CN109182457B - Complete set of reagents for preparing high-renewable nucleic acid biochip and application of complete set of reagents in mercury ion detection - Google Patents
Complete set of reagents for preparing high-renewable nucleic acid biochip and application of complete set of reagents in mercury ion detection Download PDFInfo
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Abstract
The invention discloses a complete set of reagents for preparing a high-regenerable nucleic acid biochip and application of the reagents in mercury ion detection. The kit comprises kit A, kit B, kit C, kit D and kit E; the reagent set A is amino activating solution which is (3-aminopropyl) triethoxysilane solution; the reagent set B is a cross-linking solution which is a glutaraldehyde aqueous solution; the reagent set C is a nucleic acid reaction solution, and the nucleic acid reaction solution is an amino nucleic acid solution; the reagent set D is aldehyde group confining liquid which is glycine solution; the reagent set E is interface confining liquid, and the interface confining liquid is isoelectric point confining liquid. Based on the prepared high-regenerable nucleic acid biochip, the invention designs a bivalent mercury ion nucleic acid probe rich in T basic group for detecting mercury ions in water, and inspects the detection performance of the mercury ions and tests the performance stability of the chip by taking a multi-index toxic pollutant online analyzer as a detection platform.
Description
Technical Field
The invention relates to a reagent set for preparing a high-regenerable nucleic acid biochip and application thereof in mercury ion detection, belonging to the field of detection and analysis based on biochips.
Background
The bivalent mercury ions are typical heavy metal pollutants of a water body, and the development of a novel low-cost mercury ion online detection technology has important practical value. The biosensor utilizes the biological material to identify a series of target objects, and utilizes various signal modes such as light, electricity and the like to carry out concentration quantification on the target objects, and is a quantification means with simple operation and rapid detection. In the field of heavy metal detection, the precondition of obtaining a heavy metal ion antibody with excellent performance in a classical immunosensing scheme is difficult to realize, so that a corresponding heavy metal immunodetection scheme is difficult to develop. In recent years, a sensing analysis method using a low-cost nucleic acid material as a novel biological recognition element has been rapidly developed. In the technical field of mercury ion detection, "T-Hg" was first reported in 2004 by Ono and Togashi subjects of Japan2+T' base mismatch (i.e. two originally non-complementary T bases are mismatched by mercury ions to form base pairs) and designed as a homogeneous mercury ion detection reagent by usingThe fluorescent labeling realizes the quantification of mercury ions in the system. Subsequently, "T-Hg2+T' base mismatches are widely used in the design of biosensing patterns, and a variety of mercury ion specific nucleic acid probes are designed and used. However, most of the sensing modes still stay in the homogeneous phase reaction stage, and although the sensing modes are simple and quick, only one-time testing can be realized, the reuse is difficult, and the medicine consumption is large, so that the single detection cost is high. In contrast, solid-phase sensing can be realized by means of a fixed sensing interface, the interface can be regenerated and reused, and the premise of realizing low-cost continuous online monitoring is provided.
Disclosure of Invention
The invention aims to provide a complete set of reagents for preparing a high-regenerable nucleic acid biochip and application thereof in mercury ion detection, which have the advantages of economy, simplicity, convenience and quickness and overcome the technical defects in the prior art.
Based on the prepared high-regenerable nucleic acid biochip, the invention designs a bivalent mercury ion nucleic acid probe rich in T basic group for detecting mercury ions in water, and inspects the detection performance of the mercury ions and tests the performance stability of the chip by taking a multi-index toxic pollutant online analyzer as a detection platform.
The invention provides a high-regenerable nucleic acid biochip, which comprises a chip and amino nucleic acid modified on the surface of the chip;
the sequence of the amino nucleic acid is as follows: 5' -NH2(C6)-GCAGCCCAAGAAA-3'。
The amino nucleic acid is attached to the chip via a covalent bond formed between an amino group and an aldehyde group.
Further, the invention also provides a kit for detecting mercury ions based on the T-T mismatch principle, which comprises the high-regenerable nucleic acid biochip and a mercury ion detection reagent;
the mercury ion detection reagent is a fluorescence-labeled nucleic acid probe, and the fluorescence-labeled nucleic acid probe is complementarily paired with the base of the amino nucleic acid on the reproducible nucleic acid biochip.
The sequence of the fluorescence labeled nucleic acid probe is as follows: Cy5.5-ATCCCCTTGTTTGTTTAGCCCCTATTCTTTCTTGGTCTGC.
Further, the present invention provides a kit for preparing a highly reproducible nucleic acid biochip, comprising kit A, kit B, kit C, kit D and kit E;
the reagent set A is amino activating liquid, and the amino activating liquid can be (3-aminopropyl) triethoxysilane (APTS) solution;
the reagent set B is a cross-linking solution which can be a glutaraldehyde aqueous solution;
the reagent set C is a nucleic acid reaction solution, and the nucleic acid reaction solution can be an amino nucleic acid solution;
the reagent set D is aldehyde-group confining liquid which can be glycine solution;
the reagent set E is an interface confining liquid, and the interface confining liquid can be an isoelectric point confining liquid, and specifically can be BSA isoelectric point confining liquid.
In the above reagent set, the volume fraction of the amino activating solution may be 0.5-2.5%, for example, 1%, and anhydrous toluene may be used as a solvent;
the volume fraction of glutaraldehyde in the crosslinking solution can be 0.5-2.5%, such as 1%;
the concentration of the Amino nucleic acid (Amino-DNA) in the nucleic acid reaction solution can be 0.1-1 mu M, such as 1 mu M, and a NaCl aqueous solution can be used as a solvent;
the sequence of the amino nucleic acid in the nucleic acid reaction solution is as follows: 5' -NH2(C6)-GCAGCCCAAGAAA-3'。
The concentration of glycine in the aldehyde group confining liquid can be 20-100 mM, such as 20mM, and NaCl aqueous solution can be used as a solvent;
the mass-volume concentration of the interface confining liquid can be 1-5 mg/mL, such as 2mg/mL, and a 0.1M citric acid-sodium citrate buffer solution with pH of 4.6 can be used as a solvent.
Based on the kit, the invention further provides a preparation method of the high-regenerable nucleic acid biochip, which comprises the following steps: modifying the chip by using the kit, specifically, sequentially processing the chip by using the kit A, the kit B, the kit C, the kit D and the kit E.
In the above preparation method, the treatment with the kit a comprises the following steps: and soaking the chip in the reagent set A, reacting for 1-3 hours (in a shaking table) at 20-37 ℃, then cleaning and drying, for example, soaking in anhydrous toluene for multiple times of ultrasonic cleaning, then cleaning the chip with an anhydrous toluene solution for multiple times, then blowing the chip with nitrogen, and baking the blown chip in an oven.
The treatment with the kit B comprises the following steps: immersing the chip treated by the reagent set A in the reagent set B to react for 1-3 hours (in a shaking table) at the temperature of 20-37 ℃;
the treatment with the kit C comprises the following steps: dropwise adding the reagent set C to the surface of the chip treated by the reagent set B, and reacting for 18-24 hours (in a shaking table) at 20-37 ℃;
the treatment with the kit D comprises the following steps: soaking the chip treated by the reagent set C in the reagent set D to react for 1-2 hours at 20-37 ℃; then soaking the mixture in a sodium cyanoborohydride aqueous solution to react for 1-2 hours (in a shaking table) at the temperature of 20-37 ℃;
the treatment with the kit E comprises the following steps: and soaking the chip treated by the kit reagent D in the kit reagent E to react for 1-3 hours (in a shaking table) at the temperature of 20-37 ℃.
The high-regenerable nucleic acid biochip provided by the invention is a biochip for detecting mercury ions based on a T-T mismatch principle, and a schematic diagram of the principle is shown in FIG. 1. Specifically, the surface of the sensing chip is modified with amino nucleic acid, and the amino nucleic acid can be hybridized with a probe sequence (SP-F) through base complementary pairing, so that fluorescent molecules marked on the SP-F are excited by using evanescent field energy on the surface of the chip, and a high fluorescence emission signal is generated. However, under the condition that mercury ions exist in the system, the SP-F recognizes the mercury ions in the system through T-Hg (II) -T base mismatch, and generates corresponding configuration change to form a bent hairpin structure, so that the hybridization with interface Amino-DNA is difficult to carry out through base complementation, and the collected fluorescence signal is weakened. Therefore, the fluorescence signal of the system is reduced along with the increase of the concentration of the mercury ions, and the system is in a turn-off type detection mode
In order to realize the detection of mercury ions based on the T-T mismatch principle, the invention further provides a kit comprising the kit for preparing the high-reproducible nucleic acid biochip, kit F, kit G and kit H;
the reagent set F is a mercury ion detection carrier liquid, the mercury ion detection carrier liquid is a buffer liquid formed by 3- (N-morpholinyl) propanesulfonic acid and sodium nitrate, the concentration of the 3- (N-morpholinyl) propanesulfonic acid is 5-20 mM, such as 10mM, the concentration of the sodium nitrate is 100-200 mM, such as 150mM, and the pH value of the buffer liquid is 7.0-7.4, such as 7.2;
the reagent set G is a mercury ion detection reagent, the mercury ion detection reagent is a system formed by dissolving a fluorescence-labeled nucleic acid probe (SignalProbe, SP) in a buffer solution formed by 3- (N-morpholinyl) propanesulfonic acid and sodium nitrate, the concentration of the 3- (N-morpholinyl) propanesulfonic acid in the buffer solution is 50-200 mM, such as 100mM, the concentration of the sodium nitrate is 1-2M, such as 1.5M, and the pH value of the buffer solution is 7.0-7.4, such as 7.2;
the fluorescence-labeled nucleic acid probe is complementarily paired with the base of the amino nucleic acid in the nucleic acid reaction solution;
the kit reagent H is an activating solution, the activating solution is a sodium dodecyl sulfate aqueous solution with the mass volume fraction of 0.3-1.5% (such as 0.5%), and the pH value is 1.5-2.5, such as 1.9.
The sequence of the fluorescence labeled nucleic acid probe is as follows: Cy5.5-ATCCCCTTGTTTGTTTAGCCCCTATTCTTTCTTGGTCTGC.
When the reagent set is used for detecting mercury ions, the following steps can be carried out:
preparing a high-regenerable nucleic acid biochip by using a set of reagents for preparing the high-regenerable nucleic acid biochip; mixing a reagent set G (mercury ion detection reagent) with a standard water sample containing mercury ions for pre-reaction, wherein the SP-F identifies the mercury ions in a system through T-Hg (II) -T base mismatch, and generates corresponding configuration change to form a bent hairpin structure; and reacting the liquid after the pre-reaction with the high-regenerable nucleic acid biochip, wherein the aim is to enable SP-F without a hairpin structure in the pre-reaction liquid to be hybridized with Amino-DNA on the surface of the high-regenerable nucleic acid biochip. Then washing the surface of the high-regenerable nucleic acid biochip by using a reagent set F (mercury ion detection carrier liquid), washing off non-hybridized SP-F, and then collecting a fluorescent signal; the complete set of reagent H (activating solution) is used for eluting the surface of the high-reproducible nucleic acid biochip. And (4) making a relation curve between the concentration of the mercury ions and the fluorescence signal of the standard water sample. And then, replacing the standard water sample with the water sample to be detected, repeating the process to obtain a fluorescence signal, and obtaining the concentration of mercury ions in the water sample according to the obtained relation curve.
The invention also provides a complete set of reagents for preparing the high-regenerable nucleic acid biochip, the regenerable nucleic acid biochip and application of the complete set of reagents for detecting mercury ions based on the T-T mismatch principle in mercury ion detection.
The invention achieves the following technical effects: (1) provides a complete set of reagents comprising five reagents of amino activating solution, cross-linking solution, nucleic acid reaction solution, aldehyde-based confining solution and interfacial confining solution, and the five reagents are combined to prepare a high-regenerable nucleic acid biochip; (2) provides a mercury ion specific nucleic acid probe capable of base complementary pairing hybridization with amino nucleic acid in a highly reproducible nucleic acid biochip, and utilizes T-Hg2+The T base mismatch principle specifically identifies mercury ions in water, and meanwhile, the probe changes the structure; (3) providing a mercury ion detection carrier liquid, a mercury ion detection reagent and a chip activating liquid, matching a highly reproducible nucleic acid biochip with a probe to carry out high-sensitivity 'turn-off' type mercury ion detection; (4) the provided reagent kit can realize continuous stable operation for 110 times and signal fluctuation<10％。
Drawings
FIG. 1 is a schematic diagram of the principle of mercury ion detection based on a base mismatch probe.
FIG. 2 is a process of covalently modifying amino-DNA on the surface of a chip.
FIG. 3 is a representative instrument signal obtained on a multi-index online toxic contaminant analyzer in accordance with an embodiment of the present invention.
Fig. 4 shows a mercury ion detection standard curve and related Logistic fitting parameters obtained in the embodiment of the present invention.
FIG. 5 shows the stability of the highly reproducible nucleic acid biochip of the present invention after 110 consecutive regenerations.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the following embodiments, a multi-index online analyzer (JQ MTP-online, product model) for toxic pollutants is used as an online detection platform, and Cy5.5 fluorescence labeled T-rich base DNA is used as a mercury ion detection probe.
Reagent kit
Conventional acid-base organic solvents were purchased from Beijing chemical plant, and other chemicals were purchased from Sigma-Aldrich; the nucleic acid sequence was synthesized by Biotechnology engineering (Shanghai) Inc.
Reagent set a, amino activating solution: 1 percent by volume (anhydrous toluene is used as a solvent) of (3-aminopropyl) triethoxysilane (APTS);
reagent set B, crosslinking solution: 1% volume fraction glutaraldehyde aqueous solution;
reagent set C, nucleic acid reaction solution: 50mM NaCl solution containing 1. mu.M of an Amino nucleic acid (Amino-DNA) to be modified; Amino-DNA sequence (5 '-3'): NH (NH)2(C6)-GCAGCCCAAGAAA；
Reagent set D, aldehyde group confining liquid: 20mM glycine (dissolved in 50mM sodium chloride);
reagent set E, interfacial sealant: 2mg/mL BSA isoelectric point blocking solution (dissolved in 0.1M citrate-sodium citrate buffer pH 4.6);
reagent set F, mercury ion detectionLiquid carrying: 10mM MOPS 150mM NaNO3,pH 7.2；
Reagent set G, mercury ion detection reagent: 50nM fluorescent nucleic acid probe (Signalprobe, SP) dissolved in 100mM MOPS 1.5M NaNO3(pH 7.2) buffer. Probe sequence (5 '-3'): Cy5.5-ATCCCCTTGTTTGTTTAGCCCCTATTCTTTCTTGGTCTGC;
reagent set H, chip activation solution: 0.5% by volume aqueous SDS solution, pH 1.9.
Secondly, preparing the high-reproducible nucleic acid biochip
The chip is made of K9 glass and has a length of 65 mm. 15mm wide, 1.5mm thick, 45 degree oblique angle, Beijing Ji optical processing factory.
The specific process for preparing the high-reproducible nucleic acid biochip by using the kit A-kit E modified chip is shown in FIG. 2.
Chip cleaning:
with Piranha solution (from concentrated H)2SO4With 30% (mass percent concentration) of H on the market2O2Composition, concentrated H2SO4And 30% H2O2Is 3: 1, wherein, H is concentrated2SO498% concentrated sulfuric acid produced by Beijing chemical plant), heating in an oven at 110 deg.C for 1 hour, heating and cooling to room temperature, washing the chip with ultrapure water to neutral, blowing the chip with nitrogen, and baking in an oven at 110 deg.C for at least 1 hour.
Chip modification:
(1) immersing the clean chip into an amino activating solution to react for 2 hours in a shaker (speed 90rpm) at 37 ℃; and then soaking the chip in anhydrous toluene for ultrasonic cleaning for 5 minutes for 3 times, cleaning the chip with the anhydrous toluene solution for 5 times, and then blowing the chip with nitrogen for drying. And (4) putting the dried chip into a 110 ℃ oven to be baked for 1 hour.
(2) The chips were placed in the crosslinking solution and reacted for 1 hour in a shaker (speed 90rpm) at 37 ℃.
(3) Then, the reacted chip was washed 5 times with ultrapure water, the surface was dried, 10. mu.L of the nucleic acid reaction solution was dropped onto the site to be modified on the surface, and the reaction was carried out overnight in a shaker at 37 ℃ at a speed of 90 rpm.
(4) The following day, the reacted chip was washed 5 times with clear water, and placed in aldehyde-based blocking solution to react for one hour in a shaker (speed 90rp m) at 37 ℃. Thereafter, the chip was immersed in a 20mg/mL aqueous solution of sodium cyanoborohydride and reacted in a shaker at 37 ℃ for one hour at a speed of 90 rpm; the chip was cleaned with ultrapure water, immersed in an interfacial sealing solution, sealed in a shaker at 37 deg.C (speed 90rpm) for 3 hours, and then stored in a refrigerator at 4 deg.C for a long period of time.
Third, on-line monitoring
The online detection method comprises the following specific steps:
1. initializing a detection flow: and detecting the operation conditions of each pump valve and each sampling pipeline.
2. Water sample-reagent mixing and pre-reaction: pumping water sample and mercury ion detection reagent (reagent set G) respectively and mixing the two for pre-reaction (the temperature is set to be 30 ℃).
3. Reacting the liquid with the chip after the pre-reaction: the pre-reaction solution in step 2 was reacted with the chip (the above-mentioned two prepared organisms) in order to hybridize SP-F, which did not form a hairpin structure in the pre-reaction solution, with Amino-DNA on the chip surface.
4. Reaction cell washing and signal measurement: the interface was washed by pumping in the mercury ion detection carrier (kit F) to wash away non-hybridized SP-F, followed by collection of the fluorescent signal.
5. Chip activation: the activating solution (kit H) was pumped in with the aim of eluting the surface.
Representative on-line instrument signals are shown in fig. 3, wherein A, C represents signals collected before interface reaction and after cleaning respectively; and B is a signal after the interface reaction and after washing by carrier liquid and before activation. A corresponding value of A is taken as a base line (usually 1000-2000), and a corresponding value of B-A is taken as an effective signal. C is usually used as a mark for judging whether the interface is effectively cleaned by SDS, and the interface can be considered to be cleaned when C is approximately equal to A.
Specifically, in the determination stage, a mercury ion detection reagent (kit G), standard water samples (with the concentrations of 0, 0.1, 1, 10, 100 and 1000 mu G/L respectively) containing mercury ions with different concentrations, and mercuryThe ion detection carrier liquid (kit reagent F) and the chip activating liquid (kit reagent H) are placed corresponding to the corresponding pipelines of the instrument, the measurement program of the instrument is started, the effective signals measured under the condition of each mercury ion concentration are recorded, a standard detection curve (a relation curve between the mercury ion concentration and the effective signals) is drawn, and four-parameter logistic fitting is carried out, as shown in FIG. 4. Maximum signal 10% (EC) based on fitting equation10) The detection limit was found to be 0.33. mu.g/L.
The instrument was operated continuously at a mercury ion concentration of 1 μ g/L to obtain results of 110 consecutive runs and the results were normalized to analyze the stability of the run data, as shown in fig. 5. Generally, for the biometric response, the data fluctuation within 20% of the mean was within the acceptable range, as can be seen from FIG. 5, in Hg2+The 110-time operation data on the detection chip are all in the range of 90-110% of the mean value, the fluctuation range is less than 10%, the excellent regeneration stability is shown, and a foundation is laid for the practical application of the chip in a detection site.
According to the obtained standard curve, a 1 mu g/L mercury ion water sample is tested, the average value of six measurement results is 1.018 mu g/L, the accuracy is 1.83%, and the method has high reliability.
Claims (6)
1. A kit for preparing a reproducible nucleic acid biochip, comprising kit A, kit B, kit C, kit D and kit E;
the reagent set A is amino activating solution which is (3-aminopropyl) triethoxysilane solution; the volume fraction of the amino activating solution is 0.5-2.5%;
the reagent set B is a cross-linking solution which is a glutaraldehyde aqueous solution; the volume fraction of glutaraldehyde in the crosslinking liquid is 0.5-2.5%;
the kit reagent C is a nucleic acid reaction solution, and the nucleic acid reaction solution is an amino nucleic acid solution; the concentration of the amino nucleic acid in the nucleic acid reaction solution is 0.1-1 mu M;
the reagent set D is aldehyde group confining liquid which is glycine solution; the concentration of glycine in the aldehyde group confining liquid is 20-100 mM;
the reagent set E is interface confining liquid, and the interface confining liquid is isoelectric point confining liquid; the mass-volume concentration of the interface confining liquid is 1-5 mg/mL;
the sequence of the amino nucleic acid in the nucleic acid reaction solution is as follows: 5' -NH2(C6)-GCAGCCCAAGAAA-3'；
The reproducible nucleic acid biochip is used for detecting mercury ions based on a T-T mismatch principle.
2. A method for preparing a reproducible nucleic acid biochip comprises the following steps: modifying a chip with the kit of claim 1, in particular sequentially treating said chip with said kit A, said kit B, said kit C, said kit D and said kit E;
the treatment with the kit A comprises the following steps: soaking the chip in the reagent set A, reacting for 1-3 hours at 20-37 ℃, and then cleaning and drying;
the treatment with the kit B comprises the following steps: soaking the chip treated by the reagent set A in the reagent set B to react for 1-3 hours at 20-37 ℃;
the treatment with the kit C comprises the following steps: dropwise adding the reagent set C to the surface of the chip treated by the reagent set B, and reacting for 18-24 hours at 20-37 ℃;
the treatment with the kit D comprises the following steps: soaking the chip treated by the reagent set C in the reagent set D to react for 1-2 hours at 20-37 ℃; then soaking the mixture in a sodium cyanoborohydride aqueous solution to react for 1-2 hours at the temperature of 20-37 ℃;
the treatment with the kit E comprises the following steps: and soaking the chip treated by the kit reagent D in the kit reagent E for reaction for 1-3 hours at the temperature of 20-37 ℃.
3. A regenerable nucleic acid biochip prepared by the method of claim 2.
4. A kit for detecting mercury ions based on the T-T mismatch principle, comprising the kit of claim 1, kit F, kit G and kit H;
the reagent set F is a mercury ion detection carrier liquid, the mercury ion detection carrier liquid is a buffer liquid formed by 3- (N-morpholinyl) propanesulfonic acid and sodium nitrate, the concentration of the 3- (N-morpholinyl) propanesulfonic acid is 5-20 mM, the concentration of the sodium nitrate is 100-200 mM, and the pH value of the buffer liquid is 7.0-7.4;
the kit reagent G is a mercury ion detection reagent, the mercury ion detection reagent is a system formed by dissolving a fluorescence-labeled nucleic acid probe in a buffer solution formed by 3- (N-morpholinyl) propanesulfonic acid and sodium nitrate, the concentration of the 3- (N-morpholinyl) propanesulfonic acid in the buffer solution is 5-200 mM, the concentration of the sodium nitrate is 1-2 mM, and the pH value of the buffer solution is 7.0-7.4;
the fluorescence-labeled nucleic acid probe is complementarily paired with the base of the amino nucleic acid in the nucleic acid reaction solution;
the sequence of the fluorescence labeled nucleic acid probe is as follows: Cy5.5-ATCCCCTTGTTTGTTTAGCCCCTATTCTTTCTTGGTCTGC;
the complete set of reagent H is activating solution which is 0.3-1.5% of sodium dodecyl sulfate aqueous solution by mass and volume, and the pH value is 1.5-2.5.
5. Use of the regenerable nucleic acid biochip of claim 3 for detecting mercury ions.
6. Use of a kit according to claim 1 or 4 for the detection of mercury ions.
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| CN106383101A (en) * | 2016-08-31 | 2017-02-08 | 清华大学 | Fluorescence detection method and fluorescence probe chip of mercury ions based on "off-on-off" mode |
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| CN106383101A (en) * | 2016-08-31 | 2017-02-08 | 清华大学 | Fluorescence detection method and fluorescence probe chip of mercury ions based on "off-on-off" mode |
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| Title |
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| T–T mismatch-driven biosensor using triple functional DNA-protein conjugates for facile detection of Hg2+;Ruoyu Wang 等;《Biosensors and Bioelectronics》;20160430;第78卷(第15期);图1和摘要 * |
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