CN113969282B - VEGF (vascular endothelial growth factor) 165 Protein probe and construction method and application thereof - Google Patents
VEGF (vascular endothelial growth factor) 165 Protein probe and construction method and application thereof Download PDFInfo
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Abstract
The invention provides a VEGF 165 A protein probe and a preparation method and application thereof, belonging to the technical field of biological analysis; in the invention, a self-assembled label-free magnetic nano tumor marker VEGF is constructed 165 Protein probe, designated Fe 3 O 4 /Fe 2 O 3 The linear range of the detectable concentration of the probe is 0.01-10 pg.multidot.mL ‑1 The detection limit was 0.01 pg.mL ‑1 (S/n=3), the limit of quantification was 0.03 pg.ml ‑1 (S/n=10), where S/N represents the signal-to-noise ratio; the invention also constructs an electrochemical nucleic acid sensor based on the probe, and the electrochemical nucleic acid sensor is used for detecting VEGF 165 Has good application.
Description
Technical Field
The invention belongs to the technical field of biological analysis, and particularly relates to a VEGF165 protein probe, a construction method and application thereof.
Background
Vascular Endothelial Growth Factor (VEGF) is a factor that selectively promotes vascular endothelial cell growth and has a primary physiological function of producing new blood vessels during embryonic development, after injury, and upon vascular occlusion. In addition, VEGF has been implicated in a number of diseases that occur by virtue of angiogenesis, such as diabetic retinopathy, certain inflammatory diseases, and cancer. Since VEGF has begun to synthesize during the conversion of tumor cell mass into solid tumors and is now an important period in tumor screening, VEGF can be used as a tumor screening marker.
Human expression length of VEGF is 206, 189, 183, 165, 145 and121 amino acids (aa), wherein VEGF 165 Is the most abundant and potent subtype, the predominant form of VEGF, which contains 165 amino acid residues, and is a dimer formed by disulfide bonding. Traditional VEGF 165 Many detection methods, such as ELISA, PCR, SDS-PAGE and NASBA, are available, but the detection methods have the disadvantages of complex operation, time consumption, need of training operators, expensive equipment and the like.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a VEGF165 protein probe, and a construction method and application thereof. In the invention, a self-assembled label-free magnetic nano tumor marker VEGF is constructed 165 Protein probe, designated Fe 3 O 4 /Fe 2 O 3 @Au-DNA/BSA, and an electrochemical nucleic acid sensor is constructed based on the probe, and VEGF is detected 165 Has good application.
The invention firstly provides a preparation method of a VEGF165 protein probe, which comprises the following steps:
S1.Fe 3 O 4 /Fe 2 O 3 preparation of @ Au nanocomposite:
(1) Fe (NO) 3 ) 3 ·9H 2 Standing O aqueous solution for hydrolysis reaction to obtain orange-red suspension, centrifuging, washing, dispersing in absolute ethyl alcohol to obtain ethanol suspension, calcining the ethanol suspension, and grinding after calcining to obtain magnetic Fe 3 O 4 /Fe 2 O 3 Heteroplasmic nanoparticles;
(2) Magnetic Fe 3 O 4 /Fe 2 O 3 Heterobody nano particles, ultrasonic dispersing, and then dripping HAuCl 4 The solution is heated to boiling under reflux, and sodium citrate solution is added for reaction, and then the magnetic Fe is obtained after centrifugation, washing and drying 3 O 4 /Fe 2 O 3 @au nanocomposite.
Wherein in step (1), fe (NO) 3 ) 3 ·9H 2 The concentration of the O aqueous solution is 0.05M-0.5M, and the hydrolysis condition is 60-90 DEG CHydrolyzing for 2-16 h, and dispersing the hydrolysate into 20-100 mL absolute ethyl alcohol; the calcination is performed at 200-400 ℃ for 0.5-4 h.
Further, the Fe (NO 3 ) 3 ·9H 2 The concentration of the O aqueous solution is 0.05M; the hydrolysis condition is that the hydrolysis is carried out for 8 hours at 90 ℃; the calcination was performed at 200℃for 2 hours.
In step (2), the magnetic Fe 3 O 4 /Fe 2 O 3 Heteroplasmic nanoparticles, HAuCl 4 And sodium citrate in a mass ratio of 5:20:114, wherein the reaction time is 40min.
S2.Fe 3 O 4 /Fe 2 O 3 Construction of @ Au-DNA/BSA:
(1) Fe is added to 3 O 4 /Fe 2 O 3 Dripping the suspension of the@Au nano-composite onto the surface of a clean magnetic glassy carbon electrode (magnetic glassy carbon electrode, abbreviated as MGCE), and drying at room temperature to obtain MGCE/Fe 3 O 4 /Fe 2 O 3 Modified electrode of @ Au; fe (Fe) 3 O 4 /Fe 2 O 3 the@Au is stably combined on the surface of the electrode through magnetic induction self-assembly for standby;
(2) To MGCE/Fe 3 O 4 /Fe 2 O 3 Dropwise adding a mercapto-modified DNA aptamer solution containing tris (2-carboxyethyl) phosphine on the surface of an Au modified electrode for incubation, and drying at room temperature after incubation to obtain MGCE/Fe 3 O 4 /Fe 2 O 3 An Au-DNA modified electrode; thiol-modified DNA aptamer self-assembled to Fe through Au-S bond 3 O 4 /Fe 2 O 3 @Au for standby; in the incubation process, a centrifuge tube is covered to prevent the solution from volatilizing.
(3) To MGCE/Fe 3 O 4 /Fe 2 O 3 Dripping bovine serum albumin on the surface of the@Au-DNA modified electrode to react, and drying at room temperature after the reaction is finished to obtain the self-assembled label-free magnetic nano tumor marker VEGF 165 Protein probe, designated MGCE/Fe 3 O 4 /Fe 2 O 3 @Au-DNA/BSA; in the reaction process, a centrifuge tube is covered to prevent the solution from volatilizing, and the reaction process is sealedAnd closing the non-specific sites on the surface of the electrode to prevent non-specific adsorption.
Further, in the step (1), fe 3 O 4 /Fe 2 O 3 The concentration of the @ Au nano-composite suspension is 5-30mg/mL, the dosage is 10 mu L, and the solvent is ultrapure water.
Further, in the step (2), the thiol-modified DNA aptamer solution containing tris (2-carboxyethyl) phosphine uses TE buffer as a solvent, the concentration is 0.2-1.6 mu M, and the dosage is 5 mu L; the concentration of the tris (2-carboxyethyl) phosphine is 48 μm; the sequence of the sulfhydryl modified DNA aptamer is 5' -SH- (CH) 2 ) 6 -CAATTGGGCCCGTCCGTATGGTGGGT-3’。
Further, in the step (2), the incubation condition is that the incubation is performed for 12 hours at 4 ℃;
further, in the step (3), the amount of the bovine serum albumin is 2-5 mu L, wherein the volume fraction of the bovine serum albumin is 0.25%, and the reaction condition is that the reaction is carried out for 30min at 4 ℃.
The invention also provides a VEGF165 protein probe prepared by the method, which is marked as Fe 3 O 4 /Fe 2 O 3 @Au-DNA/BSA, the probe was based on the DNA aptamer 5' -SH- (CH) 2 ) 6 CAATTGGGCCCGTCCGTATGGTGGGT-3' and the probe can detect VEGF 165 The linear range of the concentration of the protein is 0.01-10 pg.mL -1 The detection limit was 0.01 pg.mL -1 (S/n=3), the limit of quantification was 0.03 pg.ml -1 (S/n=10), where S/N represents the signal-to-noise ratio.
The invention also provides the VEGF 165 Protein probe for detecting VEGF in preparation 165 Is provided.
The invention also provides a method for detecting VEGF 165 Electrochemical nucleic acid sensor for proteins based on MGCE/Fe 3 O 4 /Fe 2 O 3 Preparation of Au-DNA/BSA, specific: VEGF to be tested 165 Drop-wise addition of protein solution to MGCE/Fe 3 O 4 /Fe 2 O 3 The surface of the @ Au-DNA/BSA is incubated to be used as a working electrode, and silver/silver chloride isA reference electrode, a platinum wire electrode as a counter electrode, and KCl-containing [ Fe (CN) 6 ] 3-/4- The solution is used as electrolyte and differential pulse voltammetry detection is performed by using an electrochemical workstation.
Further, the incubation temperature is 20-40 ℃ and the incubation time is 10-70 min.
Further, in the electrolyte, the concentration of KCl is 0.1M, [ Fe (CN) 6 ] 3-/4- The concentration of the solution was 5mM.
The electrochemical nucleic acid sensor has low price, simple and rapid operation and high sensitivity, and can realize real-time detection of complex biological systems.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a self-assembled label-free magnetic nano tumor marker VEGF (vascular endothelial growth factor) aiming at the prior detection technology 165 The protein probe overcomes the defects of the prior detection technology means, and provides the nano tumor marker detection probe which is efficient, quick, low in price, strong in specificity and good in practicability.
The invention uses Fe 3 O 4 /Fe 2 O 3 the@Au nano-composite is used for a signal amplification strategy, and the material has good conductivity and can better perform VEGF (vascular endothelial growth factor) 165 The detection was carried out with a low limit of detection (0.01 pg. ML) -1 ) Wide linear range (0.01-10 pg.multidot.mL) -1 ) And better sensitivity.
In the invention, the Fe 3 O 4 /Fe 2 O 3 the@Au nano-composite has magnetism, and Fe can be prepared by a magnetic force induced self-assembly technology 3 O 4 /Fe 2 O 3 Modified Au nano-composite to electrode surface, the magnetic core with MGCE removed can rapidly remove alpha-Fe 2 O 3 /Fe 3 O 4 The principle of Au is that the regeneration of the electrode is simple and rapid; the requirements on required equipment are low, the operation is simple, and the carrying is convenient.
The invention introduces DNA aptamer in the process of preparing protein probe, and has high stability, strong affinity, short preparation period, good specific recognition capability and high repeatabilityThe invention has the advantages of low price, easy modification, easy mass preparation in vitro, and the like, and the thiol-modified DNA aptamer is connected to Fe through Au-S bond by utilizing the self-assembly technology 3 O 4 /Fe 2 O 3 The nano Au composite surface is rapid and simple to operate, and the rest materials are not needed.
The protein probe prepared by the method does not need to be marked and pretreated; the detection process is rapid and convenient, the detection result can be obtained in a few minutes, the amount of the probe required for each detection is very small, and the cost is low; the reaction condition is mild, the process is easy to control, and the operator is not required to be trained.
Drawings
FIG. 1 is Fe 3 O 4 /Fe 2 O 3 Transmission electron micrographs of Au nanocomposites.
FIG. 2 VEGF under the conditions described in this example 165 In cyclic voltammetry at each stage in the preparation process of the protein probe, a is a bare electrode MGCE, and b is MGCE/Fe 3 O 4 /Fe 2 O 3 C is MGCE/Fe 3 O 4 /Fe 2 O 3 @Au, d is MGCE/Fe 3 O 4 /Fe 2 O 3 The @ Au-DNA, e is MGCE/Fe 3 O 4 /Fe 2 O 3 The @ Au-DNA/BSA, f is MGCE/Fe 3 O 4 /Fe 2 O 3 @Au-PNA/BSA/VEGF 165 Is a cyclic voltammogram of (c).
FIG. 3 is Fe 3 O 4 /Fe 2 O 3 Magnetic nano VEGF (vascular endothelial growth factor) of @ Au-DNA/BSA (deoxyribonucleic acid/BSA) 165 Protein probes for detecting VEGF 165 Albumin (Albumin), hemoglobin (Hemoglobin), lysozyme (Lysozyme), L-Cysteine (L-Cysteine), peak current diagram of lgG.
FIG. 4 is Fe 3 O 4 /Fe 2 O 3 Magnetic nano VEGF (vascular endothelial growth factor) of @ Au-DNA/BSA (deoxyribonucleic acid/BSA) 165 Concentration and linear range plot detectable by the protein probe.
Detailed Description
The invention will be further described with reference to the drawings and the specific embodiments, but the scope of the invention is not limited thereto.
In the present invention, recombinant human vascular endothelial growth factor 165 (VEGF) 165 ) DNA aptamers were all purchased from the biological (Shanghai) company limited; the purchased DNA aptamer sequence is 5' -SH- (CH) 2 ) 6 CAATTGGGCCCGTCCGTATGGTGGGT-3' wherein SH represents a mercapto group.
Tris (2-carboxyethyl) phosphine, TCEP for short; bovine serum albumin, BSA for short; TCEP, BSA, albumin, hemoglobin, lysozyme, L-Cysteine, lgG was purchased from Shanghai Alasdine Biochemical technologies Co., ltd.
Example 1: preparation of Fe 3 O 4 /Fe 2 O 3 Nano-particles of @ Au
4.10g of Fe (NO) 3 ) 3 ·9H 2 O is dissolved in 200mL double distilled water, stirred magnetically until a uniform solution is formed, and hydrolyzed at 90 ℃ for 8 hours to obtain orange-red suspension, then the obtained suspension is centrifuged to obtain precipitate, and the precipitate is respectively washed with water and ethanol for 3 times and then uniformly dispersed in 20mL absolute ethanol to obtain ethanol suspension. Transferring the suspension into a crucible, igniting, placing the crucible into a series of protective atmosphere resistance furnaces after flame is extinguished, calcining for 2 hours at 200 ℃, grinding after calcining is finished, and obtaining the magnetic Fe 3 O 4 /Fe 2 O 3 Heteroplasmic nanoparticles;
10mg of the above-prepared magnetic Fe was weighed 3 O 4 /Fe 2 O 3 Heterobody nano particles are dispersed in 194mL double distilled water by ultrasonic to form suspension, and then 2mL HAuCl with the concentration of 48.6mM is added 4 Dropwise adding the solution into the suspension, transferring the suspension into a three-necked flask, heating and stirring under reflux to boil, slowly dropwise adding 5mL of 155mM sodium citrate solution into the three-necked flask, and reacting for 40min under the boiling condition, wherein the color of the solution is changed from yellow to black and gradually changed into mauve; stopping heating and cooling to room temperature; centrifuging the suspension obtained by the reaction to obtain precipitate, washing the precipitate with water for 5 times, and drying to obtain magnetic Fe 3 O 4 /Fe 2 O 3 @ Au nanoparticles.
FIG. 1 shows Fe prepared under the conditions described in this example 3 O 4 /Fe 2 O 3 Transmission electron micrographs of the @ Au nanocomposites, from which several aggregated Fe can be seen 3 O 4 /Fe 2 O 3 The heterobody nano-particles are coated by gold shells, and the whole diameter is about 96nm.
Example 2: preparation of self-assembled label-free magnetic nano tumor marker VEGF 165 Protein probes and use for detection
Fe in example 1 3 O 4 /Fe 2 O 3 Dispersing the @ Au nano-composite in ultrapure water by ultrasonic waves to prepare Fe with the concentration of 5mg/mL 3 O 4 /Fe 2 O 3 The @ Au nano-composite suspension is reserved; 10 mu L of Fe with the concentration of 5mg/mL is taken 3 O 4 /Fe 2 O 3 Dripping the suspension of the@Au nano-composite onto the surface of a clean Magnetic Glassy Carbon Electrode (MGCE), and drying at room temperature to obtain MGCE/Fe 3 O 4 /Fe 2 O 3 Modified electrode of@Au for standby.
Mixing TCEP at a concentration of 48. Mu.M with a thiol-modified DNA aptamer solution at a concentration of 1.6. Mu.M to obtain a thiol-modified DNA aptamer solution comprising TCEP, and then dropping 5. Mu.L of the thiol-modified DNA aptamer solution comprising TCEP to MGCE/Fe 3 O 4 /Fe 2 O 3 Modifying the surface of the electrode by @ Au, covering a centrifuge tube, selectively incubating for 12 hours at 4 ℃, taking down the centrifuge tube after the incubation is finished, and drying at room temperature to obtain MGCE/Fe 3 O 4 /Fe 2 O 3 Modified electrode of Au-DNA.
mu.L of BSA with a volume fraction of 0.25% was added dropwise to MGCE/Fe 3 O 4 /Fe 2 O 3 Modifying the electrode surface by Au-DNA, covering a centrifuge tube, reacting for 30min at 4 ℃, closing non-specific sites on the electrode surface, preventing non-specific adsorption, taking down the centrifuge tube after incubation, and drying at room temperature to obtain the self-assembled label-free magnetic nano tumor marker VEGF 165 Protein probe, designated MGCE/Fe 3 O 4 /Fe 2 O 3 @Au-DNA/BSA。
At the time of detection, 5. Mu.L of the solution was used as the concentration0.2pg·mL -1 VEGF of (2) 165 Drop-wise addition of protein solution to MGCE/Fe 3 O 4 /Fe 2 O 3 The surface of @ Au-DNA/BSA was incubated at 30℃for 60min to allow specific binding to DNA aptamer, and the DPV peak current was measured to be 10.69. Mu.A using Differential Pulse Voltammetry (DPV) in the range of 0.1-0.6V. This is because Fe 3 O 4 /Fe 2 O 3 When the number of @ Au nanoparticles is small, the conductivity is poor, resulting in a small peak current of the DPV. When there are more DNA aptamers, the electronegativity of the DNA phosphate backbone is enhanced, and [ Fe (CN) 6 ] 3-/4- The electrostatic repulsive force between the probes increases, and the steric hindrance of the DNA becomes large, resulting in a smaller peak current of the DPV.
Example 3: preparation of self-assembled label-free magnetic nano tumor marker VEGF 165 Protein probes and use for detection
Fe in example 1 3 O 4 /Fe 2 O 3 Dispersing the @ Au nano-composite in ultrapure water by ultrasonic waves to prepare Fe with the concentration of 30mg/mL 3 O 4 /Fe 2 O 3 The @ Au nano-composite suspension is reserved; 10 mu L of Fe with the concentration of 30mg/mL is taken 3 O 4 /Fe 2 O 3 Dripping the suspension of the@Au nano-composite onto the surface of a clean Magnetic Glassy Carbon Electrode (MGCE), and drying at room temperature to obtain MGCE/Fe 3 O 4 /Fe 2 O 3 Modified electrode of@Au for standby.
Mixing TCEP at a concentration of 48. Mu.M with a thiol-modified DNA aptamer solution at a concentration of 1.4. Mu.M to obtain a thiol-modified DNA aptamer solution comprising TCEP, and then dropping 5. Mu.L of the thiol-modified DNA aptamer solution comprising TCEP to MGCE/Fe 3 O 4 /Fe 2 O 3 Modifying the surface of the electrode by @ Au, covering a centrifuge tube, selectively incubating for 12 hours at 4 ℃, taking down the centrifuge tube after the incubation is finished, and drying at room temperature to obtain MGCE/Fe 3 O 4 /Fe 2 O 3 Modified electrode of Au-DNA.
mu.L of BSA with a volume fraction of 0.25% was added dropwise to MGCE/Fe 3 O 4 /Fe 2 O 3 The surface of the electrode is modified by Au-DNA, a centrifuge tube is covered, the reaction is carried out for 30min at 4 ℃,closing the electrode surface nonspecific sites, preventing nonspecific adsorption, taking down the centrifuge tube after incubation, and drying at room temperature to obtain self-assembled label-free magnetic nanometer tumor marker VEGF 165 Protein probe, designated MGCE/Fe 3 O 4 /Fe 2 O 3 @Au-DNA/BSA。
In the detection, a concentration of 5. Mu.L was set at 10 pg.multidot.mL -1 VEGF of (2) 165 Drop-wise addition of protein solution to MGCE/Fe 3 O 4 /Fe 2 O 3 The surface of @ Au-DNA/BSA was incubated at 20℃for 50min to allow specific binding to DNA aptamer, and the DPV peak current was measured to be 2.23. Mu.A using Differential Pulse Voltammetry (DPV) in the range of 0.1-0.6V. This is because Fe 3 O 4 /Fe 2 O 3 When the number of @ Au nanoparticles is too large, the steric hindrance is large, hindering [ Fe (CN) 6 ] 3-/4- The probe reaches the electrode surface, resulting in a reduced DPV signal response.
Example 4: preparation of self-assembled label-free magnetic nano tumor marker VEGF 165 Protein probes and use for detection
Fe in example 1 3 O 4 /Fe 2 O 3 Dispersing the @ Au nano-composite in ultrapure water by ultrasonic waves to prepare Fe with the concentration of 15mg/mL 3 O 4 /Fe 2 O 3 The @ Au nano-composite suspension is reserved; 10 mu L of Fe with the concentration of 15mg/mL is taken 3 O 4 /Fe 2 O 3 Dripping the suspension of the@Au nano-composite onto the surface of a clean Magnetic Glassy Carbon Electrode (MGCE), and drying at room temperature to obtain MGCE/Fe 3 O 4 /Fe 2 O 3 Modified electrode of@Au for standby.
Mixing TCEP at a concentration of 48. Mu.M with a thiol-modified DNA aptamer solution at a concentration of 0.2. Mu.M to obtain a thiol-modified DNA aptamer solution comprising TCEP, and then dropping 5. Mu.L of the thiol-modified DNA aptamer solution comprising TCEP to MGCE/Fe 3 O 4 /Fe 2 O 3 Modifying the surface of the electrode by @ Au, covering a centrifuge tube, selectively incubating for 12 hours at 4 ℃, taking down the centrifuge tube after the incubation is finished, and drying at room temperature to obtain MGCE/Fe 3 O 4 /Fe 2 O 3 Modified Au-DNAAnd (5) decorating the electrode.
mu.L of BSA with a volume fraction of 0.25% was added dropwise to MGCE/Fe 3 O 4 /Fe 2 O 3 Modifying the electrode surface by Au-DNA, covering a centrifuge tube, reacting for 30min at 4 ℃, closing non-specific sites on the electrode surface, preventing non-specific adsorption, taking down the centrifuge tube after incubation, and drying at room temperature to obtain the self-assembled label-free magnetic nano tumor marker VEGF 165 Protein probe, designated MGCE/Fe 3 O 4 /Fe 2 O 3 @Au-DNA/BSA。
In the detection, 5. Mu.L was measured to be 0.01 pg.multidot.mL -1 VEGF of (2) 165 Drop-wise addition of protein solution to MGCE/Fe 3 O 4 /Fe 2 O 3 The surface of @ Au-DNA/BSA was incubated at 35℃for 10min to allow specific binding to DNA aptamer, and the DPV peak current was measured to be 34.23. Mu.A using Differential Pulse Voltammetry (DPV) in the range of 0.1-0.6V. This is because the DNA phosphate backbone is negatively charged and [ Fe (CN) ] 6 ] 3-/4- Electrostatic repulsion exists between the probes to prevent [ Fe (CN) 6 ] 3-/4- The probe reaches the electrode surface. The concentration of the DNA aptamer is low, the electrostatic repulsion is small, the DPV peak current is large, but VEGF can be combined 165 The amount of protein is reduced. Shorter incubation time, VEGF 165 The protein did not bind sufficiently to the DNA aptamer, resulting in a large peak current for DPV.
Example 5: preparation of self-assembled label-free magnetic nano tumor marker VEGF 165 Protein probes and use for detection
Fe in example 1 3 O 4 /Fe 2 O 3 Dispersing the @ Au nano-composite in ultrapure water by ultrasonic waves to prepare Fe with the concentration of 20mg/mL 3 O 4 /Fe 2 O 3 The @ Au nano-composite suspension is reserved; 10 mu L of Fe with concentration of 20mg/mL is taken 3 O 4 /Fe 2 O 3 Dripping the suspension of the@Au nano-composite onto the surface of a clean Magnetic Glassy Carbon Electrode (MGCE), and drying at room temperature to obtain MGCE/Fe 3 O 4 /Fe 2 O 3 Modified electrode of@Au for standby.
TCEP at 48. Mu.M and concentrationMixing thiol-modified DNA aptamer solution with a degree of 1.3. Mu.M to obtain thiol-modified DNA aptamer solution containing TCEP, and then dropping 5. Mu.L thiol-modified DNA aptamer solution containing TCEP to MGCE/Fe 3 O 4 /Fe 2 O 3 Modifying the surface of the electrode by @ Au, covering a centrifuge tube, selectively incubating for 12 hours at 4 ℃, taking down the centrifuge tube after the incubation is finished, and drying at room temperature to obtain MGCE/Fe 3 O 4 /Fe 2 O 3 Modified electrode of Au-DNA.
mu.L of BSA with a volume fraction of 0.25% was added dropwise to MGCE/Fe 3 O 4 /Fe 2 O 3 Modifying the electrode surface by Au-DNA, covering a centrifuge tube, reacting for 30min at 4 ℃, closing non-specific sites on the electrode surface, preventing non-specific adsorption, taking down the centrifuge tube after incubation, and drying at room temperature to obtain the self-assembled label-free magnetic nano tumor marker VEGF 165 Protein probe, designated MGCE/Fe 3 O 4 /Fe 2 O 3 @Au-DNA/BSA。
In the detection, 5. Mu.L of the solution was measured to be 1 pg.multidot.mL -1 VEGF of (2) 165 Drop-wise addition of protein solution to MGCE/Fe 3 O 4 /Fe 2 O 3 The surface of @ Au-DNA/BSA was incubated at 40℃for 70min to allow specific binding to DNA aptamer, and the DPV peak current was measured to be 17.62. Mu.A using Differential Pulse Voltammetry (DPV) in the range of 0.1-0.6V. This is because Fe 3 O 4 /Fe 2 O 3 The number of the@Au nano particles is excessive, the steric hindrance is large, and the [ Fe (CN) is hindered 6 ] 3-/4- The probe reaches the electrode surface, resulting in reduced DPV signal response; the incubation temperature is too high, affecting DNA aptamer activity.
Example 6: preparation of self-assembled label-free magnetic nano tumor marker VEGF 165 Protein probes and use for detection
Fe in example 1 3 O 4 /Fe 2 O 3 Dispersing the @ Au nano-composite in ultrapure water by ultrasonic waves to prepare Fe with the concentration of 15mg/mL 3 O 4 /Fe 2 O 3 The @ Au nano-composite suspension is reserved; 10 mu L of Fe with the concentration of 15mg/mL is taken 3 O 4 /Fe 2 O 3 Dripping the suspension of the@Au nano-composite onto the surface of a clean Magnetic Glassy Carbon Electrode (MGCE), and drying at room temperature to obtain MGCE/Fe 3 O 4 /Fe 2 O 3 Modified electrode of@Au for standby.
Mixing TCEP at a concentration of 48. Mu.M with a thiol-modified DNA aptamer solution at a concentration of 1.5. Mu.M to obtain a thiol-modified DNA aptamer solution comprising TCEP, and then adding 5. Mu.L of the thiol-modified DNA aptamer solution comprising TCEP dropwise to MGCE/Fe 3 O 4 /Fe 2 O 3 Modifying the surface of the electrode by @ Au, covering a centrifuge tube, selectively incubating for 12 hours at 4 ℃, taking down the centrifuge tube after the incubation is finished, and drying at room temperature to obtain MGCE/Fe 3 O 4 /Fe 2 O 3 Modified electrode of Au-DNA.
mu.L of BSA with a volume fraction of 0.25% was added dropwise to MGCE/Fe 3 O 4 /Fe 2 O 3 Modifying the electrode surface by Au-DNA, covering a centrifuge tube, reacting for 30min at 4 ℃, closing non-specific sites on the electrode surface, preventing non-specific adsorption, taking down the centrifuge tube after incubation, and drying at room temperature to obtain the self-assembled label-free magnetic nano tumor marker VEGF 165 Protein probe, designated MGCE/Fe 3 O 4 /Fe 2 O 3 @Au-DNA/BSA。
In the detection, 5. Mu.L of the sample was concentrated to 7 pg.mL -1 VEGF of (2) 165 Drop-wise addition of protein solution to MGCE/Fe 3 O 4 /Fe 2 O 3 The surface of @ Au-DNA/BSA was incubated at 37℃for 45min to allow specific binding to DNA aptamer, and the DPV peak current was measured to be 16.97. Mu.A using Differential Pulse Voltammetry (DPV) in the range of 0.1-0.6V. As the DNA aptamer is increased, the electronegativity of the DNA phosphate skeleton is enhanced, and the DNA aptamer is combined with [ Fe (CN) 6 ] 3-/4- The electrostatic repulsive force between the probes increases and the steric hindrance of the DNA becomes large, resulting in a smaller peak current of the DPV.
Example 7: preparation of self-assembled label-free magnetic nano tumor marker VEGF 165 Protein probes and use for detection
Fe in example 1 3 O 4 /Fe 2 O 3 Ultrasonic dispersion of the @ Au nanocomposite in ultra pure waterWherein, the concentration is prepared into 16mg/mL Fe 3 O 4 /Fe 2 O 3 The @ Au nano-composite suspension is reserved; 10 mu L of Fe with the concentration of 16mg/mL is taken 3 O 4 /Fe 2 O 3 Dripping the suspension of the@Au nano-composite onto the surface of a clean Magnetic Glassy Carbon Electrode (MGCE), and drying at room temperature to obtain MGCE/Fe 3 O 4 /Fe 2 O 3 Modified electrode of@Au for standby.
Mixing TCEP at a concentration of 48. Mu.M with a thiol-modified DNA aptamer solution at a concentration of 1.4. Mu.M to obtain a thiol-modified DNA aptamer solution comprising TCEP, and then dropping 5. Mu.L of the thiol-modified DNA aptamer solution comprising TCEP to MGCE/Fe 3 O 4 /Fe 2 O 3 Modifying the surface of the electrode by @ Au, covering a centrifuge tube, selectively incubating for 12 hours at 4 ℃, taking down the centrifuge tube after the incubation is finished, and drying at room temperature to obtain MGCE/Fe 3 O 4 /Fe 2 O 3 Modified electrode of Au-DNA.
mu.L of BSA with a volume fraction of 0.25% was added dropwise to MGCE/Fe 3 O 4 /Fe 2 O 3 Modifying the electrode surface by Au-DNA, covering a centrifuge tube, reacting for 30min at 4 ℃, closing non-specific sites on the electrode surface, preventing non-specific adsorption, taking down the centrifuge tube after incubation, and drying at room temperature to obtain the self-assembled label-free magnetic nano tumor marker VEGF 165 Protein probe, designated MGCE/Fe 3 O 4 /Fe 2 O 3 @Au-DNA/BSA。
In the detection, 5. Mu.L of the sample was measured to be 4 pg.multidot.mL -1 VEGF of (2) 165 Drop-wise addition of protein solution to MGCE/Fe 3 O 4 /Fe 2 O 3 The surface of @ Au-DNA/BSA was incubated at 30℃for 45min to allow specific binding to DNA aptamer, and the DPV peak current was measured to be 18.27. Mu.A using Differential Pulse Voltammetry (DPV) in the range of 0.1-0.6V. This is because the DNA aptamer increases, the electronegativity of the DNA phosphate backbone increases, and [ Fe (CN) 6 ] 3-/4- The electrostatic repulsive force between the probes increases and the steric hindrance of the DNA becomes large, resulting in a smaller peak current of the DPV.
Example 8:
the implementation isIn the example, the self-assembled mark-free Fe is examined by using a cyclic voltammetry 3 O 4 /Fe 2 O 3 Magnetic nano VEGF (vascular endothelial growth factor) of @ Au-DNA/BSA (deoxyribonucleic acid/BSA) 165 The stepwise modification process of the protein probe is shown in the following specific experimental procedures.
Wherein MGCE/Fe 3 O 4 /Fe 2 O 3 The preparation method of (2) comprises the following steps: fe is added to 3 O 4 /Fe 2 O 3 The heteroplasmic nano particles are dispersed in ultrapure water by ultrasonic to prepare Fe with the concentration of 15mg/mL 3 O 4 /Fe 2 O 3 The suspension of the heteroplasmic nano particles is taken to be 10 mu L of Fe with the concentration of 15mg/mL 3 O 4 /Fe 2 O 3 The heteroplasmic nano particle suspension liquid is dripped on the surface of a clean Magnetic Glassy Carbon Electrode (MGCE), and is dried at room temperature to obtain the MGCE/Fe 3 O 4 /Fe 2 O 3 And (3) modifying the electrode.
MGCE/Fe 3 O 4 /Fe 2 O 3 @Au、MGCE/Fe 3 O 4 /Fe 2 O 3 @Au-DNA and MGCE/Fe 3 O 4 /Fe 2 O 3 The @ Au-DNA/BSA was prepared as described in example 3.
MGCE/Fe 3 O 4 /Fe 2 O 3 @Au-PNA/BSA/VEGF 165 The preparation process of (2) is as follows: concentration of 5. Mu.L was 10 pg.mL -1 VEGF of (2) 165 Dripping protein solution into MGCE/Fe 3 O 4 /Fe 2 O 3 The surface of Au-DNA/BSA was incubated at 37℃for 40min to allow specific binding to DNA aptamer.
The measuring method comprises the following steps:
the bare electrodes MGCE and MGCE/Fe are tested by an electrochemical workstation and a three-electrode system 3 O 4 /Fe 2 O 3 、MGCE/Fe 3 O 4 /Fe 2 O 3 @Au、MGCE/Fe 3 O 4 /Fe 2 O 3 @Au-DNA、MGCE/Fe 3 O 4 /Fe 2 O 3 @Au-DNA/BSA、MGCE/Fe 3 O 4 /Fe 2 O 3 @Au-PNA/BSA/VEGF 165 Silver as working electrodeSilver chloride as reference electrode and platinum wire electrode as counter electrode, 5mM [ Fe (CN) containing 0.1M KCl therein 6 ] 3-/4- The solution was used as an electrolyte and Differential Pulse Voltammetry (DPV) was performed using an electrochemical workstation in the range of 0.1-0.6V.
FIG. 2 VEGF under the conditions described in this example 165 In the cyclic voltammetry diagram of each stage in the preparation process of the protein probe, a is a bare electrode MGCE, a clean magnetic glassy carbon electrode and b is MGCE/Fe 3 O 4 /Fe 2 O 3 C is MGCE/Fe 3 O 4 /Fe 2 O 3 @Au, d is MGCE/Fe 3 O 4 /Fe 2 O 3 The @ Au-DNA, e is MGCE/Fe 3 O 4 /Fe 2 O 3 The @ Au-DNA/BSA, f is MGCE/Fe 3 O 4 /Fe 2 O 3 @Au-PNA/BSA/VEGF 165 Is a cyclic voltammogram of (c). As can be seen from the graph, curve c has a higher current response than curve b, because of Fe 3 O 4 /Fe 2 O 3 The heteroplasmic nano-particles have larger steric hindrance and block [ Fe (CN) 6 ] 3-/4- The probe reaches the electrode surface, resulting in reduced current flow, when Fe 3 O 4 /Fe 2 O 3 After the heteroplasmon nano-particles are coated with a layer of gold shell, fe 3 O 4 /Fe 2 O 3 The conductivity of the @ Au nanoparticle is enhanced, and thus the peak current is increased; curve d is the peak current decrease after thiol-modified DNA aptamer attachment to the electrode surface due to the negatively charged DNA phosphate backbone and [ Fe (CN) 6 ] 3-/4- Electrostatic repulsion and steric hindrance of DNA are arranged between the probes; curve e peak current decreases because BSA is poorly conductive and sterically hindered; curve f is incubation VEGF 165 After protein, DNA aptamer and VEGF 165 The protein generates specific binding reaction, and the response of the electrode electric signal is weakened. The above results show that the invention successfully constructs Fe 3 O 4 /Fe 2 O 3 Magnetic nano VEGF (vascular endothelial growth factor) of @ Au-DNA/BSA (deoxyribonucleic acid/BSA) 165 A protein probe.
Example 9:
in the present embodiment, the following structureThe self-assembled label-free magnetic nano tumor marker VEGF is obtained 165 Protein probe MGCE/Fe 3 O 4 /Fe 2 O 3 And (3) taking the@Au-DNA as an electrochemical biosensor, dropwise adding an interference protein, and examining the selectivity of the electrochemical sensor.
5 mu L of the solution was concentrated to 1 pg.mL -1 VEGF of (2) 165 Drop-wise addition of protein solution to MGCE/Fe 3 O 4 /Fe 2 O 3 The mixture is incubated at 37 ℃ for 40min on the surface of the@Au-DNA/BSA to obtain MGCE/Fe 3 O 4 /Fe 2 O 3 @Au-DNA/BSA/VEGF 165 And (3) modifying the electrode.
The concentration of 5 mu L is 10 pg.mL -1 Albumin solution, hemoglobin solution, lysozyme solution, L-Cysteine solution, lgG solution drop wise to MGCE/Fe 3 O 4 /Fe 2 O 3 And (3) incubating the surface of the @ Au-DNA/BSA at 37 ℃ for 40min to obtain modified electrodes modified by different interfering proteins.
FIG. 3 is Fe 3 O 4 /Fe 2 O 3 Magnetic nano VEGF (vascular endothelial growth factor) of @ Au-DNA/BSA (deoxyribonucleic acid/BSA) 165 Protein probes for detecting VEGF 165 Peak currents of Albumin, hemoglobin, lysozyme, L-Cysteine, lgG, as seen in FIG. 3, at interferent concentrations above VEGF 165 VEGF in the case of 10-fold protein 165 The strength of the protein electric signal is only 35.2% of that of the rest interfering substances, which indicates that the electrochemical biosensor has good selectivity.
Example 10:
concentration of 5. Mu.L is 0.01-10 pg.multidot.mL -1 VEGF in between 165 The protein solution was added dropwise to the MGCE/Fe prepared in example 3 3 O 4 /Fe 2 O 3 The surface of the @ Au-DNA/BSA is incubated for 40min at 37 ℃ to enable the surface to be specifically combined with the DNA aptamer, a Differential Pulse Voltammetry (DPV) is adopted for testing within the range of 0.1-0.6V, the peak current of the DPV is measured, and the analysis performance of the electrochemical sensor is examined.
As can be seen from FIG. 4, the current value and VEGF 165 The protein concentration shows good linear correlation, and the concentration detectable by the nano probe is 0.01-10 pg.mL -1 The linear regression equation is I (μA) = -1.0689C (pg.mL) -1 ) +24.8446, correlation coefficient R 2 =0.9973. I represents current and C represents concentration. The detection limit obtained by calculation of the linear fitting data is 0.01 pg.mL -1 (S/n=3), the limit of quantification was 0.03 pg.ml -1 (S/N＝10)。
Compared with the prior art, the self-assembled label-free magnetic nano tumor marker VEGF165 protein probe has low detection limit, and when VEGF 165 The protein concentration is 0.01-10 pg.mL -1 Between the current value and VEGF 165 The protein concentration showed a good linear correlation. The preparation method is simple, has good selectivity and can realize VEGF 165 Sensitive detection of proteins.
The examples are preferred embodiments of the present invention, but the present invention is not limited to the above-described embodiments, and any obvious modifications, substitutions or variations that can be made by one skilled in the art without departing from the spirit of the present invention are within the scope of the present invention.
Claims (5)
1. VEGF (vascular endothelial growth factor) 165 The preparation method of the protein probe is characterized by comprising the following steps:
S1. Fe 3 O 4 /Fe 2 O 3 preparation of @ Au nanocomposite:
(1) Fe (NO) 3 ) 3 ·9H 2 Standing the O aqueous solution for hydrolysis reaction to obtain orange-red suspension, centrifuging, washing, dispersing in absolute ethyl alcohol to obtain ethanol suspension, calcining the ethanol suspension, and grinding to obtain magnetic Fe 3 O 4 /Fe 2 O 3 Heteroplasmic nanoparticles; the Fe (NO) 3 ) 3 ·9H 2 The concentration of the O aqueous solution is 0.05-M-0.5M; the hydrolysis condition is that the hydrolysis is carried out for 2 to 16 hours at the temperature of 60 to 90 ℃; the calcination is carried out for 0.5-h-4 hours at 200-400 ℃;
(2) Magnetic Fe 3 O 4 /Fe 2 O 3 Heteroplastid nano particles are dispersed by ultrasonic, and HAuCl is added 4 The solution is heated to boiling under reflux, sodium citrate solution is added for reaction,then centrifuging, washing and drying to obtain magnetic Fe 3 O 4 /Fe 2 O 3 Dispersing the @ Au nano-composite in ultrapure water for standby; the magnetic Fe 3 O 4 /Fe 2 O 3 Heteroplasmic nanoparticles, HAuCl 4 And sodium citrate at a mass ratio of 5:20:114; the reaction time is 40min;
S2. Fe 3 O 4 /Fe 2 O 3 construction of @ Au-DNA/BSA:
(1) Fe is added to 3 O 4 /Fe 2 O 3 Dripping the suspension of the@Au nano-composite onto the surface of the clean MGCE, and drying at room temperature to obtain the MGCE/Fe 3 O 4 /Fe 2 O 3 Modified electrode of @ Au; fe (Fe) 3 O 4 /Fe 2 O 3 The concentration of the@Au nano-composite suspension is 5-30mg/mL, and the dosage is 10 mu L;
(2) To MGCE/Fe 3 O 4 /Fe 2 O 3 Dropwise adding a mercapto-modified DNA aptamer solution containing tris (2-carboxyethyl) phosphine on the surface of an Au modified electrode for incubation, and drying at room temperature after incubation to obtain MGCE/Fe 3 O 4 /Fe 2 O 3 An Au-DNA modified electrode; the thiol-modified DNA aptamer containing tris (2-carboxyethyl) phosphine takes TE buffer solution as a solvent, the concentration is 0.2-1.6 mu M, and the dosage is 5 mu L; the concentration of the tris (2-carboxyethyl) phosphine is 48 μm; the sequence of the sulfhydryl modified DNA aptamer is 5' -SH- (CH) 2 ) 6 -CAATTGGGCCCGTCCGTATGGTGGGT-3’；
The incubation condition is that the incubation is carried out for 12 hours at 4 DEG C
(3) To MGCE/Fe 3 O 4 /Fe 2 O 3 Dripping bovine serum albumin on the surface of the@Au-DNA modified electrode to react, and drying at room temperature after the reaction is finished to obtain the self-assembled label-free magnetic nano tumor marker VEGF 165 Protein probe, designated MGCE/Fe 3 O 4 /Fe 2 O 3 @Au-DNA/BSA; the dosage of the bovine serum albumin is 2-5 mu L, wherein the volume fraction of the bovine serum albumin is 0.25%, and the reaction condition is that the reaction is carried out for 30min at 4 ℃.
2. The method according to claim 1, wherein in step S1 (1), the Fe (NO 3 ) 3 ·9H 2 The concentration of the O aqueous solution was 0.05M; the hydrolysis condition is that the hydrolysis is carried out for 8 hours at 90 ℃; the calcination was performed at 200℃for 2 hours.
3. VEGF produced by the method of any one of claims 1 or 2 165 A protein probe, characterized in that the probe is denoted as Fe 3 O 4 /Fe 2 O 3 @Au-DNA/BSA, the probe was based on the DNA aptamer 5' -SH- (CH) 2 ) 6 CAATTGGGCCCGTCCGTATGGTGGGT-3'.
4. The VEGF of claim 3 165 Protein probe for detecting VEGF in preparation 165 Is provided.
5. VEGF detection 165 An electrochemical nucleic acid sensor for proteins, characterized in that it is based on VEGF according to claim 3 165 The protein probe is prepared.
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