Chromium-based nano-particles with fluorescence tunable capability and application of chromium-based nano-particles

Abstract

The invention discloses chromium-based nano-particles with fluorescence tunable capability and application of the chromium-based nano-particles. The synthesis method provided by the invention only needs to simply mix the pre-cooled Cr < 6 + > and NaBH4 solutions for 30 minutes. The obtained spherical Cr NPs shows unique fluorescence modulation on dyes with different components and DNA (Deoxyribose Nucleic Acid). Based on the fluorescence characteristics, a CD63 aptamer-Cr NPs sensing system is constructed and is used for detecting CD63 positive TDEs, and coding and information protection can be carried out even in a real sample. In the system, a fluorescently-labeled CD63 aptamer is used as a recognition probe and an information carrier and is adsorbed to Cr NPs to form a steganography object. When the CD63 aptamer-Cr NPs is specifically combined with the CD63 or the TDEs, different fluorescence responses can be triggered, so that accurate quantitative detection and data encryption and protection are realized.

Description

Technical Field

[0001] This invention relates to nanomaterials technology, specifically to chromium-based nanoparticles with tunable fluorescence and their applications. Background Technology

[0002] In recent years, metal-based nanomaterials, due to their small size, large specific surface area, and unique physicochemical properties such as modifiability and anisotropy, have exhibited a wide range of optical, electrochemical, catalytic, and magnetic characteristics, leading to their widespread application in fields such as biological and chemical sensing, catalysis, electrochemical supercapacitors, disease treatment, antibacterial agents, and environmental protection. Besides the highly regarded noble metal nanomaterials, chromium-based nanomaterials have also attracted considerable attention due to their similar properties, showing great potential in applications such as sensing, catalysis, biomedicine (anti-cancer, antibacterial, antioxidant, anti-inflammatory, etc.), and environmental remediation. Currently, the synthesis of metal nanomaterials mainly relies on biosynthesis and physicochemical synthesis methods. While the method of synthesizing chromium nanomaterials using plants is environmentally friendly, it is difficult to achieve precise control and purify impurities. Physicochemical synthesis methods generally suffer from complex operation, high production costs, and operation at high temperatures (100-600℃), such as chemical vapor deposition (CVD), microwave plasma methods, laser vapor deposition, hydrothermal methods, high-energy ball milling, thermal decomposition, and thermal spraying. Although some methods produce materials with high purity, uniformity, and good dispersibility, numerous limitations hinder their widespread practical application. Therefore, there is an urgent need to explore an efficient, simple, and environmentally friendly synthetic route for chromium-based nanomaterials, while simultaneously investigating their potential properties (such as optical properties) and expanding their applications in new fields (such as biomedical diagnostics). Furthermore, tumor-derived exosomes (TDEs, approximately 30-150 nm in diameter), nanoscale vesicles released by tumor cells into their microenvironment, have become an emerging non-invasive biomarker. Because their surface carries various marker proteins that can reflect information such as tumor presence and metastasis, researchers are dedicated to developing nanoanalytical techniques based on affinity probes (such as CD63 antibodies and nucleic acid aptamers) targeting specific targets. Compared to antibodies, nucleic acid aptamers or peptides obtained through artificial evolutionary screening exhibit significant advantages in molecular weight, ease of synthesis, stability, and affinity, making them more readily applicable in the biomedical and analytical science fields. Therefore, the rapid and convenient detection of TDEs and their markers using small molecule affinity probes such as nucleic acid aptamers or affinity peptides has become a highly promising method for early tumor diagnosis and assessment.

[0003] Due to the high sensitivity and visualization properties of fluorescence, exploring the fluorescence properties of nanomaterials and combining them with biomolecules (such as DNA and peptides) will help develop fluorescence biosensing and imaging methods for tumor diagnosis and environmental monitoring, and establish fluorescence molecular information technologies (logic gates, information security, etc.). These fluorescence nanoquenchers (Fe-PDAN submicrobeads, MoS2 nanosheets, single / multi-walled carbon nanotubes, platinum nanoprobes, gold nanorods) and biomolecular probes are widely used in fields such as biosensing, medical diagnostics, and molecular logic computation. For example, CNS carbon nanotubes, combined with FAM-labeled MCF-7 cell surface marker protein (MUC-1) aptamers, can achieve low-toxicity, high-sensitivity, and rapid targeted cancer diagnosis; the dual-emission ratio fluorescence sensor Eu@TCBP-HOF can detect breast cancer (spermine) and ovarian cancer markers (acetylceramide) with high sensitivity in saliva and serum samples, and provide visual results in a very short time through algorithms; MIL-101 (Cr) has adsorption properties and can achieve highly sensitive detection of single and double stranded DNA with different affinities through ATP-assisted exonuclease amplification, and an AND logic gate is constructed based on this; Cr2O3 nanoparticles specifically recognize T-Hg. 2+ The -T structure was designed as a fluorescence sensing system for detecting Hg in water samples. 2+Ions were identified, and a molecular logic computing system for implementing simple logic gate operations was constructed. Furthermore, some studies combined nucleic acid aptamers with nanomaterials to construct various sensing platforms, including fluorescence, electrochemical, colorimetric, and surface-enhanced Raman spectroscopy, to detect TDEs and their labels, and to perform molecular information processing, such as constructing logic gates. For example, the synergistic interaction between Fe3O4@CD63-CLIKKPF and CD63 protein and phosphatidylserine on the surface of exosomes enables highly specific and high-yield separation of exosomes in urine, while utilizing an AND logic gate; CuCo2O4 nanorods exhibit high peroxidase-like activity when bound to CD63 aptamers. When TDEs specifically bind to CD63 aptamers, CuCo2O4 nanorods lose their hindrance, allowing electron transfer between them and the chromogenic substrate, resulting in a significant change in catalytic activity and thus triggering a color change, enabling the visual detection of exosomes; Fe-MOF also uses the same principle to detect TDEs. Compared to CuCo2O4 nanorods, which can only detect breast cancer, this method can also rapidly distinguish CD63 protein expression from breast cancer, gastric cancer, and lung cancer cell lines; Ti3C2 MXenes nanosheets adsorb Cy3-CD63 aptamers via hydrogen bonding and metal chelation, resulting in fluorescence resonance energy transfer (FRET) that rapidly quenches their fluorescence. The addition of TDEs releases the Cy3-CD63 aptamers from the nanosheets, restoring fluorescence. This platform demonstrates exceptional potential for biomarkers due to its extremely low detection limits. However, reports on utilizing the fluorescence quenching capabilities of chromium-based nanomaterials for fluorescence sensing and information protection are scarce, and their applications in combination with CD63 aptamers for sensing (particularly detecting TDEs and their markers) and cryptographic steganography remain unclear. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention discloses chromium-based nanoparticles with tunable fluorescence and their applications.

[0005] The technical solution of this invention specifically includes: chromium-based nanoparticles with tunable fluorescence, wherein the chromium-based nanoparticles with tunable fluorescence are prepared according to the following steps: pre-cooled Cr... 6+ It is obtained by mixing with NaBH4 solution and reacting in an ice bath environment.

[0006] Chromium-based nanoparticles with tunable fluorescence, wherein the Cr 6+ Mix with NaBH4 solution at a molar ratio of 1:1.

[0007] The preparation steps specifically include: mixing 50 μL of 10 mM K2Cr2O7 and an equal volume of 50 μL of 10 mM NaBH4 after pre-cooling for 10 minutes with 400 μL of pre-cooled ultrapure water, and then homogenizing the mixture; the reaction is carried out under ice bath conditions for 15 minutes, and then at room temperature for another 15 minutes, and the final pale yellow colloidal solution is the chromium-based nanoparticle solution with tunable fluorescence.

[0008] Chromium-based nanoparticles with fluorescence tunability are used in the biosensing of tumor-derived exosomes.

[0009] Applications of chromium-based nanoparticles with tunable fluorescence in molecular information protection.

[0010] The technical effects of this invention are as follows:

[0011] 1. Chromium-based nanoparticles (Cr NPs) with tunable fluorescence were prepared in a simple and rapid process.

[0012] 2. Cr NPs exhibit significantly different fluorescence modulation abilities for different dyes and DNAs with different base compositions.

[0013] 3. Cr NPs can bind to aptamers and be used for fluorescence sensing of tumor-derived exosomes.

[0014] 4. The fluorescence sensing capabilities of Cr NPs and the structural diversity of DNA make it possible to encode, encrypt, and hide information.

[0015] This invention provides a rapid and efficient synthesis of chromium-based nanoparticles (Cr NPs) with tunable fluorescence capabilities for the detection of tumor-derived exosomes (TDEs) and to achieve information security at the molecular level. The synthesis method of this invention only requires pre-cooled Cr... 6+ The mixture was simply mixed with NaBH4 solution for 30 minutes. The resulting spherical Cr NPs exhibited unique fluorescence modulation (including quenching or enhancement) to dyes and DNA of different compositions. Based on these fluorescence properties, a CD63 aptamer-Cr NPs sensing system was constructed for detecting CD63-positive TDEs, enabling encoding and information protection even in real samples. In this system, fluorescently labeled CD63 aptamers act as recognition probes and information carriers, forming steganographic objects by adsorbing onto Cr NPs. When CD63 aptamer-Cr NPs specifically bind to CD63 or TDEs, different fluorescence responses are triggered, thereby achieving accurate quantitative detection as well as data encryption and protection. This invention provides a new extension for the preparation and application of novel metal nanomaterials, offers a new platform for rapid detection of tumor biomarkers, and opens up new directions for the integration of molecular system-based sensing and information science. Attached Figure Description

[0016] Figure 1 Preparation of Cr NPs (A, B) Cr 6+ (A) Color and Tyndall effect photograph (B) of the colloid obtained by mixing (0.5 mM) and NaBH4 (0.5 mM) under ice bath and room temperature conditions. (C) Solution color, Tyndall effect photograph and absorption spectrum of Cr NPs under optimal synthesis conditions. (D) Schematic diagram of the optimal synthesis of the pale yellow colloid (Cr NPs).

[0017] Figure 2 Characterization of Cr NPs (A) TEM images of Cr NPs. Scale bars: 100 and 20 nm. (B) Statistical histogram of the diameter of spherical Cr NPs obtained by measuring TEM images using ImageJ software. The red line is a Gaussian fit to the data. (C) High-angle annular dark-field scanning electron microscope image and EDS elemental plot of the light yellow colloidal solution.

[0018] Figure 3 Characterization of Cr NPs (A, B): XRD and FTIR spectra of synthesized Cr NPs. Note: The XRD peak at 2θ = 21.11° is attributable to the SiO2 substrate. (CF)XPS was used to detect the C1s (D), O 1s (E), and Cr 2p (F) core-level spectra of Cr NPs (C).

[0019] Figure 4 Fluorescence Tunability of Cr NPs (A) Cr NPs cause changes in the fluorescence emission spectra of six common fluorescent dyes (fluorescein sodium, rhodamine B, acridine orange, calcein, fluorescein B, and erythrosine B). Illustration: Fluorescence photographs of the corresponding solutions and chemical structures of the dyes. (B) Relationship between the fluorescence change ratio (F0-F) / F0 of the six common fluorescent dyes and the concentration of Cr NPs. Fluorescein sodium, 0.5 μM; Rhodamine B, 1.25 μM; Acridine orange, 2 μM; Calcein, 0.5 μM; Fluescein B, 0.5 μM; Erythrosine B, 5 μM.

[0020] Figure 5 Fluorescence tunability of Cr NPs (A) Changes in fluorescence emission spectra of ssDNA with different base sequences (A33, T33, C33, G33, at a concentration of 100 nM) by Cr NPs. (B) Comparison of the effect of different concentrations of Cr NPs on the fluorescence quenching rate (F0-F) / F0 of FAM-labeled ssDNA with four different sequences.

[0021] Figure 6Quantitative and selective detection of CD63 (A) Fluorescence emission spectra vary with different combinations. CD63 aptamer, 200 nM; Cr NPs, 240 μM; CD63, 839 nM; buffer: 5 mM Tris-HAc (pH 7.4). (B) Schematic diagram of the interaction between CD63 aptamer, Cr NPs and CD63 protein. (C) Fluorescence selectivity response of CD63 aptamer-Cr NPs (200 nM: 240 μM) to CD63 and common interferons (CD133, BSA, EpCAM, all at 302 nM). Inset: Schematic diagram of the differential response of CD63 aptamer-Cr NPs probe to CD63 and interferon. (D) Fluorescence emission spectra of CD63 aptamer-Cr NPs (200 nM: 240 μM) after adding different concentrations of CD63 (0 μM: 839 nM). (E) Dependence of fluorescence intensity changes (F0-F) / F0 on CD63 concentration for CD63 aptamers. (F) The linear relationship between (F0-F) / F0 and CD63 is in the ranges of 12-211 nM and 302-839 nM. Buffer: 5 mM Tris-HAc (pH 7.4).

[0022] Figure 7 (A) NTA of TDEs. Inset: TEM image of TDEs. Scale bar: 200 and 100 nm. (B) Fluorescence emission spectra of CD63 aptamer-Cr NP nanoprobes (200 nM: 240 μM) after the addition of different concentrations of TDEs. (C) Schematic diagram of the interaction between CD63 aptamer, Cr NPs and TDEs. (D) Relationship between the fluorescence response (F0-F) / F0 (at 519 nm) of CD63 aptamer-Cr NPs and the concentration of TDEs. Blue dots and red lines represent the interaction at 2.06 × 10⁻⁶ nm. 6 particles / mL ~2.56×10 7 The relationship is linear within the range of particles / mL.

[0023] Figure 8 A hybrid model combining cryptography and steganography, featuring reversible encryption-decryption and steganography analysis capabilities.

[0024] Figure 9 Molecular cryptography using a nucleic acid aptamer-chromium nanoparticle sensing system: (A) A molecular cryptographic steganography sensing system based on nucleic acid aptamer-chromium nanoparticles: CD63 aptamers act as information carriers, and Cr NPs act as nanocapsules to create aptamer-Cr NP steganographic objects. CD63 or TDEs are used as molecular steganographic keys to unlock the steganographic objects and access secure information based on the aptamer sequence. (B) DNA strand structure encodes information, facilitating encryption and concealment.

[0025] Figure 10 Using steganography (A) of a nucleic acid aptamer-chromium nanoparticle sensing system, the corresponding decoding rules, charts, 6-bit or 7-bit (B) keys, and hidden encrypted secret text ("rising tides rise and miracles happen every day"). Detailed Implementation

[0026] The present invention will now be described in detail with reference to the embodiments.

[0027] 2. Experimental Section

[0028] 2.1 Materials and Reagents

[0029] Potassium dichromate (K₂Cr₂O₇), sodium borohydride (NaBH₄), sodium fluorescein, rhodamine B, acridine orange, calcein, fluorescein B, erythrosine B, tris(hydroxymethyl)aminomethane (Tris), and all metal salts used in this study were analytical grade and purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). All oligonucleotides used in this study (A33, T33, C33, G33, and CD63 aptamers (their sequences and modifications are shown in Table 1)) were synthesized by Shanghai Sangen Biotechnology Co., Ltd. The five oligonucleotides (A33, T33, C33, G33, and CD63 aptamers) have a carboxyfluorescein (FAM) tag at the 5′ end. A33, T33, and C33 represent sequences containing 33 consecutive A, T, and C nucleotides, respectively. G33 represents the sequence GGGGGGTG ...

[0030] All aqueous solutions were prepared using ultrapure water produced by a Milli-Q system (Millipore, Bedford, Massachusetts, USA, 18.2 MΩ·cm). During the reaction, the pH was controlled using a buffer solution: Tris-HAc, 5 mM, pH 7.4.

[0031] Table 1. Sequences and modifications of CD63 protein targeting and recognizing CD63 aptamers

[0032]

[0033] 2.2 Preparation and characterization of Cr NPs

[0034] Chromium nanoparticles (Cr NPs) were synthesized by adding 50 μL of K₂Cr₂O₇ (10 mM) and an equal volume (50 μL) of NaBH₄ (10 mM, pre-cooled for 10 min) to 400 μL of pre-cooled ultrapure water. The mixture was then homogenized. The reaction was carried out in an ice bath for 15 min, followed by a 15 min reaction at room temperature, ultimately forming nanoparticles. The concentration of the resulting pale yellow colloidal solution (Cr NPs) was determined to be 1 mM, based on the final molar concentration of chromium species introduced into the reaction mixture.

[0035] Absorption and fluorescence spectra were recorded using a SpectraMax M5 microplate spectrophotometer (Molecular Devices, USA). Purified chromium nanoparticle (Cr NPs) samples were placed on a copper grid, allowed to air dry, and then examined using a Tecnai G2 F20 transmission electron microscope (TEM, FE1, USA) equipped with energy-dispersive spectroscopy (EDS). At least 100 TEM images of the particles were analyzed using ImageJ software, and the particle size distribution was determined by Gaussian fitting. Furthermore, the crystal phase, surface functional groups, and elemental composition of the Cr NPs were evaluated using X-ray diffraction (XRD, Bruker D8, Germany), IS10 Fourier transform infrared spectroscopy (FTIR, Nicolet, USA), and ESCALAB250xi X-ray photoelectron spectroscopy (XPS, Thermo Scientific, USA).

[0036] Fluorescence tunability of 2.3Cr NPs

[0037] To evaluate the tunable effect of Cr NPs on fluorescence, six common fluorescent dyes and four FAM-labeled single-stranded DNA (ssDNA) sequences (T33, A33, C33, and G33) were selected for analysis. 400 μL solutions of sodium fluorescein (0.5 μM), rhodamine B (1.25 μM), acridine orange (2 μM), calcein (0.5 μM), fluorescein B (0.5 μM), and erythrosine B (5 μM), as well as FAM-labeled ssDNA (A33, T33, C33, and G33; 100 nM), were transferred to microfluorescent cuvettes equipped with 2 mm slits. The fluorescence emission spectra of these solutions were recorded in the wavelength ranges of 495–700 nm, 565–700 nm, and 510–700 nm, corresponding to different excitation wavelengths of 475, 550, 490, and 485 nm, respectively. Subsequently, different concentrations of Cr NPs were introduced into the solution, and the fluorescence emission spectra of the resulting mixtures were measured under the above conditions.

[0038] 2.4 Preparation of CD63 aptamer-Cr nanoparticle composite and detection of CD63

[0039] To obtain a stock solution, FAM-labeled CD63 aptamers were first dissolved in ultrapure water to prepare a stock solution (100 μM). A CD63 aptamer-Cr nanoparticle complex was obtained by mixing FAM-labeled CD63 aptamers (200 nM) with Cr nanoparticles (240 μM) in Tris-HAc buffer (5 mM, pH 7.4). To detect CD63, different concentrations of CD63 were added to the prepared CD63 aptamer-Cr nanoparticle complex solution (400 μL, 200 nM: 240 μM), and fluorescence spectra were collected in the 490–700 nm range. In selectivity experiments, CD133, BSA, and EpCAM were used in place of CD63 at the same concentrations.

[0040] 2.5 Cell culture, extraction, and characterization of TDEs

[0041] The SiHa cell line is a human cervical squamous cell carcinoma derived from a nationally certified cell bank (Shanghai, China). Cells were cultured in DMEM medium (Hyclone, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 100 U / mL penicillin, and 100 μg / mL streptomycin, and incubated at 37°C in a humidified environment with 5% CO2.

[0042] When SiHa cells reach 80-90% confluence, the culture medium is replaced with DMEM (C3801-0050, VivaCell) containing 10% TDEs and desaturated with serum, and incubated for 48 hours. The cell culture supernatant is then collected on ice for TDE isolation using a total TDEs extraction reagent (Thermo Fisher Scientific, USA). The procedure includes the following steps:

[0043] (1) Sample preparation: Centrifuge the cell culture medium at 2000×g for 30 minutes to remove cells and impurities. Carefully transfer the clear supernatant to a new test tube, avoiding disturbing the precipitate.

[0044] (2) TDEs isolation: Mix 2.5 mL of total TDEs separation reagent with 5 mL of treated supernatant and incubate at 2 to 8 °C for 12 hours. After incubation, centrifuge at 10,000 × g at 4 °C for one hour, discard the supernatant, and gently resuspend the precipitate in 100 μL of 1×PBS buffer to obtain a homogeneous solution. Store the isolated TDEs at 2 °C to 8 °C for short-term use (maximum one week) or for long-term storage at ≤-20 °C.

[0045] For transmission electron microscopy (TEM) morphological analysis, 10 μL of TDEs solution (8.12 × 10⁻⁶) was used. 5 The dye particles (particles / mL) were coated onto a copper grid and allowed to stand for 10 minutes. 10 μL of 2.5% uranium acetate solution was added and allowed to stand for 1 minute to remove excess dye. The grid was dried before imaging and imaged at 100 kV using a high-vacuum transmission electron microscope (JEM-1200EX, JEOL, Japan).

[0046] To analyze the particle size and concentration of TDEs, purified TDEs were diluted in 1×PBS buffer (BioIndustrial, Israel) and measured using nanoparticle tracking analysis (NTA, ZetaView PMX 110, Particle Metrix, Merbusch, Germany), analyzed using ZetaView 8.05.14SP7 software. The ZetaView system was calibrated using 110 nm polystyrene particles, and NTA measurements were performed and analyzed from at least 11 locations.

[0047] 2.6 Quantitative Detection and Actual Sample Analysis of TDEs

[0048] To quantify TDEs, FAM-labeled CD63 aptamers (200 nM) were mixed with Cr nanoparticles (240 μM) in Tris-HAc buffer (5 mM, pH 7.4). Then, different concentrations of TDEs (4.04 × 10⁻⁶) were...6 -7.55×10 7 The particles / mL were added to 400 μL of CD63 aptamer-Cr nanoparticle composite solution (200 nM: 240 μM), mixed thoroughly, and the fluorescence spectrum in the range of 495-700 nm was collected.

[0049] To evaluate the ability of CD63 aptamer-Cr nanoparticle nanoprobes to detect TDEs in real serum samples, serum standards of TDEs with known concentrations were prepared. Serum samples were collected from Hunan Normal University Hospital in accordance with the regulations of the local ethics committee. The obtained serum samples were centrifuged at 3,000g for 5 minutes and filtered through a 0.22μm filter. Standard TDEs samples (10μL) isolated from human cervical cancer SiHa cells were dissolved in 10μL of pretreated serum. Subsequently, serum standards of TDEs with known concentrations (2, 4, and 8μL) were added to 400μL of CD63 aptamer-Cr nanoparticle composite solution (200nM:240μM), mixed thoroughly, and their fluorescence spectra in the 495-700nm range were collected. Finally, the concentration of added TDEs was calculated using the corresponding linear regression equation.

[0050] 2.7 Molecular Cryptography and Steganography

[0051] In a molecular cryptography steganography sensing system based on aptamer-chromium nanoparticles, the CD63 aptamer serves as the information carrier, while the chromium nanoparticles act as a nano-coating, forming the aptamer-chromium nanoparticle steganography object. CD63 or target molecules (TDEs) serve as the molecular steganography key, used to unlock the steganography object and retrieve the protected information based on the aptamer sequence. The 32-base CD63 aptamer utilizes DNA hexamers (6 bases, 4...) 6 =4096) or heptamer (7 bases, 4 7 =16384) Encodes 27 or 26 characters from the 3′ end by shifting one base at a time. The presence of target CD63 or TDEs causes a fluorescence change, revealing the DNA sequence if (F0-F) / F0 > 40% or (F-F0) / F0 > 40%. The DNA-encoded characters can be decrypted by consulting the corresponding key table, and a coherent short text message can be obtained by combining and recognizing these characters. Two sets of key tables are provided for DNA hexamers and heptamers to encode and encrypt information.

[0052] 3. Results and Discussion

[0053] 3.1 Synthesis and Characterization of Cr NPs

[0054] We compared Cr 6+Changes in the color, Tyndall effect, and absorption spectrum of NaBH4 and its mixtures. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) 6+ Compared to NaBH4, the pale yellow mixed solution exhibits a significant Tyndall effect, accompanied by a redshift of the two absorption peaks at 258–274 nm and 352–372 nm. Figure 1 (A, 1B). To improve yield, we systematically adjusted Cr. 6+ The concentration ratio of Cr to NaBH4. 6+ The concentration of Cr was kept constant at 1 mM, while the concentration of NaBH4 was gradually increased from 0.5 mM to 2 mM, and vice versa. Through analysis of color changes, the Tyndall effect, and absorption spectra, the Cr concentration was determined to be... 6+ The optimal NaBH4 concentration ratio was 1 mM:1 mM, yielding the highest yield. Furthermore, we investigated the effect of different reaction times under ice bath and room temperature conditions. The total reaction time was set to 30 min, with the ice bath time starting at 0 min and increasing every 5 min, and the room temperature reaction time. The results showed that the optimal reaction conditions were 15 min under ice bath and 15 min at room temperature. The synthesized Cr NPs remained stable at room temperature for at least 17 days (only the duration was investigated so far). Therefore, the optimized preparation conditions for Cr NPs are: Cr... 6+ Pre-cool with NaBH4, mix in a 1 mM:1 mM ratio for 15 min, and incubate at room temperature for 15 min. Figure 1 C, 1D).

[0055] To determine the successful preparation of Cr NPs and their morphology, phase, functional group composition, and surface chemical composition, we performed a series of characterizations using UV-Vis absorption spectroscopy, TEM, XRD, FTIR, and XPS. TEM images ( Figure 2 A) It was confirmed that the obtained colloid contained irregular NPs with a particle size range of 18-56 nm and an average diameter of 29.56 ± 0.56 nm (N = 100, R...). 2 =0.988, Figure 2 B). Further high-angle annular dark-field scanning transmission electron microscopy and EDS elemental distribution results showed that NPs were mainly composed of Cr and sporadic distributions of O, K, and Na elements, while C and B elements were distributed throughout the field of view. Figure 2 C).

[0056] Analysis of the XRD spectrum revealed that the diffraction peaks at 2θ = 30.5°, 35.8°, 42.98°, 54.12°, and 63.4° correspond to the (104), (110), (113), (116), and (214) crystal planes of Cr NPs, respectively. Figure 3A), the diffraction peak at 2θ = 21.11° may correspond to the SiO2 substrate, and the peak value at 2θ = 29.55° may indicate the presence of potassium chromate during the synthesis process. FTIR functional group characteristic data ( Figure 3 B) indicates that Cr NPs were observed at 3441.68, 1642.10, 1384.22, 947.79, and 877.18 cm⁻¹. -1 Strong vibrational peaks are observed at all locations, particularly between 3400 and 2500 cm⁻¹. -1 The wider feature band and 1680-1500cm -1 The small vibrational peaks may be caused by water bending and hydroxyl groups, respectively. Located at 1384.22 cm⁻¹. -1 The nearby absorption band likely originates from CH bonds and is located at 947.79 cm⁻¹. -1 and 877.18cm -1 The characteristic peaks at these locations belong to the bending and stretching vibrations of Cr-O and O-Cr-O bonds, respectively. Figure 3 The XPS full spectrum of C showed multiple identifiable signal peaks at 285, 293, 531, 577, and 587 eV, indicating that the main components of the synthesized CrNPs were C1s, K2p, O1s, and Cr2p. To determine the elemental composition in more detail, we scanned, convolved, and fitted the high-resolution nuclear level spectra of C1s, K2p, O1s, and Cr2p. The core energy spectrum of C1s (…) Figure 3 D) can be decomposed into three different individual peaks corresponding to C=C (284.8 eV), COC (286.5 eV), and C=O (288.6 eV) bonds respectively. Figure 3 The O1s core spectrum in E has two relatively close peaks at 531.2 eV and 533.4 eV, which belong to O1s and O2, respectively. 2- H2O or OH - The K 2p core level spectrum is mainly divided into K 2p... 3 / 2 (292.7eV), K 2p 1 / 2 (295.5 eV) Two energy levels, which may originate from free K+. + K2O or KOK bond. Cr 2p core level spectrum of Cr NPs ( Figure 3 F) indicates that there are four different chromium states in Cr NPs, including Cr2O3 (577 eV), K2Cr2O7 (579.4 eV), Cr(OH)3 (587 eV), and CrO3 (582.7, 589.6 eV).

[0057] Fluorescence tunability of 3.2Cr NPs

[0058] Recently, many nanomaterials have been applied to fluorescence sensing platforms due to their unique fluorescence modulation capabilities. The effects of CrNPs on the fluorescence of several common dyes (fluorescein sodium, rhodamine B, acridine orange, calcein, fluorescein B, and erythrosine B) were measured. Figure 4 A, 4B, S5, S6). It was observed that the fluorescence of sodium fluorescein and calcein gradually increased with increasing Cr NP concentration (maximum enhancement rates were 133.1% and 88.8%, respectively). Figure 4 The fluorescence of Rhodamine B, Acridine Orange, and Fluorescein B was gradually quenched (maximum quenching rates were 82.3%, 78.9%, and 33.9%, respectively). Figure 4 B). In contrast, the fluorescence of erythrosine B remained almost unchanged. Figure 4 A, 4B). Among these dyes, Cr NPs showed relatively small fluorescence changes to Fluorescent Pink B and Erythrosine B, which may be attributed to the presence of many halogen elements in these two dyes, which is unfavorable for the interaction of Cr NPs with them. These observations indicate that Cr NPs have a broad fluorescence modulation ability (including quenching and enhancement) for a variety of fluorescent molecules. The ability of Cr NPs to alter the fluorescence of different dyes may be attributed to their ability to distinguish small chemical structural changes in these dye molecules (A, 4B). Figure 4 A) This leads to differences in their interactions. Based on Professor Zhou Xuguang's summary of the key fluorescence quenching mechanism characteristics, dynamic quenching is characterized by changes in the lifetime of fluorescent substances in the presence of a quencher, while their absorption spectra remain relatively stable, and the quenching effect strengthens with increasing temperature. Based on this, we found that the absorption spectra of Cr NPs do not completely overlap with the fluorescence emission spectra of Rhodamine B, Acridine Orange, and Luciferin B, and their absorption spectra remain stable. Furthermore, the changes in fluorescence lifetime and the effect of temperature on the quenching effect together indicate that the fluorescence quenching of these dyes by Cr NPs is achieved through a dynamic mechanism. In addition, Chris D. Geddes et al. pointed out that the main mechanisms by which chromium nanoparticles achieve metal-enhanced fluorescence (MEF) include the electric field effect and the induced plasma effect. The electric field effect can enhance the excitation and emission processes of fluorescent molecules near metal nanoparticles (<10 nm) without affecting their fluorescence lifetime. Conversely, the induced plasma effect leads to a shortened fluorescence lifetime. The results show that the lifetimes of Luciferin sodium and Calcein are almost identical in the presence or absence of Cr NPs. This indicates that the fluorescence enhancement of these two dyes by Cr NPs is mainly achieved through the electric field effect. The fluorescence quenching of Rhodamine B, Acridine Orange, and Fluorescein B caused by Cr NPs is due to dynamic quenching, while the fluorescence enhancement of sodium fluorescein and calcein is because these dyes are located near Cr NPs, absorbing more photons in the enhanced electromagnetic field, increasing the number of electrons in the excited state, and thus enhancing the fluorescence signal.

[0059] Furthermore, the fluorescence effect of Cr NPs on FAM-labeled ssDNA with different base compositions was investigated. For example... Figure 5 As shown in A and 5B, all four ssDNA sequences were quenched by Cr NPs. The degree of fluorescence quenching became more pronounced with increasing Cr NP concentration. Notably, there were significant differences in the fluorescence quenching rates of these ssDNA sequences, which may be due to the different interactions between Cr NPs and different bases. The binding priority of different bases to Cr NPs was G>A≈T>C, which provides a theoretical basis for constructing a Cr NP-based DNA sensing platform.

[0060] 3.3 Detection of CD63 and TDEs based on Cr NPs

[0061] 3.3.1 Quantitative and selective detection of CD63

[0062] We further constructed a DNA sensing platform based on Cr NPs and aptamers, which can specifically recognize CD63 (an exosome marker). Due to the presence of the FAM marker, the CD63 aptamer exhibits a strong fluorescence peak at approximately 519 nm. Figure 6 A). As Cr NPs were gradually added, the fluorescence intensity of the CD63 aptamer decreased rapidly and significantly, reaching a quenching efficiency of approximately 87.9%. Figure 6 A). This phenomenon is attributed to dynamic quenching between the FAM fluorophore and Cr NPs. Figure 6 B). Subsequently, the Stern-Volmer constant (Ksv) of CD63 aptamers-Cr NPs at different temperatures was investigated to verify the quenching mechanism. The Ksv value (0.0058 L / μmol at 25℃ and 0.0082 L / μmol at 35℃) increased with increasing temperature, indicating that the collision between FAM-labeled CD63 aptamers and Cr NPs underwent dynamic quenching. The addition of CD63 to CD63 aptamers-Cr NPs led to further fluorescence quenching ( Figure 6 (B) This may be attributed to the CD63-specifically induced changes in aptamer configuration that are more favorable for Cr NP binding (e.g., the sandwich structure of aptamer-CD63-Cr NPs). Furthermore, at the same concentration, the fluorescence change of CD63 aptamer-Cr NPs to CD63 is significantly higher than that of other common interfering agents (CD133, BSA, EpCAM), approximately seven times that of the interfering agents. Figure 6 C). This indicates that CD63 aptamer-Cr NPs exhibit a selective fluorescence response to CD63. To detect the ability of CD63 aptamer-Cr NPs to quantitatively detect CD63, we titrated with different concentrations of CD63 ( Figure 6 D). The fluorescence intensity of CD63 aptamer-Cr NPs continuously decreases with increasing CD63 concentration. Figure 6 D, 6E). Within the ranges of 12–211 nM and 302–839 nM, there are two linear relationships between the fluorescence change at 519 nm and the CD63 concentration, respectively. Figure 6 F). The two calibration equations are: y1 = 0.1772x1 + 1.8104(R) 2 =0.998, detection limit (LOD): 7.51 nM) and y2 = 0.0484x2 + 40.4409 (R 2 =0.980). These findings confirm that this aptamer-CrNPs sensing platform can perform highly sensitive and selective detection of CD63. Figure 6 ).

[0063] 3.3.2 Separation, Characterization, and Quantitative Detection of TDEs

[0064] To further investigate the potential of CD63 aptamer-Cr NPs nanoprobes in the detection of TDEs, we continued to isolate and purify TDEs from the culture medium of the human cervical cancer SiHa cell line. Transmission electron microscopy showed that the purified SiHa TDEs appeared as spherical vesicles with diameters between 50 and 250 nm. Figure 7 A). NTA further showed that the purified TDEs had an average diameter of 132.5 nm and a concentration of approximately 8.12 × 10⁵ particles / mL (A). Figure 7 A). For example Figure 7 As shown in Figure B, as the concentration of TDEs systematically increased from 0 to 7.55 × 10⁻⁶, 7 The fluorescence of CD63 aptamer-Cr NPs gradually increased over 5 min at particles / mL. A linear correlation was found between the fluorescence change at 519 nm and the TDE concentration, ranging from 2.06 × 10⁻⁶. 6 particles / mL up to 2.56 × 10 7 particles / mL ( Figure 7 D). The calibration equation is y = 2.6069 × 10 -6 x-0.5878(R 2 =0.979), LOD is 3.05×10 6particles / mL (3σ / slope). Our detection limit and linear range are comparable to, or in some cases superior to, reported TDE detection methods, including fluorescence, colorimetric, and electrochemical methods (Table 2). TDE-induced fluorescence recovery of CD63 aptamer-CrNPs is the opposite of CD63-induced fluorescence quenching. This may be due to the small size of CD63 specifically inducing conformational changes in the aptamer, which are more favorable for Cr NP binding (e.g., the sandwich structure of aptamer-CD63-CrNPs); however, since CD63 is a transmembrane protein located on the surface of exosomes, CD63 aptamer-Cr NPs interact with surface CD63 exosomes, but the large size of the exosomes increases the complex distance after interaction with the exosomes, leading to fluorescence recovery (…). Figure 8 C). To demonstrate the practicality of the CD63 aptamer-Cr NPs nanoprobe in real samples, we performed peak recovery experiments using different concentrations of TDEs in serum. The fluorescence response of the CD63 aptamer-Cr NPs nanoprobe to TDEs was approximately 14 times that of serum alone, indicating that serum alone has little interference with the signal response of the CD63 aptamer-Cr NPs nanoprobe. We then performed three parallel experiments to evaluate the performance of TDEs at three peak levels (2.02, 4.04, and 8.04 × 10⁻⁶). 6 Recovery rates were achieved at concentrations of particles / mL, ranging from 81.9% to 112.1%, with relative standard deviations (RSDs) ranging from 0.21% to 4.77% (Table 3). These results demonstrate that the CD63 aptamer-Cr NPs nanoprobes possess excellent ability to determine TDEs in serum samples.

[0065] Table 2. Comparison of detection limits and linear ranges for CD 63 or TDEs in other reports.

[0066]

[0067]

[0068] a s-SWCNTs: Single-walled carbon nanotubes

[0069] b MOF: Metal-Organic Framework

[0070] c EQCM-D: Electrochemical Quartz Crystal Microbalance with Dissipative Detection

[0071] d AlEgens: Aggregation-induced emission light source

[0072] e RLS: Resonant Light Scattering

[0073] f MIL: Materials of the Lavoisier Institute, belonging to metal-organic framework materials

[0074] 3. Detection of TDEs in serum samples based on fluorescence channels

[0075]

[0076] 3.4 Molecular information encoding, encryption, and steganography based on CD63 aptamer-Cr NPs

[0077] With the rapid development of information technology and the widespread popularity of Internet technology, information security issues have become the focus of global attention. In the digital age, information security issues are everywhere, from personal privacy protection to corporate data confidentiality and then to national security maintenance. Therefore, it has become crucial to implement encryption protection for the critical information transmitted in the shared communication channels full of risks. In fact, for centuries, physical media has been used to write information in a steganographic way to prevent information leakage. For example, as recorded in the book "Writing and Records in Ancient Egypt", the ancient Egyptians used natural substances such as lemon juice and milk as invisible inks. These substances would display the text when heated. In ancient China, to ensure the security and confidentiality of information in military communications, the information was divided into several parts and sent by different messengers. Only by collecting all the parts could the complete message be read. This method was named "yin shu" in "The Book of Han" and "The Seven Military Classics". And with the development of technology to date, steganography has also derived methods of hiding information using data media. For example, F5 steganography is an information hiding technology based on audio signals. By adjusting the frequency components of the audio signal, the secret information is embedded into ordinary audio files; in 1999, Clelland et al. used DNA microdot technology to perform DNA steganography on a text message "June 6 invasion: Normandy" and successfully recovered the original text message from the microdots. In addition, DNA steganography is also an emerging data media steganography technology that uses the high storage density and complexity of DNA molecules to hide information. These technologies not only improve the concealment and security of information hiding but also bring new development directions to the field of information security.

[0078] Cryptography and steganography are two effective means of information protection, and sometimes they can be used in combination (i.e., cryptographic steganography). Figure 8The basic cryptography and steganography model shown in Figure A comprises two parts: encryption (cryptography) and hiding (steganography). Secret information is encrypted and converted into a complex encoded text called "encrypted information." This "encrypted information" can be decrypted using a specific "key" to recover the original secret information. Furthermore, the "encrypted information" can be embedded in a "carrier / cover" to form a "steganographic object," effectively evading detection by attackers, and can then be reversibly recovered using the corresponding "steganographic key." To extend information protection technology, we have developed a molecular cryptographic steganography based on an aptamer-chromium nanoparticle sensing system for encrypting and hiding information. Figure 9 , 10 The system consists of a DNA aptamer as the information carrier, chromium nanoparticles as a nanomask, and a pair of keys: a molecular steganography key (CD63 or TDEs) and a cryptographic key (…). Figure 9 A). Figure 9 B illustrates the information-encoding structure within the aptamer's DNA strand. This 32-base DNA aptamer can be used to encode 27 or 26 characters from the 3' end in the form of DNA hexadecimals or heptameric pairs, by shifting one base at a time (…). Figure 9 B). Using a multi-letter code, a 6-base DNA sequence (4... 6 =4096) or a 7-base DNA sequence (4 7 =16384) can achieve sufficient information encoding to protect specific information. Here, we use these 6-base or 7-base DNA sequences to encode 54 common characters in a many-to-one manner. Generally, the larger the mask / carrier, the easier it is to hide the hidden information relative to the hidden information. Therefore, DNA aptamers containing encoded information can be more easily hidden in chromium nanoparticle (Cr NPs) covers / carriers, thus forming aptamer-chromium nanoparticle (steganographic object, fluorescence quenched, see Figure 9 A). The interaction between the chromium nanoparticles and the aptamer is sufficient to prevent aptamer leakage; even if an adversary discovers the steganographic object somehow, it remains difficult to view the information without a specific steganographic key. Decoding the aptamer-chromium nanoparticle (steganographic object) is only possible in the presence of the correct steganographic key (CD63 or TDEs), which will result in a significant fluorescence change ((F0-F) / F0 > 40% or (F-F0) / F0 > 40%, see... Figure 9 A, 6C, 7D). Furthermore, even if an adversary can identify the DNA sequence, it remains difficult to obtain the information without a specific cryptographic key. Only when the correct recipient uses our encryption key (see...) Figure 10Accurately decoding the information embedded in the DNA sequence reveals two distinct secrets hidden within it: "A rising tide lifts all boats" (a Chinese idiom meaning that as the foundation upon which something depends improves or strengthens, that thing also benefits) and "Miracles happen every day" (a quote from the movie *Forrest Gump*, representing the driving force behind Forrest Gump's hopeful journey through life). These molecular information security technologies demonstrate the diversity and flexibility of our approach. Due to DNA's rich structural diversity and inherent hiding properties, coupled with its specificity and high flexibility in molecular coding, it opens up vast possibilities for information encryption and concealment. Furthermore, nanosensing systems exhibit unprecedented advantages in information encoding, thus establishing a new molecular-level paradigm for information security protection.

[0079] 4. Conclusion

[0080] In summary, we have developed a simple, efficient, and low-cost method for synthesizing Cr nanoparticles (Cr NPs) by pre-cooling Cr... 6+ The reaction is completed in approximately 30 minutes with a simple mixing process with NaBH4 solution. The resulting spherical nanoparticles (average diameter approximately 30 nm) exhibit varying tunability for different fluorescent molecules (including dyes and dye-labeled DNA), attributed to two distinct phenomena: dynamic quenching and metal-enhanced fluorescence (MEF). Nucleic acid aptamer-based sensing platforms constructed using these Cr NPs enable highly sensitive and selective detection of CD63 and TDEs, even in complex serum environments. Furthermore, the fluorescence sensing capabilities of Cr NPs and the structural diversity of DNA enable information encoding, encryption, and concealment. These Cr NPs offer numerous advantages in terms of synthesis and functional properties. Their preparation is rapid, convenient, and scalable, while their high specific surface area and tunable optical properties make them ideal platforms for diverse applications. Notably, Cr NPs exhibit highly flexible fluorescence sensing capabilities, allowing for customization for analytical and biomedical purposes. Moreover, the molecular information paradigm based on Cr NPs highlights their inherent digital characteristics and immense potential in information technology applications. Inspired by this work, more types of Cr-based nanomaterials with different morphologies and compositions can be synthesized in the future, promoting their potential applications in catalysis, pharmaceuticals, and energy. By combining with other functional molecules, they also hold promise for detecting more biomarkers and enabling more advanced molecular information processing and communication. This work not only provides a new approach for the application of metal nanomaterials in information technology but also establishes a new platform for the rapid and simple detection of tumor biomarkers.

[0081] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. Chromium-based nanoparticles with tunable fluorescence, characterized in that, The chromium-based nanoparticles with tunable fluorescence were prepared according to the following steps: pre-cooled Cr... 6+ It is obtained by mixing with NaBH4 solution and reacting in an ice bath environment.

2. The chromium-based nanoparticles with tunable fluorescence according to claim 1, characterized in that, The Cr 6+ Mix with NaBH4 solution at a molar ratio of 1:

1.

3. The chromium-based nanoparticles with tunable fluorescence according to claim 1 or 2, characterized in that, The preparation steps specifically include: mixing 50 μL of 10 mM K2Cr2O7 and an equal volume of 50 μL of 10 mM NaBH4 after pre-cooling for 10 minutes with 400 μL of pre-cooled ultrapure water, and then homogenizing the mixture; the reaction is carried out under ice bath conditions for 15 minutes, and then at room temperature for another 15 minutes, and the final pale yellow colloidal solution is the chromium-based nanoparticle solution with tunable fluorescence.

4. Application of chromium-based nanoparticles with fluorescence tunability in the biosensing of tumor-derived exosomes.

5. Application of chromium-based nanoparticles with tunable fluorescence in molecular information protection.

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