CN108398419B - Method for ultrasensitively detecting thrombin by using competitive nano sensor - Google Patents
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Abstract
The invention discloses a method for detecting thrombin with ultra-sensitivity by using a competitive nano sensor. Combining BPE with gold nanoparticles to form a gold dimer, and reacting with Aptamer Aptamer-1 complementarily paired with TBA to obtain a Raman signal probe; then carrying out mixing reaction with magnetic nanoparticles modified with thrombin aptamer TBA to obtain a nanosensor system; when the thrombin to be detected exists, the structure of the nano sensor system is interrupted by competition, so that the Raman signal of the supernatant in the system is changed, and the detection of the thrombin content is realized based on the change. The method is simple to operate, good in detection stability and high in detection sensitivity; during operation, only the prepared nano sensor system is required to be sequentially added into a sample to be detected for reaction, and the detection can be immediately carried out through magnetic separation after the reaction is finished, so that the rapid quantitative detection is realized; the method can meet the requirements of food safety and environmental monitoring departments, and has wide practicability.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a method for ultrasensitively detecting thrombin by using a competitive type nano sensor based on a gold dimer substrate.
Background
Thrombin (thrombin) is an important serine proteolytic enzyme, consisting of two peptide chains linked by disulfide bonds. Thrombin has the effects of regulating blood coagulation, promoting blood circulation and the like by converting fibrinogen in blood into fibrin. Thrombin, an important multifunctional active protein, plays a key role in thrombosis, vascular proliferation, tumor growth and metastasis, etc., and is also frequently used as a therapeutic agent and a biomarker. At present, methods for detecting thrombin mainly comprise a colorimetric method, a fluorescence method and an electrochemical method, but the methods have some defects, such as poor sensitivity, complex operation, incapability of achieving measurement and detection required by modern medicine, and the like. Therefore, the establishment of a method for detecting thrombin rapidly, simply and with ultra-sensitivity has very important significance.
The Surface Enhanced Raman Spectroscopy (SERS) technology is a novel spectral labeling method, and is a current research hotspot. On one hand, the technology has the advantages of Raman spectrum, such as difficult bleaching of optical signals, small damage to biological cells and tissues, rich spectral information, narrow spectral peaks of the spectrum, easy distinguishing and the like; on the other hand, the method makes up the defects that the traditional Raman signal is weaker and is not beneficial to detection. The enhanced effect of SERS enables the spectral detection to have ultra-high sensitivity, and single-molecule level analysis research is realized at present. The SERS effect is generally generated on a rough nano metal surface or between nano metals, so that nanotechnology provides a rich technical means for constructing an SERS nano probe. The nano-probe developed based on the SERS technology shows considerable application prospect in the fields of biological imaging, protein detection, tumor identification and the like.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for ultra-sensitively detecting thrombin by using a competitive type nano sensor based on a gold dimer substrate. The nano sensor is a nano sensor adopting gold dimer nano particle assembly set immunomagnetic bead capture technology.
According to the invention, the thrombin in the sample is rapidly and highly sensitively quantitatively analyzed by the immune recognition effect of the Raman signal probe and the magnetic capture probe and the competitive reaction of the thrombin, and the problems of complex sample treatment, insufficient sensitivity and the like in the prior art are solved.
The method has the characteristics of high sensitivity, strong specificity, simple detection process, accurate quantitative detection and the like.
The purpose of the invention is realized by the following technical scheme:
a method for ultrasensitively detecting thrombin by using a competitive nanosensor based on a gold dimer substrate, comprising the following steps of:
firstly, combining BPE with gold nanoparticles to form a gold dimer, and reacting with a segment of complementary paired Aptamer Aptamer-1 of TBA to obtain a Raman signal probe; then carrying out mixing reaction with magnetic nanoparticles modified with thrombin aptamer TBA to obtain a nanosensor system; when the thrombin to be detected exists, the structure of the nano sensor system is interrupted by competition, so that the Raman signal (Raman signal probe) of the supernatant in the system is changed, and the detection of the thrombin content is realized based on the change.
The method specifically comprises the following steps:
(1) activating carboxyl of the magnetic nano-particles modified with carboxyl on the surface by using an activating agent, then adding a thrombin aptamer TBA 'hairpin' structure modified with amino, and reacting to obtain a capture probe;
(2) selecting BPE as a Raman signal molecule, combining the pyridyl at two ends of the molecule with gold nanoparticles to form a gold dimer, adding a silver nitrate solution at proper time to stop the reaction, and preventing the formation of a gold polymer, wherein the BPE is used as the Raman signal molecule and also used as an intermediate connector of the dimer;
(3) activating Aptamer Aptamer-1 which is complementarily matched with one section of TBA by using TCEP, and reacting sulfydryl on the Aptamer Aptamer-1 with the gold dimer (Au-S) in the step (2) to prepare a Raman signal probe;
(4) mixing the capture probe in the step (1) and the Raman signal probe in the step (3) for reaction, opening a TBA (tunnel boring A) hairpin structure on the capture probe by using an aptamer on the Raman signal probe, and forming a capture probe-Raman signal probe nano-sensor system through hybridization of a complementary sequence;
(5) preparing a thrombin solution by using a Tris-HCl (Tris-hydroxymethyl aminomethane-hydrochloric acid) buffer solution, and diluting the thrombin solution into a group of standard sample solution with concentration gradient;
(6) taking the nano sensor system in the step (4) with the same volume, respectively adding thrombin standard sample liquid with the same volume and different concentrations in the step (5), incubating at room temperature, carrying out signal acquisition on a free Raman signal probe in the supernatant after magnetic separation, wherein the thrombin competes with the thrombin aptamer TBA to react, and the nano sensor system is gradually interrupted along with the increase of the thrombin concentration, so that the Raman signal in the supernatant is gradually increased;
(7) according to the concentration of thrombin and the intensity of Raman signal (I)1616/I520) The standard curve is established according to the corresponding relation, so that the quantitative detection of the thrombin is carried out by applying Raman signals.
In the above method for detecting thrombin:
the thrombin is human thrombin (α -thrombin);
the magnetic nano-particles with carboxyl groups modified on the surfaces in the step (1) are ferroferric oxide Fe with carboxyl groups modified on the surfaces3O4The particle size is preferably 200 to 300 nm;
the activating agents in the step (1) are preferably 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS);
the sequence of the thrombin aptamer TBA in the step (1) is as follows:
5′-TGTCAGTGGGGTTGGACGGGATGGTGCCTGACTCTC-(CH2)7-NH2-3′；
the concentration of the thrombin aptamer TBA in the step (1) is preferably 10-100 mu M; more preferably 100. mu.M.
The BPE in step (2) is 1, 2-bis (4-pyridyl) ethylene;
the average particle size of the gold nanoparticles in the step (2) is preferably 25-35 nm; more preferably 30 nm;
the concentration of the gold nanoparticle solution in the step (2) is 0.5-1.5 mM; more preferably 1 mM.
The TCEP described in step (3) is tris (2-carboxyethyl) phosphine, and functions to activate the thrombin aptamer TBA;
the concentration of the Aptamer Aptamer-1 in the step (3) is preferably 10-100 mu M; more preferably 100. mu.M.
The Aptamer Aptamer-1 sequence in the step (3) is as follows:
5′-GGCACCATCCCGTCCAACCCCA-(CH2)6-SH-3′；
the volume mixing ratio of the capture probe and the Raman signal probe in the step (4) is 10: (6-8); more preferably 10: 8.
the Tris-HCl buffer solution in the step (5) contains 50mM NaCl, 5mM KCl and 5mM MgCl2；
The group of standard sample solutions with concentration gradients in the step (5) is 0M, 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, 1nM, 10nM, 100nM and 1 μ M, wherein 0M is a blank control;
the volume ratio of the nano sensor system to the thrombin standard sample liquid in the step (6) is 4.5-5.5; preferably 5.
The magnetic separation in the step (6) is to separate the magnetic conjugate from the supernatant by utilizing the physical action of a magnetic field, and compared with the traditional step of cleaning a solid phase substrate to remove residues, the method is more efficient;
the signal in the step (6) is measured by a micro-Raman spectrometer: the operating condition of the micro-Raman spectrometer is preferably that an excitation light source is a He-Ne laser with the wavelength of 632.8nm, the laser power reaching a sample is 1mW, and the signal collection time is 30-40 s (preferably 30 s);
the signal in the step (6) is a characteristic Raman spectrum peak (1616 cm) of a selected Raman signal molecule-1) As a quantitative peak, the thrombin concentration is plotted against the spectral intensity of the peak, and a standard curve is drawn according to the plot;
i described in step (7)1616/I520In which I1616Is BPE molecule at 1616cm-1Peak intensity of (A) in520Is a characteristic peak (520 cm) of a silicon wafer substrate for calibration-1Of (d) of the measured intensity.
The mechanism of the invention is as follows: under the condition of a certain collection time, the signal intensity and the concentration of Raman signal molecules are in positive correlation for the same Raman signal molecule; it is on this basis that quantitative detection is achieved.
Compared with the prior art, the invention has the following advantages and effects:
(1) the gold dimer subjected to immune spectrum labeling treatment is used for quantitative detection of thrombin, the signal intensity of Raman molecules is related with the concentration of a substance to be detected, and the method is similar to the Lambert beer law, so that the quantitative detection of the thrombin is realized;
(2) the Raman signal molecule adopted by the invention is BPE, and gold nanoparticles are connected together through the pyridyl groups at the two ends of the molecule to form a gold dimer, so that the strength of the signal molecule is greatly enhanced;
(3) the nano sensor system formed by the gold dimer-based Raman signal probe and the capture probe can be used for quickly and quantitatively detecting thrombin, overcomes the defects of the traditional method, and simultaneously combines the enrichment effect of the magnetic capture probe to obtain a Raman signal by detecting supernatant;
(4) the method obtains corresponding results by detecting the Raman spectrum signals of the solution, and compared with the traditional method for detecting the solid-phase substrate, the method has the advantages that the monitoring data of the Raman spectrum signals in the solution are more stable and reliable and can be repeated in a large amount;
(5) the invention has good detection stability and high detection sensitivity, and obtains a wide detection interval (1 fM-1 mu M) and a low detection limit (1 fM);
(6) the method is simple to operate, the Raman signal probe and the capture probe can be prepared in advance and can be used for preparing the nano sensor system, the nano sensor system only needs to be sequentially added into a sample to be detected for reaction during operation, and the detection can be immediately carried out through magnetic separation after the reaction is finished: collecting time for 30s, and reading out concentration from the standard curve immediately, thereby realizing rapid quantitative detection; the method can meet the requirements of food safety and environmental monitoring departments, and has wide practicability.
Drawings
FIG. 1 is a schematic flow chart of a method for rapid quantitative detection of thrombin.
FIG. 2 is a transmission electron microscope image of prepared gold nanoparticles (A), gold dimers (B), gold multimers (reaction stopped without adding silver nitrate solution) (C) and Raman signal probes (D).
FIG. 3 is a graph of Raman signal spectra obtained using different concentrations of thrombin standard solutions.
FIG. 4 is a graph of thrombin detection in a standard solution; wherein the abscissa represents the concentration of thrombin (the abscissa is log10 of the concentration) and the ordinate represents the intensity of Raman signal (I) at different concentrations of thrombin1616/I520)。
FIG. 5 is a graph of Raman signal intensity for thrombin specific detection.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
In the following examples, the experimental methods without specific conditions and environments noted are generally performed under conventional conditions or conditions recommended by the manufacturers. In the invention, BPE is Raman signal molecule 1, 2-di (4-pyridyl) ethylene; PBS means phosphate buffer; Tris-HCl represents Tris-hydroxymethyl aminomethane-hydrochloric acid buffer; EDC represents 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; NHS represents N-hydroxysuccinimide; TCEP stands for tris (2-carboxyethyl) phosphine.
The flow chart of the method for rapidly and quantitatively detecting thrombin is shown in figure 1.
Example 1:
(1) preparation of gold nanoparticles
100mL of 1mM chloroauric acid (HAuCl) are added with constant stirring4) The solution was heated to boiling and then 6ml of 38.8mm aqueous trisodium citrate solution was added. The color change of the solution at this time was: light yellow-colorless-black-purple-dark red, heating and refluxing for 15-20 min. And finally, cooling to normal temperature to prepare the gold nanoparticles with the particle size of 30nm, wherein the gold nanoparticles obtained under ideal conditions are shown in FIG. 2A.
(2) Preparation of gold dimer nanoparticles
The prepared gold nanoparticles (concentration of about 1mmol/L) were centrifuged once (8000rpm, 10min), and then resuspended with triple-distilled water (pH 7) for future use; taking the 1mL of gold nanoparticles, reacting with 30 mu L of 1mM BPE for about 10min, adding 20 mu L of 2mM silver nitrate solution before the color of the gold nanoparticles changes to terminate the reaction (10min), centrifuging (4000rpm, 20min) after the reaction is finished, and re-suspending with triple-distilled water to finally obtain gold dimer nanoparticles, wherein FIG. 2B is the gold dimer nanoparticles synthesized under ideal conditions; FIG. 2C shows the gold multimer obtained by terminating the reaction without adding silver nitrate solution.
(3) Preparation of Raman signal probe
The Aptamer Aptamer-1 is modified on the surface of the gold dimer nanoparticle by a salt deposition method: first, 50. mu.L of 100. mu.M Aptamer Aptamer-1 was activated with 3.5. mu.L of 100. mu.M freshly prepared TCEP solution for 10 minutes, and then the activated Aptamer-1 was added to the triple distilled water resuspended gold dimer nanoparticles described above. After incubation for 12h at room temperature, 100. mu.L of 100mM PBS buffer was added, 1M NaCl solution was added over 48h to a final concentration of 0.1mM, and finally, centrifugation was carried out at 4000rpm for 20 minutes, the supernatant was removed, the substrate was resuspended in 0.1M Tris-HCl buffer, and the resulting Raman signal probe was stored at 4 ℃ as shown in FIG. 2D, which is the Raman signal probe obtained under ideal conditions.
(4) Preparation of the Capture Probe
Taking 100 mu L of ferroferric oxide Fe with carboxyl modified on surface3O4Purchased from alatin reagent (shanghai) ltd, washed twice with PBS and finally made up to 500 μ L with PBS. EDC (4mg/mL) and NHS (1mg/mL) were prepared, and 30. mu.L of EDC solution was added to the bead solution, and after 30min, the same volume of NHS solution was added and activated for 90 min. Then washing the magnetic beads twice with Tris-HCl buffer solution, then resuspending with 500. mu.L Tris-HCl, then adding 50. mu.L (100. mu.M) thrombin aptamer TBA, washing with Tris-HCl after reacting for 2h to remove excessive TBA, and resuspending for later use to obtain the final productAnd (3) capturing the probe.
(5) Preparation of Standard sample solution
10 concentrations of thrombin standard solutions were selected, 0mol, 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, 1nM, 10nM, 100nM and 1. mu.M, respectively, with 0mol being blank.
(6) Nanosensor preparation and detection of thrombin
Mixing 100 mu L of the capture probe prepared in the step (4) and 80 mu L of the Raman signal probe prepared in the step (3) for reaction for 2h, and hybridizing the TBA on the capture probe with the complementary sequence on the Raman signal probe to form a 'capture probe-Raman signal probe' nano-sensor system; subsequently, 20 μ L of thrombin standard solutions with different concentrations in step (5) were added to each of the obtained nanosensor systems, and since thrombin competitively reacts with the thrombin aptamer TBA, the nanosensor systems are gradually interrupted with increasing thrombin concentration, resulting in gradually increasing raman signal in the supernatant.
(7) Measurement of Raman signals
The substrate was separated by the magnetic field of a magnet and the supernatant was retained. The Raman signal probe in the supernatant was subjected to signal collection using a micro-Raman spectrometer from Nippon Optical System, Japan, and the excitation light source was a He-Ne laser having a wavelength of 632.8nm, the laser power reaching the sample was 1mW, and the signal collection time was 30 s. After the signal acquisition is finished, baseline processing is carried out on the data through Origin software, and a clear and visual SERS spectrogram (as shown in figure 3) is obtained.
As is evident from FIG. 4, the collected SERS signal gradually increased as the thrombin concentration in the sample increased (I)1612/I520) The relationship is consistent with that Y is 0.65+1.70X, which indicates that effective quantitative analysis can be carried out in the interval.
Example 2
To illustrate the specificity of the present invention, a mouse IgG antibody standard solution, bovine serum albumin BSA, Lysozyme (Lyso) standard solution and Hemoglobin Hb standard solution were prepared, and the specific implementation steps were as described in example 1; the signal intensity obtained can be seen in FIG. 5, which illustrates the strong specificity of the present invention.
The mouse IgG antibody standard solution and bovine serum albumin BSA used were purchased from Biotechnology Ltd, Shanghai, and the Lysozyme standard solution and Hemoglobin standard solution were purchased from san Ding Xian bioscience Ltd, Shanghai.
Example 3
To test the effect of this sensor in real samples, we added thrombin to human serum samples as a simulation of real samples. Before this, a series of treatments (filtration, centrifugation, dilution) of the serum was required to eliminate the possible effects of matrix effects on the experimental results. Subsequently, thrombin was added to the treated serum at different concentrations. These samples were used for detection and analysis, respectively, and the specific implementation steps were as described in example 1; the results obtained are shown in table 1 and show that the method is very well applicable for the detection of thrombin in serum.
TABLE 1 results of measuring thrombin in simulated serum using the nanosensor system of the invention
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
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<120> a method for ultra-sensitively detecting thrombin by using competitive type nano-sensor
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
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<223> Thrombin aptamer TBA sequence
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Claims (10)
1. A method for ultra-sensitively detecting thrombin by using a competitive type nanosensor based on a gold dimer substrate, which is characterized by comprising the following steps:
firstly, combining pyridyl groups at two ends of a BPE molecule with gold nanoparticles to form a gold dimer, activating Aptamer Aptamer-1 complementarily paired with one section of TBA, and reacting a sulfydryl on the Aptamer Aptamer-1 with the gold dimer to obtain a Raman signal probe; then carrying out mixed reaction with a capture probe to obtain a nano sensor system; when thrombin exists as an object to be detected, the structure of the nano sensor system is interrupted by competition, so that the Raman signal of supernatant in the system is changed, and the detection of the thrombin content is realized based on the change;
the capture probe is obtained by activating carboxyl of magnetic nanoparticles with carboxyl modified on the surface by an activating agent, and then adding amino modified thrombin aptamer TBA for reaction;
the sequence of the thrombin aptamer TBA is as follows:
5′-TGTCAGTGGGGTTGGACGGGATGGTGCCTGACTCTC-(CH2)7-NH2-3′；
the Aptamer Aptamer-1 sequence is as follows:
5′-GGCACCATCCCGTCCAACCCCA-(CH2)6-SH-3′。
2. the method according to claim 1, characterized in that it comprises in particular the steps of:
(1) activating carboxyl of the magnetic nano-particles modified with carboxyl on the surface by using an activating agent, then adding a thrombin aptamer TBA 'hairpin' structure modified with amino, and reacting to obtain a capture probe;
(2) selecting BPE as a Raman signal molecule, combining the pyridyl at two ends of the molecule with gold nanoparticles to form a gold dimer, adding a silver nitrate solution at proper time to stop the reaction, and preventing the formation of a gold polymer, wherein the BPE is used as the Raman signal molecule and also used as an intermediate connector of the dimer;
(3) activating Aptamer Aptamer-1 complementarily paired with one section of TBA by using TCEP, and reacting sulfydryl on the Aptamer Aptamer-1 with the gold dimer in the step (2) to prepare a Raman signal probe;
(4) mixing the capture probe in the step (1) and the Raman signal probe in the step (3) for reaction, opening a TBA (tunnel boring A) hairpin structure on the capture probe by using an aptamer on the Raman signal probe, and forming a capture probe-Raman signal probe nano-sensor system through hybridization of a complementary sequence;
(5) preparing a thrombin solution by using a Tris-HCl buffer solution, and diluting the thrombin solution into a group of standard sample solution with a concentration gradient;
(6) taking the nano sensor system in the step (4) with the same volume, respectively adding thrombin standard sample liquid with the same volume and different concentrations in the step (5), incubating at room temperature, carrying out signal acquisition on a free Raman signal probe in the supernatant after magnetic separation, wherein the thrombin competes with the thrombin aptamer TBA to react, and the nano sensor system is gradually interrupted along with the increase of the thrombin concentration, so that the Raman signal in the supernatant is gradually increased;
(7) according to the concentration of thrombin and the Raman signal intensity I1616/I520Establishing a standard curve according to the corresponding relation, and performing quantitative detection on the thrombin by using a Raman signal;
said I1616/I520In which I1616Is BPE molecule at 1616cm-1Peak intensity of (A) in520Is a characteristic peak 520cm of a silicon wafer substrate for calibration-1The intensity of the spot.
3. The method according to claim 1 or 2, characterized in that:
the thrombin is human thrombin.
4. The method of claim 2, wherein:
the magnetic nano-particles with carboxyl groups modified on the surfaces in the step (1) are ferroferric oxide Fe with carboxyl groups modified on the surfaces3O4The particle size is 200-300 nm;
the activating agent in the step (1) is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.
5. The method of claim 2, wherein:
the concentration of the thrombin aptamer TBA in the step (1) is 10-100 mu M.
6. The method of claim 2, wherein:
the average particle size of the gold nanoparticles in the step (2) is 25-35 nm;
the concentration of the gold nanoparticle solution in the step (2) is 0.5-1.5 mM.
7. The method of claim 2, wherein:
the concentration of the Aptamer Aptamer-1 in the step (3) is 10-100 mu M;
the volume mixing ratio of the capture probe and the Raman signal probe in the step (4) is 10: 6-8;
and (4) the volume ratio of the nano sensor system to the thrombin standard sample liquid in the step (6) is 4.5-5.5.
8. The method of claim 2, wherein:
the set of standard sample solutions with concentration gradient in step (5) is 0M, 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, 1nM, 10nM, 100nM and 1. mu.M, wherein 0M is blank control.
9. The method of claim 2, wherein:
the signal in the step (6) is measured by a micro-Raman spectrometer: the operating conditions of the micro-Raman spectrometer are that an excitation light source is a He-Ne laser with the wavelength of 632.8nm, the laser power reaching a sample is 1mW, and the signal collection time is 30-40 s.
10. The method of claim 2, wherein:
the signal in the step (6) is a characteristic Raman spectrum peak 1616cm of a selected Raman signal molecule-1As a quantitative peak, the thrombin concentration was plotted against the spectral intensity of the peak, and a standard curve was drawn based thereon.
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