobic material is provided, and a detection area is formed within the hydrophobic isolation band.

[0007] Paper-based materials refer to sheet-like materials with a three-dimensional porous structure made from various plant fibers (including herbaceous and woody fibers) or a mixture of various plant fibers and non-plant fibers (chemical fibers), such as filter paper. Depending on the filtration speed of the paper-based material, it can be divided into fast filtration, medium-speed filtration, and slow filtration. Depending on the filtration speed of the filter paper, the filter paper can be selected as fast filter paper, medium-speed filter paper, or slow filter paper. Preferably, fast filter paper is selected.

[0008] Among them, hydrophobic materials can be graphite, colloid, paraffin, alkyl ketone dimer, octadecyltrichlorosilane, etc. They can be formed by filling the gaps in paper fibers with hydrophobic reagents such as graphite, colloid, and paraffin through physical modification to form a hydrophobic isolation band. Alternatively, they can be formed by chemical modification to bond hydrophobic reagents such as alkyl ketone dimer and octadecyltrichlorosilane to the surface of paper fibers through chemical bonds.

[0009] Another object of the present invention is to provide the application of the above-mentioned paper-based microfluidic chip for dental plaque detection in the preparation of dental plaque detection kits.

[0010] Another object of the present invention is to provide a paper-based microfluidic dental plaque detection kit, comprising the above-mentioned paper-based microfluidic chip for detecting dental plaque, a staining agent, and a rinsing agent.

[0011] The staining agent is a substance that can physically or chemically interact with dental plaque, bind to it, and produce color. The staining agent can be a solution of one or more of the following: phycoerythrone, fuchsin, iodine, mercurochrome, and basic brown. It can be an aqueous solution or other solvents, as long as the solvent can dissolve the staining agent and will not damage the paper-based material. Preferably, the staining agent is phycoerythrone or fuchsin, both commonly used dental plaque staining agents. Phycoerythrone is phycoerythrone B, sodium tetrachlorotetraiodofluorescein, and fuchsin is pararosaniline hydrochloride. Different staining agents have different binding forces with paper fibers and dental plaque, and their selectivity differs. The staining effect is best when the staining agent has a stronger binding force with dental plaque and a weaker binding force with paper fibers. More preferably, the staining agent is phycoerythrone.

[0012] Preferably, the concentration of the dye is 0.1%-10.0%. Different concentrations of the same dye result in varying binding forces with paper fibers and dental plaque; preferably, the dye concentration is 0.5%-5.0%. When the dye concentration exceeds 2%, the binding force between the dye and paper becomes too strong, making it difficult to rinse off completely. Conversely, when the dye concentration is less than 2%, the binding force between them increases with increasing dye concentration. Therefore, more preferably, the dye concentration is 2.0%.

[0013] The rinsing agent is used to wash away stains that have not bound to dental plaques when applied to paper-based materials. The rinsing agent can be distilled water, saline solution, deionized water, purified water, or other tap water that does not contain color metal ions.

[0014] Another object of the present invention is to provide a paper-based microfluidic plaque detection method, which is accomplished using the above-mentioned paper-based microfluidic plaque detection kit and includes the following steps:

[0015] S1. Take the paper-based microfluidic chip for detecting dental plaque and apply the analyte to the detection area;

[0016] S2. Use a staining agent to stain the detection area of ​​the paper-based microfluidic chip for detecting dental plaque obtained in step S1;

[0017] S3. Rinse the detection area of ​​the paper-based microfluidic chip for detecting dental plaque obtained in step S2 using a rinsing agent;

[0018] S4. Dry the paper-based microfluidic chip for detecting dental plaque obtained in step S3 to obtain a stained paper-based microfluidic chip for detecting dental plaque. Determine the amount of dental plaque in the test sample based on the stained color area.

[0019] Paper-based materials have a 3D porous structure in their paper fibers. Since the pores in dental plaque are smaller than those between paper fibers, when the analyte is applied to the detection area of ​​a paper-based microfluidic chip used for dental plaque detection, the plaque biofilm adheres to the 3D porous structure of the paper and is confined to the detection area within a hydrophobic barrier formed by a hydrophobic material. After applying a staining agent to the detection area and rinsing, the staining agent reacts with the dental plaque and binds, remaining on the paper-based microfluidic chip. In areas of the detection area without dental plaque, the staining agent is washed away by the rinsing agent, flowing through the 3D porous structure of the paper fibers and failing to remain on the paper surface. Therefore, this method, which uses dental plaque biofilm and staining agent to create a specific color area, utilizes the filtration performance of the 3D porous structure of the paper fibers and the selective staining of the paper fibers and dental plaque by the staining agent. The content of the dental plaque to be tested can be determined by the color area of ​​the reactants.

[0020] The bonding between the dye and paper fibers is gradual; the longer the time, the tighter the bond, making subsequent rinsing more difficult. Because the dye has no preferential selectivity for paper fibers, and the pores between paper fibers are large, the initial staining strength is weak, making it easily washed away by the rinsing agent. Conversely, the dye has preferential selectivity for dental plaque, and the pores of dental plaque are significantly smaller than those of paper, resulting in a stronger bond between the two. Preferably, the staining time for the detection area in step S2 is 1s-300s; more preferably, the staining time is 2s-120s; and even more preferably, the staining time is 10s.

[0021] Preferably, in step S3, the amount of rinsing agent used to rinse the detection area is 10 μl-200 μl; more preferably, it is 40 μl-120 μl; and even more preferably, it is 80 μl. The larger the amount of rinsing agent, the greater its impact on the binding of the dye to the paper fibers. Because the dye has preferential selectivity for dental plaque, under the same amount of rinsing agent, dental plaque is less likely to detach from the dye. When the amount of rinsing agent exceeds 80 μl, the effect of the rinsing agent on the binding of the paper and the dye is no longer significant, while the binding of dental plaque and the dye continues. Therefore, considering all factors, an amount of 80 μl of rinsing agent yields the best results.

[0022] Preferably, the drying time for drying the paper-based microfluidic chip for detecting dental plaque in step S4 is 0.5 min to 120 min; more preferably, the drying time is 1 min to 60 min; and even more preferably, the drying time is 10 min.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] 1. Using paper as the base material, an in vitro dental plaque detection paper-based microfluidic chip is constructed. This combines the advantages of paper material, such as low cost, easy processing, and good biocompatibility, with the miniaturization and portability of traditional microfluidic chips. The detection of dental plaque in vivo is transferred to paper. By utilizing the filtration performance of the 3D porous structure of paper fibers and the preferential selectivity of dyes on paper fibers and dental plaque, the content of the dental plaque to be tested is determined by the color region of the reactants.

[0025] 2. It can overcome the problems of conventional in vivo detection methods, such as allergic reactions due to individual differences, taste discomfort for the test subject during the test, unsightly staining of the oral cavity and tongue by the dye, and low visualization of oral cavity detection. It can also overcome the problem that existing in vitro detection methods require laboratory instruments and equipment.

[0026] 3. The dental plaque detection paper-based microfluidic chip and detection method can be further combined with visualization positioning graphic design and digital image acquisition tools to establish a quantitative relationship between the color distribution area and the amount of dental plaque in the oral cavity, making the intelligentization and remote medical application of the test strip possible. Attached Figure Description

[0027] Figure 1 This is a comparison of the detection results images of positive and negative test substances detected by the methods in Embodiments 1, 2, and 3 of this invention;

[0028] Figure 2 This is a comparison of the quantitative analysis results of the optical density of the detection area when detecting positive and negative analytes using the methods in Embodiments 1, 2, and 3 of this invention;

[0029] Figure 3 This is a comparison of the detection result images of positive and negative test substances detected by the methods in embodiments four, five, one, six, and seven of this invention;

[0030] Figure 4 This is a comparison of the quantitative analysis results of the optical density of the detection area when detecting positive and negative analytes using the methods in Embodiments 4, 5, 1, 6, and 7 of this invention;

[0031] Figure 5 This is a comparison of the detection result images of positive and negative test substances detected by the methods in embodiments eight, nine, one, ten, and eleven of this invention;

[0032] Figure 6 This is a comparison of the quantitative analysis results of the optical density of the detection area when detecting positive and negative analytes using the methods in embodiments eight, nine, one, ten, and eleven of this invention;

[0033] Figure 7This is a comparison of the detection result images of positive and negative test substances detected by the methods in Embodiments XII, I, XIII, XIV, and XV of the present invention;

[0034] Figure 8 This is a comparison of the quantitative analysis results of the optical density of the detection area when detecting positive and negative analytes using the methods in Embodiments XII, I, XIII, XIV, and XV of this invention;

[0035] Figure 9 This is a comparison of the detection result images of positive and negative test substances detected by the methods in Embodiments 16, 17, 18, and 19 of this invention;

[0036] Figure 10 This is a comparison of the quantitative analysis results of the optical density of the detection area when detecting positive and negative analytes using the methods in Embodiments 16, 17, 18, and 19 of this invention;

[0037] Figure 11 This is a comparison chart of the thickness data of the paper-based materials used in Embodiments 1, 20, and 21 of the present invention: fast filter paper, medium-speed filter paper, and slow-speed filter paper.

[0038] Figure 12 This is a comparison chart of the bulk thickness data of the paper-based materials used in Embodiments 1, 20, and 21 of the present invention, namely, fast-speed filter paper, medium-speed filter paper, and slow-speed filter paper.

[0039] Figure 13 This is a comparison diagram of the pore size distribution of the paper-based materials used in Embodiments 1, 20, and 21 of the present invention: fast-speed filter paper, medium-speed filter paper, and slow-speed filter paper.

[0040] Figure 14 This is a comparison of the detection results images of positive and negative test substances detected by the methods in Embodiments 1, 20, and 21 of this invention;

[0041] Figure 15 This is a comparison of the quantitative analysis results of optical density in the detection area when detecting positive and negative analytes using the methods in Embodiments 1, 20, and 21 of this invention;

[0042] Figure 16 This is a scanning electron microscope image of the detection area of ​​the paper-based microfluidic chip for dental plaque detection according to Embodiment 1 of the present invention after dental plaque has been added;

[0043] Figure 17 This is a scanning electron microscope image of the detection area of ​​a paper-based microfluidic chip for dental plaque detection according to Embodiment 1 of the present invention after dental plaque has been added and rinsed with a rinsing agent;

[0044] Figure 18 This is a scanning electron microscope image of the detection area of ​​the paper-based microfluidic chip for dental plaque detection according to Embodiment 1 of the present invention after only adding staining agent;

[0045] Figure 19 This is a scanning electron microscope image of the detection area of ​​a paper-based microfluidic chip for dental plaque detection according to Embodiment 1 of the present invention after adding staining agent and rinsing with rinsing agent. Detailed Implementation

[0046] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0047] Example 1

[0048] The specific implementation steps of the paper-based microfluidic-based dental plaque detection method described in this embodiment are as follows:

[0049] 1. Select commercially available rapid filter paper as the paper base material and graphite as the hydrophobic material. Use a paper cutter to cut the rapid filter paper into square pieces of 1.0cm × 1.0cm. Use a plastic ring with a diameter of 0.5cm to apply graphite to the paper pieces to create a ring with the same diameter of 0.5cm, forming a closed annular hydrophobic isolation zone. The paper base material part within the hydrophobic isolation zone serves as the detection area, thus creating a paper-based microfluidic chip for dental plaque detection.

[0050] 2. Using commercially available phycoerythritol as the dye, grind the dye into a fine powder, and then dissolve it in distilled water to prepare a 2% dye solution.

[0051] 3. Before the test begins, place an A4-sized filter paper under the square paper-based microfluidic chip to absorb any excess moisture generated during the test.

[0052] 4. Weigh 0.005g of the analyte and spread it evenly on the detection area of ​​the paper-based microfluidic chip. Use a certain amount of staining solution to stain the chip for 10 seconds.

[0053] 5. Use distilled water as a rinsing agent. After staining is complete, rinse the test area with 80ul of distilled water.

[0054] 6. After drying in a fume hood for 10 minutes, scan the front of the sensor using a scanner and save the image.

[0055] 7. Use Photoshop software to perform data analysis on the scanned detection results images. Specifically, in CMYK color mode, measure the red color density value of the detection area of ​​the paper-based microfluidic chip, and then use GraphPadPrism software to perform statistical analysis on the data.

[0056] Figure 16 A scanning electron microscope image of the detection area after dental plaque was added to the paper-based microfluidic chip for dental plaque detection in this embodiment; Figure 17 Scanning electron microscope image of the detection area after adding dental plaque to the paper-based microfluidic chip for dental plaque detection in this embodiment and rinsing it with rinsing agent; Figure 18 This is a scanning electron microscope image of the detection area of ​​the paper-based microfluidic chip for dental plaque detection in this embodiment after only adding staining agent; Figure 19 Scanning electron microscope (SEM) image of the detection area after adding staining agent to the paper-based microfluidic chip for dental plaque detection in this embodiment and rinsing with rinsing agent.

[0057] like Figure 16 Dental plaque was applied to the detection area of ​​the paper-based microfluidic chip. Before rinsing with a rinsing agent, the dental plaque biofilm was clearly visible attached to the 3D network structure formed by the paper. Several small protruding components were observed on the dental plaque biofilm, consistent with the dental plaque observed in the reference "A Method for In Vitro Detection of Dental Plaque Biofilm with Amphiphilic Fluorescent Probe".

[0058] like Figure 17 After rinsing with a rinsing agent, some smaller dental plaque biofilms may be washed away through the pores of the paper. However, most of the dental plaque biofilm remains firmly attached to the fibrous network structure. As shown in the figure, the dental plaque adheres to the paper surface, and the pores of the plaque are significantly smaller than the paper's pores. No significant changes occurred after rinsing, indicating that rinsing did not affect the adhesion of dental plaque to the paper surface.

[0059] Figure 18 For paper layers where only dye is added. Figure 19 The image shows the paper surface after dyeing and rinsing. The comparison shows that the paper has larger pores, preventing the dye from adhering to the surface, and leaving no residue after rinsing.

[0060] Depend on Figure 17 , 19 It can be seen that rinsing did not affect the state of dental plaque and staining agents on the paper fiber surface. Before rinsing, because phycoerythrone is a small molecule chemical substance, its physical form cannot be seen in the SEM scan image, and therefore it will not remain due to the 3D network structure of the paper, but can only adhere through chemical bonding. After rinsing, it will be washed away, but a small amount will remain, which is consistent with the previous test results.

[0061] As can be seen from the comparison of the above figures, because paper fibers have a 3D porous structure with significantly larger pores than dental plaque, the dye flows directly through the pores of the paper fibers without lingering. Therefore, this paper fiber can be used as a paper-based microfluidic detection chip for dental plaque.

[0062] Example 2

[0063] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that fuchsin is used as the staining agent in this embodiment.

[0064] Example 3

[0065] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1. The difference is that the staining agent used in this embodiment is a commercial dental plaque staining agent produced by Japan's Zenshikang Co., Ltd.

[0066] like Figure 1 This is a comparison of the detection results images of positive and negative analytes in Examples 1, 2, and 3. Figure 2 This is a comparison of the quantitative analysis results of optical density in the detection area when detecting positive and negative analytes in Examples 1, 2, and 3.

[0067] The results show that the contrast between positive and negative results using phycoerythritol as the staining agent was the highest when judged by the naked eye, while the contrast was lower when using fuchsin and commercial dental plaque stains produced by Japan's Quanshikang Co., Ltd. The difference in optical density between positive and negative results was greatest when using phycoerythritol as the staining agent, while the optical density values ​​for positive and negative results were similar when using fuchsin and commercial dental plaque stains produced by Japan's Quanshikang Co., Ltd. Based solely on this experiment, it can be determined that, all other things being equal, using phycoerythritol as the staining agent yields the best results and is the preferred option.

[0068] Example 4

[0069] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that the concentration of the staining agent in this embodiment is 0.5%.

[0070] Example 5

[0071] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that the concentration of the staining agent in this embodiment is 1.0%.

[0072] Example 6

[0073] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that the concentration of the staining agent in this embodiment is 3.0%.

[0074] Example 7

[0075] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that the concentration of the staining agent in this embodiment is 5.0%.

[0076] like Figure 3 This is a comparison of the detection result images from Examples 4, 5, 1, 6, and 7. Figure 4 This is a comparison of the quantitative analysis results of optical density in the detection area in Examples 4, 5, 1, 6, and 7.

[0077] The results show that the contrast between positive and negative results with a dye concentration of 2% was the highest when judged by the naked eye, while the contrast between the results with dye concentrations of 0.5% and 5.0% was the lowest. The optical density of all positive and negative tests increased with increasing dye concentration. However, the difference in average optical density between positive and negative tests was greatest at a dye concentration of 2%.

[0078] The same dye, at different concentrations, exhibits varying degrees of adhesion to paper fibers and dental plaque. Darker colors generally show stronger adhesion. Experimental results indicate that when the dye concentration exceeds 2%, the excessive adhesion makes it difficult to rinse off completely. Conversely, when the dye concentration is less than 2%, the adhesion increases with increasing concentration. Therefore, a dye concentration of 2% yields the best results and is considered the preferred solution.

[0079] Example 8

[0080] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that the amount of distilled water used as the rinsing agent in this embodiment is 40ul.

[0081] Example 9

[0082] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that the amount of distilled water used as the rinsing agent in this embodiment is 60ul.

[0083] Example 10

[0084] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that the amount of distilled water used as the rinsing agent in this embodiment is 100ul.

[0085] Example 11

[0086] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that the amount of distilled water used as the rinsing agent in this embodiment is 120ul.

[0087] like Figure 5 This is a comparison of the detection result images from Examples 8, 9, 1, 10, and 11. Figure 6 This is a comparison of the quantitative analysis results of optical density in the detection area in Examples 8, 9, 1, 10, and 11.

[0088] The results show that the contrast between positive and negative results with a rinse solution dosage of 80 μL was the highest when viewed visually, while the contrast was the lowest for paper with rinse solution dosages of 40 μL and 120 μL. The optical density of all positive tests decreased with increasing rinse solution dosage. However, with increasing rinse solution dosage, the optical density of all negative tests before 80 μL gradually decreased, while the optical density of all negative tests after 80 μL remained essentially unchanged.

[0089] This experiment alone demonstrates that, all other things being equal, before the rinsing solution dosage reaches 80 μL, the longer the staining time, the stronger the contrast between positive and negative results in plaque detection; after the rinsing solution dosage reaches 80 μL, the longer the staining time, the gradually decreasing contrast between positive and negative results in plaque detection. Therefore, in this set of data, a rinsing solution dosage of 80 μL yields the best results.

[0090] The larger the amount of rinsing agent used, the greater its impact on the binding of the dye to the paper fibers. Because the dye has preferential selectivity for dental plaque, under the same amount of rinsing agent, dental plaque is less likely to detach from the dye. However, when the amount of rinsing agent exceeds 80 μL, the effect of the rinsing agent on the binding of paper and dye becomes less significant, while the binding of dental plaque and dye continues. Therefore, considering all factors, a rinsing agent amount of 80 μL yields the best results and is the preferred option.

[0091] Example 12

[0092] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that the staining time of the staining agent in this embodiment is 2 seconds.

[0093] Example 13

[0094] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that the staining time of the staining agent in this embodiment is 30 seconds.

[0095] Example 14

[0096] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that the staining time of the staining agent in this embodiment is 1 minute.

[0097] Example 15

[0098] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that the staining time of the staining agent in this embodiment is 2 minutes.

[0099] like Figure 7 This is a comparison of the detection result images from Examples 12, 1, 13, 14, and 15. Figure 8 This is a comparison of the quantitative analysis results of optical density in the detection area in Examples 12, 1, 13, 14, and 15.

[0100] The results show that the contrast between positive and negative results with a staining time of 10 seconds was the highest when judged by the naked eye, while the contrast of the results with a staining time of 2 minutes was the lowest. All positive tests had high optical density; however, as the staining time increased, the optical density of all negative tests with a staining time before 10 seconds remained essentially unchanged, while the optical density of all negative tests with a staining time after 10 seconds increased significantly.

[0101] Based solely on this experiment, it can be determined that, all other things being equal, before 10 seconds of staining, the longer the staining time, the less effective the negative and positive contrast in plaque detection; after 10 seconds, the longer the staining time, the less effective the negative and positive contrast in plaque detection. Therefore, in this set of data, a staining time of 10 seconds yielded the best results.

[0102] The bonding between the dye and paper fibers is gradual; the longer the time, the tighter the bond, making subsequent rinsing more difficult. Because the dye has no preferential selectivity for paper fibers, and the pores between paper fibers are large, the initial dyeing effect is weak and easily washed away by rinsing agents. Conversely, the dye has preferential selectivity for dental plaque, and the pores of dental plaque are significantly smaller than those of paper, resulting in a stronger bond between the two. Considering all factors, a dyeing time of 10 seconds yields the best results and is the preferred option.

[0103] Example 16

[0104] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that the drying time in this embodiment is 1 minute.

[0105] Example 17

[0106] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that the drying time in this embodiment is 5 minutes.

[0107] Example 18

[0108] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that the drying time in this embodiment is 30 minutes.

[0109] Example 19

[0110] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that the drying time in this embodiment is 60 minutes.

[0111] like Figure 9 A comparison of the detection result images for Examples 16, 17, 1, 18, and 19. Figure 10 This is a comparison of the quantitative analysis results of optical density in the detection area in Examples 16, 17, 1, 18, and 19.

[0112] The experimental results showed that the contrast between positive and negative results with a drying time of 10 minutes was the highest when judged by the naked eye, while the contrast of the results with a drying time of 1 minute was the lowest. All positive tests had high optical density; however, as the drying time increased, the optical density of all negative tests with a drying time before 10 minutes decreased significantly, while the optical density of all negative tests with a drying time after 10 minutes remained essentially unchanged.

[0113] Based solely on this experiment, it can be determined that, under otherwise constant conditions, before 10 minutes of drying time, the longer the drying time of the test strip, the stronger the contrast between positive and negative results in plaque detection; after 10 minutes of drying time, the contrast between positive and negative results remains essentially unchanged even with longer drying times. Therefore, in this set of data, a drying time of 10 minutes yields the best results and is the preferred option.

[0114] Example 20

[0115] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that the paper-based material in this embodiment is medium-speed filter paper.

[0116] Example 21

[0117] The dental plaque detection method based on paper-based microfluidics described in this embodiment has the same implementation steps as in Embodiment 1, except that the paper-based material in this embodiment is slow-speed filter paper.

[0118] like Figure 11 , Figure 12 , Figure 13 This is a comparison chart showing the thickness, bulk, and pore size distribution of the paper-based filter materials used in Examples 1, 20, and 21: fast-speed filter paper, medium-speed filter paper, and slow-speed filter paper. Figure 14 This is a comparison of the detection result images from Examples 1, 20, and 21. Figure 15 This is a comparison of the quantitative analysis results of optical density in the detection area in Examples 1, 20, and 21.

[0119] Table 1 shows the pore structure data of the fast-speed filter paper, medium-speed filter paper, and slow-speed filter paper in Examples 1, 20, and 21.

[0120] Table 1

[0121]

[0122]

[0123] Figure 13 In the figure, the higher the peak, the more pores of that size there are, and the larger the area enclosed by the curves, the greater the porosity.

[0124] The determination of paper thickness and bulk, as well as paper porosity, shows that the greater the porosity of the paper, the more advantageous it is as a paper-based microfluidic dental plaque detection sensor for detecting the distribution and quantity of dental plaque on teeth.

[0125] The experimental results show that the paper with fast-filtering paper as its base material exhibits the highest contrast between positive and negative results as judged by the naked eye, while the paper with slow-filtering paper as its base material shows the lowest contrast. Quantitative analysis of the optical density in the detection area reveals that all positive tests have high optical density. However, as the paper filtration speed decreases, the optical density of all negative tests increases significantly.

[0126] This experiment alone demonstrates that, all other things being equal, the higher the paper filtration rate, the stronger the contrast between positive and negative results in dental plaque detection. Therefore, in this set of data, the rapid filter paper test strip showed the best performance and is the preferred option.

[0127] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A dental plaque detection kit based on paper-based microfluidics, characterized in that: The invention includes a paper-based microfluidic chip for dental plaque detection, a staining agent, and a rinsing agent. The paper-based microfluidic chip for dental plaque detection comprises a paper-based material with a closed hydrophobic isolation zone made of a hydrophobic material, within which a detection area is formed. The paper-based material is rapid filter paper; the hydrophobic material is graphite; the staining agent is phycoerythrone; the concentration of the staining agent is 2%; and the rinsing agent is distilled water. The paper-based microfluidic dental plaque detection kit is used to implement the dental plaque detection method described below: S1. Take the paper-based microfluidic chip for dental plaque detection and apply the analyte to the detection area; S2. The detection area of ​​the paper-based microfluidic chip for dental plaque detection obtained in step S1 is stained with the staining agent; the staining time of the detection area in step S2 is 10 seconds. S3. Rinse the detection area of ​​the paper-based microfluidic chip for dental plaque detection obtained in step S2 using the rinsing agent; the amount of rinsing agent used when rinsing the detection area in step S3 is 80 μl; S4. Dry the paper-based microfluidic chip for dental plaque detection obtained in step S3 for 10 minutes to obtain a stained paper-based microfluidic chip for dental plaque detection. Determine the amount of dental plaque in the test sample based on the stained color area.

Images