CN116919471A - Gingival crevicular fluid collection brush and preparation method thereof - Google Patents
Gingival crevicular fluid collection brush and preparation method thereof Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
- A61B10/0045—Devices for taking samples of body liquids
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Abstract
The invention belongs to a gingival crevicular fluid collection tool, and provides a gingival crevicular fluid collection brush and a preparation method thereof, aiming at the technical problem of low recovery efficiency of biomolecules when a paper strip absorption method which is most widely used at present is used for collecting gingival crevicular fluid. The collecting part comprises a plurality of bristles made of soft wool fibers, the collecting part is connected to the handheld part, the width and the thickness of the collecting part are gradually reduced from one end to the other end, and the small end of the collecting part is far away from the handheld part; the width and the thickness are both the length perpendicular to the extending direction of the collecting part, and the width is larger than the thickness. When the gingival crevicular fluid collection brush is prepared, the preparation of the gingival crevicular fluid collection brush is completed according to specific steps by means of a preparation mold. The collecting brush has the advantages of simple operation, low cost, rapid collection, single-point collection, high recovery rate of biomolecules and the like, and can be used as an important detection means for collecting gingival crevicular fluid and detecting periodontitis beside a chair.
Description
Technical Field
The invention belongs to a gingival crevicular fluid collection tool, and relates to a gingival crevicular fluid collection brush and a preparation method thereof.
Background
Periodontitis is an oral disease with a prevalence of up to 62.4% in middle-aged and elderly people, and can cause gingivitis, alveolar bone absorption, loosening and displacement of teeth, etc., and finally cause tooth loss, seriously endangering oral health. Meanwhile, periodontitis is also closely related to general health. Therefore, diagnosis and treatment of periodontitis is of great benefit to promote human health.
The latest classification standard of periodontal disease in 2018 suggests that: the level of biomarker in gingival crevicular fluid, saliva or serum, which is able to reflect periodontal bone loss and clinical adhesion loss, may help to improve the diagnostic accuracy of periodontitis activity and provide a decisive contribution to the grading evaluation of periodontitis. Compared with saliva and serum, gingival crevicular fluid is directly derived from periodontal tissues, can reflect inflammation conditions of single sites, and is more suitable for diagnosing periodontitis with site specificity.
Currently, there are three accepted methods for collecting gingival crevicular fluid: gingival sulcus flushing, microcapillary pipetting and paper strip absorption. The paper strip absorption method is the most widely used gingival crevicular fluid collection method because of the fact that the paper strip absorption method can be used for collecting the gingival crevicular fluid at a single point, and is short in time and simple and convenient to operate. However, the paper strip absorption method still has the defect of low recovery efficiency of biomolecules.
Disclosure of Invention
Aiming at the technical problem that the recovery efficiency of biomolecules is low when the gingival crevicular fluid is collected by the paper strip absorption method which is most widely used at present, the invention provides a gingival crevicular fluid collection brush and a preparation method thereof.
In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:
a gingival crevicular fluid collection brush, which comprises a handheld part and a collection part;
the collecting part comprises a plurality of bristles made of soft wool fibers and is connected to the handheld part;
the width and the thickness of the collecting part are gradually reduced from one end to the other end, and the small end of the collecting part is far away from the handheld part; the width and the thickness are both the length perpendicular to the extending direction of the collecting part, and the width is larger than the thickness.
Further, the width of the small end of the collecting part is 0.5-1.5mm, and the width of the large end is 1.5-2.5mm.
Further, the width of the small end of the collecting part is 1.5mm, and the width of the large end is 2.0mm.
Further, the collecting part comprises 20-60 bristles made of soft hair fibers.
Further, the collecting part includes 40 bristles made of soft wool fibers.
Further, the thickness of the large end of the collecting part is 0.75-0.77mm.
The invention also provides a preparation method of the gingival crevicular fluid collection brush, which comprises the following steps:
s1, arranging a plurality of bristles made of soft wool fibers, so that the lengths of all the bristles are equal and the two ends of the bristles are aligned;
s2, placing a plurality of bristles in a groove of a collecting part of a preparation mold, and enabling two ends of all the bristles to respectively contact with the side wall of the groove of the collecting part;
the preparation mould is a plate-type mould, a pressing plate groove and at least one collecting part groove are formed in one surface of the preparation mould, the shape and the size of each collecting part groove are matched with those of a collecting part to be prepared, and the pressing plate groove partially covers all the collecting part grooves;
s3, placing a pressing plate in the pressing plate groove to enable all bristles to be kept in the collecting part groove; the shape and the size of the pressing plate are matched with the pressing plate groove;
s4, fixing the large ends of the plurality of bristles, and enabling the plurality of bristles to form a whole through the fixation of the large ends to finish the manufacture of the acquisition part;
s5, connecting the acquisition part and the handheld part to finish the preparation.
Further, in step S4, the fixing the large ends of the plurality of bristles is specifically fixing the large ends of the plurality of bristles by epoxy structural adhesive.
Further, the collecting part groove is trapezoidal in shape.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention provides a gingival crevicular fluid collection brush, wherein a collection part comprises a plurality of bristles made of soft fibers, the soft fibers have good biocompatibility, no biotoxicity, the bristles are fine and soft, no irritation is caused to gums, capillary force similar to microtubes can be formed among the plurality of bristles, and fluid can rise along the bristles, so that the purpose of collecting gingival crevicular fluid and being used for subsequent periodontitis detection is realized, and the gingival crevicular fluid collection brush has the advantages of simplicity and convenience in operation, low cost, rapidness in collection, single-point collection and the like. The bristles of the collecting part are gathered to form a plurality of micro-capillary channels, the pipe diameter is small, the capillary force is large, and the time consumption is short when liquid is sucked. In addition, because the pores in the bristle fiber are tiny, biomolecules in gingival crevicular fluid are not easy to stay and easy to elute, so that the recovery rate of the biomolecules is remarkably improved, and a foundation is provided for detecting periodontitis through the content of the biomolecules in the follow-up process.
2. According to the invention, according to the specific application scene of the collecting brush, the width, thickness and bristle number of the collecting part are optimally designed according to the gingival crevicular fluid collection, and experiments prove that under the preferable parameter setting of the invention, the recovery rate, the liquid absorbing capacity and the like of the collecting brush can reach better effects.
3. The invention also provides a preparation method of the gingival crevicular fluid collection brush, and the preparation mold specially designed for the collection brush can be used for conveniently and rapidly completing the preparation, ensures that the structure of the collection brush meets the preparation requirement, is a preparation method specially designed for the collection brush, and is convenient to popularize and use.
Drawings
For a clearer description of the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic view of a gingival crevicular fluid collection brush according to a first embodiment of the present disclosure;
fig. 2 is a schematic surface structure of a preparation mold provided with a groove of a collecting part in a fourth embodiment of the present invention;
FIG. 3 is a graph showing the comparison of the results of the recovery rate experiments of the third embodiment and the periodic paper strip for collecting different standard antigens when the initial volumes of the samples are the same and the initial concentrations are different; wherein, (a) is IL-1 beta, (b) is TNF-alpha, (c) is MMP-8, (d) is ds DNA, (e) is IL-1 beta ssDNA, and (f) is microRNA146 a;
FIG. 4 is a graph showing the comparison of the results of the recovery rate experiments of the third embodiment and the periodic paper strip for collecting different standard antigens when the initial concentration and the initial volume of the sample are the same; wherein (a) is IL-1 beta, (b) is TNF-alpha, (c) is MMP-8, and (d) is ds DNA.
Wherein: 1-hand-held part, 2-collection part, 3-clamp plate recess, 4-collection part recess, 5-preparation mould.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further definition or explanation thereof is necessary in the following figures.
In the description of the embodiments of the present invention, it should be noted that, if the terms "upper," "lower," "horizontal," "inner," and the like indicate an azimuth or a positional relationship based on the azimuth or the positional relationship shown in the drawings, or the azimuth or the positional relationship in which the inventive product is conventionally put in use, it is merely for convenience of describing the present invention and simplifying the description, and does not indicate or imply that the apparatus or element to be referred to must have a specific azimuth, be configured and operated in a specific azimuth, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and the like, are used merely to distinguish between descriptions and should not be construed as indicating or implying relative importance.
Furthermore, the term "horizontal" if present does not mean that the component is required to be absolutely horizontal, but may be slightly inclined. As "horizontal" merely means that its direction is more horizontal than "vertical", and does not mean that the structure must be perfectly horizontal, but may be slightly inclined.
In the description of the embodiments of the present invention, it should also be noted that, unless explicitly specified and limited otherwise, the terms "disposed," "mounted," "connected," and "connected" should be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific circumstances.
The invention is described in further detail below with reference to the attached drawing figures:
the invention provides a gingival crevicular fluid collecting brush and a preparation method thereof, which can collect gingival crevicular fluid conveniently and rapidly and has high recovery efficiency of biological molecules by utilizing the characteristics of good flexibility, good water absorption, no toxicity and the like of bristles of a commercial toothbrush.
Example 1
As shown in fig. 1, the invention provides a gingival crevicular fluid collection brush, which comprises a handheld part 1 and a collection part 2. The collection portion 2 is connected on handheld portion 1, and collection portion 2 is including the many brush hair of material for soft hair fibre, and width and the thickness of collection portion 2 all reduce gradually from one end to the other end, and handheld portion 1 is kept away from to the tip of collection portion 2, forms the gathering form brush hair cluster of similar writing brush nib.
The width and thickness of the collecting portion 2 refer to a length perpendicular to the extending direction of the collecting portion 2, and the width is larger than the thickness. Such a gathered bristle tuft may have various shapes, such as a prism shape, a truncated cone shape, and a cone shape, and if a prism shape is used, a plane perpendicular to the axis of the collecting portion 2 is used as a cross section, and the cross section of the collecting portion 2 is obtained as a rectangle, and the length of the long side of the rectangle is the width, and the length of the short side is the thickness. In other shapes, the thickness can be analogically estimated according to the width being larger than the thickness.
The shape and structure of the hand-held part 1 are not limited, and the hand-held part 1 has the function of being convenient for an operator to hold the gingival crevicular fluid collection brush when carrying out gingival crevicular fluid collection. The type of bristles used by the collection unit 2 can be adjusted as needed, as long as the collection effect can be ensured.
The multiple bristles of the collecting part 2 are gathered to form a structure with one large end and one small end, and a micro-tube cavity is formed between the bristles, so that the collecting part has strong capillary force and can rapidly absorb liquid. In addition, the brush hair in the collection part 2 is soft and does not damage the gum, and meanwhile, the fiber inside of the brush hair is compact, so that the recovery rate of biomolecules is high, the subsequent detection of the biomolecule content in gingival crevicular fluid is facilitated, and the accurate diagnosis of periodontal health condition is facilitated. In order to make the collection portion 2 have a large end and a small end, the bristles themselves may have a large end and a small end, or the bristles may have the same diameter in the axial direction, but after the collection portion is made, the bristles are formed into bristle tufts having a large end and a small end.
Example two
On the basis of the first embodiment, the structural parameters of the collecting part 2 are optimized, so that the width of the small end of the collecting part 2 is 0.5-1.5mm, the width of the large end of the collecting part 2 is 1.5-2.5mm, and meanwhile, the collecting part 2 comprises 20-60 bristles made of soft hair fibers.
The liquid absorption performance of the gingival crevicular fluid collection brush is tested:
adding ultrapure water into a centrifuge tube, and weighing the total mass. Then, ultrapure water in the centrifuge tube was sucked up by using a gingival crevicular fluid collection brush, and the mass of the centrifuge tube was weighed again at 15s, 30s, 45s, and 60s of suction time, respectively. The mass difference was calculated and based on the mass to volume relationship of water (1 g=1 mL), the liquid volumes of 15s, 30s, 45s, 60s of gingival crevicular fluid collection brush were obtained as shown in table 1.
Table 1 gingival crevicular fluid collection brush imbibition volume table
| Imbibition time/s | 15s | 30s | 45s | 60s |
| Liquid suction volume/. Mu.L | 4.71±0.66 | 5.40±0.57 | 6.08±0.74 | 6.66±0.84 |
As shown in Table 1, the liquid suction amount of the collecting brush in 15 seconds can reach 4.71 mu L, and the clinical requirement (< 2 mu L) can be met, so that the gingival crevicular fluid collecting brush is expected to reduce the collecting time to 15 seconds, and the discomfort of a patient is greatly reduced.
Example III
The gingival crevicular fluid collection brush further optimizes the structural parameters of the collection portion 2 on the basis of the first embodiment, so that the width of the small end of the collection portion 2 is 1.5mm (see position B in fig. 1), the width of the large end is 2.0mm (see position a in fig. 1), the thickness of the large end is 0.75-0.77mm, and meanwhile, the collection portion 2 comprises 40 bristles made of soft hair fibers.
Test the structure of the collection brush in example three:
1. effect of bristle count on liquid absorption properties:
collecting brushes with 20, 30, 40, 50 and 60 bristles are respectively used as a collecting part 2, ultra-pure water is added into a centrifuge tube, the total mass is weighed, then ultra-pure water in the centrifuge tube is sucked by using a gingival crevicular fluid collecting brush, and the mass of the centrifuge tube is weighed again when 15s, 30s, 45s and 60s are sucked respectively. The mass difference was calculated and based on the mass to volume relationship of water (1 g=1 mL), the liquid volumes of 15s, 30s, 45s, 60s of gingival crevicular fluid collection brush were obtained as shown in table 2.
Table 2 liquid suction volume table for collecting gingival crevicular fluid with different bristle numbers
It can be seen from table 2 that the number of bristles and the volume of liquid absorbed at the same liquid absorbing time are not simply monotonically related, and a turn occurs in the middle. In addition, in the case where other parameters are the same, the liquid suction volume of 60 bristles is the largest in the same liquid suction time.
2. Since the gingival crevicular fluid collection brush of the present invention is used clinically, the thickness of the collection portion 2 including the different number of bristles is measured with reference to the thickness of the periodontal probe, which is generally less than 1mm, and the measurement results are shown in table 3:
TABLE 3 gingival crevicular fluid collection brush collection portion large end thickness measuring meter with different bristle counts
The "coefficient of variation" in table 3 refers to the difference in the measured values in the group of the plurality of collection brush measurements, which are obtained by performing the plurality of collection brush measurements, and the same number of bristles, when the "thickness measured value" is measured. The thickness measurement value is obtained by measuring the large end of the acquisition brush by using a vernier caliper.
3. Effect of bristle count on liquid absorption Rate
Table 4 gingival crevicular fluid collection brush liquid suction speed meter with different bristle numbers
As can be seen from Table 4, the liquid suction rate was faster at 60 bristles, and the liquid suction volume was also maximized at 60 bristles in combination with the previous study. However, the collecting brush should be too thick in practical application, considering the practical applicability of clinical application. Therefore, the invention combines the liquid absorption performance and the large end thickness of the collecting part, and takes the structural parameters in the third embodiment as the optimal parameter combination.
Example IV
Aiming at the sampling brush structure, the invention also provides a preparation method of the gingival crevicular fluid collection brush, which comprises the following specific steps:
s1, arranging a plurality of bristles made of soft wool fibers, so that the lengths of all the bristles are equal and the two ends of the bristles are aligned. Specifically, the sources of the bristles may be different, in this embodiment, the bristles are large at one end and small at one end, for example, the bristles are taken from other products, or the bristles themselves are long, and each bristle can be cut off into two or more bristles from the middle, so that the required number of bristles is counted, and the two ends of the bristles are opposite to each other.
S2, when preparing the brush hair, in order to ensure the preparation quality of the collecting brush, the preparation of the collecting brush is completed by means of the preparation mold 5. As shown in fig. 2, as a preferred embodiment, the preparation mold 5 has a rectangular plate structure, and the upper surface of the preparation mold 5 is provided with a pressing plate groove 3 and a collecting part groove 4, wherein the pressing plate groove 3 is used for accommodating the pressing plate in the subsequent step, and the collecting part groove 4 is used for accommodating the bristles in the subsequent step. In the fourth embodiment, the number of the grooves is specifically 4, and each collecting portion groove 4 is trapezoidal and is used for accommodating the collecting portion 2. The shape and the size of the collecting part groove 4 are matched with the collecting part 2 to be prepared. The manufacture of the preparation mould 5 and the pressing plate can be completed by using a laser cutting machine, and the shape and the size of the pressing plate are matched with those of the pressing plate groove 3.
The plurality of bristles are placed in the collecting part groove 4, so that two ends of all the bristles respectively contact the side wall of the collecting part groove.
S3, placing the pressing plate in the pressing plate groove 3, so that all bristles are kept in the collecting part groove 4, and the bristles are prevented from falling out.
S4, mixing two parts of Devcon epoxy resin structural adhesive in equal proportion, coating the uniformly stirred epoxy resin structural adhesive on the large ends of the bristles, waiting for solidification, fixing the large ends of the plurality of bristles, and fixing the plurality of bristles through the large ends to form a whole to finish the manufacture of the acquisition part 2.
S5, connecting the collecting part 2 with the handheld part 1 to finish the preparation of the whole gingival crevicular fluid sampling brush.
After the gingival crevicular fluid sampling brush is prepared by adopting the method, the parameters of the obtained product are verified. The front photographs of the straight ruler and 8 gingival crevicular fluid collection brushes were taken at a fixed distance, the photographs were processed using ImageJ software, the widths of the small and large ends of the collection brushes were measured with the length of 1cm in the straight ruler as a standard, and the measurements were repeated 3 times for each and averaged, as shown in table 5.
Table 5 example four preparation of statistical table of collecting brush small end and large end widths
From the data in table 5, the variation coefficients of the small end width and the large end width of the collecting brush are controlled below 15%, which shows that the preparation method of the invention can play a role in standardizing the shape of the collecting brush, and the consistency of the shape of the prepared collecting brush is good.
The technical effects of the invention are further illustrated below by means of a comparative example (using the method of collecting gingival crevicular fluid by means of a strip of a Periopaper according to the prior art):
1. sample initial volume was the same and recovery rate was measured at different initial concentrations:
verification experiment one:
(1) Standard antigen solutions of different concentrations were prepared by diluting recombinant human IL-1 beta, TNF-alpha and MMP-8 standard antigen stock with deionized water according to the instructions of the Periopaper strip. IL-1 beta concentration after dilution is 0.8ng/mL,4ng/mL and 8ng/mL respectively; the concentration of the TNF-alpha standard antigen solution after dilution is 4ng/mL,8ng/mL and 16ng/mL respectively; the concentration of MMP-8 standard antigen solution after dilution is 50ng/mL,100ng/mL and 200ng/mL respectively;
(2) 1.5 mu L of standard antigen solution is dripped on the sampling brush in the third embodiment, the sampling brush is rinsed 1 time by using 120 mu L of PBSA buffer (PBS containing 0.5% w/v BSA), the sampling brush is centrifuged by using a desktop centrifuge for 30s, and the flushing fluid and the centrifugate are mixed for standby;
(3) 1.5. Mu.L of standard antigen solution was added dropwise to a Periopaper strip and placed in a centrifuge tube containing 120. Mu.L of LPBSA buffer (PBS containing 0.5% w/v BSA) and allowed to stand in a refrigerator at 4℃for 30min. Centrifuging at high speed (12000 rpm, 4deg.C, 10 min), and collecting supernatant;
(4) For the control group, 1.5 μl of three standard antigen solutions of different concentrations were directly added to 120 μl LPBSA buffer and mixed well;
(5) The content of recombinant human IL-1 beta, TNF-alpha and MMP-8 standard antigen in the supernatant extracted from the washing centrifugal mixed solution of the sampling brush and the Periopaper strip is detected by using an IL-1 beta, TNF-alpha and MMP-8ELISA kit, and all the operations are carried out according to the specification;
(6) Absorbance of each sample was recorded using a Tecan Spark 10M microplate reader, and each set of measurements was repeated three times;
(7) And determining the recovery rate of the recombinant human IL-1 beta, TNF-alpha and MMP-8 standard antigen with different initial concentrations by using the sampling brush and the Periopaper strip according to the ratio of the absorbance of the extracted sample to the absorbance of the control group.
And (3) verification experiment II:
(1) According to the instruction, using sterile and sterile water to dissolve standard double-stranded DNA (dsDNA) dry powder, synthesized IL-1β single-stranded DNA (ssDNA) dry powder and microRNA146a (mir-146 a) dry powder in the total nucleic acid quantification kit, the concentration of the ds DNA and IL-1β ssDNA after dissolution is 4 μg/mL,8 μg/mL and 16 μg/mL respectively; the concentration of the dissolved microRNA146a solution is 5 mug/mL, 10 mug/mL and 20 mug/mL respectively;
(2) For recovery of dsDNA and IL-1. Beta. SsDNA, 1.5. Mu.L of standard solution was added dropwise to the sampling brush, and 120. Mu.L of sterile and non-sterile solution was usedWashing the sampling brush for 1 time by using enzyme water, centrifuging the sampling brush for 30s by using a tabletop centrifuge, collecting flushing liquid and centrifugate, and uniformly mixing for later use; for recovery of dsDNA and IL-1. Beta. SsDNA, 1.5. Mu.L of standard solution was added dropwise to a strip of Periopaper and placed in a centrifuge tube containing 120. Mu.L of sterile, enzyme-free water and allowed to stand in a refrigerator at 4℃for 30min. Centrifuging at high speed (12000 rpm, 4deg.C, 10 min), and collecting supernatant; for the control group, 1.5. Mu.L of dsDNA and IL-1. Beta. SsDNA solutions with different concentrations are directly added into 120. Mu.L of sterile water and uniformly mixed; using Helixyte TM The green fluorescence total nucleic acid quantitative kit tests the contents of dsDNA and IL-1 beta ssDNA in the supernatant extracted by the washing centrifugal mixed solution of the sampling brush and the period paper strip, and all the operations are carried out according to the specification;
(3) For the recovery of mir-146a, 1.5 μl of standard solution was added dropwise to the sampling brush, the sampling brush was rinsed 1 time with 120 μl of sterile enzyme-free water containing RNase inhibitor, the sampling brush was centrifuged with a tabletop centrifuge for 30s, and the rinse solution and the centrifugate were mixed for use; 1.5. Mu.L of standard solution was added dropwise to a strip of Periopaper and placed in a centrifuge tube containing 120. Mu.L of sterile, enzyme-free water, which was placed in a refrigerator at 4℃for 30min. Centrifuging at high speed (12000 rpm, 4deg.C, 10 min), and collecting supernatant; for the control group, 1.5. Mu.L of mir-146a solution with different concentrations is directly added into 120. Mu.L of sterile enzyme-free water and uniformly mixed; using Qubit TM The microRNA detection kit tests the content of microRNA146a in the supernatant extracted by the washing centrifugal mixed solution of the sampling brush and the Periopaper strip, and all operations are carried out according to the specification;
(4) The fluorescence value of each sample is recorded by using a Tecan Spark 10M enzyme-labeled instrument, and each group of detection is repeated three times;
(5) And determining recovery rates of ds DNA, IL-1 ss DNA and microRNA146a with different initial concentrations, wherein the initial volumes of the sampling brush and the period paper strip are the same according to the ratio of the fluorescence value of the extracted sample to the fluorescence value of the control group.
As shown in fig. 3, for the control plots of the recovery rate measurement performed with the same initial volume of the sample and different initial concentrations, in each control group of the respective plots of fig. 3, "Brush" represents the acquisition Brush test result of example three, and "period" represents the period paper strip test result. Wherein, (a) shows the comparison of the results of the diluted recombinant human IL-1 beta standard antigen concentrations of 0.8ng/mL,4ng/mL and 8ng/mL in the first verification experiment, (b) shows the comparison of the results of the diluted TNF-alpha standard antigen solutions of 4ng/mL,8ng/mL and 16ng/mL in the first verification experiment, (c) shows the comparison of the results of the diluted MMP-8 standard antigen solutions of 50ng/mL,100ng/mL and 200ng/mL in the first verification experiment, (d) shows the comparison of the results of the dissolved ds DNA concentrations of 4 mug/mL, 8 mug/mL and 16 mug/mL in the second verification experiment, (e) shows the comparison of the results of the dissolved IL-1 beta ssDNA concentrations of 4 mug/mL, 8 mug/mL and 16 mug/mL in the second verification experiment, and f) shows the comparison of the results of the dissolved microRNA146a concentrations of 5 mug/mL, 10 mug/mL and 20 mug/mL in the second verification experiment. As can be seen from fig. 3, the recovery rate is significantly better with the sampling brush of the present invention.
2. Sample initial concentration was the same and recovery rate was measured at different initial volumes:
verification experiment one:
(1) According to the application instruction, deionized water is used for diluting recombinant human IL-1 beta, TNF-alpha and MMP-8 standard antigen storage solution to prepare standard antigen solutions with different concentrations, the concentration of the diluted IL-1 beta standard antigen solution is 6ng/mL, the concentration of the TNF-alpha standard antigen solution is 12ng/mL, and the concentration of the MMP-8 standard antigen solution is 120ng/mL;
(2) Dripping 0.5 mu L,1 mu L and 1.5 mu L of standard antigen solution on a sampling brush, rinsing the sampling brush for 1 time by using 120 mu L of PBSA buffer solution, centrifuging the sampling brush for 30 seconds by using a tabletop centrifuge, collecting flushing liquid and centrifugate, and uniformly mixing for later use;
(3) 0.5. Mu.L, 1. Mu.L and 1.5. Mu.L of standard antigen solution were added dropwise to the Periopaper strip, respectively, and placed in a centrifuge tube containing 120. Mu.L of deionized water, and allowed to stand in a refrigerator at 4℃for 30min. Centrifuging at high speed (12000 rpm, 4deg.C, 10 min), and collecting supernatant;
(4) For the control group, three standard antigen solutions of different concentrations of 0.5 μl,1 μl and 1.5 μl were directly added to 120 μl PBSA buffer and mixed well;
(5) The content of recombinant human IL-1 beta, TNF-alpha and MMP-8 standard antigen in the supernatant extracted from the washing centrifugal mixed solution of the sampling brush and the Periopaper strip is detected by using an IL-1 beta, TNF-alpha and MMP-8ELISA kit, and all the operations are carried out according to the specification;
(6) Absorbance of each sample was recorded using a Tecan Spark 10M microplate reader, and each set of measurements was repeated three times;
(7) And determining the recovery rate of recombinant human IL-1 beta, TNF-alpha and MMP-8 standard antigen with the same initial concentration and different initial volumes by the sampling brush and the Periopaper strip according to the ratio of the absorbance of the extracted sample to the absorbance of the control group.
And (3) verification experiment II:
(1) Preparing an IL-1 beta ssDNA standard solution with a concentration of 12 mug/mL by using sterile and sterile water according to the instruction of use;
(2) Dripping 0.5 mu L,1 mu L and 1.5 mu L IL-1 beta ssDNA standard solution on a sampling brush, rinsing the sampling brush for 1 time by using 120 mu L sterile enzyme-free water, centrifuging the sampling brush for 30s by using a tabletop centrifuge, and mixing flushing fluid and centrifugate for later use;
(3) 0.5. Mu.L, 1. Mu.L and 1.5. Mu.L of standard solution were added dropwise to the Periopaper strip, respectively, and placed in a centrifuge tube containing 120. Mu.L of sterile, enzyme-free water, and allowed to stand in a refrigerator at 4℃for 30min. Centrifuging at high speed (12000 rpm, 4deg.C, 10 min), and collecting supernatant;
(4) For the control group, 0.5. Mu.L, 1. Mu.L and 1.5. Mu.L of standard solutions with different concentrations are directly added into 120. Mu.L of sterile enzyme-free water and uniformly mixed;
(5) Using Helixyte TM The green fluorescence total nucleic acid quantitative kit tests the content of IL-1 ss DNA in the supernatant extracted by the washing centrifugal mixed solution of the sampling brush and the period paper strip, and all operations are carried out according to the specification;
(6) The fluorescence value of each sample is recorded by using a Tecan Spark 10M enzyme-labeled instrument, and each group of detection is repeated three times;
(7) And determining the recovery rate of the IL-1 ss DNA with the same initial concentration and different initial volumes by the sampling brush and the period paper strip according to the ratio of the fluorescence value of the sampling to the fluorescence value of the control group.
As shown in FIG. 4, for the control plots of the recovery rate measurement with the same initial concentration and different initial volume of the sample, in each control group of the plots of FIG. 4, "Brush" represents the acquisition Brush test result of example three, and "Periopaper" represents the Periopaper strip test result. Wherein, (a) represents a comparison of results of 0.5. Mu.L, 1. Mu.L and 1.5. Mu.L of recombinant human IL-1β standard antigen volumes, respectively, after dilution in validation experiment one, (b) represents a comparison of results of 0.5. Mu.L, 1. Mu.L and 1.5. Mu.L of TNF- α standard antigen volumes, respectively, after dilution in validation experiment one, (c) represents a comparison of results of 0.5. Mu.L, 1. Mu.L and 1.5. Mu.L of MMP-8 standard antigen volumes, respectively, after dilution in validation experiment one, and (d) represents a comparison of results of 0.5. Mu.L, 1. Mu.L and 1.5. Mu.L of ds DNA volumes, after solubilization, respectively, in validation experiment two. As can be seen from fig. 4, the recovery rate is significantly better with the sampling brush of the present invention.
The Periopaper strip has larger pores, can retain biomolecules, is not easy to elute, and has low recovery rate of biomolecules. The gingival crevicular fluid collection brush provided by the invention has compact bristle fiber, tiny pores and difficult retention of biomolecules, and can remarkably improve the recovery efficiency of the biomolecules.
Through principle analysis and numerous experimental verification results, the gingival crevicular fluid collection brush provided by the invention has the advantages of short collection time and capability of reducing uncomfortable feeling of a patient. Meanwhile, the gingival crevicular fluid collection brush biomolecule recovery rate is high, and the method can be expected to be matched with a sensitive and efficient biomolecule detection method, can be applied to aspects of early screening, daily monitoring, rapid diagnosis and the like of periodontitis, and further can promote providing a powerful detection means for oral health of all human beings.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A gingival crevicular fluid collection brush which is characterized in that: comprises a hand-held part (1) and an acquisition part (2);
the collecting part (2) comprises a plurality of bristles made of soft wool fibers, and the collecting part (2) is connected to the handheld part (1);
the width and the thickness of the collecting part (2) are gradually reduced from one end to the other end, and the small end of the collecting part (2) is far away from the handheld part (1); the width and the thickness are both the length perpendicular to the extending direction of the collecting part, and the width is larger than the thickness.
2. The gingival crevicular fluid collection brush of claim 1, wherein: the width of the small end of the collecting part (2) is 0.5-1.5mm, and the width of the large end is 1.5-2.5mm.
3. A gingival crevicular fluid collection brush according to claim 2, wherein: the width of the small end of the collecting part (2) is 1.5mm, and the width of the large end is 2.0mm.
4. A gingival crevicular fluid collection brush according to any one of claims 1 to 3, wherein: the collecting part (2) comprises 20-60 bristles made of soft hair fibers.
5. The gingival crevicular fluid collection brush of claim 4, wherein: the collecting part (2) comprises 40 bristles made of soft hair fibers.
6. The gingival crevicular fluid collection brush of claim 5, wherein: the thickness of the large end of the collecting part is 0.75-0.77mm.
7. A method of preparing a gingival crevicular fluid collection brush according to any one of claims 1 to 6, comprising the steps of:
s1, arranging a plurality of bristles made of soft wool fibers, so that the lengths of all the bristles are equal and the two ends of the bristles are aligned;
s2, placing a plurality of bristles in the collecting part groove (4) of the preparation mold (5) so that two ends of all the bristles respectively contact with the side wall of the collecting part groove (4);
the preparation mould (5) is a plate-type mould, a pressing plate groove (3) and at least one collecting part groove (4) are formed in one surface of the preparation mould (5), the shape and the size of the collecting part groove (4) are matched with those of the collecting part (2) to be prepared, and the pressing plate groove (3) partially covers all the collecting part grooves (4);
s3, placing a pressing plate in the pressing plate groove (3) to enable all bristles to be kept in the collecting part groove (4); the shape and the size of the pressing plate are matched with the pressing plate groove (3);
s4, fixing the large ends of the plurality of bristles, and enabling the plurality of bristles to form a whole through the fixation of the large ends to finish the manufacture of the acquisition part (2);
s5, connecting the acquisition part (2) with the handheld part (1) to finish the preparation.
8. The method according to claim 7, wherein in the step S4, the large ends of the plurality of bristles are fixed specifically by epoxy structural adhesive.
9. A method of manufacturing a gingival crevicular fluid collection brush according to claim 8, wherein the collection portion recess (4) is trapezoidal in shape.
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