Kits for detecting content of fluoride ions in microsamples

Abstract

Disclosed is a kit for detecting content of fluoride ions in a microsample, including: at least one 96-well plate, reagent A, reagent B, reagent C, reagent D, reagent E and a fluoride standard solution having a concentration of 2.5 mg/L. The kit can be used to effectively overcome the uncertainties in the existing methods for detecting fluoride ions, and also involves rapid and convenient operation. Moreover, this method involves simple and rapid operation, the use of a small amount of a sample and simultaneous detection of multiple samples. This kit provides a more standardized detection to lower the human error, thereby allowing for a more reliable result and for a suitable application in the on-site detection of content of fluoride ions in various environments such as in water quality engineering or in the laboratory.

Claims

1. A kit for detecting content of fluoride ions in a microsample, comprising: at least one 96-well plate; and a detecting liquid; wherein the at least one 96-well plate comprises 8 main wells which are wells A, B, C, D, E, F, G and H, respectively; the 8 main wells are filled with fluoride solution with different concentrations of fluoride ions; concentrations of the fluoride ions in the 8 main wells are 0 mg/L in well A, 0.039 mg/L in well B, 0.078 mg/L in well C, 0.156 mg/L in well D, 0.313 mg/L in well E, 0.625 mg/L in well F, 1.25 mg/L in well G and 2.5 mg/L in well H, respectively; the detecting liquid is a mixture of 7 parts by volume of reagent A, 2 parts by volume of reagent B, 2 parts by volume of reagent C, 2 parts by volume of reagent D and 2 parts by volume of reagent E; wherein the reagent A is an analytical acetone; the reagent B is an analytical acetylacetone; the reagent C is a solution having a pH of 5.0 and containing 0.05 mol/L of alizarin complexone; the reagent D is a solution having a pH of 4.1 and containing sodium acetate; and the reagent E is a solution having a pH of 4.1 and containing 0.05 mol/L of lanthanum nitrate.

2. The kit of claim 1, wherein the reagent C is prepared by a method comprising the following steps: adding 1.927 g of the alizarin complexone to a 100 mL beaker; adding 5 mL of deionized water by a micropipette; then dropwise adding 5-15 mL of 1 mol/L sodium hydroxide solution to dissolve the alizarin complexone; after the alizarin complexone is dissolved, adding 0.625 g of sodium acetate to produce a mixture, and adjusting the mixture to pH 5.0 with 1 mol/L hydrochloric acid followed by dilution to 100 mL with deionized water to produce the reagent C containing 0.05 mol/L of the alizarin complexone.

3. The kit of claim 1, wherein the reagent D is prepared by a method comprising the following steps: dissolving 3.5 g of sodium acetate in 80 mL of deionized water; adding 7.5 mL of glacial acetic acid followed by dilution to 100 mL with deionized water to produce a mixture; and adjusting the mixture to pH 4.1 with an acetic acid solution or a sodium hydroxide solution using a pH meter to produce the reagent D.

4. The kit of claim 1, wherein the reagent E is prepared by a method comprising the following steps: weighing 2.215 g of solid lanthanum nitrate; dropwise adding 3-8 mL of a hydrochloric acid solution to dissolve the solid lanthanum nitrate to produce a mixture; and adjusting the mixture to pH 4.1 with 1 mol/L sodium acetate followed by dilution to 100 mL with deionized water to obtain the reagent E containing 0.05 mol/L of the lanthanum nitrate.

Description

DETAILED DESCRIPTION OF EMBODIMENTS

(1) The application will be further described below with reference to the embodiments, and the advantages and features of the application will become more apparent. However, these embodiments are merely illustrative of the invention and are not intended to limit the invention. It should be understood that any modifications and replacements made by those skilled in the art without departing from the spirit and scope of the invention should still fall within the scope of the invention.

Example 1 Preparation of a Kit

(2) The kit included a 96-well plate, reagent A, reagent B, reagent C, reagent D, reagent E and a fluoride standard solution having a concentration of 2.5 mg/L, where the reagent A was an analytical acetone and the reagent B was an analytical acetylacetone.

(3) The reagent C was prepared as follows. 1.927 g of alizarin complexone was weighed in a 100 mL beaker, added with 5 mL of deionized water by a micropipette, and then dropwise added with 5-15 mL of 1 mol/L sodium hydroxide solution to dissolve the alizarin complexone. After the alizarin complexone was dissolved, 0.625 g of sodium acetate was added to produce a mixture. The mixture was adjusted to pH 5.0 with 1 mol/L hydrochloric acid and diluted to 100 mL with deionized water to produce the reagent C containing 0.05 mol/L of the alizarin complexone.

(4) The reagent D was prepared as follows. 3.5 g of sodium acetate was dissolved in 80 mL of deionized water, added with 7.5 mL of glacial acetic acid, diluted to 100 mL with deionized water and adjusted to pH 4.1 with an acetic acid solution or a sodium hydroxide solution using a pH meter to produce the reagent D.

(5) The reagent E was prepared as follows. 2.215 g of solid lanthanum nitrate was weighed, dropwise added with 3-8 mL of a hydrochloric acid solution, adjusted to pH 4.1 with a sodium acetate solution (1 mol/L) and diluted to 100 mL with deionized water to produce the reagent E containing 0.05 mol/L of the lanthanum nitrate.

Example 2

(6) A fluoride standard solution having a concentration of 0.5 mg/L was used herein as sample 1 and the kit prepared in Example 1 was employed to detect the content of fluoride ions in sample 1, where 8 main wells were selected for the plotting of a standard curve and 20 secondary wells were selected for the detection of sample 1.

(7) (1) Preparation of a Detecting Liquid

(8) 2,100 μL of the reagent A, 600 μL of the reagent B, 600 μL of the reagent C and 600 μL of the reagent D were mixed uniformly, added with 600 μL of the reagent E and mixed uniformly to produce the detecting liquid for use.

(9) (2) Loading 8 wells in a 96-well microplate were selected as main wells, where the last well, i.e., the well H, was added with 200 μL of the 2.5 mg/L fluoride standard solution and the wells A-G were respectively added with 100 μL of deionized water. Then 100 μL of the fluoride standard solution in the well H was accurately transferred to the well G by a micropipette and mixed to produce a mixture and 100 μL of the mixture G was accurately transferred by a micropipette to the well F and mixed to produce a mixture F. The rest main wells were sequentially treated in the same manner, until 100 μL of the mixture B was discarded instead of transferring it to the well A. Thus, the liquid in each main well was 100 μL, and fluoride ion concentrations in the wells A-H were 0 mg/L, 0.039 mg/L, 0.078 mg/L, 0.156 mg/L, 0.313 mg/L, 0.625 mg/L, 1.25 mg/L and 2.5 mg/L, respectively.

(10) 20 wells among the rest wells in the 96-well microplate were selected as secondary wells and respectively added with 100 μL of the sample 1 (a fluoride standard solution having a known concentration of 0.5 mg/L).

(11) (3) Detection of Content of Fluoride Ions

(12) 150 μL of the detecting liquid prepared in (2) was separately added to all of the 28 wells for reaction. After 5 min, the 96-well microplate was transferred to a microplate reader, and the absorbance was measured at 650 nm within 20 min. The results were shown in Table 1.

(13) TABLE-US-00001 TABLE 1 Absorbance of the sample 1 at 650 nm Column 2 Column 3 Column 4 Column 1 (main (secondary (secondary (secondary ID wells) wells) wells) wells) A 0.092 0.141 0.142 0.141 B 0.096 0.142 0.142 0.143 C 0.099 0.142 0.141 0.142 D 0.107 0.142 0.142 0.141 E 0.121 0.141 0.141 F 0.152 0.142 0.141 G 0.212 0.143 0.142 H 0.338 0.141 0.143

(14) A standard curve was plotted based on the fluoride contents in the wells A-H in the column 1 (respectively 0 mg/L, 0.039 mg/L, 0.078 mg/L, 0.156 mg/L, 0.313 mg/L, 0.625 mg/L, 1.250 mg/L and 2.500 mg/L) and the corresponding absorbance, where the regression equation was y=0.0982x-0.0008 with a regression coefficient R.sup.2 of 0.9998.

(15) The fluoride contents in the 20 secondary wells were calculated according to the above equation and shown in Table 2.

(16) TABLE-US-00002 TABLE 2 Fluoride contents (mg/L) of the sample 1 Column 2 Column 3 Column 4 Column 1 (main (secondary (secondary (secondary ID wells) wells) wells) wells) A 0 0.500 0.510 0.500 B 0.039 0.510 0.510 0.520 C 0.078 0.510 0.500 0.510 D 0.156 0.510 0.510 0.500 E 0.313 0.500 0.500 F 0.625 0.510 0.500 G 1.250 0.520 0.510 H 2.500 0.500 0.520

(17) According to the above results, the average fluoride content of the sample 1 was calculated as 0.508 mg/L with standard deviation of 0.007 mg/L and variation coefficient of 1.38%.

Example 3

(18) A fluoride standard solution having a concentration of 1.0 mg/L was used herein as sample 2 and the kit prepared in Example 1 was employed to detect the content of fluoride ions in sample 2, where 8 main wells were selected for the plotting of a standard curve and 20 secondary wells were selected for the detection of sample 2.

(19) (1) Preparation of a Detecting Liquid

(20) 2,100 μL of the reagent A, 600 μL of the reagent B, 600 μL of the reagent C and 600 μL of the reagent D were mixed uniformly, added with 600 μL of the reagent E and mixed uniformly to produce the detecting liquid for use.

(21) (2) Loading

(22) 8 wells in a 96-well microplate were selected as main wells, where the last main well, i.e., the well H, was added with 200 μL of the 2.5 mg/L fluoride standard solution and the wells A-G were respectively added with 100 μL of deionized water. Then 100 μL of the fluoride standard solution in the well H was accurately transferred to the well G by a micropipette and mixed to produce a mixture G, and 100 μL of the mixture G was accurately transferred by a micropipette to the well F and mixed to produce a mixture F. The rest main wells were sequentially treated in the same manner, until 100 μL of the mixture B was discarded instead of transferring it to the well A. Thus, the liquid in each main well was 100 μL, and fluoride ion concentrations in the wells A-H were 0 mg/L, 0.039 mg/L, 0.078 mg/L, 0.156 mg/L, 0.313 mg/L, 0.625 mg/L, 1.25 mg/L and 2.5 mg/L, respectively.

(23) 20 wells among the rest wells in the 96-well microplate were selected as secondary wells and respectively added with 100 μL of the sample 2 (a fluoride standard solution having a known concentration of 1.0 mg/L).

(24) (3) Detection of Content of Fluoride Ions

(25) 150 μL of the detecting liquid prepared in (2) was separately added to all of the 28 wells for reaction. After 5 min, the 96-well microplate was transferred to a microplate reader, and the absorbance was measured at 650 nm within 20 min. The results were shown in Table 3.

(26) TABLE-US-00003 TABLE 3 Absorbance of the sample 2 at 650 nm Column 2 Column 3 Column 4 Column 1 (main (secondary (secondary (secondary ID wells) wells) wells) wells) A 0.086 0.180 0.179 0.180 B 0.089 0.180 0.180 0.179 C 0.093 0.179 0.181 0.179 D 0.102 0.180 0.180 0.180 E 0.114 0.181 0.180 F 0.143 0.180 0.180 G 0.205 0.181 0.180 H 0.321 0.181 0.181

(27) A standard curve was plotted based on the fluoride contents in the wells A-H in the column 1 (respectively 0 mg/L, 0.039 mg/L, 0.078 mg/L, 0.156 mg/L, 0.313 mg/L, 0.625 mg/L, 1.250 mg/L and 2.500 mg/L) and the corresponding absorbance, where the regression equation was y=0.0938x-0.0009 with a regression coefficient R.sup.2 of 0.9997.

(28) The fluoride contents in the 20 secondary wells were calculated according to the above equation and shown in Table 4.

(29) TABLE-US-00004 TABLE 4 Fluoride contents of the sample 2 (mg/L) Column 2 Column 3 Column 4 Column 1 (main (secondary (secondary (secondary ID wells) wells) wells) wells) A 0.000 1.000 0.990 1.000 B 0.039 1.000 1.000 0.990 C 0.078 0.990 1.010 0.990 D 0.156 1.000 1.000 1.000 E 0.313 1.010 1.000 F 0.625 1.000 1.000 G 1.250 1.010 1.000 H 2.500 1.010 1.010

(30) According to the above results, the average fluoride content of the sample 2 was calculated as 1.001 mg/L with standard deviation of 0.007 mg/L and variation coefficient of 0.669%.

Example 4

(31) A fluoride standard solution having a concentration of 1.5 mg/L was used herein as sample 3 and the kit prepared in Example 1 was employed to detect the content of fluoride ions in sample 3, where 8 main wells were selected for the plotting of a standard curve and 20 secondary wells were selected for the detection of sample 3.

(32) (1) Preparation of a Detecting Liquid

(33) 2,100 μL of the reagent A, 600 μL of the reagent B, 600 μL of the reagent C and 600 μL of the reagent D were mixed uniformly, added with 600 μL of the reagent E and mixed uniformly to produce the detecting liquid for use.

(34) (2) Loading

(35) 8 wells in a 96-well microplate were selected as main wells, where the last well, i.e., the well H, was added with 200 μL of the 2.5 mg/L fluoride standard solution and the wells A-G were respectively added with 100 μL of deionized water. Then 100 μL of the fluoride standard solution in the well H was accurately transferred to the well G by a micropipette and mixed to produce a mixture and 100 μL of the mixture G was accurately transferred by a micropipette to the well F and mixed to produce a mixture F. The rest main wells were sequentially treated in the same manner, until 100 μL of the mixture B was discarded instead of transferring it to the well A. Thus, the liquid in each main well was 100 μL, and fluoride ion concentrations in the wells A-H were 0 mg/L, 0.039 mg/L, 0.078 mg/L, 0.156 mg/L, 0.313 mg/L, 0.625 mg/L, 1.25 mg/L and 2.5 mg/L, respectively.

(36) 20 wells among the rest wells in the 96-well microplate were selected as secondary wells and respectively added with 100 μL of the sample 3 (a fluoride standard solution having a known concentration of 1.5 mg/L).

(37) (3) Detection of Content of Fluoride Ions

(38) 150 μL of the detecting liquid prepared in step (2) was separately added to all of the 28 wells for reaction. After 5 min, the 96-well microplate was transferred to a microplate reader, and the absorbance was measured at 650 nm within 20 min. The results were shown in Table 5.

(39) TABLE-US-00005 TABLE 5 Absorbance of the sample 3 at 650 nm Column 2 Column 3 Column 4 Column 1 (main (secondary (secondary (secondary ID wells) wells) wells) wells) A 0.092 0.239 0.240 0.237 B 0.096 0.240 0.239 0.239 C 0.099 0.239 0.237 0.239 D 0.107 0.239 0.238 0.238 E 0.121 0.240 0.239 F 0.152 0.238 0.240 G 0.212 0.239 0.237 H 0.338 0.241 0.239

(40) A standard curve was plotted based on the fluoride contents in the wells A-H in the column 1 (respectively 0 mg/L, 0.039 mg/L, 0.078 mg/L, 0.156 mg/L, 0.313 mg/L, 0.625 mg/L, 1.250 mg/L and 2.500 mg/L) and the corresponding absorbance, where the regression equation was y=0.0982x-0.0008 with a regression coefficient R.sup.2 of 0.9998.

(41) The fluoride contents in the 20 secondary wells were calculated according to the above equation and shown in Table 6.

(42) TABLE-US-00006 TABLE 6 Fluoride contents (mg/L) of the sample 3 Column 2 Column 3 Column 4 Column 1 (main (secondary (secondary (secondary ID wells) wells) wells) wells) A 0.000 1.500 1.510 1.480 B 0.039 1.510 1.500 1.500 C 0.078 1.500 1.480 1.500 D 0.156 1.500 1.489 1.489 E 0.313 1.510 1.500 F 0.625 1.489 1.510 G 1.250 1.500 1.480 H 2.500 1.520 1.500

(43) According to the above results, the average fluoride content of the sample 2 was calculated as 1.498 mg/L with standard deviation of 0.011 mg/L and variation coefficient of 0.717%.

Example 5

(44) A fluoride standard solution having a concentration of 2.0 mg/L was used herein as sample 4 and the kit prepared in Example 1 was employed to detect the content of fluoride ions in sample 4, where 8 main wells were selected for the plotting of a standard curve and 20 secondary wells were selected for the detection of sample 4.

(45) (1) Preparation of a Detecting Liquid

(46) 2,100 μL of the reagent A, 600 μL of the reagent B, 600 μL of the reagent C and 600 μL of the reagent D were mixed uniformly, added with 600 μL of the reagent E and mixed uniformly to produce the detecting liquid for use.

(47) (2) Loading

(48) 8 wells in a 96-well microplate were selected as main wells, where the last well, i.e., the well H, was added with 200 μL of the 2.5 mg/L fluoride standard solution and the wells A-G were respectively added with 100 μL of deionized water. Then 100 μL of the fluoride standard solution in the well H was accurately transferred to the well G by a micropipette and mixed to produce a mixture and 100 μL of the mixture G was accurately transferred by a micropipette to the well F and mixed to produce a mixture F. The rest main wells were sequentially treated in the same manner, until 100 μL of the mixture B was discarded instead of transferring it to the well A. Thus, the liquid in each main well was 100 μL, and fluoride ion concentrations in the wells A-H were 0 mg/L, 0.039 mg/L, 0.078 mg/L, 0.156 mg/L, 0.313 mg/L, 0.625 mg/L, 1.25 mg/L and 2.5 mg/L, respectively.

(49) 20 wells among the rest wells in the 96-well microplate were selected as secondary wells and respectively added with 100 μL of the sample 4 (a fluoride standard solution having a known concentration of 2.0 mg/L).

(50) (3) Detection of Content of Fluoride Ions

(51) 150 μL of the detecting liquid four prepared in step (2) was separately added to all of the 28 wells for reaction. After 5 min, the 96-well microplate was transferred to a microplate reader, and the absorbance was measured at 650 nm within 20 min. The results were shown in Table 7.

(52) TABLE-US-00007 TABLE 7 Absorbance of the sample 4 at 650 nm Column 2 Column 3 Column 4 Column 1 (main (secondary (secondary (secondary ID wells) wells) wells) wells) A 0.090 0.282 0.282 0.283 B 0.094 0.281 0.283 0.282 C 0.097 0.281 0.282 0.281 D 0.104 0.282 0.281 0.280 E 0.119 0.283 0.280 F 0.150 0.282 0.282 G 0.210 0.280 0.282 H 0.331 0.280 0.283

(53) A standard curve was plotted based on the fluoride contents in the wells A-H in the column 1 (respectively 0 mg/L, 0.039 mg/L, 0.078 mg/L, 0.156 mg/L, 0.313 mg/L, 0.625 mg/L, 1.250 mg/L and 2.500 mg/L) and the corresponding absorbance, where the regression equation was y=0.0965x-0.0005 with a regression coefficient R.sup.2 of 1.

(54) The fluoride contents in the 20 secondary wells were calculated according to the above equation and shown in Table 8.

(55) TABLE-US-00008 TABLE 8 Fluoride contents of the sample 4 (mg/L) Column 2 Column 3 Column 4 Column 1 (main (secondary (secondary (secondary ID wells) wells) wells) wells) A 0.000 2.000 2.000 2.010 B 0.039 1.989 2.010 2.000 C 0.078 1.989 2.000 1.989 D 0.156 2.000 1.989 1.980 E 0.313 2.010 1.980 F 0.625 2.000 2.000 G 1.250 1.980 2.000 H 2.500 1.980 2.010

(56) According to the above results, the average fluoride content of the sample 2 was calculated as 1.996 mg/L with standard deviation of 0.010 mg/L and variation coefficient of 0.518%.

(57) It can be seen from the above examples that the kit of the invention for detecting the content of fluoride ions in a microsample has desirable accuracy and reproducibility.