A fluorescence / electrochemical biosensor for GP73 detection based on fluorescence resonance energy transfer

Abstract

A fluorescence / electrochemical biosensor for GP73 detection based on fluorescence resonance energy transfer was constructed, which used nitrogen-doped graphene quantum dots modified with GP73 aptamers as fluorescence resonance donors and molybdenum disulfide@ferrocene@palladium nanoparticles as fluorescence resonance acceptors. Apt Fluorescence resonance energy transfer phenomenon between N‑GQDs‑GP73 and MoS2@Fc@PdNPs Apt The MoS2@Fc@PdNPs fluorescence / electrochemical biosensor uses a fluorescence / electrochemical dual-mode biosensor to detect GP73 content, based on the relationship between fluorescence intensity recovery before and after the addition of GP73 protein and the change in electrochemical current. This method, which achieves two signal detection modes through a single-step reaction, offers a streamlined, rapid, sensitive, and highly selective detection process, demonstrating strong practicality.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and specifically relates to a fluorescence / electrochemical dual-mode biosensor based on fluorescence resonance energy transfer for realizing high-sensitivity detection of GP73. Background Art

[0002] Primary liver cancer, also known as HCC, is an epithelial malignancy that originates in the liver. Over 90% of liver cancers are hepatocellular carcinomas (HCCs). Golgi protein 73 (GP73) is a novel serum marker used in recent years in clinical medicine for the early diagnosis and screening of HCC. Fluorescence resonance energy transfer (FRET) refers to the energy transfer between a fluorescent molecule when the emission wavelength of the fluorescent molecule overlaps with the excitation wavelength of another substance. In clinical testing, detection methods based on fluorescence and electrochemistry have become the mainstream. Invention patent CN202211636825.3 discloses a fluorescent immunoassay kit. This kit modifies an alpha-fetoprotein antigen in a mold channel, adds a fluorescein-labeled antibody to the analyte, forms an antibody-analyte-antibody fluorescent complex, and measures the relative luminescence intensity to achieve fluorescence diagnosis of HCC. Invention patent CN109682875B discloses a nucleic acid electrochemical detection system and method for HCC screening. This method relies on a catalytic hairpin assembly process to achieve specific recognition and signal amplification of the target miRNA-500. It then leverages click chemistry to facilitate the transfer of an electrochemical signal reporter molecule from the solution to the electrode interface, enabling electrochemical detection of miRNA-500. This invention offers advantages such as high sensitivity, strong specificity, and simple operation. Currently, combining multiple modalities for detection is a popular area in the detection field, necessitating the development of a novel fluorescence / electrochemical biosensor leveraging both fluorescence and electrochemistry. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a fluorescence / electrochemical biosensor for GP73 detection, which is constructed based on nitrogen-doped graphene quantum dots (N-GQDs) with high-efficiency fluorescence properties and molybdenum disulfide@ferrocene@palladium nanoparticles (MoS2@Fc@PdNPs) with good electrochemical activity and quenching effect.

[0004] In order to solve this technical problem, N-GQDs were used as fluorescent substances and N-GQDs were combined with the amino-GP73 aptamer GP73. Apt Combined through amide bonds to form N-GQDs-GP73 Apt Fluorescent probe. In N-GQDs-GP73 AptMoS2@Fc@PdNPs, N-GQDs-GP73 were added to the fluorescent probe Apt Through van der Waals force and hydrogen bonding, fluorescence resonance energy transfer (FRET) occurs on the surface of MoS2@Fc@PdNPs. Apt The fluorescence of the system was quenched, and the fluorescence intensity of the whole system decreased. In addition, due to the adsorption of N-GQDs-GP73Apt on the surface of MoS2@Fc@PdNPs, the redox activity of Fc in MoS2@Fc@PdNPs was inhibited, thereby forming N-GQDs-GP73 Apt / MoS2@Fc@PdNPs fluorescence / electrochemical biosensor; after adding GP73 protein, due to GP73 Apt Specificity for GP73, GP73 preferentially binds to N-GQDs-GP73 Apt Combination to form N-GQDs-GP73 Apt The interaction between N-GQDs-GP73Apt and MoS2@Fc@PdNPs is significantly weakened, disrupting FRET and restoring fluorescence in the system with high fluorescence intensity. The electroactivity of the released MoS2@Fc@PdNPs is then restored, enhancing the Fc redox activity within the MoS2@Fc@PdNPs. A GP73 working curve was established based on the relationship between the fluorescence intensity recovery before and after the addition of GP73 protein and the change in the electrochemical current and GP73, enabling rapid, sensitive, and selective quantitative detection of GP73.

[0005] The present invention is carried out according to the following steps:

[0006] Step 1: Fluorescence resonance donor N-GQDs-GP73 Apt Preparation

[0007] (1) Preparation of N-GQDs: Weigh citric acid and urea, add pure water to the volume and stir evenly, heat at high temperature for a certain time, cool and add ethanol to mix and stir, after stirring completely, dialyze for a period of time, and freeze-dry the dialyzed solution to obtain N-GQDs powder;

[0008] (2)N-GQDs-GP73 Apt Preparation: Measure N-GQDs and GP73 aptamer GP73 Apt Activate with EDAC / NHS crosslinker, incubate with stirring for a certain time at room temperature and in the dark to obtain N-GQDs-GP73 Apt solution.

[0009] Step 2: Preparation of fluorescence resonance receptor MoS2@Fc@PdNPs and construction of fluorescence / electrochemical biosensor

[0010] (1) Weigh MoS2 powder, add N,N-dimethylformamide (DMF) solution to the final volume, and crush in an ultrasonic cell disrupter until the MoS2 powder is completely dispersed in DMF to obtain a MoS2 dispersion. Weigh mercaptoethylamine, add it to the MoS2 dispersion, stir thoroughly, and centrifuge. Wash the separated precipitate and dry it to obtain MoS2-NH2 powder.

[0011] (2) MoS2-NH2 powder and ferrocene (Fc) powder are weighed and mixed to form a solution, a crosslinker solution is added, the mixture is stirred thoroughly and then centrifuged, the separated precipitate is washed and dried to obtain MoS2-Fc powder.

[0012] (3) MoS2-Fc powder, Na2PdCl6, ascorbic acid (AA) and sodium carboxymethyl cellulose (CMC) were weighed and prepared into solutions respectively. The prepared solutions were mixed and stirred for a period of time. The obtained solution was centrifuged, and the separated precipitate was washed and dried to obtain MoS2@Fc@PdNPs powder.

[0013] (4) Weigh the MoS2@Fc@PdNPs powder and prepare a solution. Mix the MoS2@Fc@PdNPs solution and N-GQDs-GP73 Apt The solutions were mixed and incubated at a certain temperature for a period of time to form N-GQDs-GP73 Apt / MoS2@Fc@PdNPs fluorescence / electrochemical biosensor. After centrifugation, the upper layer was collected and scanned using a fluorescence spectrophotometer, recording its fluorescence intensity (F0). The lower layer was dropped onto the activated screen-printed electrode and scanned using a DPV electrochemical workstation, recording its peak current (C0).

[0014] Step 3: Drawing the GP73 working curve

[0015] (1) Add different concentrations of GP73 solution to N-GQDs-GP73 Apt The / MoS2@Fc@PdNPs FRET fluorescence / electrochemical biosensor was incubated at a certain temperature for a period of time and then centrifuged. The upper layer of liquid was scanned using a fluorescence spectrophotometer to record its fluorescence intensity F1. The lower layer of liquid was dropped onto the activated screen-printed electrode and scanned using the DPV of an electrochemical workstation to record its peak current value C1.

[0016] (2) Using (F1-F0) / F0 as the ordinate and GP73 concentration as the abscissa, a working curve was drawn to calculate the minimum detection limit of the fluorescence / electrochemical biosensor in fluorescence detection; using △C, i.e., C1-C0 value, as the ordinate and GP73 concentration as the abscissa, a working curve was drawn to calculate the minimum detection limit of the fluorescence / electrochemical biosensor in electrochemical detection.

[0017] Step 4: Detection of GP73 in actual serum samples

[0018] (1) Collect serum samples with known GP73 concentrations and add the serum samples to be tested to the N-GQDs-GP73 in step 2. Apt The / MoS2@Fc@PdNPs FRET fluorescence / electrochemical biosensor was incubated at a certain temperature for a period of time and then centrifuged. The upper layer of liquid was scanned using a fluorescence spectrophotometer with an excitation wavelength of 348 nm to measure the fluorescence intensity at 438 nm. The lower layer of liquid was dropped onto the activated screen-printed electrode and scanned using a DPV electrochemical workstation to record its peak current value.

[0019] (2) Calculate the concentration of GP73 in the serum sample to be tested based on the GP73 working curve obtained in step 3.

[0020] Furthermore, in step 1, the amount of citric acid is 2.1 g, and the amount of urea is 1.8 g;

[0021] Furthermore, in step 1, pure water was added and the volume was adjusted to 15 mL;

[0022] Furthermore, in step 1, heating is performed at 180° C. for 8 h;

[0023] Furthermore, the volume of ethanol added in step 1 is 30 mL;

[0024] Furthermore, in step 1, the dialysis bag with a molecular weight of 300 was used for dialysis for 8 hours;

[0025] Furthermore, the concentration of N-GQDs in step 1 is 1.0 mg / mL, GP73 Apt The concentration was 1 μM;

[0026] Furthermore, the GP73 in step 1 Apt The DNA sequence is 5′-NH2-C6-GCAGTTGATCCTTTGGATACCCTGG-3′, and the concentration is 1.5 μM;

[0027] Furthermore, the cross-linking agent solution in step 1 and step 2 contains carbodiimide (EDAC) and N-hydroxysuccinimide (NHS), wherein the concentration of EDAC is 0.7668 mg / mL and the concentration of NHS is 2.1713 mg / mL;

[0028] Furthermore, in step 1, the N-GQDs solution and GP73 Apt The volume ratio of solution and cross-linker solution was 10:10:1;

[0029] Furthermore, the incubation temperature in step 1 is 25° C. and the time is 1 h;

[0030] Furthermore, in step 2, the concentration of MoS2 in the mixed solution is 1.0 mg / mL, the concentration of mercaptoethylamine is 2.0 mg / mL, and the stirring time is 12 h;

[0031] Furthermore, in step 2, the centrifuge was centrifuged at a speed of 6000 r / min for 5 minutes;

[0032] Furthermore, in step 2, the concentration of MoS2-NH2 in the mixed solution is 1.0 mg / mL, and the concentration of Fc is 3.0 mg / mL;

[0033] Furthermore, in step 2, the volume ratio of the mixed solution to the cross-linking agent solution is 10:1, and the stirring time is 24 hours;

[0034] Furthermore, the centrifugal speed in steps 2, 3 and 4 is 6000 r / min and the centrifugal time is 5 min;

[0035] Furthermore, in step 2, the concentration of the MoS2-Fc solution is 1.0 mg / mL; the concentration of the Na2PdCI6 solution is 1.826 mg / mL; the concentration of the ascorbic acid (AA) solution is 17.612 mg / mL; and the concentration of the sodium carboxymethyl cellulose (CMC, 800–1200 mPas) solution is 24.216 mg / mL;

[0036] Furthermore, in step 2, the volume ratio of the MoS2-Fc solution, the Na2PdCI6 solution, the ascorbic acid (AA) solution, and the sodium carboxymethyl cellulose (CMC, 800–1200 mPas) aqueous solution is 1000:1:5:1; the stirring time is 30 minutes;

[0037] Furthermore, the concentration of the MoS2@Fc@PdNPs solution in step 2 was 1.0 mg / mL, and 1 mL of MoS2@Fc@PdNPs solution and 100 μL of N-GQDs-GP73 were taken. Apt The solution is prepared into a mixed solution;

[0038] Furthermore, the incubation temperature in step 2 is 25°C and the incubation time is 30 min;

[0039] Furthermore, in step 2, step 3 and step 4, the DPV linear scanning range is -0.4V to 1.0V, and the scanning rate is 0.01V / s.

[0040] Furthermore, the excitation wavelength of fluorescence detection in step 2, step 3 and step 4 is 348 nm, and the emission wavelength is 438 nm;

[0041] Furthermore, the incubation temperature of the GP73 protein in steps 3 and 4 is 25° C. and the incubation time is 1 h;

[0042] Step 1 provides a blue fluorescent N-GQDs and N-GQDs-GP73 Apt The probe provides a fluorescent donor for fluorescence resonance energy transfer in step 2. Step 2 provides MoS2@Fc@PdNPs nanomaterials with both fluorescence quenching properties and electroactive capabilities, which serve as receptors for fluorescence resonance energy transfer and carriers of electrochemical signals. Apt There are van der Waals forces and hydrogen bonds between N-GQDs-GP73 and MoS2@Fc@PdNPs, which makes Apt The close proximity of the MoS2@Fc@PdNPs and the MoS2@Fc@PdNPs leads to the FRET phenomenon, which results in the fluorescence quenching of the system and weak fluorescence intensity. In addition, due to the adsorption of N-GQDs-GP73 on the surface of MoS2@Fc@PdNPs, the fluorescence intensity of the system is weakened. Apt , which inhibited the Fc redox activity in MoS2@Fc@PdNPs and obtained a lower electrochemical signal. Step 3 is a further extension of Step 2. When GP73 protein is added to N-GQDs-GP73 Apt In the / MoS2@Fc@PdNPs system, due to the GP73 Apt Preferentially binds to GP73 to form GP73-GP73 Apt Complex, changed its original conformation, N-GQDs-GP73 Apt The interaction between N-GQDs-GP73 and MoS2@Fc@PdNPs was greatly weakened, interrupting the FRET phenomenon. AptSeparated from MoS2@Fc@PdNPs, the fluorescence in the system is restored and the fluorescence intensity is high; and the electroactivity of the separated MoS2@Fc@PdNPs is restored, which enhances the Fc redox activity in MoS2@Fc@PdNPs and obtains a higher electrochemical signal. Based on the relationship between the fluorescence intensity recovery value before and after the addition of GP73 protein and the current change value of the electrochemical signal and GP73, a working curve of GP73 was established. The working curve of GP73 in step 3 provides a calculation basis for the determination of GP73 concentration in the actual sample in step 4. It can be seen that steps 1-4 support each other and work together to utilize N-GQDs-GP73. Apt Fluorescence resonance energy transfer phenomenon between N-GQDs-GP73 and MoS2@Fc@PdNPs was established to detect GP73. Apt / MoS2@Fc@PdNPs fluorescence / electrochemical biosensor.

[0043] Compared with the prior art, the present invention has the following advantages:

[0044] 1. Taking advantage of the abundant fluorescence of N-GQDs and the excellent fluorescence quenching ability and electroactivity of MoS2@Fc@PdNPs, a fluorescence / electrochemical dual-mode sensor was successfully constructed. This sensor can compare and judge the measurement results of the two aspects, greatly avoiding the occurrence of false detection and improving the accuracy of the sensor.

[0045] 2. The sensor built with aptamers as the identification element has the characteristics of anti-interference and strong adaptability, ensuring its practicality;

[0046] 3. MoS2@Fc@PdNPs has good conductivity and high specific surface area, and has strong adsorption capacity with aptamers, and has a strong adsorption capacity for N-GQDs-GP73 Apt It has a good quenching effect; at the same time, the Fc in MoS2@Fc@PdNPs has good electroactive activity and is a good in situ electrical signal probe, making the detection process simple and the results accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagram of the fluorescence / electrochemical biosensor constructed based on N-GQDs and MoS2@Fc@PdNPs for the detection of GP73.

[0048] Figure 2 A is the transmission electron microscopy image of N-GQDs; B is the transmission electron microscopy image of N-GQDs and N-GQDs-GP73 Apt Fluorescence spectrum of

[0049] Figure 3A is a scanning electron microscopy image of MoS2@Fc@PdNPs; B is an X-ray photoelectron spectroscopy image of MoS2@Fc@PdNPs, and the inset is the elemental content analysis;

[0050] Figure 4 SEM characterization of fluorescence / electrochemical biosensors; A: N-GQDs-Apt / MoS2@Fc@PdNPs / SPE; B: GP73 / NGQDs-Apt / MoS2-Fc-PdNPs / SPE;

[0051] Figure 5 Fluorescence spectra (A) and electrochemical DPV spectra (B) of the fluorescence / electrochemical biosensor detecting different GP73 concentrations. DETAILED DESCRIPTION

[0052] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0053] A fluorescence / electrochemical biosensor for detecting GP73 based on fluorescence resonance energy transfer of N-GQDs / MoS2@Fc@PdNPs combined with aptamers. The detection principle is shown in Figure 1 In N-GQDs-GP73 Apt MoS2@Fc@PdNPs, N-GQDs-GP73 were added to the fluorescent probe Apt Through van der Waals force and hydrogen bonding, fluorescence resonance energy transfer (FRET) occurs on the surface of MoS2@Fc@PdNPs. Apt The fluorescence of the system was quenched, and the fluorescence intensity of the whole system decreased. In addition, due to the adsorption of N-GQDs-GP73Apt on the surface of MoS2@Fc@PdNPs, the redox activity of Fc in MoS2@Fc@PdNPs was inhibited, thereby forming N-GQDs-GP73 Apt / MoS2@Fc@PdNPs fluorescence / electrochemical biosensor; after adding GP73 protein, due to GP73 Apt Specificity for GP73, GP73 preferentially binds to N-GQDs-GP73 Apt Combination to form N-GQDs-GP73 Apt-GP73 complex, the interaction between N-GQDs-GP73Apt and MoS2@Fc@PdNPs is greatly weakened, interrupting FRET, thereby restoring fluorescence in the system with high fluorescence intensity; the electroactivity of the detached MoS2@Fc@PdNPs is restored, enhancing the Fc redox activity in the MoS2@Fc@PdNPs. Based on the relationship between the fluorescence intensity recovery value before and after the addition of GP73 protein and the current change value of the electrochemical signal and GP73, a GP73 working curve was established, achieving rapid, sensitive, and selective quantitative detection of GP73. The implementation steps are as follows:

[0054] 1. Fluorescence resonance donor N-GQDs-GP73 Apt Preparation

[0055] (1) 2.1 g of citric acid and 1.8 g of urea were diluted to 15 mL with pure water, stirred until completely dissolved, and placed in a high-temperature reactor lined with Teflon. The reactor was then placed in a forced air drying oven and heated at 180°C for 8 h.

[0056] (2) After the reaction solution was cooled to room temperature, 30 mL of ethanol was added, and the mixture was thoroughly shaken and stirred. The solution was dialyzed for 8 h using a dialysis bag with a molecular weight of 300. The dialyzed solution was freeze-dried to obtain N-GQDs freeze-dried powder. Figure 2 A is a transmission electron microscopy image of N-GQDs. The prepared N-GQDs are uniform in size and well dispersed, with a particle size of about 7 nm.

[0057] (3) Weigh 7.668 mg of carbodiimide (EDAC) and 21.713 mg of N-hydroxysuccinimide (NHS), mix them and add pure water to 10 mL, stir evenly to obtain a crosslinker solution. Weigh 10 mg of N-GQDs and add pure water to 10 mL to obtain a 1.0 mg / mL N-GQDs solution. Measure 200 μL of N-GQDs and 200 μL of 1 μM GP73 Apt and 20 μL crosslinker solution, mixed evenly, and placed in a dark environment at room temperature of 25°C and shaken evenly, incubated for 1 hour to obtain the fluorescently labeled complex N-GQDs-GP73 Apt solution. Figure 2 B is N-GQDs and N-GQDs-GP73 Apt The fluorescence spectra of the two are basically the same, with the maximum excitation wavelength at 348nm and the maximum emission wavelength at 438nm. Apt The fluorescence intensity of N-GQDs is smaller than that of N-GQDs, indicating that N-GQDs and GP73 Apt Successfully connected.

[0058] 2. Preparation of fluorescence resonance receptor MoS2@Fc@PdNPs and construction of fluorescence / electrochemical biosensors

[0059] (1) 30 mg of MoS2 solid was weighed using a precision electronic balance and placed in a beaker. N,N-dimethylformamide solution (DMF) was added to a constant volume of 30 mL. The beaker was placed in an ultrasonic cell disruptor for 1 h. After the disruption was complete, a MoS2 dispersion with a concentration of 1 mg / mL was obtained. 60 mg of mercaptoethylamine was weighed and mixed with the MoS2 dispersion and stirred continuously for 12 h. The mixed liquid was removed and placed in a centrifuge for centrifugation at 6000 r / min for 5 min. After centrifugation, the resulting precipitate was repeatedly washed with pure water and dried in a vacuum low-temperature drying oven to obtain a black MoS2-NH2 solid.

[0060] (2) 30 mg of MoS2-NH2 solid and 90 mg of Fc powder were weighed into a beaker using a precision electronic balance. Pure water was added to make the volume 30 mL, and then 3 mL of cross-linker solution was added. The solution was then placed on a magnetic stirrer and stirred continuously for 24 h. The mixed liquid was taken out and placed in a centrifuge for centrifugation at 6000 r / min for 5 min. After centrifugation, the resulting precipitate was repeatedly washed with pure water and dried in a vacuum low-temperature drying oven to obtain a black MoS2-Fc solid.

[0061] (3) Use a precision electronic balance to weigh 10 mg MoS2-Fc, 18.26 mg Na2PdCl6 powder, 176.12 mg ascorbic acid (AA), and 242.16 mg sodium carboxymethyl cellulose (CMC, 800–1200 mPas) and place them in a beaker respectively. Add pure water to make the volume to 10 mL. Then, the MoS2-Fc solution, Na2PdCl6 solution, AA solution, and CMC aqueous solution are mixed in a volume ratio of 1000:1:5:1 and stirred for 30 minutes. Then, the mixed liquid is taken out and placed in a centrifuge for centrifugation at a speed of 6000 r / min for 5 minutes. After centrifugation, the resulting precipitate is repeatedly washed with pure water and placed in a vacuum low-temperature drying oven for low-temperature drying to obtain black MoS2@Fc@PdNPs solid. Figure 3 A is the scanning electron microscopy image of MoS2@Fc@PdNPs. It can be seen that particles of different sizes are deposited on the surface of the wrinkled sheet structure, which proves the successful modification of Fc and PdNPs. Figure 3 B is the X-ray photoelectron spectrum of MoS2@Fc@PdNPs. The spectrum shows that in addition to the common C1s (283.4eV) and O1s (543.1eV) elements, the measured material also has corresponding Mo3d (229.5eV), S2p (110eV), Pd3d (335.3eV) and Fe2p (710.6eV) elements, providing support for the existence of Fe and Pd. Figure 3 The inset in B shows that the material is rich in Fe and Pd elements, with Fe accounting for 6.48% and Pd accounting for 29.89%, which proves that the synthesis of MoS2-Fc-PdNPs is successful.

[0062] (4) Weigh 20 mg of MoS2@Fc@PdNPs solid using a precision electronic balance and place it in a beaker. Add pure water to make the volume 20 mL. Take 1 mL of MoS2@Fc@PdNPs solution and 100 μL of N-GQDs-GP73 Apt The solution was prepared into a mixed solution and incubated in a dark environment at 25°C for 30 minutes to form the N-GQDs-GP73Apt / MoS2@Fc@PdNPs fluorescence / electrochemical biosensor. The solution was placed in a centrifuge and centrifuged at 6000 rpm for 5 minutes. After centrifugation, the upper layer was scanned using a fluorescence spectrophotometer with an excitation wavelength of 348 nm. The fluorescence intensity F0 at 438 nm was measured. The lower layer was dropped onto the activated screen-printed electrode SPE and the electrode was characterized by scanning electron microscopy. Figure 4 As shown in A, it can be seen that the electrode surface is covered with granular and coated particles of different sizes, which is N-GQDs-GP73 Apt Adsorbed on the electrode surface modified with MoS2@Fc@PdNPs. Using the DPV scanning of the electrochemical workstation, the scanning range was -0.4V~1.0V, and the scanning rate was 0.01V / s, and the peak current C0 was recorded.

[0063] 3. Drawing of GP73 working curve

[0064] (1) Add 200 μL of GP73 protein solution (2.0 ng / mL, 4.0 ng / mL, 6.0 ng / mL, 8.0 ng / mL, 10.0 ng / mL) to 200 μL of the N-GQDs-GP73Apt / MoS2@Fc@PdNPs solution prepared in step 2, shake and mix evenly, and incubate in a dark environment at 25°C for 60 min. Place it in a centrifuge and centrifuge at a speed of 6000 r / min for 5 min. After centrifugation, take the upper layer and scan it with a fluorescence spectrophotometer. The excitation wavelength is fixed at 348 nm, and the fluorescence intensity F1 at 438 nm is measured; the lower layer is dropped on the activated screen-printed electrode, and the electrode is characterized by scanning electron microscopy, as shown in FIG. Figure 4 B. Comparison Figure 4 A shows that the particles on the electrode surface are reduced and there are more wrinkles, indicating that N-GQDs-GP73 Apt The detachment of the MoS2@Fc@PdNPs resulted in the surface exposure of the MoS2@Fc@PdNPs. The DPV scanning of the electrochemical workstation was performed in the range of -0.4V to 1.0V and the scanning rate was 0.01V / s, and the peak current value C1 was recorded. Figure 5 The fluorescence spectra of different GP73 concentrations detected by fluorescence / electrochemical biosensor ( Figure 5 A) and electrochemical DPV scanning diagram ( Figure 5 B). As can be seen, as the concentration of GP73 increases, the fluorescence intensity and electrochemical current value of the sensor both increase.

[0065] (2) Using the fluorescence recovery value (F1-F0) / F0 as the ordinate and the GP73 concentration as the abscissa, a working curve of fluorescence detection was drawn. When the GP73 protein concentration was in the range of 1.0-10.0 ng / mL, the relationship between the fluorescence recovery value (F1-F0) / F0 of the sensor and the GP73 concentration was linear. The working curve was Y=0.00604X+0.14327 (Y represents the fluorescence recovery value, X represents the concentration of GP73 protein), and the correlation coefficient was R 2 =0.98074, the minimum detection limit is 0.812 ng / mL (S / N=3); at the same time, the peak current change value △C, i.e., C1-C0 value, is used as the ordinate and the GP73 concentration is used as the abscissa to draw the working curve of electrochemical detection. When the GP73 protein concentration is in the range of 1.0-10.0 ng / mL, the peak current change value △C of the sensor is linearly related to the GP73 concentration. The working curve is Y=0.00375X-0.0011 (Y represents the peak current change value, X represents the GP73 protein concentration), and the correlation coefficient is R 2 =0.9941, and the minimum detection limit was 0.0425 ng / mL (S / N=3).

[0066] 4. Detection of GP73 in actual serum samples

[0067] (1) Three types of serum samples were collected, including serum from normal subjects, serum from patients with cirrhosis, and serum from patients with liver cancer, with 3 samples from each type of serum. The GP73 levels in the three types of serum were determined using the ELISA method for clinical detection of GP73. The GP73 concentrations in the serum of normal subjects were 1.31 ng / mL, 2.03 ng / mL, and 2.43 ng / mL, respectively; the GP73 concentrations in the serum of patients with cirrhosis were 28.49 ng / mL, 23.9 ng / mL, and 36.72 ng / mL; and the GP73 concentrations in the serum of patients with liver cancer were 126.68 ng / mL, 93.76 ng / mL, and 151.94 ng / mL. 200 μL of each serum sample was added to 200 μL of N-GQDs-GP73. AptThe sample was mixed with the MoS2@Fc@PdNPs fluorescence / electrochemical biosensor solution by vortexing and incubating at 25°C in a dark environment for 60 minutes. The sample was centrifuged at 6000 rpm for 5 minutes. After centrifugation, the fluorescence intensity and electrochemical peak current were measured using the method for measuring GP73 protein in step 3. Each serum sample was measured three times.

[0068] (2) Based on the working curve Y = 0.00375X-0.0011 for electrochemical detection and the working curve Y = 0.02079X+0.06762 for fluorescence detection obtained in step 3, the corresponding concentration of GP73 in the actual serum sample can be calculated. The results of fluorescence detection are shown in Table 1, and the results of electrochemical detection are shown in Table 2. As can be seen from Table 1, the relative error of the fluorescence / electrochemical biosensor for the fluorescence detection of GP73 in the actual serum sample is between 1.58% and 7.28%, and the relative standard deviation is between 1.62% and 5.62%, respectively, compared with the ELISA method. As can be seen from Table 2, the relative error of the fluorescence / electrochemical biosensor for the electrochemical detection of GP73 in the actual serum sample is between 0.38% and 7.87%, and the relative standard deviation is between 0.15% and 6.62%, respectively, compared with the ELISA method. Both detection results are in line with expectations, indicating that the fluorescence / electrochemical biosensor can be applied to the detection of GP73 in actual serum samples.

[0069] Table 1 Fluorescence detection results of GP73 in actual serum samples

[0070]

[0071]

[0072] Table 2 Electrochemical detection results of GP73 in actual serum samples

[0073]

[0074] (Note: Serum samples were obtained from the Guangxi Key Laboratory of Metabolic Disease Research, 924th Hospital of the Chinese People's Liberation Army (Guilin, China), and followed the requirements of the Ethics Committee of the Guangxi Key Laboratory of Metabolic Disease Research, 924th Hospital of the Chinese People's Liberation Army.)

Claims

1. A fluorescence / electrochemical biosensor for the detection of Golgi protein GP73 based on fluorescence resonance energy transfer for non-diagnostic purposes is constructed, which is carried out in the following steps: Step 1: Fluorescence resonance donor N-GQDs-GP73 Apt Preparation (1) Preparation of nitrogen-doped graphene quantum dots N-GQDs: Weigh citric acid and urea, add pure water to the volume and stir evenly, heat at high temperature for a certain time, cool and add ethanol to mix and stir, after stirring completely, dialyze for a period of time, and freeze-dry the dialyzed solution to obtain N-GQDs powder; (2)N-GQDs-GP73 Apt Preparation: Measure N-GQDs and GP73 aptamer GP73 Apt Activate with carbodiimide EDAC / N-hydroxysuccinimide NHS crosslinker, incubate with stirring for a certain time at room temperature and in the dark to obtain N-GQDs-GP73 Apt solution; Step 2: Preparation of fluorescence resonance receptor molybdenum disulfide@ferrocene@palladium nanoparticles MoS2@Fc@PdNPs and construction of fluorescence / electrochemical biosensor (1) Weighing MoS2 powder, adding N,N-dimethylformamide solution (DMF) to the fixed volume, and crushing it in an ultrasonic cell crusher until the MoS2 powder is completely dispersed in DMF to obtain a MoS2 dispersion; Weigh mercaptoethylamine, add it to the MoS2 dispersion, stir thoroughly, centrifuge, wash the separated precipitate, and dry it to obtain MoS2-NH2 powder; (2) Weighing MoS2-NH2 powder and ferrocene (Fc) powder, mixing them into a solution, adding a crosslinker solution, stirring thoroughly, and centrifuging. The separated precipitate is washed and dried to obtain MoS2-Fc powder; (3) MoS2-Fc powder, Na2PdCl6, ascorbic acid (AA), and sodium carboxymethyl cellulose (CMC) were weighed and prepared into solutions, the prepared solutions were mixed and stirred for a period of time, the resulting solution was centrifuged, and the separated precipitate was washed and dried to obtain MoS2@Fc@PdNPs powder; (4) Weigh the MoS2@Fc@PdNPs powder and prepare a solution. Mix the MoS2@Fc@PdNPs solution and N-GQDs-GP73 Apt The solutions were mixed and incubated for a period of time in a dark environment at a certain temperature to form N-GQDs-GP73 Apt / MoS2@Fc@PdNPs fluorescence / electrochemical biosensor; centrifuge, take the upper layer of liquid and scan it with a fluorescence spectrophotometer to record its fluorescence intensity F0, drop the lower layer of liquid on the activated screen-printed electrode, use the DPV scanning of the electrochemical workstation to record its peak current value C0; Step 3: Drawing the GP73 working curve (1) Add different concentrations of GP73 solution to N-GQDs-GP73 Apt / MoS2@Fc@PdNPs FRET fluorescence / electrochemical biosensor solution, incubated in a dark environment at a certain temperature for a period of time, centrifuged, and the upper layer liquid was scanned using a fluorescence spectrophotometer to record its fluorescence intensity F1; the lower layer liquid was dropped onto the activated screen-printed electrode, and the DPV scanning of the electrochemical workstation was used to record its peak current value C1; (2) Using (F1-F0) / F0 as the ordinate and the GP73 concentration as the abscissa, a working curve was drawn to calculate the minimum detection limit of the fluorescence / electrochemical biosensor in fluorescence detection; using ΔC (i.e., C1-C0 value) as the ordinate and the GP73 concentration as the abscissa, a working curve was drawn to calculate the minimum detection limit of the fluorescence / electrochemical biosensor in electrochemical detection; Step 4: Detection of GP73 in actual serum samples (1) Collect serum samples with known GP73 concentrations and add the serum samples to be tested to the N-GQDs-GP73 in step 2. Apt The FRET fluorescence / electrochemical biosensor solution was incubated in a dark environment at a certain temperature for a period of time, and then centrifuged. The upper layer was scanned using a fluorescence spectrophotometer with an excitation wavelength of 348 nm to measure the fluorescence intensity at 438 nm. The lower layer was dropped onto the activated screen-printed electrode and scanned using a DPV electrochemical workstation to record the peak current value. (2) Calculate the concentration of GP73 in the serum sample to be tested based on the GP73 working curve obtained in step 3.

2. The detection method of the fluorescence / electrochemical biosensor according to claim 1, characterized in that: The molecular weight of the dialysis bag used for dialysis in step 1 is 300, and the dialysis time is 6 hours.

3. The detection method of the fluorescence / electrochemical biosensor according to claim 1, characterized in that: The concentration of N-GQDs in step 1 is 1.0 mg / mL; the GP73 Apt The concentration is 1.0 μM; the cross-linking agent contains carbodiimide EDAC and N-hydroxysuccinimide NHS, wherein the concentration of EDAC is 0.7668 mg / mL and the concentration of NHS is 2.1713 mg / mL; N-GQDs solution, GP73 Apt The volume ratio of solution to cross-linker solution was 10:10:1, and the incubation time was 1 h.

4. The detection method of the fluorescence / electrochemical biosensor according to claim 1, characterized in that: In step 2, the concentration of the MoS2-Fc solution is 1.0 mg / mL; the concentration of the Na2PdCI6 solution is 1.826 mg / mL; the concentration of the ascorbic acid solution is 17.612 mg / mL; and the concentration of the sodium carboxymethyl cellulose aqueous solution is 24.216 mg / mL.

5. The detection method of the fluorescence / electrochemical biosensor according to claim 1, characterized in that: In step 2, step 3 and step 4, the incubation temperature is 25° C. and the incubation time is 60 min.

6. The detection method of the fluorescence / electrochemical biosensor according to claim 1, characterized in that: In steps 2, 3, and 4, the excitation wavelength of the fluorescence spectrophotometer is 348 nm, and the emission wavelength is 438 nm; the DPV linear scanning range is -0.4 V to 1.0 V, and the scanning rate is 0.01 V / s.

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