CN115876853A - Light addressing potential sensor for detecting low-density lipoprotein based on nanocomposite and aptamer - Google Patents
Light addressing potential sensor for detecting low-density lipoprotein based on nanocomposite and aptamer Download PDFInfo
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Abstract
A light-addressable potentiometric sensor based on a nanocomposite-bound aptamer and used for detecting low-density lipoprotein takes an LDL aptamer as an identification probe, and the nanocomposite based on reduced graphene oxide-polyaniline-Hemin (RGO-PANI-Hemin) has good electron transfer effect and excellent load capacity, the LDL aptamer can specifically identify and bind with LDL protein, so that a novel aptamer sensor capable of specifically identifying and quantitatively analyzing the LDL protein is constructed and used for detecting the content of LDL in serum. The method has the advantages of simple operation, time saving and low cost, and the lowest detection limit is 0.8989 mu g/mL.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to an optical addressing potential sensor for detecting low-density lipoprotein based on a nanocomposite material and a suitable ligand.
Background
The detection method of Low Density Lipoprotein (LDL) in serum mainly comprises an ultracentrifugation method, a Friedewald formula calculation method, a chemical precipitation method and the like. The invention patent publication No. CN111647641B relates to a method for quantitatively determining the content of low-density lipoprotein (LDL) based on the change in absorbance value using a kit, which is long-lasting and expensive. The invention patent of publication No. CN101482570 relates to a method for obtaining specific LDL content in serum by taking a heparin-magnesium reagent as a substrate, centrifuging the heparin-magnesium reagent and the serum, reacting, precipitating, and then comparing and measuring absorbance. These methods are complicated, time-consuming and technically demanding, and there is a strong need for a rapid, sensitive, easy-to-operate method for detecting LDL.
Disclosure of Invention
The invention aims to solve the technical problem of providing a nano composite material based on reduced graphene oxide-polyaniline-Hemin RGO-PANI-Hemin, which is combined with a specific recognition molecule LDL aptamer LDL apt The surface of the chip of the light-addressable potentiometric sensor is modified to construct a biosensor which can improve the detection efficiency of LDL and can carry out portable detection.
The detection principle of the invention is as follows: RGO-PANI-Hemin is modified on the surface of the silicon-based LAPS chip by adopting physical action. Among them, RGO has a large specific surface area and strong conductivity, heme can improve the stability of RGO, PANI further improves the conductivity. The RGO-PANI-Hemin nano composite material can enhance the detection signal of the LAPS sensor, thereby improving the sensitivity of the sensor. LDL binding by non-covalent binding and intermolecular forces apt The aptamer is loaded on the surface of the RGO-PANI-Hemin material, is connected with the composite material in a single-chain form due to the unstable space structure of the aptamer, and can be further modified on a LAPS chip. After LDL is added to the biosensing interface, the LDL apt Can be specifically combined with LDL protein to form a protein-aptamer complex which is in a stable spatial structure and is orderly arranged on the surface of the modified LAPS chip, and the change of the surface potential of the LAPS chip is caused. Is not limited toThe change of surface potential caused by LDL of the same concentration causes a certain shift of an I-V curve, and the detection of LDL of different concentrations is realized by monitoring the shift of the I-V curve. Compared with the existing method, the method has the advantages of relatively simple operation and high sensitivity, realizes portable detection, and can reach the detection limit of 0.8989 mu g/mL.
The invention is carried out according to the following steps:
step 1: preparation of RGO-PANI-Hemin and Au NPs composite material
(1) Preparation of Reduced Graphene Oxide (RGO)
Weighing single-layer Graphene Oxide (GO), adding ultrapure water, and crushing by using an ultrasonic crusher to disperse uniformly; ascorbic Acid (AA) was then added and stirred on a thermostated magnetic stirrer to give an RGO solution.
(2) Preparation of reduced graphene oxide-heme (RGO-Hemin)
Preparing hemin solution, mixing hemin solution and RGO solution at equal ratio, adding hydrazine hydrate (N) 2 H 4 ·H 2 O), and placing the mixture in a constant-temperature water bath kettle for heating and stirring for a period of time in a water bath, and then centrifuging the mixture to remove the supernatant to obtain RGO-Hemin.
(3) Preparation of reduced graphene oxide-polyaniline-heme (RGO-PANI-Hemin)
Adding Polyaniline (PANI) solution into RGO-Hemin, mixing uniformly, then adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) solution, stirring, centrifugally washing, dissolving in ultrapure water again, and freeze-drying to obtain the RGO-PANI-Hemin nanocomposite.
(4) Preparation of Nanogold (Au NPs) solution
The chloroauric acid solution is put into a beaker and heated and stirred until boiling. Then, sodium citrate (C) 6 H 5 Na 3 O 7 ) The solution was slowly added to the chloroauric acid solution. And continuously stirring until the solution turns from slight yellow to wine red, and naturally cooling to room temperature to obtain the Au NPs solution.
And 2, step: modification of sensitive units of LAPS sensors
(1) Pretreatment of LAPS chips
And (2) taking a cleaned silicon-based LAPS chip, dropwise adding a sodium hydroxide (NaOH) solution on the surface of the silicon-based LAPS chip for activation, then dropwise adding a Mercaptopropyltriethoxysilane (MPTES) solution, standing, performing silanization on the silicon chip, and naturally drying to obtain the silanized LAPS chip.
(2) Modification of LAPS chip by RGO-PANI-Hemin/Au NPs nano material
And (3) dripping AuNPs solution on the silicon-based LAPS chip after silanization treatment, continuously dripping RGO-PANI-Hemin nano composite material after surface drying, standing and drying to obtain the RGO-PANI-Hemin/Au NPs sensor sensitive membrane.
(3)LDL apt Construction of/RGO-PANI-Hemin/Au NPs/LAPS sensitive unit
Treating LDL apt Dropping the solution on the LAPS silicon wafer, placing in an incubator, and incubating to obtain the product with LDL apt LAPS sensitive unit interface of/RGO-PANI-Hemin/Au NPs/LAPS.
And step 3: working Curve plotting of LDL
(1) And (3) dropwise adding the standard LDL solution to the sensitive unit interface of the LAPS chip obtained in the step (2), and incubating to prepare the LAPS working electrode.
(2) And (3) putting the prepared LAPS working electrode into a LAPS detection system, adding Phosphate Buffer Solution (PBS) supporting liquid and a reference electrode into a detection pool, detecting by adopting the LAPS system, and recording an I-V curve of the LAPS working electrode. The I-V curve was normalized and compared to a blank sample to calculate the voltage offset.
(3) LDL of different concentrations was detected, and the lowest detection limit of this method was calculated by plotting an operation curve with the LDL concentration as the abscissa and the voltage shift amount as the ordinate.
And 4, step 4: detection of LDL in actual sample
And (3) dripping an actual sample to be tested on the LAPS sensitive unit interface obtained in the step (2), and incubating to obtain the working electrode. And (3) placing the LAPS working electrode into a LAPS detection system, adding a PBS buffer solution and a reference electrode into the detection cell, detecting by adopting the LAPS system, and recording an I-V curve of the detection cell. The I-V curve was normalized and compared with a blank sample to determine the voltage offset.
And (4) obtaining the concentration of LDL in the actual sample to be measured according to the voltage deviation value by using the working curve obtained in the step (3).
Further, in the step 1, GO is 30mg, and AA is 300mg.
Further, the hemin solution in step 1 was 1.0mg/mL.
Further, the mass fraction of the hydrazine hydrate solution in the step 1 is 80%.
Further, the PANI concentration in the step 1 is 1.0mg/mL.
Further, the volume ratio of the EDC/NHS solution in the step 1 is 4.
Further, in the step 1, the concentration of the chloroauric acid is 0.01%, and the concentration of the sodium citrate is 1%.
Further, the concentration of NaOH in the step 2 is 1.0mol/L.
Preferably, the amount of Au NPs used in step 2 is 25. Mu.L.
Preferably, the dosage of rGO-PANI-Hemin in the step 2 is 25 mu L.
Preferably, the LDLapt concentration in step 2 is 1.0. Mu. Mol/L.
Preferably, the concentration of PBS in step 2 and step 3 is 0.2mol/L and the pH value is 7.4.
Preferably, the incubation temperature in step 2 and step 3 is 25 ℃ and the incubation time is 1h.
Wherein, the step 1 provides the step 2 with a high-conductivity nano composite material RGO-PANI-Hemin to cause the sensing interface to respond quickly. Step 2 constitutes a biosensing interface that specifically recognizes LDL and contributes to the efficiency of electron transfer. The construction of biosensing interface in step 2 is an essential key step in the detection of LDL by the LAPS sensor in step 3 and step 4. The working curve for LDL in step 3 provides a basis for calculation for measuring the LDL concentration in the actual sample in step 4. It can be seen that the steps 1-4 support each other and act together to achieve the detection of LDL using the RGO-PANI-Hemin composite and the LDL aptamer as recognition probes.
Compared with the prior art, the invention has the following advantages:
1. the method successfully prepares the RGO-PANI-Hemin composite material with good biocompatibility and strong conductivity by utilizing the property of enhancing the electron transfer effect of polyaniline particles and the effective current signal amplification effect and excellent load capacity of reduced graphene oxide. And forming a biological sensitive membrane on a silicon-based LAPS chip by using the RGO-PANI-Hemin composite material in combination with LDL apt The probe forms a new photoaddressable potentiometric sensor that specifically detects LDL.
2. The existing methods for detecting LDL at home and abroad have the problems of complicated operation or the need of professional personnel for operation and the like, and the light-addressable potential sensor modified by the RGO-PANI-Hemin nano composite material has the advantages of simple and convenient operation, high precision and realization of portable detection. The lowest detection limit is 0.9898 mu g/mL, and the relative error is between 0.32 and 6.79 percent when the serum sample with the actual known LDL level is subjected to LDL detection by using a direct measurement method.
Drawings
FIG. 1 is a schematic diagram of the detection of LDL by an RGO-PANI-Hemin based photo-addressable potentiometric sensor;
FIG. 2 TEM images of RGO-Hemin (A) and RGO-PANI-Hemin (B);
FIG. 3 is a Scanning Electron Microscope (SEM) image of various stages in the construction of a LAPS chip; wherein: (A) bare LAPS chip, (B) MPTES-LAPS chip, (C) AuNPs/LAPS chip, (D) RGO-PANI-Hemin/Au NPs/LAPS chip, (E) LDL apt /RGO-PANI-Hemin/Au NPs/LAPS chip, (F) LDL/LDL apt the/RGO-PANI-Hemin/Au NPs/LAPS chip.
FIG. 4 is a graph of the operation of an RGO-PANI-Hemin-based photo-addressable potentiometric sensor for detecting different LDL concentrations.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
A potential sensor for detecting LDL is constructed based on RGO-PANI-Hemin nano composite material, and the detection principle is shown in figure 1. Firstly, naOH is dripped on the surface of a silicon-based LAPS chip for activation, and then Mercaptopropyltriethoxysilane (MPTES) is added to form a mercapto silanized LAPS chip. Au NPs are fixed on the surface of the sulfydryl silanized LAPS chip through Au-S action. RGO-PANI-Hemin was added to the AuNPs modified LAPS chip surface. Incubating the LDL aptamer to enable the LDL aptamer to be covalently bonded with RGO-PANI-Hemin on the surface of the electrode, and connecting the LDL aptamer with the composite material in a single-chain form due to a relatively unstable spatial structure of the LDL aptamer, so that the LDL aptamer can be fixed on the surface of the modified LAPS chip. After LDL is added, the LDL aptamer can be specifically combined with LDL protein to form a protein-aptamer complex to form a stable spatial structure, so that the protein-aptamer complex is orderly arranged on the surface of the electrode. The change of the electrochemical signal of the sensor is detected by the LAPS system, so that the quantitative analysis of the LDL protein can be effectively realized.
The implementation steps are as follows:
1: preparation of RGO-PANI-Hemin/Au NPs composite material
(1) 30mg of GO is added into 30mL of pure water, the mixture is crushed for 1h by an ultrasonic crusher to be uniformly dispersed, and then 300mg of ascorbic acid is added and stirred for 12h under a constant-temperature magnetic stirrer to obtain 1.0mg/mL of RGO solution. Putting 20mg of hemin into a beaker, adding 10 mu L of ammonia water and 20mL of pure water, stirring to dissolve the hemin uniformly, and standing in a refrigerator overnight to obtain a 1.0mg/mL hemin solution. 10mL of hemin solution and 5.0mL of LRGO solution are mixed, 10 mu L of hydrazine hydrate is added, and the mixture reacts for 4 hours in a water bath at 60 ℃. Centrifuging the reaction solution at 10000r/min for 5min, and removing the supernatant to obtain RGO-Hemin solution. Pouring 10mL of the solution of LRGO-Hemin into a beaker, adding 10mL of 1.0mg/mL of PANI solution, and mixing uniformly. That is, EDC/NHS buffer (volume ratio 4, 1, concentration 1M) was added later, and the reaction was stirred under a constant temperature magnetic stirrer for 3h. Then centrifuging for 3 times (9000 r/min,10 min), removing supernatant, and freeze drying subnatant to obtain RGO-PANI-Hemin composite material.
The RGO-PANI-Hemin nano material was characterized by JEM-1200EX transmission electron microscope, as shown in FIG. 2. FIG. 2A is a TEM image of RGO-Hemin, which is relatively smooth and wrinkled. FIG. 2B is a TEM image of RGO-PANI-Hemin with many particles on the rugate surface, indicating successful binding of PANI to RGO-Hemin.
(2) 50ml of a 0.01% chloroauric acid solution are taken in a clean beaker and the water bath is continuously heated and stirred to a temperature of 100 ℃. Then 1.5mL of a 0.1% sodium citrate solution was slowly added to the chloroauric acid solution. Stirring was continued at 100 ℃ until the solution changed from pale yellow to wine red. Naturally cooling to room temperature to obtain Au NPs solution, and refrigerating at 4 ℃ for later use.
2: modification of sensitive units of LAPS sensors
(1) First, the silicon wafer is placed in a solution (H) 2 O 2 And rich H 2 SO 4 Soaking for 10min in a volume ratio of 3).
(2) Dripping 5 mu L NaOH solution (1.0 mol/L) on the working surface of the chip, cleaning after 30min, then leading the surface of the LAPS chip to be subjected to MPTES sulfydryl silylation, terminating the surface with-SH groups to obtain a sulfydryl silylated LAPS chip (MPTES-LAPS chip), placing the chip in a refrigerator at 4 ℃ for 12 hours, and cleaning with pure water for three times.
(3) And (3) dripping 20 mu LAuNPs solution into the MPTES-LAPS chip, continuously dripping 20 mu L of 1.0 mg/mLRGO-PANI-Hemin solution after the surface is completely dried, and incubating in a constant temperature incubator (25 ℃) for 1h and then cleaning to obtain the RGO-PANI-Hemin/AuNPs/LAPS chip.
(4) 5.0. Mu.L of 1.0. Mu.M LDL was dropped on the above chip apt Incubating the solution in a shaking incubator (25 deg.C) for 1h, and naturally drying to obtain LDL apt the/RGO-PANI-Hemin/AuNPs/LAPS sensitive unit interface.
FIG. 3 is a Scanning Electron Microscope (SEM) image of each stage of the LAPS chip. In the graph a, a bare chip SEM image shows that the surface is smooth and flat. Panel B is an SEM image of an MPTES-LAPS chip with a significant surface coating of particulate material. Panel C is an SEM image of an AuNPs/LAPS chip, and obvious luminescent particles can be seen, which indicates that AuNPs particles are successfully modified on the chip through Au-S bonds. Panel D is an SEM image of the RGO-PANI-Hemin/AuNPs/LAPS chip, where some membranous material was clearly visible in the gaps between the granular AuNPs particles, indicating that the RGO-PANI-Hemin nanocomposite had been immobilized on the LAPS chip. FIG. E is LDL apt SE of/RGO-PANI-Hemin/AuNPs/LAPS chipM plot, the modifying substance on the chip surface became much thicker compared to plot D, because of LDL apt Is a disordered single-stranded DNA.
3: working Curve plotting of LDL
(1) Dripping 2.0 μ L LDL solution on the LAPS chip sensitive unit interface constructed in step 2, incubating for 30min at 25 deg.C to obtain LDL/LDL apt the/RGO-PANI-Hemin/AuNPs/LAPS chip (LAPS working electrode).
FIG. 3F is LDL/LDL apt SEM image of/RGO-PANI-Hemin/AuNPs/LAPS chip. In contrast to FIG. 3E, a new layer of plate-like material was coated on the chip surface due to LDL apt And the specific combination with GPC3 is carried out to cover the surface of the chip, thus forming a LAPS working electrode.
(2) And (3) putting the prepared LAPS working electrode into a LAPS detection system, adding a phosphate buffer solution (PBS, pH 7.4) buffer solution and a reference electrode into a detection pool, detecting by adopting the LAPS system, and recording an I-V curve of the LAPS working electrode. The I-V plot of different LDL concentrations is shown in FIG. 4, with the I-V curve shifting to the right by a greater magnitude as the LDL concentration increases. The I-V curve was normalized and compared to a blank sample to calculate the voltage offset. When the LDL concentration is in the range of 1.0-100.0 mug/mL, the relationship between the voltage offset (Y) and the LDL concentration (X) of the sensor is linear, Y =4.17911X +128.67823 (wherein Y represents the difference between the voltage offset at the concentration and the voltage offset of an LDL blank group, and X represents the concentration of LDL), and the correlation coefficient R 2 =0.97826. LOD =3S according to the calculation formula b /b(S b Representing the standard deviation obtained for the blank control in the experiment and b representing the slope of the standard curve), a minimum detection limit of 0.8989 μ g/mL was calculated.
4: detection of LDL in actual sample
(1) LDL serum samples at 2 different concentrations (19.72. Mu.g/mL, 80.43. Mu.g/mL) were measured by direct methods. The collection and treatment of the serum sample all meet the requirements of ethical committees of key laboratories in the research of metabolic diseases in Guangxi province. LDL obtained in step 2 apt The interface of/RGO-PANI-Hemin/AuNPs/LAPS sensitive unit is dropped with 2.0 mu L serum sample containing LDL and incubated at the temperature of 25 DEG CAnd culturing for 30min to obtain LAPS working electrode.
(2) And (3) putting the prepared LAPS working electrode into a LAPS detection system, adding a phosphate buffer solution (PBS, pH 7.4) buffer solution and a reference electrode into a detection pool, detecting by adopting the LAPS system, and recording an I-V curve of the LAPS working electrode. The I-V curve was normalized and compared to a blank sample to calculate the voltage offset.
(3) The actual concentration of LDL in the serum to be measured was obtained from the voltage deviation value based on the operation curve obtained in step 3, and the results are shown in Table 1. The relative error of the detection of the serum samples by the LAPS sensor by the direct measurement method is between 0.32 and 6.79 percent, and the relative standard deviation values are 0.54 percent and 1.14 percent. These results indicate that the developed LDL sensor is expected to have a good application prospect in medical diagnosis.
TABLE 1 results of LDL detection in actual serum samples
Claims (7)
1. An optical addressing potential sensor for detecting low-density lipoprotein based on a nano composite material combined with an aptamer comprises the following steps:
step 1: preparation of reduced graphene oxide-polyaniline-heme RGO-PANI-Hemin and nanogold AuNPs composite material
(1) Preparation of Reduced Graphene Oxide (RGO)
Weighing single-layer graphene oxide GO, adding ultrapure water, and crushing with an ultrasonic crusher to disperse uniformly; adding ascorbic acid AA, and stirring on a constant-temperature magnetic stirrer to obtain an RGO solution;
(2) Preparation of reducing graphene oxide-heme RGO-Hemin
Preparing hemin solution, mixing the hemin solution and RGO solution at equal mass ratio, adding hydrazine hydrate N 2 H 4 ·H 2 O, placing the mixture in a constant-temperature water bath kettle for water bath heating and stirring; centrifuging, removing supernatant to obtainTo RGO-Hemin;
(3) Preparation of reduced graphene oxide-polyaniline-heme RGO-PANI-Hemin
Adding polyaniline PANI solution into RGO-Hemin solution, mixing well, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide EDC/NHS solution, stirring; centrifuging, washing, dissolving in ultrapure water, and freeze drying to obtain RGO-PANI-Hemin composite material;
(4) Preparation of nano-gold AuNPs solution
Heating and stirring the chloroauric acid solution until the chloroauric acid solution is boiled; adding the sodium citrate solution into the chloroauric acid solution, and continuing stirring until the solution turns from slight yellow to wine red; cooling to obtain AuNPs solution;
step 2: modification of sensitive units of LAPS sensors
(1) Pretreatment of LAPS chips
Taking a cleaned silicon-based LAPS chip, dropwise adding a sodium hydroxide NaOH solution on the surface of the silicon-based LAPS chip for activation, then dropwise adding a Mercaptopropyltriethoxysilane (MPTES) solution, and standing; performing silanization treatment on the silicon chip, and naturally drying to obtain a silanized LAPS chip;
(2) Modification of LAPS chip by RGO-PANI-Hemin/AuNPs nano material
Dripping AuNPs solution on the silanized silicon-based LAPS chip, drying the surface, dripping RGO-PANI-Hemin nano composite material, standing and drying to obtain RGO-PANI-Hemin/AuNPs sensor sensitive membrane;
(3)LDL apt construction of/RGO-PANI-Hemin/AuNPs/LAPS sensitive unit
Bringing LDL into contact with apt Dropping the solution on the RGO-PANI-Hemin/AuNPs sensor sensitive membrane, and incubating to obtain the sensor with LDL apt LAPS sensitive unit interface of/RGO-PANI-Hemin/AuNPs/LAPS;
and 3, step 3: working Curve plotting of LDL
(1) Dropwise adding the standard LDL solution to the sensitive unit interface of the LAPS chip obtained in the step (2), and incubating to prepare a LAPS working electrode;
(2) Putting the prepared LAPS working electrode into a LAPS detection system, adding phosphate buffer solution PBS buffer solution and a reference electrode into a detection pool, detecting by adopting the LAPS system, and recording an I-V curve of the detection pool; normalizing the I-V curve, comparing with a blank sample, and calculating the voltage offset;
(3) Respectively detecting LDL (low-density lipoprotein) with different concentrations, drawing a working curve by taking the concentration of the LDL as an abscissa and the voltage offset as an ordinate, and calculating the lowest detection limit of the method;
and 4, step 4: detection of LDL in actual sample
Dripping the actual sample to be tested on the LAPS sensitive unit interface obtained in the step 2, and incubating to obtain a working electrode; putting the LAPS working electrode into a LAPS detection system, adding a PBS buffer solution and a reference electrode into the detection pool, detecting by adopting the LAPS system, and recording an I-V curve of the detection pool; normalizing the I-V curve, and comparing with a blank sample to obtain the voltage offset;
and (4) obtaining the concentration of LDL in the actual sample to be measured according to the voltage deviation value by using the working curve obtained in the step (3).
2. The sensor of claim 1, wherein: the Hemin solution in the step 1 is 1.0mg/mL; the RGO solution is 1.0mg/mL; the mass fraction of the hydrazine hydrate solution is 80%; the PANI concentration is 1.0mg/mL; the volume ratio of the EDC/NHS solution is 4, and the concentration is 10mol/L.
3. The sensor of claim 1, wherein: the concentration of NaOH in the step 2 is 1.0mol/L; the concentration of MPTES is 1%; the AuNPs are used in an amount of 20. Mu.L.
4. The sensor of claim 1, wherein: the RGO-PANI-Hemin in step 2 is used in an amount of 20. Mu.L, and the concentration is 1.0mg/mL.
5. The sensor of claim 1, wherein: the LDL in step 2 apt The concentration was 1. Mu.M, and the amount was 5.0. Mu.L.
6. The sensor of claim 1, wherein: the PBS buffer solution in step 3 and step 4 has a pH of 7.4 and a concentration of 0.2mol/L.
7. The sensor of claim 1, wherein: the incubation temperature in step 3 and step 4 is 25 ℃, and the incubation time is 1h.
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