CN114354914B - Preparation method and application of fluorescent biosensor for detecting transgenic protein - Google Patents

Preparation method and application of fluorescent biosensor for detecting transgenic protein Download PDF

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CN114354914B
CN114354914B CN202111535937.5A CN202111535937A CN114354914B CN 114354914 B CN114354914 B CN 114354914B CN 202111535937 A CN202111535937 A CN 202111535937A CN 114354914 B CN114354914 B CN 114354914B
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CN114354914A (en
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郭智勇
陈小双
郝婷婷
王照亮
郭文博
曹国洲
邬杨波
王忠琦
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Ningbo University
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Abstract

The invention discloses a preparation method and application of a fluorescence biosensor for detecting transgenic protein, which is characterized by comprising the following steps: mixing 3,4,9, 10-perylene tetracarboxylic acid solution with the concentration of 1mg/mL and silver nanocluster solution according to the volume ratio of 5:1, and stirring for 4 hours at normal temperature to obtain AgNCs/PTCA solution; adding the EDC/NHS mixed solution into the AgNCs/PTCA solution in equal volume, keeping out of the sun at room temperature, continuously stirring for 6 h, and adding a second antibody Ab of the transgenic protein to be detected in equal volume with the EDC/NHS mixed solution 2 Incubated at 37 ℃ for 2 h and centrifuged to remove free secondary antibody Ab 2 The super-pure water is used for washing the product for 3 times and is re-dispersed into PBS buffer solution with the same volume as the EDC/NHS mixed solution to obtain the super-molecular high-fluorescence material, and the super-molecular high-fluorescence material has the advantages of strong specificity, high sensitivity and good accuracy.

Description

Preparation method and application of fluorescent biosensor for detecting transgenic protein
Technical Field
The invention relates to a fluorescence immunosensor, in particular to a preparation method and application of a fluorescence biosensor for detecting transgenic protein.
Background
The PAT protein is a homodimer encoded and expressed by the bar gene and having a molecular weight of about 23 KDa, and consists of 183 amino acids. The PAT protein belongs to an acetyl transferase family, has relatively the same structure and function as acetyl transferase, and has the physiological effect of acetylating the herbicide glufosinate and inhibiting the activity of glufosinate so as to detoxify glufosinate. With the widespread use of transgenic products, people pay more and more attention to some potential risks possibly brought by the transgenic products. Therefore, the development of accurate, sensitive, convenient and fast detection methods for transgenic crops and foods is urgently needed.
The most mature detection methods for transgenic products are mainly based on polymerase chain technology (PCR). however, PCR analysis requires complex procedures, usually several hours to complete a sample detection. Furthermore, this method requires strict and reliable quality control procedures to avoid cross-contamination, and rapid detection in the field cannot be achieved using PCR equipment. Therefore, protein immunoassay-based methods have attracted much attention due to their rapid detection and simple operation. The miniaturization of photoelectric detection equipment becomes a hot spot of great attention, but the miniaturization of equipment often brings low sensitivity, and in order to improve the problem, if the performance of a photoelectric material can be further improved, a photoelectric sensing miniature equipment capable of realizing transgenic crops can be developed, so that the rapid high-sensitivity field detection of transgenic products can be realized.
3,4,9, 10-perylenetetracarboxylic acid (PTCA) is a hydrolysis product of 3,4,9, 10-perylenetetracarboxylic dianhydride (PTCDA). It is a carboxyl-rich 5-ring polycyclic aromatic hydrocarbon, a typical stackable organic perylene dye, has excellent photochemical stability and satisfactory photoelectric properties, and is widely considered to be one of the most promising advanced material research fields. It has been found that PTCDA and PTCA can strongly interact with metals such as Ag, Au, Cu and the like through electron transfer, and Ag NCs/PTCA has higher and stronger fluorescence emission compared with PTCA monomer.
Disclosure of Invention
The invention aims to provide a preparation method and application of a supermolecule high-fluorescence material for detecting transgenic protein, which has strong specificity, high sensitivity and good accuracy.
The technical scheme adopted by the invention for solving the technical problems is as follows: a preparation method of a fluorescence biosensor for detecting transgenic protein comprises the following steps:
(1) synthesis of 3,4,9, 10-perylene tetracarboxylic acid (PTCA)
Adding 8-10 mg of 3,4,9,10 perylene tetracarboxylic dianhydride (PTCDA) into 10-12 mL of a 1 mol/LNaOH solution, carrying out ultrasonic treatment for 15 min under the ultrasonic frequency of 80-100 kHz, heating the solution until the PTCDA is completely dissolved, adding 1 mol/L HCl solution into the solution after the solution color becomes yellow-green, carrying out centrifugal washing after the mixture color becomes red completely, washing the collected precipitate alternately with absolute ethyl alcohol and ultrapure water to obtain a PTCA precipitate, carrying out vacuum drying for 3 h at 40 ℃, and dispersing the precipitate in ultrapure water;
(2) synthesis of silver nanoclusters
Adding 4.2-4.5 mg of lipoic acid powder into 2.0mL of ultrapure water, then adding 0.01-0.015 mL of 2 mol/L sodium borohydride solution, violently stirring for 30 min, then enabling the solution to become clear and transparent, slowly adding 0.04-0.05 mL of 0.05mol/L silver nitrate solution and 0.03-0.04 mL of 2 mol/L sodium borohydride solution under rapid stirring, continuously reacting for 90 min, stopping stirring, and obtaining silver nanocluster (Ag NCs) solution, placing the silver nanocluster (Ag NCs) solution in a refrigerator, and storing the silver nanocluster solution for more than 24 h at 4 ℃;
(3) synthesis of Ag NCs/PTCA
Mixing the PTCA solution with the concentration of 1mg/mL obtained in the step (1) and the silver nanocluster solution obtained in the step (2) according to the volume ratio of 5:1, and stirring for 4 hours at normal temperature to obtain an AgNCs/PTCA solution; the silver nanoclusters are combined with PTCA (protein-coupled carrier) through chemical bonding, and the PTCA is induced to self-assemble to obtain a spiral flower-shaped structure;
(4) signal unit Ab 2 Synthesis of/AgNCs/PTCA
Adding the EDC/NHS mixed solution into the AgNCs/PTCA solution obtained in the step (3) in the same volume, keeping the mixture away from light at room temperature, continuously stirring for 6 h, and adding a second antibody Ab of 10 mu g/mL transgenic protein to be detected in the same volume with the EDC/NHS mixed solution 2 Incubated at 37 ℃ for 2 h and centrifuged to remove free secondary antibody Ab 2 And washing the product with ultrapure water for 3 times, and re-dispersing the product into PBS buffer solution with the same volume as the EDC/NHS mixed solution and the pH =7.4 to finally obtain a signal unit, namely obtaining the fluorescent biosensor for detecting the transgenic protein.
The concentration of EDC in the EDC/NHS mixed solution is 0.4 mol/L, and the concentration of NHS is 0.1 mol/L.
The method for rapidly detecting the transgenic protein on site by using the prepared fluorescent biosensor comprises the following steps: 0.1mL of first antibody Ab of 1ug/mL of transgenic protein to be detected and diluted by coating solution is added into each hole of the enzyme label plate 1 The solution was incubated in a 37 ℃ water bath for 3 hours at 4 ℃ overnight and stored in a refrigerator; removing the coating solution, and washing the enzyme-labeled hole for 3 times by using washing buffer solution containing 0.05 percent of Tween-20 after 5 minutes each time; adding 0.1mL PBS solution containing 2wt% BSA to block other active sites and washing for 3 times, then adding 0.1mL of test specimen containing the transgenic protein antigen to be detected and with different concentrations, which is diluted by a dilution buffer solution, into each concave hole, and acting for 1 hour at 37 ℃; finally, 0.2 mL of supramolecular high-fluorescence material is added into each concave hole, the wells are washed for 3 times, and 0.2 mL of PBS solution with the concentration of 0.1 mol/L and the pH = 8.0 is added as detection buffer for detection.
The transgenic protein to be detected is PAT/bar protein, and the second antibody Ab 2 Is PAT/bar pAb, and the first antibody Ab 1 PAT/bar mAb.
The transgenic protein to be detected is Cry1Ab protein, and the second antibody Ab 2 Cry1Ab pAb, the first antibody Ab 1 Is Cry1Ab mAb.
The invention principle is as follows: the invention uses PAT/bar anti-Ab 1 Fixed on a micro-porous plate, reacts with the target transgenic protein PAT/bar specifically and then reacts with a signal unit PTCA/Ag Ncs-Ab 2 Bind to form "Ab 1 -PAT/bar-signaling unit "complex. The supermolecule nano material prepared by the invention has large specific surface area and can load more Ab 2 . Secondly, due to the plasma resonance effect of Ag NCs, the composite material has good photoluminescence performance, the Ag NCs can transfer hot electrons and hot holes to PTCA, so that the rapid recombination of electron holes is realized, the strong photoluminescence phenomenon is generated, stable fluorescence signal peaks appear at 480 nm and 510 nm, the change of the fluorescence intensity is closely related to the concentration of PAT/bar protein in the system, and the super-photoluminescence performance can be realizedDetection and analysis of low concentration of transgenic protein.
Compared with the prior art, the invention has the advantages that:
(1) PTCA/Ag Ncs in the signal unit has strong fluorescence signals, and after the immune composite structure is constructed, the signal unit can be lapped on the surface of the microporous plate, and the supermolecular structure brings large FL signal intensity;
(2) the Ag Ncs itself has good size effect and photocatalytic properties to achieve secondary enhancement of PTCA photoluminescence signals.
In conclusion, the fluorescent material with high fluorescence intensity is prepared for the first time, so that the high-sensitivity detection of the PAT/bar protein is realized, the fluorescent material has the advantages of high sensitivity, simplicity, rapidness, easiness in operation, low experiment cost and the like, the detection of the PAT/bar protein with ultra-low concentration can be realized, and the fluorescent material has a good application prospect.
Drawings
FIG. 1 is a flow chart of the preparation of Ag Ncs/PTCA of the present invention;
FIG. 2 is a schematic diagram of the process for preparing and detecting the sensor of the present invention;
FIG. 3 is a mechanism diagram of fluorescence enhancement of Ag Ncs/PTCA material of the sensor of the present invention;
FIG. 4 is a graph of FL assay for different transgenic PAT/bar protein concentrations;
FIG. 5 is a graph of the linear dependence of the concentration of different PAT/bar on the fluorescence value;
FIG. 6 shows the fluorescence intensity values of the immunosensor for PL detection of blank, CP4-EPSPS, Cry1Ab, respectively.
Detailed Description
The invention is described in further detail below with reference to the following examples of the drawings.
Detailed description of the preferred embodiment
A method for preparing a fluorescence biosensor for detecting transgenic proteins, as shown in FIG. 1, comprises the following steps:
(1) synthesis of 3,4,9, 10-perylenetetracarboxylic acid (PTCA)
Adding 9 mg of 3,4,9,10 perylene tetracarboxylic dianhydride (PTCDA) into 11 mL of 1 mol/LNaOH solution, carrying out ultrasonic treatment for 15 min at the ultrasonic frequency of 90kHz, heating the solution until the PTCDA is completely dissolved, adding 1 mol/L of HCl solution into the solution after the color of the solution is changed into yellow green, carrying out centrifugal washing after the color of the mixture is completely changed into red, alternately washing the collected precipitate with absolute ethyl alcohol and ultrapure water to obtain PTCA precipitate, carrying out vacuum drying for 3 h at the temperature of 40 ℃, and dispersing in the ultrapure water;
(2) synthesis of silver nanoclusters
Adding 4.3 mg of lipoic acid powder into 2.0mL of ultrapure water, then adding 0.012mL of 2 mol/L sodium borohydride solution, violently stirring for 30 min, then enabling the solution to become clear and transparent, then slowly adding 0.045mL of 0.05mol/L silver nitrate solution and 0.035 mL of 2 mol/L sodium borohydride solution under rapid stirring, continuing to react for 90 min, and then stopping stirring to obtain silver nanocluster (Ag NCs) solution, placing the silver nanocluster (Ag NCs) solution in a refrigerator, and storing the silver nanocluster (Ag NCs) solution for more than 24 hours at 4 ℃;
(3) synthesis of Ag NCs/PTCA
Mixing the PTCA solution with the concentration of 1mg/mL obtained in the step (1) and the silver nanocluster solution obtained in the step (2) according to the volume ratio of 5:1, and stirring for 4 hours at normal temperature to obtain an AgNCs/PTCA solution; the silver nanoclusters are combined with PTCA through chemical bonding, and the PTCA is induced to self-assemble to obtain a spiral flower-shaped structure;
(4) signal unit Ab 2 Synthesis of/AgNCs/PTCA
Adding the EDC/NHS mixed solution into the AgNCs/PTCA solution obtained in the step (3) in the same volume, keeping the mixture in the dark at room temperature, continuously stirring for 6 h, adding 10 mu g/mL PAT/bar pAb (second antibody of PAT/bar transgenic protein) in the same volume with the EDC/NHS mixed solution, incubating for 2 h at 37 ℃, and centrifuging to remove free Ab 2 (PAT/bar pAb), washing the product with ultrapure water for 3 times, and dispersing the product into PBS buffer solution with pH =7.4 and the same volume as the mixed solution of EDC/NHS, finally obtaining a signal unit, namely obtaining the fluorescence biosensor for detecting the transgenic protein, wherein the concentration of EDC in the mixed solution of EDC/NHS is 0.4 mol/L, and the concentration of NHS is 0.1 mol/L.
Example 2
The difference from the above example 1 is that: step (1) in the synthesis of 3,4,9, 10-perylenetetracarboxylic acid (PTCA): adding 8 mg of 3,4,9,10 perylene tetracarboxylic dianhydride (PTCDA) into 10 mL of 1 mol/LNaOH solution, and carrying out ultrasonic treatment for 15 min at the ultrasonic frequency of 80 kHz; step (2) in the synthesis of the silver nanoclusters: adding 4.2 mg of lipoic acid powder into 2.0mL of ultrapure water, then adding 0.01 mL of 2 mol/L sodium borohydride solution, violently stirring for 30 min, then enabling the solution to become clear and transparent, then slowly adding 0.04mL of 0.05mol/L silver nitrate solution and 0.03 mL of 2 mol/L sodium borohydride solution under rapid stirring, continuing to react for 90 min, then stopping stirring, and finally obtaining silver nanocluster solution, placing the silver nanocluster solution in a refrigerator, and storing the silver nanocluster solution for more than 24 hours at 4 ℃.
Example 3
The difference from the above example 1 is that: step (1) in the synthesis of 3,4,9, 10-perylenetetracarboxylic acid (PTCA): adding 10 mg of 3,4,9,10 perylene tetracarboxylic dianhydride (PTCDA) into 12mL of 1 mol/LNaOH solution, and carrying out ultrasonic treatment for 15 min at the ultrasonic frequency of 100 kHz; step (2) in the synthesis of the silver nanoclusters: adding 4.5 mg of lipoic acid powder into 2.0mL of ultrapure water, then adding 0.015mL of 2 mol/L sodium borohydride solution, violently stirring for 30 min, then enabling the solution to become clear and transparent, then slowly adding 0.05mL of 0.05mol/L silver nitrate solution and 0.04mL of 2 mol/L sodium borohydride solution under rapid stirring, continuously reacting for 90 min, stopping stirring, and obtaining silver nanocluster solution, placing the silver nanocluster solution in a refrigerator, and storing the silver nanocluster solution for more than 24 hours at 4 ℃.
Detailed description of the preferred embodiment
A method for rapidly detecting a transgenic protein on site by using a fluorescence biosensor prepared in the first embodiment is shown in FIG. 2, and comprises the following steps:
adding 0.1mL of PAT/bar mAb solution with concentration of 1ug/mL diluted by coating solution into each hole of the ELISA plate, water bathing at 37 deg.C for 3 hr, standing overnight at 4 deg.C, and storing in refrigerator; removing the coating solution, and washing the enzyme-labeled hole with a washing buffer solution containing 0.05% Tween-20 for 3 times, 5 minutes each time; adding 0.1mL PBS solution containing 2wt% BSA to block other active sites and washing for 3 times, then adding 0.1mL test specimen containing PAT/bar protein antigen with different concentrations diluted by dilution buffer into each concave hole, and acting for 1 hour at 37 ℃; finally, 0.2 mL of supramolecular high-fluorescence material is added into each concave hole, the wells are washed for 3 times, and 0.2 mL of PBS solution with the concentration of 0.1 mol/L and the pH = 8.0 is added as detection buffer for detection. Wherein the coating buffer is a carbonate buffer with pH = 9.6 and a concentration of 0.05 mol/L; the washing buffer is PBS buffer containing 0.05% Tween-20 and having pH =7.4 and a concentration of 0.1 mol/L; diluting buffer solution and washing buffer solution; blocking solution was 2 g BSA dissolved in 100 mL PBS solution pH = 7.4.
As shown in fig. 3, under 440 nm xenon lamp illumination, electrons at the HOMO of the PTCA material can be injected into the LUMO (process a). The surface plasmon resonance (LSPR) -induced local electric field amplification inherent to Ag Ncs noble metals can facilitate photon absorption in the visible region of PTCA, and the thermionic transfer (HET) mechanism in LSPR can transfer thermions into the LUMO of PTCA (process b) and accelerate the electron and hole recombination velocity (process c), thus generating a strong fluorescence signal.
As shown in FIG. 4, under the irradiation of a xenon lamp at 440 nm, the fluorescence detection of PAT/bar proteins with different concentrations (0.001-50 ng/mL) has different fluorescence signals, and the fluorescence signal value increases with the increase of the PAT/bar concentration.
As shown in FIG. 5, there is a good linear relationship between the logarithm of concentration of the fluorescence signal PAT/bar, and the linear equation is: y = 2977 + 818
Figure 287956DEST_PATH_IMAGE001
The correlation coefficient is R 2 = 0.995, where y denotes PL intensity (a.u.) and x denotes concentration of PAT/bar (ng mL _) -1 ). Based on the signal-to-noise ratio S/N = 3, the detection Limit (LOD) of the method for detecting the PAT/bar protein is about 0.3 pg mL -1 The sensor has ultrahigh sensitivity and accuracy.
Detailed description of the invention
As shown in FIG. 6, blanks, CP4-EPSPS, Cry1Ab, PAT/bar and transgenic protein mixed solution (containing Cry1Ab, CP 4-EPS) were set up respectivelyPS and PAT/bar-protein) four control experiments. Wherein the concentration of each transgenic protein used as control is 1 ng mL -1 . As can be seen from the experimental results, Cry1Ab and CP4-EPSPS, blank control have almost no fluorescence signal, and the signal intensity of the transgenic protein mixed solution containing PAT/bar protein is almost the same as that of PAT/bar protein, which shows that the sensor has excellent selectivity.
In addition to the above examples, the detection target is replaced by Cry1Ab protein (antigen), and the corresponding antibody is Cry1Ab mAb (Ab) 1 And Cry1Ab pAb (Ab) 2 ）。
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.

Claims (5)

1. A preparation method of a fluorescence biosensor for detecting transgenic protein is characterized by comprising the following steps:
(1) synthesis of 3,4,9, 10-perylenetetracarboxylic acid
Adding 8-10 mg of 3,4,9,10 perylene tetracarboxylic dianhydride into 10-12 mL of 1 mol/LNaOH solution, carrying out ultrasonic treatment for 15 min at the ultrasonic frequency of 80-100 kHz, heating the solution until the PTCDA is completely dissolved, adding 1 mol/L HCl solution into the solution after the color of the solution is changed into yellow green, carrying out centrifugal washing after the color of the mixture is completely changed into red, alternately washing the collected precipitate with absolute ethyl alcohol and ultrapure water to obtain PTCA precipitate, carrying out vacuum drying for 3 h at 40 ℃, and dispersing in the ultrapure water;
(2) synthesis of silver nanoclusters
Adding 4.2-4.5 mg of lipoic acid powder into 2.0mL of ultrapure water, then adding 0.01-0.015 mL of 2 mol/L sodium borohydride solution, violently stirring for 30 min to ensure that the solution becomes clear and transparent, then slowly adding 0.04-0.05 mL of 0.05mol/L silver nitrate solution and 0.03-0.04 mL of 2 mol/L sodium borohydride solution under rapid stirring, continuously reacting for 90 min, and stopping stirring to obtain a silver nanocluster solution;
(3) synthesis of Ag NCs/PTCA
Mixing the PTCA solution with the concentration of 1mg/mL obtained in the step (1) and the silver nanocluster solution obtained in the step (2) according to the volume ratio of 5:1, and stirring for 4 hours at normal temperature to obtain an AgNCs/PTCA solution;
(4) signal unit Ab 2 Synthesis of/AgNCs/PTCA
Adding the EDC/NHS mixed solution into the AgNCs/PTCA solution obtained in the step (3) in the same volume, keeping the mixture in the dark at room temperature, continuously stirring for 6 h, and adding a second antibody Ab of 10 mu g/mL transgenic protein to be detected in the same volume with the EDC/NHS mixed solution 2 Incubated at 37 ℃ for 2 h and centrifuged to remove free secondary antibody Ab 2 The product was washed 3 times with ultrapure water and redispersed in an equal volume of PBS buffer pH =7.4 to the EDC/NHS mixed solution to finally obtain a signal unit, i.e. a supramolecular superfluorescent material.
2. The method for preparing a fluorescence biosensor for detecting transgenic protein according to claim 1, wherein: the concentration of EDC in the EDC/NHS mixed solution is 0.4 mol/L, and the concentration of NHS is 0.1 mol/L.
3. A method for rapidly detecting a transgenic protein in situ using the fluorescent biosensor prepared according to claim 1 or 2, comprising the steps of: 0.1mL of first antibody Ab of the transgenic protein to be detected with the concentration of 1ug/mL and diluted by coating solution is added into each hole of the ELISA plate 1 The solution was incubated in a 37 ℃ water bath for 3 hours at 4 ℃ overnight and stored in a refrigerator; removing the coating solution, and washing the enzyme-labeled hole for 3 times by using washing buffer solution containing 0.05 percent of Tween-20 after 5 minutes each time; adding 0.1mL PBS solution containing 2wt% BSA to block other active sites and washing for 3 times, then adding 0.1mL of test specimen containing the transgenic protein antigen to be detected and with different concentrations, which is diluted by a dilution buffer solution, into each concave hole, and acting for 1 hour at 37 ℃; finally, 0.2 mL of supramolecular superfluorescent material was added to each well, washed 3 times again, and 0.2 mL of supramolecular superfluorescent material was added at 0.1 mol/L, pH = 8.0The PBS solution was used as the detection buffer for detection.
4. The method of claim 3, wherein the fluorescent biosensor is used for rapid in-situ detection of the transgenic protein, and the method comprises the following steps: the transgenic protein to be detected is PAT/bar protein, and the second antibody Ab 2 Is PAT/bar pAb, and the first antibody Ab 1 PAT/bar mAb.
5. The method of claim 3, wherein the fluorescent biosensor is used for rapid in-situ detection of the transgenic protein, and the method comprises the following steps: the transgenic protein to be detected is Cry1Ab protein, and the second antibody Ab 2 Cry1Ab pAb, the first antibody Ab 1 Is Cry1Ab mAb.
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