Paper-based colorimetric sensor for saliva detection

Abstract

The present invention relates to a colorimetric sensor for saliva detection comprising a test strip impregnated with G3-CNP (2-chloro-4-nitrophenyl-α-D-maltotrioside) and a method for saliva detection using the colorimetric sensor. By changing the color of the colorimetric sensor, the presence or absence of saliva in a sample can be confirmed in a short time even in a small amount of sample. Furthermore, since the colorimetric sensor in which saliva is detected can be used as a sample for STR profile analysis, it can be utilized for economical, simple, and rapid saliva detection and identification, and thus can be usefully applied in the field of forensic science.

Description

Technology Field

[0001] The present invention relates to a paper-based colorimetric sensor for saliva detection. Background Technology

[0003] It is difficult to visually identify whether many stains found at forensic investigation scenes are caused by beverages, exhaled blood, or other bodily fluids. Accordingly, confirming the presence of bodily fluids through preliminary testing before DNA analysis can improve the efficiency of investigations by rapidly identifying only valid samples. The three representative bodily fluids are blood, semen, and saliva; while there are many types of preliminary testing methods for blood and semen, preliminary testing methods for saliva are relatively limited.

[0004] Existing preliminary test methods for saliva using amylase activity include the starch-iodine test and Phadebas ® Test method, SALIgAE ® Testing Methods and RSID ™ There is the Saliva test method. The starch-iodine test utilizes the principle that starch appears blue in the presence of iodine, and that salivary amylase breaks down starch to cause a color change. However, since the color of the starch-iodine test varies depending on the amount of amylase activity, it is difficult to determine the presence or absence of saliva. In addition, Phadebas ® The test method is a chemical for saliva detection based on an insoluble starch complex to which amylase activity and color molecules are attached; color is released when amylase breaks down the complex. However, these Phadebas ® The test method has the problem of producing false positive reactions to hand cream, lotion, urine, and stool, and taking a long time to react. SALIgAE ® The test method can determine the presence of saliva by observing a color change from colorless to yellow in the liquid phase if saliva is present. However, SALIgAE ®The testing method has the problem that it is very expensive and difficult to verify quickly because the detailed detection method is not disclosed. In addition, RSID ™ The Saliva test is a method that uses a lateral flow immunochromatographic strip test to detect the presence of alpha-amylase (α-amylase) in human saliva. However, this RSID ™ The Saliva test also has the problem that it does not react specifically to human saliva.

[0005] Accordingly, the inventors have completed the present invention by developing a colorimetric sensor for saliva detection that is convenient to use at forensic science sites without complex experimental procedures and allows for rapid confirmation of the presence or absence of saliva by observing a change in color visually. Prior art literature

[0007] Korean Registered Patent No. 10-1847475 The problem to be solved

[0008] The object of the present invention is to provide a colorimetric sensor for saliva detection comprising a test strip impregnated with G3-CNP (2-chloro-4-nitrophenyl-α-D-maltotrioside).

[0009] Another objective of the present invention is to provide a composition for saliva detection comprising a test strip impregnated with G3-CNP.

[0010] Another objective of the present invention is to provide a saliva detection method comprising the steps of: contacting a sample with a colorimetric sensor for saliva detection comprising a test paper impregnated with the G3-CNP; and checking whether there is a color change of the colorimetric sensor. means of solving the problem

[0012] The present invention provides a colorimetric sensor for saliva detection comprising a test paper impregnated with G3-CNP.

[0013] In addition, the present invention provides a composition for detecting saliva comprising a test strip impregnated with G3-CNP.

[0014] In addition, the present invention provides a saliva detection method comprising the steps of: contacting a sample with a colorimetric sensor for saliva detection comprising a test paper impregnated with the G3-CNP; and checking whether there is a color change of the colorimetric sensor. Effects of the invention

[0016] The colorimetric sensor for saliva detection according to the present invention can confirm the presence or absence of saliva in a short time even in a small amount of sample, and the colorimetric sensor in which saliva is detected can be used as a sample for STR profile analysis, thereby enabling economical, simple, and rapid saliva detection and identification, and thus can be usefully utilized in the field of forensic science. Brief explanation of the drawing

[0018] Figure 1 shows Gal-G2-CNP, G3-CNP, and SALIgAE ® This is the result of observing the color change after reacting with various concentrations of alpha-amylase (α-amylase, 20, 50, 75, 100, 200, 300, 400, and 500 μg / mL) for 3 minutes. Figure 2 shows the results of confirming the difference in reactivity according to the pH of the buffer. Figure 3 shows the results of reacting paper (glass fiber filter and Whatman Chromatography paper) impregnated with a buffer containing G3-CNP with various concentrations of alpha-amylase (20, 50, 75, 100, 200, 300, 400, and 500 μg / mL) and observing the color change of the paper. Figure 4 shows the results of reacting paper impregnated with buffers containing various concentrations of G3-CNP (5, 10, 15, and 20 mg / mL) with various concentrations of alpha-amylase (20, 50, 70, 100, 200, 300, 400, and 500 μg / mL) and observing the color change of the paper. Figure 5 is a schematic diagram showing the fabrication process of a paper-based colorimetric sensor (Sal-ID). Figure 6 shows the results of analyzing the sensitivity of a paper-based colorimetric sensor. Figure 7 shows the results of analyzing the specificity of a paper-based colorimetric sensor. Figure 8 shows the results of analyzing a mixed sample using a paper-based colorimetric sensor. Figure 9 shows the results of analyzing the STR full profile from the sample eluted from the colorimetric sensor. Specific details for implementing the invention

[0019] The present invention will be described in detail below.

[0020] The terms used in this invention have been selected based on currently widely used general terms whenever possible, taking into account the functions of the invention; however, these terms may vary depending on the intent of those skilled in the art or the emergence of new technologies. Additionally, in specific cases, terms may be selected arbitrarily, and in such cases, their meanings will be described in detail in the description section of the relevant embodiments. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0021] In the present invention, when a component or step is described as "comprising," this means that, unless specifically stated otherwise, it does not exclude other components or steps but may include additional components or steps.

[0023] The present invention provides a colorimetric sensor for saliva detection comprising a test strip impregnated with G3-CNP (2-chloro-4-nitrophenyl-α-D-maltotrioside).

[0024] In the present invention, the test sheet may be impregnated with G3-CNP on a substrate.

[0025] In the present invention, the G3-CNP may be included in a buffer composed of at least one of sodium chloride, calcium acetate, potassium thiocyanate, and sodium azide in the MES buffer, but is not limited thereto.

[0026] The above MES buffer is one of the commonly used buffers and can be used to extend the shelf life of the buffer. Additionally, sodium chloride and potassium thiocyanate can be used in relation to amylase activity, calcium acetate can be used to increase the yield of amylase contained in saliva, and sodium azide can be used as an activator and preservative. The pH of the buffer can be adjusted with 1M sodium hydroxide (NaOH).

[0027] In the present invention, the pH of the buffer may be pH 5.5 to pH 7.0, preferably pH 6.0 to pH 6.7, and more preferably pH 6.5, but is not limited thereto.

[0028] In the above, MES may be included in an amount of 80 to 120 mmol / L, preferably 90 to 110 mmol / L, more preferably 100 mmol / L, but is not limited thereto. In addition, sodium chloride may be included in an amount of 300 to 400 mmol / L, preferably 310 to 390 mmol / L, 320 to 380 mmol / L, 330 to 370 mmol / L, more preferably 360 mmol / L, but is not limited thereto. In addition, calcium acetate may be included in an amount of 4 to 8 mmol / L, preferably 5 to 7 mmol / L, more preferably 6 mmol / L, but is not limited thereto. In addition, potassium thiocyanate may be included in an amount of 700 to 1200 mmol / L, preferably 800 to 1100 mmol / L, and more preferably 900 mmol / L, but is not limited thereto. Sodium azide may preferably be included in an amount of 0.1% or less.

[0029] In the present invention, the G3-CNP may be included at a concentration of 5 mg / mL or more, preferably at a concentration of 6 mg / mL or more, 7 mg / mL or more, 8 mg / mL or more, 9 mg / mL or more, and more preferably at a concentration of 10 mg / mL or more. There is no particular technical significance to limit the upper limit, but it may preferably be included at a concentration of 30 mg / mL or less, 25 mg / mL or less, or 20 mg / mL or less.

[0030] In the present invention, the above material may be paper or fiber.

[0031] In the present invention, the colorimetric sensor may detect saliva through a reaction with an alpha-amylase (α-amylase) active enzyme contained in the sample.

[0032] In the present invention, the colorimetric sensor may detect saliva through a color change to yellow when saliva is present in the sample, that is, through a color change from colorless to yellow.

[0033] In the present invention, the colorimetric sensor may be one in which saliva is detected and can be used as a sample for gene or DNA analysis, and in particular, may be one in which it can be used as a sample for personal identification through STR profile analysis.

[0034] In addition, the present invention provides a composition for detecting saliva comprising a test strip impregnated with G3-CNP.

[0035] In addition, the present invention provides a saliva detection method comprising the steps of: contacting a colorimetric sensor for saliva detection, comprising a test strip impregnated with G3-CNP, with a biological sample; and checking whether there is a color change of the colorimetric sensor.

[0036] In the present invention, the method may be confirmed through a color change to yellow when saliva is present in the sample, that is, a color change from colorless to yellow.

[0037] In the present invention, the method may involve confirming a color change within 5 minutes, preferably within 3 minutes, through visual inspection or image collection, but is not limited thereto.

[0039] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited to these examples.

[0041] Example 1. Substrate selection for detection of alpha-amylase (α-amylase) active enzyme

[0042] For substrate selection for alpha-amylase activity detection, Gal-G2-CNP (α-(2-chloro-4-nitrophenyl)-β-1,4-galactopyranosylmaltoside), G3-CNP (2-chloro-4-nitrophenyl-α-D-maltotrioside), and SALIgAE ® Color changes were observed after reacting (Abacus Diagnostics) with various concentrations of alpha-amylase (20, 50, 75, 100, 200, 300, 400, and 500 μg / mL) for 3 minutes. Images of the color changes were collected using an Epson Perfection V600 photo scanner, and RGB colors were extracted using PowerPoint. The extracted values ​​were plotted on a graph using the Euclidean distance equation.

[0043] As a result, as shown in Figure 1, G3-CNP exhibited a rapid color change even at lower concentrations of alpha-amylase compared to Gal-G2-CNP, and showed a pattern similar to that of the positive control, SALIgAE. Thus, it was confirmed that G3-CNP can be used as a substrate for the fabrication of a colorimetric sensor for saliva detection in the present invention.

[0045] Example 2. Preparation of a liquid buffer

[0046] For the liquid buffer, sodium chloride, calcium acetate, potassium thiocyanate, and sodium azide were added to MES buffer. MES extends the shelf life of the buffer, sodium chloride and potassium thiocyanate influence amylase activity, calcium acetate increases salivary amylase yield, and sodium azide acts as an activator and preservative. The pH of the buffer was adjusted with 1M sodium hydroxide (NaOH). The composition of the liquid buffer is shown in Table 1 below.

[0048] Reactive Ingredients Concentration Sodium Chloride 330-370 mmol / L Calcium Acetate 5-7 mmol / L Potassium Thiocyanate 800-1100 mmol / L Sodium Azide < 0.1% MES buffer 90-110 mmol / L 1M NaOH 0.001 mmol / L

[0050] Since the pH of the buffer can affect the reactivity between the substrate and amylase, color changes were observed after reacting with alpha-amylase in buffers containing G3-CNP at various pH conditions (pH 5.0, pH 5.5, pH 5.7, pH 6.0, pH 6.3, pH 6.5, pH 6.7, and pH 7.0). Images of the color changes were collected using an Epson Perfection V600 photo scanner, and RGB colors were extracted using PowerPoint. The extracted values ​​were then plotted on a graph using the Euclidean distance equation.

[0051] As a result, as shown in Figure 2, the buffer containing G3-CNP showed the best reactivity at pH 6.5, decreased to about 61% at pH 6.0, pH 6.3, and pH 6.7, and decreased to about 35% or less at pH 5.3, pH 5.7, and pH 7.0. Thus, the pH of the buffer containing G3-CNP was adjusted to pH 6.0 to pH 6.7.

[0053] Example 3. Paper Selection

[0054] Paper (glass fiber filter and Whatman Chromatography paper) impregnated with a buffer containing G3-CNP was reacted with various concentrations of alpha-amylase (20, 50, 75, 100, 200, 300, 400, and 500 μg / mL), and the color change of the paper was observed. Images of the color changes were collected using an Epson Perfection V600 photo scanner, and RGB colors were extracted using PowerPoint. The extracted values ​​were plotted on a graph using the Euclidean distance equation.

[0055] As a result, as shown in Figure 3, it was confirmed that saliva can be detected even on paper by reacting with a G3-CNP substrate and alpha-amylase. In particular, it was confirmed that in Whatman Chromatography paper, the reaction proceeded even in a sample containing a low concentration of alpha-amylase of 50 μg / mL. Thus, the present invention has confirmed that saliva can be detected economically, simply, and rapidly by using paper as a substrate for fabricating a colorimetric sensor for saliva detection.

[0057] Example 4. Optimization of substrate concentration

[0058] Paper impregnated with buffers containing various concentrations of G3-CNP (5, 10, 15, and 20 mg / mL) was reacted with various concentrations of alpha-amylase (20, 50, 70, 100, 200, 300, 400, and 500 μg / mL), and the color change of the paper was observed. Images of the color changes were collected using an Epson Perfection V600 photo scanner, and RGB colors were extracted using PowerPoint. The extracted values ​​were plotted on a graph using the Euclidean distance equation.

[0059] As a result, as shown in Figure 4, the G3-CNP substrate reacted with alpha-amylase in a concentration-dependent manner. In particular, for samples containing 300 μg / mL or more of alpha-amylase, the best reactivity was observed with 20 mg / mL of G3-CNP, whereas for samples containing 200 μg / mL or less of alpha-amylase, similar reactivity was observed with G3-CNP substrates ranging from 10 to 20 mg / mL. Thus, it was confirmed that a colorimetric sensor can be fabricated using G3-CNP substrates with a concentration of 5 mg / mL or more, preferably 10 mg / mL or more.

[0061] Example 5. Fabrication of a paper-based colorimetric sensor (Sal-ID)

[0062] In the present invention, as an example of a paper-based colorimetric sensor (Sal-ID), paper (Whatman Chromatography paper) was impregnated with a liquid buffer containing G3-CNP while shaking for one day, and then air-dried at room temperature. Subsequently, the paper was prepared with an area (1.5 × 1.5 cm) large enough for a cotton swab to be imprinted. 2 A paper-based colorimetric sensor was prepared by cutting into (Fig. 5).

[0063] In addition, the cover of the paper-based colorimetric sensor (Sal-ID) was made of black paper to prevent contamination of evidence and remain unaffected by light, while the interior was designed to include a saliva test paper, a control group for determining positive or negative results, and a memo section for writing evidence information.

[0065] Example 6. Sensitivity and Specificity Analysis

[0066] To verify the validity of the paper-based colorimetric sensor, experiments were conducted to confirm its sensitivity and specificity. First, to verify the sensitivity of the paper-based colorimetric sensor, saliva samples were prepared by testing samples from 15 subjects twice, resulting in diluted saliva at 1:16, 1:32, 1:64, and 1:128. Subsequently, the saliva samples were reacted with the fabricated paper-based colorimetric sensor, and the color change of the paper was observed.

[0067] As a result, as shown in Fig. 6, positive reactions were observed in the undiluted saliva sample (Neat) and saliva samples diluted to 1:16 and 1:32. In addition, it was confirmed that some positive reactions were observed in saliva samples diluted to 1:64 and 1:128. Thus, it was confirmed that the paper-based colorimetric sensor of the present invention can detect saliva even in saliva samples diluted to 1:128, and has high sensitivity with a detection limit of 0.8% (=1 / 128*100).

[0068] Next, to verify the specificity of the paper-based colorimetric sensor, samples of semen, tears, runny nose, sweat, cat saliva, and dog saliva were prepared. Subsequently, the samples were reacted with the fabricated paper-based colorimetric sensor for 3 minutes, and the color change of the paper was observed. As a result, as shown in Figure 7, no color change was observed in any of the samples when the samples were reacted with the fabricated paper-based colorimetric sensor for 3 minutes.

[0069] In addition, samples of saliva and blood mixed in various ratios (10:1, 5:1, 2:1, 1:1, 1:2, 1:5, and 1:10) were prepared, reacted with the fabricated paper-based colorimetric sensor, and the color change of the paper was observed. Images of the color change were collected using an Epson Perfection V600 photo scanner, and RGB colors were extracted using PowerPoint. The extracted values ​​were then plotted on a graph using the Euclidean distance equation.

[0070] As a result, as shown in Fig. 8, the presence of saliva could be confirmed visually through a color change in samples diluted with saliva and blood in a ratio of 10:1 to 1:1. In addition, when RGB colors were extracted and verified, the presence of saliva could be confirmed up to samples diluted with saliva and blood in a ratio of 1:2.

[0072] Example 7. Results of Short Tandem Repeat (STR) profile analysis from a sample eluted from a colorimetric sensor

[0073] An experiment was conducted to verify whether STR profile analysis was possible from samples eluted by a paper-based colorimetric sensor. After dispensing 20 μL of a subject's saliva onto a cotton swab and observing the color change using a paper-based colorimetric sensor (Sal-ID), the dried saliva sample was punched and eluted using a Spin Basket (Promega Corporation). Subsequently, Direct PCR was performed using a PowerPlex® Fusion System (Promega Corporation), and STRs were analyzed.

[0074] As a result, it was confirmed that STR full profile analysis is possible from the sample eluted from the colorimetric sensor (Table 2 and Fig. 9). Thus, the present invention has confirmed that saliva can be detected from a paper-based colorimetric sensor even when only a trace amount of sample is present, and that personal identification is also possible through STR profile analysis from the same sample eluted therefrom.

[0076] STR Locus Allele Amelogenin X D3S1358 17, 18 D1S1656 14, 15 D2S441 11 D10S1248 13, 16 D13S317 10, 12 Penta E 12, 16 D16S539 11, 14 D18S51 16, 22 D2S1338 18, 19 CSF1PO 11, 13 Penta D 9 TH01 7, 9 vWA 14, 16 D21S11 31, 31.2 D7S820 10, 12 D5S818 10, 13 TPOX 8 DYS391 - D8S1179 11, 14 D12S391 16, 21 D19S433 13, 15.2 FGA 23, 25 D22S1045 16, 17

Claims

Claim 1 A colorimetric sensor for saliva detection comprising a test strip impregnated with G3-CNP (2-chloro-4-nitrophenyl-α-D-maltotrioside), wherein the G3-CNP is contained in an MES buffer containing 330-370 mmol / L sodium chloride, 5-7 mmol / L calcium acetate, 800-1100 mmol / L potassium thiocyanate, and sodium azide, and wherein the colorimetric sensor has a structure in which a sample eluted by punching a test strip in which a colorimetric reaction occurs upon contact with saliva has a structure that can be used for STR profile analysis through PCR. Claim 2 A colorimetric sensor according to claim 1, wherein the test sheet is a substrate impregnated with G3-CNP. Claim 3 delete Claim 4 A colorimetric sensor according to claim 1, wherein the G3-CNP is included at a concentration of 5 mg / mL or more. Claim 5 A colorimetric sensor according to claim 2, wherein the above description is paper or fiber. Claim 6 In claim 1, the colorimetric sensor detects saliva through a reaction with an alpha-amylase (α-amylase) active enzyme contained in a sample. Claim 7 In claim 1, the colorimetric sensor detects saliva through a yellow color change when saliva is present in a sample. Claim 8 delete Claim 9 delete Claim 10 A saliva detection method comprising: a step of contacting a colorimetric sensor according to any one of claims 1, 2, 4 to 7 with a sample; and a step of checking whether there is a color change of the colorimetric sensor. Claim 11 In claim 10, the method is a method of confirming the presence of saliva in a sample through a color change to yellow. Claim 12 In claim 10, the method is to confirm a color change within 5 minutes through visual inspection or image collection.

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