Fluorescent aptamer sensor based on few-layered v2ctx as quencher and application thereof

Abstract

This invention discloses a method based on few-slice V2CT x A fluorescent aptamer sensor for use with a quencher and its application includes a quencher, an aminated PSA aptamer, and carboxylated graphene quantum dots, wherein the quencher, the aminated PSA aptamer, and the carboxylated graphene quantum dots are dispersed in a solvent; the quencher is a few-layer V2CT. x This invention, through comparison of different quenching platforms, concludes that FL-V2CT-based... x The fluorescence recovery intensity of the PSA fluorescent aptamer sensor is significantly improved compared to existing technologies. This aptamer sensor has high selectivity for PSA detection and can be practically applied to the detection of PSA in human serum samples.

Description

Technical Field

[0001] This invention relates to the field of specific antigen detection technology, specifically to a method based on few-slice V2CT. x Fluorescent aptamer sensor for quenching agent and its application in prostate-specific antigen detection. Background Technology

[0002] Prostate cancer is one of the most common cancers among men worldwide. Prostate-specific antigen (PSA), a serine protease produced by prostate tissue, is one of the most reliable biomarkers for early-stage prostate cancer. Prostate cancer causes PSA to be released into the circulation, increasing its level in the blood. When the PSA concentration in a person's serum exceeds 4 ng / mL, the likelihood of having prostate cancer is high. Currently, many methods are used to detect PSA, such as enzyme-linked immunosorbent assay (ELISA), surface-enhanced Raman scattering (SERS), and electrochemical and fluorescent aptamer sensors. Among these methods, fluorescent aptamer sensors have attracted much attention due to their simplicity, efficiency, and high sensitivity.

[0003] The performance of PSA fluorescent aptamer sensors depends on the fluorophore and the quencher, which can effectively quench the fluorophore. Currently, many nanomaterials have been developed as quenchers for fluorescent aptamer sensors and applied to the detection of PSA: such as graphene oxide (GO), MoS2 nanosheets, metal-organic frameworks (MOFs), and Au nanoclusters.

[0004] Multilayer MXenes are typically prepared by etching MAX phase precursors. Multilayer MXenes (ML-MXenes) possess large specific surface areas, high conductivity, good biocompatibility, and excellent hydrophilicity. MXenes have been used in batteries, supercapacitors, surface-enhanced Raman scattering, fluorescent aptamer sensors, and electrochemical sensors. Among these applications, fluorescent aptamer sensors have attracted increasing attention due to their fast response, simplicity, and high sensitivity. ML-Ti3C2T, with its abundant hydrophilic functional groups, is a particularly promising candidate. x MXenes have been used to develop fluorescent aptamer sensors. For example, Zhang and colleagues (Q. Zhang, F. Wang, H. Zhang, Y. Zhang, M. Liu, Y. Liu, Universal Ti3C2 MXenes based self-standard ratiometric fluorescence resonance energy transfer platform for highly sensitive detection of exosomes, Anal. Chem. 90 (2018) 12737-12744.) constructed a fluorescent aptamer sensor in which anthocyanin 3-labeled aptamer and ML-Ti3C2Tx As a quencher for exosome detection, Cui et al. (H.Cui,X.Fu,L.Yang,S.Xing,XFWang, 2D titanium carbide nanosheets based fluorescent aptasensor for sensitive detection of thrombin, Talanta 228(2021)122219.) designed a quencher based on ML-Ti3C2T. x A fluorescent aptamer sensor was used to detect thrombin. Furthermore, Wang et al. designed a chimeric DNA-functionalized ML-Ti3C2T... x (S.Wang, S.Wei, S.Wang, X.Zhu, C.Lei, Y.Huang, Chimeric DNA-functionalized titanium carbide MXenes for simultaneous mapping of dual cancer biomarkers in living cells, Anal.Chem.91(2019)1651-1658.) This is used to detect biomarkers in living cells.

[0005] Few-layer MXenes (FL-MXenes) are prepared by layering ML-MXenes with tetramethylammonium hydroxide (TMAOH). FL-MXenes have been applied in surface-enhanced Raman scattering, sensors, and lithium-ion capacitors. However, few-layer V2CT... x and Ti3C2T x MXene has not yet been reported as a quencher in fluorescent aptamer sensors. Summary of the Invention

[0006] This invention provides a method based on few-slice V2CT x It is a fluorescent aptamer sensor for quenchers, characterized by ease of construction, high sensitivity, and selectivity.

[0007] A method based on few-slice V2CT x A fluorescent aptamer sensor with a quencher includes a quencher, an aminated PSA aptamer, and carboxylated graphene quantum dots, wherein the quencher, aminated PSA aptamer, and carboxylated graphene quantum dots are dispersed in a solvent; the quencher is a few-layer V2CT. x .

[0008] Optionally, the concentration of the quencher solution prepared by dispersing the quencher in a solvent is 20–30 mg / mL. –1 .

[0009] Optional, few-slice V2CT x (FL-V2CT x Preparation method of ) by layering multiple V2CTs with tetramethylammonium hydroxide (TMAOH). x (ML-V2CT x It was prepared by [method name missing].

[0010] A specific preparation method: The preparation of the quencher includes:

[0011] Multi-layer V2CT x Add to an aqueous solution of tetramethylammonium hydroxide to obtain a mixture; stir the resulting mixture at 20–30°C for 10–15 hours, then wash, adjust the pH to neutral, and dry to obtain the few-layer V2CT. x denoted as FL-V2CT x ;

[0012] Multislice V2CT x The mass-to-volume ratio of the tetramethylammonium hydroxide solution added is 0.15–0.5 g: 20 mL, and the mass percentage concentration of the tetramethylammonium hydroxide solution is 20–30%.

[0013] More specifically, 0.2g ML-V2CT x Add to 20 mL of TMAOH solution (25% in water); stir the mixture at 25 °C for 12 hours; after centrifugation, wash the resulting product with water and adjust the pH to 7; dry the product overnight at 65 °C under vacuum to obtain FL-V2CT. x .

[0014] Optionally, the concentration of the aptamer solution obtained by dispersing the aminated PSA aptamer in a solvent is 1–2 μM.

[0015] Optionally, the concentration of the quantum dot solution obtained by dispersing the carboxylated graphene quantum dots in a solvent is 15–2.5 mg / mL. –1 .

[0016] Optionally, the base sequence of the PSA aptamer is shown in SEQ ID NO: 1.

[0017] The base sequence of the PSA aptamer is: 5'-AATTAAAGCTCGCCCATCAAATAGC(SEQ ID NO: 1)-3'.

[0018] The sequence of the amino-modified PSA aptamer:

[0019] 5'-NH2C6-AATTAAAGCTCGCCCATCAAATAGC (SEQ ID NO: 1)-3'.

[0020] Optionally, it also includes EDC and NHS. The concentration of the EDC solution obtained by dispersing the EDC in a solvent is 25–30 mg / mL. –1 The concentration of the NHS solution obtained by dispersing NHS in a solvent is 15–20 mg / mL. –1 .

[0021] Optionally, the solvent may be PBS, such as PBS (10mM, pH 7.4).

[0022] Furthermore, the concentration of the quencher solution is 24–26 mg / mL. –1 The concentration of the carboxylated graphene quantum dot solution is 1.8–2.2 mg / mL. –1 The concentration of the aminated PSA aptamer solution is 1.4–1.6 μM.

[0023] Furthermore, the concentration of the quencher solution is 25 mg / mL. –1 The concentration of the carboxylated graphene quantum dot solution is 2 mg / mL. –1 The concentration of the aminated PSA aptamer solution is 1.5 μM.

[0024] Optionally, the carboxylated graphene quantum dots can be obtained commercially.

[0025] Optionally, the hydroxylated PSA aptamer can be commercially available or synthesized by a contract manufacturer.

[0026] The present invention also provides an application of the fluorescent aptamer sensor as described above in the preparation of PSA early diagnostic products.

[0027] The present invention also provides a method for detecting PSA concentration in vitro for non-diagnostic purposes using the aforementioned fluorescent aptamer sensor, comprising:

[0028] (1) The aminated PSA aptamer and the carboxylated graphene quantum dots are mixed and the aminated PSA aptamer and the carboxylated graphene quantum dots are connected by a condensation reaction to obtain the first reaction solution.

[0029] (2) Add the test solution containing PSA to the first reaction solution and incubate the mixture at 37°C for 40-60 min to obtain the second reaction solution;

[0030] (3) Add the quencher to the second reaction solution and incubate at 37°C for 10-30 min to obtain the third reaction solution;

[0031] (4) The fluorescence recovery intensity ΔF of the third reaction solution was measured at an excitation wavelength of 327 nm and an emission wavelength of 446 nm. The obtained fluorescence recovery intensity ΔF was substituted into the standard curve to calculate the PSA concentration in the test solution.

[0032] Optionally, the fluorescence recovery intensity ΔF is calculated as follows:

[0033] ΔF = F - F0;

[0034] Where F0 is the fluorescence intensity of the fluorescent aptamer sensor at 446 nm without PSA, and F is the fluorescence intensity of the reaction solution at 446 nm after the addition of PSA.

[0035] Optionally, the condensation reaction is carried out by adding EDC and NHS.

[0036] Specifically, the carboxylated graphene quantum dot solution is first mixed with EDC solution and NSH solution to obtain a mixture, and then the mixture is mixed with the aminolated PSA aptamer solution and incubated overnight at 37°C.

[0037] Optionally, the amount of quencher added is such that its final concentration in the detection system is 1.8 mg / mL. -1 The PSA concentration in the test solution ranges from 0.1 to 20 ng / mL. -1 Within this ratio range, the fluorescence recovery intensity (ΔF) corresponds to a PSA concentration of 0.1 to 20 ng / mL. -1 A good linear relationship was observed within the range.

[0038] Optionally, the pH of the detection system is controlled at 7.4 throughout the detection process.

[0039] Optionally, the incubation time in step (2) is 60 min, and the incubation time in step (3) is 30 min.

[0040] Carboxylated graphene quantum dots (CGQDs) and aminated PSA aptamers are linked via a conjugation reaction, after which apt-CGQDs are strongly adsorbed onto FL-V2CT. x The fluorescence of CGQDs is quenched through a photoinduced electron transfer process. Upon addition of PSA, the fluorescence of apt-CGQDs recovers. This is compared to four other quenching materials, namely ML-V2CT. x FL-Ti3C2T x ML-Ti3C2T x Compared to GO, FL-V2CT x It exhibits the highest fluorescence recovery intensity, indicating that FL-V2CT x It has the advantage of a large specific surface area.

[0041] This invention uses few-slice V2CT x (FL-V2CT x A novel prostate-specific antigen (PSA) fluorescent aptamer sensor was constructed using tetramethylammonium hydroxide (TMAOH) as a quencher. First, a multilayer V2CT sensor was constructed using tetramethylammonium hydroxide (TMAOH). x (ML-V2CT x Preparation of FL-V2CT x Then, an aminated PSA aptamer and a carboxylated graphene quantum dot were linked by a condensation reaction to prepare aptamer-carboxylated graphene quantum dot probes (apt-CGQDs); finally, the apt-CGQDs were adsorbed onto FL-V2CT via hydrogen bonding. x On the surface, this leads to a decrease in the fluorescence of apt-CGQDs due to photoinduced energy transfer. When used for PSA detection, upon the addition of PSA, the PSA-apt-CGQDs complex is released from FL-V2CT. x The fluorescence is released from the surface and greatly restored.

[0042] The invention constructs a FL-V2CT-based x The sensing platform exhibits high sensitivity, with a linear range of 0.1 ng / mL. -1 Up to 20 ng / mL -1 The detection limit is 0.03 ng / mL. -1 This invention, through comparison of different quenching platforms, concludes that FL-V2CT-based... x The fluorescence recovery intensities of the PSA fluorescent aptamer sensor are respectively ML-V2CT x FL-Ti3C2T x ML-Ti3C2T x The 5.6, 3.7, 7.7, and 5.4 times higher values ​​compared to GO demonstrate the advantages of fewer layers. This aptamer sensor exhibits high selectivity for PSA detection and can be practically applied to the detection of PSA in human serum samples.

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

[0044] This invention uses FL-V2CT x MXene is a fluorescent aptamer sensor for detecting PSA (phosphoassay). FL-V2CT x It is composed of multi-layer V2CT x (ML-V2CT x It was prepared by layering with tetramethylammonium hydroxide (TMAOH). Furthermore, carboxylated graphene quantum dots (CGQDs) and aminated PSA aptamers were linked via a conjugation reaction. Subsequently, apt-CGQDs were strongly adsorbed onto FL-V2CT. xThe fluorescence of CGQDs is quenched through a photoinduced electron transfer process. Upon addition of PSA, the fluorescence of apt-CGQDs recovers. This is compared to four other quenching materials, namely ML-V2CT. x FL-Ti3C2T x ML-Ti3C2T x Compared to GO, FL-V2CT x It exhibits the highest fluorescence recovery intensity. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the fluorescent aptamer sensor for PSA detection according to the present invention;

[0046] Figure 2 For ML-V2CT x (A) and FL-V2CT x (B) SEM image; ML-V2CT x (C) and FL-V2CT x (D) TEM image;

[0047] Figure 3 For ML-V2CT x (a) and FL-V2CT x (b) EDS spectrum;

[0048] Figure 4 For ML-V2CT x (a) and FL-V2CT x (b) XPS spectrum;

[0049] Figure 5 The fluorescence spectra used to verify the feasibility of the fluorescent aptamer sensor for detecting PSA in this invention are shown below. From top to bottom, the curves are a(apt-CGQDs) and b(PSA+apt-CGQDs+FL-V2CT). x ), c(apt-CGQDs+FL-V2CT x The fluorescence spectrum of )

[0050] Figure 6 For different concentrations of FL-V2CT x Fluorescence changes in PSA detected by the prepared fluorescent aptamer sensor, from top to bottom FL-V2CT x The concentrations were, in order: a (0 mg / mL), b (0.3 mg / mL). -1 c (0.6 mg mL) –1 ), d (1.2 mg mL) –1 e (1.5 mg / mL) –1 f(1.8mg mL) –1), g (2.1 mg mL) –1 );

[0051] Figure 7 FL-V2CT x Relationship between concentration and detected fluorescence intensity, FL-V2CT x The concentrations are as follows: a(0 mg mL) –1 b (0.3 mg mL) –1 c (0.6 mg mL) –1 ), d (1.2 mg mL) –1 ), e (1.5 mg mL) –1 f(1.8mg mL) –1 ), g (2.1 mg mL) –1 );

[0052] Figure 8 FL-V2CT x Relationship between fluorescence recovery response value ΔF;

[0053] Figure 9 For a(apt-CGQDs), b(apt-CGQDs+FL-V2CT x ), c(FL-V2CT x The visible absorption spectrum of )

[0054] Figure 10 Graph showing the relationship between different types of quenchers and different PSA concentrations and fluorescence recovery response value ΔF;

[0055] Figure 11 This is a graph showing the relationship between pH value and fluorescence recovery response value ΔF.

[0056] Figure 12 This is a graph showing the relationship between recovery time after quenching and fluorescence recovery response value ΔF.

[0057] Figure 13 A graph showing the relationship between incubation time and fluorescence recovery response value ΔF;

[0058] Figure 14 The fluorescence emission spectra of the fluorescent aptamer sensor at different concentrations of PSA are shown, with the concentrations from bottom to top being a(0 ng / mL). –1 b (0.1 ng mL) –1 c(0.5ng mL) –1 ), d(3ng mL –1 ), e(5ng mL –1 f(10ng mL) –1 ), g (15ng mL) –1 h(20ng mL) –1);

[0059] Figure 15 This is a standard curve of PSA concentration versus fluorescence recovery response value ΔF;

[0060] Figure 16 The fluorescence recovery response of PSA, CEA, AFP, CA125, HSA and BSA was detected using a fluorescent aptamer sensor; the concentrations of PSA and other interfering substances were 15 ng / mL.

[0061] Figure 17 The graph shows the effect of the fluorescent aptamer sensor on the fluorescence recovery response of PSA in the presence of 3 times the concentrations of CEA, AFP, CA125, HSA, and BSA, where the PSA concentration is 15 ng / mL.

[0062] Figure 18 Infrared spectra of CGQDs;

[0063] Figure 19 Transmission electron microscopy (TEM) images of CGQDs;

[0064] Figure 20 The fluorescence (a) excitation (b) emission spectra of CGQDs are shown. Detailed Implementation

[0065] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0067] Example 1: Preparation of FL-V2CT x

[0068] 0.2 g of ML-V2CTx (commercially available) was added to 20 mL of TMAOH solution (25 wt% in water); the mixture was stirred at 25 °C for 12 hours; after centrifugation, the resulting product was washed with water and the pH was adjusted to 7; the product was dried overnight at 65 °C under vacuum to obtain FL-V2CTx.

[0069] Example 2: Fluorescence detection of PSA

[0070] The following solutions were prepared in PBS (10 mM, pH 7.4): 28 mg / mL –1 EDC, 16mg mL –1 NHS, 2 mg / mL –1 Carboxylated CGQD and 1.5 μM aminolated PSA aptamer (5'-NH2C6-AATTAAAGCTCGCCCATCAAATAGC(SEQID NO: 1)-3').

[0071] A total of 980 μL of carboxylated CGQDs solution (2 mg / mL) was prepared. –1 ) and 10.5 μL EDC (28 mg mL) –1 ) and 11.0 μL NHS (16 mg mL) –1 Mix and sonicate for 40 minutes. Then, add 70 μL of the resulting mixture to 50 μL of aminoated PSA aptamer (1.5 μM) and incubate overnight at 37 °C to obtain apt-CGQD solution.

[0072] Different concentrations of PSA (non-aminated, as the target) were added to the apt-CGQDs solution, and the mixture was incubated at 37°C for 60 minutes.

[0073] Then, 30 μL of FL-V2CT was added. x (25mg mL –1 The sample was diluted to 400 μL with PBS. After incubation at 37 °C for 10 minutes, the fluorescence spectrum was measured at an excitation wavelength of 327 nm (excitation slit width: 10 nm, emission slit width: 10 nm).

[0074] The fluorescence recovery intensity ΔF is determined using the formula: ΔF = F - F0, where F0 is the fluorescence intensity of apt-CGQDs-FL-V2CT without PSA. x The fluorescence intensity at 446 nm, F represents the fluorescence intensity of apt-CGQDs-FL-V2CT with added PSA. x Fluorescence intensity at 446 nm.

[0075] Detection mechanism:

[0076] Figure 1 FL-V2CT was displayed x Preparation and FL-V2CT based x The mechanism of PSA detection using a fluorescent aptamer sensor. First, the ML-V2CT... x The reaction with TMAOH resulted in layering to obtain FL-V2CT. xAmination-modified PSA aptamers are functionalized onto CGQDs to obtain apt-CGQDs. apt-CGQDs are then attached to FL-V2CT. x On the surface, the fluorescence of apt-CGQDs is reduced due to photoinduced energy transfer. After the addition of PSA, the binding of PSA to apt-CGQDs leads to FL-V2CT... x The PSA-apt-CGQDs complex is released and fluorescence is restored. Therefore, PSA is detected based on fluorescence changes, and an FL-V2CT-based detection method is constructed. x Fluorescent aptamer sensor.

[0077] TEM and SEM characterization:

[0078] SEM images show that ML-V2CT x It exhibits a multi-layered and accordion-like structure. Figure 2 (A) and the synthesized FL-V2CT x It has a sheet-like and multi-layered structure. Figure 2 (Prepared in Example 1, B). TEM images show that, compared with ML-V2CT... x Compared to TEM ( Figure 2 (D), FL-V2CT x Electron transmission electron microscopy (TEM) Figure 2 (C) shows FL-V2CT x Thin-section features. SEM and TEM results indicate that ML-V2CT... x Successfully stratified.

[0079] Elemental composition characterization:

[0080] Obtaining EDS spectra to monitor ML-V2CT x and FL-V2CT x The elements that make up the composition, such as Figure 3 As shown, from ML-V2CT x ( Figure 3 a) and FL-V2CT x ( Figure 3 The presence of O, F, V, and C was observed in the EDS spectrum of the sample prepared in Example b (Prepared in Example 1); the presence of O, F, V, and C was observed in the XPS spectrum of the sample from ML-V2CT. x ( Figure 4 a) and FL-V2CT x ( Figure 4 The presence of O1s, V2p, C1s, and F1s responses in b) is clearly demonstrated by EDS and XPS results. x The chemical properties did not change after the layers were separated.

[0081] Functional group characteristics:

[0082] The present invention also tested the FTIR spectra of carboxylated CGQDs to determine the functional groups contained therein, at 1734 and 1407 cm⁻¹. -1 The absorption bands at these locations correspond to the C=O and -OH vibrations of the carboxyl group in CGQDs, respectively. Figure 18 Carboxylated CGQDs are spherical and well dispersed, with a size of 5-10 nm visible in TEM. Figure 19 Furthermore, this invention investigated the fluorescence excitation and emission spectra of carboxylated CGQDs. The maximum excitation and emission wavelengths of carboxylated CGQDs were located at 322 nm and 442 nm, respectively. Figure 20 ).

[0083] Based on FL-V2CT x The fluorescent aptamer sensor is used for PSA detection and feasibility testing of fluorescence quenching mechanisms:

[0084] Based on Examples 1 and 2, this invention studies the use of FL-V2CT x Feasibility of PSA fluorescent aptamer sensors as quenchers ( Figure 5 In the figure, the curves from top to bottom are a(apt-CGQDs) and b(PSA+apt-CGQDs+FL-V2CT), respectively. x ), c(apt-CGQDs+FL-V2CT x ), d(FL-V2CT x ), e (aminated PSA aptamer).

[0085] The results showed that FL-V2CT x The aminated PSA aptamer showed no fluorescence emission in the wavelength range of 390 nm to 600 nm, while apt-CGQDs exhibited a strong fluorescence signal at approximately 446 nm (corresponding to figure a). The addition of FL-V2CT... x In the case of FL-V2CT x Effective quenching (corresponding to c in the figure). And when PSA is combined with apt-CGQDs and FL-V2CT... x In the constructed fluorescence sensor system, due to the specific interaction between PSA and the aptamer, the PSA-apt-CGQDs complex is released from FL-V2CT. x Surface detachment is macroscopically manifested as fluorescence recovery (corresponding to b in the figure). This indicates that the FL-V2CT constructed in this paper... x Fluorescent aptamer sensors, acting as quenchers, can be used to detect PSA.

[0086] FL-V2CT x The effect of concentration on sensor sensitivity:

[0087] Due to FL-V2CT x The concentration of [a specific substance] has a significant impact on the sensitivity of the sensor. This invention first optimized it by adjusting the FL-V2CT [process] in Example 2. x The dosage was adjusted so that the final concentration in the detection system was a(0 mg / mL). -1 b (0.3 mg mL) -1 c (0.6 mg mL) -1 ), d (1.2 mg mL) -1 ), e (1.5 mg mL) -1 f(1.8mg mL) -1 ), g (2.1 mg mL) -1 ).

[0088] The results are as follows Figure 6 As shown. From top to bottom, FL-V2CT x The concentrations are as follows: a(0 mg mL) -1 b (0.3 mg mL) -1 c (0.6 mg mL) -1 ), d (1.2 mg mL) -1 ), e (1.5 mg mL) -1 f(1.8mg mL) -1 ), g (2.1 mg mL) -1 The results showed that after the addition of PSA, apt-CGQDs could bind with PSA to form a stable structure, thus achieving a stable structure at 1.8 mg / mL. -1 FL-V2CT x A relatively high fluorescence signal was observed at the concentration ( Figure 6 (f). However, when FL-V2CT x The concentration was 2.1 mg / mL. -1 hour( Figure 6 The fluorescence recovery rate was low in the medium g), indicating that high concentrations of FL-V2CT... x The stronger interaction force with the aptamer complex makes it difficult for the PSA-bound aptamer complex to detach from FL-V2CT. x Therefore, 1.8 mg / mL -1 As FL-V2CT x The optimized concentration will be used for subsequent experiments.

[0089] Furthermore, the time dependence of fluorescence quenching indicates that apt-CGQDs and FL-V2CT x The interaction between them reaches equilibrium after about 10 minutes. Figure 8 This sudden quenching can be attributed to the transition from apt-CGQD to FL-V2CT. xPhotoinduced electron transfer, apt-CGQDs via carboxyl groups and FL-V2CT x The O (or F) atoms interact with FL-V2CT through hydrogen bonding. x Combined, FL-V2CT x It accepts electrons from CGQDs and quenches their fluorescence.

[0090] apt-CGQDs and FL-V2CT x This interaction can be confirmed by ultraviolet-visible spectroscopy. Figure 9 , a(apt-CGQDs), b(apt-CGQDs+FL-V2CT x ), c(FL-V2CT x )). apt-CGQD and the addition of FL-V2CT x The apt-CGQD spectrum exhibits absorption peaks at 332 nm and 258 nm, respectively. The difference between the two spectra indicates that apt-CGQD and FL-V2CT... x There is a strong interaction between them.

[0091] Comparison of different quenching materials:

[0092] To explore the synthetic FL-V2CT x The present invention studies fluorescence aptamer sensors based on different quenchers for different PSA concentrations (i.e., the concentration of PSA added during detection) and different PSA concentrations. Figure 10 ). (Using FL-Ti3C2T) x The aptamer sensor for the quencher showed the highest fluorescence recovery signal at different PSA concentrations. FL-V2CT x Aptamer sensor for 15 ng mL -1 The fluorescence recovery intensities of PSA were ML-V2CT. x FL-Ti3C2T x ML-Ti3C2T x Compared to GO, the values ​​were 5.6, 3.7, 7.7, and 5.4 times higher. Compared to ML-V2CT... x Compared to FL-V2CT x The performance has been greatly improved; compared with FL-Ti3C2T x Compared to FL-V2CT x It exhibits higher fluorescence recovery because the strongly metallic Ti-based FL-MXene demonstrates a stronger interaction than V-based FL-MXene. Therefore, it is adsorbed onto FL-Ti3C2T... xapt-CGQDs on the material surface are more difficult to separate from the aptamer sensor. The GO-based aptamer sensor exhibits poor fluorescence recovery, which can be attributed to the strong interaction between the two graphene materials. The results indicate that the FL-V2CT-based... x The fluorescent aptamer sensor exhibited excellent performance in PSA assays.

[0093] Condition optimization:

[0094] This paper optimizes various experimental parameters, including pH, quenching time, and FL-V2CT. x The incubation time. The fluorescence recovery intensity was highest at pH 7.4. Figure 11 Therefore, pH 7.4 was chosen for the experiment. Before 30 minutes, the fluorescence recovery intensity increased with increasing quenching time. Then, apt-CGQDs and FL-V2CT... x Incubation time increased from 30 to 50 minutes, and fluorescence recovery decreased over time. Figure 12 Therefore, a fixed recovery time of 30 minutes was used in the experiment. The incubation time of apt-CGQDs and PSA was also an important factor. The fluorescence recovery intensity of apt-CGQDs and PSA was highest at an incubation time of 60 minutes. Figure 13 Therefore, 60 minutes was chosen as the incubation time.

[0095] Based on FL-V2CT x Fluorescent aptamer sensors are used for PSA detection:

[0096] The PSA was measured using the FL-V2CT-based method designed and constructed in this invention. x The fluorescence intensity increased with increasing PSA concentration (the PSA concentration in the test solution at the time of detection) using a fluorescent aptamer sensor. Figure 14 The concentrations from bottom to top are a(0 ng / mL). –1 b (0.1 ng mL) –1 c(0.5ng mL) –1 ), d(3ng mL –1 ), e(5ng mL –1 f(10ng mL) –1 ), g (15ng mL) –1 h(20ng mL) –1 ).

[0097] The results showed that the fluorescence recovery intensity (ΔF) was related to the PSA concentration from 0.1 to 20 ng / mL. -1 A good linear relationship was observed within the range ( Figure 15 The linear equation is as follows: F = 137.14CPSA +86.23 (R = 0.9994). The detection limit of this aptamer sensor is 0.03 ng / mL. –1 (S / N = 3). This invention also compared this aptamer sensor with previous PSA fluorescent aptamer sensors (Table 1), showing that the aptamer sensor proposed in this invention has the highest sensitivity to PSA. Therefore, based on apt-CGQDs as fluorescent probes and FL-V2CT... x Aptamer sensors, as quenchers, have advantages.

[0098] Table 1. Comparison of this method with other methods for detecting fluorescent aptamers of PSA.

[0099]

[0100] a metal-organic framework; b fluorescein amidite (FAM); c metal-organic g

[0101] In Table 1, Tb-MOFa / gold nanoparticles, DNA-Ag nanocluster, Zn / DNAzyme, FAMaptamerb / Cu-MOGc, Carbon dots / graphene oxide, and Glucose oxidase / Au@Ag@SiO2 were prepared according to [1-6] respectively:

[0102] [1] F.Qu, Y.Ding,

[0103] [2]B.Y.Fang,J.An,B.Liu,Y.D.Zhao,Hybridization induced fluorescenceenhanced DNA-Ag nanocluster / aptamer probe for detection of prostate-specificantigen,Colloid.Surface.B,175(2019)358-364.

[0104] [3]B.Li,J.Liu,H.Zhou,Amplified fluorescence detection of serumprostate specific antigen based on metal-dependent DNAzyme assistantnanomachine,Anal.Chim.Acta,1008(2018)96-102.

[0105] [4]T.T.Zhao,Z.W.Peng,D.Yuan,S.J.Zhen,C.Z.Huang,Y.F.Li,Metal-organicgel enhanced fluorescence anisotropy for sensitive detection of prostatespecific antigen,Spectrochim.Acta.A Mol.Biomol.Spectrosc.192(2018)328-332.

[0106] [5]J.H.He,Y.Y.Cheng,Q.Q.Zhang,H.Liu,C.Z.Huang,Carbon dots-basedfluorescence resonance energy transfer for the prostate specific antigen(PSA)with high sensitivity,Talanta 219(2020)121276.

[0107] [6] C. Jiang, Y. Huang, T. He, P. Huang, J. Lin, A dual-round signal amplification strategy for colorimetric / photoacoustic / fluorescence triple read-out detection of prostate specific antigen, Chem. Commun. 56 (2020) 4942-4945.

[0108] Selectivity:

[0109] To confirm the selectivity of the fluorescent aptamer sensor, several proteins and tumor markers, such as BSA, HSA, CEA, CA125, and AFP, were tested under the same experimental conditions. 15 ng / mL of the solution was added. -1 The PSA sample showed significant fluorescence recovery, while the fluorescence recovery intensity of other proteins and tumor markers was very low. Figure 16 Furthermore, no interference with PSA detection was found in the presence of 3 times the levels of BSA, HSA, CEA, CA125, and AFP. Figure 17 These results demonstrate the high selectivity of this aptamer sensor for PSA.

[0110] PSA detection in human serum samples:

[0111] To verify the practical application of this aptamer sensor in serum detection, human serum was diluted 100 times (serum sample provided by Zhejiang Xin'an International Hospital). The fluorescent aptamer sensor of this invention was then used to measure PSA in the diluted serum. The results are shown in Table 2. The detection results of this aptamer sensor are consistent with those obtained by the hospital's Archtecti 2000sr fully automated chemiluminescence immunoassay analyzer. The results show that this fluorescent aptamer sensor can be used to detect PSA in the serum of prostate cancer patients with high accuracy, indicating that the fluorescent aptamer sensor constructed in this invention has potential clinical applications.

[0112] Table 2 shows the detection of PSA in human serum (n=3) using the fluorescent aptamer sensor of the present invention.

[0113]

[0114] In summary, this invention establishes a method based on CGQDs as fluorophores and FL-V2CT. x A fluorescent aptamer sensor acting as a quencher is used to detect PSA. Apta-CGQDs are adsorbed onto FL-V2CT via hydrogen bonding. xThe upper part was subjected to photo-induced electron transfer by FL-V2CT x Rapid quenching. Fluorescence recovery of the fluorescent aptamer sensor was observed using the target PSA. (Compared with ML-V2CT) x Compared to FL-V2CT x The performance has been improved. This is due to the high metallicity of FL-Ti3C2T. x It exhibits strong interactions with apt-CGQDs, making it difficult for apt-CGQDs to escape from FL-Ti3C2T. x Separation from FL-Ti3C2T. Therefore, with FL-Ti3C2T... x Compared to FL-V2CT x It exhibits higher fluorescence recovery efficiency, confirming the efficacy of FL-V2CT. x The superiority of the material. This sensor can accurately measure PSA in the serum of prostate cancer patients. The fluorescent aptamer sensor proposed in this invention is characterized by its speed, convenience, low cost, high sensitivity, and high selectivity. Importantly, the concept of few-layer MXene as a quencher in fluorescent aptamer sensors can open up new application avenues for MXene materials.

[0115] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Use of a fluorescent aptamer sensor in the manufacture of a PSA early diagnosis product, characterized in that, The fluorescent aptamer sensor comprises a quencher, an aminoated PSA aptamer and a carboxylated graphene quantum dot, wherein the quencher, the aminoated PSA aptamer and the carboxylated graphene quantum dot are dispersed in solvents respectively; the quencher is a few-layer V2CT x ; the concentration of the quencher solution obtained by dispersing the quencher in the solvent is 20-30 mg / mL –1 ; the concentration of the aptamer solution obtained by dispersing the aminoated PSA aptamer in the solvent is 1-2 µM; the concentration of the quantum dot solution obtained by dispersing the carboxylated graphene quantum dot in the solvent is 1.5-2.5 mg / mL –1 ; and the base sequence of the PSA aptamer is shown in SEQ ID NO:

1.

2. Use according to claim 1, characterized in that, The preparation of the quencher comprises: The multi-layer V2CT x is added into the aqueous solution of tetramethylammonium hydroxide to obtain a mixture; the obtained mixture is stirred at 20-30°C for 10-15 hours, and then is washed, adjusted to neutral pH value and dried in sequence to obtain the few-layer V2CT x , denoted as FL-V2CT x ; Multi-layer v2ct x The mass-volume ratio of the added to the aqueous solution of tetramethylammonium hydroxide is 0.15-0.5 g:20 mL, and the mass percentage concentration of the aqueous solution of tetramethylammonium hydroxide is 20-30%.

3. A method for in vitro detection of PSA concentration for non-diagnostic purposes using a fluorescent aptamer sensor, characterized by, The fluorescent aptamer sensor comprises a quencher, an aminoated PSA aptamer and a carboxylated graphene quantum dot, and the quencher, the aminoated PSA aptamer and the carboxylated graphene quantum dot are dispersed in solvents respectively; the quencher is a few-layer V2CT x ; the concentration of the quencher solution obtained by dispersing the quencher in the solvent is 20-30 mg mL –1 ; the concentration of the aptamer solution obtained by dispersing the aminoated PSA aptamer in the solvent is 1-2 µM; the concentration of the quantum dot solution obtained by dispersing the carboxylated graphene quantum dot in the solvent is 1.5-2.5 mg mL –1 ; the base sequence of the PSA aptamer is shown in SEQ ID NO:1; The method comprises: (1) mixing the aminoated PSA aptamer and carboxylated graphene quantum dots, connecting the aminoated PSA aptamer and carboxylated graphene quantum dots through a condensation reaction to obtain a first reaction solution; (2) adding a solution to be detected containing PSA to the first reaction solution, and incubating the mixed solution at 37 DEG C for 40-60 min to obtain a second reaction solution; (3) adding the quenching agent to the second reaction solution, and incubating at 37°C for 10-30 min to obtain a third reaction solution; the quenching agent is added in an amount such that the final concentration thereof in the detection system is 1.8 mg / mL -1 ; the PSA concentration in the solution to be detected is 0.1-20 ng / mL -1 . (4) determining the fluorescence recovery intensity ΔF of the third reaction solution under an excitation wavelength of 327 nm and an emission wavelength of 446 nm; and substituting the obtained fluorescence recovery intensity ΔF into a standard curve to obtain the PSA concentration in the solution to be detected.

4. The method of claim 3, wherein, The fluorescence recovery intensity ΔF is calculated as follows: ΔF = F - F0; wherein F0 is the fluorescence intensity of the fluorescence aptamer sensor at 446 nm in the absence of the target PSA to be detected, and F is the fluorescence intensity of the reaction solution obtained after the target PSA to be detected is added.

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