CN112210588A - Dual-signal amplification probe, sensor, detection method and application - Google Patents
Dual-signal amplification probe, sensor, detection method and application Download PDFInfo
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Abstract
The invention belongs to the field of biochemical analysis and detection, and particularly relates to a dual-signal amplification probe, a sensor, a detection method and application, wherein the dual-signal amplification probe comprises the following components: the aptamer probe is formed by self-assembling at least 3 single-stranded sequences, and the sequence S1 comprises a sequence ST complementary to the aptamer sequence S3 and a first binding sequence; sequence S2 includes sequence ST complementary to aptamer sequence S3 and a sequence complementary to the first binding sequence; aptamer sequence S3; a hairpin probe comprising a second binding sequence, a G-quadruplex sequence, and a sequence ST which is partially complementary to sequence ST; the second binding sequence is partially complementary to the G-quadruplex sequence; the second binding sequence is partially complementary to sequence ST ', forming a protruding end at the 3 ' end of sequence ST '; (ii) a blunt end at the 3' end of sequence ST when sequence ST is complementary to sequence ST; the dual-signal amplification probe detects a target by adopting an exonuclease-assisted signal amplification method, and has the advantages of excellent system stability, high detection sensitivity and strong specificity.
Description
Technical Field
The invention relates to the field of biochemical analysis and detection, in particular to a dual-signal amplification probe, a sensor, a detection method and application.
Background
Organophosphorus pesticides are a class of phosphorus-containing organic compound pesticides such as the commonly used trichlorfon, dichlorvos, omethoate and malathion (Mal). In the organophosphorus pesticide family, malathion is one of the most common commercial broad-spectrum insecticides and is widely used as a protectant for the prevention and control of crop pests. Despite the enormous economic benefits of malathion in food production, its threat to human health cannot be underestimated. Malathion is reported to inhibit acetylcholinesterase (AChE) activity, resulting in dyspnea, headache and dizziness, and in severe cases, respiratory paralysis and death. The Chinese national food safety standard (GB 2763-2019) stipulates that the residual limit of malathion in grains is 0.1-8mg/kg, and the residual limit of fruits such as oranges, pears, apples and the like is 2 mg/kg. In the United states, the maximum residual limit of malathion in food products is 8 mg/kg. In addition, the food code Committee (CAC) specifies a limit of 0.01-25mg/kg of malathion in various seeds, grains, fruits and vegetables.
In the last decade, new biological detection methods based on aptamer sensors or immunosensors have emerged in the field of trace detection of biomolecules and monitoring of environmental pollutants. Aptamers are single-stranded oligonucleotides screened from a particular pool of oligonucleotides using exponential enrichment ligand evolution techniques. They have the advantages of simple screening, wide range of target substances, high affinity and specificity, easy modification and the like. At present, the successful construction of various aptamer sensors such as electrochemiluminescence, fluorescence, colorimetry, chemiluminescence and the like greatly promotes the development of pesticide residue detection technology. In these aptamer sensors, the use of nucleic acid amplification strategies (such as exponential amplification reaction (EXPAR), Catalytic Hairpin Assembly (CHA) and Exonuclease Assisted Signal Amplification (EASA) play an important role in improving the sensitivity of the detection methodHydrolysis of the 3, 5-phosphodiester linkage at the end (3 'or 5') gives a nucleotide. Based on the differences in the exonuclease hydrolysis patterns (different hydrolysis directions or different substrates), a variety of exonuclease-assisted signal amplification strategies have been developed and ideally integrated into the construction of aptamer sensors. G-quadruplexes/hemin DNases formed from guanine-rich nucleic acid quadruplexes and heme are the most commonly used biocatalytic DNases, which have horseradish peroxidase-like activity and can pass H in the presence of the cofactor hemin2O2Catalyze the oxidation of a variety of substrates such as 2,2 ' -diaza-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 3 ', 5,5 ' -Tetramethylbenzidine (TMB), and 3-aminophthalide (luminol). Compared to conventional proteases, the G quadruplex/hemin DNases have significant advantages, including relatively low cost, ease of preparation and modification, and high thermostability. Thus, G-quadruplex/hemin DNases have been widely used in the highly sensitive detection of nucleic acids, proteins, metal ions and small molecules.
However, there are currently no reports of aptamer sensors that combine exonuclease-assisted dual signal amplification with G-quadruplex/hemin dnase to detect malathion.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a dual-signal amplification probe, a sensor, a detection method and application, wherein the dual-signal amplification probe, the sensor and the detection method can be used for detecting malathion with high sensitivity and high specificity.
Therefore, the invention provides the following technical scheme:
a dual signal amplification probe comprising:
an aptamer probe self-assembled from at least 3 single-stranded sequences, the single-stranded sequences including sequence S1, sequence S2, and aptamer sequence S3 for binding to a target; the sequence S1 comprises a sequence ST complementary to the aptamer sequence S3 and a first binding sequence from 5 'end to 3' end; the sequence S2 comprises a sequence ST complementary to the aptamer sequence S3 and a complementary sequence of the first binding sequence in sequence from 5 'end to 3' end;
a hairpin probe comprising, in order from 5 'to 3', a second binding sequence, a G-quadruplex sequence, and a sequence ST which is partially complementary to sequence ST; the second binding sequence is partially complementary to the 3' terminal sequence of the G-quadruplex sequence; the second binding sequence is partially complementary to sequence ST and forms a protruding end at the 3' end of sequence ST; when sequence ST is complementary to sequence ST, a blunt end is formed at the 3' end of sequence ST.
The sequence S1 of the dual-signal amplification probe sequentially comprises a sequence a, a sequence b, a sequence c, a sequence d, a sequence e and a sequence f from a 5 'end to a 3' end; the sequence a, the sequence b, the sequence c and the sequence d are a sequence ST; sequence e and sequence f are first binding sequences;
the sequence S2 sequentially comprises a sequence a, a sequence b, a sequence c, a sequence d, a sequence f complementary to the sequence f and a sequence e complementary to the sequence e from the 5 'end to the 3' end; sequence f and sequence e are the complements of the first binding sequences
Aptamer sequence S3 sequentially includes, from 5 'to 3', sequence d complementary to sequence d, sequence c complementary to sequence c, sequence b complementary to sequence b, and sequence a complementary to sequence a;
the hairpin probe sequentially comprises a sequence b, a sequence g, a sequence h, a sequence b and a sequence a from the 5 'end to the 3' end; sequence b and sequence G are the second binding sequence, sequence h is the G-quadruplex sequence, sequence b and sequence a are sequence ST; the sequence g is complementary to the 3' terminal sequence of the sequence h.
The length of the G-quadruplex sequence of the double-signal amplification probe is 18-22 basic groups;
alternatively, the length of the G-quadruplex sequence is 18 bases;
optionally, the second binding sequence has a number of bases that is partially complementary to the 3' end sequence of the G-quadruplex sequence of less than or equal to 1/3 of the total number of bases of the G-quadruplex sequence; preferably, the number of bases of the second binding sequence which are partially complementary to the 3' -end sequence of the G-quadruplex sequence is 5;
optionally, the bases of the second binding sequence that are partially complementary to the sequence at the 3' end of the G-quadruplex sequence include at least 3 guanine bases G.
In the dual-signal amplification probe, the nucleotide sequence of the sequence S1 is shown as SEQ ID NO.1, the nucleotide sequence of the sequence S2 is shown as SEQ ID NO.2, the nucleotide sequence of the aptamer sequence S3 is shown as SEQ ID NO.3, and the nucleotide sequence of the hairpin probe is shown as SEQ ID NO. 4.
The invention provides a double-signal amplification detection kit, which comprises a double-signal amplification probe;
optionally, the molar ratio of the aptamer probe to the hairpin probe is (1-3) to (1-5);
optionally, the molar ratio of the aptamer probe to the hairpin probe is 2: 3;
optionally, the kit also comprises exonuclease Exo I, wherein the dosage ratio of the exonuclease Exo I to the aptamer probe is (10-20) to (0.01-0.03), and the proportional relation is U/nmol;
optionally, the dosage ratio of the exonuclease Exo I to the aptamer probe is 15:0.02, and the proportional relation is U/nmol;
optionally, the kit also comprises exonuclease Exo III, wherein the dosage ratio of the exonuclease Exo III to the hairpin probe is (15-30) to (0.01-0.05), and the proportional relation is U/nmol;
optionally, the dosage ratio of the exonuclease Exo III to the hairpin probe is 20:0.03, and the proportion relation is U/nmol.
The invention provides a dual-signal amplification detection sensor, which comprises a dual-signal amplification probe or a dual-signal amplification detection kit;
optionally, in the sensor, the concentration of the aptamer probe is 50-150nM, the amount of Exo I is 10-20U, the concentration of the hairpin probe is 50-250nM, and the amount of Exo III is 15-20U;
optionally, in the sensor, the concentration of the aptamer probe is 100nM, the amount of Exo I is 15U, the concentration of the hairpin probe is 150nM, and the amount of Exo III is 20U;
in the dual signal amplification detection sensor, the total volume is 200 μ L, and the dual signal amplification detection sensor comprises:
a first reaction liquid comprising:
aptamer probe with concentration of 1-3 μ M and volume of 10 μ L;
exo I with a concentration of 5-10U/. mu.L and a volume of 2. mu.L;
a first buffer in a volume of 41 μ L;
a second reaction solution comprising:
hairpin probe, concentration 2/3-10/3 u M, volume 15L;
exo III with a concentration of 7.5-15U/. mu.L and a volume of 2. mu.L;
first buffer, volume 30. mu.L.
Optionally, the first reaction solution includes:
aptamer probe with concentration of 2 μ M and volume of 10 μ L;
exo I at a concentration of 7.5U/. mu.L in a volume of 2. mu.L;
first buffer, volume 41. mu.L.
Optionally, the second reaction solution includes:
hairpin probe, concentration of 2. mu.M, volume of 15. mu.L;
exo III at a concentration of 10U/. mu.L in a volume of 2. mu.L;
first buffer, volume 30. mu.L.
Optionally, the method further comprises a third reaction solution:
hemin with a concentration of 2 μ M and a volume of 10 μ L;
a second buffer in a volume of 40 μ L;
luminol at a concentration of 1mM in a volume of 25 μ L;
H2O2at a concentration of 1mM, in a volume of 25. mu.L.
Optionally, the first buffer is 1 XNEBuffer 3.1 (containing 50mM Tris HCl, 100mM NaCl, 10mM MgCl)2,100μg/mL BSA,pH 7.9)。
Optionally, the second buffer is HEPES buffer (25mM, pH 7.4) containing 200mM NaCl,20mM KCl, and volume percent 1% DMSO.
The dual signal amplification probe, the dual signal amplification detection kit, or the dual signal amplification detection sensor is used for detecting a target;
optionally, the target is malathion;
optionally, the application of the malathion in detection of traditional Chinese medicines or grains.
A method of dual signal amplification detection of a target comprising using the dual signal amplification probe, the dual signal amplification detection kit, or the dual signal amplification detection sensor.
The method comprises the following steps:
adding a sample to be detected into a mixed solution containing an aptamer probe and exonuclease I, incubating, heating to inactivate the exonuclease I, and cooling to obtain a first amplification reaction product;
adding the obtained first amplification reaction product into a mixed solution containing the hairpin probe and exonuclease III, and incubating to obtain a second amplification reaction product;
optionally, in the first amplification reaction product preparation, the incubation time is 45-75 minutes; the incubation temperature is 35-45 ℃;
optionally, in the first amplification reaction product preparation, the incubation time is 60 minutes; the incubation temperature was 37 ℃;
optionally, in the preparation of the first amplification reaction product, the Exo I is inactivated by heating to 80-90 ℃ for 15-20 minutes;
optionally, in the preparation of the second amplification reaction product, the incubation time is 60-120 minutes; the incubation temperature is 35-45 ℃;
optionally, in the preparation of the second amplification reaction product, the incubation time is 90 minutes; the incubation temperature was 37 ℃;
optionally, preparing the second amplification reaction product into G-quadruplex/hemin DNase, and performing chemiluminescence detection;
alternatively, in preparing the G-quadruplex/hemin DNase, hemin, the second amplification reaction product and a buffer are mixed and incubated at room temperature for 60 to 75 minutes.
The technical scheme of the invention has the following advantages:
1. the invention provides a dual signal amplification probe, which comprises: an aptamer probe self-assembled from at least 3 single-stranded sequences, the single-stranded sequences including sequence S1, sequence S2, and aptamer sequence S3 for binding to a target; the sequence S1 comprises a sequence ST complementary to the aptamer sequence S3 and a first binding sequence from 5 'end to 3' end; the sequence S2 comprises a sequence ST complementary to the aptamer sequence S3 and a complementary sequence of the first binding sequence in sequence from 5 'end to 3' end; a hairpin probe comprising, in order from 5 'to 3', a second binding sequence, a G-quadruplex sequence, and a sequence ST which is partially complementary to sequence ST; the second binding sequence is partially complementary to the 3' terminal sequence of the G-quadruplex sequence; the second binding sequence is partially complementary to sequence ST and forms a protruding end at the 3' end of sequence ST; (ii) a blunt end is formed at the 3' end of sequence ST when sequence ST is complementary to sequence ST; when the double-signal amplification probe is used for detection under the assistance of exonuclease, when a target (malathion) exists in a sample to be detected, an aptamer sequence S3 in the aptamer probe is specifically combined with the target to form a compound Mal-S3, the compound is separated from the aptamer probe, a duplex S1-S2 is left, the compound Mal-S3 is recognized by exonuclease I (Exo I), the sequence S3 in the compound is gradually removed, the target malathion is released, the released target malathion can be freely combined with a new aptamer probe, and the first double-signal amplification assisted by the exonuclease I is started. The first heavy signal amplification, which produces more duplexes S1-S2, is treated with exonuclease III (Exo III) and converted to a larger number of secondary targets ST, which interact with the hairpin probe H1 to form complexes H1-ST, which complexes H1-ST are further digested by exonuclease III and liberate secondary targets ST. The released secondary target ST is recycled to start exonuclease III assisted second signal amplification. After complete amplification, a large amount of G-quadruplex sequence is produced which can be prepared as a G-quadruplex/hemin DNase. These DNases catalyze luminol-H2O2Oxidation of the system, resulting in an intense chemiluminescent signal. Under the best experimental conditions, the prepared dual-signal amplification probe has malathion in the range of 1pM to 100nMHas excellent linear response, detection limit of 0.47pM, high sensitivity and strong specificity.
Furthermore, the main structure of the aptamer probe designed by the invention is provided with at least two support arms, different aptamer sequences can be respectively combined on the support arms, so that different targets can be detected, when the two single-chain support arms are combined with the same aptamer sequence, more secondary targets ST can be generated during first heavy signal amplification, and the detection sensitivity is further improved;
furthermore, the invention only comprises 2 probes, so that the sensing system during detection is further simplified, and the potential interaction between different probes in the same solution is reduced, thereby improving the stability of the sensing system; meanwhile, the reduction of the number of the probes reduces the chemiluminescence background signal of a sensing system, and is beneficial to the improvement of the detection sensitivity.
2. The number of bases of the second binding sequence which are partially complementary to the G-quadruplex sequence is not more than 1/3 of the total number of bases of the G-quadruplex sequence; since the G-quadruplex sequence is a single-stranded DNA sequence rich in guanine base G, the length is generally 18-22 bases. When the single-stranded structure is in a free single-stranded state, 4 bases G can be connected through Hoogsteen hydrogen bonds to form a circular plane, and a G-quadruplex structure is further formed through pi-pi stacking, namely the G-quadruplex sequence in the single-stranded state can be folded into the G-quadruplex structure in molecules. In the present invention, the G-quadruplex sequence is immobilized by controlling the number of bases of the second binding sequence partially complementary to the G-quadruplex sequence to be not more than 1/3 of the total number of bases of the entire G-quadruplex sequence, meanwhile, on one hand, the method avoids the problems that the hairpin conformation stability of the hairpin probe is too strong due to too many base numbers for hybridization and fixation, the opening of the hairpin probe is not facilitated in the reaction process, the amplification efficiency of the amplification reaction is reduced, and in severe cases, the hairpin probe cannot be opened, which results in failure of the whole amplification reaction, and on the other hand, the number of bases for hybridization immobilization is too small, the immobilization effect cannot be achieved, when the hydrogen bonding effect in the G-quadruplex molecule is larger than that between the base pairs, the hairpin probe loses significance, when no target detection object exists, a G-quadruplex structure is formed, so that the whole detection system fails;
furthermore, the G-quadruplex sequence in the invention has 18 bases in total, one part is positioned in the circular region (13 bases) of the hairpin probe, and the other part is positioned in the stem region (5 bases) of the hairpin. When the hairpin probe is not opened, the G-quadruplex sequence is fixed by 5 bases (including 3 GGG bases), so that the hairpin probe cannot fold into a G-quadruplex structure without being opened, and cannot bind haem to form G-quadruplex/haem DNase. Only when the hairpin probe is opened will the entire G-quadruplex sequence be exposed (or released) and further bound to hemin to form a G-quadruplex/hemin DNase.
3. The invention provides a dual-signal amplification detection sensor, which comprises an aptamer probe, a hairpin probe, exonuclease Exo I and exonuclease Exo III; the method adopts an exonuclease I and exonuclease III assisted signal amplification (EASA) strategy, namely the first heavy signal amplification and the second heavy signal amplification belong to the same amplification technology, the reaction controllability is higher, the system stability is better, the method is obviously superior to the existing catalytic hairpin self-assembly (CHA) combined exonuclease III assisted signal amplification (EASA) strategy, the organic combination of the two different amplification technologies needs to be unified into a reaction system, and the stability of a sensing system is not favorable.
4. The invention provides a method for detecting a target by double signal amplification, which is characterized in that an aptamer probe, a hairpin probe, exonuclease Exo I and exonuclease Exo III are utilized, and the target is detected by an exonuclease I and exonuclease III assisted signal amplification (EASA) strategy, so that the method is high in detection sensitivity, strong in specificity, higher in reaction controllability and better in system stability.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram of the design of aptamer probes S1-S2-S3 and hairpin probe H1 of example 1 of the invention;
FIG. 2 is a schematic representation of the exonuclease assisted dual signal amplification strategy used in the detection of malathion chemiluminescence in example 11 of the present invention;
FIG. 3 is a graph showing the results of the feasibility study of the exonuclease assisted dual signal amplification strategy in Experimental example 1 of the present invention; FIG. A is a schematic representation of the assay of malathion using aptamer probe S1-S2-S3 and hairpin probe H1; panel B is a schematic of the Exo I-assisted signal amplification strategy used in the malathion assay; in the figure, C is a chemiluminescence response diagram, and the curve: a: S1-S2-S3+ H1, b: malathion + S1-S2-S3+ H1 (no amplification), c: malathion + S1-S2-S3+ Exo I + H1 (single signal amplification), d malathion + S1-S2-S3+ Exo I + Exo III + H1 (double signal amplification);
FIG. 4 shows the effect of the concentration of aptamer probes S1-S2-S3 on the sensing system in Experimental example 2 of the present invention; error bars in the figure represent Standard Deviation (SD) of three independent experiments;
FIG. 5 shows the effect of the amount of exonuclease I used in Experimental example 2 of the present invention on the sensing system; error bars in the figure represent Standard Deviation (SD) of three independent experiments;
FIG. 6 shows the effect of Exo I-assisted amplification time on the sensing system in Experimental example 2 of the present invention; error bars in the figure represent Standard Deviation (SD) of three independent experiments;
FIG. 7 shows the effect of hairpin probe H1 concentration on the sensing system in Experimental example 2 of the invention; error bars in the figure represent Standard Deviation (SD) of three independent experiments;
FIG. 8 shows the effect of the amount of exonuclease III used in Experimental example 2 of the present invention on the sensing system; error bars in the figure represent Standard Deviation (SD) of three independent experiments;
FIG. 9 shows the effect of Exo III-assisted amplification time on the sensing system in Experimental example 2 of the present invention; error bars in the figure represent Standard Deviation (SD) of three independent experiments;
FIG. 10 shows the results of the analysis performance of the sensor in Experimental example 3 of the present invention; in the figure, A is a chemiluminescence spectrum under different concentration conditions of malathion; in the figure, B is the chemiluminescence intensity (. lamda.)em422nm) and malathion concentration, with the inset: chemiluminescence intensity (lambda)em422nm) and the log of the malathion concentration; error bars represent Standard Deviation (SD) of three independent experiments;
FIG. 11 shows the results of selective detection by a sensor in Experimental example 4 of the present invention; in the figure, A is the selectivity of the aptamer sensor to different organophosphorus pesticide molecules, including chlorpyrifos, phoxim, methyl parathion, omethoate and malathion, and an error bar in the figure represents the Standard Deviation (SD) of three independent experiments; in the figure, B is the chemical structure of five organophosphorus pesticide molecules.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
The oligonucleotide sequences (S1, S2, S3, H1, H2, H3) used in the present invention were synthesized by Nanjing Kingsry Biotech Co., Ltd and purified by HPLC. The sequences of these oligonucleotides are listed in table 1. Malathion, chlorpyrifos, phoxim, methyl parathion and omethoate were purchased from Beijing Zhongke quality inspection Biotech, Inc. Exonuclease I (Exo I), exonuclease III (Exo III), and DEPC treated water are supplied by Biotechnology engineering (Shanghai) Inc. 10 XNEBuffer 3.1 was purchased from NY British Biotechnology (Beijing) Ltd. Radix Angelicae sinensis, radix Codonopsis and radix astragaliPurchased from traditional Chinese medicine institute of Wuxi city. Rice and wheat were purchased from local high-care supermarkets. Tris (hydroxymethyl-1) aminomethane (Tris), 2- (4- (2-hydroxyethyl) piperazin-1-yl) ethanesulfonic acid (HEPES), 3-aminophthalic hydrazide (luminol), hemin (hemin) and H2O2Purchased from Shanghai Aladdin Biotechnology Ltd. Other chemicals (analytical grade) were ordered from the national pharmaceutical group chemical agents limited.
Preparing a malathion standard substance: purchased from Beijing Zhongke Biotech limited. The specification is 250 mg/bottle, colorless to light yellow oily liquid. Taking out a proper amount of malathion, adding a proper amount of acetone to dissolve the malathion, and preparing into a malathion solution with the concentration of 10 mu M. Subsequently, 10. mu.M malathion solution was diluted stepwise with phosphate buffered saline (PBS, pH 7.4) to a concentration of 100nM,10nM,1nM,100pM,10pM,1pM malathion solution (μ M, nM, pM are concentration units, all referred to as μmol/L, nmol/L, pmol/L). Other standards, such as chlorpyrifos, phoxim, methyl parathion, and omethoate, are also available from beijing midkine quality testing biotechnology limited, either solid or liquid in character. The solution was diluted to 10. mu.M with acetone and further diluted to the desired concentration with phosphate buffered saline (PBS, pH 7.4).
The instrument comprises the following steps: chemiluminescence emission spectra were collected and recorded by a SpectraMax M5e multifunctional microplate reader at room temperature (meigu molecular instruments ltd) in combination with a 96-well transparent microplate reader (corning). Collecting the chemiluminescence spectra in the range of 370-510nm by taking 2nm as a step size. The maximum emission wavelength was 422 nm.
The room temperature ranges referred to in the following examples are 20-25 ℃.
Example 1 design of aptamer probes S1-S2-S3 and hairpin probe H1
This example designed aptamer probe S1-S2-S3 and hairpin probe H1, including:
an aptamer probe self-assembled from at least 3 single-stranded sequences, the single-stranded sequences including sequence S1, sequence S2, and aptamer sequence S3 for binding to a target; the sequence S1 comprises a sequence ST complementary to the aptamer sequence S3 and a first binding sequence from 5 'end to 3' end; the sequence S2 comprises a sequence ST complementary to the aptamer sequence S3 and a complementary sequence of the first binding sequence in sequence from 5 'end to 3' end;
a hairpin probe comprising, in order from 5 'to 3', a second binding sequence, a G-quadruplex sequence, and a sequence ST which is partially complementary to sequence ST; the second binding sequence is partially complementary to a sequence at the 3' end of the G-quadruplex sequence; the second binding sequence is partially complementary to sequence ST and forms a protruding end at the 3' end of sequence ST; when sequence ST is complementary to sequence ST, a blunt end is formed at the 3' end of sequence ST.
Specifically, the sequence S1 sequentially includes a sequence a, a sequence b, a sequence c, a sequence d, a sequence e and a sequence f from the 5 'end to the 3' end; the sequence a, the sequence b, the sequence c and the sequence d are a sequence ST; sequence e and sequence f are first binding sequences;
the sequence S2 sequentially comprises a sequence a, a sequence b, a sequence c, a sequence d, a sequence f complementary to the sequence f and a sequence e complementary to the sequence e from the 5 'end to the 3' end; sequence f and sequence e are the complements of the first binding sequence;
aptamer sequence S3 sequentially includes, from 5 'to 3', sequence d complementary to sequence d, sequence c complementary to sequence c, sequence b complementary to sequence b, and sequence a complementary to sequence a;
the hairpin probe sequentially comprises a sequence b, a sequence g, a sequence h, a sequence b and a sequence a from the 5 'end to the 3' end; sequence b and sequence G are the second binding sequence, sequence h is the G-quadruplex sequence, sequence b and sequence a are sequence ST; the sequence g is complementary to the 3' terminal sequence of the sequence h.
Further, the length of the G-quadruplex sequence is 18 to 22 bases, and in this example, the length of the G-quadruplex sequence is 18 bases.
In a preferred embodiment, the number of bases of the second binding sequence that are partially complementary to the G-quadruplex sequence is 1/3 or less based on the total number of bases of the G-quadruplex sequence; in a more preferred embodiment, the number of bases of the second binding sequence that are partially complementary to the G-quadruplex sequence is 5.
In a preferred embodiment, the bases of the second binding sequence that are partially complementary to the G-quadruplex sequence comprise at least 3 guanine bases G.
The nucleotide sequence of the sequence S1 is shown as SEQ ID NO.1, the nucleotide sequence of the sequence S2 is shown as SEQ ID NO.2, the nucleotide sequence of the aptamer sequence S3 is shown as SEQ ID NO.3, and the nucleotide sequence is shown as a hairpin probe (H1 for short) shown as SEQ ID NO. 4.
For the reaction of each segment in the specific sequence of the probe described above, see table 1 below, the G-quadruplex sequence (abbreviated GQ), as shown in table 1 below:
TABLE 1 HPLC-pure oligonucleotide sequences used in this example
The sequences corresponding to a, b, c, d, e, f, g, h, a, b, c, d, e, f in table 1 represent the sequences a, b, c, d, e, f, g, h, a, b, c, d, e, f, respectively.
The design principle of the above-mentioned dual signal probe, as shown in figure 1,
the aptamer probe is self-assembled by three single-stranded sequences S1, S2 and S3, wherein S3 is an aptamer sequence of a target malathion. All probe designs were derived from aptamer sequence S3. In order to simplify the design difficulty, a segmented design concept is introduced in the design of the probe. Aptamer sequence S3(72 bases) was first divided into four numbered domains a, b, c, and d, and the four domains (a, b, c, and d) in aptamer sequence S3 were identified, and the complementary domains (a, b, c, and d) in S1 and S2 were identified accordingly. The remaining domains e and f in S1 are complementary to domains e and f in S2, which form a portal conformation upon self-assembly. The sequence of these domains was determined using the in-line program OligoAnalyzer (IDT, version 3.1). According to the dual signal amplification strategy of the present invention, hairpin probe H1 functions in two ways, one is to perform the second signal amplification and the other is to output an optical signal, so that the G-quadruplex sequence (GQ, 18 bases) is encoded into hairpin probe H1, in which most of the sequence is encoded in the loop region of the hairpin and the rest of the sequence is encapsulated in the stem region of the hairpin by hybridization with domain G. Thus, in the initial state, the G-quadruplex sequence is restricted from binding the cofactor heme to form the G-quadruplex/heme DNase. The aptamer probe (abbreviated as S1-S2-S3) and the hairpin probe H1 were designed to be activated in the presence of malathion and with the aid of exonuclease to perform a dual signal amplification reaction.
Example 2
This example provides methods for preparing aptamer probes S1-S2-S3 and hairpin probes H1, H2, H3 designed in example 1
Preparation method of hairpin probe H1: oligonucleotide powder H1 was centrifuged at 12000rpm (Edwarder centrifuge 5417R) for 5 minutes and then dissolved in 20mM Tris-HCl buffer (200mM NaCl,20mM KCl,2mM MgCl)2pH 7.4) to prepare a 10. mu.M stock solution. The stock solution of H1 was then heated to 95 ℃ for 10 minutes and then slowly cooled to room temperature to form a hairpin structure. Hairpin probes H2 and H3 were prepared in the same manner.
The preparation method of the aptamer probe S1-S2-S3 comprises the following steps: the oligonucleotide powders S1, S2 and S3 were centrifuged at 12000rpm for 5 minutes, respectively, and then dissolved in 20mM Tris-HCl buffer (pH 7.4) to give 100. mu.M stock solutions. Then 10. mu.L of sequence S1 (100. mu.M), 10. mu.L of sequence S2 (100. mu.M) and 20. mu.L of sequence S3 (100. mu.M) were added to 60. mu.L of 20mM Tris-HCl buffer (pH 7.4) and incubated at 37 ℃ for 30 minutes. After the sequences S1, S2 and S3 were completely self-assembled, the aptamer probe S1-S2-S3 was formed at a concentration of 10. mu.M.
EXAMPLE 3 Dual Signal amplification detection kit
This example provides a dual signal amplification assay kit, comprising aptamer probes S1-S2-S3 and hairpin probe H1 of example 1.
Further, the aptamer probe S1-S2-S3 was present in a 2:3 molar ratio to hairpin probe H1.
Further, the kit also comprises exonuclease Exo I, the dosage ratio of the exonuclease Exo I to the aptamer probes S1-S2-S3 is 15:0.02, and the proportion relation is U/nmol.
Furthermore, the kit also comprises exonuclease Exo III, wherein the dosage ratio of the exonuclease Exo III to the hairpin probe H1 is 20:0.03, and the ratio relation is U/nmol.
EXAMPLE 4 Dual Signal amplification detection kit
This example provides a dual signal amplification assay kit, comprising aptamer probes S1-S2-S3 and hairpin probe H1 of example 1.
Further, the aptamer probe S1-S2-S3 was present in a 1:1 molar ratio to hairpin probe H1.
Further, the kit also comprises exonuclease Exo I, the exonuclease Exo I and the aptamer probe S1-S2-S3, wherein the dosage ratio is 10:0.01, and the ratio relation is U/nmol.
Furthermore, the kit also comprises exonuclease Exo III, wherein the dosage ratio of the exonuclease Exo III to the hairpin probe H1 is 15:0.01, and the ratio relation is U/nmol.
Example 5 Dual Signal amplification detection kit
This example provides a dual signal amplification assay kit, comprising aptamer probes S1-S2-S3 and hairpin probe H1 of example 1.
Further, the aptamer probe S1-S2-S3 was present in a molar ratio of 3:5 to hairpin probe H1.
Further, the kit also comprises exonuclease Exo I, the exonuclease Exo I and the aptamer probe S1-S2-S3 in a dosage ratio of 20:0.03 in a proportion relationship of U/nmol.
Furthermore, the kit also comprises exonuclease Exo III, wherein the dosage ratio of the exonuclease Exo III to the hairpin probe H1 is 30:0.05, and the ratio relation is U/nmol.
EXAMPLE 6 Dual Signal amplification detection sensor
The present embodiment provides a dual signal amplification detection sensor, which comprises, based on a total volume of 200 μ L:
the first reaction solution includes:
aptamer probes S1-S2-S3 at a concentration of 1. mu.M in a volume of 10. mu.L;
exo I at a concentration of 5U/. mu.L in a volume of 2. mu.L;
a first buffer in a volume of 41 μ L;
the second reaction solution includes:
hairpin probe H1 at a concentration of 2/3. mu.M in a volume of 15. mu.L;
exo III at a concentration of 7.5U/. mu.L in a volume of 2. mu.L;
a first buffer in a volume of 30 μ L;
further, the method also comprises a third reaction solution:
hemin with a concentration of 2 μ M and a volume of 10 μ L;
a second buffer in a volume of 40 μ L;
luminol at a concentration of 1mM in a volume of 25 μ L;
H2O2at a concentration of 1mM, in a volume of 25. mu.L.
The first buffer was 1 XNEBuffer 3.1 (containing 50mM Tris HCl, 100mM NaCl, 10mM MgCl)2,100μg/mL BSA,pH 7.9)。
The second buffer was HEPES buffer (25mM, pH 7.4) containing 200mM NaCl,20mM KCl and 1% DMSO by volume.
Example 7 Dual Signal amplification detection sensor
The present embodiment provides a dual signal amplification detection sensor, which comprises, based on a total volume of 200 μ L:
the first reaction solution includes:
aptamer probes S1-S2-S3 at a concentration of 3. mu.M in a volume of 10. mu.L;
exo I at a concentration of 10U/. mu.L in a volume of 2. mu.L;
a first buffer in a volume of 41 μ L;
the second reaction solution includes:
hairpin probe H1 at a concentration of 10/3. mu.M in a volume of 15. mu.L;
exo III at a concentration of 15U/. mu.L in a volume of 2. mu.L;
a first buffer in a volume of 30 μ L;
further, the method also comprises a third reaction solution:
hemin with a concentration of 2 μ M and a volume of 10 μ L;
a second buffer in a volume of 40 μ L;
luminol at a concentration of 1mM in a volume of 25 μ L;
H2O2at a concentration of 1mM, in a volume of 25. mu.L.
The first buffer was 1 XNEBuffer 3.1 (containing 50mM Tris HCl, 100mM NaCl, 10mM MgCl)2,100μg/mL BSA,pH 7.9)。
The second buffer was HEPES buffer (25mM, pH 7.4) containing 200mM NaCl,20mM KCl and 1% DMSO by volume.
Example 8 Dual Signal amplification detection sensor
The present embodiment provides a dual signal amplification detection sensor, which comprises, based on a total volume of 200 μ L:
the first reaction solution includes:
aptamer probes S1-S2-S3 at a concentration of 2. mu.M in a volume of 10. mu.L;
exo I at a concentration of 7.5U/. mu.L in a volume of 2. mu.L;
a first buffer in a volume of 41 μ L;
the second reaction solution includes:
hairpin probe H1 at a concentration of 2. mu.M in a volume of 15. mu.L;
exo III at a concentration of 10U/. mu.L in a volume of 2. mu.L;
a first buffer in a volume of 30 μ L;
further, the method also comprises a third reaction solution:
hemin with a concentration of 2 μ M and a volume of 10 μ L;
a second buffer in a volume of 40 μ L;
luminol at a concentration of 1mM in a volume of 25 μ L;
H2O2at a concentration of 1mM, in a volume of 25. mu.L.
The first buffer was 1 XNEBuffer 3.1 (containing 50mM Tris HCl, 100mM NaCl, 10mM MgCl)2,100μg/mL BSA,pH 7.9)。
The second buffer was HEPES buffer (25mM, pH 7.4) containing 200mM NaCl,20mM KCl and 1% DMSO by volume.
Example 9A method for detecting a target by dual signal amplification
This example provides a method for dual signal amplification of a target, comprising the steps of:
(1) mu.L of a test sample, which may contain the malathion of interest, was added to a first signal amplification system comprising 10. mu.L of aptamer probe S1-S2-S3 (1. mu.M), 2. mu.L of Exo I (5U/. mu.L) and 41. mu.L of 1 XNEBuffer 3.1(50mM Tris HCl, 100mM NaCl, 10mM MgCl)2100. mu.g/mL BSA, pH 7.9). Incubate at 35 ℃ for 45 minutes for Exo I-assisted signal amplification. Then, Exo I was inactivated by heating to 85 ℃ for 18 minutes and slowly cooled to room temperature.
(2) mu.L of the amplification reaction product was added to a second signal amplification system comprising 15. mu.L of hairpin probe H1 (2/3. mu.M), 2. mu.L of Exo III (7.5U/. mu.L) and 30. mu.L of 1 XNEBuffer 3.1. The mixture was incubated at 35 ℃ for 60 minutes for Exo III-assisted signal amplification.
(3) mu.L of hemin (2. mu.M) and 101. mu.L of the above double amplification reaction product were added to 40. mu.L of HEPES buffer (25mM, pH 7.4) containing 200mM NaCl,20mM KCl and 1% DMSO by volume to prepare G-quadruplex/hemin DNase. The mixture was incubated at room temperature for 68 minutes to form the G-quadruplex/hemin DNase. Thereafter, 25. mu.L of luminol (1mM) and 25. mu. L H were added2O2(1mM) to a final solution volume of 201. mu.L. Chemiluminescence spectra were collected using a microplate reader (SpectraMax M5 e).
Example 10A method for detecting a target by dual signal amplification
This example provides a method for dual signal amplification of a target, comprising the steps of:
(1) mu.L of a test sample, which may contain the malathion of interest, was added to a first signal amplification system comprising 10. mu.L of aptamer probe S1-S2-S3 (3. mu.M), 2. mu.L of Exo I (10U/. mu.L) and 41. mu.L of 1 XNEBuffer 3.1(50mM Tris HCl, 100mM NaCl, 10mM MgCl)2100. mu.g/mL BSA, pH 7.9). Incubate at 45 ℃ for 75 minutes for Exo I-assisted signal amplification. Then, Exo I was inactivated by heating to 90 ℃ for 20 minutes and slowly cooled to room temperature.
(2) mu.L of the amplification reaction product was added to a second signal amplification system comprising 15. mu.L of hairpin probe H1 (10/3. mu.M), 2. mu.L of Exo III (15U/. mu.L) and 30. mu.L of 1 XNEBuffer 3.1. The mixture was incubated at 45 ℃ for 120 minutes for Exo III-assisted signal amplification.
(3) mu.L of hemin (2. mu.M) and 101. mu.L of the above double amplification reaction product were added to 40. mu.L of HEPES buffer (25mM, pH 7.4) containing 200mM NaCl,20mM KCl and 1% DMSO by volume to prepare G-quadruplex/hemin DNase. The mixture was incubated at room temperature for 75 minutes to form G-quadruplex/hemin DNase. Thereafter, 25. mu.L of luminol (1mM) and 25. mu. L H were added2O2(1mM) to a final solution volume of 201. mu.L. Chemiluminescence spectra were collected using a microplate reader (SpectraMax M5 e).
Example 11A method for detecting a target by dual signal amplification
This example provides a method for dual signal amplification of a target, comprising the steps of:
(1) mu.L of the target malathion (at different concentrations) was added to a first signal amplification system comprising 10. mu.L of aptamer probe S1-S2-S3 (2. mu.M), 2. mu.L of Exo I (7.5U/. mu.L) and 41. mu.L of 1 XNEBuffer 3.1(50mM Tris HCl, 100mM NaCl, 10mM MgCl)2100. mu.g/mL BSA, pH 7.9). Incubate at 37 ℃ for 60 minutes for Exo I-assisted signal amplification. Then, Exo I was inactivated by heating to 80 ℃ for 15 minutes and slowly cooled to room temperature.
(2) mu.L of the amplification reaction product was added to a second signal amplification system comprising 15. mu.L of hairpin probe H1 (2. mu.M), 2. mu.L of Exo III (10U/. mu.L) and 30. mu.L of 1 XNEBuffer 3.1. The mixture was incubated at 37 ℃ for 90 minutes for Exo III-assisted signal amplification.
(3) mu.L of hemin (2. mu.M) and 101. mu.L of the above double amplification reaction product were added to 40. mu.L of HEPES buffer (25mM, pH 7.4) containing 200mM NaCl,20mM KCl and 1% DMSO by volume to prepare G-quadruplex/hemin DNase. The mixture was incubated at room temperature for 60 minutes to form G-quadruplexes/heminsA DNA enzyme. Thereafter, 25. mu.L of luminol (1mM) and 25. mu. L H were added2O2(1mM) to a final solution volume of 201. mu.L. Chemiluminescence spectra were collected using a microplate reader (SpectraMax M5 e).
EXAMPLE 1 feasibility study of exonuclease-assisted Dual Signal amplification strategy
The working principle of the amplification strategy of the exonuclease-assisted dual-signal amplification probe designed by the invention is as follows:
FIG. 2 shows a schematic diagram of the exonuclease assisted dual signal amplification strategy of the probe, sensor or method of the invention for highly sensitive detection of malathion. The detection system consists of aptamer probes S1-S2-S3, hairpin probe H1, Exo I and Exo III. In the absence of the target malathion, the aptamer probe S1-S2-S3 is protected from exonuclease I cleavage by having a matched double stranded structure, and both are stably co-present in solution. After exonuclease I is inactivated and exonuclease III is added, the double-stranded aptamer probe S1-S2-S3 is catalyzed by exonuclease III to hydrolyze into nucleotide fragments. That is, when the target is not present, the aptamer probe S1-S2-S3 fails to trigger a first amplification reaction, is then completely cleaved by exonuclease III, and fails to trigger a second amplification reaction. As a result, the entire detection system shows a relatively low background signal. After introduction of the target malathion, the aptamer sequence S3 in the aptamer probe S1-S2-S3 binds with high specificity to the target to form a complex Mal-S3 due to the high affinity between the aptamer probe S1-S2-S3 and detaches from the aptamer probe S1-S2-S3, leaving the duplex S1-S2 with two free single-stranded arms. The newly formed complex Mal-S3 was further recognized by Exo I, where the aptamer sequence S3 was removed by stepwise hydrolysis of single nucleotides by Exo I, thereby releasing the target malathion. The released target is free to bind a new aptamer probe S1-S2-S3 and initiate a new round of amplification reaction (Cycle I) with the aid of exonuclease I. After the cycle I completely reacts, inactivating the exonuclease I and adding the exonuclease III. The addition of exonuclease III serves two purposes: one is to completely hydrolyze excess aptamer probe S1-S2-S3 into nucleotide fragments, and the other is to convert duplex S1-S2Is the secondary target ST. The single-stranded secondary target ST can further serve as a primer to initiate exonuclease III assisted signal amplification. Specifically, based on competitive hybridization, domains (a and b) in ST can hybridize to domains (a and b) in hairpin H1 to form complex H1-ST. Complex H1-ST has a 3 '-blunt end and a 5' -protruding end, making it the most suitable substrate for exonuclease III. Notably, the opening of hairpin H1 resulted in the exposure of the G-quadruplex sequence (domain H). Exonuclease III catalyzes hydrolysis of complex H1-ST, releasing secondary target ST and single-stranded G-quadruplex sequence. The released secondary target ST can bind to the new hairpin H1, initiating a new round of amplification reaction (Cycle II) with the aid of exonuclease III. Following this mechanism, trace amounts of target can undergo multiple rounds of exonuclease-assisted amplification reactions, producing large amounts of G-quadruplex sequences. These G-quadruplex sequences further fold into barb-like hairpins and are held together by tetrahydrogenated guanine bases and then bind hemin to form G-quadruplex/hemin dnase. These formed dnases can act as luminol-H2O2An effective catalyst for a chemiluminescent system produces enhanced chemiluminescence for detection of trace amounts of malathion.
In order to verify the feasibility of the dual signal amplification strategy designed by the present invention, the chemiluminescent signals of malathion under different experimental conditions were recorded. The experimental conditions are shown in table 2 below:
TABLE 2 Experimental conditions
Note: replacing the malathion concentration of the sample to be detected with phosphate buffer solution (PBS, pH 7.4) with the same volume; replacing the Exo I concentration of 0 with equal volume of DEPC treated water; exo III concentration of 0 conditions were replaced with an equal volume of DEPC treated water.
The experimental conditions of schemes a-d in table 2 above were substituted into the following detection methods, respectively:
adding 1 μ L of the sample to be testedInto the first Signal amplification System, which contained 10. mu.L of aptamer probe S1-S2-S3, 2. mu.L of Exo I and 41. mu.L of 1 XNEBuffer 3.1(50mM Tris HCl, 100mM NaCl, 10mM MgCl)2100. mu.g/mL BSA, pH 7.9). Incubate at 37 ℃ for 60 minutes. Then, the mixture was heated to 80 ℃ for 15 minutes and slowly cooled to room temperature.
Next, 54. mu.L of the amplification reaction product was added to a second signal amplification system comprising 15. mu.L of hairpin probe H1, 2. mu.L of Exo III and 30. mu.L of 1 XNEBuffer 3.1. The mixture was incubated at 37 ℃ for 60 minutes to obtain a double amplification reaction product.
mu.L of hemin (2. mu.M) and 101. mu.L of the above double amplification reaction product were added to 40. mu.L of HEPES buffer (25mM, pH 7.4) containing 200mM NaCl,20mM KCl and 1% DMSO by volume to prepare G-quadruplex/hemin DNase. The mixture was incubated at room temperature for 60 minutes to form G-quadruplex/hemin DNase. Thereafter, 25. mu.L of luminol (1mM) and 25. mu. L H were added2O2(1mM) to a final solution volume of 201. mu.L. Chemiluminescence spectra were collected using a microplate reader (SpectraMax M5 e).
The results are shown in FIG. 3, where A is a schematic representation of the assay for malathion using aptamer probes S1-S2-S3 and hairpin probe H1, B is a schematic representation of the Exo I-assisted signal amplification strategy used for the assay for malathion, and C is a chemiluminescence response plot. From plot a in FIG. 3, it can be seen that under the experimental conditions of scheme a, only a weak chemiluminescent signal was observed, indicating that no interaction occurred between aptamer probe S1-S2-S3 and hairpin probe H1 in the absence of the target malathion. From plot b in plot C of FIG. 3, it can be seen that under the experimental conditions of scheme b, after addition of the target malathion, a clear chemiluminescent signal is detectable as compared to plot a, which, as can be seen in conjunction with plot A of FIG. 3, is primarily due to hybridization of the target malathion with the aptamer probe S1-S2-S3, the sequence S3 is separated from the aptamer probe S1-S2-S3, leaving the duplex S1-S2, the duplex S1-S2 further hybridizes with the hairpin probe H1 to form a complex S1-S2-H1, the free G-quadruplex sequence in the complex S1-S2-H1 folds into a G-quadruplex structure, and further binds with hemin to form a G-quadruplex/hemin DNase, which catalyzes oxidation of luminol and produces an optical signal, it is noteworthy that there is no signal amplification throughout the reaction. Under the experimental conditions of scheme C, i.e. after introduction of Exo I, as shown in panel B of figure 3, Exo I can hydrolyze the complex Mal-S3 to release the target malathion to initiate the Exo I-assisted signal amplification reaction, yielding more duplex S1-S2, and as more duplex S1-S2 is present in the sensor system, more G-quadruplex/hemin dnase is produced, which catalyzes oxidation of luminol and produces a stronger optical signal, and thus, as shown in curve C of figure 3, the chemiluminescence intensity also increases, which can be considered as exonuclease I-assisted single-plex signal amplification. More importantly, as shown by curve d in panel C of fig. 3, under the experimental conditions of scheme d, i.e. when Exo i and Exo III were introduced, the chemiluminescent intensity increased significantly, since exonuclease III could hydrolyze duplexes 1-S2, releasing secondary target ST which further served as a primer to trigger the exonuclease III assisted signal amplification reaction. Based on the Exo i and Exo iii assisted dual signal amplification, G-quadruplex/hemin dnase is produced exponentially, resulting in a significant enhancement of the chemiluminescent intensity. In summary, comparing the three different reaction strategies (curve b, no amplification; curve c, single signal amplification; curve d, double signal amplification), it is clear that the double signal amplification strategy has the highest amplification efficiency, with a signal enhancement of 204.2% (curve d vs b) and 84.4% (curve d vs c), respectively. These results strongly suggest that exonuclease-assisted dual signal amplification strategies can be used for highly sensitive detection of malathion.
Experimental example 2 optimization of experimental conditions
luminol-H2O2The chemiluminescence signal generated by the system depends on the amount of G-quadruplex/hemin DNA enzyme, and the experimental research finds that the chemiluminescence signal is greatly influenced by the concentration of the probe, the dosage of exonuclease and the amplification reaction time. Therefore, the present experiment was systematically studied on these experimental conditions.
1. Effect of aptamer Probe S1-S2-S3 concentration on sensing System
The first heavy amplification reaction (Exo I-assisted signal amplification) involved mainly aptamer probes S1-S2-S3 and exonuclease I, and the second heavy amplification reaction (Exo III-assisted signal amplification) involved mainly hairpin probe H1 and exonuclease III. In order to study the influence of the parameters of the first amplification reaction on the sensing system, the conditions of the second amplification reaction were fixed, and the specific detection method was as follows:
(1) mu.L of the target malathion (100nM) was added to the first signal amplification system containing 10. mu.L of aptamer probe S1-S2-S3, 2. mu.L of Exo I (7.5U/. mu.L) and 41. mu.L of 1 XNEBuffer 3.1(50mM Tris HCl, 100mM NaCl, 10mM MgCl 2100. mu.g/mL BSA, pH 7.9). Incubate at 37 ℃ for 60 minutes for Exo I-assisted signal amplification. Then, Exo I was inactivated by heating to 80 ℃ for 15 minutes and slowly cooled to room temperature.
(2) mu.L of the amplification reaction product was added to a second signal amplification system comprising 15. mu.L of hairpin probe H1 (4/3. mu.M), 2. mu.L of Exo III (10U/. mu.L) and 30. mu.L of 1 XNEBuffer 3.1. The mixture was incubated at 37 ℃ for 60 minutes for Exo III-assisted signal amplification.
(3) mu.L of hemin (2. mu.M) and 101. mu.L of the above double amplification reaction product were added to 40. mu.L of HEPES buffer (25mM, pH 7.4) containing 200mM NaCl,20mM KCl and 1% DMSO by volume to prepare G-quadruplex/hemin DNase. The mixture was incubated at room temperature for 60 minutes to form G-quadruplex/hemin DNase. Thereafter, 25. mu.L of luminol (1mM) and 25. mu. L H were added2O2(1mM) to a final solution volume of 201. mu.L. Chemiluminescence spectra were collected using a microplate reader (SpectraMax M5 e).
The concentrations of the aptamer probes S1-S2-S3 added in step (1) in the above detection method were selected to be 1. mu.M, 1.5. mu.M, 2. mu.M, 2.5. mu.M, and 3. mu.M, respectively. In this experiment, the total volume of the liquid sensor was 200. mu.L, which was calculated as follows: mu.l (final volume) -1. mu.l (target malathion) ═ 200. mu.l. The final concentration of the aptamer probe added, S1-S2-S3, in the liquid sensor was calculated as follows: the added volume (10. mu.L). times.the concentration of the aptamer probe S1-S2-S3 solution/200. mu.L, was calculated to have a final concentration of the aptamer probe S1-S2-S3 in the liquid sensor of 50nM, 75nM, 100nM, 125nM, 150nM, respectively. And respectively detecting the chemiluminescence spectrums of the target malathion in the sample to be detected under the conditions of existence and non-existence.
As shown in FIG. 4, the optimal signal-to-noise ratio (I/I) was obtained when the final concentration of the aptamer probe S1-S2-S3 in the liquid sensor was 100nM0) Wherein I and I0Chemiluminescence intensity of the sensing system with and without target malathion, respectively, thus, the optimal final concentration of aptamer probe S1-S2-S3 in the liquid sensor was 100nM and was used for subsequent experiments.
2. Effect of exonuclease I dosage on sensing systems
In order to study the influence of the parameters of the first amplification reaction on the sensing system, the conditions of the second amplification reaction were fixed, and the specific detection method was as follows:
(1) mu.L of the target malathion (100nM) was added to the first signal amplification system containing 10. mu.L of aptamer probe S1-S2-S3 (2. mu.M), 2. mu.L of Exo I and 41. mu.L of 1 XNEBuffer 3.1(50mM Tris HCl, 100mM NaCl, 10mM MgCl 3.1)2100. mu.g/mL BSA, pH 7.9). Incubate at 37 ℃ for 60 minutes for Exo I-assisted signal amplification. Then, Exo I was inactivated by heating to 80 ℃ for 15 minutes and slowly cooled to room temperature.
(2) mu.L of the amplification reaction product was added to a second signal amplification system comprising 15. mu.L of hairpin probe H1 (4/3. mu.M), 2. mu.L of Exo III (10U/. mu.L) and 30. mu.L of 1 XNEBuffer 3.1. The mixture was incubated at 37 ℃ for 60 minutes for Exo III-assisted signal amplification.
(3) mu.L of hemin (2. mu.M) and 101. mu.L of the above double amplification reaction product were added to 40. mu.L of HEPES buffer (25mM, pH 7.4) containing 200mM NaCl,20mM KCl and 1% DMSO by volume to prepare G-quadruplex/hemin DNase. The mixture was incubated at room temperature for 60 minutes to form G-quadruplex/hemin DNase. Thereafter, 25. mu.L of luminol (1mM) and 25. mu. L H were added2O2(1mM) to a final solution volume of 201. mu.L. Using a microplate reader (SpectraMax M5e)The chemiluminescence spectra were collected.
The amounts of exonuclease I added in step (1) in the detection method are respectively selected from 10, 12.5, 15.0, 17.5 and 20.0U.
As a result, as shown in FIG. 5, it was found that the chemiluminescence intensity increased with the amount of Exo I used. In view of the cost-effectiveness of exonuclease I, 15U was determined as the optimum amount of exonuclease I to be used.
Effect of Exo I-assisted amplification time on sensing System
In order to study the influence of the parameters of the first amplification reaction on the sensing system, the conditions of the second amplification reaction were fixed, and the specific detection method was as follows:
(1) mu.L of the target malathion (100nM) was added to a first signal amplification system containing 10. mu.L of aptamer probe S1-S2-S3 (2. mu.M), 2. mu.L of Exo I (7.5U/. mu.L) and 41. mu.L of 1 XNEBuffer 3.1(50mM Tris HCl, 100mM NaCl, 10mM MgCl 2100. mu.g/mL BSA, pH 7.9). Incubate at 37 ℃ for Exo I-assisted signal amplification. Then, Exo I was inactivated by heating to 80 ℃ for 15 minutes and slowly cooled to room temperature.
(2) mu.L of the amplification reaction product was added to a second signal amplification system comprising 15. mu.L of hairpin probe H1 (4/3. mu.M), 2. mu.L of Exo III (10U/. mu.L) and 30. mu.L of 1 XNEBuffer 3.1. The mixture was incubated at 37 ℃ for 60 minutes for Exo III-assisted signal amplification.
(3) mu.L of hemin (2. mu.M) and 101. mu.L of the above double amplification reaction product were added to 40. mu.L of HEPES buffer (25mM, pH 7.4) containing 200mM NaCl,20mM KCl and 1% DMSO by volume to prepare G-quadruplex/hemin DNase. The mixture was incubated at room temperature for 60 minutes to form G-quadruplex/hemin DNase. Thereafter, 25. mu.L of luminol (1mM) and 25. mu. L H were added2O2(1mM) to a final solution volume of 201. mu.L. Chemiluminescence spectra were collected using a microplate reader (SpectraMax M5 e).
The incubation times in "incubation at 37 ℃ for Exo I-assisted signal amplification" in step (1) of the above detection method were selected to be 30, 45, 60, 75, and 90 minutes, respectively.
As shown in FIG. 6, the chemiluminescence intensity increased with the increase in the reaction time. When the reaction time exceeded 60 minutes, the increase in chemiluminescent intensity shifted to a plateau indicating the end of the amplification reaction. Thus, the optimal Exo-I assisted amplification time was determined to be 60 minutes and used for subsequent experiments.
4. Effect of hairpin Probe H1 concentration on the sensing System
After the first amplification reaction is optimized, the conditions of the second amplification reaction are further optimized, and the specific method comprises the following steps:
(1) mu.L of the target malathion (100nM) was added to a first signal amplification system containing 10. mu.L of aptamer probe S1-S2-S3 (2. mu.M), 2. mu.L of Exo I (7.5U/. mu.L) and 41. mu.L of 1 XNEBuffer 3.1(50mM Tris HCl, 100mM NaCl, 10mM MgCl 2100. mu.g/mL BSA, pH 7.9). Incubate at 37 ℃ for 60 minutes for Exo I-assisted signal amplification. Then, Exo I was inactivated by heating to 80 ℃ for 15 minutes and slowly cooled to room temperature.
(2) mu.L of the amplification reaction product was added to a second signal amplification system comprising 15. mu.L of hairpin probe H1, 2. mu.L of Exo III (10U/. mu.L) and 30. mu.L of 1 XNEBuffer 3.1. The mixture was incubated at 37 ℃ for 60 minutes for Exo III-assisted signal amplification.
(3) mu.L of hemin (2. mu.M) and 101. mu.L of the above double amplification reaction product were added to 40. mu.L of HEPES buffer (25mM, pH 7.4) containing 200mM NaCl,20mM KCl and 1% DMSO by volume to prepare G-quadruplex/hemin DNase. The mixture was incubated at room temperature for 60 minutes to form G-quadruplex/hemin DNase. Thereafter, 25. mu.L of luminol (1mM) and 25. mu. L H were added2O2(1mM) to a final solution volume of 201. mu.L. Chemiluminescence spectra were collected using a microplate reader (SpectraMax M5 e).
The concentrations of the hairpin probe H1 solution added in step (2) in the above detection method were selected to be 2/3. mu.M, 4/3. mu.M, 2. mu.M, 8/3. mu.M, and 10/3. mu.M, respectively. In this experiment, the total volume of the liquid sensor was 200. mu.L, which was calculated as follows: mu.l (final volume) -1. mu.l (target malathion) ═ 200. mu.l. The final concentration of added hairpin probe H1 in the liquid sensor was calculated as follows: the volume added (15. mu.L). times.hairpin probe H1 solution concentration/200. mu.L was calculated to give final concentrations of hairpin probe H1 in the liquid sensor of 50nM, 100nM, 150nM, 200nM, 250nM, respectively. And respectively detecting the chemiluminescence spectrums of the target malathion in the sample to be detected under the conditions of existence and non-existence.
As shown in FIG. 7, the best signal-to-noise ratio (I/I) was obtained when hairpin probe H1 was present in the liquid sensor at a final concentration of 150nM0) Wherein I and I0Chemiluminescence intensity of the sensing system with and without target malathion, respectively, therefore, hairpin probe H1 was optimally at a final concentration of 150nM in the liquid sensor and used in subsequent experiments.
5. Effect of exonuclease III dose on sensing System
After the first amplification reaction is optimized, the conditions of the second amplification reaction are further optimized, and the specific method comprises the following steps:
(1) mu.L of the target malathion (100nM) was added to a first signal amplification system containing 10. mu.L of aptamer probe S1-S2-S3 (2. mu.M), 2. mu.L of Exo I (7.5U/. mu.L) and 41. mu.L of 1 XNEBuffer 3.1(50mM Tris HCl, 100mM NaCl, 10mM MgCl 2100. mu.g/mL BSA, pH 7.9). Incubate at 37 ℃ for 60 minutes for Exo I-assisted signal amplification. Then, Exo I was inactivated by heating to 80 ℃ for 15 minutes and slowly cooled to room temperature.
(2) mu.L of the amplification reaction product was added to a second signal amplification system comprising 15. mu.L of hairpin probe H1 (2. mu.M), 2. mu.L of Exo III and 30. mu.L of 1 XNEBuffer 3.1. The mixture was incubated at 37 ℃ for 60 minutes for Exo III-assisted signal amplification.
(3) mu.L of hemin (2. mu.M) and 101. mu.L of the above double amplification reaction product were added to 40. mu.L of HEPES buffer (25mM, pH 7.4) containing 200mM NaCl,20mM KCl and 1% DMSO by volume to prepare G-quadruplex/hemin DNase. The mixture was incubated at room temperature for 60 minutes to form G-quadruplex/hemin DNase. Then, 25. mu.L of luminol was added(1mM) and 25. mu. L H2O2(1mM) to a final solution volume of 201. mu.L. Chemiluminescence spectra were collected using a microplate reader (SpectraMax M5 e).
In the step (2) of the detection method, 10, 15, 20, 25 and 30U of the exonuclease III is respectively selected.
As shown in FIG. 8, the chemiluminescence intensity was observed to increase with increasing amounts of Exo III. In view of the cost-effectiveness of exonuclease III, 20U was determined as the optimum amount of exonuclease III to be used.
Effect of Exo III-assisted amplification time on sensing systems
After the first amplification reaction is optimized, the conditions of the second amplification reaction are further optimized, and the specific method comprises the following steps:
(1) mu.L of the target malathion (100nM) was added to a first signal amplification system containing 10. mu.L of aptamer probe S1-S2-S3 (2. mu.M), 2. mu.L of Exo I (7.5U/. mu.L) and 41. mu.L of 1 XNEBuffer 3.1(50mM Tris HCl, 100mM NaCl, 10mM MgCl 2100. mu.g/mL BSA, pH 7.9). Incubate at 37 ℃ for 60 minutes for Exo I-assisted signal amplification. Then, Exo I was inactivated by heating to 80 ℃ for 15 minutes and slowly cooled to room temperature.
(2) mu.L of the amplification reaction product was added to a second signal amplification system comprising 15. mu.L of hairpin probe H1 (2. mu.M), 2. mu.L of Exo III (10U/. mu.L) and 30. mu.L of 1 XNEBuffer 3.1. The mixture was incubated at 37 ℃ for Exo III-assisted signal amplification.
(3) mu.L of hemin (2. mu.M) and 101. mu.L of the above double amplification reaction product were added to 40. mu.L of HEPES buffer (25mM, pH 7.4) containing 200mM NaCl,20mM KCl and 1% DMSO by volume to prepare G-quadruplex/hemin DNase. The mixture was incubated at room temperature for 60 minutes to form G-quadruplex/hemin DNase. Thereafter, 25. mu.L of luminol (1mM) and 25. mu. L H were added2O2(1mM) to a final solution volume of 201. mu.L. Chemiluminescence spectra were collected using a microplate reader (SpectraMax M5 e).
In the detection step (2), the incubation times in "incubation of the mixture at 37 ℃ for Exo III-assisted signal amplification" were selected to be 45, 60, 75, 90, 105, and 120 minutes, respectively.
As shown in FIG. 9, the chemiluminescence intensity increased with the increase in the reaction time. When the reaction time exceeds 90 minutes, the increase in the chemiluminescence intensity shifts to a plateau, indicating the end of the amplification reaction. Thus, the optimal Exo-III assisted amplification time was determined to be 90 minutes and used for subsequent experiments.
Experimental example 3 analytical Properties of sensor
In order to verify the ability of the sensor prepared by the invention to detect the target with high sensitivity, malathion with various concentrations is analyzed under the optimal experimental conditions. The specific method comprises the following steps:
(1) mu.L of the test sample was added to a first signal amplification system comprising 10. mu.L of aptamer probe S1-S2-S3 (2. mu.M), 2. mu.L of Exo I (7.5U/. mu.L) and 41. mu.L of 1 XNEBuffer 3.1(50mM Tris HCl, 100mM NaCl, 10mM MgCl 2100. mu.g/mL BSA, pH 7.9). Incubate at 37 ℃ for 60 minutes for Exo I-assisted signal amplification. Then, Exo I was inactivated by heating to 80 ℃ for 15 minutes and slowly cooled to room temperature.
(2) mu.L of the amplification reaction product was added to a second signal amplification system comprising 15. mu.L of hairpin probe H1 (2. mu.M), 2. mu.L of Exo III (10U/. mu.L) and 30. mu.L of 1 XNEBuffer 3.1. The mixture was incubated at 37 ℃ for 90 minutes for Exo III-assisted signal amplification.
(3) mu.L of hemin (2. mu.M) and 101. mu.L of the above double amplification reaction product were added to 40. mu.L of HEPES buffer (25mM, pH 7.4) containing 200mM NaCl,20mM KCl and 1% DMSO by volume to prepare G-quadruplex/hemin DNase. The mixture was incubated at room temperature for 60 minutes to form G-quadruplex/hemin DNase. Thereafter, 25. mu.L of luminol (1mM) and 25. mu. L H were added2O2(1mM) to a final solution volume of 201. mu.L. Chemiluminescence spectra were collected using a microplate reader (SpectraMax M5 e).
In the above detection step (1), the concentration of the target malathion in 1. mu.L of the sample to be detected is selected to be 1pM,10pM,100pM,1nM,10nM,100nM, respectively, and the blank is 1uL of phosphate buffer solution (PBS, pH 7.4).
The results are shown in FIG. 10, in which the chemiluminescence responses of malathion at various concentrations in the range of 0 to 100nM are shown in Panel A of FIG. 10, and a gradual increase in the intensity of chemiluminescence is observed with increasing concentration of malathion. At the same time, a good exponential correspondence between the chemiluminescence intensity and the concentration of malathion can be observed in panel B of fig. 10, which is consistent with the working principle of the exonuclease-assisted dual signal amplification strategy of the present invention, i.e., the more malathion that is added to the system, the more G-quadruplex/heme dnase is generated and, therefore, the stronger the chemiluminescence intensity. In addition, as shown in the inset of the B plot of FIG. 10, the log of the chemiluminescence intensity versus the concentration of malathion showed a good linear correlation between 1pM and 100 nM. The linear regression equation is that I is 137749.65+33512.19log10C, correlation coefficient 0.9971, where I and C represent chemiluminescence intensity and malathion concentration, respectively. The detection Limit (LOD) was found to be 0.47pM by calculation. Table 3 gives a comprehensive comparison of the analytical performance between this experimental method and other reported malathion detection methods. It can be seen that the present experimental method has excellent analytical performance, which is mainly attributed to the high amplification efficiency of exonuclease-assisted dual signal amplification.
TABLE 3 comparison of Malathion detection based on different methods
SERS, surface enhanced Raman scattering; AgNPs silver nanoparticles; AuNPs gold nanoparticles; QDs are quantum dots; LC is liquid crystal; PDDA poly (dimethyldiallylammonium chloride)
(Nie et al.,2018):Nie,Y.,Teng,Y.,Li,P.,Liu,W.,Shi,Q.,Zhang,Y.,2018.Spectrochim.Acta A 191,271-276.
(Li et al.,2019):Liu,J.,Zhang,Y.,Xie,H.,Zhao,L.,Zheng,L.,Ye,H.,2019.Small 15,1902989.
(Bala et al.,2018):Bala,R.,Swami,A.,Tabujew,I.,Peneva,K.,Wangoo,N.,Sharma,R.K.,2018.Biosens.Bioelectron.104,45-49.
(Kim Hong and Jang,2020):Kim Hong,P.T.,Jang,C.H.,2020.Anal.Biochem.593,113589.
(Bala et al.,2017):Bala,R.,Dhingra,S.,Kumar,M.,Bansal,K.,Mittal,S.,Sharma,R.K.,Wangoo,N.,2017.Chem.Eng.J.311,111-116.
(Bala et al.,2016):Bala,R.,Kumar,M.,Bansal,K.,Sharma,R.K.,Wangoo,N.,2016.Biosens.Bioelectron.85,445-449.
Experimental example 4 sensor Selectivity
To evaluate the specificity of the sensors of the invention, four non-specific organophosphorus pesticide molecules (chlorpyrifos, phoxim, methyl parathion and omethoate) were selected as negative controls under optimal experimental conditions. The specific method comprises the following steps:
(1) mu.L of the test sample was added to a first signal amplification system comprising 10. mu.L of aptamer probe S1-S2-S3 (2. mu.M), 2. mu.L of Exo I (7.5U/. mu.L) and 41. mu.L of 1 XNEBuffer 3.1(50mM Tris HCl, 100mM NaCl, 10mM MgCl 2100. mu.g/mL BSA, pH 7.9). Incubate at 37 ℃ for 60 minutes for Exo I-assisted signal amplification. Then, Exo I was inactivated by heating to 80 ℃ for 15 minutes and slowly cooled to room temperature.
(2) mu.L of the amplification reaction product was added to a second signal amplification system comprising 15. mu.L of hairpin probe H1 (2. mu.M), 2. mu.L of Exo III (10U/. mu.L) and 30. mu.L of 1 XNEBuffer 3.1. The mixture was incubated at 37 ℃ for 90 minutes for Exo III-assisted signal amplification.
(3) mu.L of hemin (2. mu.M) and 101. mu.L of the above double amplification reaction product were added to 40. mu.L of HEPES buffer (25mM, pH 7.4) containing 200mM NaCl,20mM KCl and 1% DMSO by volume to prepare G-quadruplex/hemin DNase. The mixture was incubated at room temperature for 60 minutes to form G-quadruplex/hemin DNase. Thereafter, 25. mu.L of luminol (1mM) and 25. mu. L H were added2O2(1mM) to a final solution volume of 201. mu.L. Chemiluminescence spectra were collected using a microplate reader (SpectraMax M5 e).
In the detection step (1), 1 μ L of the sample to be detected is 1 μ L of a solution of 5 organophosphorus pesticide molecules (the concentrations of chlorpyrifos, phoxim, methyl parathion, omethoate and malathion in the solution are 100nM respectively), and the blank control adopts a solvent which does not contain any organophosphorus pesticide molecules, namely 1 μ L of phosphate buffer solution (PBS, pH 7.4).
The detection results are shown in fig. 11 (graph a is the chemiluminescence intensity detection result, graph B is the chemical structure of five organophosphorus pesticide molecules), and as shown in graph a of fig. 11, under the same experimental conditions, the target malathion can detect a significant chemiluminescence signal, while no significant chemiluminescence signal is observed for chlorpyrifos, phoxim, methyl parathion and omethoate. The signal intensity of these non-specific target molecules was almost the same or slightly increased compared to the blank control solution. The results show that the method provided by the invention has good specificity for detection of malathion, and the excellent specificity of the method is derived from high specificity of an aptamer probe for a target to a great extent.
Experimental example 5 detection of malathion in spiked grain and traditional Chinese medicine samples
To evaluate the applicability of the sensor of the present invention to real samples, recovery tests were performed using standard addition methods. Three traditional Chinese medicine samples (angelica, codonopsis pilosula and astragalus) and two grain samples (rice and wheat) are selected and respectively pretreated, and the detailed sample pretreatment process is as follows: first, 2g of the sample was accurately weighed and transferred to a 15mL centrifuge tube. Then, 10mL of an extraction solvent (methanol: water ═ 6:4(v/v)) was added, and the mixture was soaked for 60 minutes and then sonicated for 45 minutes. Subsequently, the supernatant was collected into a new centrifuge tube and centrifuged at 3500rpm for 5 minutes. After centrifugation, 1mL of the supernatant was removed and filtered through a sterile syringe filter (0.22 μm, Mercury). Finally, the resulting filtrate was diluted 10-fold with 1 × NEBuffer 3.1. Malathion (10 and 100nM) was added to the sample solution at two concentrations, respectively, and measured, and concentration detection was performed under the optimum experimental conditions, in the same manner as in experimental example 4. The malathion detection amounts are shown in table 4 below. The recovery rate is the detected amount/added standard amount multiplied by 100 percent; the calculated recoveries and relative standard deviations are shown in table 4.
Table 4 detection of malathion in spiked grain and traditional Chinese medicine samples (n ═ 3)
In table 4 above, the recovery of malathion was 94.4% to 108.7% and the Relative Standard Deviation (RSD) was less than 6.6%, indicating the potential use of the sensor of the present invention in the detection of pesticide residues.
Conclusion of the experiment
The invention develops a novel label-free and high-sensitivity chemiluminescent aptamer sensor based on exonuclease-assisted double signal amplification and G-quadruplex/heme DNA enzyme. Under the ingenious matching of the aptamer probe, the hairpin probe, the exonuclease I and the exonuclease III, the target malathion successfully triggers two tandem amplification reactions, so that the obvious amplification of signals is realized. Under the optimal experimental conditions, the prepared sensor has high sensitivity for the determination of malathion, and the detection limit is as low as 0.47 pM. Meanwhile, the sensor shows excellent specificity to malathion, can detect the malathion in the labeled grain and traditional Chinese medicine samples, and has a satisfactory result. Therefore, the sensor has a good application prospect in the field of pesticide residue detection.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
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Claims (10)
1. A dual signal amplification probe, comprising:
an aptamer probe self-assembled from at least 3 single-stranded sequences, the single-stranded sequences including sequence S1, sequence S2, and aptamer sequence S3 for binding to a target; the sequence S1 comprises a sequence ST complementary to the aptamer sequence S3 and a first binding sequence from 5 'end to 3' end; the sequence S2 comprises a sequence ST complementary to the aptamer sequence S3 and a complementary sequence of the first binding sequence in sequence from 5 'end to 3' end;
a hairpin probe comprising, in order from 5 'to 3', a second binding sequence, a G-quadruplex sequence, and a sequence ST which is partially complementary to sequence ST; the second binding sequence is partially complementary to the 3' terminal sequence of the G-quadruplex sequence; the second binding sequence is partially complementary to sequence ST and forms a protruding end at the 3' end of sequence ST; when sequence ST is complementary to sequence ST, a blunt end is formed at the 3' end of sequence ST.
2. The dual signal amplification probe of claim 1, wherein the sequence S1 comprises, in order from 5 'to 3', sequence a, sequence b, sequence c, sequence d, sequence e, and sequence f; the sequence a, the sequence b, the sequence c and the sequence d are a sequence ST; sequence e and sequence f are first binding sequences;
the sequence S2 sequentially comprises a sequence a, a sequence b, a sequence c, a sequence d, a sequence f complementary to the sequence f and a sequence e complementary to the sequence e from the 5 'end to the 3' end; sequence f and sequence e are the complements of the first binding sequence;
aptamer sequence S3 sequentially includes, from 5 'to 3', sequence d complementary to sequence d, sequence c complementary to sequence c, sequence b complementary to sequence b, and sequence a complementary to sequence a;
the hairpin probe sequentially comprises a sequence b, a sequence g, a sequence h, a sequence b and a sequence a from the 5 'end to the 3' end; sequence b and sequence G are the second binding sequence, sequence h is the G-quadruplex sequence, sequence b and sequence a are sequence ST; the sequence g is complementary to the 3' terminal sequence of the sequence h.
3. The dual signal amplification probe of claim 1 or 2, wherein the G-quadruplex sequence is 18 to 22 bases in length;
alternatively, the length of the G-quadruplex sequence is 18 bases;
optionally, the second binding sequence has a number of bases that is partially complementary to the 3' end sequence of the G-quadruplex sequence of less than or equal to 1/3 of the total number of bases of the G-quadruplex sequence; preferably, the number of bases of the second binding sequence which are partially complementary to the 3' -end sequence of the G-quadruplex sequence is 5;
optionally, the bases of the second binding sequence that are partially complementary to the sequence at the 3' end of the G-quadruplex sequence include at least 3 guanine bases G.
4. The dual signal amplification probe of any one of claims 1-3, wherein the nucleotide sequence of sequence S1 is set forth in SEQ ID No.1, the nucleotide sequence of sequence S2 is set forth in SEQ ID No.2, the nucleotide sequence of aptamer sequence S3 is set forth in SEQ ID No.3, and the nucleotide sequence of the hairpin probe is set forth in SEQ ID No. 4.
5. A dual signal amplification test kit comprising the dual signal amplification probe of any one of claims 1-4;
optionally, the molar ratio of the aptamer probe to the hairpin probe is (1-3) to (1-5);
optionally, the molar ratio of the aptamer probe to the hairpin probe is 2: 3;
optionally, the kit also comprises exonuclease Exo I, wherein the dosage ratio of the exonuclease Exo I to the aptamer probe is (10-20) to (0.01-0.03), and the proportional relation is U/nmol;
optionally, the dosage ratio of the exonuclease Exo I to the aptamer probe is 15:0.02, and the proportional relation is U/nmol;
optionally, the kit also comprises exonuclease Exo III, wherein the dosage ratio of the exonuclease Exo III to the hairpin probe is (15-30) to (0.01-0.05), and the proportional relation is U/nmol;
optionally, the dosage ratio of the exonuclease Exo III to the hairpin probe is 20:0.03, and the proportion relation is U/nmol.
6. A dual signal amplification detection sensor comprising a dual signal amplification probe according to any one of claims 1 to 4, or a dual signal amplification detection kit according to claim 5;
optionally, in the sensor, the concentration of the aptamer probe is 50-150nM, the amount of Exo I is 10-20U, the concentration of the hairpin probe is 50-250nM, and the amount of Exo III is 15-20U;
optionally, in the sensor, the aptamer probe concentration is 100nM, the amount of Exo I is 15U, the hairpin probe concentration is 150nM, and the amount of Exo III is 20U.
7. The dual signal amplification detection sensor of claim 6, comprising, in a total volume of 200 μ L:
a first reaction liquid comprising:
aptamer probe with concentration of 1-3 μ M and volume of 10 μ L;
exo I with a concentration of 5-10U/. mu.L and a volume of 2. mu.L;
a first buffer in a volume of 41 μ L;
a second reaction solution comprising:
hairpin probe, concentration 2/3-10/3 u M, volume 15L;
exo III with a concentration of 7.5-15U/. mu.L and a volume of 2. mu.L;
a first buffer in a volume of 30 μ L;
optionally, the method further comprises a third reaction solution:
hemin with a concentration of 2 μ M and a volume of 10 μ L;
a second buffer in a volume of 40 μ L;
luminol at a concentration of 1mM in a volume of 25 μ L;
H2O2at a concentration of 1mM, in a volume of 25. mu.L.
8. Use of the dual signal amplification probe of any one of claims 1-4, the dual signal amplification detection kit of claim 5, or the dual signal amplification detection sensor of claim 6 or 7 for detecting a target;
optionally, the target is malathion;
optionally, the application of the malathion in detection of traditional Chinese medicines or grains.
9. A method for dual signal amplification target detection, comprising using the dual signal amplification probe of any one of claims 1-4, the dual signal amplification detection kit of claim 5, or the dual signal amplification detection sensor of claim 6 or 7.
10. The method of claim 9, comprising:
adding a sample to be detected into a mixed solution containing an aptamer probe and exonuclease I, incubating, heating to inactivate the exonuclease I, and cooling to obtain a first amplification reaction product;
adding the obtained first amplification reaction product into a mixed solution containing the hairpin probe and exonuclease III, and incubating to obtain a second amplification reaction product;
optionally, in the first amplification reaction product preparation, the incubation time is 45-75 minutes; the incubation temperature is 35-45 ℃;
optionally, in the preparation of the first amplification reaction product, the Exo I is inactivated by heating to 80-90 ℃ for 15-20 minutes;
optionally, in the preparation of the second amplification reaction product, the incubation time is 60-120 minutes; the incubation temperature is 35-45 ℃;
optionally, preparing the second amplification reaction product into G-quadruplex/hemin DNase, and performing chemiluminescence detection;
alternatively, in preparing the G-quadruplex/hemin DNase, hemin and the second amplification reaction product are mixed and incubated at room temperature for 60-75 minutes.
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CN113322256A (en) * | 2021-06-22 | 2021-08-31 | 江苏省原子医学研究所 | Probe set, sensor, detection method and application thereof |
CN114032290A (en) * | 2021-10-27 | 2022-02-11 | 中山大学 | Aptamer functionalization-based SERS-FL sensor and preparation method and application thereof |
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