Hybrid chain reaction fluorescent biosensor as well as preparation method and application thereof

Abstract

The invention discloses a hybridization chain reaction fluorescent biosensor and a preparation method and application thereof, red cell membrane coated gold nanoparticles are used as a reaction substrate, the surface of the gold nanoparticles is covalently connected with a sulfydryl modification initiation probe aiming at target miRNA through an Au-S bond, and the red cell membrane coated gold nanoparticles are treated by Fe < 3 + >. According to the method, the red cell membrane completely coats the gold nanoparticles, so that common false positive signals in a complex body fluid environment are remarkably reduced; the hybridization chain reaction is limited on the surface of the cell membrane, so that the local concentration of the hairpin probe is improved, the chain amplification process is accelerated, and the detection time is shortened; sufficient sites are provided for probe fixation and HCR polymerization by utilizing the large specific surface area of the gold nanoparticles, and the signal amplification factor is further enhanced; fe < 3 + > is introduced to perform surface modification on the red cell membrane coated gold nanoparticles, so that non-specific binding is effectively inhibited, blank background fluorescence is remarkably reduced, and the signal-to-noise ratio is increased.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to a hybridization chain reaction fluorescent biosensor, its preparation method, and its application. Background Technology

[0002] MicroRNAs (miRNAs) are a class of non-coding RNAs approximately 18-25 nucleotides in length, playing crucial regulatory roles in physiological processes such as cell differentiation, proliferation, and apoptosis. Numerous studies have shown that the expression levels of various miRNAs in the plasma, serum, and urine of cancer patients differ significantly from those in healthy individuals, and their expression profiles are cancer type-specific, thus making them highly promising tumor markers for liquid biopsy. Compared to traditional tissue biopsies, miRNA-based liquid biopsies offer advantages such as being non-invasive, repeatable, and dynamically reflecting tumor status, making them an important area of ​​development for early cancer screening, postoperative recurrence monitoring, and treatment efficacy evaluation.

[0003] However, current miRNA detection methods still have significant limitations, restricting their clinical translation. First, the absolute concentration of circulating miRNAs in body fluids is extremely low (fM to aM level). While traditional real-time quantitative PCR is the gold standard, it requires reverse transcription and enzymatic amplification, which is cumbersome, costly, and susceptible to inhibitors. Second, while fluorescent and electrochemical sensors based on isothermal amplification (e.g., rolling circle amplification (RCA), exponential amplification (EXPAR)) or enzyme-free amplification (e.g., hybridization chain reaction (HCR), catalytic hairpin assembly (CHA)) simplify operation, they generally face problems such as severe non-specific adsorption, high background signal, and insufficient amplification efficiency due to low local probe concentration in complex body fluid environments, making reliable detection of ultra-low abundance difficult. Furthermore, exposed nanomaterials are easily covered by protein corona in serum, causing probe masking or signal quenching, further reducing the specificity and reproducibility of the detection. These problems collectively restrict the sensitivity, specificity, and practical application of miRNA liquid biopsy technology, necessitating the development of a new detection strategy that can simultaneously achieve high amplification efficiency and low background interference in complex biological matrices. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a hybridization chain reaction fluorescent biosensor.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned hybridization chain reaction fluorescent biosensor.

[0006] Another object of the present invention is to provide the application of the above-mentioned hybridization chain reaction fluorescent biosensor.

[0007] The technical solution of the present invention is as follows:

[0008] A hybridization chain reaction fluorescent biosensor uses gold nanoparticles coated with erythrocyte membranes as the reaction substrate. The surface of the gold nanoparticles is covalently linked with thiol-modified probes targeting miRNAs via Au-S bonds. Furthermore, the erythrocyte membrane-coated gold nanoparticles are subjected to Fe... 3+ deal with.

[0009] In a preferred embodiment of the present invention, the gold nanoparticles have a particle size of 15-15 nm.

[0010] In a preferred embodiment of the present invention, the thickness of the red blood cell membrane coating is 3-6 nm.

[0011] In a preferred embodiment of the present invention, the gold nanoparticles have a particle size of 18-20 nm, and the red blood cell membrane coating has a thickness of 4-5 nm.

[0012] In a preferred embodiment of the present invention, the Fe 3+ The process involved incubating gold nanoparticles coated with red blood cell membranes in FeCl3 solution with shaking at room temperature for 30-60 minutes, followed by centrifugation, discarding the supernatant, and washing with ultrapure water.

[0013] The preparation method of the above-mentioned hybridization chain reaction fluorescent biosensor includes the following steps:

[0014] (1) Preparation of gold nanoparticles;

[0015] (2) Preparation of red blood cell membranes;

[0016] (3) The red blood cell membrane obtained in step (2) is coated onto the gold nanoparticles obtained in step (1) to obtain the red blood cell membrane coated gold nanoparticles.

[0017] (4) The gold nanoparticles coated with red blood cell membranes obtained in step (3) were subjected to Fe³⁺ treatment;

[0018] (5) The thiol-modified initiation probe is covalently linked to the surface of the material obtained in step (4) via Au-S bond to obtain the material.

[0019] In a preferred embodiment of the present invention, the coating in step (3) is achieved by mixing vesicularized red blood cell membranes with gold nanoparticles and then subjecting the mixture to ice bath sonication.

[0020] In a preferred embodiment of the present invention, before covalently linking Au-S bonds in step (5), the thiol-modified initiation probe is first reduced using TCEP.

[0021] The above-mentioned hybridization chain reaction fluorescent biosensor is used in the preparation of a miRNA detection kit in body fluids.

[0022] A kit for detecting miRNA in body fluids, which has the above-mentioned hybridization chain reaction fluorescent biosensor.

[0023] The beneficial effects of this invention are:

[0024] 1. This invention uses gold nanoparticles completely coated with red blood cell membranes to endow the sensor with natural resistance to non-specific protein adsorption and long-cycle characteristics, which significantly reduces false positive signals commonly found in complex body fluid environments.

[0025] 2. This invention confines the hybridization chain reaction to the cell membrane surface, significantly increasing the local concentration of the hairpin probe, accelerating the chain amplification process, and shortening the overall detection time.

[0026] 3. This invention utilizes the large specific surface area of ​​gold nanoparticles to provide sufficient sites for probe immobilization and HCR polymerization, further enhancing the signal amplification factor.

[0027] 4. This invention introduces Fe 3+ Surface modification of gold nanoparticles coated on erythrocyte membranes effectively inhibited the non-specific binding of SYBR Green I dye to nanomaterials, significantly reduced blank background fluorescence, and improved the signal-to-noise ratio.

[0028] 5. The entire detection process of this invention does not require enzyme participation, isothermal reaction, and is simple to operate. It can be completed with only conventional centrifugation and fluorescence spectrophotometer, and is easy to convert into portable devices.

[0029] 6. The present invention maintains excellent anti-interference ability and accurate spike recovery performance even in diluted human serum, proving its reliability in real clinical samples. Attached Figure Description

[0030] Figure 1 These are experimental results from various stages of the fabrication process of the Au@MFe-P fluorescent biosensor in Example 1 of this invention. Wherein: Figure 1 a represents the ultraviolet absorption spectrum of the prepared AuNPs; Figure 1 b is a transmission electron microscope image of AuNPs; Figure 1 c is the ultraviolet absorption spectrum of the RBCm solution; Figure 1 d is a transmission electron microscope image of the prepared Au@M.

[0031] Figure 2 The figures show step (7) of Embodiment 1 and the experimental results of Embodiment 2 of the present invention. Wherein: Figure 2 a represents the fluorescence curves of different concentrations of miR-155 detected by the Au@M biosensor; Figure 2b is a graph showing the linear relationship between the fluorescence intensity difference (ΔF) and the negative logarithm (-logC) of the corresponding concentration in the Au@Me-P detection of miR-155 between the experimental and control groups. Figure 2 c represents the fluorescence curves of different concentrations of miR-155 detected by Au@MFe-P; Figure 2 d is a graph showing the linear relationship between the fluorescence intensity difference (ΔF) and the negative logarithm (-logC) of the corresponding concentration in the Au@MFe-P detection of miR-155 between the experimental and control groups. Figure 2 e represents the fluorescence curves of Au@MFe-P in 1% human serum at different concentrations of miR-155; Figure 2 f is a graph showing the linear relationship between the fluorescence intensity difference (ΔF) and the negative logarithm (-logC) of the corresponding concentration in the Au@MFe-P assay for miR-155 detection in 1% human serum between the experimental group and the control group.

[0032] Figure 3 The figure shows the experimental results of Embodiment 3 of the present invention. Detailed Implementation

[0033] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0034] Example 1: Fabrication of Au@MFe-P fluorescent biosensor and detection of miR-155

[0035] (1) Preparation of gold nanoparticles (AuNPs)

[0036] Add 50 mL of ultrapure water to a 100 mL three-necked flask, then add 250 μL of 50 mM chloroauric acid solution (HAuCl4·4H2O). Heat to boiling with magnetic stirring. After boiling, quickly add 3.5 mL of 1 wt% sodium citrate solution, and continue stirring while boiling for 15 min. The solution color changes from pale yellow to wine red. Stop heating, allow to cool naturally to room temperature, and store at 4 ℃ protected from light for later use. The UV-Vis absorption spectra of the obtained AuNPs are shown below. Figure 1 a (characteristic absorption peak at 520 nm); transmission electron microscopy characterization is shown in [reference needed]. Figure 1 b. AuNPs exhibit regular spherical shapes, good dispersibility, and uniform particle size, with an average particle size of 18.87±4.4 nm.

[0037] (2) Preparation of red blood cell membrane (RBCm)

[0038] Take 10 mL of fresh sheep whole blood anticoagulated with heparin, centrifuge at 3500 rpm for 5 min, and remove the supernatant plasma and white membrane layer. Wash the lower red blood cells three times with PBS (pH 7.4) containing 1 mM EDTA-2Na. Add 5 times the original blood volume of 0.1×PBS (containing 1 mM EDTA-2Na), vortex vigorously for 10 s, and then place in an ice bath for lysis for 30 min. After complete lysis, centrifuge at 12000 rpm and 4 ℃ for 15 min, and discard the supernatant. Wash the precipitate repeatedly with PBS containing 1 mM EDTA-2Na until the supernatant is colorless and transparent and the precipitate is pale pink. Finally, resuspend the obtained red blood cell membrane in PBS containing 0.2 mM EDTA-2Na and store at 4 ℃ for later use. The UV-Vis absorption spectrum of the obtained red blood cell membrane suspension is shown in [Figure number missing]. Figure 1 c. Characteristic absorption peaks of membrane proteins and residual heme appear at 280 nm and 410 nm. The peak at around 280 nm corresponds to the ultraviolet absorption of conjugated double bonds in residues such as tryptophan and tyrosine contained in membrane proteins, while the peak at 410 nm is the characteristic absorption peak of the porphyrin ring in a small amount of residual heme in the erythrocyte membrane.

[0039] (3) Preparation of gold nanoparticles (Au@M) coated on red blood cell membranes

[0040] Take 2 mL of the above RBCm suspension and sonicate it with a probe (200 W, 3 s on, 2 s off, 10 min total) to form uniform vesicles. Mix the vesicled RBCm with 20 mL of AuNPs solution and sonicate it with a probe under ice bath (90% power, 4 s on, 2 s off, 15 min total). After sonication, centrifuge at 10000 rpm and 4 ℃ for 8 min, discard the supernatant, and remove excess cell membranes. Wash the precipitate once with ultrapure water and resuspend it in 1 mL of ultrapure water to obtain the Au@M nanocomposite. Transmission electron microscopy shows that the Au@M nanocomposite exhibits a distinct core-shell structure (see...). Figure 1 d), consistent with the particle size distribution of AuNPs above, the gold nucleus diameter is about 20 nm and the thickness of the red blood cell membrane coating layer is about 4 nm.

[0041] (4) Fe 3+ Preparation of surface-modified Au@MFe

[0042] Take 500 μL of the above Au@M suspension, add 100 μL of 100 μM FeCl3 solution, and incubate with shaking at room temperature for 30 min. After incubation, centrifuge at 10000 rpm for 8 min, discard the supernatant, and wash twice with ultrapure water to remove free Fe. 3+ Finally, it was resuspended in 200 μL of ultrapure water to obtain Au@MFe.

[0043] (5) Covalent linking of thiol-initiating probe (SH-Probe) with Au@MFe

[0044] Take 10 μL of 1 μM SH-Probe: SH-tttttttttttt aac ccc tat cac gat tag cattaa cca att tta atg cta atc ​​gtg (as shown in SEQ ID NO.01, which is a 5′ thiol-modified initiating chain designed for miR-155), add 1 μL of 100 mM TCEP, and incubate at 37 ℃ for 10 min to reduce any possible disulfide bonds. Mix the reduced SH-Probe with the above Au@MFe, and incubate at 37 ℃ with shaking for 30 min to allow them to be firmly linked by Au-S bonds. After the reaction is complete, centrifuge at 10000 rpm for 8 min, discard the supernatant, wash with ultrapure water and centrifuge 3 times to remove unreacted SH-Probe. Finally, resuspend the precipitate in 100 μL of ultrapure water to obtain the fluorescent biosensor Au@MFe-P.

[0045] (6) Hybridization chain reaction (HCR) based on cell membrane surface

[0046] Take 20 μL of the above Au@MFe-P (equivalent to approximately 0.1 mg Au@MFe), centrifuge at 10000 rpm for 5 min, discard the supernatant, and resuspend in 175 μL of ultrapure water. Add the following in sequence:

[0047] 2 μL 5 μM H1:tta atg cta atc ​​gtg gaa agt cac gat tag cat taa aat tgg (SEQ ID NO.02, hairpin probe).

[0048] 2 μL 5 μM H2:act ttc cac gat tag cat taa cca att tta atg cta atc ​​gtg (SEQ ID NO.03, hairpin probe).

[0049] 1 μL 1 μM target miR-155: uua aug cua auc gug aua ggg guu (RNA, SEQ ID NO. 04, different concentration gradients can be set optionally),

[0050] 20 μL 10× PB buffer (800 mM Na) + (pH 7.4).

[0051] After mixing thoroughly, the mixture was reacted at a constant temperature of 37 °C for 60 min.

[0052] (7) Fluorescence detection

[0053] After the reaction in step (6) was completed, the sample was centrifuged at 10,000 rpm for 8 min, the supernatant was discarded, and the sample was washed with ultrapure water and centrifuged three times to remove unreacted H1, H2, and free DNA strands. The final precipitate was resuspended in 100 μL of 1× SYBR Green I (diluted from 10,000× stock solution), transferred to a 96-well black plate, and measured using a fluorescence spectrophotometer (λex = 488 nm, λem = 500–650 nm, peak around 520 nm). The fluorescence intensity F was recorded, and ΔF = F – F0 was calculated using the fluorescence intensity F0 of the blank group without miR-155.

[0054] The fluorescence emission spectra of different concentrations of miR-155 (0 M, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM) in a pure buffer system are shown in [reference needed]. Figure 2 a. Fluorescence intensity increased significantly with increasing miR-155 concentration. Specifically: the vertical axis ΔF represents the difference (F-F0) between the fluorescence intensity of the experimental group and the corresponding control group, with miR-155 concentrations from top to bottom being 10 nM, 1 nM, 100 pM, 10 pM, 1 pM, and 100 fM.

[0055] The corresponding linear relationship between ΔF and the negative logarithm (-lgC) of miR-155 concentration is shown in the figure. Figure 2 Figure b shows the linear relationship between the fluorescence intensity difference (ΔF) and the negative logarithm (-logC) of the corresponding concentration in the Au@M detection of miR-155 between the experimental and control groups. The detection limit of Au@M for miR-155 is as low as 6.3 × 10⁻⁶. -14 M, linear range 10 -13 Up to 10 -8 M.

[0056] Fe 3+ The fluorescence emission spectra of the treated Au@MFe-P at the same concentration gradient are shown in the figure. Figure 2 c. Background fluorescence in the blank group was significantly lower than that in the untreated group; the corresponding linear relationship is shown in [see figure]. Figure 2 d. Specifically: the vertical axis ΔF represents the difference (F-F0) between the fluorescence intensity of the experimental group and the corresponding control group. From top to bottom, the miR-155 concentrations are 1 nM, 100 pM, 10 pM, 1 pM, 100 fM, and 10 fM, respectively. It can be seen that after Fe... 3+ After processing, its detection limit for miR-155 was further reduced to 1.6 × 10⁻⁶. -15 M, linear range is 10 -14 Up to 10 -9 M.

[0057] Example 2: Detection performance verification in 1% human serum

[0058] Take mixed serum from healthy individuals and dilute it 100 times with PBS to prepare a 1% serum matrix. Replace 175 μL of ultrapure water in step (6) of Example 1 with 175 μL of 1% human serum, and keep all other operations exactly the same. Add miR-155 standards to final concentrations of 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, and 10 nM respectively, and perform HCR reaction and fluorescence measurement. At the same time, set up three groups of spiked recovery experiments (adding known amounts of miR-155), and calculate the recovery rate and relative standard deviation (RSD, n=3).

[0059] The fluorescence emission spectra of miR-155 at different concentrations in a 1% human serum matrix are shown below. Figure 2 e. The fluorescence intensity still increases with concentration gradient. The vertical axis ΔF is the difference between the fluorescence intensity of the experimental group and the corresponding control group (F-F0). From top to bottom, the miR-155 concentrations are 10 nM, 1 nM, 100 pM, 10 pM, 1 pM, and 100 fM.

[0060] The corresponding linear relationship between ΔF and the negative logarithm of concentration is shown in the figure. Figure 2 Figure f shows the linear relationship between the fluorescence intensity difference (ΔF) and the negative logarithm (-logC) of the corresponding concentration in the Au@MFe-P detection of miR-155 in 1% human serum between the experimental group and the control group. It is evident that even in a serum environment containing a large amount of protein, the Au@MFe-P prepared in Example 1 maintains good signal response and linearity.

[0061] The results showed that Au@MFe-P maintained good linear response and anti-interference ability in a complex matrix of 1% human serum, and could still achieve a resolution of 4.3 × 10⁻⁶. -14 The detection limit of M and the recovery rate are between 99.4% and 102.7%, with an RSD ≤ 11.7%, proving that the present invention can achieve reliable detection in real clinical samples.

[0062] Example 3 Specificity Verification

[0063] Experiments were conducted under the exact same conditions as in Example 1 using equal concentrations (10 nM) of the following mismatched sequences: single-base mismatched sequence (mismatch 1: uua aug cua auc gugaua ggc guu, RNA, SEQ ID NO. 05), double-base mismatched sequence (mismatch 2: uua aug cua auc cugaua ggc guu, RNA, SEQ ID NO. 06), triple-base mismatched sequence (mismatch 3: uua aug gua auc guc auacgg guu, RNA, SEQ ID NO. 07), and miR-145: guc cag uuu ucc cag gaa ucc cu (RNA, SEQ ID NO. 08, replacing miR-155). The results are as follows: Figure 3 As shown, only the perfectly matched miR-155 produced significant fluorescence enhancement, while the fluorescence intensity of the other sequences was significantly different from that of the miR-155 group, proving that the Au@MFe-P prepared in Example 1 has excellent sequence-specific recognition ability.

[0064] The above embodiments demonstrate that the preparation method of the hybridization chain reaction fluorescent biosensor provided by the present invention has good reproducibility, clear steps, and mild conditions. It can achieve high sensitivity and high specificity detection of miR-155 in both pure buffer and diluted human serum, fully achieving the expected technical effect. It can be widely used for early non-invasive cancer screening, postoperative recurrence monitoring, and targeted therapy efficacy evaluation.

[0065] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A hybridization chain reaction fluorescent biosensor, characterized in that: Using erythrocyte membrane-coated gold nanoparticles as the reaction substrate, the surface of these gold nanoparticles is covalently linked with thiol-modified probes targeting miRNA via Au-S bonds. Furthermore, the erythrocyte membrane-coated gold nanoparticles are treated with Fe... 3+ deal with.

2. The hybridization chain reaction fluorescent biosensor as described in claim 1, characterized in that: The gold nanoparticles have a particle size of 15-15 nm.

3. The hybridization chain reaction fluorescent biosensor as described in claim 1, characterized in that: The thickness of the erythrocyte membrane coating is 3-6 nm.

4. The hybridization chain reaction fluorescent biosensor as described in claim 1, characterized in that: The gold nanoparticles have a particle size of 18-20 nm, and the red blood cell membrane coating has a thickness of 4-5 nm.

5. A hybridization chain reaction fluorescent biosensor as described in any one of claims 1 to 4, characterized in that: The Fe 3+ The process involved incubating gold nanoparticles coated with red blood cell membranes in FeCl3 solution with shaking at room temperature for 30-60 minutes, followed by centrifugation, discarding the supernatant, and washing with ultrapure water.

6. A method for preparing a hybridization chain reaction fluorescent biosensor according to any one of claims 1 to 5, characterized in that: Includes the following steps: (1) Preparation of gold nanoparticles; (2) Preparation of red blood cell membranes; (3) The red blood cell membrane obtained in step (2) is coated onto the gold nanoparticles obtained in step (1) to obtain the red blood cell membrane coated gold nanoparticles. (4) The gold nanoparticles coated with red blood cell membranes obtained in step (3) were subjected to Fe³⁺ treatment; (5) The thiol-modified initiation probe is covalently linked to the surface of the material obtained in step (4) via Au-S bond to obtain the material.

7. The preparation method according to claim 6, characterized in that: The coating in step (3) is achieved by mixing vesicularized red blood cell membranes with gold nanoparticles and then subjecting the mixture to ice bath ultrasound.

8. The preparation method according to claim 6, characterized in that: Before performing Au-S covalent linkage in step (5), the thiol-modified initiation probe is first reduced using TCEP.

9. Use of the hybridization chain reaction fluorescent biosensor according to any one of claims 1 to 4 in the preparation of a miRNA detection kit in body fluids.

10. A kit for detecting miRNA in body fluids, characterized in that: It has the hybridization chain reaction fluorescent biosensor as described in any one of claims 1 to 4.

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