CN115876853B - Light addressing potential sensor for detecting low density lipoprotein based on nano composite material junction proper ligand - Google Patents
Light addressing potential sensor for detecting low density lipoprotein based on nano composite material junction proper ligand Download PDFInfo
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Abstract
An optical addressing potential sensor for detecting low density lipoprotein based on a nanocomposite binding proper ligand uses an LDL aptamer as a recognition probe, and the nanocomposite based on reduced graphene oxide-polyaniline-chlorhexidine (RGO-PANI-Hemin) has good electron transfer effect and excellent loading capacity, so that the LDL aptamer can specifically recognize and bind with LDL protein, and a novel aptamer sensor capable of specifically recognizing and quantitatively analyzing 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 minimum detection limit is 0.8989 mug/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 binding proper ligand.
Background
The method for detecting the low density lipoprotein (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 of publication No. CN111647641B relates to a method for quantitatively determining the content of low-density lipoprotein (LDL) according to the change of absorbance value by using a kit, which has long duration and high cost. The invention patent of publication No. CN101482570 relates to a method for obtaining the specific LDL content in serum by comparing absorbance after reaction precipitation after centrifugation of heparin-magnesium reagent serving as a substrate and serum, wherein the method requires a plurality of reagents which are expensive and not easy to obtain. 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 technical problem to be solved by the invention is to provide a nano composite material based on reduced graphene oxide-polyaniline-chlorhexidine RGO-PANI-Hemin, and a specific recognition molecule LDL aptamer LDL apt is combined to modify the surface of an optical addressing potential sensor chip, so that a biosensor for improving the detection efficiency of LDL and portable detection is constructed.
The detection principle of the invention is as follows: RGO-PANI-Hemin is modified on the surface of a silicon-based LAPS chip by adopting physical action. Wherein RGO has large specific surface area and strong conductivity, heme can improve the stability of RGO, and PANI further improves the conductivity. The RGO-PANI-Hemin nanocomposite can enhance the detection signal of the LAPS sensor, thereby improving the sensitivity of the sensor. LDL apt is loaded on the surface of RGO-PANI-Hemin material through non-covalent binding and intermolecular acting force, and the aptamer is connected with the composite material in a single-chain form due to a relatively unstable space structure, so that the aptamer can be modified on a LAPS chip. After addition of LDL at the biosensing interface, LDL apt will specifically bind to LDL protein, form a protein-aptamer complex and take on a stable spatial structure, which is orderly arranged on the surface of the modified LAPS chip, which will cause a change in the surface potential of the LAPS chip. The surface potential changes caused by the LDL with different concentrations are different, so that the I-V curve is offset to a certain extent, and the offset of the I-V curve is monitored, so that the LDL with different concentrations is detected. 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 mug/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, crushing by using an ultrasonic crusher, and uniformly dispersing; ascorbic Acid (AA) was then added and stirred on a constant temperature magnetic stirrer to give an RGO solution.
(2) Preparation of reduced graphene oxide-heme (RGO-Hemin)
Preparing a Hemin solution, mixing the Hemin solution and the RGO solution in equal proportion, adding hydrazine hydrate (N 2H4·H2 O), placing in a constant-temperature water bath, heating and stirring for a period of time, centrifuging, and removing 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, redissolving in ultrapure water, and freeze-drying to obtain the RGO-PANI-Hemin nanocomposite.
(4) Preparation of gold nanoparticles (Au NPs) solution
The chloroauric acid solution is placed in a beaker and heated and stirred until boiling. Then, the sodium citrate (C 6H5Na3O7) solution was slowly added to the chloroauric acid solution. Stirring is continued until the solution turns from yellowish to reddish wine, and the temperature is naturally reduced to room temperature, thus obtaining the Au NPs solution.
Step 2: modification of LAPS sensor sensing unit
(1) Pretreatment of LAPS chip
And (3) taking a cleaned silicon-based LAPS chip, dropwise adding a sodium hydroxide (NaOH) solution on the surface of the cleaned silicon-based LAPS chip for activation, then dropwise adding a mercaptopropyl triethoxysilane (MPTES) solution, standing, performing silanization treatment 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) dropwise adding an AuNPs solution to the silanized silicon-based LAPS chip, continuously dropwise adding the RGO-PANI-Hemin nanocomposite after surface drying, standing and drying to obtain the RGO-PANI-Hemin/Au NPs sensor sensitive film.
(3) Construction of LDL apt/RGO-PANI-Hemin/Au NPs/LAPS sensitive units
And (3) dripping the LDL apt solution on the LAPS silicon wafer, and placing the LAPS silicon wafer into an incubator for incubation to obtain the LAPS sensitive unit interface with LDL apt/RGO-PANI-Hemin/Au NPs/LAPS.
Step 3: plotting of working curves of LDL
(1) And (3) dropwise adding the standard LDL solution to the LAPS chip sensitive unit interface obtained in the step (2), and incubating to prepare the LAPS working electrode.
(2) And (3) placing the prepared LAPS working electrode into a LAPS detection system, adding Phosphate Buffer Solution (PBS) supporting 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 is normalized, and a blank sample is used as a reference to calculate the voltage offset.
(3) LDL of different concentrations is detected respectively, the concentration of LDL is taken as an abscissa, the voltage offset is taken as an ordinate, a working curve is drawn, and the lowest detection limit of the method is calculated.
Step 4: detection of LDL in actual sample
And (3) dropwise adding an actual sample to be detected into the LAPS sensitive unit interface obtained in the step (2), and incubating to obtain a working electrode. The LAPS working electrode is put into a LAPS detection system, PBS buffer solution and a reference electrode are added into a detection pool, the LAPS system is adopted for detection, and the I-V curve of the LAPS working electrode is recorded. And normalizing the I-V curve, and comparing blank samples to obtain the voltage offset.
And (3) obtaining the concentration of the LDL in the actual sample to be detected according to the voltage offset 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 chlorhexidine solution in step 1 is 1.0mg/mL.
Further, the mass fraction of the hydrazine hydrate solution in the step 1 is 80%.
Further, the PANI concentration in the step1 is 1.0mg/mL.
Further, the EDC/NHS solution volume ratio in step 1 was 4:1, with a concentration of 10M each.
Further, the chloroauric acid concentration in the step 1 is 0.01%, and the sodium citrate concentration is 1%.
Further, the concentration of NaOH in the step 2 is 1.0mol/L.
Preferably, the Au NPs are used in an amount of 25. Mu.L in step 2.
Preferably, the rGO-PANI-Hemin is used in step 2 in an amount of 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 is 7.4.
Preferably, the incubation temperature in step 2 and step 3 is 25℃and the incubation time is 1h.
Step 1 provides a nanocomposite RGO-PANI-Hemin with high conductivity to the step 2 to cause a rapid response of the sensing interface. Step 2 constitutes a biosensing interface specifically recognizing LDL and facilitates electron transfer efficiency. The construction of the biosensing interface in step 2 is an essential key step in step 3 and step 4 for the LAPS sensor to detect LDL. The working curve of LDL in step 3 provides a basis for the determination of LDL concentration in the actual sample in step 4. It can be seen that steps 1-4 support each other and act together to realize detection of LDL by using RGO-PANI-Hemin composite material and LDL aptamer as recognition probes.
Compared with the prior art, the invention has the following advantages:
1. The method utilizes the property of polyaniline particles for enhancing electron transfer effect, and the effective current signal amplifying effect and excellent loading capacity of the reduced graphene oxide to successfully prepare the RGO-PANI-Hemin composite material with good biocompatibility and strong electric conductivity. And RGO-PANI-Hemin composite material is formed into a biological sensitive film on a silicon-based LAPS chip, and matched with an LDL apt probe, a novel optical addressing potential sensor for specifically detecting LDL is formed.
2. The existing methods for detecting LDL at home and abroad have the problems of complex operation or need of professional operation, etc., and the optical addressing potential sensor modified by RGO-PANI-Hemin nano composite material has simple and convenient operation and high precision, thereby realizing portable detection. The lowest limit of detection is 0.9898 mug/mL, and the serum sample with the actually known LDL level is subjected to LDL detection by using a direct measurement method, and the relative error is between 0.32 and 6.79 percent.
Drawings
FIG. 1 is a schematic diagram of an RGO-PANI-Hemin based optical addressing potentiometric sensor for detecting LDL;
FIG. 2RGO-Hemin (A) and RGO-PANI-Hemin (B) transmission electron microscopy images;
FIG. 3 is a Scanning Electron Microscope (SEM) image of various stages of 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/RGO-PANI-Hemin/Au NPs/LAPS chip.
FIG. 4 is a graph of the operation of an RGO-PANI-Hemin based optical addressing potentiometric sensor to detect different LDL concentrations.
Detailed Description
The invention will be described in detail below with reference to the drawings and the detailed description.
An electric 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 mercaptopropyl triethoxysilane (MPTES) is added to form the mercapto silanized LAPS chip. Au NPs are fixed on the surface of the mercapto-silanized LAPS chip through Au-S action. RGO-PANI-Hemin is added to the surface of the AuNPs modified LAPS chip. And incubating the LDL aptamer, so that the LDL aptamer can be covalently bound with RGO-PANI-Hemin on the surface of the electrode, and is connected with the composite material in a single-chain form due to a relatively unstable space structure, so that the LDL aptamer can be fixed on the surface of the modified LAPS chip. After addition of LDL, LDL aptamer specifically binds to LDL protein, forms a protein-aptamer complex and takes a stable spatial structure, thereby being orderly arranged on the surface of an electrode. The quantitative analysis of LDL proteins can be effectively realized by detecting the change of the electrochemical signal of the sensor through the LAPS system.
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, crushed for 1 hour by an ultrasonic crusher, uniformly dispersed, and then 300mg of ascorbic acid is added and stirred for 12 hours under a constant temperature magnetic stirrer, thus obtaining 1.0mg/mL of RGO solution. 20mg of hemin is taken and put into a beaker, 10 mu L of ammonia water and 20mL of pure water are added, the solution is stirred to be uniformly dissolved, and the solution is put into a refrigerator for standing overnight to obtain 1.0mg/mL of hemin solution. 10mL of a hemin solution and 5.0mLRGO were mixed, 10. Mu.L of hydrazine hydrate was added, and the mixture was reacted in a water bath at 60℃for 4 hours. Centrifuging the reaction solution at 10000r/min for 5 min, and removing supernatant to obtain RGO-Hemin solution. 10mLRGO-Hemin solution was poured into a beaker, 10mL of 1.0mg/mL PANI solution was added and mixed well. 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 3 hours. And centrifuging for 3 times (9000 r/min,10 min), removing the supernatant, and freeze-drying the lower layer liquid to obtain the RGO-PANI-Hemin composite material.
RGO-PANI-Hemin nanomaterials were characterized using a JEM-1200EX transmission electron microscope as shown in FIG. 2. FIG. 2A is a TEM image of RGO-Hemin with a relatively smooth surface and a wrinkled appearance. FIG. 2B is a TEM image of RGO-PANI-Hemin with a pleated surface having a plurality of particles, indicating that PANI is successfully bound to RGO-Hemin.
(2) 50Ml of 0.01% chloroauric acid solution was taken in a clean beaker and stirred in a water bath with continued heating to 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 reddish-wine. Naturally cooling to room temperature to obtain Au NPs solution, and refrigerating at 4 ℃ for standby.
2: Modification of LAPS sensor sensing unit
(1) Firstly, placing a silicon wafer in a solution (H 2O2 and concentrated H 2SO4 in a volume ratio of 3:7) for soaking for 10min, then placing the silicon wafer in ethanol, acetone and pure water in sequence, ultrasonically cleaning for 5min in an ultrasonic cleaning machine, and finally, standing for 30min and then cleaning with pure water.
(2) 5 Mu LNaOH solution (1.0 mol/L) is dripped on the working surface of the chip, the chip is cleaned after 30min, then the surface of the LAPS chip is subjected to MPTES sulfhydrylation and silanization, the surface is terminated by-SH groups, and the sulfhydrylation silanization LAPS chip (MPTES-LAPS chip) is obtained, and the chip is placed in a refrigerator at 4 ℃ for 12 hours and is cleaned with pure water for three times.
(3) And (3) dropwise adding 20 mu LAuNPs of solution into the MPTES-LAPS chip, continuously dropwise adding 20 mu L of 1.0 mg/mLRGO-PANI-Hemin solution after the surface is completely dried, and cleaning after incubating for 1h in a constant temperature incubator (25 ℃) to obtain the RGO-PANI-Hemin/AuNPs/LAPS chip.
(4) And (3) dropwise adding 5.0 mu L of 1.0 mu M LDL apt solution on the chip, placing the chip in a shake incubator (25 ℃) for incubation for 1h, and naturally airing the chip to obtain the LDL apt/RGO-PANI-Hemin/AuNPs/LAPS sensitive unit interface.
Fig. 3 is a Scanning Electron Microscope (SEM) image of various stages of the LAPS chip. Wherein, the graph A is a bare chip SEM graph, and the smooth and even surface can be seen. FIG. B is an SEM image of an MPTES-LAPS chip, with a surface significantly covered with a layer of particulate material. Panel C is an SEM image of an AuNPs/LAPS chip, and obvious luminescent particles can be seen, indicating that AuNPs particles were successfully modified on the chip by Au-S bonds. Panel D is an SEM image of RGO-PANI-Hemin/AuNPs/LAPS chips, some membranous material can be seen clearly in the granular AuNPs particle gap, which indicates that RGO-PANI-Hemin nanocomposite has been immobilized on LAPS chips. FIG. E is an SEM image of an LDL apt/RGO-PANI-Hemin/AuNPs/LAPS chip, the modified substance on the chip surface became much thicker compared to FIG. D, since LDL apt is a disordered single-stranded DNA.
3: Plotting of working curves of LDL
(1) And 2.0 mu L of LDL solution is dripped into the sensitive unit interface of the LAPS chip constructed in the step 2, and incubated for 30min at 25 ℃ to obtain the LDL/LDL apt/RGO-PANI-Hemin/AuNPs/LAPS chip (LAPS working electrode).
FIG. 3F is an SEM image of an LDL/LDL apt/RGO-PANI-Hemin/AuNPs/LAPS chip. In contrast to FIG. 3E, a new layer of sheet material was coated on the chip surface, since LDL apt specifically bound to GPC3 to coat the chip surface, forming a LAPS working electrode.
(2) And (3) placing 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 graph for 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 is normalized, and a blank sample is used as a reference to calculate the voltage offset. When the LDL concentration is in the range of 1.0-100.0 μg/mL, the relationship between the voltage offset (Y) of the sensor and the LDL concentration (X) is linear, y=4.17911x+128.67823 (where Y represents the difference between the voltage offset value at this concentration and the LDL-blank voltage offset value, X represents the concentration of LDL), and the correlation coefficient R 2 = 0.97826. The minimum detection limit was calculated to be 0.8989 μg/mL according to the calculation formula lod=3s b/b(Sb representing the standard deviation obtained for the blank control group in the experiment, and b representing the slope of the standard curve.
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 using the direct method. The collection and treatment of the serum sample meet the requirements of the ethical committee of the important laboratory for the research of the Guangxi metabolic diseases. And 2.0 mu L of serum sample containing LDL is dripped at the interface of the LDL apt/RGO-PANI-Hemin/AuNPs/LAPS sensitive unit obtained in the step 2, and incubated for 30min at 25 ℃ to obtain the LAPS working electrode.
(2) And (3) placing 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 is normalized, and a blank sample is used as a reference to calculate the voltage offset.
(3) And (3) obtaining the concentration of LDL in the actual serum to be tested according to the voltage offset value according to the working curve obtained in the step (3), wherein the result is shown in a table 1. The relative error of the detection of the serum sample by using the LAPS sensor by adopting a direct measurement method is between 0.32% and 6.79%, and the relative standard deviation value is between 0.54% and 1.14%. These results indicate that the developed LDL sensor is expected to have good application prospects in medical diagnosis.
TABLE 1 detection results of LDL in actual serum samples
Claims (7)
1. A method for constructing an optical addressing potential sensor based on a nanocomposite junction proper ligand for detecting low-density lipoprotein comprises the following steps:
step 1: preparation of reduced graphene oxide-polyaniline-heme RGO-PANI-Hemin and nano-gold AuNPs composite material
(1) Preparation of reduced graphene oxide RGO
Weighing single-layer graphene oxide GO, adding ultrapure water, crushing by using an ultrasonic crusher, and uniformly dispersing; adding ascorbic acid AA, and stirring on a constant-temperature magnetic stirrer to obtain RGO solution;
(2) Preparation of reduced graphene oxide-heme RGO-Hemin
Preparing a hemin solution, mixing the hemin solution and the RGO solution in equal mass ratio, adding hydrazine hydrate N2H2H2.H2O, placing in a constant-temperature water bath kettle, heating in a water bath, and stirring; centrifuging, and removing supernatant to obtain RGO-Hemin;
(3) Preparation of reduced graphene oxide-polyaniline-heme RGO-PANI-Hemin
Adding polyaniline PANI solution into RGO-Hemin solution, mixing uniformly, then adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide EDC/NHS solution, stirring; centrifuging, washing, redissolving in ultrapure water, and freeze-drying to obtain the RGO-PANI-Hemin composite material;
(4) Preparation of gold nano AuNPs solution
Heating chloroauric acid solution, and stirring to boil; adding the sodium citrate solution into the chloroauric acid solution, and continuing stirring until the solution turns from yellowish to reddish wine; cooling to obtain AuNPs solution;
Step 2: modification of LAPS sensor sensing unit
(1) Pretreatment of LAPS chip
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 mercaptopropyl triethoxysilane MPTES solution, and standing; performing silanization treatment on the silicon wafer, and naturally drying to obtain a silanized LAPS chip;
(2) Modification of RGO-PANI-Hemin/AuNPs nano material to the LAPS chip, dripping AuNPs solution on the silanized silicon-based LAPS chip, dripping RGO-PANIHEMIN nano composite material after surface drying, standing and drying to obtain RGO-PANI-Hemin/AuNPs sensor sensitive film;
(3) Construction of LDLapt/RGO-PANI-Hemin/AuNPs/LAPS sensitive unit
Dropping LDLapt solution on the RGO-PANI-Hemin/AuNPs sensor sensitive film, and incubating to obtain a LAPS sensitive unit interface with LDLapt/RGOPANI-Hemin/AuNPs/LAPS;
step 3: plotting of working curves of LDL
(1) Dropwise adding the standard LDL solution to the LAPS chip sensitive unit interface obtained in the step 2, and incubating to prepare a LAPS working electrode;
(2) Placing 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 using the LAPS system, and recording an I-V curve; normalizing the I-V curve, and calculating the voltage offset by taking a blank sample as a reference;
(3) Detecting LDL with different concentrations respectively, drawing a working curve by taking the concentration of LDL as an abscissa and the voltage offset as an ordinate, and calculating the lowest detection limit of the method;
step 4: detection of LDL in actual sample
Dropwise adding an actual sample to be detected into the LAPS sensitive unit interface obtained in the step 2, and incubating to obtain a working electrode; placing the LAPS working electrode into a LAPS detection system, adding PBS buffer solution and a reference electrode into a detection pool, detecting by using the LAPS system, and recording an I-V curve of the LAPS working electrode; normalizing the I-V curve, and comparing with a blank sample to obtain a voltage offset; and (3) obtaining the concentration of the LDL in the actual sample to be detected according to the voltage offset value by using the working curve obtained in the step (3).
2. The method according to claim 1, characterized in that: 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 4:1, and the concentration is 10mol/L.
3. The method according to claim 1, characterized in that: the concentration of NaOH in the step 2 is 1.0mol/L; MPTES concentration was 1%; the amount of AuNPs used was 20. Mu.L.
4. The method according to claim 1, characterized in that: RGO-PANI-Hemin in step 2 was used in an amount of 20. Mu.L at a concentration of 1.0mg/mL.
5. The method according to claim 1, characterized in that: the LDLapt concentration in step 2 was 1. Mu.M and the amount was 5.0. Mu.L.
6. The method according to claim 1, characterized in that: the PBS buffer solution in the step 3 and the step 4 has a pH value of 7.4 and a concentration of 0.2 mol/L.
7. The method according to claim 1, characterized in that: the incubation temperature in the step 3 and the step 4 is 25 ℃ and the incubation time is 1h.
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