A fluorescence / photothermal dual-mode imaging composite nanoprobe for detecting N-terminal pro-B-type natriuretic peptide and preparation and application thereof

Abstract

The application discloses a fluorescence / photothermal dual-mode imaging composite nanoprobe for detecting N-terminal B-type natriuretic peptide and preparation and application thereof, and relates to the technical field of medicine and biochemistry. The composite nanoprobe is prepared by loading a fluorescent dye Cy7 modified nucleic acid aptamer on the surface of AuAgPt NPs, and realizes fluorescence signal regulation by using nanometal surface energy transfer and internal filter effect. The composite nanoprobe can quantitatively and highly sensitively analyze the concentration of N-terminal B-type natriuretic peptide, and the detection limit is as low as 0.13 pg / mL. The probe integrates near-infrared fluorescence / photothermal dual-mode imaging to realize dynamic monitoring of N-terminal B-type natriuretic peptide in an in-vivo organ. The composite nanoprobe can be used for accurate and highly sensitive detection of N-terminal B-type natriuretic peptide in vitro, and provides a favorable method for early diagnosis of heart failure. In addition, the composite nanoprobe can be used for dynamic monitoring of N-terminal B-type natriuretic peptide in vivo, and provides a very promising new tool for pathological research and individualized diagnosis and treatment of heart failure and other diseases.

Description

Technical Field

[0001] This invention relates to the fields of medicine and biochemistry, and in particular to a fluorescent / photothermal dual-mode imaging composite nanoprobe for detecting N-terminal pro-B-type natriuretic peptide, its preparation and application. Background Technology

[0002] N-terminal pro-Brain natriuretic peptide (NT-proBNP) levels are significantly associated with cardiac stress response and the presence and severity of heart failure (HF). In the American College of Cardiology / American Heart Association (ACSA) heart failure guidelines, NT-proBNP is considered the gold standard biomarker for diagnosing HF. Currently, various methods exist for in vitro detection of NT-proBNP levels in blood. Common methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CIA), and fluorescence immunochromatography (FIC) typically rely on unstable antibodies as recognition elements, and cross-reactivity between antigens and antibodies can lead to biased specificity and insufficient sensitivity. Furthermore, although accurate detection of NT-proBNP concentration in blood is crucial for assessing cardiac function, NT-proBNP concentration is influenced by various factors such as age, sex, and renal function, sometimes weakening its correlation with the patient's actual cardiac function status, posing a significant challenge to NT-proBNP-based HF diagnosis. Therefore, establishing a method for real-time monitoring of NT-proBNP dynamics in vivo using visualization imaging technology is of great clinical significance for the early diagnosis and individualized treatment of HF.

[0003] Although various imaging methods have been developed for the dynamic monitoring of biomarkers in vivo, fluorescence imaging technology has attracted much attention due to its high sensitivity. However, due to the low abundance and high interference characteristics of biomarkers, fluorescence imaging technology alone still has limitations and technical bottlenecks in the in vivo imaging of heart failure biomarkers. It is impossible to monitor the organ distribution characteristics of NT-proBNP in vivo using fluorescence imaging, which limits its clinical application in the early diagnosis and treatment of heart failure. Summary of the Invention

[0004] The purpose of this invention is to provide a fluorescence / photothermal dual-mode imaging composite nanoprobe for detecting N-terminal pro-B-type natriuretic peptide, its preparation and application, so as to solve the problems existing in the prior art.

[0005] This invention develops a highly sensitive and specific probe for recognizing NT-proBNP based on the synergistic inner filter effect (IFE) fluorescence quenching mechanism of nanometal surface energy transfer (NSET) between gold-silver-platinum nanoparticles (AuAgPt NPs) and Cy7-modified NT-proBNP nucleic acid aptamers (Cy7-apt), as well as the specific recognition ability of the aptamer. This probe exhibits high sensitivity and strong specificity in in vitro detection of NT-proBNP. Furthermore, this invention provides a method for preparing the probe and its application in in vivo imaging. Through near-infrared fluorescence / photothermal dual-mode imaging, this probe can achieve dynamic in vivo monitoring of NT-proBNP. Therefore, this probe is expected to support personalized diagnosis of HF.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] One of the technical solutions of this invention is to provide a fluorescent / photothermal dual-mode imaging composite nanoprobe for detecting N-terminal pro-B natriuretic peptide. The composite nanoprobe is obtained by combining gold, silver, and platinum nanoparticles (AuAgPt NPs) and an N-terminal pro-B natriuretic peptide nucleic acid aptamer modified with the fluorescent dye Cy7 (Cy7-apt).

[0008] The sequence of the N-terminal B-type natriuretic peptide pronucleotide aptamer is 5'-CAG GGG GAC GGG TCG GGT TACAGC TGA GTG TGG CC-3'.

[0009] In this invention, the gold, silver, and platinum nanoparticles possess a broad localized surface plasmon resonance absorption peak in the 500-900 nm range, and this absorption peak overlaps with the excitation and emission spectra of the fluorescent dye Cy7. When the two are close together, with AuAgPt NPs acting as the energy acceptor and Cy7-apt as the energy donor, NSET and IFE occur between them, leading to fluorescence quenching and a "closed" fluorescence signal. When NT-proBNP is present in the system, the specific recognition of NT-proBNP by Cy7-apt increases the distance between Cy7-apt and AuAgPt NPs. The fluorescence emission wavelength of this probe is located in the near-infrared region, exhibiting a high signal-to-noise ratio and strong tissue penetration, thereby enhancing the sensitivity and imaging effect of this composite probe for monitoring NT-proBNP in vivo. Therefore, this fluorescent / photothermal dual-mode nanoprobe will provide a powerful imaging and detection tool for early diagnosis and treatment based on low-abundance NT-proBNP and for personalized diagnosis of HF in pathological process studies.

[0010] The second technical solution of the present invention provides a method for preparing the above-mentioned fluorescence / photothermal dual-mode imaging composite nanoprobe, comprising the following steps:

[0011] (1) Preparation of AuAgPt NPs:

[0012] a. In the presence of a surfactant, gold nanoparticles, silver ions and a reducing agent are mixed in a solution system, so that the silver ions are reduced and deposited on the surface of the gold nanoparticles to form a gold-silver binary alloy nanostructure.

[0013] b. Add platinum ions and a reducing agent to the reaction system of step a, so that the platinum ions are reduced and combined with the gold-silver binary alloy nanostructure, and then separated to obtain the AuAgPt NPs.

[0014] (2) Preparation of fluorescence / photothermal dual-mode imaging composite nanoprobe for detecting N-terminal pro-B natriuretic peptide: The AuAgPt NPs and Cy7-apt were mixed and reacted in a solution system to obtain the fluorescence / photothermal dual-mode imaging composite nanoprobe.

[0015] As a further preferred embodiment of the present invention, the gold nanoparticles are obtained by reducing gold ions in a solution system using a reducing agent.

[0016] The third technical solution of the present invention provides a method for detecting NT-proBNP using the above-mentioned fluorescence / photothermal dual-mode imaging composite nanoprobe, comprising the following steps:

[0017] (1) Construction of reaction system: The fluorescence / photothermal dual-mode imaging composite nanoprobe was mixed with a sample solution containing NT-proBNP to construct the reaction system;

[0018] (2) Signal detection: By detecting the changes in fluorescence signal of the reaction system, signal parameters related to the NT-proBNP concentration are obtained;

[0019] (3) Quantitative analysis: Based on the correspondence between fluorescence signal and NT-proBNP concentration, the content of NT-proBNP in the sample to be tested is determined.

[0020] As a further preferred embodiment of the present invention, during detection, the fluorescence / photothermal dual-mode imaging composite nanoprobe is mixed with standard solutions of NT-proBNP at different concentrations, and allowed to stand in the dark for 30 min. The fluorescence spectrum in the range of 760~820 nm is plotted. The concentration of NT-proBNP in the sample is positively correlated with the degree of fluorescence recovery. Based on the fluorescence intensity change at 773~777 nm, a corresponding linear regression equation is constructed with the fluorescence recovery efficiency of the composite nanoprobe and the concentration of NT-proBNP.

[0021] The composite nanoprobe described in this invention has a response time of 10~60 min to NT-proBNP.

[0022] The fluorescence / photothermal dual-mode imaging composite nanoprobe of this invention is a probe that enables real-time dynamic monitoring of NT-proBNP in living organisms.

[0023] The fourth technical solution of the present invention provides the application of the above-mentioned fluorescence / photothermal dual-mode imaging composite nanoprobe in the preparation of NT-proBNP fluorescence imaging detection reagent.

[0024] The fifth technical solution of the present invention provides the application of the above-mentioned fluorescence / photothermal dual-mode imaging composite nanoprobe in the preparation of NT-proBNP photothermal imaging reagent.

[0025] The sixth technical solution of this invention: providing the application of the above-mentioned fluorescence / photothermal dual-mode imaging composite nanoprobe in the preparation of diagnostic reagents for heart failure.

[0026] The present invention discloses the following technical effects:

[0027] 1. This invention constructs a fluorescence / photothermal dual-mode imaging composite nanoprobe. By precisely matching the local surface plasmon resonance absorption peak of AuAgPtNPs with the excitation and emission wavelengths of Cy7-apt, the overlap area of ​​the donor-acceptor spectra is increased. Based on the synergistic and efficient fluorescence quenching of NSET and IFE, the detection sensitivity of the composite nanoprobe is improved. Its detection range is 0.2~15 pg / ml, and the detection limit is as low as 0.13 pg / ml.

[0028] 2. This invention utilizes nucleic acid aptamers as target recognition elements, which can effectively avoid antibody-antigen cross-reaction and improve detection specificity.

[0029] 3. Based on the ingenious design of the composite probe, this invention integrates fluorescence / photothermal dual-mode imaging, and for the first time realizes the in vivo dynamic monitoring of NT-proBNP, breaking through the limitations of a single fluorescence imaging mode, and providing a powerful imaging and detection tool for early diagnosis and treatment of HF, personalized diagnosis and treatment, and pathological process research. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is the energy-dispersive X-ray spectral distribution of elemental surface of AuAgPt NPs.

[0032] Figure 2 The image shows the UV-Vis absorption spectrum of AuAgPt NPs.

[0033] Figure 3 The curves show the temperature changes of AuAgPt NPs under 808 nm laser irradiation with time and the linear fitting curves of cooling time t versus -ln(q).

[0034] Figure 4 To explore the feasibility of detecting NT-proBNP using composite nanoprobes.

[0035] Figure 5 To investigate the fluorescence quenching mechanism of the composite nanoprobe; A shows the UV-Vis absorption spectrum of AuAgPt NPs and the fluorescence excitation and emission spectra of Cy7-apt; B shows the fluorescence lifetime of the system under different conditions.

[0036] Figure 6 To optimize the detection performance of the composite nanoprobe for NT-proBNP; A shows the fluorescence quenching and recovery effects of the system with different concentrations of AuAgPt NPs; B shows the fluorescence quenching effect of the system with different quenching times; C shows the fluorescence recovery effect of the system with different recovery times; D shows the fluorescence recovery effect of the system with different pH values.

[0037] Figure 7 To investigate the detection performance of composite nanoprobes on NT-proBNP; A shows the fluorescence recovery spectra of the system with different concentrations of NT-proBNP; B shows the linear fitting graph of the fluorescence recovery efficiency of the system with the concentration of NT-proBNP; C shows the fluorescence recovery efficiency of NT-proBNP or other different interfering substances; D shows the fluorescence recovery efficiency of a mixture of NT-proBNP and other different interfering substances.

[0038] Figure 8 Evaluation of the cytotoxicity of AuAgPt NPs.

[0039] Figure 9 To investigate the in vivo monitoring ability of composite nanoprobes on NT-proBNP in HF mice using fluorescence imaging; A shows in vivo fluorescence imaging of mice at different time points after injection of different component solutions; B shows in vitro fluorescence imaging of the mouse heart, liver, spleen, lung, kidney, and brain after imaging; C shows in vitro fluorescence quantification of the mouse heart, liver, spleen, lung, kidney, and brain after imaging.

[0040] Figure 10To investigate the in vivo monitoring ability of composite nanoprobes on NT-proBNP in HF mice using photothermal in vivo imaging; A and B are imaging images of the temperature changes of the heart and brain of mice injected with different component solutions over time; C and D are bar charts of temperature difference-time changes in the heart and brain of mice injected with different component solutions; E~H are bar charts of temperature difference-time changes in the liver, kidney, spleen, and lungs of mice injected with different component solutions, respectively. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0047] The first aspect of the present invention is to provide a fluorescent / photothermal dual-mode imaging composite nanoprobe for detecting N-terminal pro-B natriuretic peptide, wherein the composite nanoprobe is obtained by combining gold, silver and platinum nanoparticles (AuAgPt NPs) and an N-terminal pro-B natriuretic peptide nucleic acid aptamer modified with the fluorescent dye Cy7 (Cy7-apt);

[0048] The sequence of the N-terminal B-type natriuretic peptide pronucleotide aptamer is 5'-CAG GGG GAC GGG TCG GGT TACAGC TGA GTG TGG CC-3'.

[0049] In this invention, the gold, silver, and platinum nanoparticles possess a broad localized surface plasmon resonance absorption peak in the 500-900 nm range, and this absorption peak overlaps with the excitation and emission spectra of the fluorescent dye Cy7. When the two are close together, with AuAgPt NPs acting as the energy acceptor and Cy7-apt as the energy donor, NSET and IFE occur between them, leading to fluorescence quenching and a "closed" fluorescence signal. When NT-proBNP is present in the system, the specific recognition of NT-proBNP by Cy7-apt increases the distance between Cy7-apt and AuAgPt NPs. The fluorescence emission wavelength of this probe is located in the near-infrared region, exhibiting a high signal-to-noise ratio and strong tissue penetration, thereby enhancing the sensitivity and imaging effect of this composite probe for monitoring NT-proBNP in vivo. Therefore, this fluorescent / photothermal dual-mode nanoprobe will provide a powerful imaging and detection tool for early diagnosis and treatment based on low-abundance NT-proBNP and for personalized diagnosis of HF in pathological process studies.

[0050] A second aspect of the present invention is to provide a method for preparing the above-mentioned fluorescence / photothermal dual-mode imaging composite nanoprobe, comprising the following steps:

[0051] (1) Preparation of AuAgPt NPs:

[0052] a. In the presence of a surfactant, gold nanoparticles, silver ions and a reducing agent are mixed in a solution system, so that the silver ions are reduced and deposited on the surface of the gold nanoparticles to form a gold-silver binary alloy nanostructure.

[0053] b. Add platinum ions and a reducing agent to the reaction system of step a, so that the platinum ions are reduced and combined with the gold-silver binary alloy nanostructure, and then separated to obtain the AuAgPt NPs.

[0054] (2) Preparation of fluorescence / photothermal dual-mode imaging composite nanoprobe for detecting N-terminal pro-B natriuretic peptide: The AuAgPt NPs and Cy7-apt were mixed and reacted in a solution system to obtain the fluorescence / photothermal dual-mode imaging composite nanoprobe.

[0055] Furthermore, the gold nanoparticles are obtained by reducing gold ions in a solution system using a reducing agent.

[0056] Furthermore, the preferred method for preparing the fluorescence / photothermal dual-mode imaging composite nanoprobe of the present invention is to employ the following steps:

[0057] (1) Add 5 mL of trimethyloctadecyl ammonium chloride solution (10 mM) and 6 mL of chloroauric acid (0.5 mM) to a 25 mL round-bottom flask, place it in an ice-water mixture for an ice bath, and let it stand for 10 min. Then, add 1 mL of ascorbic acid (10 mM) and let it stand for 30 min to obtain a gold nanoparticle dispersion.

[0058] (2) The gold nanoparticle dispersion was placed in a 60 ℃ water bath, and then 25 μL of potassium iodide solution (10 mM), 200 μL of potassium hydroxide solution (100 mM), 75 μL of silver nitrate solution (2 mM), and 625 μL of ascorbic acid solution (10 mM) were added sequentially. The reaction was allowed to proceed for 1 h. Finally, 75 μL of chloroplatinic acid (10 mM) was added, and the reaction was allowed to proceed for 4 h. After the reaction was completed, the resulting dispersion was centrifuged (10000 r / min, 10 min). After centrifugation, the supernatant was discarded, and the precipitate was resuspended in 3 mL of ultrapure water in the centrifuge tube to obtain the AuAgPt NPs dispersion.

[0059] (3) Mix AuAgPt NPs dispersion with Cy7-apt in a vortex and let stand at room temperature in the dark for 25 min to obtain fluorescence / photothermal dual-mode imaging composite nanoprobe.

[0060] A third aspect of the present invention is to provide a method for detecting NT-proBNP using the above-described fluorescence / photothermal dual-mode imaging composite nanoprobe, comprising the following steps:

[0061] (1) Construction of reaction system: The fluorescence / photothermal dual-mode imaging composite nanoprobe was mixed with NT-proBNP sample solution to construct the reaction system;

[0062] (2) Signal detection: By detecting the changes in fluorescence signal of the reaction system, signal parameters related to the NT-proBNP concentration are obtained;

[0063] (3) Quantitative analysis: Based on the correspondence between fluorescence signal changes and NT-proBNP concentration, the content of NT-proBNP in the sample to be tested is determined.

[0064] Furthermore, during detection, the fluorescence / photothermal dual-mode imaging composite nanoprobe was mixed with standard solutions of NT-proBNP at different concentrations, and allowed to stand in the dark for 30 min. Its fluorescence spectrum in the range of 760~820 nm was plotted. The concentration of NT-proBNP in the sample was positively correlated with the degree of fluorescence recovery. Based on the fluorescence intensity change at 773~777 nm, a corresponding linear regression equation was constructed between the fluorescence recovery efficiency of the composite nanoprobe and the concentration of NT-proBNP.

[0065] Furthermore, the standard solutions of NT-proBNP can be 0.2, 0.4, 0.8, 1.5, 3.0, 5.0, 8.0, 12, or 15 pg / mL.

[0066] The fluorescence / photothermal dual-mode imaging composite nanoprobe described in this invention has a response time of 10~60 min to NT-proBNP.

[0067] The fluorescence / photothermal dual-mode imaging composite nanoprobe of this invention is a probe that enables real-time dynamic monitoring of NT-proBNP in living organisms.

[0068] The fourth aspect of this invention is to provide the application of the above-mentioned fluorescence / photothermal dual-mode imaging composite nanoprobe in the preparation of NT-proBNP fluorescence imaging detection reagents.

[0069] The fifth aspect of this invention is to provide the application of the above-mentioned fluorescence / photothermal dual-mode imaging composite nanoprobe in the preparation of NT-proBNP photothermal imaging reagents.

[0070] The sixth aspect of this invention is to provide the application of the above-mentioned fluorescence / photothermal dual-mode imaging composite nanoprobe in the preparation of diagnostic reagents for heart failure.

[0071] Example 1: Preparation of AuAgPt NPs Dispersion

[0072] In a 25 mL round-bottom flask, 5 mL of trimethyloctadecylammonium chloride solution (10 mM) and 6 mL of chloroauric acid (0.5 mM) were added sequentially. The flask was placed in an ice-water bath and allowed to stand for 10 min. Then, 1 mL of ascorbic acid (10 mM) was added, and the mixture was allowed to stand for 30 min to obtain an Au NPs dispersion. The Au NPs dispersion was then placed in a 60 °C water bath, and 25 μL of potassium iodide solution (10 mM), 200 μL of potassium hydroxide solution (100 mM), 75 μL of silver nitrate solution (2 mM), and 625 μL of ascorbic acid solution (10 mM) were added sequentially. The reaction was allowed to proceed for 1 h. Finally, 75 μL of chloroplatinic acid (10 mM) was added, and the reaction was allowed to proceed for 4 h. After the reaction was complete, the resulting dispersion was centrifuged (10000 r / min, 10 min). After centrifugation, the supernatant was discarded, and 3 mL of ultrapure water was added to the centrifuge tube to resuspend the precipitate, thus obtaining the AuAgPt NPs dispersion (concentration of 1000 μM).

[0073] Energy-dispersive X-ray spectral elemental distribution of AuAgPt NPs:

[0074] The elemental distribution of AuAgPt NPs was characterized using energy-dispersive X-ray spectroscopy. The energy-dispersive X-ray spectra of the prepared AuAgPt NPs are shown below. Figure 1 As shown in the energy-dispersive X-ray diffraction (EDXRD) elemental distribution pattern, the material is composed of Au, Ag, and Pt. Au and Ag atoms are uniformly distributed in the central region, while Pt atoms are more densely distributed at the edges. This indicates that Pt-edge-modified AuAgPt NPs have been successfully prepared.

[0075] UV-Vis absorption spectra of AuAgPt NPs:

[0076] A 250 μM dispersion of AuAgPt NPs was prepared, and its absorption intensity in the 200–900 nm range was scanned using a UV-Vis spectrophotometer. The UV-Vis absorption spectrum of the prepared AuAgPt NPs is shown below. Figure 2 As shown, it can be seen that it has a very broad local surface plasmon resonance absorption peak in the range of 500-900 nm.

[0077] Photothermal property characterization of AuAgPt NPs:

[0078] 350 μL AuAgPt NPs (final concentration 62.5 μM) were placed in a 96-well plate and sprayed at 0.33 W / cm². 2An 808 nm laser was used to irradiate the sample area, with the laser spot diameter covering the entire sample region. Once the solution temperature reached its maximum steady-state temperature, the laser was turned off, allowing it to cool naturally. During this period, a thermal imager was used to monitor the solution temperature at 20-second intervals. A linear fit was then performed with cooling time t as the vertical axis and -Ln(q) as the horizontal axis to calculate the slope τ. The photothermal conversion efficiency (η) of AuAgPtNPs was then calculated using the formula.

[0079] The formula for calculating photothermal conversion efficiency is as follows:

[0080]

[0081] Where A is the surface area for radiative heat transfer, and ΔT max The maximum steady-state temperature of AuAgPt NPs at room temperature is given by h, where h is the heat transfer coefficient. Let I be the maximum steady-state temperature of H2O at room temperature, and A be the power density. 808 The absorbance of the AuAgPt NPs dispersion at 808 nm is given.

[0082] The temperature change curves of AuAgPt NPs dispersion during heating and cooling, and the linear relationship between the cooling stage time t-ln(q) are shown in the figure. Figure 3 The slope τ was calculated to be 217.29. Combining this with the above photothermal conversion efficiency formula, the η of AuAgPt NPs was calculated to be 67.6%. This indicates that the synthesized AuAgPt NPs possess excellent photothermal properties.

[0083] Example 2: Preparation of AuAgPt NPs-Cy7-apt composite nanoprobes

[0084] Add AuAgPt NPs dispersion (final concentration 5 μM), 100 µL PBS buffer (pH 7.4), and 40 µL Cy7-apt solution (200 nM) sequentially to an EP tube, and vortex to mix. Let stand at room temperature for 25 min to obtain the fluorescence / photothermal dual-mode imaging composite nanoprobe (AuAgPt NPs-Cy7-apt).

[0085] 1. Feasibility verification of composite nanoprobe detection of NT-proBNP

[0086] Experimental group setup: Six control groups and one experimental group were set up, with the specific composition of each group as follows:

[0087] Control group 1 (Cy7-apt): A solution containing only the Cy7-modified NT-proBNP aptamer (Cy7-apt); the Cy7-apt concentration was 20 nM.

[0088] Control group 2 (Cy7-apt+NT-proBNP): a mixed solution of Cy7-apt and NT-proBNP; Cy7-apt concentration was 20 nM, and NT-proBNP concentration was 40 pg / mL;

[0089] Control group 3 (Cy7-apt + AuAgPt NPs): a mixed solution of Cy7-apt and AuAgPt NPs; Cy7-apt concentration was 20 nM, and AuAgPt NPs concentration was 4.2 μM;

[0090] Control group 4 (AuAgPt NPs +NT-proBNP): a mixed solution of NT-proBNP and AuAgPt NPs; the concentration of NT-proBNP was 40 pg / mL, and the concentration of AuAgPt NPs was 4.2 μM;

[0091] Control group 5 (NT-proBNP): a solution containing only NT-proBNP; the NT-proBNP concentration was 40 pg / mL;

[0092] Control group 6 (AuAgPt NPs): a dispersion containing only AuAgPt NPs; the concentration of AuAgPt NPs was 4.2 μM;

[0093] Experimental group (Cy7-apt+AuAgPt NPs+NT-proBNP): a mixed solution of Cy7-apt, AuAgPt NPs and NT-proBNP; Cy7-apt concentration was 20 nM, AuAgPt NPs concentration was 4.2 μM, and NT-proBNP concentration was 40 pg / mL.

[0094] NT-proBNP (final concentration of 40 pg / mL) was added to the experimental group sample, and the system was allowed to stand in the dark for 60 min. After the reaction, ultrapure water was added to the experimental group system to make up the total volume to 400 μL. Then, the fluorescence intensity of the system in the wavelength range of 760~820 nm was measured using the corresponding detection equipment.

[0095] The results are as follows Figure 4 As shown, when Cy7-apt and AuAgPt NPs are present simultaneously in the system, the fluorescence of Cy7-apt is effectively quenched, with a quenching efficiency as high as 88.3%. After the addition of NT proBNP, Cy7-apt specifically binds to NT proBNP, causing Cy7-apt to detach from the surface of AuAgPt NPs, and the fluorescence of the system is restored. This indicates that the method for detecting NT proBNP is feasible.

[0096] 2. Investigation into the quenching mechanism of composite nanoprobes

[0097] The sequence of the NT-proBNP aptamer synthesized by Sangon Biotech (Shanghai) Co., Ltd. is: 5'-CAG GGG GAC GGG TCG GGT TAC AGC TGA GTG TGG CC-3' (SEQ ID NO.1).

[0098] Add 29.3 μL of ultrapure water to Cy7-apt lyophilized powder to prepare a stock solution with a concentration of 100 μM, and then dilute it with 0.01 M phosphate buffer (pH 7.4) to the corresponding concentration for use.

[0099] a. Prepare a 20 nM Cy7-apt solution and measure its fluorescence excitation and emission spectra; prepare a 250 μM AuAgPt NPs dispersion and measure its UV-Vis absorption spectra.

[0100] from Figure 5 As can be seen from A, AuAgPt NPs have a broad local surface plasmon resonance absorption peak in the range of 600 nm-900 nm, and it highly overlaps with the excitation and emission spectra of Cy7-apt, indicating that AuAgPt NPs and Cy7-apt have the conditions for NSET and IFE to occur.

[0101] b. Prepare a 20 nM Cy7-apt solution and determine its fluorescence lifetime. Simultaneously, prepare a composite nanoprobe according to Example 4 and determine its fluorescence lifetime.

[0102] The results are as follows Figure 5 As shown in Figure B, the fluorescence lifetime τ of Cy7-apt alone is 1.05 ns, while the fluorescence lifetime τ is shortened to 0.93 ns after the addition of AuAgPt NPs, indicating that the fluorescence quenching of Cy7-apt is due to NSET between the two. Further calculations showed that the energy transfer efficiency was 11.4% < 88.3%, indicating that the fluorescence quenching was not only caused by NSET, but also by IFE.

[0103] 3. Optimization of various detection conditions for NT-proBNP detection based on composite nanoprobes

[0104] a. Concentration optimization of AuAgPt NPs

[0105] The concentration of AuAgPt NPs in the composite nanoprobe was adjusted to achieve final concentrations of 3.1, 3.6, 4.2, 5.0, and 6.3 μM in the detection system. Its fluorescence intensity in the 773 nm–777 nm range was then measured.

[0106] The results are as follows Figure 6 As shown in Figure A, the fluorescence quenching efficiency of AuAgPt NPs on Cy7-apt gradually decreases with decreasing AuAgPt NPs concentration (from 89.3% to 66.5%). When the material concentration is 5 μM, the fluorescence quenching efficiency of the composite nanoprobe is 87.0%, and the fluorescence recovery efficiency is as high as 63.2%. Therefore, 5 μM is selected as the optimal material concentration for the reaction.

[0107] b. Optimization of system fluorescence quenching time

[0108] The final concentration of AuAgPt NPs was fixed at 5.0 μM, the final concentration of Cy7-apt was 20 nM, the pH of the detection system was 7.4, and the quenching times were adjusted to 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, and 35 min. The fluorescence intensity in the 773 nm–777 nm range was measured.

[0109] The results are as follows Figure 6 As shown in Figure B, the fluorescence quenching efficiency of the system gradually increases with the extension of the reaction time between AuAgPt NPs and Cy7-apt. When the quenching time is 25 min, the fluorescence quenching efficiency reaches 88.9% and tends to stabilize. Therefore, 25 min was subsequently selected as the optimal reaction time for fluorescence quenching of the system.

[0110] c. Optimization of system fluorescence recovery time

[0111] The final concentration of AuAgPt NPs was fixed at 5.0 μM, the final concentration of Cy7-apt was 20 nM, the pH of the detection system was 7.4, the quenching time was 25 min, and six different recovery times were set: 10 min, 15 min, 20 min, 30 min, 45 min, and 60 min. The fluorescence intensity in the range of 773 nm to 777 nm was tested.

[0112] The results are as follows Figure 6 As shown in Figure C, under the condition that other conditions remain the same, the fluorescence recovery efficiency of the system gradually increases with the extension of the incubation time of the composite nanoprobe and NT-proBNP. When the incubation time is 30 min, the fluorescence recovery efficiency of the system is as high as 65.5% and remains stable. Therefore, 30 min was selected as the optimal incubation time for fluorescence recovery in subsequent experiments.

[0113] d. Optimization of system pH

[0114] The concentration of AuAgPt NPs suspension was fixed at 5.0 μM, the final concentration of Cy7-apt was 20 nM, the quenching time was 25 min, and the recovery time was 30 min. Five pH buffer systems were set up: pH 6.0, 6.5, 7.0, 7.4, and 8.0. The fluorescence intensity in the 773 nm–777 nm range was measured.

[0115] The results are as follows Figure 6 As shown in Figure D, when the pH of the system is in the range of 6 to 8, there is no significant difference in the fluorescence quenching efficiency of the composite nanoprobe. In order to facilitate the application of the composite nanoprobe to the detection of actual samples, pH 7.4 was selected as the optimal reaction pH of the system.

[0116] 4. Establishment of a standard curve for the detection of NT-proBNP using composite nanoparticles

[0117] Based on the optimization results described above, NT-proBNP was detected. Eleven groups with different concentrations of NT-proBNP were set up as experimental groups, from low to high: 0.2, 0.4, 0.8, 1.5, 3.0, 5.0, 8.0, 12, 15, 30, and 40 pg / mL. A standard curve was constructed corresponding to the fluorescence recovery efficiency of the system and the NT-proBNP concentration.

[0118] The results are as follows Figure 7 As shown in A and 7B, the fluorescence recovery efficiency of the composite nanoprobe showed a good linear correlation with the NT-proBNP concentration in the range of 0.2–15 pg / mL. The obtained linear regression equation was: (F2–F1) / (F0–F1) = 1.77c + 15.6, r = 0.991 (F0 is the initial fluorescence intensity of Cy7-apt; F1 is the fluorescence intensity of the AuAgPt NPs-Cy7-apt composite nanoprobe; F2 is the fluorescence intensity recovered after the addition of NT-proBNP; c is the concentration of NT-proBNP). The calculated detection limit was 0.13 pg / mL, indicating that the method has high sensitivity for detecting NT-proBNP.

[0119] 5. Selectivity test for NT-proBNP detection

[0120] Based on the optimization results described above, the selectivity of the composite nanoprobe for detecting NT-proBNP was evaluated. Seventeen other substances, including C-type natriuretic peptide (CNP), C-reactive protein (CRP), hemoglobin (Hb), and pro-brain natriuretic peptide (proBNP), were used as interfering substances for detection. The final concentration of NT-proBNP and each interfering substance was 10 pg / mL.

[0121] The results are as follows Figure 7 As shown in Figure C, when a certain amount of NT-proBNP is present in the system, the fluorescence recovery efficiency of the composite nanoprobe is greater than 30%. In contrast, the fluorescence recovery efficiency of the composite nanoprobe is less than 8% for other interfering substances, indicating that the constructed composite nanoprobe has high specificity for the detection of NT-proBNP.

[0122] 6. Testing the anti-interference capability of NT-proBNP

[0123] Based on the optimization results above, the anti-interference ability of the composite nanoprobe to detect NT-proBNP was evaluated. Three groups of solutions were set up for the experiment: Group 1 was a mixture of bovine serum albumin, atrial natriuretic peptide, pre-B-type natriuretic peptide, hemoglobin, and lactoferrin; Group 2 was NT-proBNP alone; and Group 3 was a mixture of Group 1 with the same concentration of NT-proBNP added. The concentration of NT-proBNP and each interfering substance in the system was 10.0 pg / mL.

[0124] from Figure 7 As can be seen from D, the fluorescence recovery efficiency of the system is low when only interfering substances are present. Moreover, under the condition that NT-proBNP and interfering substances coexist, the fluorescence recovery efficiency of the composite nanoprobe is comparable to that when only NT-proBNP is present, indicating that the composite nanoprobe has a strong anti-interference ability for NT-proBNP detection.

[0125] 7. Spiked recovery experiment of NT-proBNP in actual serum samples using composite nanoprobes

[0126] Three groups of NT-proBNP spikes were set up for experiments: low (1.0 pg / mL), medium (5.0 pg / mL), and high (10 pg / mL). Serum samples from suspected HF patients (collected from the University City Hospital Affiliated to Chongqing Medical University) were used as samples. The serum samples were diluted according to clinical detection values ​​(samples 1 and 2 were diluted 3000-fold, and sample 3 was diluted 2000-fold). The concentration of NT-proBNP in each group was measured using a composite nanoprobe. Each group was tested in triplicate, and the spiked recovery rate was calculated.

[0127]

[0128] Table 1. Spike recovery experiments of this method in actual plasma samples.

[0129]

[0130] The experimental results are shown in Table 1. The recovery rate of NT-proBNP spiked in serum by this method was 89.2% to 113%, and the relative standard deviation obtained from three parallel experiments was 3.70% to 10.3%, indicating that the method has good accuracy in detecting NT-proBNP levels in actual serum samples.

[0131] 8. Cytotoxicity evaluation of AuAgPt NPs

[0132] Different concentrations of AuAgPt NPs (5.00, 10.0, 16.6, 25.0, 41.7, 62.5, 125 μM) were mixed with human umbilical vein endothelial cells, and triplet wells were prepared. The mixtures were incubated at 37 °C with 5% CO2 for 24 h. After the incubation period, 20 μL of CCK-8 reagent was added to each well, and incubation continued for another 1 h. After incubation, the absorbance of the 96-well plate was measured using a microplate reader, and the average value of the replicates was recorded. The absorbance value (OD) was read at 450 nm. 450 ), calculate cell viability.

[0133] Cell viability (%) = (As - A) b ) / (A c -A b )

[0134] Among them, As and A b A c The absorbance values ​​at 450 nm are for the experimental group, control group, and blank group, respectively.

[0135] The results are as follows Figure 8As shown, after co-incubation with different concentrations of AuAgPt NPs for 24 h, the cell survival rate of each experimental group was >80%, indicating that the nanomaterial does not affect the growth status of cells in the concentration range of 5~125 μM and has low cytotoxicity.

[0136] 9. Ability of composite nanoprobes to perform near-infrared fluorescence / photothermal imaging of NT-proBNP at the in vivo level

[0137] (1) First, the HF mouse model was constructed. C57BL / 6j mice (7-9 weeks old, male) were purchased from the Experimental Animal Center of Chongqing Medical University. The animal handling procedures were approved by the Animal Experimental Animal Management and Use Committee of Chongqing Medical University (No. IACUC-CQMU-2024-0122). The model mice with different degrees of HF were constructed in reverse chronological order: C57BL / 6j mice (8-9 weeks old, male) were injected intraperitoneally daily with doxorubicin (Dox, 1 mg / kg / day) to construct HF-4W mice. At the same time, healthy mice were injected intraperitoneally daily with an equal volume of 0.9% NaCl solution. HF-2W mice were constructed two weeks after the start of modeling, and HF-1W mice were constructed three weeks after the start of modeling. After modeling, healthy and HF mice were grouped as follows: In fluorescence imaging, groups I, II, III, IV, V, VI, and VII were respectively injected with PBS, Cy7-apt, Cy7-apt + AuAgPt NPs, Cy7-apt, HF-1W, Cy7-apt + AuAgPt NPs, Cy7-apt + AuAgPt NPs, and Cy7-apt + AuAgPt NPs into healthy mice, respectively; in photothermal imaging, groups I, II, III, IV, V, VI, and VII were respectively injected with PBS, AuAgPt NPs, AuAgPt NPs, Cy7-apt + AuAgPt NPs, Cy7-apt + AuAgPt NPs, and Cy7-apt + AuAgPt NPs into healthy mice, respectively. AuAgPt NPs were injected into HF-4W mice with Cy7-apt + AuAgPt NPs. All mice were anesthetized with ether before imaging.

[0138] (2) Fluorescence Imaging: PBS, Cy7-apt solution, and AuAgPt NPs-Cy7-apt composite nanoprobes were injected into each group of mice via the tail vein to ensure that the concentration of AuAgPt NPs in the mice was 25 μM and the concentration of Cy7-apt was 100 nM. Fluorescence images of each group of mice were acquired using an In Vivo Smart-LF fluorescence imaging system (VIEWORKS) at 0, 0.5, 1, 2, and 3 h after administration, with imaging parameters kept constant (light source: ICG, laser intensity: middle). After in vivo imaging, the mice were euthanized by cervical dislocation, and then dissected. Heart, liver, spleen, lung, kidney, and brain tissue samples were collected sequentially. Fluorescence images of the isolated organs were acquired using an In Vivo Smart-LF fluorescence imaging system (VIEWORKS) (light source: ICG, laser intensity: middle), and the fluorescence intensity of each tissue was quantitatively analyzed.

[0139] from Figure 9 As shown in Figure A, the PBS group mice exhibited only weak fluorescence, while the fluorescence intensity of Healthy and HF mice injected via the tail vein into Cy7-apt was significantly higher than that of other groups. Notably, because the concentration of NT-proBNP in HF mice was much higher than in healthy mice, the fluorescence signal intensity of HF mice injected with the AuAgPt NPs-Cy7-apt composite nanoprobe was higher than that of Healthy mice injected with the same probe. To further investigate the distribution of Cy7-apt in vivo, in vitro organ imaging was performed (…). Figure 9 (B, 9C). The results showed that the fluorescence intensity of all organs in the HF group mice injected with the composite nanoprobe was higher than that in the Healthy group mice injected with the composite nanoprobe, and the fluorescence intensity of each organ increased with the degree of heart failure. Furthermore, the distribution of fluorescence signal intensity varied among organs, with the liver and kidneys showing the strongest fluorescence signals. This distribution characteristic suggests that the possible binding products of the composite nanoprobe after specific recognition with NT-proBNP are mainly metabolized and cleared via the liver and kidneys. In summary, this indicates that the AuAgPt NPs-Cy7-apt composite nanoprobe can reflect changes in NT-proBNP levels in HF mice through fluorescence signal intensity.

[0140] (3) Photothermal imaging: AuAgPt NPs and a mixture of AuAgPt NPs and Cy7-apt were injected via the tail vein to ensure that the concentration of AuAgPt NPs in mice was 50 μM. Precise photothermal imaging of various organs of mice was performed using an 808 nm laser at 0.5, 1.5, 3, and 5 h after administration (5 min, 0.5 W / cm²). 2The temperature changes of each organ were recorded. After in vivo imaging, the mice in each group were euthanized using the cervical dislocation method.

[0141] The results are as follows Figure 10 As shown, the ΔT values ​​in various organs of mice in the PBS group were lower than those in other groups. Furthermore, it can be observed that with prolonged injection time, except for the PBS group, the ΔT values ​​in the heart and brain of mice in the other HF and healthy groups generally showed a trend of first increasing and then decreasing, reaching a peak at 1.5 h. The ΔT values ​​in the heart of mice injected with the composite nanoprobe in the HF-1W, HF-2W, and HF-4W groups were 1.00–1.67 times higher than the average ΔT values ​​of the HF+AuAgPt NPs group and 1.02–2.06 times higher than those of the Healthy+AuAgPt NPs-Cy7-apt group. Figure 10 A, C). In addition, the ΔT in the brains of mice injected with the composite nanoprobes in the HF-1W, HF-2W, and HF-4W groups was 1.14–1.80 times that of the HF+AuAgPt NPs group, and 1.41–1.84 times that of the Healthy+AuAgPt NPs-Cy7-apt group. Figure 10 (B, D). The results showed that the ΔT in the heart and brain of HF mice injected with the composite nanoprobe was not only higher than that in other healthy mice, but also higher than that in HF mice injected with AuAgPt NPs alone. At the same time, ΔT was positively correlated with the degree of HF in the mice, indicating that the difference in ΔT was due to the specific recognition of NT-proBNP by the composite nanoprobe.

[0142] Photothermal imaging was performed on the liver, kidneys, lungs, and spleen of mice in each group, and the results are as follows: Figure 10 E and Figure 10In mice, the ΔT values ​​in the liver and kidneys showed a trend of increasing and then decreasing, reaching a peak at 3 h. Compared with other control groups, the ΔT values ​​in the HF-1W, HF-2W, and HF-4W mice injected with the composite nanoprobe group were 1.02–1.39 times higher than those in the HF+AuAgPt NPs group and 1.12–1.74 times higher than those in the Healthy+AuAgPt NPs-Cy7-apt group. The renal ΔT values ​​in the HF-1W, HF-2W, and HF-4W mice injected with the composite nanoprobe group were the highest, reaching 0.97–2.12 times higher than those in the HF+AuAgPt NPs group and 0.91–1.79 times higher than those in the Healthy+AuAgPt NPs-Cy7-apt group. There was no significant difference in temperature increase between heart failure and healthy mice, which may be because AuAgPt NPs are metabolized by the liver, therefore the ΔT value in the liver is mainly affected by the metabolism of AuAgPt NPs themselves. Meanwhile, since NT-proBNP is metabolized by the kidneys, the difference in ΔT between healthy mice and heart failure mice was more pronounced in the kidneys than in the liver. The lungs and spleen are also important components of the circulatory system; therefore, the spleen of each group of mice was analyzed. Figure 10 G) and lungs ( Figure 10 H) Photothermal imaging was performed. Compared with other organs, the lungs and spleen showed no other regular changes in ΔT except for a trend of first increasing and then decreasing.

[0143] This invention leverages the synergistic fluorescence quenching effect of AuAgPt NPs on the NSET and IFE of the Cy7-modified NT-proBNP aptamer, along with the aptamer's specific recognition capability, to achieve "on-off" modulation of the Cy7 fluorescence signal. This enables highly sensitive and specific determination of NT-proBNP levels, with a detection limit as low as 0.13 pg / mL. Furthermore, the composite nanoprobe, combining the excellent photothermal effect of AuAgPt NPs with the near-infrared fluorescence properties of Cy7, can be used for fluorescence / photothermal dual-mode imaging of NT-proBNP in vivo. Therefore, this composite probe holds promise for supporting personalized diagnosis of heart failure.

[0144] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A fluorescence / photothermal dual-mode imaging composite nanoprobe for detecting N-terminal pro-B-type natriuretic peptide, characterized in that, It was obtained by combining N-terminal B-type natriuretic peptide pronucleotide aptamers modified with gold, silver and platinum nanoparticles and fluorescent dye Cy7; The gold, silver and platinum nanoparticles have a broad local surface plasmon resonance absorption peak in the range of 500~900nm, and this absorption peak overlaps with the excitation and emission spectra of the fluorescent dye Cy7. The nucleotide sequence of the N-terminal B-type natriuretic peptide pronucleotide aptamer is shown in SEQ ID NO.1; The preparation method of the fluorescence / photothermal dual-mode imaging composite nanoprobe includes the following steps: (1) Preparation of gold, silver and platinum nanoparticles: a. In the presence of a surfactant, gold nanoparticles, silver ions and a reducing agent are mixed in a solution system, so that the silver ions are reduced and deposited on the surface of the gold nanoparticles to form a gold-silver binary alloy nanostructure. b. Add platinum ions and a reducing agent to the reaction system of step a, so that the platinum ions are reduced and combined with the gold-silver binary alloy nanostructure, and then separated to obtain the gold-silver-platinum nanoparticles. (2) Preparation of fluorescence / photothermal dual-mode imaging composite nanoprobe for detecting N-terminal pro-B natriuretic peptide: The gold, silver and platinum nanoparticles and the N-terminal pro-B natriuretic peptide nucleic acid aptamer modified with the fluorescent dye Cy7 were mixed and reacted in a solution system to obtain the fluorescence / photothermal dual-mode imaging composite nanoprobe.

2. The fluorescence / photothermal dual-mode imaging composite nanoprobe according to claim 1, characterized in that, The gold nanoparticles are obtained by reducing gold ions in a solution system using a reducing agent.

3. The application of the fluorescence / photothermal dual-mode imaging composite nanoprobe as described in claim 1 in the preparation of a fluorescence imaging detection reagent for N-terminal pro-B-type natriuretic peptide.

4. The application of the fluorescence / photothermal dual-mode imaging composite nanoprobe as described in claim 1 in the preparation of N-terminal pro-B-type natriuretic peptide photothermal imaging reagent.

5. The application of the fluorescence / photothermal dual-mode imaging composite nanoprobe as described in claim 1 in the preparation of diagnostic reagents for heart failure.

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