Optical fiber biosensor based on nucleic acid aptamer and cell detection application thereof

Abstract

The invention relates to the field of biochemical sensing, in particular to a nucleic acid aptamer biosensor, an optical fiber sensor and cell detection application. The nucleic acid aptamer biosensor comprises a matrix, a connecting structure and TDN-coated AuNP, the matrix and the TDN-coated AuNP are combined through the connecting structure, and the connecting structure is an amino group, PolyA or a functionalized complementary chain. Through a special tetrahedral nanogold structure, the problem that nanogold is gathered due to the fact that the surface of the matrix is modified with nanogold is solved, and the biosensor has a good application prospect. Therefore, high-sensitivity detection is realized, and the method has extraordinary significance in the whole LSPR field.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemical sensing, in particular to a fiber-optic biosensor based on aptamer and its application in cell detection. BACKGROUND

[0002] The principle of localized surface plasmon resonance (LSPR) is that when nano-metal particles are made on a transparent substrate, they will produce surface plasmon resonance on the surface of the nano-metal particles under the excitation of incident light. Since the frequency and intensity of this resonance are easily affected by the surrounding environment, changes in wavelength or signal intensity occur, so changes in local dielectric constant can be used for analyte detection. Therefore, as long as the analyte is bonded near the particles, optical changes can be measured by optical instruments. Gold nanoparticles (AuNPs) have the characteristics of simple modification, large dielectric constant, and good biocompatibility, and are commonly used in fiber-optic localized surface plasmon resonance (LSPR) as a nano-layer. In order to improve the sensitivity of LSPR, those skilled in the art have obtained some results by changing the material, shape of the matrix, or changing the type, shape, size, and modification method of the nano-metal particles, but this process is only part of the preparation of the sensing device. If LSPR is applied to the field of biochemical sensing, AuNPs usually need to be modified to detect specific analytes. However, in the AuNPs modification process, salt ions and unreacted cross-linking reagents can easily induce AuNPs to aggregate, especially in the process of modifying AuNPs on the substrate, which can easily cause AuNPs to aggregate on the surface of the substrate. Gold nanoparticle aggregation can change the resonance frequency and make the full width at half maximum (FWHM) wider, thereby significantly reducing the sensing sensitivity of the fiber-optic LSPR sensor. This reduction has a universal impact on any LSPR applied to the field of biochemical detection. Therefore, it is crucial to seek a sensor that does not cause aggregation of nano-gold on the surface of the substrate during the modification process due to AuNPs modification or other environmental changes, which can revolutionize the entire LSPR sensing field. SUMMARY

[0003] Based on the above purpose, the present application provides a nucleic acid aptamer biosensor which does not affect the sensitivity of LSPR due to AuNPs modification or other environmental changes.

[0004] Spherical nucleic acid (SNA) is a kind of DNA@AuNP multivalent conjugate, and the structure is that the gold nanoparticles are wrapped on the surface in a dense radial arrangement. This kind of SNA can improve the salt resistance under complex conditions. Tetrahedral DNA (TDN) is a self-assembled supramolecular structure constructed by using sequence programmable DNA nanotechnology. Compared with single-stranded nucleic acid, TDN structure is rigid, and the size controllability is high at nanoscale. Therefore, it is speculated that tetrahedral DNA can be used as a spacer to spatially regulate the distance between particles in the gold nanoparticle nanolayer on the substrate surface.

[0005] The present application uses TDN@AuNP as a controllable supramolecular nanolayer strategy, realizes the uniform distribution of AuNPs on the substrate surface and the resistance of the environment through specific connection with the substrate, alleviates the aggregation problem of gold nanoparticles on the substrate surface, and further improves the sensitivity of the LSPR biosensor.

[0006] Specifically as follows: The nucleic acid aptamer-based fiber optical biosensor comprises a substrate, a connecting structure and TDN@AuNP, the substrate and TDN@AuNP are combined through the connecting structure, the connecting structure is amino, PolyA or functionalized complementary chain, and the substrate is an optical fiber.

[0007] The substrate of the present application is mainly used to provide a reaction site for generating LSPR, and any material capable of causing LSPR reaction can be used.

[0008] Further, the main material of the substrate is silica or plastic, preferably silica.

[0009] The “main material” of the present application can contain a small amount or no other components, for example, it can be a full silica material, or glass, most of which is silica, and also contains a small amount of other oxides (such as sodium oxide, calcium oxide, etc.).

[0010] Further, the optical fiber is an Ω-shaped optical fiber.

[0011] The Ω-shaped optical fiber of the present application refers to the sensing part of the optical fiber in the shape of Ω.

[0012] The present application takes the optical fiber as an example, and is not limited to the optical fiber itself, and other substrate materials capable of causing LSPR effect and suitable for the scope of the present application are also included in the scope of the present application.

[0013] Preferably, the number of PolyA bases is 10-40.

[0014] Further, the number of PolyA bases is 15-35, preferably, the number of PolyA bases is 15-20, more preferably, the number of PolyA bases is 15.

[0015] The AuNP particle size is 13 nm.

[0016] The amino group is electrostatically adsorbed to the AuNPs in the TDN@AuNP; the ployA is covalently combined with the AuNPs in the TDN@AuNP; and the functional complementary strand can be combined with part of the sequence in the TDN through DNA-DNA base complementary interaction, so as to realize specific binding of the TDN@AuNP.

[0017] The TDN@AuNP combines the advantages of gold nanoparticles and tetrahedral DNA, and is easy to be conjugated on the surface. The citrate-coated bare gold nanoparticles have negative charges, and can be easily adsorbed on the surface of the optical fiber with positive charges through electrostatic interaction.

[0018] The TDN@AuNP not only has excellent salt resistance and crosslinker resistance, but also can act as a spacer layer to prevent the aggregation of the nanolayer on the surface of the optical fiber.

[0019] For convenience, the biosensor with an amino group as a connecting structure is named as a SAM biosensor, the biosensor with a PolyA as a connecting structure is named as a PolyA biosensor, and the biosensor with a functional complementary strand as a connecting structure is named as a CS biosensor.

[0020] Further, the number of TDN in the TDN@AuNP is greater than the number of AuNPs.

[0021] Further, in the nucleic acid aptamer biosensor with an amino group as a connecting structure, TDN:AuNPs≤5; in the nucleic acid aptamer biosensor with a PolyA as a connecting structure, TDN:AuNPs≤10; and in the nucleic acid aptamer biosensor with a functional complementary strand as a connecting structure, 1≤TDN:AuNPs≤5, and the ratio is a molar ratio.

[0022] Further, the TDN contains sequences TDN-A, TDN-B, TDN-C and TDN-D, the structure of the TDN-A is a-AA-b-TC-c-polyA, the structure of the TDN-B is d-GC-b'-TT-e-polyA, the structure of the TDN-C is a'-CA-f-AG-e'-polyA, and the structure of the TDN-D is d'-AC-f'-GA-c'-TTT-capture strand.

[0023] The a-a', b-b', c-c' and d-d' are complementary sequences.

[0024] The capture strand can specifically capture the target substance, such as cells, bacteria, etc.

[0025] In some embodiments, the sequences a-f are as shown in Seq NO: 1-6, and the sequences a'-f' are as shown in Seq NO: 7-12.

[0026] In another aspect, the present application provides a biological detection device, which contains the aptamer biosensor or the optical fiber sensor.

[0027] Further, the biological detection device further comprises a light source, a spectrometer and an output device, wherein the light source is connected to the biosensor, and the output device outputs the signal measured by the spectrometer.

[0028] In some embodiments, the output device can be a computer.

[0029] In another aspect, the present application provides a preparation method of the aptamer biosensor or the optical fiber sensor, comprising the following steps: (1) modifying a connection structure on the surface of the substrate; (2) reacting the TDN@AuNP with the connection structure.

[0030] In another aspect, the present application discloses the application of the aptamer biosensor or the optical fiber sensor in cell detection, wherein the cells include tumor cells, cancer cells or bacterial cells.

[0031] The term "functional complementary strand" refers to the ability to combine with part of the sequence in TDN through DNA-DNA base complementary interaction, so as to realize the specific binding of TDN@AuNP. Since the structure of TDN@AuNP is that the TDN is densely arranged in a radial manner on the surface of the gold nanoparticle core, the amount of TDN is much larger than that of gold nanoparticles, and only part of the aptamer sequence on the TDN is complementary combined with the functional complementary strand, thereby playing a role in connecting TDN@AuNP to the substrate. In the present application, the functional complementary strand is combined with the capture strand, and in some embodiments, the capture strand is an aptamer sequence.

[0032] The term "Ω-shaped optical fiber" refers to the sensing part of the optical fiber being Ω-shaped, i.e., the silica part after removing the cladding is integrally formed into an Ω shape by high-temperature sintering. For details, reference can be made to the Ω-shaped optical fiber prepared in the previous research of the inventors.

[0033] The term "DNA@AuNP" (DNA functionalized gold nanoparticles) refers to a composite structure in which DNA is attached to the surface of gold nanoparticles (AuNP) by chemical or physical methods. This structure combines the optical properties of gold nanoparticles and the biological functions and programmability of DNA, and is considered as a programmable atomic equivalent (PAEs). DNA@AuNP is usually composed of three parts: an inorganic nanoparticle core: gold nanoparticles provide optical properties; an organic functional layer: DNA chains are attached to the surface of gold nanoparticles by physical adsorption or chemical bonding; a programmable interface: the ends of the DNA chains carry specific sequences, and self-assembly or replication can be achieved through strand displacement reaction.

[0034] The term "AuNPs modification" refers to the process of chemical modification on the surface of gold nanoparticles, etc., which changes the structure or properties of AuNPs.

[0035] In the present application, "gold nanoparticles", "AuNP" and "AuNPs" all represent spherical or spherical-like gold nanoparticles.

[0036] The term "bare fiber" or "bare optical fiber" refers to an optical fiber that has not been modified with gold nanoparticles.

[0037] Beneficial effects The present application first proposes to modify DNA@AuNP on the surface of the substrate for preparing LSPR sensors, and provides different DNA@AuNP modification modes, which can solve the agglomeration problem caused by gold nanoparticle modification during the preparation of LSPR, thereby ensuring the modification efficiency and overall sensitivity of the sensor. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is a performance diagram of three states of gold nanoparticles, a is a numerical simulation diagram, wherein (1) is a straight optical fiber modified with uniform gold nanoparticles (AuNP); (2) is a curved optical fiber modified with uniform AuNP; (3) is a curved optical fiber modified with aggregated AuNP; b is an absorption spectrum diagram, the absorption spectrum of uniform gold nanoparticles on a straight optical fiber (black curve), the absorption spectrum of uniform gold nanoparticles on a curved optical fiber (red curve), and the absorption spectrum of aggregated gold nanoparticles on a curved optical fiber (blue curve).

[0039] Figure 2 is a schematic diagram of the experiment, wherein (a) is a process for preparing TDN@AuNPs using annealing and salt aging steps; (b) is a biological sensor with different connection structures, and the connection structures from left to right are based on SAM, polyA and cs; (c) is a detection device of the optical fiber biosensor.

[0040] Figure 3 is a verification diagram of TDN synthesis gel.

[0041] Figure 4 UV-vis absorption spectrum and actual photo (inset) of gold nanoparticles (AuNPs). The absorption peak is at 520 nm.

[0042] Figure 5 TEM image (a) and particle size statistics (b) of AuNPs. The scale bar of TEM image is 100 nm. The average diameter of AuNPs is 13.25 nm.

[0043] Figure 6 Principle diagram of preparation of TDN@AuNPs Figure 7 Preparation of TDN@AuNPs under high salt conditions. TDN was self-assembled in 1×TM buffer (20 mM Tris-HCl, 50 mM MgCl2, pH=8.0). Subsequently, TDN was added into AuNPs solution at different molar ratios (TDN:AuNPs=1:4, 5, 6, 7, 10, 12, 15 and control group), and the mixed system was aged by adding PBS buffer. (a): Initial mixing of TDN and AuNPs; (b): First addition of PBS; (c): Fifth addition of PBS. The concentration of AuNPs was set to 10 nM.

[0044] Figure 8 Tolerance of gold nanoparticles to different dilution ratios of TM buffer. (a) Color change of gold nanoparticles. (b) UV absorption spectrum of gold nanoparticles after incubation with TM buffer overnight. (c) Maximum absorption value at 520 nm of gold nanoparticles after incubation with TM buffer overnight. Different dilution ratios of TM buffer in 1-6: 1×TM, 0.02×TM, 0.01×TM, 0.002×TM.

[0045] Figure 9 Synthesis of TDN using different TM buffers. (a) Gel electrophoresis diagram of TDN synthesis using different concentrations of TM solution. Lane 1: TDN-A; Lane 2: TDN-B; Lane 3: TDN-C; Lanes 4-8: results of TDN synthesis using 0.02×TM, 0.05×TM, 0.1×TM, 0.5×TM and 1×TM buffer, respectively.

[0046] Figure 10 Agarose gel electrophoresis of TDN stability under different dilution multiples in TM buffer. Lane 1: Marker; Lanes 2-8: 0, 1, 2.5, 5, 10, 25 and 50-fold water dilution.

[0047] Figure 11UV-Vis spectra of TDN@AuNPs with different molar concentrations (0-50), where the inset is the photo of the corresponding TDN@AuNPs. Figure 12 TEM images of TDN@AuNPs, scale bar is 100 nm.

[0048] Figure 13 Size distribution of TDN@AuNPs with different molar concentrations (0-50); resistance of bare AuNPs (1-7) and TDN@AuNPs (8-13) to different dilution of TM buffer, where 1: no TM buffer (control); 2 and 8: 0.02x TM; 3 and 9: 0.05x TM; 4 and 10: 0.1x TM; 5 and 11: 0.2x TM; 6 and 12: 0.5x TM; 7 and 13: 1x TM.

[0049] Figure 14 UV-Vis spectra and their photos (inset is the photo) of AuNPs and TDN@AuNPs for their crosslinker resistance ability.

[0050] Figure 15 Diameter of TDN@AuNPs measured by dynamic light scattering.

[0051] Figure 16 Spatial distribution of refractive index sensitivity in bare fiber under the same conditions.

[0052] Figure 17 (a) Spectra of directly modified TDN@AuNPs. (b) Refractive index sensitivity of directly modified TDN@AuNPs.

[0053] Figure 18 Process diagram of gold nanoparticles modification by different methods.

[0054] Figure 19 TDN was prepared under different substrate concentrations and its corresponding effect on the refractive index sensitivity of fiber optic (FO)-based localized surface plasmon resonance (LSPR) was studied. (a) Lane 1: DNA ladder; Lanes 2-10: 1.0 μM, 2.5 μM, 5.0 μM, 7.5 μM, 10 μM. (b) Substrate concentration range is 1.0-10.0 μM.

[0055] Figure 20Performance comparison of fiber-optic based LSPR biosensor based on SAM, polyA and CS biosensor. Characterization of SAM modification (a-d), polyA modification (e-h) and CS modification (i-l) method. Schematic diagram of SAM modification (a), polyA modification (e) and CS modification (i) method; Absorption spectrum of fiber-optic based LSPR modified by SAM (b), polyA (f) and CS (j) modification method using TDN@AuNPs with different TDN:AuNPs molar ratio. Time-dependent increase curve of LSPR signal of TDN@AuNPs prepared by SAM (c), polyA (g) and CS (k) modification method. Response intensity of fiber-optic based LSPR modified by TDN@AuNPs by SAM (d), polyA (h) and CS (l) modification method. The molar ratio of TDN: AuNPs ranges from 0 to 50:1.

[0056] Figure 21 Acid-base stability sensitivity experiment of the sensor of the application.

[0057] Figure 22 Gel verification of acid-base stability of the sensor of the application.

[0058] Figure 23 Fluid stability (left) and long-term stability experiment (right) of the sensor of the application.

[0059] Figure 24 Performance evaluation of LSPR biosensor based on SAM method and CS method. Time-dependent enhancement of sensing signal of FO-based LSPR based on SAM method (a) and CS method (d). Linear relationship between sensing signal of FO-based LSPR and MCF-7 cell concentration based on SAM method (b) and CS method (e). Detection limit of biosensor in LSPR biosensor based on SAM method (c) and CS method (f) depends on the molar ratio of TDN to AuNPs and detection time. Both of the two LSPR-based biosensors have the advantage of time and molar ratio dependent sensing sensitivity. DETAILED DESCRIPTION

[0060] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme of the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments.

[0061] The biosensor of the application comprises a matrix, a connecting structure and TDN@AuNPs, wherein the matrix is connected with the TDN@AuNPs through the connecting structure.

[0062] The present application does not have special requirements for the substrate, as long as it can occur LSPR reaction. Since the aggregation of gold nanoparticles affects the LSPR reaction effect, the present application focuses on solving the problem of gold nanoparticle aggregation.

[0063] Based on the related research of the present inventors in the early stage, the optical fiber LSPR, especially the Ω-shaped optical fiber LSPR, has high sensitivity and simple preparation process, and the performance of the optical fiber LSPR can be directly evaluated by the refractive index sensitivity. Therefore, if the sensor is affected by the environment, the high sensitivity can also be very intuitive to analyze the small changes, which is used to show the effect of the present application in the examples.

[0064] The refractive index sensitivity evaluation is as follows: the optical fiber probe is immersed in sucrose solutions with mass volume concentrations (w / v%) of 0%, 4%, 8%, 12%, 16%, and 20%, respectively, and the corresponding absorption spectra are collected. A linear relationship curve between the absorbance value at the characteristic peak and the refractive index is established, and the slope of the curve is defined as the refractive index sensitivity.

[0065] Example 1 Influence of gold nanoparticle (AuNP) state on performance The gold nanoparticles (AuNP) on the surface of the optical fiber can enhance the evanescent field, thereby exciting strong localized surface plasmon resonance (LSPR) to meet the needs of biosensing. The state of the AuNP on the surface of the optical fiber plays a crucial role in the sensing sensitivity. Previous studies have shown that AuNP aggregation can change the resonance frequency and broaden the full width at half maximum (FWHM), thereby significantly reducing the refractive index sensitivity. However, there is no reasonable theoretical explanation for this. The present application further explores the causes and uses finite element method for numerical simulation.

[0066] The specific numerical simulation process is as follows: The finite element method (FEM) is used to analyze the modal of the optical fiber, and three FO-based LSPR models are constructed: (1) Control group: straight optical fiber with uniform gold nanoparticles (AuNP) on the surface; (2) Experimental group: curved optical fiber with uniform AuNP on the surface, and curved optical fiber with aggregated AuNP on the surface.

[0067] Among them, the curved optical fiber with aggregated AuNP on the surface is designed to analyze the influence of the state of AuNP on the refractive index sensitivity. The size of AuNP in the simulation is set to 13 nm. According to the following formula, the curved optical fiber is converted into an equivalent straight optical fiber by conformal mapping:

[0068] where n represents the refractive index profile in the equivalent straight fiber, x represents the displacement, and R represents the bending radius of the fiber. To simplify the model, the core radius and cladding thickness were set to 1.5 μm and 3 μm, respectively; the refractive indices of the core and cladding were set to 1.4438 and 1.4378, respectively.

[0069] After the parameter scanning at different wavelengths, the imaginary part of the effective refractive index of the fundamental mode (LP01 mode) was calculated, and then the optical loss was calculated using the following formula:

[0070] where λ represents the wavelength, Im(neff) represents the imaginary part of the effective refractive index.

[0071] The results are shown in Figure 1 a. The bending region of the fiber changes the transmission mode of light, causing the fundamental mode to leak from the core to the cladding. As can be seen from Figure 1 a, the electric field intensity of the fiber modified with uniform AuNPs is higher than that of the fiber modified with aggregated AuNPs. As shown in Figure 1 b, when the AuNPs remain in a uniform distribution state, the LSPR signal intensity generated by the bent fiber is 2 times that of the straight fiber; when the fiber surface is modified with aggregated AuNPs, the LSPR peak becomes wide, resulting in no obvious identifiable LSPR peak. In addition, the imaginary part of the effective refractive index of the aggregated AuNPs is smaller than that of the uniform AuNPs. Therefore, developing a new modification method that can inhibit the aggregation of AuNPs on the surface of the fiber is of great significance for constructing a high-sensitivity fiber-based LSPR sensor.

[0072] Example 2 Preparation of TDN@AuNPs.

[0073] The principle of preparation of TDN@AuNPs is shown in Fig. 2a. After the tetrahedral DNA is self-assembled by the annealing process, it is assembled onto the surface of gold nanoparticles using the salt aging technique. Specifically as follows: (1) TDN preparation: TDN was synthesized by annealing steps. The DNA substrates of TDN-A, TDN-B, TDN-C, and TDN-D were mixed in TM buffer at an equimolar concentration. The TM buffer consists of 20 mM Tris-HCl and 50 mM MgCl2. In a PCR cycler (SimpliAmp, ABI, USA), heat at 95℃ for 5 min, and quickly cool to 4℃ at a rate of 4℃ / s. Finally, the mixture was kept at 4℃ for at least 6 hours to form TDN.

[0074] For the convenience of effect display, MCF-7 cells are captured by the sensor of the application for display, wherein the mucin 1 (MUC1) aptamer can specifically capture MCF-7 cells, therefore, in the embodiment, the MUC1 aptamer is selected for verification, wherein the sequence of the MUC1 aptamer is shown as SEQ NO: 13, and the sequence related to TDN is shown in Table 1. Table 1 TDN related sequence ; The green and italic oligonucleotide is the sequence of the MUC1 aptamer.

[0075] If other cells, bacteria, etc. need to be captured by those skilled in the art, corresponding specific aptamers can be selected, wherein the synthetic sequence of TDN can refer to the sequence disclosed in the embodiment, and only needs to replace the above green italic sequence, or a suitable sequence capable of synthesizing a tetrahedral structure can be designed according to the prior art.

[0076] The results are shown in Figure 3 It can be seen that the electrophoretic migration rate gradually slows down after the addition of the substrate DNA; the gel electrophoresis result further proves the successful synthesis of TDN.

[0077] (2) Preparation of AuNPs: Gold nanoparticles were prepared by citric acid reduction method. First, 50 mL ultrapure water was mixed with 0.5 mL 1% HAuCl4 solution, and then boiled. Once the solution starts to boil, 2 ml 1% sodium citrate (Na3Ct) solution is rapidly added. The solution color changes from light yellow to wine red, indicating the formation of AuNPs. The reaction lasted for about 10 minutes until the color remained stable. AuNPs were collected and stored at 4°C for subsequent experiments. The characteristics of AuNPs were analyzed by ultraviolet-visible spectroscopy (UV-vis) and transmission electron microscopy (TEM). The molar concentration of AuNPs was calculated by ultraviolet-visible spectroscopy.

[0078] The results are shown in Figures 4-5 It can be seen that the electrophoretic migration rate gradually slows down after the addition of the substrate DNA; the gel electrophoresis result further proves the successful synthesis of TDN.

[0079] (3) Preparation of TDN@AuNP: Citrate-stabilized gold nanoparticles (AuNP) and tetrahedral DNA (TDN) were incubated at 4°C for 16 hours. After incubation, 10 mM PBS and 0.1 M sodium chloride solution were added to the mixed system and left to stand for 40 hours to promote further binding of AuNP and TDN.

[0080] The preparation principle is shown in Figure 6As shown, TDN is assembled onto the AuNP surface via salt aging. Specifically, the modified tetrahedral DNA (TDN) self-assembles after rapid quenching in TM buffer. Three vertices of the TDN are modified with polyadenylate (polyA) tail DNA to effectively immobilize the TDN on the AuNP surface. The remaining vertex is connected to an aptamer to achieve specific capture of cancer cells.

[0081] (4) Preparation of TDN@AuNP with good dispersibility The results are as follows Figure 7 As shown, when TDN in TM buffer (20 mM Tris-HCl and 50 mM MgCl2) was added to the AuNP solution, the color of the AuNP solution quickly changed from wine red to purple-blue. This is because the high salt concentration in the TM buffer induced AuNP aggregation.

[0082] Experiments showed that diluting TM buffer 100 times (i.e., 0.01×TM buffer) was suitable for the preparation of TDN@AuNP. Figure 8 Meanwhile, in diluted TM buffer, TDN can still effectively self-assemble and exhibits high stability. Figures 9-10 In diluted TM buffer, TDN was incubated with AuNP at different molar ratios. Salt aging treatment was then used to conjugate TDN to the AuNP surface, forming TDN@AuNP, as shown below. Figure 11 The observed slight color change and wavelength shift indicate that TDN@AuNP has been successfully synthesized. Figure 12 Transmission electron microscopy (TEM) images show that the dispersibility of TDN@AuNP in water improves with increasing TDN to AuNP molar ratio. Figure 13 As shown, 100-fold dilution of TM buffer induces aggregation of naked AuNPs, while TDN@AuNPs maintain good stability in this buffer. Figure 14 The results showed that in the 3-aminopropyltriethoxysilane (APTES) crosslinking agent, only naked AuNPs aggregated, while TDN@AuNPs did not exhibit aggregation. These results indicate that TDN@AuNPs can enhance salt resistance and resistance to crosslinking agents, thereby inhibiting AuNP aggregation. Based on the number of complementary bases, theoretical calculations yielded... Figure 6 The height of the TDN is 3.44 nm. Figure 15 The results show that as the amount of TDN modified on the AuNP surface increases, the particle size of TDN@AuNP increases from 12.6 nm to 18.6 nm, consistent with theoretical calculations. This result confirms that TDN can act as a spacer layer, effectively preventing AuNP aggregation. Figures 14-15TDN@AuNPs were synthesized under different molar ratios (0-50) of TDN to AuNPs.

[0083] In summary, in the solution state, the TDN@AuNPs of the present application will not agglomerate due to the influence of salt or crosslinking agent, and have very good dispersibility, which ensures the performance of the TDN@AuNPs, so that subsequent TDN@AuNPs modification on the substrate surface will not agglomerate on the substrate surface.

[0084] Example 3 Preparation of a biosensor The schematic diagram of the three biosensors is shown in Figure 1 The detection device is shown in Figure 2 c), and the specific sensor preparation method is as follows.

[0085] To avoid measurement errors caused by differences in fiber structure, in this study, bare fibers with similar refractive index sensitivity were selected for the exploration of different sensors, and the selection of bare fibers is shown in Figure 16 .

[0086] After incubating the selected bare fibers with TDN@AuNP, the results are shown in Figure 17 : the intensity of the characteristic absorption peak increases with the extension of the incubation time. Under a specific modification time, the absorption peak intensity can reflect the density of TDN@AuNP on the surface of the fiber. After the modification of TDN@AuNP is completed, the fiber probe is immersed in different concentrations of sucrose solution, and the characteristic absorption peak will change. Then, the linear fitting is performed with the refractive index as the variable, and the slope of the obtained fitting curve is defined as the refractive index sensitivity.

[0087] (1) SAM sensor SAM refers to the assembly of a monolayer, i.e. the connecting structure is amino. The specific mechanism of action is that the negatively charged bare AuNP is adsorbed on the surface of the amino-functionalized fiber through electrostatic interaction.

[0088] The Ω-shaped fiber probe is immersed in aqua regia for 20 minutes, and then ultrasonically cleaned in ultrapure water for 5 minutes to remove surface impurities. After drying, the sensing area of the Ω-shaped fiber is immersed in a 5% APTMS solution for 20 minutes to achieve amino-functionalized modification, i.e. amino is modified on the surface of the fiber. Finally, the amino-functionalized fiber is immersed in the TDN@AuNP solution to complete the modification.

[0089] The comparative example is to replace TDN@AuNP with AuNPs prepared in Example 2 The results are shown in Figure 18TDN@AuNP modification process, left side is the comparative example, right side is the present application, it can be seen that the TDN@AuNP modification in the present application does not occur agglomeration in the process of modifying the surface of the optical fiber, so that the solution changes from red to blue, while the solution color changes in the AuNPs modification process, indicating that agglomeration has occurred.

[0090] (2) polyA sensor That is, the connection structure is polyA. The specific mechanism of action is that the polyA on the surface of the optical fiber is constructed by using the characteristics of being combined with AuNP through Au-N bond.

[0091] First, the amino-functionalized optical fiber (according to the method in (1) above) is soaked in a 5% glutaraldehyde (GA) solution for 1 hour; after washing with deionized water for 3 times and drying, it is immersed in a 2 μM PolyA-NH2 DNA solution for 2 hours; then the optical fiber is incubated with TDN@AuNP solution to complete the modification.

[0092] The comparative example is to replace TDN@AuNP with AuNPs prepared in Example 2 The results are shown in Figure 18 TDN@AuNP modification process, left side is the comparative example, right side is the present application, it can be seen that the TDN@AuNP modification in the present application does not occur agglomeration in the process of modifying the surface of the optical fiber, so that the solution changes from red to blue, while the solution color changes in the AuNPs modification process, indicating that agglomeration has occurred.

[0093] (3) CS sensor That is, the connection structure is functionalized complementary strand. The specific mechanism of action is that the complementary strand on the surface of the optical fiber hybridizes with the aptamer on TDN@AuNP, thereby realizing the fixation of TDN@AuNP Replace 2 μM PolyA-NH2 DNA solution in the polyA-based modification method with 2 μM amino-functionalized complementary strand (NH2-functionalized complementary strand), and the rest of the experimental conditions are exactly the same as the polyA-based modification method.

[0094] The comparative example is to replace TDN@AuNP with AuNPs prepared in Example 2 The results are shown in Figure 18 TDN@AuNP modification process, left side is the comparative example, right side is the present application, it can be seen that the TDN@AuNP modification in the present application does not occur agglomeration in the process of modifying the surface of the optical fiber, so that the solution changes from red to blue, while the solution color changes in the AuNPs modification process, indicating that agglomeration has occurred.

[0095] In summary, the three sensors prepared by the method of the present application have TDN@AuNP uniformly dispersed on the surface of the optical fiber substrate without agglomeration.

[0096] Example 4 Performance study of the biosensor 1. Effect of TDN:AuNP on the sensitivity of the sensor The molar ratio of TDN to AuNP changes the density of TDN on the surface of AuNP, which in turn significantly affects the binding efficiency of the above three methods. Therefore, comparative experiments were conducted on the three modification methods under different TDN:AuNP molar ratios. Before the comparative experiments, the synthesis concentration of TDN was first optimized. The results, as shown in Figure 19 , show that substrates of different concentrations can successfully self-assemble to form TDN Figure 19 (a); and as the synthesis concentration of TDN increases, the refractive index sensitivity gradually increases Figure 19 (b). Finally, TDN with a high synthesis concentration was selected for subsequent comparative experiments.

[0097] In the SAM-based and polyA-based modification methods, the binding efficiency depends on the exposed surface area of AuNP. TDN can shield the surface charge of AuNP, inhibiting the binding of TDN@AuNP to the surface of the optical fiber. The results, as shown in Figure 20 a-h, show that in these two methods, the absorption peak intensity and the refractive index sensitivity are negatively correlated with the TDN:AuNP molar ratio.

[0098] However, in the CS-based modification method, the binding efficiency depends on the molar concentration of TDN on the surface of AuNP: when the TDN:AuNP molar ratio increases from 1:1 to 2:1, the absorption peak intensity and the refractive index sensitivity both increase Figure 20 i-l); if the TDN:AuNP molar ratio continues to increase, unbound free TDN will compete with the complementary DNA on the surface of the optical fiber for hybridization, resulting in a decrease in the density of TDN@AuNP on the surface of the optical fiber. Therefore, when the TDN:AuNP molar ratio exceeds 2:1, the absorption peak intensity and the refractive index sensitivity both decrease significantly.

[0099] In summary, when TDN@AuNP is used as a nanolayer to modify the surface of an optical fiber, the FO-based LSPR sensor exhibits a significant advantage: its refractive index sensitivity is dependent on the TDN:AuNP molar ratio.

[0100] 3. Stability evaluation Stability is a key performance indicator of FO-based LSPR sensors.

[0101] (1) Acid-base stability The chemical stability of the three sensors in Example 3 under pH conditions was also analyzed.

[0102] The results show that the same rule is obtained by the three methods, and the results are shown in Figures 21-22 Under acidic conditions, the amino groups (-NH2) on the surface of the optical fiber will combine with protons to form ammonium ions (NH4 + ), which is conducive to the adsorption of citrate-coated gold nanoparticles (AuNP) on the surface of the optical fiber. On the contrary, under alkaline conditions, the amino groups are basically neutral or slightly negatively charged; at the same time, the unactivated silicon hydroxyl groups (Si-OH) on the surface of the optical fiber also carry negative charges, which will produce repulsion with negatively charged citrate-coated gold nanoparticles.

[0103] Therefore, in the modification methods based on SAM and polyA, under acidic conditions, SAM and ployA change the charge, and the acidic conditions can make TDN@AuNP stably adsorbed on the surface of the optical fiber, and the CS destroys the binding ability of double-stranded DNA.

[0104] As shown in Figure 21 When the pH is lower than 5.0, the refractive index sensitivity and absorption peak intensity of the sensor are both low. This is because under strong acidic conditions, the structure of tetrahedral DNA (TDN) will be destroyed and degraded into single-stranded DNA, Figure 22 The gel electrophoresis results also verify this phenomenon. In summary, the three modification methods based on SAM, polyA and CS can all maintain high chemical stability in weakly acidic or neutral environments.

[0105] (2) Fluid environment stability In this study, the optical fiber probe was flushed with phosphate buffered saline (PBS) at a flow rate of 5 mm / s to evaluate the binding stability of the three modification methods, and the results are shown in Figure 23 The left figure. After flushing, the refractive index sensitivity of the modification methods based on SAM, polyA and CS decreased to 55.4%, 76.9% and 87.3% of the initial value, respectively, which indicates that the modification method based on CS has the highest binding stability. Good binding stability means that the modification method based on CS has great application potential in fluid environments such as the circulatory system.

[0106] (3) Long-term storage stability In the long-term stability evaluation, the optical fiber probe was stored at room temperature for 24 hours. The results are shown in Figure 23As shown in the right graph, the refractive index sensitivity of the SAM-based, polyA-based and CS-based modification methods after storage correspondingly decreased to 74.2%, 86.1% and 97.0% of the initial value, indicating that the polyA-based and CS-based modification methods both exhibited excellent long-term stability. In addition, the refractive index sensitivity (RIS) of the CS-based modification method was higher than that of the other two methods, regardless of long-term storage or washing treatment. Therefore, the CS-based modification method is an ideal choice for constructing a high-sensitivity fiber-based LSPR biosensor.

[0107] Example 5: Biosensor application The TDN@AuNP was synthesized in Example 2 and was labeled with MUC1 aptamer, so this example was selected for application to the detection of MCF-7 cells to verify the biosensing performance of each sensor.

[0108] Since the ployA-based method had the lowest refractive index sensitivity, two modification methods, SAM-based and complementary strand (CS)-based, were used to construct biosensors. Different molar ratios of TDN@AuNP would affect the density of MUC1 aptamer on the surface of gold nanoparticles (AuNP); the density of MUC1 aptamer determined its multivalent state, which in turn affected the affinity of the fiber surface to MCF-7 cells. In addition, the molar ratio of TDN@AuNP also changed the number of TDN@AuNP adsorbed on the fiber surface, thereby significantly affecting the refractive index sensitivity.

[0109] The present application evaluated the sensing sensitivity of the sensor by conjugating complexes with different molar ratios of TDN:AuNP to the fiber surface. The results showed that the LSPR spectrum of the bare fiber changed weakly, while the fiber-based LSPR sensor modified with TDN@AuNP exhibited a significant signal enhancement over time, as shown in Figure 24 a and Figure 24 d.

[0110] Under different binding times, the logarithmic value of the cell concentration showed a good linear relationship with the absorbance, as shown in Figure 24 b and Figure 24 e. The limit of detection (LOD) was calculated according to the formula 3δ / S, where δ represents the standard deviation of the LSPR signal, and S represents the slope of the linear curve. From Figure 24 c and Figure 24It is known that the sensing sensitivity is largely dependent on the detection time and TDN:AuNP molar ratio. Previous studies have demonstrated that the sensitivity of the fiber-optic based LSPR sensor is time-enhanced. In addition, the sensing sensitivity of the fiber-optic based LSPR sensor is found to be molar-ratio-dependent by the SAM and CS based modification methods. When the TDN:AuNP molar ratio increases from 1 to 5, the multivalent state of the aptamer is increased, resulting in the enhanced affinity of the aptamer to the MCF-7 cancer cells. However, when the TDN:AuNP molar ratio further increases from 5 to 50, the absorption intensity is decreased, resulting in the decreased density of TDN@AuNP on the fiber surface and the refractive index sensitivity, and thus the significantly increased detection limit. As shown in Table 2, the sensing sensitivity of the sensor based on the CS and polyA modification methods is consistent with the change of the detection time and TDN:AuNP molar ratio. Among them, the sensor based on the CS modification method has the lowest detection limit, which can reach 12 cells / mL, lower than the reported detection limit level in the recent related studies (Table 3).

[0111] Table 2 Working detection limit of the fiber-optic LSPR biosensor ; Table 3 Sensing sensitivity comparison between the present application and the reported works ; In summary, in addition to the time-enhanced sensitivity, the fiber-optic LSPR (FOLSPR) based biosensor exhibits a new advantage, i.e., the sensing sensitivity is molar-ratio-dependent.

[0112] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, all possible combinations of the technical features in the above-described embodiments are not described, however, as long as the combinations of the technical features do not contradict each other, they should be considered within the scope of the present disclosure.

[0113] The above-described embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be noted that for those skilled in the art, without departing from the concept of the present application, some modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present application should be subject to the appended claims.

Claims

1. A fiber optic biosensor based on nucleic acid aptamers, characterized in that, The nucleic acid aptamer biosensor includes a matrix, a linker structure, and a TDN@AuNP. The matrix and the TDN@AuNP are bound together by the linker structure, which is an amino group, a polyA group, or a functionalized complementary chain. The matrix is ​​an optical fiber.

2. The fiber optic biosensor as described in claim 1, characterized in that, The number of PolyA bases is 10-40; preferably, the number of PolyA bases is 15-35.

3. The fiber optic biosensor as described in claim 1 or 2, characterized in that, The number of TDNs in TDN@AuNP is greater than that in AuNPs.

4. The fiber optic biosensor as described in claim 3, characterized in that, In the nucleic acid aptamer biosensor with an amino group as the linkage structure, TDN:AuNPs≤5; in the nucleic acid aptamer biosensor with a PolyA linkage structure, TDN:AuNPs≤10; in the nucleic acid aptamer biosensor with a functionally complementary chain linkage structure, 1≤TDN:AuNPs≤5, and the ratio is a molar ratio.

5. The fiber optic biosensor as described in any one of claims 1, 2, or 4, characterized in that, The TDN contains sequences TDN-A, TDN-B, TDN-C, and TDN-D. The structure of TDN-A is a-AA-b-TC-c-polyA, the structure of TDN-B is d-GC-b'-TT-e-polyA, the structure of TDN-C is a'-CA-f-AG-e'-polyA, and the structure of TDN-D is d'-AC-f'-GA-c'-TTT-capture chain. Among these, a-a', b-b', c-c', and d-d' are complementary sequences.

6. The fiber optic biosensor as described in claim 5, characterized in that, The sequences a-f are shown in SEQ ID NO: 1-6, and the sequences a'-d' are shown in SEQ ID NO: 7-12.

7. The fiber optic biosensor as described in claim 1 or 2, characterized in that, The optical fiber is an Ω-type optical fiber.

8. A biological detection device, characterized in that, The biological detection device contains a fiber optic biosensor as described in any one of claims 1-7.

9. A method for fabricating an optical fiber sensor as described in any one of claims 1-7, characterized in that, Includes the following steps: (1) Modify the bonding structure on the surface of the matrix; (2) React the TDN@AuNP with the connection structure.

10. The application of the fiber optic sensor as described in any one of claims 1-7 in cell detection.

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