Triple base stack mediated colorimetric nano-probe for pH monitoring and application of triple base stack mediated colorimetric nano-probe

Abstract

The invention relates to a triple base stack mediated colorimetric nanoprobe for pH monitoring and an application of the triple base stack mediated colorimetric nanoprobe. The pyrimidine chain HPy is HPy-1, HPy-2 or HPy-3 (shown in SEQ ID NO.1-3), the purine chain HPu is HPu-1, HPu-2 or HPu-3 (shown in SEQ ID NO.4-6), during use, the pyrimidine chain HPy and the purine chain HPu which have the same TAT proportion need to be hybridized to form double chains, and then the double chains are matched with the AuNPs modified by the thiolated pyrimidine chain HPy with the same TAT proportion to be used. When the colorimetric nanoprobe is used as an indicator for classical acid-base titration, the color change point is closer to a stoichiometric point, the inherent titration error caused by the colorimetric nanoprobe is reduced by two orders of magnitude compared with that caused by a traditional indicator, and the colorimetric nanoprobe is more accurate compared with traditional test paper, methyl red, phenolphthalein and other indicating dyes. The colorimetric nanoprobe is small in pH error and higher in anti-interference capability, the response range can be reasonably and flexibly adjusted, and the colorimetric nanoprobe can be widely applied to various accurate pH detections.

Description

Technical Field

[0001] The invention relates to a triple base stacking-mediated colorimetric nanoprobe for pH monitoring and application thereof, and belongs to the technical field of biological detection. Background Art

[0002] pH is the negative logarithm of hydrogen ion activity or concentration, which can evaluate the acidity or alkalinity of aqueous solutions. pH analysis is the foundation and key of the field of chemistry. Basically, the solubility or form of many chemicals or biomolecules in solution, as well as the reaction rate and extent, depend on pH. In addition, changes in pH may indicate abnormal conditions, such as acidification of rainwater and food spoilage. Naked eye colorimetric determination is the simplest method to measure pH, with minimal dependence on instruments. Commonly used pH test paper will show a specific color after contact with the liquid, but it is necessary to obtain a rough pH by comparing with the standard color, and it is difficult to have high accuracy due to the interference of the matrix in the liquid. Indicator dyes such as methyl orange and phenolphthalein change conformation with the activity of hydrogen ions and can indicate pH changes, but their color change points cannot be changed, and there are inherent errors in application scenarios such as titration analysis. Therefore, new colorimetric pH indicators are still in demand.

[0003] Gold nanoparticles (AuNPs) have molar extinction coefficients several orders of magnitude higher than organic dyes and visible absorption that depends on the interparticle distance, making them promising colorimetric sensors. However, AuNPs themselves do not respond well to changes in pH, for example, citrate-capped AuNPs are stable in the pH range of 6.6 to 11.1. At the same time, bare AuNPs can only remain stable in solutions with low ionic strength. To obtain reliable pH response, AuNPs are functionalized with ligands such as synthetic molecules and polymers, which can be aggregated by proton-mediated ligand pairing or ligand charge reduction. Since the molecular structure of the ligand is determined, it is usually responsive to a specific pH. Compared with ligands, DNA oligonucleotides modified onto AuNPs and protecting AuNPs from matrix interference with high stability and can be more flexible through base conversion. In particular, some types of DNA oligonucleotides are responsive to pH, such as cytosine-rich sequences that can form i-motif structures under acidic conditions, and homopurine / homopyrimidine sequences that can form triplex-like structures at low pH. When DNA is modified onto AuNPs, the dispersibility of the nanoparticles changes with pH due to changes in the DNA structure. However, its response time usually takes 30 min or longer, or requires additional heating and cooling steps. Therefore, it is more applied to pH-regulated DNA-AuNPs self-assembly but rarely used for pH monitoring. This may be because the aggregation of AuNPs is achieved by cross-linking of oligonucleotide hybridization and structure formation between particles, which needs to overcome the obstacles in the densely packed DNA layer. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a triple base stacking-mediated colorimetric nanoprobe for pH monitoring and applications thereof.

[0005] The technical solution of the present invention is as follows:

[0006] A triple base stacking-mediated colorimetric nanoprobe for pH monitoring, including a pyrimidine chain HPy and a purine chain HPu;

[0007] The pyrimidine chain HPy is HPy-1, HPy-2 or HPy-3, and the sequences are shown in SEQ ID NOs. 1 to 3 respectively;

[0008] The purine chain HPu is HPu-1, HPu-2 or HPu-3, and the sequences are shown in SEQ ID NOs. 4 to 6 respectively.

[0009] A composition for pH monitoring comprises a double chain formed by hybridization of a pyrimidine chain HPy and a purine chain HPu, and AuNPs modified with thiolated pyrimidine chain HPy; wherein the TAT ratio of the pyrimidine chain HPy and the purine chain HPu is the same.

[0010] Preferably according to the present invention, the composition for pH monitoring includes three situations, namely: a double chain formed by hybridization of a pyrimidine chain HPy-1 and a purine chain HPu-1, and AuNPs modified with a thiolated pyrimidine chain HPy-1; a double chain formed by hybridization of a pyrimidine chain HPy-2 and a purine chain HPu-2, and AuNPs modified with a thiolated pyrimidine chain HPy-2; a double chain formed by hybridization of a pyrimidine chain HPy-3 and a purine chain HPu-3, and AuNPs modified with a thiolated pyrimidine chain HPy-3.

[0011] Preferably, according to the present invention, the AuNPs modified with thiolated pyrimidine chains HPy are prepared according to the following method:

[0012] a. Heat 45 mL of ultrapure water to boiling, then add 5 mL of 10 mM chloroauric acid to the ultrapure water while stirring vigorously, and quickly add 5 mL of 38.8 mM sodium citrate solution 2 minutes later, and heat to boil for 10 minutes; stop heating, and cool the solution to room temperature while stirring slowly; filter the cooled solution with a 0.22 μm filter membrane to obtain gold nanoparticles (AuNPs);

[0013] b. According to a molar ratio of 1:50, the thiolated pyrimidine chain HPy was incubated with TCEP for 2 hours to reduce the disulfide bond to obtain the thiolated pyrimidine chain HPy treated with TCEP; then, the thiolated pyrimidine chain HPy treated with TCEP was mixed with gold nanoparticles according to a molar ratio of 100:1, incubated at room temperature in the dark for 16 hours, and then a saline solution was added. The solution was continued to be slowly stirred in the dark for 24 hours, and the solution was centrifuged at 14000 rpm and 4°C for 30 minutes. After washing 3 times, AuNPs modified with thiolated pyrimidine chain HPy were obtained.

[0014] Preferably, according to the present invention, in step b, the composition of the salt solution is: 55.55 mM Tris-HCl, 1110 mM NaCl, pH 8.0; the salt buffer solution is added in 10 times, with a time interval of 40 min between each addition, so that the final concentration of Tris is 5 mM and the final concentration of NaCl is 0.1 M.

[0015] A kit for pH monitoring comprises the above composition for pH monitoring and MgCl2.

[0016] The method for using the above-mentioned kit for pH monitoring comprises the following steps:

[0017] Adding AuNPs modified with a double chain formed by hybridization of a pyrimidine chain HPy and a purine chain HPu, thiolated pyrimidine chain HPy, and MgCl2 to deionized water, and mixing them evenly to obtain a reaction system; then adding the sample to be tested to the reaction system, incubating at room temperature for 5 to 15 minutes, visually observing, and determining the pH value of the sample to be tested according to the color development;

[0018] Among them, the double chain formed by the hybridization of pyrimidine chain HPy-1 and purine chain HPu-1, and the AuNPs modified with thiolated pyrimidine chain HPy-1 showed color changes between pH 5.0 and pH 6.0; the double chain formed by the hybridization of pyrimidine chain HPy-2 and purine chain HPu-2, and the AuNPs modified with thiolated pyrimidine chain HPy-2 showed color changes between pH 6.0 and pH 7.0; the double chain formed by the hybridization of pyrimidine chain HPy-3 and purine chain HPu-3, and the AuNPs modified with thiolated pyrimidine chain HPy-3 showed color changes between pH 7.0 and pH 9.0.

[0019] Preferably according to the present invention, the concentration of the double chain formed by hybridization of pyrimidine chain HPy and purine chain HPu in the reaction system is 2.5 μM, the concentration of AuNPs modified with thiolated pyrimidine chain HPy is 80 nM, the concentration of MgCl2 is 100 mM, and the total volume is 100 μL.

[0020] Application of the triple base stacking-mediated colorimetric nanoprobe for pH monitoring, the composition for pH monitoring or the kit for pH monitoring in environmental pH monitoring and food pH monitoring.

[0021] According to the present invention, the thiolation of the pyrimidine chain HPy is a conventional prior art and is commercially available, as indicated in Table 1 of the specific embodiment, and is labeled as thiolated HPy-1 to 3.

[0022] The detection principle and technical features of the present invention are as follows:

[0023] like Figure 1 As shown, the present invention provides a colorimetric nanoprobe composed of a pyrimidine chain HPy and a purine chain HPu, wherein the pyrimidine chain HPy is HPy-1, HPy-2 or HPy-3 (shown in SEQ ID NO.1-3), and the purine chain HPu is HPu-1, HPu-2 or HPu-3 (SEQ ID NO.4-6). When used, the pyrimidine chain HPy and the purine chain HPu with the same TAT ratio need to be hybridized to form a double strand, and then used in conjunction with AuNPs modified with thiolated pyrimidine chain HPy with the same TAT ratio. Since the AuNPs modified with thiolated pyrimidine chain HPy have a negatively charged DNA phosphate backbone and a flexible end, the AuNPs are always in a dispersed state. When the pH is lowered, the cytosine in the thiolated pyrimidine chain HPy modified on the surface of the AuNPs is protonated to form a C+GC triplex with the double strand, forming a non-classical base pairing. This non-classical base pairing does not disrupt Watson-Crick hydrogen bonds, but instead forms two additional Hoogsteen hydrogen bonds for each nucleotide between AuNPs modified with thiolated pyrimidine chains HPy and the purine-rich duplex. This leads to the formation of parallel DNA triplexes on the AuNP surface. Because the ends of the DNA triplex are blunt, this can produce base stacking interactions with the DNA triplex on another AuNP, inducing the aggregation of nanoparticles in a non-crosslinking manner. Since inter-particle chain hybridization is not required, the aggregation kinetics of AuNPs are faster, so changes in pH can be indicated within 10 minutes.

[0024] The beneficial effects of the present invention are as follows:

[0025] 1. The present invention provides a triple base stacking-mediated colorimetric nanoprobe for pH monitoring, wherein the pyrimidine chain HPy is HPy-1, HPy-2 or HPy-3 (shown in SEQ ID NO.1-3), and the purine chain HPu is HPu-1, HPu-2 or HPu-3 (SEQID NO.4-6). When used, the pyrimidine chain HPy and the purine chain HPu with the same TAT ratio need to be hybridized to form a double chain, and then used in conjunction with AuNPs modified with thiolated pyrimidine chain HPy with the same TAT ratio. When the colorimetric nanoprobe is used as an indicator for classical acid-base titration, its color change point is closer to the stoichiometric point, which is two orders of magnitude less than the inherent titration error caused by traditional indicators. Compared with traditional test paper, methyl red, phenolphthalein and other indicator dyes are more accurate. Moreover, the pH of the colorimetric nanoprobe not only has a small error, but also has a stronger anti-interference ability, and the response range can also be reasonably and flexibly adjusted, and can be widely used in various precise pH detections.

[0026] 2. The composition for pH monitoring and the kit for pH monitoring provided by the present invention have few reagents, low cost, simple operation, high sensitivity, and stronger anti-interference ability, and are easy to promote and use in various application scenarios such as acid rain monitoring and spoiled fruit detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the detection principle of the triple base stacking-mediated colorimetric nanoprobe for pH monitoring of the present invention.

[0028] Figure 2 The polyacrylamide gel electrophoresis diagrams of different pyrimidine chains HPy, purine chains HPu and hybrid double chains under different pH conditions;

[0029] In the figure: A is the electrophoresis results of HPy-1, HPu-1 and hybrid double-stranded HPy / HPu-1 at pH 5.0 and 7.0, lane 1: DNA Marker; lane 2: HPu-1; lane 3: hybrid double-stranded HPy / HPu-1; lane 4: hybrid double-stranded HPy / HPu-1+HPy-1; lane 5: HPy-1; B is the electrophoresis results of HPy-2, HPu-2 and hybrid double-stranded HPy / HPu-2 at pH 6.0 and 8.0, lane 1: DNA Marker; lane 2: HPu-2; lane 3: hybrid double-stranded HPy / HPu-2; lane 4: hybrid double-stranded HPy / HPu-2+HPy-2; lane 5: HPy-2; C is the electrophoresis results of HPy-3, HPu-3, hybrid double-stranded HPy / HPu-3 at pH Electrophoresis results at 7.0 and 9.0, lane 1: DNA Marker; lane 2: HPu-3; lane 3: hybrid double-stranded HPy / HPu-3; lane 4: hybrid double-stranded HPy / HPu-3 +HPy-3; lane 5: HPy-3.

[0030] Figure 3 Characterization of the response of composition 1 to changes in pH value and the response time to pH;

[0031] In the figure, A is the UV-visible absorption spectrum of composition 1 at different pH values; B is the change of absorbance of composition 1 at 520 nm with pH value; C is the change of absorbance of composition 1 at 520 nm with time when pH is adjusted from 7.0 to 5.0, and the inset is the visual observation of color change; D is the transmission electron microscopy characterization of composition 1 at pH 7.0 and pH 5.0.

[0032] Figure 4 It is the derivative curve of the absorbance of composition 1 at 520 nm as a function of pH.

[0033] Figure 5 Characterization of the response of composition 2 to changes in pH value and the response time to pH;

[0034] In the figure, A is the UV-visible absorption spectrum of composition 2 at different pH values; B is the change of absorbance of composition 2 at 520 nm with pH value; C is the change of absorbance of composition 2 at 520 nm with time when pH is adjusted from 8.0 to 6.0, and the inset is the visual observation of color change.

[0035] Figure 6 The derivative curve of the absorbance of composition 2 at 520 nm as a function of pH.

[0036] Figure 7 Characterization of composition 3 in response to pH changes and response time to pH;

[0037] In the figure, A is the UV-visible absorption spectrum of composition 3 at different pH values; B is the change of absorbance of composition 3 at 520 nm with pH value; C is the change of absorbance of composition 3 at 520 nm with time when pH is adjusted from 9.0 to 7.0, and the inset is the visual observation of color change.

[0038] Figure 8 The derivative curve of the absorbance of composition 3 at 520 nm as a function of pH.

[0039] Fig. 9 The normalized graphs and visual observation graphs of compositions 1 to 3 in response to changes in pH values.

[0040] In the figure, A is a normalized graph of the absorbance change at 520 nm of compositions 1 to 3 versus pH, and B is a visual observation graph of compositions 1 to 3 at pH 5.0, 6.0, 7.0, 8.0, and 9.0.

[0041] Fig.10 It is the visual color change of Composition 1 in the narrow pH range of 5.00 to 5.50.

[0042] Fig.11 The absorbance at 520 nm changes with time for cross-linking and aggregation of DNA-modified AuNPs based on inter-particle hybridization.

[0043] Fig.12 The visual observations of Composition 4 and Composition 5 at pH 5.0 are shown.

[0044] Fig.13 This is an acid-base titration diagram using methyl red, composition 3 and phenolphthalein as indicators. The solid triangle represents the color change point.

[0045] Fig.14 Results of testing simulated acid rain, local natural rain and ultrapure water for composition 1;

[0046] In the figure, A is the visual observation diagram, and B is the UV-visible absorption spectrum.

[0047] Fig.15 The result graph of composition 2 detecting different concentrations of 3-NPA and;

[0048] In the figure, A is the UV-visible absorption spectrum of composition 2 in the presence of different concentrations of 3-NPA; B is the linear relationship between the absorbance change value of composition 2 at 520 nm and the concentration of 3-NPA; C is the visual observation diagram of composition 2 detecting different concentrations of 3-NPA. D is the response of composition 2 to 3-NPA and its coexisting analogs; E is the detection of composition 2 to 10% sugarcane juice with or without 3-NPA.

[0049] Fig.16 The detection limit for composition 2 was determined for 3-NPA. DETAILED DESCRIPTION

[0050] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

[0051] Reagents and materials:

[0052] The DNA sequences (Table 1) were synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China).

[0053] Tris base, 40% acrylamide / bisacrylamide (19:1) solution, ammonium persulfate (APS), N,N,N,N′-tetramethyleneethylenediamine (TEMED), disodium ethylenediaminetetraacetate (EDTA), serine, alanine, glucose, sucrose, and fructose were obtained from Shanghai Shenggong Biotechnology Co., Ltd. Gold chloride trihydrate (HAuCl4·3H2O), sodium citrate (Na3C6H5O7·2H2O), and tris(2-carboxyethyl)phosphate hydrochloride (TCEP) were purchased from Sigma (St. Louis, MO, USA). SYBR gold nucleic acid stain was ordered from Thermo Fisher Scientific, and 3-nitropropionic acid (3-NPA) was purchased from Aladdin Biochemical Technology Co., Ltd., Shanghai, China. All other reagents were of analytical grade and used as received. Ultrapure water (18.25 MΩ·cm) from UP water purification system was used throughout the experiments.

[0054] The formula of TE buffer is: 10 mM Tris, 1 mM EDTA, pH 8.

[0055] UV-visible absorption spectra were recorded on a TU-1901 spectrometer (Persee, China). pH measurements were performed with a FE28 pH meter (Mettler Toledo, Switzerland). Polyacrylamide gels were imaged on a GelDocTM XR+ imaging system (Bio-RAD Laboratories Inc., USA). Transmission electron microscopy (TEM) characterization was performed using a JSM-6700F transmission electron microscope (JEOL, Japan).

[0056] Table 1. Oligonucleotide sequences used in the present invention

[0057]

[0058] Adenine and thymine nucleotides accounting for the A / T content ratio are underlined. Poly T spacers used to separate the homopyrimidine chain from the AuNP surface are in italics. Overhanging bases that interfere with the blunt end are in bold.

[0059] HPy-1 (40% A / T), HPy-2 (60% A / T), HPy-3 (80% A / T), HPu-1 (40% A / T), HPu-2 (60% A / T), HPu-3 (80% A / T) in Table 1 are referred to as HPy-1, HPy-2, HPy-3, HPu-1, HPu-2 and HPu-3 in the following examples, respectively. HPu-A protrusion is referred to as ProtrudingHPu-A, and HPu-AA protrusion is referred to as ProtrudingHPu-AA.

[0060] Example 1. Preparation of AuNPs and hybrid double chains modified with thiolated pyrimidine chains HPy

[0061] 1. AuNPs modified with thiolated pyrimidine chains HPy were prepared as follows:

[0062] a. Heat 45 mL of ultrapure water to boiling, then add 5 mL of 10 mM chloroauric acid to the ultrapure water while stirring vigorously, and quickly add 5 mL of 38.8 mM sodium citrate solution 2 minutes later, and heat to boil for 10 minutes; stop heating, and cool the solution to room temperature while stirring slowly; filter the cooled solution with a 0.22 μm filter membrane to obtain gold nanoparticles (AuNPs);

[0063] b. According to a molar ratio of 1:50, the thiolated pyrimidine chains HPy-1, HPy-2, and HPy-3 were incubated with TCEP for 2 hours to reduce the disulfide bonds, thereby obtaining TCEP-treated thiolated pyrimidine chains HPy-1, HPy-2, and HPy-3; then, the TCEP-treated thiolated pyrimidine chains HPy-1, HPy-2, and HPy-3 were mixed with gold nanoparticles at a molar ratio of 100:1, and incubated at room temperature in the dark for 16 hours, and then 100 μL of sodium chloride solution (55.55 mM Tris-HCl, 1110 mM NaCl, pH 8.0) was added. The salt buffer solution was added 10 times, with an interval of 40 minutes between each addition, so that the final concentration of Tris was 5 mM and the final concentration of NaCl was 0.1 M; then, the solution was stirred slowly in the dark for 24 hours, and the solution was centrifuged at 14000 rpm and 4°C for 30 minutes, and the supernatant was removed; the collected red precipitate was washed with 10 mM After washing three times with Tris-HCl buffer (pH 8.0), AuNPs modified with thiolated pyrimidine chain HPy-1, AuNPs modified with thiolated pyrimidine chain HPy-2, and AuNPs modified with thiolated pyrimidine chain HPy-3 were obtained.

[0064] The AuNPs modified with thiolated pyrimidine chain HPy-1, the AuNPs modified with thiolated pyrimidine chain HPy-2, and the AuNPs modified with thiolated pyrimidine chain HPy-3 were resuspended in 10 mM Tris-HCl buffer (pH 8.0) and stored at 4°C for later use.

[0065] 2. The hybrid double strands were prepared as follows:

[0066] The pyrimidine chain and purine chain with the same TAT ratio were grouped together to obtain three combinations, namely HPy-1+HPu-1, HPy-2+HPu-2 and HPu-3+HPy-3. The three groups of HPy chains and HPu chains were then mixed with TE buffer in an equal molar ratio, heated at 95°C for 10 min, and then slowly cooled to room temperature to hybridize into double chains, obtaining hybrid double chains HPy / HPu-1, HPy / HPu-2 and HPy / HPu-3.

[0067] Then, the hybrid double-stranded HPy-1 / ProtrudingHPu-A and hybrid double-stranded HPy-1 / ProtrudingHPu-AA were obtained according to the same method and used as controls later. The sequences of ProtrudingHPu-A and ProtrudingHPu-AA are shown in Table 1.

[0068] The pyrimidine chains HPy-1, HPy-2, HPy-3, the purine chains HPu-1, HPu-2, HPu-3 and the hybrid double chains HPy / HPu-1, HPy / HPu-2, HPy / HPu-3 were further grouped, and then the groups were mixed with HPy and the resulting double chains in TM buffer (20 mM Tris, 10 mM MgCl2, pH adjusted with hydrochloric acid) with pH values ​​of 5.0, 6.0, 7.0, 8.0, and 9.0 to form DNA triplexes. After the reaction solution was incubated at room temperature for 10 minutes, it was subjected to polyacrylamide gel electrophoresis at 200 V for 2 h, stained with SYBR gold for 40 minutes and imaged. The results are shown in Figure 2 shown.

[0069] The specific groups were as follows: Group 1: ①HPu-1 (1 μM), ②hybridized double-stranded HPy / HPu-1 (200 nM), ③HPy / HPu-1 (200 nM) + HPy-1 (200 nM), ④HPy-1 (5 μM);

[0070] Group 2: ①HPu-2 (1μM), ②hybrid double-stranded HPy / HPu-2 (200 nM), ③HPy / HPu-2 (200 nM) + HPy-2 (200 nM), ④HPy-2 (5μM);

[0071] Group 3: ①HPu-3 (1μM), ②hybrid double-stranded HPy / HPu-3 (200 nM), ③HPy / HPu-3 (200 nM) + HPy-3 (200 nM), ④HPy-3 (5μM).

[0072] Depend on Figure 2 It can be seen that when the A / T content is 40% ( Figure 2A), the hybridization of HPu-1 chain (lane 2) and HPy-1 chain (lane 5) leads to the formation of double-stranded HPy / HPu-3 (lane 3), which shows a delayed electrophoretic mobility. At pH 7.0, the double strand does not interact with the single-stranded HPy (lane 4), but when the pH is reduced to 5.0, a DNA triplex can be formed, which appears as the slowest migrating band on the gel. This is because the C in the single-stranded HPy is protonated at a lower pH and forms a C + GC triplex with the GC base pair in the double strand. At the same time, although T is not protonated, it can form a TA-T triplex with the AT base pair in the double strand through Hoogsteen hydrogen bonding. These two types of triplexes induce the generation of triplexes with blunt ends. In the case of an A / T content of 60% ( Figure 2 B), DNA triplex cannot form at pH 8.0, but can form at pH 6.0. The average pKa of thymine deprotonation is ~10, and above this pH the TA-T triplex is unstable. The average value of cytosine protonation is ~6.5, and above this pH the C + GC triplex is unstable. Therefore, as the proportion of TA-T triplexes increases and the proportion of C + GC triplexes decreases, the dependence of triplex formation on acidic pH decreases. When the A / T content increases to 80%, DNA triplexes can also be formed at pH 7.0 ( Figure 2 C). Therefore, by changing the ratio of A / T content in the DNA chain, reasonable adjustment of triplex formation at different pH values ​​can be achieved.

[0073] Example 2: Verification of the function of the composition for pH monitoring

[0074] The AuNPs modified with the thiolated pyrimidine chain HPy prepared in Example 1 and the hybrid double-chain HPy / HPu were mixed in groups, specifically:

[0075] Composition 1: AuNPs modified with thiolated pyrimidine chain HPy-1 (80 nM) + hybrid double-chain HPy / HPu-1 (2.5 μM);

[0076] Composition 2: AuNPs modified with thiolated pyrimidine chain HPy-2 (80 nM) + hybrid double-chain HPy / HPu-2 (2.5 μM);

[0077] Composition 3: AuNPs modified with thiolated pyrimidine chain HPy-3 (80 nM) + hybrid double-chain HPy / HPu-3 (2.5 μM);

[0078] Composition 4: AuNPs modified with thiolated pyrimidine chain HPy-1 (80 nM) + hybrid double-stranded HPy-1 / ProtrudingHPu-A (2.5 μM);

[0079] Composition 5: AuNPs modified with thiolated pyrimidine chain HPy-1 (80 nM) + hybrid double-stranded HPy-1 / ProtrudingHPu-AA (2.5 μM).

[0080] 1. In order to determine the pH response range of the probe, according to the configuration of the above compositions 1 to 3, 3 μL of AuNPs modified with thiolated pyrimidine chain HPy at a concentration of 80 nM and 3 μL of hybrid double-stranded HPy / HPu at a concentration of 2.5 μM were mixed to obtain three compositions; the three compositions were added to 94 μL of TM buffer with a pH range of 5.0 to 9.0 to obtain a reaction system; the reaction system was incubated at room temperature for 10 minutes, and visual detection and UV-visible absorption spectrum were measured; then compositions 1 to 3 were tested at five identical pH values ​​of pH 5.0, 6.0, 7.0, 8.0, and 9.0; finally, composition 1 was subjected to a visual color change test in a narrow pH range of 5.00 to 5.50 (pH 5.0, 5.07, 5.17, 5.25, 5.30, and 5.50). The specific results of the above experiments are as follows:

[0081] ①, the characterization of the response of composition 1 to pH value change and the derivative curve of absorbance at 520 nm with pH change are as follows: Figure 3 A. Figure 3 B and Figure 4 shown.

[0082] Depend on Figure 3 A, B and Figure 4 It can be seen that when the pH changes from 7.0 to 5.0, the absorbance at 520 nm of the gold nanoparticles dispersed in composition 1 gradually decreases, and AuNPs gradually aggregate ( Figure 3 A). Figure 3 B is the absorbance variation of composition 1 at 520 nm as a function of pH value, where the variation value (△A 520 ) is the absolute value of the decrease in absorbance compared to pH 7.0. It can be seen from the figure that as the pH increases from 5.0 to 7.0, the absorbance changes show a Boltzmann decreasing curve, and by calculating the differential coefficient, it is found that the change trend is the largest at pH 5.36 ( Figure 4 ).

[0083] ②, the characterization of the response of composition 2 to pH value change and the derivative curve of absorbance at 520 nm with pH change are as follows: Figure 5 A. Figure 5 B and Figure 6 shown.

[0084] Depend on Figure 5 A, B and Figure 6It can be seen that as the pH of composition 2 decreases from 8.0 to 6.0, the characteristic absorbance of dispersed AuNPs gradually decreases, indicating that there is aggregation phenomenon ( Figure 5 A). Compared with composition 1, composition 2 showed sensitive changes in absorbance with pH in a higher range ( Figure 5 B), and by calculating the differential coefficient, the change trend is the largest when the pH value is 6.26 ( Figure 6 ).

[0085] ③. Characterization of the response of composition 3 to pH value and the derivative curve of absorbance at 520 nm with pH value are shown in Figure 7 A. Figure 7 B and Figure 8 shown.

[0086] Depend on Figure 7 A, B and Figure 8 It can be seen that composition 3 shows a pH-dependent decrease in absorbance in the range of 9.0 to 7.0 ( Figure 7 A), has a sensitive response to the highest range of pH ( Figure 7 B) and the derivative inflection point at pH 7.36 ( Figure 8 ).

[0087] ④, continue to analyze the absorbance changes of compositions 1 to 3 at 520 nm and pH ( Figure 3 B. Figure 5 B and Figure 7 B) Normalization. A normalized value of 1 represents aggregated AuNPs, and a normalized value of 0 represents dispersed AuNPs. The results are shown in Fig. 9 As shown in A.

[0088] Depend on Fig. 9 As can be seen, nanoprobe compositions 1 to 3 all aggregate as the pH decreases. However, it is obvious that their response pH belongs to different ranges. The nanoprobe with an A / T content of 40% (composition 1) shows a color change between pH 5.0 and pH 6.0, while the nanoprobe with an A / T content of 60% (composition 2) shows a color change between pH 6.0 and pH 7.0, and the nanoprobe with an A / T content of 80% (composition 3) shows a color change between pH 8.0 and pH 9.0. As the A / T content ratio increases, its response pH is more alkaline.

[0089] In addition, when the change value reaches half, the number of dispersed nanoprobes should be equal to the number of aggregated nanoprobes. The corresponding pH values ​​of nanoprobe compositions 1 to 3, i.e., pH = 5.36 for composition 2, pH = 6.26 for composition 2, and pH = 7.36 for composition 3, are consistent with the pH point of the maximum change trend of the nanoprobe dispersion state calculated by the differential coefficient. This can be regarded as a point where a slight increase or decrease in pH value will cause a difference in the dispersion and aggregation of AuNPs, thereby producing the most sensitive absorbance change.

[0090] The visual observation results of compositions 1 to 3 at pH 5.0, 6.0, 7.0, 8.0, and 9.0 are as follows: Fig. 9 As shown in B.

[0091] Depend on Fig. 9 B It can be seen that compositions 1 to 3 show different responses.

[0092] ⑤. The visual color change results of composition 1 in the narrow pH range of 5.00 to 5.50 are as follows: Fig.10 shown.

[0093] Depend on Fig.10 It can be seen that Composition 1 can accurately detect a narrow pH range.

[0094] In summary, the pH range of composition 1 is 5.0-7.0, the pH range of composition 2 is 6.0-8.0, and the pH range of composition 3 is 7.0-9.0. This shows that users can select a specific type of nanoprobe to achieve the desired rapid colorimetric detection.

[0095] 2. To investigate the response time of the probe to pH, according to the configuration of the above compositions 1 to 3, 3 μL of AuNPs modified with thiolated pyrimidine chain HPy at a concentration of 80 nM and 3 μL of hybrid double-stranded HPy / HPu at a concentration of 2.5 μM were mixed to obtain three compositions; composition 1 was added to 94 μL of TM buffer at a pH of 7.0, followed by the addition of hydrochloric acid to immediately adjust the pH of the solution to 5.0, and the UV-visible absorption spectrum was recorded every 2 min to monitor the response of the probe to pH in real time; composition 2 was added to 94 μL of TM buffer at a pH of 8.0, followed by the addition of hydrochloric acid to immediately adjust the pH of the solution to 6.0, and the UV-visible absorption spectrum was recorded every 2 min to monitor the response of the probe to pH in real time; composition 3 was added to 94 μL of TM buffer at a pH of 9.0, followed by the addition of hydrochloric acid to immediately adjust the pH of the solution to 7.0, and the UV-visible absorption spectrum was recorded every 2 min to monitor the response of the probe to pH in real time. The results are as follows: Figure 3 As shown in C, 5C, and 7C.

[0096] Depend on Figure 3C, 5C, and 7C show that the response time of the nanoprobe to pH is relatively fast, and the absorbance change can reach saturation within 10 minutes. In addition, AuNPs modified with thiolated pyrimidine chains HPy appear purple at low pH and red at high pH.

[0097] AuNPs modified with thiolated pyrimidine chains HPy-1 and HPu chains-1 were mixed at pH 5.0 and then the absorbance was measured to obtain the time-dependent absorbance variation at 520 nm of the cross-linked aggregation of DNA-modified AuNPs based on interparticle hybridization. Fig.11 .

[0098] Depend on Fig.11 It can be seen that the response time of nanoprobe compositions 1 to 3 to pH is faster than this method, and the absorbance change can reach saturation within 10 minutes, which is much shorter than the aggregation time of this DNA-modified AuNPs based on cross-linking.

[0099] 3. Taking composition 1 as an example, 3 μL of AuNPs modified with thiolated pyrimidine chain HPy-1 with a concentration of 80 nM, 3 μL of hybrid double-chain HPy / HPu-1 with a concentration of 2.5 μM and 10 μL of 100 mM MgCl2 were mixed to obtain composition 1; composition 1 was added to 84 μL of hydrochloric acid solution with a pH value of 5.0 and 84 μL of deionized water with a pH value of 5.0, incubated at room temperature for 10 min, and TEM measurements were performed to reflect the aggregation state of gold nanoparticles under two pH conditions. The results are shown in Figure D.

[0100] At the same time, the visual changes of composition 4 and composition 5 were observed under the same conditions and methods. The results are as follows: Fig.12 shown. Fig.12 Here, “Nanoprobe” represents composition 1, “AProtruded” represents composition 4, and “AAProtruded” represents composition 5.

[0101] As shown in Figure D, the nanoprobes in composition 1 remained dispersed at pH 7.0 but became aggregated when the pH dropped to 5.0. In particular, the spacing between the aggregated AuNPs was reduced, which may be caused by the stacked DNA layers.

[0102] Depend on Fig.12 It can be seen that the nanoprobes in Compositions 4 and 5 cannot aggregate because the protruding bases break the π-π stacking between adjacent aromatic groups.

[0103] Above (Figure D and Fig.12 Together, we demonstrated that base stacking interactions between blunt ends play an important role in nanoprobe aggregation.

[0104] Example 3, indication of acid-base titration

[0105] HCl of equal molar concentration was dripped into 50 μL NaOH, and then the composition 3 in Example 2 (AuNPs modified with thiolated pyrimidine chain HPy-3 at a concentration of 80 nM, hybrid double-stranded HPy / HPu-3 at a concentration of 2.5 μM, Nanoprobe) was used to indicate the titration endpoint as the experimental group. Methyl red and phenolphthalein were used to indicate the titration endpoint as the control group. Photos were taken when the total amount of HCl dripped reached 20, 45, 50, 110, and 120 μL. The results are shown in the figure. Fig.13 shown.

[0106] Depend on Fig.13 It can be seen that when the amount of hydrochloric acid added is equal to the amount of sodium hydroxide added, the colorimetric nanoprobe of the present invention will change color, and the indication is more accurate than methyl red and phenolphthalein. Methyl red changes color at a more acidic pH value, resulting in excessive consumption of the titrant, which leads to a higher analysis result. Phenolphthalein changes color at a more alkaline pH value, resulting in insufficient consumption of the titrant and a lower analysis result. In addition, the titration error was calculated using the Linbang error equation, and it was found that the titration error using the colorimetric nanoprobe of the present invention as an indicator was significantly reduced by two orders of magnitude.

[0107] The specific calculation process and formula are as follows:

[0108]

[0109]

[0110]

[0111]

[0112] Example 4: Monitoring of acid rain

[0113] According to the acid rain type of sulfuric acid and nitric acid compound in China, sulfuric acid and nitric acid were added to deionized water in a mass ratio of 2:1 to obtain simulated acid rain (SAR) with a pH of 5.0.

[0114] Then, using the composition 1 in Example 2, 3 μL of AuNPs modified with thiolated pyrimidine chain HPy-1 with a concentration of 80 nM, 3 μL of hybrid double-stranded HPy / HPu-1 with a concentration of 2.5 μM and 10 μL of 100 mM MgCl2 were mixed to obtain a composition; the composition was added to 84 μL of simulated acid rain, 84 μL of rain collected in Jinan and 84 μL of ultrapure water, respectively, and incubated at room temperature for 10 minutes, and then visually observed and UV-visible absorption spectra were recorded. The results are as follows: Fig.14 shown.

[0115] Depend on Fig.14 It can be seen that after adding the colorimetric nanoprobe of the present invention, the color of the acid rain changes to purple ( Fig.14 A), which indicates that the aggregation of the nanoprobes is due to the formation of blunt-ended triplexes at pH 5.0 and the base stacking between the modified DNA layers on the AuNPs surface. For rainwater and deionized water collected locally, the solution remained red after adding the colorimetric nanoprobe of the present invention. Correspondingly, the absorbance of the nanoprobe at 520 nm dropped sharply in the presence of acid rain ( Fig.14 B).

[0116] Example 4, Detection of 3-nitropropionic acid (3-NPA)

[0117] 1. 3-Nitropropionic acid (3-NPA) is a secondary metabolite of arsenic fungi in spoiled sugarcane, which can cause central nervous system disorders and even brain damage in humans. 3-NPA can be stably present during the industrial saccharification of sugarcane and further food production, so the detection of 3-NPA in spoiled sugarcane is crucial to prevent it from entering the food consumption chain and maintaining food safety.

[0118] In order to study the concentration response range of the probe to 3-NPA, 3 μL of 80 nM thiolated pyrimidine chain HPy-2-modified AuNPs, 3 μL of 2.5 μM hybrid double-stranded HPy / HPu-2, 10 μL of 100 mM MgCl2 and 1 μL of 1 mM HCl were mixed to obtain composition 2; composition 2 was added to 83 μL of deionized water to obtain a reaction system; then 3-NPA was added to the reaction system so that the 3-NPA in the reaction system was a gradient concentration of 0, 10, 20, 40, 50, 60, 80, 100, 110, 120, 140 μM, and incubated at room temperature for 10 min. Spectral test and colorimetric analysis were performed. The results are shown in Figure 2. Fig.15 As shown in A~C.

[0119] Depend on Fig.15 From A to C, it can be seen that as the concentration of 3-NPA increases, the absorbance at 520 nm gradually decreases ( Fig.15 A), indicating the aggregation of nanoprobes; in the range of 3 to 140 μM, the absorbance change value showed a good linear relationship with the 3-NPA concentration ( Fig.15 B); By testing the background fluctuation and 3-NPA signal, the detection limit was directly measured (not extrapolated) to be 3 μM. As the concentration of 3-NPA increased, the color of the solution gradually changed from red to purple ( Fig.15 C).

[0120] 2. In order to explore the ability of the probe to selectively detect 3-NPA, 100 μM sucrose, serine, alanine, glucose, and fructose were added to the reaction system for detecting 3-NPA in step 1 to conduct competition experiments. The UV-visible spectra of the above reaction solutions were recorded and compared with the results of 3-NPA (100 μM) at the same concentration. The results are as follows: Fig.15 As shown in D.

[0121] Depend on Fig.15 D shows that the absorbance of the probe at 520 nm does not decrease significantly in the presence of substances commonly found in sugarcane, such as sucrose, serine, alanine, glucose and fructose, and 3-NPA can be easily distinguished from these analogs.

[0122] 3. In order to evaluate the feasibility of the probe in actual samples, 3-NPA was added to simulate spoiled sugarcane. According to the Chinese Food Supplement Inspection Method BJ202205, 2 g of chopped sugarcane was dissolved in 15 mL of deionized water and then ultrasonicated for 10 minutes. The supernatant was collected and washed with deionized water and centrifuged at 10,000 rpm for 10 minutes. After repeating once, the sugarcane concentrate was obtained. Finally, the sugarcane concentrate was added to deionized water to prepare 10% sugarcane juice for subsequent use.

[0123] 3 μL of 80 nM thiolated pyrimidine chain HPy-2 modified AuNPs, 3 μL of 2.5 μM hybrid double-stranded HPy / HPu-2, 10 μL of 100 mM MgCl2 and 1 μL of mM HCl were mixed to obtain composition 2; composition 2 was added to 83 μL of 10% sugarcane juice and 10% sugarcane juice containing 100 μM 3-NPA, respectively, and visual observation was performed and UV-visible absorption spectra were recorded. The results are shown in FIG. Fig.15 As shown in E.

[0124] Depend on Fig.15 E shows that after adding 3-NPA to 10% sugarcane juice, the presence of 3-NPA can be clearly determined by absorbance changes or visual observation.

[0125] 4. Measure at least 3 times in the absence of 3-NPA (same experimental components as in step 1 of Example 4, but 3-NPA concentration is 0 μM), and calculate the average absorbance at 520 nm (average absorbance, recorded as M) minus 3σ (3 times the standard deviation). We gradually increase (starting from 0 μM) the concentration of 3-NPA and measure its absorbance. When the concentration of 3-NPA rises to 3 μM, its absorbance is still lower than (M-3σ), indicating that the decrease in absorbance is caused by the presence of 3-NPA. As a directly measured detection limit, the results are as follows Fig.16 shown.

[0126] Depend on Fig.16 It can be seen that the detection limit was 3 μM, which was directly measured (not extrapolated) by testing the background fluctuation and 3-NPA signal.

[0127] In summary, the present invention provides a triple base stacking-mediated colorimetric nanoprobe for pH monitoring, wherein the pyrimidine chain HPy is HPy-1, HPy-2 or HPy-3, and the purine chain HPu is HPu-1, HPu-2 or HPu-3. At a lower pH, the conjugated homopyrimidine chain on the surface of AuNPs binds to the homopurine-homopyrimidine double strand through Hoogsteen hydrogen bonds to form a DNA triplex with blunt ends. During the collision process of the nanoprobes, the blunt ends of the outer layer of AuNPs are prone to base stacking interactions, inducing them to aggregate in a non-crosslinked manner. Compared with the crosslinking method based on interparticle hybridization, the polymerization time is greatly shortened. Using the characteristic that the hydrogen ion concentration required for triplex formation depends on the ratio of A / T content, three nanoprobes with different pH response ranges are demonstrated. Due to its reasonable adjustability, it can be further applied to various analytical scenarios, such as an accurate indicator in classical titration, visual determination of acid rain, and colorimetric detection of 3-NPA.

Claims

1. A triple base stacking-mediated colorimetric nanoprobe for pH monitoring, characterized in that: Includes pyrimidine chain HPy and purine chain HPu; The pyrimidine chain HPy is HPy-1, HPy-2 or HPy-3, and the sequences are shown in SEQ ID NOs. 1 to 3 respectively; The purine chain HPu is HPu-1, HPu-2 or HPu-3, and the sequences are shown in SEQ ID NOs. 4 to 6 respectively.

2. A composition for pH monitoring, characterized in that It comprises a double chain formed by hybridization of the pyrimidine chain HPy described in claim 1 and the purine chain HPu described in claim 1, and AuNPs modified with thiolated pyrimidine chain HPy.

3. The composition for pH monitoring according to claim 2, characterized in that The composition for pH monitoring includes three situations, namely: a double chain formed by hybridization of a pyrimidine chain HPy-1 and a purine chain HPu-1, and AuNPs modified with a thiolated pyrimidine chain HPy-1; a double chain formed by hybridization of a pyrimidine chain HPy-2 and a purine chain HPu-2, and AuNPs modified with a thiolated pyrimidine chain HPy-2; a double chain formed by hybridization of a pyrimidine chain HPy-3 and a purine chain HPu-3, and AuNPs modified with a thiolated pyrimidine chain HPy-3.

4. The composition for pH monitoring according to claim 2, characterized in that The AuNPs modified with thiolated pyrimidine chains HPy were prepared as follows: a. Heat 45 mL of ultrapure water to boiling, then add 5 mL of 10 mM chloroauric acid to the ultrapure water while stirring vigorously, and quickly add 5 mL of 38.8 mM sodium citrate solution 2 minutes later, and heat to boil for 10 minutes; stop heating, and cool the solution to room temperature while stirring slowly; filter the cooled solution with a 0.22 μm filter membrane to obtain gold nanoparticles (AuNPs); b. According to a molar ratio of 1:50, the thiolated pyrimidine chain HPy was incubated with TCEP for 2 hours to reduce the disulfide bond to obtain the thiolated pyrimidine chain HPy treated with TCEP; then, the thiolated pyrimidine chain HPy treated with TCEP was mixed with gold nanoparticles according to a molar ratio of 100:1, and incubated in the dark for 16 hours at room temperature, and then a saline solution was added. The solution was slowly stirred in the dark for 24 hours, and the solution was centrifuged at 14000 rpm and 4°C for 30 minutes. After washing 3 times, AuNPs modified with the thiolated pyrimidine chain HPy were obtained.

5. The composition for pH monitoring according to claim 4, characterized in that In step b, the composition of the salt solution is: 55.55mM Tris-HCl, 1110mM NaCl, pH 8.0; the salt buffer solution is added in 10 times, with a time interval of 40 minutes between each addition, so that the final concentration of Tris is 5mM and the final concentration of NaCl is 0.1M.

6. A kit for pH monitoring, characterized in that The invention comprises the composition for pH monitoring according to claim 2 and MgCl2.

7. The method for using the kit for pH monitoring according to claim 6, characterized in that: The steps include: Adding AuNPs modified with a double chain formed by hybridization of a pyrimidine chain HPy and a purine chain HPu, thiolated pyrimidine chain HPy, and MgCl2 to deionized water, and mixing them evenly to obtain a reaction system; then adding the sample to be tested to the reaction system, incubating at room temperature for 5 to 15 minutes, visually observing, and determining the pH value of the sample to be tested according to the color development; Among them, the double chain formed by the hybridization of pyrimidine chain HPy-1 and purine chain HPu-1, and the AuNPs modified with thiolated pyrimidine chain HPy-1 showed color changes between pH 5.0 and pH 6.0; the double chain formed by the hybridization of pyrimidine chain HPy-2 and purine chain HPu-2, and the AuNPs modified with thiolated pyrimidine chain HPy-2 showed color changes between pH 6.0 and pH 7.0; the double chain formed by the hybridization of pyrimidine chain HPy-3 and purine chain HPu-3, and the AuNPs modified with thiolated pyrimidine chain HPy-3 showed color changes between pH 7.0 and pH 9.

0.

8. The method of use according to claim 1, characterized in that: In the reaction system, the concentration of the double chain formed by the hybridization of the pyrimidine chain HPy and the purine chain HPu is 2.5 μM, the concentration of the AuNPs modified with the thiolated pyrimidine chain HPy is 80 nM, the concentration of MgCl2 is 100 mM, and the total volume is 100 μL.

9. Use of the triple base stacking-mediated colorimetric nanoprobe for pH monitoring according to claim 1, the composition for pH monitoring according to claim 2, or the kit for pH monitoring according to claim 6 in environmental pH monitoring and food pH monitoring.

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