CN115876853A - Light addressing potential sensor for detecting low-density lipoprotein based on nanocomposite and aptamer - Google Patents

Abstract

A light-addressable potential sensor based on nanocomposite materials combined with suitable ligands for detecting low-density lipoproteins, using LDL aptamers as recognition probes, based on reduced graphene oxide-polyaniline-hemin (RGO ‑PANI‑Hemin) nanocomposite material has good electron transfer effect and excellent loading capacity, LDL aptamer can specifically recognize and bind LDL protein, and construct a new type of aptamer that can specifically recognize and quantitatively analyze LDL protein The ligand sensor is used to detect the content of LDL in serum. The method is simple, time-saving and low-cost, and the minimum detection limit is 0.8989 μg/mL.

Description

A photo-addressable potentiometric sensor based on nanocomposite combined with aptamer for detecting low-density lipoprotein

Technical Field

The invention belongs to the field of biological detection, and in particular relates to a light-addressable potential sensor for detecting low-density lipoprotein based on a nanocomposite material combined with an aptamer.

Background Art

The main methods for detecting low-density lipoprotein (LDL) in serum include ultracentrifugation, Friedewald formula calculation method, chemical precipitation method, etc. The invention patent with publication number CN111647641B relates to a method of quantitatively determining the content of low-density lipoprotein (LDL) by using a kit according to the change in absorbance value. This method takes a long time and is costly. The invention patent with publication number CN101482570 relates to a method of using heparin-magnesium reagent as a substrate, reacting with serum after centrifugation and precipitation, and then comparing the absorbance to obtain the specific LDL content in serum. This method requires many reagents, which are expensive and difficult to obtain. These methods are complicated, time-consuming and technically demanding. There is an urgent need for a rapid, sensitive and easy-to-operate LDL detection method.

Summary of the invention

The technical problem to be solved by the present invention is to provide a nanocomposite material based on reduced graphene oxide-polyaniline-hemin RGO-PANI-Hemin, and to modify the surface of a light-addressable potential sensor chip by combining a specific recognition molecule LDL aptamer LDL _apt to construct a biosensor for improving the detection efficiency of LDL and portable detection.

The detection principle of the present invention is: RGO-PANI-Hemin is modified on the surface of silicon-based LAPS chip by physical action. Among them, RGO has a large specific surface area and strong conductivity, heme can improve the stability of RGO, and PANI further improves the conductivity. RGO-PANI-Hemin nanocomposite material can enhance the detection signal of LAPS sensor, thereby improving the sensitivity of sensor. LDL _apt is loaded on the surface of RGO-PANI-Hemin material through non-covalent binding and intermolecular force, and the aptamer is connected to the composite material in a single chain form due to its relatively unstable spatial structure, and then can be modified to LAPS chip. After adding LDL to the biosensor interface, LDL _apt will specifically bind to LDL protein, form a protein-aptamer complex and present a stable spatial structure, which is orderly arranged on the surface of the modified LAPS chip, which will cause the change of the surface potential of LAPS chip. The surface potential changes caused by different concentrations of LDL are different, resulting in a certain offset of the IV curve. By monitoring the offset of the IV curve, the detection of LDL of different concentrations is realized. Compared with existing methods, the operation is relatively simple, the sensitivity is high, and portable detection is realized, which can achieve a detection limit of 0.8989 μg/mL.

The present invention is carried out according to the following steps:

Step 1: Preparation of RGO-PANI-Hemin and Au NPs composites

(1) Preparation of reduced graphene oxide (RGO)

Weigh a single layer of graphene oxide (GO), add ultrapure water, and crush it with an ultrasonic crusher to disperse it evenly; then add ascorbic acid (AA) and stir it on a constant temperature magnetic stirrer to obtain an RGO solution.

(2) Preparation of reduced graphene oxide-hemin (RGO-Hemin)

Prepare the hemin solution, take equal proportions of the hemin solution and RGO solution to mix, add hydrazine hydrate (N ₂ H ₄ ·H ₂ O), place in a constant temperature water bath for heating and stirring for a period of time, then centrifuge, remove the supernatant, and obtain RGO-Hemin.

(3) Preparation of reduced graphene oxide-polyaniline-hemin (RGO-PANI-Hemin)

Polyaniline (PANI) solution was added to RGO-Hemin and mixed evenly, followed by addition of 1-ethyl-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS) solution, stirring, centrifugal washing and redissolving in ultrapure water, and freeze-drying to obtain RGO-PANI-Hemin nanocomposite.

(4) Preparation of gold nanoparticles (Au NPs) solution

The chloroauric acid solution is placed in a beaker, heated and stirred until boiling. Then, the sodium citrate (C ₆ H ₅ Na ₃ O ₇ ) solution is slowly added to the chloroauric acid solution. The stirring is continued until the solution changes from light yellow to wine red, and the AuNPs solution is obtained after the solution is naturally cooled to room temperature.

Step 2: Modification of the LAPS sensor sensitive unit

(1) LAPS chip preprocessing

A cleaned silicon-based LAPS chip was taken, and sodium hydroxide (NaOH) solution was added to the surface for activation, followed by addition of mercaptopropyltriethoxysilane (MPTES) solution. The silicon chip was allowed to stand for silanization, and the silanized LAPS chip was obtained after natural drying.

(2) Modification of LAPS chip by RGO-PANI-Hemin/Au NPs nanomaterials

The AuNPs solution was dripped onto the silicon-based LAPS chip after silanization treatment. After the surface was dried, the RGO-PANI-Hemin nanocomposite material was continued to be dripped and allowed to stand and dry to obtain the RGO-PANI-Hemin/Au NPs sensor sensitive membrane.

(3) Construction of LDL _apt /RGO-PANI-Hemin/Au NPs/LAPS sensitive unit

The LDL _apt solution was dropped onto the above-mentioned LAPS silicon wafer and placed in an incubator for incubation to obtain a LAPS sensitive unit interface having LDL _apt /RGO-PANI-Hemin/Au NPs/LAPS.

Step 3: Draw the working curve of LDL

(1) Add the standard LDL solution dropwise to the interface of the LAPS chip sensitive unit obtained in step 2, incubate, and prepare the LAPS working electrode.

(2) Place the prepared LAPS working electrode into the LAPS detection system, add phosphate buffer solution (PBS) support solution and reference electrode into the detection cell, use the LAPS system to detect, and record its I-V curve. Normalize the I-V curve, use a blank sample as a control, and calculate the voltage offset.

(3) Different concentrations of LDL were tested respectively, and a working curve was drawn with the concentration of LDL as the horizontal axis and the voltage offset as the vertical axis to calculate the minimum detection limit of the method.

Step 4: Detection of LDL in actual samples

At the interface of the LAPS sensitive unit obtained in step 2, the actual sample to be tested is added and incubated to obtain a working electrode. The LAPS working electrode is placed in the LAPS detection system, PBS buffer solution and reference electrode are added to the detection cell, and the LAPS system is used for detection to record its I-V curve. The I-V curve is normalized and compared with a blank sample to obtain the voltage offset.

Using the working curve obtained in step 3, the concentration of LDL in the actual sample to be tested is obtained according to the voltage offset value.

Furthermore, in step 1, GO is 30 mg and AA is 300 mg.

Furthermore, in step 1, the hemin solution is 1.0 mg/mL.

Furthermore, the mass fraction of the hydrazine hydrate solution in step 1 is 80%.

Furthermore, in step 1, the concentration of PANI is 1.0 mg/mL.

Furthermore, in step 1, the volume ratio of EDC/NHS solution is 4:1, and the concentration of each is 10M.

Furthermore, in step 1, the concentration of chloroauric acid is 0.01%, and the concentration of sodium citrate is 1%.

Furthermore, the concentration of NaOH in step 2 is 1.0 mol/L.

Preferably, the amount of Au NPs used in step 2 is 25 μL.

Preferably, the amount of rGO-PANI-Hemin used in step 2 is 25 μL.

Preferably, the LDLapt concentration in step 2 is 1.0 μmol/L.

Preferably, the concentration of PBS in step 2 and step 3 is 0.2 mol/L and the pH value is 7.4.

Preferably, the incubation temperature in step 2 and step 3 is 25° C. and the incubation time is 1 h.

Among them, step 1 provides a high-conductivity nanocomposite material RGO-PANI-Hemin for step 2 to cause a rapid response of the sensing interface. Step 2 constitutes a biosensing interface that specifically recognizes LDL and is beneficial to the transfer efficiency of electrons. The construction of the biosensing interface in step 2 is an essential key step in the detection of LDL by the LAPS sensor in steps 3 and 4. The working curve of LDL in step 3 provides a calculation basis for the determination of the LDL concentration in the actual sample in step 4. It can be seen that steps 1-4 support each other and work together to achieve the detection of LDL using the RGO-PANI-Hemin composite material and the LDL aptamer as the recognition probe.

Compared with the prior art, the present invention has the following advantages:

1. This method uses the properties of polyaniline particles to enhance electron transfer, as well as the effective current signal amplification and excellent load capacity of reduced graphene oxide, to successfully prepare RGO-PANI-Hemin composite materials with good biocompatibility and strong conductivity. The RGO-PANI-Hemin composite material is formed into a biosensitive membrane on a silicon-based LAPS chip, and combined with an LDL _apt probe to form a new light-addressable potential sensor for specific detection of LDL.

2. The current methods for detecting LDL at home and abroad have problems such as complex operation or need for professional operation, while the light-addressable potential sensor modified by RGO-PANI-Hemin nanocomposite is easy to operate, high in precision, and realizes portable detection. The minimum detection limit is 0.9898μg/mL. The direct measurement method is used to detect LDL in serum samples with actual known LDL levels, and the relative error is between 0.32% and 6.79%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the principle of detecting LDL based on the light-addressable potentiometric sensor of RGO-PANI-Hemin;

Figure 2 Transmission electron microscopy images of RGO-Hemin (A) and RGO-PANI-Hemin (B);

Figure 3 Scanning electron microscope (SEM) images of various stages of constructing the LAPS chip; including: (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.

Figure 4 Working curve of the light-addressable potentiometric sensor based on RGO-PANI-Hemin for detecting different LDL concentrations.

DETAILED DESCRIPTION

The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

A potentiometric sensor for detecting LDL based on RGO-PANI-Hemin nanocomposite material is constructed. The detection principle is shown in Figure 1. First, NaOH is added dropwise to the surface of the silicon-based LAPS chip for activation, and then mercaptopropyltriethoxysilane (MPTES) is added to form a mercaptosilane LAPS chip. Au NPs are fixed on the surface of the mercaptosilane LAPS chip through the action of Au-S. RGO-PANI-Hemin is added to the surface of the AuNPs-modified LAPS chip. The LDL aptamer is incubated so that the LDL aptamer can covalently bind to the RGO-PANI-Hemin on the electrode surface, and because of its relatively unstable spatial structure, it is connected to the composite material in a single chain form, and can then be fixed on the surface of the modified LAPS chip. After adding LDL, the LDL aptamer will specifically bind to the LDL protein, forming a protein-aptamer complex with a stable spatial structure, and thus orderly arranged on the electrode surface. By detecting the changes in the electrochemical signal of the sensor through the LAPS system, the quantitative analysis of the LDL protein can be effectively achieved.

The implementation steps are as follows:

1: Preparation of RGO-PANI-Hemin/Au NPs composites

(1) Take 30 mg of GO and add it to 30 mL of pure water. Use an ultrasonic crusher to crush it for 1 hour to make it evenly dispersed. Then add 300 mg of ascorbic acid and stir it under a constant temperature magnetic stirrer for 12 hours to obtain a 1.0 mg/mL RGO solution. Take 20 mg of hemin and put it into a beaker. Add 10 μL of ammonia water and 20 mL of pure water, stir it to dissolve it evenly, and put it in a refrigerator to stand overnight to obtain a 1.0 mg/mL hemin solution. Take 10 mL of hemin solution and 5.0 mL of RGO solution, mix them, add 10 μL of hydrazine hydrate, and react in a 60°C water bath for 4 hours. Centrifuge the reaction solution at a speed of 10000 r/min for 5 minutes, remove the supernatant, and obtain RGO-Hemin solution. Take 10 mL of RGO-Hemin solution and pour it into a beaker. Add 10 mL of 1.0 mg/mL PANI solution and mix evenly. Then, EDC/NHS buffer (volume ratio 4:1, concentration 1M) was added and stirred for 3 h under a constant temperature magnetic stirrer. Then, the mixture was centrifuged 3 times (9000 r/min, 10 min), the supernatant was removed, and the lower layer was freeze-dried to obtain the RGO-PANI-Hemin composite material.

The RGO-PANI-Hemin nanomaterials were characterized using a JEM-1200EX transmission electron microscope, as shown in Figure 2. Figure 2A is a TEM image of RGO-Hemin, which has a relatively smooth surface and a wrinkled shape. Figure 2B is a TEM image of RGO-PANI-Hemin, which has many particles on the wrinkled surface, indicating that PANI and RGO-Hemin are successfully combined.

(2) Take 50 ml of 0.01% chloroauric acid solution in a clean beaker and heat and stir in a water bath until the temperature reaches 100°C. Then slowly add 1.5 mL of 0.1% sodium citrate solution to the chloroauric acid solution. Continue stirring at 100°C until the solution changes from light yellow to wine red. Cool naturally to room temperature to obtain the Au NPs solution, which is refrigerated at 4°C for later use.

2: Modification of the sensitive unit of the LAPS sensor

(1) First, soak the silicon wafer in a solution ( _{H2O2 and} concentrated _{H2SO4 ,} volume ratio 3:7) for 10 minutes, then place the silicon wafer in ethanol, acetone and pure water in turn, ultrasonically clean it in an ultrasonic cleaner for 5 minutes, and finally let it stand for 30 minutes before cleaning it with pure water.

(2) Add 5 μL of NaOH solution (1.0 mol/L) to the working surface of the chip and clean it after 30 minutes. Then, the surface of the LAPS chip is silanized with MPTES mercapto groups and terminated with -SH groups to obtain a mercaptosilanized LAPS chip (MPTES-LAPS chip). Place it in a refrigerator at 4°C for 12 hours and wash it three times with pure water.

(3) Add 20 μL of AuNPs solution to the MPTES-LAPS chip. After the surface is completely dry, continue to add 20 μL of 1.0 mg/mL RGO-PANI-Hemin solution. Incubate in a constant temperature incubator (25°C) for 1 hour and then clean it to obtain the RGO-PANI-Hemin/AuNPs/LAPS chip.

(4) 5.0 μL of 1.0 μM LDL _apt solution was added to the above chip, and the chip was placed in a shaking incubator (25°C) for incubation for 1 h and allowed to dry naturally to obtain an LDL _apt /RGO-PANI-Hemin/AuNPs/LAPS sensitive unit interface.

Figure 3 shows the scanning electron microscope (SEM) images of the LAPS chip at various stages. Figure A is a bare chip SEM image, and it can be seen that the surface is smooth and flat. Figure B is a SEM image of the MPTES-LAPS chip, and the surface is obviously covered with a layer of granular substances. Figure C is a SEM image of the AuNPs/LAPS chip, and obvious luminous particles can be seen, indicating that the AuNPs particles are successfully modified on the chip through the Au-S bond. Figure D is a SEM image of the RGO-PANI-Hemin/AuNPs/LAPS chip, and some membrane-like substances can be clearly seen in the gaps between the granular AuNPs particles, indicating that the RGO-PANI-Hemin nanocomposite material has been fixed on the LAPS chip. Figure E is a SEM image of the LDL _apt /RGO-PANI-Hemin/AuNPs/LAPS chip. Compared with Figure D, the modified substance on the chip surface has become much thicker, which is because LDL _apt is a disordered single-stranded DNA.

3: Drawing of the working curve of LDL

(1) Add 2.0 μL of LDL solution to the interface of the sensitive unit of the LAPS chip constructed in step 2 and incubate at 25°C for 30 min to obtain an LDL/LDL _apt /RGO-PANI-Hemin/AuNPs/LAPS chip (LAPS working electrode).

Figure 3F is a SEM image of the LDL/LDL _apt /RGO-PANI-Hemin/AuNPs/LAPS chip. Compared with Figure 3E, there is a new layer of flaky material covering the surface of the chip. This is because LDL _apt specifically binds to GPC3 and covers the surface of the chip, forming a LAPS working electrode.

(2) The prepared LAPS working electrode was placed in the LAPS detection system, and a phosphate buffer solution (PBS, pH 7.4) buffer solution and a reference electrode were added to the detection cell. The LAPS system was used for detection and the IV curve was recorded. The IV curves of different LDL concentrations are shown in FIG4 . As the LDL concentration increases, the IV curve shifts to the right more. The IV curve was normalized and the blank sample was used as a control to calculate the voltage offset. When the LDL concentration was in the range of 1.0 to 100.0 μg/mL, the relationship between the voltage offset (Y) of the sensor and the LDL concentration (X) was linear, Y=4.17911X+128.67823 (where Y represents the difference between the voltage offset value at this concentration and the voltage offset value of the LDL blank group, and X represents the LDL concentration), and the correlation coefficient R ² =0.97826. According to the calculation formula LOD = 3S _b /b (S _b represents the standard deviation of the blank control group in the experiment, and b represents the slope of the standard curve), the minimum detection limit was calculated to be 0.8989 μg/mL.

4: Detection of LDL in actual samples

(1) Two different concentrations (19.72 μg/mL and 80.43 μg/mL) of LDL serum samples were measured by direct method. The collection and processing of serum samples met the requirements of the ethics committee of Guangxi Key Laboratory of Metabolic Disease Research. 2.0 μL of serum sample containing LDL was added to the LDL _apt /RGO-PANI-Hemin/AuNPs/LAPS sensitive unit interface obtained in step 2, and incubated at 25°C for 30 minutes to obtain the LAPS working electrode.

(2) Place the prepared LAPS working electrode into the LAPS detection system, add phosphate buffer solution (PBS, pH 7.4) buffer solution and reference electrode into the detection cell, use the LAPS system to detect, and record its I-V curve. Normalize the I-V curve, use a blank sample as a control, and calculate the voltage offset.

(3) According to the working curve obtained in step 3, the concentration of LDL in the actual serum to be tested is obtained according to the voltage offset value, and the results are shown in Table 1. The relative error of the serum sample detected by the LAPS sensor using the direct measurement method is between 0.32% and 6.79%, and the relative standard deviation values are 0.54% and 1.14%. These results show 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 light-addressable potentiometric sensor for detecting low-density lipoprotein based on a nanocomposite material combined with an aptamer, comprising the following steps: Step 1: Preparation of reduced graphene oxide-polyaniline-hemin RGO-PANI-Hemin and gold nanoparticles AuNPs composites (1) Preparation of reduced graphene oxide RGO Weigh a single layer of graphene oxide GO, add ultrapure water, and crush it with an ultrasonic crusher to disperse it evenly; add ascorbic acid AA, and stir it on a constant temperature magnetic stirrer to obtain an RGO solution; (2) Preparation of reduced graphene oxide-hemin RGO-Hemin Prepare the hemin solution, take the hemin solution and RGO solution in equal proportion by mass, mix them, add hydrazine hydrate N ₂ H ₄ ·H ₂ O, place them in a constant temperature water bath for heating, stir; centrifuge, remove the supernatant, and obtain RGO-Hemin; (3) Preparation of reduced graphene oxide-polyaniline-hemin RGO-PANI-Hemin Adding the polyaniline PANI solution to the RGO-Hemin solution, mixing evenly, then adding the 1-ethyl-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide EDC/NHS solution, stirring; centrifuging, washing, and redissolving in ultrapure water, and freeze-drying to obtain the RGO-PANI-Hemin composite material; (4) Preparation of AuNPs solution Heat the chloroauric acid solution and stir until it boils; add the sodium citrate solution to the chloroauric acid solution and continue stirring until the solution changes from light yellow to wine red; cool to obtain an AuNPs solution; Step 2: Modification of the LAPS sensor sensitive unit (1) LAPS chip preprocessing Take a cleaned silicon-based LAPS chip, add sodium hydroxide NaOH solution on its surface for activation, then add mercaptopropyl triethoxysilane MPTES solution, and let it stand; perform silanization on the silicon chip, and obtain a silanized LAPS chip after natural drying; (2) Modification of LAPS chip by RGO-PANI-Hemin/AuNPs nanomaterials The AuNPs solution was dripped onto the silicon-based LAPS chip after silanization treatment. After the surface was dried, the RGO-PANI-Hemin nanocomposite material was dripped, and the surface was allowed to stand and dried to obtain the RGO-PANI-Hemin/AuNPs sensor sensitive film. (3) Construction of LDL _apt /RGO-PANI-Hemin/AuNPs/LAPS sensitive unit The LDL _apt solution was dropped onto the sensitive membrane of the RGO-PANI-Hemin/AuNPs sensor and incubated to obtain a LAPS sensitive unit interface having LDL _apt /RGO-PANI-Hemin/AuNPs/LAPS; Step 3: Draw the working curve of LDL (1) adding a standard LDL solution dropwise to the interface of the LAPS chip sensitive unit obtained in step 2, incubating, and preparing a LAPS working electrode; (2) placing the prepared LAPS working electrode into the LAPS detection system, adding phosphate buffer solution (PBS) and a reference electrode into the detection cell, using the LAPS system for detection, and recording its I-V curve; normalizing the I-V curve, using a blank sample as a control, and calculating the voltage offset; (3) Detecting LDL of different concentrations respectively, plotting the working curve with the concentration of LDL as the abscissa and the voltage offset as the ordinate, and calculating the minimum detection limit of the method; Step 4: Detection of LDL in actual samples Add the actual sample to be tested to the interface of the LAPS sensitive unit obtained in step 2, incubate, and obtain a working electrode; put the LAPS working electrode into the LAPS detection system, add PBS buffer solution and reference electrode into the detection cell, use the LAPS system to detect, and record its I-V curve; normalize the I-V curve, and use a blank sample as a control to calculate the voltage offset; Using the working curve obtained in step 3, the concentration of LDL in the actual sample to be tested is obtained according to the voltage offset value. 2. The sensor according to claim 1 is characterized in that: the Hemin solution in step 1 is 1.0 mg/mL; the RGO solution is 1.0 mg/mL; the mass fraction of the hydrazine hydrate solution is 80%; the PANI concentration is 1.0 mg/mL; the volume ratio of the EDC/NHS solution is 4:1, and the concentrations are all 10 mol/L. 3. The sensor according to claim 1, characterized in that: in step 2, the concentration of NaOH is 1.0 mol/L; the concentration of MPTES is 1%; and the amount of AuNPs used is 20 μL. 4. The sensor according to claim 1, characterized in that: in step 2, the amount of RGO-PANI-Hemin used is 20 μL and the concentration is 1.0 mg/mL. 5 . The sensor according to claim 1 , wherein the LDL _apt concentration in step 2 is 1 μM and the dosage is 5.0 μL. 6. The sensor according to claim 1, characterized in that the pH value of the PBS buffer solution in step 3 and step 4 is 7.4 and the concentration is 0.2 mol/L. 7 . The sensor according to claim 1 , wherein the incubation temperature in step 3 and step 4 is 25° C. and the incubation time is 1 hour.

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