CN114350658B - Tetrahedral frame nucleic acid SERS probe, sensor, preparation method and application thereof - Google Patents

Tetrahedral frame nucleic acid SERS probe, sensor, preparation method and application thereof Download PDF

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CN114350658B
CN114350658B CN202210255008.7A CN202210255008A CN114350658B CN 114350658 B CN114350658 B CN 114350658B CN 202210255008 A CN202210255008 A CN 202210255008A CN 114350658 B CN114350658 B CN 114350658B
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吴萍
祝晶甜
郏雯雨
方宁宁
蔡称心
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Abstract

The invention discloses a tetrahedral framework nucleic acid SERS probe, a sensor, a preparation method and application thereof, wherein the probe comprises a single-chain probe Tetra-A, a single-chain probe Tetra-B, Tetra-C and a single-chain probe Tetra-D for modifying cyanine dye 5; the sensor is formed by the assembly of probes on a long-range SERS substrate and hybridization of ampicillin aptamers. The probe is uniformly distributed on the surface of the substrate, is uniformly oriented, can provide high-reproducibility SERS signals, and has good specificity; the long-range surface enhanced Raman scattering characteristic of the long-range substrate overcomes the limitation of the tFNA probe in SERS detection due to the large size of the tFNA probe, so that the probe can still obtain a strong Raman signal on the surface of the SERS substrate. The invention combines the tFNA probe and the long-range surface enhanced Raman substrate for the first time, and develops a novel ampicillin detection sensor with high sensitivity, high selectivity and high repeatability.

Description

Tetrahedral frame nucleic acid SERS probe, sensor, preparation method and application thereof
Technical Field
The invention belongs to biological Raman detection, and particularly relates to a tetrahedral framework nucleic acid SERS probe, a sensor, a preparation method and application thereof.
Background
Ampicillin, also known as ampicillin, is a beta-lactam antibiotic that has been widely used to treat various bacterial infections in humans, livestock, poultry and aquaculture due to its wide applicability to bacterial infections, good activity, good tolerability, low toxicity, low cost and good clinical efficacy. In recent years, although ampicillin as a growth regulator or an anti-infective drug has promoted rapid development of livestock and feed processing industries, illegal and excessive use in animals and livestock directly causes contamination and residue of antifungal drugs during production of foods of human and animal origin, causing health and safety problems to be increasingly prominent. Currently, the maximum residual ampicillin content is generally specified to be 10 ng/mL. Therefore, in order to guarantee the health of people, it is important to establish a reliable and rapid ampicillin detection method.
At present, technologies for detecting ampicillin exist, such as high performance liquid chromatography, electrochemical analysis, mass spectrometry, capillary electrophoresis, microbiological methods and the like, and the methods have some defects, such as expensive detection instruments, long consumed time, complex operation, large errors and the like. Although SERS methods have been used to detect ampicillin, the linear range in those methods is small; the detection limit is high; and the direct detection requires the pretreatment of the sample; in particular, the metal nanoparticles have poor enhancement effect and are easy to agglomerate, which causes instability of detection signals and can not obtain a Raman spectrogram with good enhancement effect and good reproducibility. The tetrahedral frame nucleic acid probe (tFNA probe) has the characteristics of uniform distribution and uniform orientation on the surface of a substrate, so that a high-reproducibility SERS signal can be provided, but the tFNA probe has a large size and is hardly used in the SERS field. Because SERS is a near-field effect, the effective range of the enhancement field of the hot spot region is 5 nm, and if the side length of the tFNA probe is 5.8 nm and the height is 8.67 nm, which exceeds 5 nm, the SERS signal of the tFNA probe is weak. Therefore, how to realize ampicillin detection by using the tFNA probe through the SERS method is an urgent problem to be solved.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a tetrahedral frame nucleic acid (tFNA) SERS probe, which is applied to ampicillin detection for the first time, constructs a sensor for detecting ampicillin in a water environment based on Surface Enhanced Raman Spectroscopy (SERS), and establishes a method for detecting ampicillin in a water environment based on Surface Enhanced Raman Spectroscopy (SERS), so that ampicillin can be detected quickly and efficiently, and the method has the advantages of high sensitivity, good reproducibility, simplicity in operation and low cost.
The invention also provides a preparation method and application of the tetrahedral framework nucleic acid (tFNA) SERS probe.
A third object of the present invention is to provide a sensor comprising tetrahedral framework nucleic acid (tFNA) SERS probes and applications thereof.
The technical scheme is as follows: in order to achieve the purpose, the tetrahedral framework nucleic acid SERS probe comprises a single-stranded probe Tetra-A, a single-stranded probe Tetra-B, a single-stranded probe Tetra-C and a single-stranded probe Tetra-D of a modified cyanine dye 5, wherein the sequences of the single-stranded probe Tetra-A, the single-stranded probe Tetra-B, the single-stranded probe Tetra-C and the single-stranded probe Tetra-D are respectively shown in SEQ ID NO. 1-4.
Wherein, the single-chain probe Tetra-A (SEQ ID NO.1 sequence: Cy 5-CCGCTATACAACCGGGCGCAAAAAAAAACATTCCTAAGTCTGAAACATTACAGCTTGCTACACGAGAAGAGCCGCCATAGTA), the single-chain probe Tetra-B (SEQ ID NO.2 sequence: SH-TATCACCAGGCAGTTGACAGTGTAGCAAGCTGTAATAGATGCGAGGGTCCAATAC), the single-chain probe Tetra-C (SEQ ID NO.3 sequence: SH-TCAACTGCCTGGTGATAAAACGACACTACGTGGGAATCTACTATGGCGGCTCTTC) and the single-chain probe Tetra-D (SEQ ID NO.4 sequence: SH-TTCAGACTTAGGAATGTGCTTCCCACGTAGTGTCGTTTGTATTGGACCCTCGCAT) of the modified cyanine dye 5 (Cy 5).
The probe of the invention designs a specific stem-loop structure with a Raman signal molecule Cy5 modified at the top end so as to be capable of hybridizing with an ampicillin aptamer.
Wherein the tetrahedral framework nucleic acid SERS probe comprises a single-stranded probe Tetra-A, a single-stranded probe Tetra-B, a single-stranded probe Tetra-C and a single-stranded probe Tetra-D of the modified cyanine dye 5 in equimolar amount.
The tetrahedral framework nucleic acid SERS probe is provided with a tetrahedral structure, the side length of the tetrahedral framework nucleic acid SERS probe is 5.8 nm, the height of the tetrahedral framework nucleic acid SERS probe is 8.67 nm, thiol groups are modified on the three top points of the bottom of the tetrahedral framework nucleic acid SERS probe, 8 adenine nucleotides are used as spacers on the top of the tetrahedral framework nucleic acid SERS probe to modify a stem-loop structure with a Raman signal molecule Cy5, and the stem-loop structure can be identified and hybridized with an aptamer of ampicillin.
The preparation method of the tetrahedral framework nucleic acid SERS probe comprises the following steps:
mixing single-stranded probe Tetra-A, single-stranded probe Tetra-B, single-stranded probe Tetra-C and single-stranded probe Tetra-D for modifying cyanine dye 5 according to equimolar ratio, heating, and cooling to obtain the cyanine dye.
Preferably, the single-stranded probe Tetra-A, the single-stranded probe Tetra-B, the single-stranded probe Tetra-C and the single-stranded probe Tetra-D of the modified cyanine dye 5 are mixed in equimolar amounts, heated at 92-95 ℃ for 8-10 minutes, and cooled at 2-4 ℃ for 30-50 seconds.
The sensor of the tetrahedral framework nucleic acid SERS probe is characterized in that the sensor is formed by assembling the tetrahedral framework nucleic acid SERS probe on a long-range SERS substrate and hybridizing an ampicillin aptamer.
Preferably, three vertexes of the tetrahedral frame nucleic acid SERS probe are connected to the long-range SERS substrate in a self-assembly mode through Au-S bonds to obtain the SERS substrate of the surface-assembled tetrahedral frame nucleic acid SERS probe, and then ampicillin aptamer solution is dropwise added to the SERS substrate of the surface-assembled tetrahedral frame nucleic acid SERS probe for incubation to enable the ampicillin aptamer to fully react with the probe.
Wherein, the ampicillin aptamer (SEQ ID NO.5 sequence: GCGGGCGGTTGTATAGCGG).
Preferably, the tFNA probe is self-assembled on the surface of the long-range SERS substrate by Au-S bond, and incubated with ampicillin aptamer (100. mu.L, 1. mu.M) at 37 ℃ for 2 hours to allow sufficient hybridization, and TM buffer (containing 20 mM Tris-HCl and 50 mM MgCl. TM.)2•6H2O, pH 8.0) to remove non-hybridized aptamers and record the raman response signal of the fna probe after hybridization.
The invention relates to an application of a tetrahedral framework nucleic acid SERS probe or a sensor in detecting ampicillin.
The sensor is soaked in ampicillin solutions with different concentrations, after incubation, unreacted ampicillin is washed away, the Raman spectrum of the sensor is measured, and ampicillin is detected.
Preferably, the SERS detection of ampicillin is carried out by immersing the long-range substrate adsorbed with hybridized tFNA probe in different concentrations (10)−14 ～ 10−9mol/L) of ampicillin, incubating for 1 hour at 37 ℃, washing with TM buffer solution to remove unreacted ampicillin, and measuring Raman spectrum to obtain a standard working curve. Examination ofAnd when the environmental sample is measured, judging whether ampicillin is contained according to the change of the SERS signal of the tFNA probe, and obtaining the concentration of ampicillin according to a standard working curve.
Specifically, a working curve for detecting the concentration of ampicillin is established: the response signal of the blank standard is I0The response signal containing ampicillin standard sample is IiDifference of response signalsΔI is defined as the response signal I containing ampicillin standardiSubtracting the response signal I of the blank standard0(ii) a Will be described inΔLog of the concentration of ampicillin and ICIs drawn intoΔI-Log CWorking curve and then linear regression methodΔI-Log CAnd (3) a linear regression equation is established, and a working curve of ampicillin in the water body environment such as detection of the sensor based on the tFNA probe is established.
When the concentration of ampicillin in a sample to be detected is detected, the long-range substrate adsorbed with the hybridized tFNA probe is soaked in the sample to be detected, incubation is carried out for 1 hour at 37 ℃, TM buffer solution is used for washing away unreacted sample to be detected, the Raman spectrum of the tFNA probe is detected through a Raman spectrometer, and a Raman response signal is recorded. And judging whether ampicillin is contained according to the SERS signal change of the tFNA probe, and obtaining the concentration of ampicillin according to a standard working curve.
Further, in the detection process, the excitation wavelength of the raman spectrometer is 785 nm, the integration time is 10 s, and the cycle number is 3 times.
The invention aims at the problems that the tFNA probe has large size, the SERS signal of the tFNA probe is weak and the like. The long-range SERS substrate is used for exciting the tFNA probe, and the effective enhancement signal of the long-range SERS substrate can reach 25 nm and far exceeds the size of the tFNA probe, so that the limitation of the tFNA probe in SERS detection due to the large size of the tFNA probe is overcome, and the tFNA probe can also have a stronger SERS signal on the surface of the long-range SERS substrate.
The tFNA probe used by the invention has the characteristics of uniform distribution and uniform orientation on the surface of the substrate, thereby being capable of providing high-reproducibility SERS signals; the tFNA probe contains a nucleic acid sequence capable of highly hybridizing with an ampicillin aptamer, so that the detection specificity is greatly improved; meanwhile, the long-range surface enhanced Raman scattering characteristic of the long-range substrate overcomes the limitation of the tFNA probe in SERS detection due to the large size of the tFNA probe, so that the probe can still obtain a strong Raman signal on the surface of the SERS substrate. The invention combines the tFNA probe and the long-range surface enhanced Raman substrate for the first time, and develops a novel ampicillin sensor with high sensitivity, high selectivity and high repeatability.
The detection principle of the invention is as follows: firstly, preparing a tFNA probe by a one-step synthesis method of a DNA nano self-assembly technology, self-assembling the tFNA probe on a long-range SERS active substrate, wherein the top end of the tFNA probe is of a stem-loop structure with a Raman signal molecule Cy5, the signal molecule Cy5 is close to the substrate, and the Raman signal is strong; when the ampicillin aptamer is subjected to hybridization reaction, the stem-loop structure is opened to be a straight-chain structure, a tFNA probe-aptamer compound is formed, the signal molecule Cy5 is far away from the substrate, and the Raman signal is weakened; when the ampicillin is incubated with ampicillin solution, ampicillin is specifically combined with an aptamer of the ampicillin, the aptamer competes from the tFNA probe-aptamer compound, the tFNA probe restores a stem-loop structure, and a Raman signal is restored. A method for quantitatively detecting ampicillin by adopting a Raman spectrum method is provided by utilizing competition reaction between a tFNA probe and ampicillin aptamer and between the ampicillin aptamer and ampicillin. The concentration of ampicillin in the detection sample is calculated through the change of the Raman signal intensity of the tFNA probe, so that quick and sensitive qualitative detection of ampicillin can be realized, and quantitative determination can also be carried out.
In the present invention, a specific tetrahedral sequence is selected to form a tetrahedral structure of a specific size, and in the present invention, tFNAs with side lengths of 17 bp (-5.8 nm) are selected because they provide a lateral spacing of about 6 nm when assembled on the substrate surface. At such intervals, the DNA hybridization efficiency reached saturation (82%) 5-fold higher than ssDNA (16%). The specific tetrahedral probe has the characteristics of high hybridization rate (obviously higher than that of tetrahedral probes with other sizes), less amount of aptamers required during detection, higher sensitivity, uniform distribution on the surface of a substrate and uniform orientation. However, due to the size effect, the SERS signal of the tFNA probe exceeding 5 nm is weak, and the long-range SERS substrate is used for exciting the specific tFNA probe to effectively enhance the signal, so that the tFNA probe can have a strong SERS signal on the surface of the long-range SERS substrate, and the method is effectively applied to ampicillin detection. Meanwhile, in order to ensure that the probe can be hybridized with ampicillin aptamer, a DNA sequence with a Raman signal molecule Cy5 is modified at the top end of the tetrahedron, and in order to ensure that the beacon molecule Cy5 on the tetrahedron is close to the surface of the substrate and has strong Raman signals, the DNA sequence is designed to have a stem-loop structure (which can be proved by NUPACK software). Also, in order for ampicillin to compete with the aptamer, the DNA sequence was designed to have two mismatched bases with the ampicillin aptamer.
The top end of the probe is modified with a stem-loop structure with a Raman signal molecule Cy5, and the DNA sequence of the stem-loop structure is designed to be a sequence with two mismatched bases with an ampicillin aptamer. The stem-loop structure of the invention leads the beacon molecules to be closer to the surface of the substrate and has strong Raman signals, and experiments show that the hybridization affinity of the tFNA probe which modifies the stem-loop structure and the ampicillin aptamer is 386 nmol/L, and the hybridization affinity of the ampicillin aptamer and the ampicillin is 13.4 nmol/L, so that the ampicillin can easily compete the aptamer. In SERS detection, it is a very challenging problem to achieve both high sensitivity and reproducibility. In indirect SERS detection, signal fluctuations are caused by diffusion and orientation changes of probe molecules. The present invention thus relies on tFNA as a SERS probe because it has the characteristic of uniform distribution and orientation on the substrate surface, thereby providing a highly reproducible SERS signal. However, SERS is a near-field effect, the effective range of the enhancement field of a hot spot area is 5 nm, the side length of a tFNA probe is 5.8 nm, the height of the tFNA probe is 8.67 nm and exceeds 5 nm, meanwhile, a beacon molecule (Cy 5) forms size limitation on a DNA tetrahedron, and a Raman signal of the tFNA probe can not be detected by a common SERS substrate. Therefore, the long-range SERS substrate is used for exciting the tFNA probe, the effective enhanced signal of the long-range SERS substrate can reach 25 nm and far exceeds the size of the tFNA probe, so that the limitation of the tFNA probe in SERS detection caused by the large size of the tFNA probe is overcome, and the tFNA probe can also have a stronger SERS signal on the surface of the long-range SERS substrate. The invention combines the tFNA probe and the long-range surface enhanced Raman substrate, and develops a novel ampicillin sensor with high sensitivity, high selectivity and high repeatability. The tFNA probe and the long-range surface enhanced Raman substrate are combined for the first time to be applied to the detection of ampicillin.
Has the advantages that: compared with the prior art, the invention has the following advantages:
1. compared with a single-stranded DNA probe, the tFNA probe adopted by the invention has a stable tetrahedral structure, thiol groups can be modified at the bottom of the tetrahedron, the surface of the Au nano material is stably anchored through Au-S, and each tetrahedron has the same orientation. By taking advantage of the rigid structure, nanoscale addressability, and versatility of the tFNA, the type of biomolecule probe attached to a single tFNA can be customized to detect a particular molecule. The tFNA is fixed on the surface of the substrate in an ordered orientation and stable coverage density, so that signal fluctuation caused by probe molecule diffusion and orientation change can be avoided, the spatial positioning range and accessibility of the probe on the surface are improved, the biological identification efficiency is improved, and the nonspecific adsorption of biomolecules is reduced. Therefore, the invention realizes the detection of the antibiotic ampicillin with high sensitivity, high selectivity and high repeatability by the specific tetrahedral framework nucleic acid (tFNA) SERS probe.
2. The long-range SERS substrate is used for exciting the tFNA probe, and the effective enhanced signal of the long-range SERS substrate can reach 25 nm and far exceeds the size of the tFNA probe, so that the limitation of the tFNA probe in SERS detection caused by the large size of the tFNA probe is overcome, and the probe can still obtain a strong Raman signal on the surface of the SERS substrate.
3. In the detection method, competition reaction between the tFNA probe and the aptamer and between the aptamer and ampicillin is adopted, and the method is simple to operate and does not need expensive instruments, and the characteristic of high ampicillin specificity of the tFNA probe is reflected.
4. The inventionThe tFNA probe and the long-range surface enhanced Raman substrate are combined for use and applied to ampicillin detection for the first time, and ampicillin is detected quickly and efficiently by a Raman analysis method. In the detection of ampicillin, the detection limit can be as low as 9.1 multiplied by 10−15 mol/L is far superior to other detection methods in the prior art. In addition, the linear range is wide and is 10−14 ～ 10−9 And mol/L, ampicillin in the water environment can be detected.
5. The probe and the sensor are simple to prepare and convenient to use, and the tFNA probe solution is only dripped on the long-range SERS substrate when the sensor is prepared, and the tFNA probe is self-assembled on the surface of the long-range SERS substrate by utilizing an Au-S bond; then, the ampicillin aptamer is dripped on the assembled substrate, and the ampicillin aptamer and the assembled substrate are incubated to be fully hybridized.
Drawings
FIG. 1 is a gel electrophoresis image of a tFNA probe prepared according to the present invention;
FIG. 2 is a schematic diagram of the detection of ampicillin in the present invention;
FIG. 3 is a Raman spectrum of the tFNA probe prepared according to the present invention showing the variation of Raman intensity with ampicillin concentration;
FIG. 4 is a graph showing the operation of ampicillin concentration detection according to the present invention;
FIG. 5 is a schematic representation of the reproducibility of ampicillin detection in the present invention;
FIG. 6 is a schematic diagram showing the specificity of ampicillin detection in the present invention;
FIG. 7 is a schematic diagram showing detection of ampicillin in a practical sample of the present invention;
fig. 8 is a raman spectrum of the fna probe recorded from the long-range SERS substrate and the conventional SERS substrate according to the present invention.
Detailed Description
The present invention will be further described with reference to the following examples and the accompanying drawings.
The experimental methods described in the examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
The single-stranded probe sequence and the aptamer sequence were synthesized by Biotechnology engineering (Shanghai) GmbH.
Example 1
Preparation of tFNA Probe
mu.L of 100. mu.M each of Tetra-A, Tetra-B, Tetra-C and Tetra-D, 10. mu.L of 30 mM Tris (2-carboxyethyl) phosphine (TCEP) and 86. mu.L of TM buffer (prepared from 20 mM Tris-HCl, 50 mM MgCl. RTM2•6H2O, pH 8.0) and mixing. After heating at 95 ℃ for 10 minutes, the temperature is rapidly reduced to 4 ℃ for more than 30 seconds, and the temperature is controlled by a PCR instrument to obtain the tFNA probe containing the functionalized nucleic acid with the final concentration of 1 mu M. The gel electrophoresis image is used for characterization, and a Tetra-A single-stranded solution, a Tetra-B single-stranded solution, a Tetra-C single-stranded solution, a Tetra-D single-stranded solution, a solution obtained by hybridizing two strands of Tetra-A and Tetra-B, a solution obtained by hybridizing three strands of Tetra-A, Tetra-B and Tetra-C, a solution obtained by hybridizing four strands of Tetra-A, Tetra-B, Tetra-C and Tetra-D (namely tFNA) and a DNA Marker solution are respectively arranged in the left to the right of a graph 1. Because the molecular weight of tFNA is relatively large, tFNA moves relatively slowly, thereby demonstrating the successful synthesis of tFNA probes. Successful synthesis of the tFNA probe was demonstrated as shown in FIG. 1.
Example 2
Preparation of Long-Range substrates
A100 nm thick gold film was deposited on a glass substrate having a length and width of 1 cm X1 cm on a 2X 10 glass substrate by a thermal evaporator-6 The thickness of the Au film was controlled at a deposition rate of 0.02 nm/s at a pressure of mbar. Then MgF with a thickness of 200 nm2Thermally evaporating and coating a film on the gold surface to form MgF2Au mirror/glass multilayer structure (the synthetic references ACS appl. mater. Interfaces 2021, 13(15), 18301-. 6 mL of gold nanoplatticed plate particles (synthesis reference adv. Optical Mater. 2016, 4, 76-85) having an Optical density of 2, which had been prepared, were taken and added to a 10 mL beaker, 2 mL of n-hexane was added to form a water/n-hexane interface, and 1-2 mL of ethanol was added dropwise to the above solution at a rate of 0.1 mL/min. At the moment, discontinuous gold luster hexagonal plate islands appear on a water/normal hexane interface, and then the hexagonal plate islands shrink rapidly under the action of surface tension to form a larger surfaceThe accumulated continuous hexagonal plate monolayer film floated on the water surface after hexane evaporated. Mixing the prepared MgF2The Au mirror/glass multilayer structure is immersed in the solution and slowly lifted up, and the single-layer gold nano hexagonal plate array thin film floating on the gas-liquid interface is shoveled (the gold nano hexagonal plate is transferred to the MgF)2/Au mirror/glass multilayer Structure reference Langmuir 2007, 23, 10505-10510), thin film supported on multilayer structure to form gold nano hexagonal plate thin film/MgF2Au mirror/glass, thereby obtaining a long-range SERS substrate (LR-SERS substrate) with a length and width of 1 cm × 1 cm.
Example 3
Assembly of tFNA probe on a long-range SERS substrate
100 μ L of 1 μ M tFNA probe prepared in example 1 was dropped on the LR-SERS substrate prepared in example 2, and incubated at 37 ℃ for 2 hours, so that three apexes of the tFNA probe were self-assembled by Au-S bonds to the long-range SERS substrate, the unbound tFNA probe was removed by washing with TM buffer, and a Raman response signal was recorded because a Raman signal of Cy5 on the tFNA probe molecule was recorded, at which time, Cy5 was closer to the substrate surface, the peak intensity was about 10795 cps, and the Raman signal was strong.
Example 4
Hybridization of tFNA Probe with ampicillin aptamer
Dissolving the ampicillin aptamer in a TM buffer solution to obtain an ampicillin aptamer solution with the concentration of 1 mu M. And dropwise adding 100 mu L of ampicillin aptamer solution onto the SERS substrate of the surface-assembled tFNA probe obtained in the example 3, incubating for 2 hours at 37 ℃ to ensure that the ampicillin aptamer and the tFNA probe fully react, washing with TM buffer solution to remove unhybridized aptamers to obtain a functionalized SERS substrate sensor of the surface-assembled tFNA probe, and recording a Raman response signal, wherein the peak intensity is about 3677 cps, and the Raman signal is weakened.
Example 5
Preparing ampicillin solution and reacting with the functionalized SERS substrate
Dissolving ampicillin in deionized water to obtain ampicillin solution with concentration of 10−9 mol/L. The product obtained in example 4 was transferred toSoaking the sensor in 1mL of ampicillin solution, incubating for 1 hour at 37 ℃, utilizing the specific combination of ampicillin and aptamer, competing ampicillin aptamer from the tFNA probe, washing with TM buffer solution to remove unreacted ampicillin from the sensor, recording a Raman response signal, wherein the peak intensity is 8809 cps, and the Raman signal is recovered.
Examples 1-5 reflect the construction and detection principles of the sensor of the present invention (as shown in fig. 2), and the processes of collecting raman signals and detecting in examples 3-5 are as follows: the Raman spectrometer is adopted for measurement, the excitation wavelength is 785 nm, the integration time is 10 s, and the cycle time is 3 times.
Example 6
Establishing a working curve of the tFNA probe sensor for detecting the concentration of ampicillin
Preparing a group of standard solutions with different ampicillin concentrations including a blank standard sample, wherein the ampicillin concentration of the blank standard sample is 0 mol/L, and the concentrations of the other ampicillin standard solutions are 10−14 ～ 10−9mol/L, the sensor obtained in example 4 is soaked in 1mL ampicillin solution with different concentrations, incubated for 1 hour at 37 ℃, and washed by TM buffer solution; raman signals of a series of ampicillin standard solutions with different concentrations were detected by a Raman spectrometer with an excitation wavelength of 785 nm, an integration time of 10 s, and a cycle number of 3 times, as shown in FIG. 3.
The response signal I containing ampicillin standard sampleiResponse signal I to blank standard0Difference of (2)ΔLog Log of ampicillin concentration in I and Standard solutionCIs drawn intoΔI-Log CThe working curve (as shown in FIG. 4) is fitted linearly to obtain a linear equation ofΔI = 1456 Log C+ 20897. Accordingly, a working curve of the Raman intensity and the ampicillin concentration is established, and the linear range of the working curve is 10−14 ～ 10−9 mol/L, detection limit of 9.1 × 10−15 mol/L; the sensor constructed by the invention has high detection sensitivity and wide detection range on ampicillin.
Example 7
The sensor obtained in example 4 was immersed in1 mL，10−11Incubating in mol/L ampicillin solution at 37 deg.C for 1 hr, washing with TM buffer solution, collecting Raman signals of 20 different regions on sensor, detecting, and analyzing for 1367 cm−1The variation of SERS intensity fluctuation was found to be 3.12% as shown in fig. 5, which is due to the stable adsorption and constant orientation of the fna probe, indicating that the method of the present invention is more reproducible.
Example 8
The sensor obtained in example 4 was immersed in 1mL of 10−9Incubating at 37 ℃ for 1 hour in mol/L ampicillin solution, amoxicillin solution, erythromycin solution, cephalotin solution, ciprofloxacin solution and chloramphenicol solution, washing with TM buffer solution, recording Raman response signals, and analyzing for 1367 cm−1Raman intensity (as in figure 6). Compared with ampicillin, SERS signals of the non-target samples are recovered negligibly, which shows that the developed sensor has good ampicillin specificity recognition capability, and the results prove that the reliability of ampicillin quantitative analysis of the invention.
Example 9
Ampicillin was added to 1mL of river water, 5% diluted milk and 10% diluted human serum to prepare three samples to be tested with different concentrations (1, 10 and 100 pM), the sensor obtained in example 4 was immersed in the above samples to be tested, incubated at 37 ℃ for 1 hour, washed with TM buffer, and raman response signals were recorded to obtain SERS spectra of the river water, milk and serum samples with different concentrations of antibiotics (see fig. 7). The recovery rates of ampicillin in river water, milk and serum are 97.00% -101.89%, 99.00% -100.90% and 97.48% -102.00%, respectively, which shows that the developed sensor is highly sensitive to trace ampicillin, and the measured SERS signal and the established calibration curve are well fitted, so that the method can be effectively used for detecting ampicillin in an actual sample.
Example 10
The raman spectra of the fna probe were recorded separately using a long-range SERS substrate and a common SERS substrate. The method comprises the following specific steps:
mu.L of 1. mu.M tFNA probe solution prepared in example 1 was taken out and collectedDropping on a long-range SERS substrate and a common SERS substrate (the preparation method is the same as that of example 2, except that MgF is added2Au mirror/glass multilayer replaced by glass), incubated at 37 ℃ for 2 hours, washed with TM buffer to remove unbound tFNA probe, and the raman spectrum of the tFNA probe was recorded separately (see a in fig. 8). Then 100 mu L of ampicillin aptamer solution is respectively dripped on a long-range SERS substrate and a common SERS substrate with a tFNA probe assembled on the surface, incubation is carried out for 2 hours at 37 ℃, ampicillin aptamer and the tFNA probe are fully reacted, TM buffer solution is used for washing to remove non-hybridized aptamer, Raman spectra (shown as B in figure 8) of the hybridized tFNA probe are respectively recorded, the Raman spectrometer is adopted for measurement, the excitation wavelength is 785 nm, the integration time is 10 s, and the cycle number is 3 times.
In a of fig. 8, a curve a is a tFNA probe raman spectrum recorded by a general SERS substrate, and a very weak signal can be seen, and a curve b is a tFNA probe raman spectrum recorded by a long-range SERS substrate, and a very strong signal can be seen, and the signal intensity is increased by about 5.4 times. In B of fig. 8, curve a is the raman spectrum of the fna probe after hybridization recorded by the conventional SERS substrate, and the raman signal disappears completely, and curve B is the raman spectrum of the fna probe after hybridization recorded by the long-range SERS substrate, and a significant signal can still be seen. Therefore, the substrate has a long-range effect, can be used together with the tFNA probe, and overcomes the defect of large size of the tFNA probe.
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Claims (7)

1. A sensor of a tetrahedral framework nucleic acid SERS probe, which is characterized in that the sensor is formed by assembling the tetrahedral framework nucleic acid SERS probe on a long-range SERS substrate and hybridizing an ampicillin aptamer; the tetrahedral framework nucleic acid SERS probe comprises a single-chain probe Tetra-A, a single-chain probe Tetra-B, a single-chain probe Tetra-C and a single-chain probe Tetra-D for modifying cyanine dye 5, wherein the sequences of the single-chain probe Tetra-A, the single-chain probe Tetra-B, the single-chain probe Tetra-C and the single-chain probe Tetra-D are respectively shown as SEQ ID NO. 1-4; the long-range SERS substrate is a gold nano hexagonal plate film/magnesium fluoride/gold mirror/glass; thiol groups are modified at three vertexes of the single-chain probe Tetra-B, the single-chain probe Tetra-C and the single-chain probe Tetra-D; the method comprises the steps of dropwise adding the tetrahedral frame nucleic acid SERS probe onto a long-range SERS substrate, incubating, connecting three vertexes of the tetrahedral frame nucleic acid SERS probe to the long-range SERS substrate through Au-S bond self-assembly to obtain the SERS substrate with the tetrahedral frame nucleic acid SERS probe assembled on the surface, dropwise adding ampicillin aptamer solution onto the SERS substrate with the tetrahedral frame nucleic acid SERS probe assembled, and incubating to enable the ampicillin aptamer to fully react with the probe.
2. The sensor of tetrahedral frame nucleic acid SERS probes according to claim 1, wherein the tetrahedral frame nucleic acid SERS probes comprise equimolar amounts of the single-stranded probe Tetra-a, the single-stranded probe Tetra-B, the single-stranded probe Tetra-C and the single-stranded probe Tetra-D of the modified cyanine dye 5.
3. The sensor of a tetrahedral framework nucleic acid SERS probe, according to claim 1, wherein the tetrahedral framework nucleic acid SERS probe has a tetrahedral structure, the side length of the tetrahedral structure is 5.8 nm, the height of the tetrahedral structure is 8.67 nm, and the top end of the tetrahedral framework nucleic acid SERS probe uses 8 adenine nucleotides as a spacer to modify a stem-loop structure with Raman signal molecules Cy 5.
4. The sensor of tetrahedral frame nucleic acid SERS probe according to claim 1, wherein the preparation method of the tetrahedral frame nucleic acid SERS probe comprises the following steps:
and mixing the single-chain probe Tetra-A, the single-chain probe Tetra-B, the single-chain probe Tetra-C and the single-chain probe Tetra-D which are used for modifying the cyanine dye 5 according to the equal molar ratio, heating and cooling to form the cyanine dye.
5. The sensor of the tetrahedral framework nucleic acid SERS probe, according to claim 4, wherein the single-stranded probe Tetra-A, the single-stranded probe Tetra-B, the single-stranded probe Tetra-C and the single-stranded probe Tetra-D of the modified cyanine dye 5 are mixed in an equimolar manner, heated at 92-95 ℃ for 8-10 minutes, and cooled at 2-4 ℃ for 30-50 seconds.
6. Use of a sensor according to claim 1 for detecting ampicillin.
7. The use according to claim 6, wherein the sensor is immersed in an ampicillin solution, incubated and washed to remove unreacted ampicillin, and subjected to Raman spectroscopy to detect ampicillin.
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Publication number Priority date Publication date Assignee Title
CN104651491A (en) * 2014-12-23 2015-05-27 中国科学院上海高等研究院 DNA tetrahedral nano-structure signal probe and application thereof
CN110672851A (en) * 2019-08-19 2020-01-10 上海理工大学 Kanamycin identification/sensing integrated probe, preparation method and detection method

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104651491A (en) * 2014-12-23 2015-05-27 中国科学院上海高等研究院 DNA tetrahedral nano-structure signal probe and application thereof
CN110672851A (en) * 2019-08-19 2020-01-10 上海理工大学 Kanamycin identification/sensing integrated probe, preparation method and detection method

Non-Patent Citations (1)

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Preparation of a Novel Raman Probe and Its Application in the Detection of Circulating Tumor Cells and Exosomes;Xiaoru Zhang 等;《ACS Appl. Mater. Interfaces》;20190718;第11卷;图2、第28672页左栏最后一段-右栏最后一段 *

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