Electrochemical staged detection method for multiple Helicobacter pylori single-base mutants

Abstract

The present invention belongs to the field of biological detection technology and relates to an electrochemical staged detection method for multiple Helicobacter pylori single-base mutants. The electrochemical sensor used in the method includes a sensing electrode, auxiliary DNA, sgRNA, and Cas9 nuclease; the sensing electrode consists of a conductive substrate, gold nanoparticles, probe DNA, and nano-CdS. The two ends of the probe DNA are respectively connected to the gold nanoparticles and nano-CdS, and the conductive substrate is connected to the gold nanoparticles; the probe DNA, the auxiliary DNA, and the target DNA can hybridize with each other to form a Y-shaped structure; the sgRNA and the Cas9 nuclease can form a Cas9 / sgRNA complex, and the Cas9 / sgRNA complex can destroy the Y-shaped structure, causing the nano-CdS to separate from the Y-shaped structure. The present invention realizes a comprehensive assessment of the Helicobacter pylori infection status and drug resistance status by detecting the total nucleic acid amount and single-base mutants.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and relates to an electrochemical phased detection method for multiple Helicobacter pylori single-base mutants. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily to be regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Helicobacter pylori ( H. pylori ) is a Gram-negative bacillus, and its infection is closely related to many digestive system diseases. The detection techniques commonly used in the clinical diagnosis of H. pylori include gastroscopy, 13C / 14C urea breath test, serum test, fecal test, and urine test. At present, the detection techniques for SNV mainly include: melting curve analysis for SNV detection, SNV detection using enhanced hybridization probes, and protein-assisted SNV detection. However, there is still no method that can simultaneously analyze the total nucleic acid amount and detect a single genotype. Therefore, it is particularly important to further study a new method for high-throughput and highly selective detection of Helicobacter pylori DNA and single-base mutants. Summary of the Invention

[0004] In order to solve the deficiencies of the prior art and actual needs, the purpose of the present invention is to provide an electrochemical detection method for multiple Helicobacter pylori single-base mutants, and through the detection of the total nucleic acid amount and single-base mutants, a comprehensive assessment of the Helicobacter pylori infection status and drug resistance status can be achieved.

[0005] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0006] On the one hand, an electrochemical sensor includes a sensing electrode, auxiliary DNA, sgRNA, and Cas9 nuclease;

[0007] The sensing electrode consists of a conductive substrate, gold nanoparticles, probe DNA, and nano-CdS. The probe DNA is in a hairpin structure, and both ends of the probe DNA are respectively connected to gold nanoparticles and nano-CdS, and the conductive substrate is connected to the gold nanoparticles;

[0008] The probe DNA, the auxiliary DNA, and the target DNA can hybridize with each other to form a Y-shaped structure;

[0009] The sgRNA and the Cas9 nuclease can form a Cas9 / sgRNA complex, and the Cas9 / sgRNA complex can destroy the Y-shaped structure, causing nano-CdS to separate from the Y-shaped structure.

[0010] On the other hand, an electrochemical detection method for multiple Helicobacter pylori single-base mutants provides the above-mentioned electrochemical sensor, and includes the following steps:

[0011] Add the auxiliary DNA and the target DNA to be detected to the sensing electrode for incubation, and detect the ECL signal;

[0012] Pre-incubate the sgRNA and Cas9 nuclease to obtain the Cas9 / sgRNA complex;

[0013] Add the obtained Cas9 / sgRNA complex to the incubated sensing electrode, continue to incubate, and detect the ECL signal;

[0014] Detect the target DNA to be detected through the ECL signal difference;

[0015] The target DNA is a Helicobacter pylori single-base mutant.

[0016] In the third aspect, an electrochemical detection kit for Helicobacter pylori single-base mutants includes the above-mentioned electrochemical sensor and buffer solution.

[0017] Since the probe DNA is in a hairpin structure, the gold nanoparticles and nano-CdS approach to produce a resonance energy transfer effect, so there is no ECL signal at this time. When the target DNA exists, it can hybridize with the auxiliary DNA and the probe DNA to form a Y-shaped structure. This change in spatial conformation breaks the resonance energy transfer effect and generates an ECL signal. The signal intensity is positively correlated with the nucleic acid concentration. Due to the limited reduction in the binding efficiency of a single base mismatch in the long-chain reaction, in the first stage, both mutants and non-mutants can form a Y-shaped structure to generate signals, and the detection of total Helicobacter pylori nucleic acid can be completed through this low-specificity mode. In the second stage, the highly sequence-specific cas9 endonuclease is used to detect mutants. The sgRNA binds to Cas9 to form the Cas9 / sgRNA complex, which excises nucleic acids of specific genotypes and destroys the Y-shaped structure, and different single-base mutants are detected by the signal difference method. All mutant phenotypes are detected by specific genotypes.

[0018] The beneficial effects of the present invention are as follows:

[0019] 1. The present invention first completes the detection of the total amount of Helicobacter pylori nucleic acid. The target sequence breaks the resonance energy transfer effect of the hairpin probe by forming a Y-shaped DNA structure, generating an electrochemiluminescence (ECL) signal, and the signal intensity is positively correlated with the nucleic acid concentration. In the second stage, through the highly specific recognition and cleavage action of Cas9, the corresponding guide RNA is used to excise nucleic acids of specific genotypes, and different single-base mutants are detected by the signal difference method. The infection status and drug resistance status of Helicobacter pylori are comprehensively evaluated through the detection of two stages with different specificities.

[0020] 2. The present invention can use indium tin oxide (ITO) conductive glass as a substrate, integrate an electrode array on the chip, and improve the detection throughput. Each electrode unit can complete the analysis of the sample, and the transparent glass substrate is conducive to visual detection, realizing the visual detection of nucleic acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0022] Figure 1 It is a schematic diagram of the detection principle of the embodiment of the present invention.

[0023] Figure 2 It is a high-magnification transmission electron microscope image of the embodiment of the present invention. a is a transmission electron microscope photograph of AuNPs prepared in the embodiment of the present invention; b is a transmission electron microscope photograph of CdS prepared in the embodiment of the present invention; c is a high-magnification transmission electron microscope photograph of CdS combined with AuNPs prepared in the embodiment of the present invention.

[0024] Figure 3 It is a gel electrophoresis characterization diagram of the embodiment of the present invention. a is a gel electrophoresis diagram for verifying the formation of the Y-shaped structure in the embodiment of the present invention; Figure 3 b is a gel electrophoresis diagram for verifying the cleavage of the Y-shaped structure by CRISPR / Cas9 enzyme in the embodiment of the present invention.

[0025] Figure 4 It is a diagram of the condition optimization results of the embodiment of the present invention. a is a curve of the change in ECL signal with different Na+ concentrations, b is a curve of the change in ECL signal with different Mg2+ concentrations, c is a curve of the change in ECL signal with time after using different buffers, and d is a bar chart of the change in ECL signal intensity with different Help chain concentrations.

[0026] Figure 5 It is a logarithmic linear relationship diagram between the concentrations of a series of target DNAs at different stages and the ECL signal. The sensitivity detection results of the embodiment of the present invention. a is a calibration curve of the logarithm of different target DNA concentrations in the first stage and the ECL signal; Figure b is a calibration curve of the logarithm of different target DNA concentrations in the second stage and the ECL signal;

[0027] Figure 6 a is a curve diagram of the ECL signal corresponding to the mixed DNA sample of wtDNA and mutDNA with different molar ratios in the first stage of the embodiment of the present invention; b is a curve diagram of the ECL signal corresponding to the mixed DNA sample of wtDNA and mutDNA with different molar ratios in the second stage of the embodiment of the present invention.

[0028] Figure 7Specific detection results of the embodiments of the present invention.

[0029] Figure 8 Schematic diagram of the high-throughput ITO array electrode chip template prepared by the present invention. Detailed implementation manners

[0030] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0031] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary implementation manners according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0032] In view of the current lack of a method that can simultaneously analyze the total amount of nucleic acids and detect a single genotype, the present invention proposes an electrochemical detection method for multiple Helicobacter pylori single-base mutants.

[0033] A typical implementation manner of the present invention provides an electrochemical sensor, including a sensing electrode, auxiliary DNA, sgRNA, and Cas9 nuclease;

[0034] The sensing electrode consists of a conductive matrix, gold nanoparticles, probe DNA, and nano-CdS. The probe DNA is in a hairpin structure, and both ends of the probe DNA are respectively connected to gold nanoparticles and nano-CdS, and the conductive matrix is connected to the gold nanoparticles;

[0035] The probe DNA, the auxiliary DNA, and the target DNA can hybridize with each other to form a Y-shaped structure;

[0036] The sgRNA and the Cas9 nuclease can form a Cas9 / sgRNA complex, and the Cas9 / sgRNA complex can destroy the Y-shaped structure, causing nano-CdS to separate from the Y-shaped structure.

[0037] In some embodiments, one end of the probe DNA is modified with a thiol group, and the gold nanoparticles are connected to the probe DNA through Au-S bonds.

[0038] In some embodiments, one end of the probe DNA is modified with an amino group, and nano-CdS is connected to the probe DNA through an amide bond.

[0039] In some embodiments, the conductive matrix is a glassy carbon electrode or conductive glass. The conductive glass is, for example, ITO conductive glass.

[0040] In some embodiments, the probe DNA is as shown in SEQ ID NO.2.

[0041] In some embodiments, the auxiliary DNA is as shown in SEQ ID NO.3.

[0042] In some embodiments, the target DNA is as shown in SEQ ID NO.1, 4, and / or 5.

[0043] In some embodiments, the sgRNA is as shown in SEQ ID NO.11, 12, and / or 13.

[0044] Another embodiment of the present invention provides an electrochemical detection method for multiple Helicobacter pylori single-base mutants, providing the above electrochemical sensor; comprising the following steps:

[0045] Add the auxiliary DNA and the target DNA to be detected to the sensing electrode for incubation, and detect the ECL signal;

[0046] Pre-incubate the sgRNA and Cas9 nuclease to obtain a Cas9 / sgRNA complex;

[0047] Add the obtained Cas9 / sgRNA complex to the incubated sensing electrode, continue the incubation, and detect the ECL signal;

[0048] Detect the target DNA to be detected through the difference in ECL signals;

[0049] The target DNA is a Helicobacter pylori single-base mutant.

[0050] In some embodiments, the probe DNA and gold nanoparticles are added to the surface of the conductive matrix, and the probe DNA is fixed on the surface of the conductive matrix by Au-S bonds.

[0051] In one or more embodiments, cadmium sulfide is activated using an EDC / NHS mixed solution, and then the activated cadmium sulfide is subjected to an amidation reaction with the probe DNA immobilized on the surface of the conductive matrix.

[0052] When drawing the working curve, two types of target DNA, wild-type and mutant, were used, and only one sgRNA that perfectly matches the wild-type was used for detection. When performing staged detection, the reason for using array detection is that each array uses different sgRNAs (each perfectly matching DNA of different genotypes). In this way, when there is a perfect match, cleavage occurs, resulting in a large signal difference, thus completing the detection of a specific genotype.

[0053] The third embodiment of the present invention provides an electrochemical detection kit for Helicobacter pylori single-base mutants, including the above-mentioned electrochemical sensor and buffer solution.

[0054] Specifically, the buffer solution is PBS buffer solution.

[0055] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0056] Embodiment

[0057] The detection principle of the embodiment is as Figure 1 shown. First, gold nanoparticles are synthesized and can be labeled on a DNA strand modified with mercapto and amino groups at both ends. AuNPs are modified on the electrode surface through Au-S bonds, and CdS is further connected through an amide reaction. Then, the target strand and the auxiliary DNA are modified on the electrode to assemble into a Y-shaped structure. This change in spatial conformation breaks the resonance energy transfer effect and generates an ECL signal. sgRNA can bind to Cas9 to form an activated Cas9 / sgRNA complex, excise nucleic acids of specific genotypes, destroy the Y-shaped structure, and reduce the ECL signal. Different single-base mutants are detected by the signal difference method.

[0058] Preparation of cadmium sulfide (CdS): The whole reaction is carried out in a nitrogen environment. Add 50 mL of 0.01 M CdCl2·5 / 2H2O solution to a 100 mL three-necked flask, add 250 μL of MPA, heat and stir, and adjust the pH to 11 with saturated NaOH. Slowly add 5.5 mL of 0.1 M Na2S and reflux at 110 °C for 4 h. Then stop heating. After the solution cools naturally, add an appropriate amount of isopropanol and centrifuge at a speed of 10000 r / min for 10 min. The particles sink to the bottom of the centrifuge tube, and the supernatant is discarded. Transfer the purified CdS sample to a centrifuge tube and store it in a refrigerator at 4 °C for later use. The purified particles can be used for dynamic light scattering analysis and TEM characterization.

[0059] Preparation of gold nanoparticles (AuNPs): 99 mL of pure water and 1 mL of HAuCl4 were mixed and added to a 250 mL three-necked flask. After stirring and heating to full reflux, 3.5 mL of 38.8 mmol / L sodium citrate was added, and heating and stirring were continued for 15 min. The solution gradually changed from light yellow to wine red, and the reaction was terminated. The gold nanoparticle sample cooled to room temperature was transferred to a centrifuge tube, 1×PBS (0.1 M NaCl, 5 mM MgCl2) was added, and centrifugation was carried out at 14000 r / min for 15 min. The particles sank to the bottom of the centrifuge tube, the supernatant was discarded, and centrifugal washing was carried out 2 times. Finally, it was dispersed with pure water. It was stored in a -4 °C refrigerator.

[0060] Assembly of Y-shaped structure and electrode modification:

[0061] (1) The glassy carbon electrode was polished successively with alumina powders of different particle sizes, and ultrasonically treated successively in ethanol and pure water to remove surface residues. Using 0.1 M KCl (0.05 M potassium ferricyanide) as the electrolyte, the cyclic voltammetry curve was scanned.

[0062] (2) The probe DNA (Probe DNA, hairpin structure) labeled with amino group and thiol group at both ends was modified onto the electrode treated in step (1), and left standing at 4 °C for 16 hours, so as to fix Probe DNA on the surface of the glassy carbon electrode by using Au-S bond.

[0063] (3) Cadmium sulfide was activated with an EDC / NHS mixed solution, and then cadmium sulfide was modified onto the electrode treated in step (2) for amidation reaction. Specifically: 20 μL of 0.2M NHS was added dropwise to the electrode, 0.04 M EDC was used to activate 1 mL of CdS, and the reaction was carried out at room temperature for 30 min. 20 uL of activated CdS was added dropwise and left overnight at 4 °C. The ECL quenching signal was tested.

[0064] (4) The target strand and the helper DNA (Help DNA, 1 μM) were modified (at 37 °C, 60 min) onto the electrode treated in step (3) to form a Y-shaped structure, and the ECL recovery signal was tested.

[0065] (5) Cas9 nuclease and sgRNA were mixed and pre-incubated in a reaction buffer (10 μL of 1×buffer buffer containing 50 nM Cas9 nuclease and 50 nM sgRNA was pre-cultured at 25 °C for 10 minutes), with a final concentration of 50 nM. 10 μL of the mixed solution was taken and reacted at 37 °C for 15 min for RNP complex assembly, and then dropped onto the electrode treated in step (4) and reacted at 37 °C for 20 min, and the ECL cleavage signal was tested.

[0066] The ECL detection solution is a 1×TE buffer solution containing 0.05 M K2S208. Electrochemical measurement: All electrochemical tests were read out through a Chenhua CHI660E electrochemical workstation. A three-electrode system was used: a glassy carbon electrode as the working electrode, a platinum wire electrode as the counter electrode, and an Ag / AgCl electrode as the reference electrode. The ECL signal was detected at a potential from -1.25 V to 0 V, and the photomultiplier tube (PMT) was set to -480 V.

[0067] The prepared AuNPs aqueous solution, CdS aqueous solution, and the solution of the mixed reaction (obtained from step (3) of this example above) were dropped onto a copper mesh to observe the morphology. The high-magnification transmission electron microscope photos are as Figure 2 shown in a, b, and c in

[0068] As Figure 3 shown in a, it is a gel electrophoresis diagram of the assembled Y-shaped structure. The bands from left to right are different DNA sequences (where M is the 20bp DNA marker, 1 is the target DNA, 2 is the Help DNA, 3 is the Probe DNA, 4 is the targetDNA / Help DNA, 5 is the target DNA / Probe DNA, 6 is the Probe DNA / Help DNA, 7 is the target DNA / Probe DNA / Help DNA, 8 is the target DNA / Probe DNA / Help DNA / RNP). By comparing the sizes of the DNA bands, it can be seen that the Y-shaped structure was gradually assembled successfully. Figure 3 b is the gel electrophoresis diagram of verifying the CRISPR / Cas9 cleavage of the Y-shaped structure in the example of the present invention.

[0069] Figure 4 It shows that after optimization, when the Na+ concentration is 0.1 M, the Mg2+ concentration is 5 mM, the buffer solution is PBS, and the help strand concentration is 1 μM, the experimental conditions reach the optimum.

[0070] The target DNA at different concentrations in different stages was detected by the method of this example, as Figure 5As shown, it can be seen from Figure 5a that the target DNA concentration and the ECL signal show a logarithmic linear relationship in the concentration range of 0.01 - 500 nM. In the concentration range of 0.01 - 500 nM, the linear equation is y = 0.0285logC(nmol / L) + 0.0867, the correlation coefficient R2 is 0.9994, and the detection limit with a signal-to-noise ratio of 3 is 8 pM. Figure 5b is the calibration curve of the logarithm of different target DNA concentrations and the ECL signal in the second stage, y = -0.02027logC(nmol / L) + 0.09164, R2 is 0.99544. Among them, in the process of obtaining this curve, the total amount of fixed nucleic acid (there are two genotypes of nucleic acid, one is wild type and the other is single-base mutant) is 1.1 μM, and the working curve of the correlation between the concentration of the genotype that is completely matched with sgRNA and the ECL signal is obtained.

[0071] To further study the ability to distinguish mutant DNA and normal wtDNA, mixed DNA samples with different molar ratios for quenching signals (a) 0.1%:99.9%, (b) 1%:99%, (c) 10%:90%, (d) 50%:50%, (e) 90%:10%, (f) 99%:1%, (g) 99.9%:0.1% with a total concentration of 100 nM were prepared as test objects. As Figure 6 shown, from Figure 6 a, it can be seen that in the mixed DNA sample, the ECL signal in the first stage does not change with the increase in the mutant DNA concentration, Figure 6 and b shows that the ECL signal in the second stage decreases dynamically with the increase in the mutant DNA concentration.

[0072] In this example, H. Pylori, Shigella, S. Aureus, Salmonella and E. coli strains of different genera were also used to test the ECL response of the electrochemical biosensor system to evaluate the specificity of this method. The results are as Figure 7 shown, H. Pylori triggered a strong ECL response, while Shigella, S. Aureus, Salmonella and E. coli were below the minimum detection limit of the target bacteria, indicating that this method can specifically distinguish Helicobacter pylori from pathogenic bacteria of different genera, and this electrochemical detection technology has high specificity for Helicobacter pylori.

[0073] Pretreatment of ITO Glass and Preparation of PDMS Film: The ITO glass was ultrasonically treated with acetone, ethanol, and pure water for 15 min in sequence, and then dried with nitrogen for standby. The liquid PDMS matrix and the curing agent were mixed evenly at a mass ratio of 10:1. After stirring evenly with a glass rod, the bubbles were removed. PDMS was poured on the ITO glass and cured at 60 °C for 3 h to form a PDMS film. The PDMS film was cut into the required shape, such as Figure 8 as shown. Each electrode unit integrated on the ITO chip can complete the analysis and visualization detection of the sample.

[0074] The nucleic acid sequences used in this embodiment are shown in the following table:

[0075]

[0076] Among them, the underlined bases are mismatched bases.

[0077] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An electrochemical detection method for multiple Helicobacter pylori single-base mutants, characterized in that, It includes the following steps: Add the auxiliary DNA and the target DNA to be detected to the sensing electrode for incubation, and detect the ECL signal; Pre-incubate the sgRNA and the Cas9 nuclease to obtain the Cas9 / sgRNA complex; Add the obtained Cas9 / sgRNA complex to the incubated sensing electrode, continue incubation, and detect the ECL signal; Detect the target DNA to be detected through the difference in ECL signals; The target DNA is a single-base mutant of Helicobacter pylori; The sensing electrode consists of a conductive substrate, gold nanoparticles, probe DNA, and nano-CdS. The probe DNA is in a hairpin structure, and the two ends of the probe DNA are respectively connected to the gold nanoparticles and nano-CdS, and the conductive substrate is connected to the gold nanoparticles; The probe DNA, the auxiliary DNA, and the target DNA can hybridize with each other to form a Y-shaped structure; The sgRNA and the Cas9 nuclease can form a Cas9 / sgRNA complex, and the Cas9 / sgRNA complex can destroy the Y-shaped structure, causing the nano-CdS to separate from the Y-shaped structure.

2. The electrochemical detection method of the multiple Helicobacter pylori single-base mutants as described in claim 1, characterized in that, Add the probe DNA and gold nanoparticles to the surface of the conductive substrate, and fix the probe DNA on the surface of the conductive substrate using the Au-S bond.

3. The electrochemical detection method for multiple Helicobacter pylori single-base mutants according to claim 2, characterized in that, Activate cadmium sulfide using an EDC / NHS mixed solution, and then perform an amidation reaction between the activated cadmium sulfide and the probe DNA fixed on the surface of the conductive substrate.

4. An electrochemical sensor, characterized in that, Provided by the electrochemical detection method according to claim 1, including a sensing electrode, auxiliary DNA, sgRNA, and Cas9 nuclease; The sensing electrode consists of a conductive substrate, gold nanoparticles, probe DNA, and nano-CdS. The probe DNA is in a hairpin structure, and the two ends of the probe DNA are respectively connected to the gold nanoparticles and nano-CdS, and the conductive substrate is connected to the gold nanoparticles; The probe DNA, the auxiliary DNA, and the target DNA can hybridize with each other to form a Y-shaped structure; The sgRNA and the Cas9 nuclease can form a Cas9 / sgRNA complex, and the Cas9 / sgRNA complex can destroy the Y-shaped structure, causing the nano-CdS to separate from the Y-shaped structure.

5. The electrochemical sensor according to claim 4, characterized in that, One end of the probe DNA is modified with a mercapto group, and the gold nanoparticles are connected to the probe DNA through the Au-S bond.

6. The electrochemical sensor according to claim 4, characterized in that, One end of the probe DNA is modified with an amino group, and the nano-CdS is connected to the probe DNA through an amide bond.

7. The electrochemical sensor according to claim 4, characterized in that, The conductive substrate is a glassy carbon electrode or conductive glass.

8. The electrochemical sensor according to claim 4, characterized in that, The probe DNA is as shown in SEQ ID NO.2; Or, the auxiliary DNA is as shown in SEQ ID NO.3; Or, the target DNA is as shown in SEQ ID NO.1, 4, and / or 5; Or, the sgRNA is as shown in SEQ ID NO.11, 12, and / or 13.

9. An electrochemical detection kit for a single-base mutant of Helicobacter pylori, characterized in that, It includes the electrochemical sensor and buffer solution according to any one of claims 4 to 8.

10. The electrochemical detection kit for Helicobacter pylori single-base mutants according to claim 9, characterized in that, The buffer solution is a PBS buffer solution.

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