Preparation of coumarin derivatives and method for ratiometric detection of cysteine

Abstract

The present invention provides a method for preparing a coumarin derivative and detecting cysteine ​​by ratiometric analysis, belonging to the technical field of coumarin derivatives. The coumarin derivatives provided herein can target lysosomes and detect Cys in lysosomes. Furthermore, as ratiometric probes, by observing the ratio of two non-interfering fluorescence signals of the ratiometric probe, interferences such as concentration, instrument fluctuations, and background fluorescence can be minimized. The method exhibits advantages such as high sensitivity, good selectivity, and a long response time. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the technical field of coumarin derivatives, in particular to a coumarin derivative, a preparation method and application thereof, and a method for ratiometric detection of cysteine. Background Art

[0002] A growing body of research indicates that lysosomes, acting as intracellular sensors, can receive signals from other intracellular organelles or metabolic pathways, make adjustments, and send signals to the cytoplasm and nucleus to regulate specific metabolic processes within the cell. Maintaining an acidic pH plays a crucial role in the two basic functions of lysosomes: degradation and recycling of substances. Cystinosis is the first documented genetic disease caused by abnormal lysosomal transport function. Lysosomal cystine is the main source of intracellular cysteine, and a pH gradient is required for cysteine ​​to enter lysosomes. Therefore, designing a fluorescent probe that can accurately detect changes in cysteine ​​content in lysosomes has important research significance and application value for understanding the process of cysteine ​​entering lysosomes and abnormal lysosomal transport function. Summary of the Invention

[0003] In view of this, the present invention aims to provide a coumarin derivative and its preparation method and application, as well as a method for ratiometric detection of cysteine. The coumarin derivative provided by the present invention can target lysosomes and detect Cys in lysosomes.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a coumarin derivative having a structure shown in Formula I:

[0006]

[0007] The present invention also provides a method for preparing the coumarin derivatives described in the above technical solution, comprising the following steps:

[0008] mixing 7-(diethylamino)coumarin with an organic solvent to obtain a 7-(diethylamino)coumarin solution;

[0009] Mixing POCl3 with an organic solvent to obtain a formylating agent;

[0010] adding the 7-(diethylamino)coumarin solution dropwise to the formylation reagent to carry out a formylation reaction to obtain 7-(diethylamino)coumarin-3-carboxaldehyde;

[0011] The 7-(diethylamino)coumarin-3-carboxaldehyde, 3-morpholine-3-oxopropionitrile, piperidine and an organic solvent are mixed to carry out a Knoevenagel condensation reaction to obtain the coumarin derivative.

[0012] Preferably, the molar ratio of 7-(diethylamino)coumarin to POCl3 is 1:2 to 1:3.

[0013] Preferably, the formylation reaction is carried out at a temperature of 55 to 65° C. and for a time of 8 to 16 hours.

[0014] Preferably, the molar ratio of 7-(diethylamino)coumarin-3-carboxaldehyde to 3-morpholine-3-oxopropionitrile is 1:1 to 1:2.

[0015] Preferably, the Knoevenagel condensation reaction is carried out at a temperature of 70 to 90° C. and for a time of 4 to 6 hours.

[0016] The present invention also provides the use of the coumarin derivative described in the above technical solution or the coumarin derivative prepared by the preparation method described in the above technical solution in the qualitative detection of cysteine.

[0017] The present invention also provides the use of the coumarin derivative described in the above technical solution or the coumarin derivative prepared by the preparation method described in the above technical solution in ratiometric detection of cysteine.

[0018] The present invention provides a method for ratiometric detection of cysteine, comprising the following steps:

[0019] After mixing a test solution, a PBS buffer solution, and a coumarin derivative solution, fluorescence detection is performed at 490 nm and 572 nm, respectively, and the ratio of the fluorescence intensities at 490 nm and 572 nm is calculated, wherein the coumarin derivative in the coumarin derivative solution is the coumarin derivative described in the above technical solution or the coumarin derivative prepared by the preparation method described in the above technical solution, and the test solution contains cysteine;

[0020] The concentration of cysteine ​​in the solution to be tested is calculated using a standard curve, wherein the standard curve uses the concentration of cysteine ​​as the horizontal axis and the concentration of F as the horizontal axis. 490 / F 572 Is the vertical axis.

[0021] Preferably, the pH value of the PBS buffer solution is 4.0-8.0.

[0022] The present invention provides a coumarin derivative. Compared with the prior art, the present invention has the following advantages and effects:

[0023] The coumarin derivatives provided by the present invention can target lysosomes and detect Cys in lysosomes. As ratiometric probes, by observing the ratio of two non-interfering fluorescence of the ratiometric probe, interferences such as concentration, instrument fluctuations, and background fluorescence can be minimized. The coumarin derivatives have the advantages of high sensitivity, good selectivity, and long response time.

[0024] The present invention also provides a method for preparing the coumarin derivatives. The method has simple synthesis steps, low cost and low toxicity.

[0025] The present invention also provides a method for ratiometric detection of cysteine. The detection method of the present invention is simple and quick and can be implemented with the aid of a fluorescence spectrometer. The present invention adopts dual-channel detection, and the detection signal is obvious and highly specific. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the H NMR spectrum of the coumarin derivative ECMA prepared in Example 1;

[0027] Figure 2 This is the C NMR spectrum of the coumarin derivative ECMA prepared in Example 1;

[0028] Figure 3 This is the mass spectrum of the coumarin derivative ECMA prepared in Example 1;

[0029] Figure 4 This is the fluorescence emission diagram of the coumarin derivative ECMA interacting with Cys at 572 nm;

[0030] Figure 5 This is the fluorescence emission diagram of the coumarin derivative ECMA interacting with Cys at 490 nm;

[0031] Figure 6 is the fluorescence intensity ratio of coumarin derivative ECMA (F 490 / F 572 ) and the linear relationship between the Cys concentration;

[0032] Figure 7 This is a histogram of the fluorescence intensity changes at 490 nm when the coumarin derivative ECMA reacts with various interfering substances;

[0033] Figure 8 This is the kinetic diagram of the interaction between the coumarin derivative ECMA and Cys;

[0034] Figure 9 This is the fluorescence emission diagram of the coumarin derivative ECMA interacting with Cys at different pH;

[0035] Figure 10 This is the fluorescence emission diagram of the coumarin derivative ECMA and Cys addition product reacting with H2O2 at 490nm;

[0036] Figure 11 This is a cell imaging diagram for the determination of Cys by ECMA of coumarin derivatives;

[0037] Figure 12This is a cell imaging diagram of the interaction between the coumarin derivative ECMA and Cys addition product and H2O2;

[0038] Figure 13 This is a map of the lysosomal localization of the coumarin derivative ECMA. DETAILED DESCRIPTION

[0039] The present invention provides a coumarin derivative having a structure shown in Formula I:

[0040]

[0041] The coumarin derivative provided by the present invention has the Chinese name (E)-3-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-2-(morpholine-4-carbonyl)acrylonitrile, and the English name (E)-3-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-2-(morpholine-4-carbonyl)acrylonitrile, named ECMA. The probe is a lysosome-targeted ratiometric cysteine ​​(Cysteine, Cys) fluorescent probe, which has the advantages of high sensitivity, good selectivity and fast response speed for lysosomal cysteine ​​detection.

[0042] The present invention also provides a method for preparing the coumarin derivatives described in the above technical solution, comprising the following steps:

[0043] mixing 7-(diethylamino)coumarin with an organic solvent to obtain a 7-(diethylamino)coumarin solution;

[0044] Mixing POCl3 with an organic solvent to obtain a formylating agent;

[0045] adding the 7-(diethylamino)coumarin solution dropwise to the formylation reagent to carry out a formylation reaction to obtain 7-(diethylamino)coumarin-3-carboxaldehyde;

[0046] The 7-(diethylamino)coumarin-3-carboxaldehyde, 3-morpholine-3-oxopropionitrile, piperidine and an organic solvent are mixed to carry out a Knoevenagel condensation reaction to obtain the coumarin derivative.

[0047] In the present invention, unless otherwise specified, the raw materials used are commercially available products in the art.

[0048] The invention mixes 7-(diethylamino)coumarin with an organic solvent to obtain a 7-(diethylamino)coumarin solution.

[0049] In the present invention, the organic solvent is preferably N,N-dimethylformamide (DMF).

[0050] In the present invention, the 7-(diethylamino)coumarin is preferably a commercially available product with a CAS number of 20571-42-0.

[0051] Preferably, 0.48 g of 7-(diethylamino)coumarin is mixed with 4 mL of DMF.

[0052] The present invention mixes POCl3 with an organic solvent to obtain a formylating agent.

[0053] In the present invention, 0.6 mL of POCl 3 is preferably added dropwise to 0.8 mL of DMF, followed by stirring and mixing to obtain the formylating agent.

[0054] The present invention has no particular limitation on the stirring time and speed, as long as the raw materials can be mixed evenly.

[0055] After obtaining a 7-(diethylamino)coumarin solution and a formylating agent, the present invention adds the 7-(diethylamino)coumarin solution dropwise to the formylating agent to carry out a formylation reaction (Vilsmeier formylation reaction) to obtain 7-(diethylamino)coumarin-3-carboxaldehyde. The principle of the formylation reaction is shown in the following formula:

[0056]

[0057] In the present invention, the molar ratio of 7-(diethylamino)coumarin to POCl3 is preferably 1:2 to 1:3, more preferably 1:2.8.

[0058] In the present invention, the dripping rate is preferably 1 to 3 drops / second. The purpose of the dripping is to prevent the reactants from agglomerating due to the formylation reaction, which is not conducive to further reaction.

[0059] In the present invention, the temperature of the formylation reaction is preferably 55 to 65° C., more preferably 60° C., and the time is preferably 8 to 16 hours, more preferably 12 hours.

[0060] After the formylation reaction is completed, the present invention preferably sequentially quenches the reaction with ice water, adjusts the pH value to 5-6, filters the crude product, washes with water, and vacuum-dries to obtain the formylation product.

[0061] The present invention is not particularly limited to the specific methods of quenching the reaction, adjusting the pH value, filtering, washing with water, and vacuum drying, and methods well known to those skilled in the art can be used. In a specific embodiment of the present invention, the pH value is preferably adjusted using a sodium hydroxide solution, and the concentration of the sodium hydroxide solution is preferably 20 wt%.

[0062] After obtaining 7-(diethylamino)coumarin-3-carboxaldehyde, the present invention mixes the 7-(diethylamino)coumarin-3-carboxaldehyde, 3-morpholine-3-oxopropionitrile, piperidine and an organic solvent to carry out a Knoevenagel condensation reaction to obtain the coumarin derivative.

[0063] In the present invention, the 3-morpholine-3-oxopropionitrile is preferably a commercially available product with a CAS number of 15029-32-0.

[0064] In the present invention, the molar ratio of 7-(diethylamino)coumarin-3-carboxaldehyde to 3-morpholine-3-oxopropionitrile is preferably 1:1 to 1:2, more preferably 1:1.5.

[0065] In the present invention, the usage ratio of 7-(diethylamino)coumarin-3-carboxaldehyde to piperidine is preferably 1 mmol:15 μL.

[0066] In the present invention, the organic solvent is preferably anhydrous ethanol.

[0067] In the present invention, the usage ratio of 7-(diethylamino)coumarin-3-carboxaldehyde to anhydrous ethanol is preferably 1 mmol:10 mL.

[0068] In the present invention, the temperature of the Knoevenagel condensation reaction is preferably 70 to 90° C., more preferably 80° C., and the time is preferably 4 to 6 hours, more preferably 5 hours. The principle of the Knoevenagel condensation reaction is shown in the following formula:

[0069]

[0070] After the Knoevenagel condensation reaction is completed, the present invention preferably performs natural cooling to room temperature, vacuum distillation and column chromatography separation in sequence to obtain the coumarin derivative.

[0071] The present invention has no particular limitation on the specific method of the vacuum distillation, and any method well known to those skilled in the art may be used.

[0072] In the present invention, the eluent for column chromatography separation is preferably a methanol-dichloromethane mixture, and the volume ratio of methanol to dichloromethane in the methanol-dichloromethane mixture is preferably 1:50.

[0073] The present invention also provides the use of the coumarin derivative described in the above technical solution or the coumarin derivative prepared by the preparation method described in the above technical solution in the qualitative detection of cysteine.

[0074] In the present invention, the application preferably comprises the following steps:

[0075] After the test solution, PBS buffer solution and coumarin derivative solution are mixed, fluorescence detection is performed. If the fluorescence intensity at 572 nm decreases and the fluorescence intensity at 490 nm gradually increases, it is said that the test solution contains cysteine.

[0076] In the present invention, the pH value of the PBS buffer solution is preferably 5.

[0077] The present invention also provides the use of the coumarin derivative described in the above technical solution or the coumarin derivative prepared by the preparation method described in the above technical solution in ratiometric detection of cysteine.

[0078] The present invention also provides a method for ratiometric detection of cysteine, comprising the following steps:

[0079] After mixing a test solution, a PBS buffer solution, and a coumarin derivative solution, fluorescence detection is performed at 490 nm and 572 nm, respectively, and the ratio of the fluorescence intensities at 490 nm and 572 nm is calculated, wherein the coumarin derivative in the coumarin derivative solution is the coumarin derivative described in the above technical solution or the coumarin derivative prepared by the preparation method described in the above technical solution, and the test solution contains cysteine;

[0080] The concentration of cysteine ​​in the solution to be tested is calculated using a standard curve, wherein the standard curve uses the concentration of cysteine ​​as the horizontal axis and the concentration of F as the horizontal axis. 490 / F 572 Is the vertical axis.

[0081] In the present invention, the pH value of the PBS buffer solution is preferably 4.0 to 8.0, more preferably 5.0 to 7.0.

[0082] In the present invention, the concentration of cysteine ​​in the test solution is preferably 0 to 200 μM.

[0083] The present invention has no particular limitation on the method for obtaining the standard curve, and any method well known to those skilled in the art may be used.

[0084] In order to further illustrate the present invention, the method provided by the present invention is described in detail below with reference to examples, but they should not be construed as limiting the scope of protection of the present invention.

[0085] Example 1

[0086] Preparation and characterization of ECMA

[0087] At room temperature, 0.6 mL of POCl₃ was slowly added dropwise to 0.8 mL of DMF. After stirring for 2 h, the formylation reagent was prepared. 7-(Diethylamino)coumarin (0.48 g, 2.2 mmol) was dissolved in 4 mL of DMF and added dropwise to the formylation reagent to obtain a suspension. Refluxed at 60°C for 12 h. After the reaction, the mixture was poured into 200 mL of ice water and the pH was adjusted to 5-6, resulting in a large precipitate. The crude product was filtered, washed with water, and dried under vacuum to obtain 7-(diethylamino)coumarin-3-carboxaldehyde as an orange solid. The crude product was used directly in subsequent experiments without further purification.

[0088] 7-(Diethylamino)coumarin-3-carboxaldehyde (0.49 g, 2 mmol) and 3-morpholino-3-oxopropionitrile (0.46 g, 3 mmol) were added to a round-bottom flask containing 20 mL of anhydrous ethanol, followed by 30 μL of piperidine. The mixture was refluxed at 80°C for 5 h, cooled to room temperature, and evaporated under reduced pressure. The solvent was then removed by vortexing to yield a crude product. Column chromatography using methanol / dichloromethane (1 / 50, v / v) as the eluent yielded 0.32 g of an orange-red solid, the target probe, for a yield of 41.9%.

[0089] Structural characterization:

[0090] Proton spectrum: 1 H NMR (600MHz, DMSO-d6) δ8.65(s,1H),7.97(s,1H),7.68(s,1H),7.59(d,J=9.0Hz,1H),7.56(d,J=9.0Hz,1H),7.44(s,1H),6.82(dd,J=9.1,2.5H z,1H),6.79(dd,J=9.0,2.5Hz,1H),6.64(d,J=2.6Hz,1H),6.59(d,J=2.4Hz,1H),3.64-3.59(m,12H),3.53-3.49(m,12H),1.16-1.13(m,12H).( Figure 1 ).

[0091] Carbon spectrum: 13 C NMR (151MHz, DMSO-d6) δ162.99,160.55,157.56,153.22,145.77,144.67,143.56,142.60,132.10,131.86,116. 93,111.07,110.83,110.64,108.12,108.08,103.16,97.02,96.76,66.36,66.05,47.19,44.95,42.31,12.83.( Figure 2 ).

[0092] Mass spectrum: [M+H] + Theoretical value 382.1761, test value 382.1757 ( Figure 3 ).

[0093] Example 2

[0094] Prepare pH=5 PBS buffer solution, prepare 2mM ECMA DMSO solution, prepare 20mM Cys aqueous solution, take 2mL pH=5 PBS buffer solution and 10μL ECMA DMSO solution and add them to the fluorescence cuvette. With the addition of Cys (0-200μM), the fluorescence intensity at 572nm decreases and the fluorescence intensity at 490nm gradually increases. The fluorescence emission graph is shown in Figure 2. Figure 4 、 Figure 5 shown.

[0095] Example 3

[0096] Prepare 20mM Cys solution with distilled water, add pH=5 PBS buffer solution to 2mL fluorescence cuvette, and perform fluorescence titration experiment with different concentrations of Cys. Measure on fluorescence spectrophotometer, with Cys concentration as horizontal axis and F as horizontal axis. 490 / F 572 The ordinate is used to draw a graph and the ratio of Cys concentration to fluorescence intensity (F 490 / F 572 ) has a linear relationship. Fluorescence intensity ratio (F 490 / F 572 The linear relationship between ) and Cys concentration is shown in the figure Figure 6 shown.

[0097] Example 4

[0098] Prepare pH = 5 PBS buffer solution, prepare 2mM ECMA DMSO solution, prepare 20mM Cys aqueous solution; in a fluorescence cuvette, add 2mL pH = 5 PBS buffer solution and 10μL ECMA DMSO solution, then add 10 times equivalent of other analytes and Cys: Gly, Hcy, Glu, Pro, Arg, Asp, Met, Tyr, Lys, Ile, Trp, Ser, Thr, His, Leu, GSH, NaHS, Cys aqueous solution, detect on a fluorescence spectrophotometer, and draw a graph showing the change in fluorescence intensity at 490nm after ECMA reacts with different analytes as shown below. Figure 7 As shown in Figure 3, Cys enhanced the fluorescence intensity of the detection system at 490 nm, while other analytes did not cause any changes in the fluorescence intensity of the detection system.

[0099] Example 5

[0100] Prepare a PBS buffer solution with pH = 5. When 10 μL of ECMA DMSO solution is added to 2 mL of PBS solution, and then 200 μM Cys is added, the fluorescence intensity at 490 nm begins to rise immediately after addition and reaches equilibrium in about 300 seconds. The kinetic diagram of the interaction between ECMA and Cys is shown in Figure 2. Figure 8 shown.

[0101] Example 6

[0102] Prepare PBS buffer solutions of different pH values, take 2mL of PBS buffer solution and 10μL of ECMA DMSO solution and add them to the cuvette. Detect the fluorescence signal of the probe, which remains basically stable in the pH range of 4.0 to 8.0. Add Cys to the probe solution, and the fluorescence signal at 490nm slowly increases between pH 4.0 and 8.0. The fluorescence emission graphs of ECMA and Cys at different pH values ​​are shown in Figure 2. Figure 9 shown.

[0103] Example 7

[0104] 2mL of pH 5 PBS buffer solution and 10μL of ECMA in DMSO were added to a cuvette, and then 200μM Cys was added. Subsequently, H2O2 was gradually added to the reaction system, and the fluorescence intensity at 490nm gradually decreased. The fluorescence emission of the ECMA and Cys addition product reacting with H2O2 at 490nm is shown in the figure. Figure 10 As shown, ECMA can reversibly recognize Cys at pH = 5 and can reflect the redox balance in real time. When the active oxygen species such as hydrogen peroxide in the system increase, the addition of the probe to Cys will proceed in reverse, causing the fluorescence intensity at 490 nm to weaken.

[0105] Example 8

[0106] Prepare a PBS buffer solution with a pH of 7.4, a 2mM ECMA DMSO solution, and a 20mM Cys aqueous solution; add 10μL of the ECMA DMSO solution to 2mL of the PBS buffer solution; add the probe solution to the HeLa cell culture medium to make its concentration 10μM, incubate for 5min and 20min, and collect the channel 1: 542-602nm (λ ex =488nm) (orange light) and channel 2: 460~520nm (λ ex =405nm) (blue light) fluorescence signal. Laser confocal fluorescence imaging showed that the fluorescence of the blue channel increased and the fluorescence of the orange channel decreased, indicating that the probe can detect endogenous cysteine ​​in cell lysosomes, see Figure 11 .

[0107] Example 9

[0108] Prepare PBS buffer solution with pH=7.4, prepare 2mM ECMA DMSO solution; prepare 20mM H2O2 aqueous solution, add 10μL ECMA DMSO solution to 2mL PBS buffer solution; add it to HeLa cell culture medium, incubate at 37℃ for 10min, after adding H2O2, the fluorescence signal in the orange channel increases with time (0-10min), and the fluorescence signal in the blue channel decreases significantly, as shown Figure 12 .

[0109] Example 10

[0110] Prepare a PBS buffer solution with a pH of 7.4, a 2mM ECMA DMSO solution, and a 20mM Cys aqueous solution; add 10μL of the ECMA DMSO solution to 2mL of the PBS buffer solution; add the probe solution to the HeLa cell culture medium to make its concentration 10μM, and incubate with the HeLa cells at 37°C for 10 minutes; observe the system under a fluorescence imager; then add 0.2μM of the lysosomal staining reagent LTR to the system and incubate at 37°C for 30 minutes. Observe the system under a fluorescence imager and calculate the colocalization rate of the obtained fluorescence image. The colocalization coefficient is 0.90, see Figure 13 .

[0111] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation thereto. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications shall also be considered within the scope of protection of the present invention.

Claims

1. A coumarin derivative having the structure shown in Formula I:

2. The method for preparing the coumarin derivative according to claim 1, characterized in that: The following steps are involved: mixing 7-(diethylamino)coumarin with an organic solvent to obtain a 7-(diethylamino)coumarin solution; Mixing POCl3 with an organic solvent to obtain a formylating agent; adding the 7-(diethylamino)coumarin solution dropwise to the formylation reagent to carry out a formylation reaction to obtain 7-(diethylamino)coumarin-3-carboxaldehyde; The 7-(diethylamino)coumarin-3-carboxaldehyde, 3-morpholine-3-oxopropionitrile, piperidine and an organic solvent are mixed to carry out a Knoevenagel condensation reaction to obtain the coumarin derivative.

3. The preparation method according to claim 2, characterized in that The molar ratio of the 7-(diethylamino)coumarin to POCl3 is 1:2 to 1:

3.

4. The preparation method according to claim 2, characterized in that The temperature of the formylation reaction is 55-65° C., and the time is 8-16 hours.

5. The preparation method according to claim 2, characterized in that The molar ratio of the 7-(diethylamino)coumarin-3-carboxaldehyde to 3-morpholine-3-oxopropionitrile is 1:1 to 1:

2.

6. The preparation method according to claim 2, characterized in that The Knoevenagel condensation reaction temperature is 70-90° C. and the reaction time is 4-6 hours.

7. Use of the coumarin derivative according to claim 1 or the coumarin derivative prepared by the preparation method according to any one of claims 2 to 6 in the qualitative detection of cysteine.

8. Use of the coumarin derivative according to claim 1 or the coumarin derivative prepared by the preparation method according to any one of claims 2 to 6 in ratiometric detection of cysteine.

9. A method for ratiometric detection of cysteine, characterized in that: The following steps are involved: After mixing a test solution, a PBS buffer solution, and a coumarin derivative solution, fluorescence detection is performed at 490 nm and 572 nm, respectively, and the ratio of the fluorescence intensities at 490 nm and 572 nm is calculated, wherein the coumarin derivative in the coumarin derivative solution is the coumarin derivative according to claim 1 or the coumarin derivative prepared by the preparation method according to any one of claims 2 to 6, and the test solution contains cysteine; The concentration of cysteine ​​in the solution to be tested is calculated using a standard curve, wherein the standard curve uses the concentration of cysteine ​​as the horizontal axis and the concentration of F as the horizontal axis. 490 / F 572 Is the vertical axis.

10. The method according to claim 9, characterized in that The pH value of the PBS buffer solution is 4.0-8.0.

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