Biosensor chip based on NanoSPR technology and its application in detecting gene delivery vectors

Abstract

The present invention discloses a biosensor chip based on NanoSPR technology and its application in detecting gene delivery vector, belonging to the field of qualitative and quantitative detection technology of gene delivery vector. The biosensor chip provided by the present invention includes a substrate and a protein, antibody or functional group modified on the substrate; the substrate includes a substrate, a titanium film, a silver film and a gold film from bottom to top, and the substrate surface matrix is ​​arranged with nanopores; the thickness of the titanium film is 2-30nm, the thickness of the silver film is 5-100nm, and the thickness of the gold film is 2-100nm; protein, antibody or functional group specifically refers to a protein, antibody or functional group specifically bound to a gene delivery vector, and a gene delivery vector refers to a viral vector (such as adenovirus, adeno-associated virus, slow virus, etc.) and a non-viral vector (lipid nanoparticles, exosomes, etc.). The present invention also provides a modification method of the above-mentioned chip, and an application method, with high detection sensitivity, fast detection speed and high throughput, which can be used for high-throughput quantification or screening quality control in the field of gene delivery.

Description

Technical Field

[0001] The invention belongs to the technical field of qualitative and quantitative detection of gene delivery vectors, in particular to a method for rapid detection of gene delivery vectors based on NanoSPR technology. Background Art

[0002] A gene delivery vector is a medium necessary for assembling a target gene and introducing it into a target cell or a corresponding host cell to express the target gene. Common gene delivery vectors include adenovirus, adeno-associated virus, lentivirus, and non-viral vectors (e.g., lipid nanoparticles LNP, exosomes, etc.). Adenovirus (Adv) is a particle with a diameter of 70-90 nm without an envelope and is the most common gene delivery vector. Adenovirus can infect the respiratory tract, gastrointestinal tract, urethra and bladder, eyes, liver, etc. About 1 / 3 of the known serotypes of human adenovirus are usually associated with human diseases, but one serotype can cause different clinical diseases, and different serotypes can also cause the same disease.

[0003] Nanoplasma chip technology (NanoSPR) is different from the two technical modes of plasma chip technology (SPR) and localized surface plasmon resonance technology (LSPR). Due to its unique three-dimensional structure, it is different from the SPR effect of the planar model and the LSPR effect of metal nanoparticles. It can support both SPR and LSPR modes at the same time.

[0004] The plasma resonance effect of the nanopore array biosensor can be directly incident on the nanopore metal structure, and the surface light field is immediately excited, so there is no need for complex optical paths and large optical instruments. NanoSPR technology retains many advantages of traditional SPR sensors, such as real-time, no labeling, no background interference, and high resolution, and retains the advantages of LSPR sensors in performance modification; by adjusting the aperture, depth, shape, period of the nanopore array, and the type and thickness of the surface metal, high-quality chips that can capture the strongest LSPR signal are selected, and stronger signals can be obtained without a large spectrometer.

[0005] In the prior art, the US patent US8158343B2 "Method for Detecting Virus-Related Immune Markers for Diagnosing Respiratory Infections" mentions that SPR biosensor chips can be used to qualitatively detect proteins related to respiratory viruses (such as human Parainfluenza virus 1, human Parainfluenza virus 2, human Parainfluenza virus 3, respiratory syncytial virus, influenza virus A, influenza virus B, and adenovirus) in serum samples. However, the detection of adenovirus using ordinary SPR technology is cumbersome, time-consuming, and the sensitivity needs to be improved. Summary of the invention

[0006] In view of the above shortcomings of the prior art, the present invention provides a biosensor chip based on NanoSPR technology and its application in detecting gene delivery vectors, invents a biosensor chip that can quickly detect gene delivery vectors, has high detection sensitivity, and uses the biosensor chip to detect gene delivery vectors such as adenovirus, which has the advantages of high throughput, short time consumption, and accurate detection results. It is specifically achieved through the following technologies.

[0007] A biosensor chip based on NanoSPR technology comprises a substrate and a protein, antibody or functional group modified on the substrate; the substrate comprises a base, a titanium film, a silver film and a gold film from bottom to top, and the surface of the substrate is embossed with nanopores arranged in a matrix; the diameter of the nanopores is 50-800nm, the height is 100-800nm, and the spacing between two adjacent nanopores is 200-1000nm; the thickness of the titanium film is 2-30nm, the thickness of the silver film is 5-100nm, and the thickness of the gold film is 2-100nm; the protein, antibody or functional group specifically refers to a protein, antibody or functional group that specifically binds to a gene delivery vector. For example, CAR protein (coxsackievirus adenovirus receptor that can bind to adenovirus), Fx protein (i.e. FactorX coagulation factor), adenovirus hexon protein antibody, adeno-associated virus receptor (AAVR), biotinylated heparin; P24 capsid protein antibody that can bind to lentivirus (HIV-1), lentivirus cell binding receptor protein, etc.; CD63, CD81, CD9, TSG101, HSP27 antibodies that can bind to exosomes, etc. The gene delivery vector refers to viral vectors (adenovirus, adeno-associated virus, lentivirus, etc.) and non-viral vectors (lipid nanoparticles LNP, exosomes, etc.); the functional group is an anionic, cationic or zwitterionic group that can bind to charged lipid nanoparticles.

[0008] Preferably, the thickness of the titanium film is 15 nm, the thickness of the silver film is 70 nm, and the thickness of the gold film is 20 nm.

[0009] The present invention also provides a method for preparing the modified biosensor chip based on NanoSPR technology, comprising the following steps:

[0010] S1. Wash the substrate, add 1-50 μl of 5-50 μg / ml CAR protein and / or Fx protein solution, incubate at 4-37° C. for 1.5-24 h, and then wash the substrate coated with CAR protein and / or Fx protein several times with PBST solution.

[0011] S2, adding a blocking solution to the surface of the substrate obtained in step S1, incubating at 25-37° C. for 1-4 hours, and removing excess blocking solution;

[0012] S3, dripping protective liquid on the surface of the substrate obtained in step S2, incubating at 25-37° C. for 0.5-1 h, removing excess protective liquid, and drying to obtain a biosensor chip.

[0013] The blocking solution is a complex solution with a concentration of 10 μg / ml prepared by dissolving bovine serum albumin in a CBS solution; the CBS solution contains 4-6 g / L potassium carbonate, 7.5-10 g / L sodium bicarbonate, and 2.5-5 ml / L of preservative Proclin300;

[0014] The protective solution is a complex solution with a concentration of 10-200 μg / ml prepared by dissolving sugar in PBST solution; the sugar is one of dextran, glucose, sucrose, and trehalose;

[0015] Preferably, the PBST solution contains 2-3 g / L disodium hydrogen phosphate dodecahydrate, 0.2-0.5 g / L sodium dihydrogen phosphate, 0.1-0.3 g / L potassium chloride, 6-8 g / L sodium chloride, 0.5-1 ml / L Tween-20, and 0.5-1 ml / L preservative Proclin300.

[0016] The present invention also provides a chip microplate, which integrates the above-mentioned biosensor chip. The chip microplate can be selected from various types of microplates such as 1-hole, 48-hole, and 96-hole.

[0017] The present invention also provides a gene delivery vector detection kit, which includes the above-mentioned biosensor chip, or includes the above-mentioned chip microplate. The kit can be equipped with other different reagents based on different detection methods (label-free detection, sandwich method).

[0018] Preferably, the above detection kit further comprises washing solution, gene delivery vector purification reference, colloidal gold solution, gene delivery vector sample. The gene delivery vector antibody, CAR protein and / or Fx protein can bind to the gene delivery vector at different epitopes.

[0019] The first method of using the above chip microplate or detection kit to detect the concentration of the gene delivery vector in the sample to be tested does not require the use of luminescent reagents such as colloidal gold, but directly inserts the chip microplate into a full-spectrum microplate reader for detection. It has very good sensitivity and specificity for gene delivery vector detection. The specific method is:

[0020] P1. Add 30-50 μL of washing solution to each well of the chip microplate, place it in the microplate reader, record the initial value, and then discard the liquid in the well;

[0021] P2. Use diluent to dilute the purified reference standard of gene delivery vector to obtain different concentrations. Add 30-200 μl of diluted samples with different dilution multiples to each well, shake the plate to react, and discard the liquid in the well;

[0022] P3, add 100-200 μl of washing solution to each well of the chip microplate, wash the plate, tap the plate, and spin dry; after shaking the plate for reaction, put it into the microplate reader, record the endpoint value, and draw a standard curve;

[0023] P4. Take another new chip microplate after the operation in step P1, repeat the operation method in steps P2-P4 to add 30-200 μl of the diluted sample to be tested into each well of the chip microplate, record the endpoint value again, and read it against the standard curve to obtain the concentration of the sample to be tested.

[0024] The second method of using the above chip microplate or detection kit to detect the concentration of the gene delivery vector in the sample to be tested requires first using luminescent reagents such as colloidal gold to bind to CAR protein, Fx protein, adenovirus hexon protein antibody, biotinylated heparin and other compounds that can specifically bind to the gene delivery vector on the chip, and then inserting the chip microplate into a full-spectrum microplate reader for detection. It has very good sensitivity and specificity for gene delivery vector detection. The specific method is:

[0025] Q1. Add 30-50 μL of washing solution to each well of the chip microplate, place it in the microplate reader, record the initial value, and then discard the liquid in the well;

[0026] Q2, add 30-200 μl of purified reference gene delivery vector to each well of the chip microplate, shake the plate for 0.5-1h, and discard the liquid in the well;

[0027] Q3. Add 100-200 μl of washing solution to each well of the chip microplate, wash the plate, tap the plate, and spin dry;

[0028] Q4. Add 30-50 μl of the diluted colloidal gold solution to each well of the chip microplate treated in step P3, shake the plate at 500-700 rpm for 5-15 min, put it into the microplate reader, record the endpoint value, and draw a standard curve;

[0029] Q5. Take another new chip microplate after the operation in step P1, add 30-200 μl of the diluted sample to each well of the chip microplate, repeat the operation method of steps P2-P4, record the endpoint value again, read it against the standard curve, and you can get the concentration of the sample to be tested.

[0030] The samples to be tested in the above two detection methods can be collected from human or animal serum, and can also be used in the production and preparation of reagents such as vaccines or biological preparations using gene delivery vectors as carriers to detect the concentration of gene delivery vectors. Therefore, whether for the purpose of diagnosis or treatment or for non-diagnosis or treatment, the above methods can be used to perform qualitative and quantitative detection of gene delivery vectors with very good sensitivity and specificity.

[0031] The diluent for diluting the purified reference of the gene delivery vector or the sample to be tested can be a common Hanksbuffer, PBS buffer or Tris buffer solution containing 0.5-4% sucrose or glucose, 0.02-2.5% PEG, 0.03-0.1% P300, and a pH value of 5-8.

[0032] Compared with the prior art, the present invention is beneficial in that: based on NanoSPR technology, the present invention provides a biosensor chip that can be used to detect gene delivery vectors, a chip microplate integrating the above biosensor chip, and a corresponding detection kit. The biosensor chip, chip microplate and detection kit provided by the present invention can complete the detection of gene delivery vectors in just one step, with high detection sensitivity, fast detection speed and high throughput, and can be used for high-throughput quantification or screening quality control in the field of gene delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a full spectrum diagram of the detection of 7 concentration gradients of adenovirus purification reference products in Example 1;

[0034] Figure 2 Based on Figure 1 The OD values ​​of the purified adenovirus reference products with 7 concentration gradients were obtained;

[0035] Figure 3 The standard curve obtained in Example 1;

[0036] Figure 4 This is a full spectrum diagram of the detection of 7 concentration gradients of adenovirus purification reference products in Example 2;

[0037] Figure 5 Based on Figure 4 The OD values ​​of the purified adenovirus reference products with 7 concentration gradients were obtained;

[0038] Figure 6 This is a full spectrum diagram of the detection of 7 concentration gradients of adenovirus purification reference products in Example 3;

[0039] Figure 7 Based on Figure 6The OD values ​​of the purified adenovirus reference products with 7 concentration gradients were obtained;

[0040] Figure 8 The standard curve obtained in Example 3;

[0041] Fig. 9 This is a full spectrum diagram of the detection of 7 concentration gradients of adenovirus purification reference products in Example 4;

[0042] Fig.10 Based on Figure 8 The OD values ​​of the purified adenovirus reference products with 7 concentration gradients were obtained;

[0043] Fig.11 The standard curve obtained in Example 4;

[0044] Fig.12 This is the full spectrum of the detection of the exosome purification reference product with 7 concentration gradients in Example 5;

[0045] Fig.13 Based on Fig.10 The OD values ​​of the 7 concentration gradients of exosome purification reference products were obtained;

[0046] Fig.14 The standard curve obtained in Example 5;

[0047] Fig.15 This is the full spectrum of the detection of the exosome purification reference product with 7 concentration gradients in Example 6;

[0048] Fig.16 Based on Fig.12 The OD values ​​of the 7 concentration gradients of exosome purification reference products were obtained; Fig.17 The standard curve obtained in Example 6;

[0049] Fig.18 It is the full spectrum of detection of 7 concentration gradients of adenovirus purification reference in Comparative Example 1;

[0050] Fig.19 Based on Fig.15 The OD values ​​of the purified adenovirus reference products with 7 concentration gradients were obtained;

[0051] Fig. 20 This is the standard curve obtained for Comparative Example 1. DETAILED DESCRIPTION

[0052] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] In the following examples and comparative examples, the Coxsackie virus adenovirus receptor (CAR protein) used was purchased from Beijing Baipusaisi Biotechnology Co., Ltd., FactorX coagulation factor (Fx protein), CD9 antibody, and CD63 antibody were purchased from Beijing Yiqiao Shenzhou Company; bovine serum albumin was purchased from Wuhan Qianzesui Biotechnology Co., Ltd.; the protective solution was prepared by dissolving sucrose in PBST solution at a concentration of 100 μg / ml. The CAR protein and Fx protein solutions used were prepared by diluting the above-mentioned CAR protein and Fx protein with CBS solution to the corresponding concentrations.

[0054] The raw materials and preparation method of PBST solution are: 2.5g / L disodium hydrogen phosphate dodecahydrate, 0.3g / L sodium dihydrogen phosphate, 0.2g / L potassium chloride, 7.5g / L sodium chloride, 0.8ml / L Tween-20, 0.8ml / L preservative Proclin300, add double distilled water and mix well.

[0055] The CBS solution is prepared by mixing 5.5 g / L potassium carbonate, 8.5 g / L sodium bicarbonate, 4 ml / L preservative Proclin 300, and purified water.

[0056] PBST solution was used as washing solution; adenovirus purification reference products were purchased from Shandong Weizhen Biotechnology Co., Ltd.; exosome purification reference products were purchased from Heyuan Biotechnology (Shanghai) Co., Ltd.

[0057] Example 1: One-step label-free detection of adenovirus using CAR protein-coated microplates

[0058] In this embodiment, the preparation method of the chip microplate (96 holes) of the nanoplasmonic resonance biochip is as follows: (1) photolithography is performed on an oxidized silicon wafer to produce a nanopore mold; (2) the nanostructure on the 12-inch silicon wafer nanomold is transferred to a polymer flexible material substrate (PET thin plate substrate) by a nanoimprint method to form an inverse wafer-level nanodevice structure; (3) a titanium film layer (thickness of 15 nm), a silver film layer (thickness of 70 nm), and a gold film layer (thickness of 20 nm) are sequentially plated on its surface to form a 96-hole chip microplate integrated with a plasma sensor chip substrate;

[0059] 1. Modification and preparation of biosensor chips

[0060] (1) Clean the microplate substrate and add 50 μl of 10 μg / ml CAR protein solution to each well; incubate at 37°C for 12 h, and then wash the CAR protein-coated substrate several times with PBST solution;

[0061] (2) Add blocking solution at a volume of 150 μl / well onto the surface of the substrate, incubate in a 37°C oven for 1 h, and wash with PBST solution to remove excess blocking solution;

[0062] (3) Add protective solution at a volume of 150 μl / well, incubate in a 37°C oven for 30 min, wash with PBST solution to remove excess protective solution, and continue to place in a 37°C oven for 10 min to ensure that the chip surface is dry, thereby obtaining a biosensor chip.

[0063] 2. Draw the standard curve by label-free method for standard sample detection

[0064] (1) Use a pipette to add 150 μl of PBST solution to each well of the 96-well microarray plate and wash three times, then add 50 μl of PBST solution, place in a microplate reader, record the initial value, and discard the liquid in the well;

[0065] (2) Dilute the purified adenovirus reference sample of different concentrations with diluent, add 50 μl of the diluted sample to the well, shake the plate at 37° for 30 minutes, and discard the liquid in the well;

[0066] The diluent formula is 1% NaCl, 0.8% sucrose, 2.5% PEG, 0.1% P300, pH = 5.3; the concentration of the adenovirus purification reference after dilution is: 3×10 6 , 6.26×10 6 , 12.5×10 6 , 25×10 6 , 50×10 6 , 100×10 6 , 200×10 6 TCID50 / ml these 7 concentration gradients (4-fold concentration dilution), the dilutions were used as blank controls;

[0067] (3) Add 150 μl of PBST solution to each well of the chip microplate, wash the plate, tap the plate, and spin dry, and repeat the washing three times; at this time, send the above 7 concentration gradient chip microplates (without adding colloidal gold) into the full spectrum microplate reader to obtain the corresponding full spectrum graph and OD value, as shown in Figure 1 , 2 As shown; draw a standard curve, such as Figure 3 The standard curve equation is Y = (AD) / [1 + (X / C) B ]+D; A=0.21105, B=-1.02018, C=12.66572, D=-0.00074, r 2 =0.99938.

[0068] Figure 1 In the graph, the horizontal axis is the wavelength of light and the vertical axis is the absorbance. Figure 2 In the figure, the horizontal axis is the different concentration gradients, and the vertical axis is the OD value. Figure 2 The OD value is Figure 1 The difference between the peak and the trough of the curve of the purified adenovirus reference at each dilution concentration; Figure 3 For the standard curve.

[0069] Example 2: Label-free detection using Fx protein coated chip microplate

[0070] The method and steps of this embodiment are basically the same as those of embodiment 1, the only difference being that the antibody used to modify the biosensor chip is replaced by the Fx protein instead of the CAR protein of embodiment 1; and the dilution concentration of the purified adenovirus reference product is 0.245×10 4 , 0.98×10 4 , 3.9×10 4 , 15.6×10 4 , 62.5×10 4 , 250×10 4 , 1000×10 4 TCID50 / ml, and the diluent was used as blank control.

[0071] The obtained 7 concentration gradient chip microplates (without colloidal gold) were sent to the full spectrum microplate reader to obtain the corresponding full spectrum graphs and OD values ​​as shown in the following figure: Figure 4 , 5 shown.

[0072] Example 3: Sandwich assay using Fx protein coated chip microplate

[0073] The method for modifying and preparing the biosensor chip and the chip microplate (96 wells) of the nanoplasmon resonance biochip in this embodiment is the same as that in Example 1; the only difference is that the antibody for modifying and preparing the biosensor chip is replaced by the Fx protein instead of the CAR protein in Example 1;

[0074] (1) The same as step (1) of Example 1;

[0075] (2) The same as step (2) of Example 1; the dilution concentration is 0.245×10 4 , 0.98×10 4 , 3.9×10 4 , 15.6×10 4 , 62.5×10 4 , 250×10 4 , 1000×10 4 TCID50 / ml these 7 concentration gradients (4-fold concentration dilution), the dilutions were used as blank controls;

[0076] (3) Add 100-200 μl of washing solution to each well of the chip microplate, wash the plate, tap the plate, and spin dry;

[0077] (4) Add 30 μl of diluted colloidal gold solution (25 μl PBST solution to dissolve 5 μl gold particles) to each well of the chip microplate, and shake the plate at 700 rpm for 15 min; at this time, send the above 7 concentration gradient chip microplates (with colloidal gold added) into the well to obtain the corresponding full spectrum and OD value, as shown in Figure 2. Figure 6 , 7 As shown; put the chip microplate into the microplate reader and record the end point value. Draw a standard curve, as shown Figure 8 The standard curve equation is Y = (AD) / [1 + (X / C) B ]+D; A=0.90862, B=-1.69968, C=4867.44126, D=0.41422, r 2 =0.99097.

[0078] Example 4: Sandwich detection using a microplate chip coated with an antibody against adenovirus hexon protein

[0079] The method steps of this embodiment are basically the same as those of embodiment 3, the only difference being that the Fx protein is replaced with an adenovirus hexon protein antibody (Abcam).

[0080] The obtained 7 concentration gradient chip microplates (with colloidal gold added) were sent to the full spectrum microplate reader to obtain the corresponding full spectrum graph and OD value as shown in the figure. Fig. 9 , 10 The obtained standard curve equation is shown as Fig.11 As shown, the standard curve equation is Y = (AD) / [1 + (X / C) B ]+D; A=0.17102, B=-0.48030, C=25.24172, D=-0.00144, r 2 =0.98987.

[0081] Example 5: One-step label-free detection of exosomes using CD9 antibody-coated microplates

[0082] In this embodiment, the preparation method of the chip microplate (96 holes) of the nanoplasmonic resonance biochip is as follows: (1) photolithography is performed on an oxidized silicon wafer to produce a nanopore mold; (2) the nanostructure on the 12-inch silicon wafer nanomold is transferred to a polymer flexible material substrate (PET thin plate substrate) by a nanoimprint method to form an inverse wafer-level nanodevice structure; (3) a titanium film layer (thickness of 15 nm), a silver film layer (thickness of 70 nm), and a gold film layer (thickness of 20 nm) are sequentially plated on its surface to form a 96-hole chip microplate integrated with a plasma sensor chip substrate;

[0083] 1. Modification and preparation of biosensor chips

[0084] (1) Clean the microplate substrate and add 50 μl of 15 μg / ml CD9 antibody solution to each well; incubate at 37°C for 12 h, and then wash the CD9 antibody-coated substrate several times with PBST solution;

[0085] (2) Add blocking solution at a volume of 150 μl / well onto the surface of the substrate, incubate in a 37°C oven for 1 h, and wash with PBST solution to remove excess blocking solution;

[0086] (3) Add protective solution at a volume of 150 μl / well, incubate in a 37°C oven for 30 min, wash with PBST solution to remove excess protective solution, and continue to place in a 37°C oven for 10 min to ensure that the chip surface is dry, thereby obtaining a biosensor chip.

[0087] 2. One-step label-free method to detect exosome purification standard samples and draw standard curve

[0088] Dilute the exosome purification reference with different concentrations using diluent, take 50 μl of the diluted sample and add it to the well, and immediately put it into the microplate reader for kinetic detection, and the detection time is 5 min;

[0089] The diluent formula is PBST solution, 0.5% sucrose, 4% PEG, 0.5% P300, pH = 7.4; the concentration of the exosome purification reference after dilution is: 1.0×10 8 , 0.5×10 8 , 0.25×10 8 , 0.125×10 8 , 0.0625×10 8 , 0.03125×10 8 , 0.015625×10 8 exosomes / mL these 7 concentration gradients (2-fold concentration dilution), the dilutions served as blank controls;

[0090] Obtain the corresponding kinetic diagram and OD value bar graph, such as Fig.12 ,13 As shown; draw the standard curve as Fig.14 As shown, the equation is Y = A + (BA) / [1 + e^ (-a*x + b)]; A = -0.05406, B = 0.07984, a = 7.65925, b = 0.47089, r 2 =0.99905.

[0091] Example 6: Using CD9 antibody to coat the chip microplate and CD63 antibody to label colloidal gold for sandwich detection of exosomes

[0092] The method for modifying and preparing the biosensor chip and the chip microplate (96 wells) of the nanoplasmon resonance biochip in this embodiment is the same as that in Example 5; the only difference is that 5ul of colloidal gold solution labeled with CD63 antibody is added at the same time as the sample for detection;

[0093] Dilute the exosome purification reference with different concentrations with diluent, take 50 μl of diluted sample and add it to the well, add 5 ul of CD63 antibody-labeled colloidal gold solution at the same time, and immediately put it into the microplate reader for kinetic detection, and the detection time is 5 min;

[0094] The diluent formula is PBST solution, 0.5% sucrose, 4% PEG, 0.0.5% P300, pH = 7.4; the concentration of the exosome purification reference after dilution is: 1.0×10 6 , 0.5×10 6 , 0.25×10 6 , 0.125×10 6 , 0.0625×10 6 , 0.03125×10 6 , 0.015625×10 6 exosomes / mL these 7 concentration gradients (2-fold concentration dilution), the dilutions served as blank controls;

[0095] Obtain the corresponding kinetic diagram and OD value bar graph, such as Fig.15 , 16 As shown; draw a standard curve, such as Fig.17 The standard curve equation is Y = (AD) / [1 + (X / C) B ]+D; A=0.29959, B=-1.17405, C=0.12844, D=-0.01748, r 2 =0.99660. The detection sensitivity of the sandwich method of this embodiment can be improved by two orders of magnitude.

[0096] Comparative Example 1: Replace the diluent used to dilute the adenovirus standard and perform sandwich detection

[0097] The method steps of this embodiment are basically the same as those of Example 2, with the only difference being that the diluent formula for diluting the adenovirus standard is replaced with 10 mM Tris, 0.02% PEG20000, and 0.03% P300; and the dilution concentrations of the adenovirus standard are 0.25×100, 1×100, 4×100, 16×100, 64×100, 250×100, and 1000×100 TCID50 / ml.

[0098] Sandwich assay results: The obtained 7 concentration gradient chip microplates were placed in an ELISA reader to obtain the corresponding full spectrum and OD values ​​as shown in Fig.18 , 19 The obtained standard curve equation is shown as Fig. 20 As shown, the standard curve equation is, Y = (AD) / [1 + (X / C) B ]+D; A=0.17102, B=-0.48030, C=25.24172, D=-0.00144, r 2 =0.98987.

[0099] From the test results of Examples 1-5 above, it can be seen that when the chip is coated with CAR protein, Fx protein or adenovirus hexon protein antibody, it has very high accuracy and specificity for adenovirus.

Claims

1. A biosensor chip based on NanoSPR technology, characterized in that: The invention comprises a substrate and a protein, antibody or functional group modified on the substrate; the substrate comprises a base, a titanium film, a silver film and a gold film in order from bottom to top, and the surface of the substrate is imprinted with nanopores arranged in a matrix; the diameter of the nanopores is 50-800nm, the height is 100-800nm, and the spacing between two adjacent nanopores is 200-1000nm; the thickness of the titanium film is 15-30nm, the thickness of the silver film is 70-100nm, and the thickness of the gold film is 2-20nm; the protein, antibody or functional group specifically refers to a protein, antibody or functional group that specifically binds to a gene delivery vector.

2. The biosensor chip based on NanoSPR technology according to claim 1, characterized in that: The thickness of the titanium film is 15 nm, the thickness of the silver film is 70 nm, and the thickness of the gold film is 20 nm.

3. The method for preparing a modified biosensor chip based on NanoSPR technology according to claim 1 or 2, characterized in that: The following steps are involved: S1. Wash the substrate, add 1-50 μl of 5-50 μg / ml CAR protein and / or Fx protein solution; incubate at 4-37° C. for 1.5-24 h, and then wash the substrate coated with CAR protein and / or Fx protein several times with PBST solution; S2, adding a blocking solution to the surface of the substrate obtained in step S1, incubating at 25-37° C. for 1-4 hours, and removing excess blocking solution; S3, dripping protective liquid on the surface of the substrate obtained in step S2, incubating at 25-37° C. for 0.5-1 h, removing excess protective liquid, and drying to obtain a biosensor chip.

4. The method for preparing a modified biosensor chip based on NanoSPR technology according to claim 3, characterized in that: The blocking solution is a complex solution with a concentration of 10 μg / ml prepared by dissolving bovine serum albumin in a CBS solution; the CBS solution contains 4-6 g / L potassium carbonate, 7.5-10 g / L sodium bicarbonate, and 2.5-5 ml / L of preservative Proclin300; The protective solution is a complex solution with a concentration of 10-200 μg / ml prepared by dissolving sugar in PBST solution; the sugar is one of dextran, glucose, sucrose and trehalose.

5. The method for preparing a modified biosensor chip based on NanoSPR technology according to claim 3, characterized in that: The PBST solution contains 2-3 g / L of disodium hydrogen phosphate dodecahydrate, 0.2-0.5 g / L of sodium dihydrogen phosphate, 0.1-0.3 g / L of potassium chloride, 6-8 g / L of sodium chloride, 0.5-1 ml / L of Tween-20, and 0.5-1 ml / L of preservative Proclin300.

6. A chip microplate integrated with the biosensor chip according to claim 1 or 2.

7. A gene delivery vector detection kit, characterized in that: The biosensor chip comprises the biosensor chip according to claim 1 or 2, or the chip microplate according to claim 6.

8. The gene delivery vector detection kit according to claim 7, characterized in that: Also included are washing solution, gene delivery vector purification reference, colloidal gold solution, and gene delivery vector sample.

9. A method for detecting a gene delivery vector not for diagnosis and treatment, characterized in that: The detection is performed using the chip microplate according to claim 6, or using the detection kit of the gene delivery vector according to claim 7 or 8, wherein the specific steps are: P1. Add 30-50 μL of washing solution to each well of the chip microplate, place it in the microplate reader, record the initial value, and then discard the liquid in the well; P2. Use diluent to dilute the purified reference standard of gene delivery vector to obtain different concentrations. Add 30-200 μl of diluted samples with different dilution multiples to each well, shake the plate to react, and discard the liquid in the well; P3, add 100-200 μl of washing solution to each well of the chip microplate, wash the plate, tap the plate, and spin dry; after shaking the plate for reaction, put it into the microplate reader, record the endpoint value, and draw a standard curve; P4. Take another new chip microplate after the operation in step P1, repeat the operation method in steps P2-P4 to add 30~200μl of the diluted sample to be tested into each well of the chip microplate, record the endpoint value again, and read it against the standard curve to obtain the concentration of the sample to be tested.

10. A method for detecting a gene delivery vector not for diagnosis and treatment, characterized in that: The detection is performed using the chip microplate according to claim 6, or using the detection kit of the gene delivery vector according to claim 7 or 8, wherein the specific steps are: Q1. Add 30-50 μL of washing solution to each well of the chip microplate, place it in the microplate reader, record the initial value, and then discard the liquid in the well; Q2. Add 30-200 μl of purified reference gene delivery vector to each well of the chip microplate, shake the plate for 0.5-1 hour, and discard the liquid in the well; Q3. Add 100-200 μl of washing solution to each well of the chip microplate, wash the plate, tap the plate, and spin dry; Q4. Add 30-50 μl of the diluted colloidal gold solution to each well of the chip microplate treated in step Q3, shake the plate at 500-700 rpm for 5-15 min, put it into the microplate reader, record the endpoint value, and draw a standard curve; Q5. Take another new chip microplate after the operation in step Q1, add 30~200μl of the diluted sample to each well of the chip microplate, repeat the operation methods of steps Q2-Q4, record the endpoint value again, and read it against the standard curve to obtain the concentration of the sample to be tested.

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