DNA-silver nanoclusters and preparation method and application thereof

Abstract

The application relates to the technical field of detection analysis, and particularly discloses a DNA-nano silver cluster, a preparation method and application thereof. The application rapidly identifies the everted level of phosphatidylserine in blood cells through a composite nano silver cluster fluorescence sensing array technology, thereby realizing real-time prediction of disseminated intravascular coagulation of sepsis, filling the blank of the sepsis DIC prediction method, and avoiding the lag of the existing sepsis DIC diagnosis, so that a new time window is provided for sepsis DIC treatment.

Description

Technical Field

[0001] This invention belongs to the field of detection and analysis technology, specifically relating to a DNA-silver nanocluster, its preparation method, and its application. Background Technology

[0002] Sepsis is a systemic inflammatory response syndrome (SIRS) characterized by a dysregulated host response to infection. It is often accompanied by coagulation abnormalities and frequently progresses to disseminated intravascular coagulation (DIC), which can lead to multiple organ dysfunction and ultimately death. However, current diagnosis and treatment of sepsis-related DIC rely solely on existing methods such as ELISA to detect relevant coagulation markers. The results obtained reflect patient signs from several hours prior, while sepsis progresses extremely rapidly. These current methods severely delay diagnosis and treatment, significantly limiting the effectiveness of treatment. Currently, there is no method for rapid, point-of-care testing of any marker to predict sepsis-related DIC.

[0003] The inventors demonstrated through previous research that in sepsis, Caspase-11-mediated gasdermin D (GSDMD) pores induce the extravasation of phosphatidylserine (PS) residues in macrophages, thereby enhancing tissue factor (TF) activity and subsequently promoting the coagulation cascade, ultimately leading to disseminated intracellular coagulation (DIC) in sepsis. Normally, PS resides on the inner side of the cell membrane and is not typically bound by extracellular antibodies or other molecules that bind PS. However, the inventors' research revealed that cellular PS extravasates to the outer side of the cell in sepsis, at which point PS can be detected in blood cells.

[0004] Electro-optical biosensor systems are an organic fusion of fluorescence aptamer biosensor and electro-optical nanopore-sequencing (EON) technology based on controlled signal release. Aptamers are short polypeptide or oligonucleotide sequences that specifically bind to the target being analyzed. When bound to specific molecular beacon probes, they excite a fluorescence reaction, thereby detecting the presence of the relevant marker. EON, based on optical waveguides and measurement spectroscopy, selectively converts the biomarkers and biochemical information to be analyzed into optical signals that the analytical system can receive and sense through continuous transmission. The signal intensity detection and simulation are then transformed into visual information, and the transmission between single molecules or small molecules is fed back as perforated current pulses, thereby improving the specificity and anti-interference capability of the recognition component. Precise detection is achieved through targeted signal amplification. The electro-optical biosensor system, composed of fluorescence aptamer sensing and electro-optical EON, features ease of operation, high sensitivity and specificity, and high throughput. Using metal nanoclusters to construct fluorescent probes has unparalleled advantages. By using aggregates composed of metal atoms, the probe size is greatly reduced to the nanoscale, enabling it to respond quickly and accurately to the target, thereby mediating a more stable and higher-yield fluorescent signal. Summary of the Invention

[0005] This invention aims to solve the technical problems existing in the prior art. To this end, this invention proposes a DNA-silver nanocluster, its preparation method, and its application. This DNA-silver nanocluster can rapidly detect the outward turning level of PS in sepsis-related disseminated intravascular coagulation (DIC) blood cells within 5 minutes, thereby predicting the occurrence of DIC in advance and providing a time window for early intervention in DIC.

[0006] In a first aspect, the present invention provides an oligo-stranded DNA fragment, the nucleotide sequence of which is 5'-CCCCCCCCCCCC-ATC CGT CCT TAT ACA CAA TTG TTT TTC TCT TAA CTT CTT GAG TGT TGGCTC CCG TAT-3' (SEQ ID NO.1).

[0007] This invention uses the C-rich sequence "CCCCCCCCCCCC" as a template for synthesizing DNA-AgNCs, which can form stable C-Ag. + -C structure; in addition, this oligo-stranded DNA fragment can bind to PS. The formed DNA-AgNCs, after binding to PS, will produce a strong fluorescent signal under 488nm laser irradiation.

[0008] A second aspect of the present invention provides a method for preparing DNA-silver nanoclusters, comprising the following steps:

[0009] The aqueous solution of the above oligo-stranded DNA fragment is mixed with AgNO3 solution, and then NaBH4 solution is added. The reaction is carried out to obtain the final product.

[0010] Ag + Ag has a strong affinity for the N3 group on the C base of DNA. Under the action of the reducing agent NaBH4, Ag... + Restored, Ag 0 Clustering and nucleation result in stable fluorescence properties.

[0011] In some embodiments of the present invention, the molar ratio of the oligo-stranded DNA fragment, AgNO3 and NaBH4 is 1:1 to 1.5:0.2 to 0.4; preferably 1:1.2 to 1.5:0.2 to 0.3; more preferably 1:1.2:0.24.

[0012] In some embodiments of the present invention, the concentration of the oligostranded DNA fragment is 5 to 15 mmol / L, preferably 10 mmol / L.

[0013] In some embodiments of the present invention, the concentration of the AgNO3 solution is 10-30 mmol / L, preferably 20 mmol / L.

[0014] In some embodiments of the present invention, the concentration of the NaBH4 solution is 0.5 to 2 mmol / L, preferably 1 mmol / L.

[0015] In some embodiments of the present invention, the mixing time is 5 to 15 minutes, preferably 10 minutes.

[0016] In some embodiments of the present invention, the reaction time is 5 to 15 minutes, preferably 10 minutes.

[0017] In some embodiments of the present invention, the NaBH4 solution is a freshly prepared NaBH4 solution ready for use.

[0018] A third aspect of the present invention provides a DNA-silver nanocluster prepared by the above-described preparation method.

[0019] In some embodiments of the present invention, the particle size of the DNA-silver nanoclusters is 1 to 3 nm.

[0020] In some embodiments of the present invention, the DNA-silver nanoclusters are excited with a wavelength of 488 nm, and the maximum fluorescence emission intensity is obtained at 620-640 nm.

[0021] In a fourth aspect, the present invention provides the use of the above-described oligochain DNA fragments or DNA-silver nanoclusters in the preparation of a reagent for detecting phosphatidylserine, a marker of disseminated intravascular coagulation in sepsis.

[0022] In this invention, the reagent for detecting PS eversion is a DNA-silver nanocluster that specifically targets PS. After binding to PS, it can emit fluorescence of a specific wavelength under specific laser irradiation, thus being detected.

[0023] In some embodiments of the present invention, the phosphatidylserine is PS located on the cell membrane of blood cells.

[0024] In some embodiments of the present invention, the phosphatidylserine is OMV-mediated everted phosphatidylserine.

[0025] In some embodiments of the present invention, the sepsis is sepsis caused by bacterial infection.

[0026] In some embodiments of the present invention, the bacteria include Gram-negative bacteria.

[0027] The Gram-negative bacteria include, but are not limited to, Shigella dysenteriae, Salmonella typhi, Escherichia coli, Proteus, Pseudomonas aeruginosa, Bordetella pertussis, Vibrio cholerae, and Neisseria meningitidis.

[0028] PS eversion is a key step in initiating tissue factor activation, leading to excessive coagulation and ultimately sepsis-induced disseminated intravascular coagulation (DIC). In severe Gram-negative bacterial infections, the pathogenic component lipopolysaccharide (LPS, also known as endotoxin) enters the cytoplasm via various pathways, activating its intracellular receptor Caspase-11 (human Caspase-4 / 5). Activated Caspase-11 cleaves GSDMD, releasing its N-terminal active moiety. The N-terminal active moiety of GSDMD spontaneously assembles into pores of approximately 20 nanometers on the cell membrane, promoting calcium ion influx. Under the action of calcium kinases, this causes the PS on the inner side of the cell membrane to evert to the outer side, triggering tissue factor activation and the coagulation cascade. Therefore, PS eversion is a crucial node in the initiation of DIC. At this point, the everted PS is easily detected, which is the fundamental theoretical basis of this invention.

[0029] In some embodiments of the present invention, the method of using the reagent includes: adding the sample to be tested to the above-mentioned DNA-silver nanoclusters and then performing detection.

[0030] In some embodiments of the present invention, the sample to be tested is blood cells, blood, serum or plasma; preferably blood cells.

[0031] Beneficial effects:

[0032] Based on the fact that PS eversion mediates coagulation and DIC initiation, the inventors explored a DNA-silver nanocluster probe based on an electro-optical biosensor system and a probe that can specifically bind to PS. This DNA-silver nanocluster can rapidly detect the level of PS eversion in blood cells with sepsis DIC within 5 minutes, providing a new and faster method for the clinical diagnosis of sepsis DIC.

[0033] Terminology in this application:

[0034] "Silver nanoclusters," or AgNCs, are composed of several to dozens of silver atoms. Their ultra-small size is close to the Fermi wavelength of electrons, thus their continuous energy band is separated into multiple independent energy levels, exhibiting many molecular properties such as strong fluorescence. Silver nanoclusters possess characteristics such as high fluorescence quantum yield, good optical stability, and small nanoscale size, and their fluorescence behavior is sensitive to changes in their own size and the surrounding microenvironment. Therefore, silver nanoclusters are often widely used as a fluorescence signal source in biomedical imaging and bioanalytical detection.

[0035] Sepsis refers to a systemic inflammatory response syndrome caused by the invasion of the body by pathogenic microorganisms such as bacteria. It includes sepsis-related diseases such as septicemia, endotoxemia, severe sepsis, and septic shock. All are caused by systemic bacterial infection, leading to symptoms such as multi-organ dysfunction, and their causes and treatments are similar. Severe sepsis often progresses to a later stage where pathogenic bacteria invade the bloodstream, multiply, and eventually cause sepsis (i.e., bacteria enter the bloodstream). In addition to the manifestations of systemic inflammatory response syndrome and primary infection focus, severely ill patients often exhibit organ insufficiency.

[0036] "OMV (outer membrane vesicles)" are spherical vesicles that bulge out and release from the outer membrane of Gram-negative bacteria. They contain LPS. Due to OMV-mediated bacterial-host interactions, LPS is transported in the host, thereby triggering a Caspase-11-dependent inflammatory pathway, inducing uncontrolled immune response to infection and an inflammatory-coagulation positive feedback loop in the host.

[0037] "PS eversion" is the flipping of PS from the inside to the outside of the cell membrane mediated by calcium ion influx caused by cell membrane pores.

[0038] "Blood cells" refers to all blood cells in anticoagulated blood, including but not limited to monocytes and macrophages, lymphocytes, granulocytes, etc. Attached Figure Description

[0039] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0040] Figure 1 This is a diagram illustrating the detection mechanism of the present invention;

[0041] Figure 2 This is a transmission electron microscope image of the silver nanoclusters in Example 1 of the present invention;

[0042] Figure 3 This is a characterization diagram of DNA-silver nanoclusters alone or after binding with PS in Example 1 of the present invention;

[0043] Figure 4 The fluorescence changes of DNA-silver nanoclusters reacted with different concentrations of PS standards in Example 2 of the present invention are shown in the graph (curves from a to i represent PS standards from 0.1 nM to 1 mM, namely 0.1 nM, 0.5 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, and 1 mM respectively).

[0044] Figure 5 This is a diagram showing the effect of DNA-silver nanoclusters detecting PS in Example 3 of the present invention based on a cell model.

[0045] Figure 6 This is a diagram showing the effect of DNA-silver nanoclusters detecting PS in Example 4 of the present invention based on an animal model.

[0046] Figure 7 This is a diagram illustrating the effect of DNA-silver nanoclusters detecting PS in Example 5 of the present invention, based on actual clinical samples. Detailed Implementation

[0047] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0048] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in this technical field.

[0049] Reagents, materials, or instruments required for the experiment:

[0050] The reagents AgNO3 (99.99%), NaBH4 (98%), Na2S, NaOH, NaSiO3, Na2CO3, NaCl, NaClO4, NaF4, NaHCO3, NaHSO4, NaNO3, CH3COONa, Na2SO3, and NaHSO3 were all purchased from Acros (USA), Aldrichchemical Co., Ltd. (USA), and J&K Scientific Ltd. (China).

[0051] The DNA template sequence 5'-CCCCCCCCCCCC-ATC CGT CCT TAT ACA CAA TTG TTT TTC TCTTAA CTT CTT GAG TGT TGG CTC CCG TAT-3' (SEQ ID NO.1) was synthesized by Shanghai Sangon Biotech.

[0052] All colorimetric analyses were recorded using the following method: the absorption spectra of DNA-AgNCs (from 300 nm to 700 nm) were recorded using a UV-Vis spectrophotometer (Shimadzu, UV-2450).

[0053] The prepared silver nanoclusters were analyzed using a Titan G2 60-300 transmission electron microscope.

[0054] In addition, a high-performance liquid chromatograph (ACQUITY UPLC H-Class, WATERS, USA) was used, including a binary solvent system (BSM), an automated sampling manager (SM), a diode array detector (PDA), and an Empower3 chromatography workstation.

[0055] The detection mechanism of this invention is as follows: the DNA aptamer portion of DNA-AgNCs specifically binds to PS, and AgNCs, after binding to the target analyte, fluoresce under a 488nm laser. Therefore, DNA-AgNCs can specifically bind to PS to produce fluorescence under a 488nm laser and be detected, thus it can be used for PS detection, specifically as follows... Figure 1 As shown.

[0056] Example

[0057] Example 1: Characterization of DNA-silver nanoclusters alone or in combination with their target substance (PS).

[0058] I. Experimental Procedure

[0059] (1) Synthesis of DNA-silver nanoclusters

[0060] PS-specific binding aptamers were used to construct rDNA oligonucleotides or molecular beacon detectors from 5 kbp double-stranded DNA (dsDNA, sequence same as SEQ ID NO. 1). Other rDNA oligonucleotides and molecular beacon detectors were synthesized using DNA Technology. Vectors were prepared using biotinylated DNA probes or MB, and DNA oligonucleotides were diluted with binding buffer and hybridization was performed according to ThermoScientific's relevant instructions.

[0061] The silver nanoclusters were synthesized using an optimized method. 35 μL of an aqueous solution of 10 mM DNA was mixed with 21 μL of a 20 mM AgNO3 solution and incubated for 10 minutes. Then, 84 μL of freshly prepared 1 mM NaBH4 solution was added, and the mixture was incubated in the dark for another 10 minutes. The synthesized silver nanoclusters were then characterized using transmission electron microscopy.

[0062] (2) Specific binding of DNA-silver nanoclusters to PS

[0063] Nanotube tips were prepared by fabricating capillaries with an inner diameter of 0.5 mm, an outer diameter of 1.0 mm, and a length of 7.5 cm. After plasma cleaning and contamination removal, any remaining residue was removed using a P-2000 Pipette Puller. Testing was then conducted using a custom confocal microscope (1.20 nA, UPLSAPO 60XW). A 488 nm sapphire laser was used with a 60x or higher objective lens to illuminate the nanotube tip and collect fluorescence through the tube. PerkinElmer dichroism was used for detection via two APDs, and the data were recorded. The optimal focal length for the nanotube tip and confocal microscope was then selected, and scattering originating from the tip was observed using an emCCD camera for appropriate adjustments. Nanoparticle photoelectric translocation experiments and simultaneous detection were performed according to the EON-System instructions. Poisson fitting was used to determine and adjust the background signal-to-noise level of the field of view, and the detection data were recorded. The amplification effect of the quantitative target was calculated and statistically analyzed. In a 10 nM DNA-AgNC system, fluorescence intensity was detected under 488 nm laser excitation with or without the addition of PS (10 nM).

[0064] II. Experimental Results:

[0065] In this embodiment, AgNC appears as follows under a transmission electron microscope: Figure 2 As shown, the size is approximately 1–3 nanometers, and it exhibits good dispersibility. The fluorescence change upon binding with PS is as follows: Figure 3 As shown, the fluorescence intensity varies significantly with and without PS, indicating good specificity.

[0066] Example 2: Fluorescence changes after DNA-silver nanoclusters react with PS standards of different concentrations

[0067] I. Experimental Procedure

[0068] PS was diluted with ultrapure water to a concentration gradient from 0.1 nM to 1 mM, and 1 nM DNA-silver nanoclusters were added to each. The fluorescence intensity was measured under 488 nm laser excitation.

[0069] II. Experimental Results

[0070] like Figure 4 As shown, in PS buffer with gradually increasing concentration, DNA-silver nanoclusters exhibit the same increasing fluorescence intensity when bound to PS, that is, the fluorescence intensity is positively correlated with the PS concentration, indicating that PS is suitable for quantitative detection of PS concentration.

[0071] Example 3: Verification of the effect of DNA-silver nanoclusters in detecting PS based on cell model

[0072] I. Experimental Procedure

[0073] (1) OMV extraction

[0074] Take 1 μL of *E. coli* (purchased from ATCC) preserved in glycerol and add it to 100 μL of LB liquid medium. Incubate on a shaker (37℃, 220 rpm) for 5 min to revive. Pour the revive culture into an LB agar plate and streak. Incubate overnight at 37℃. Pick a single *E. coli* colony and add it to 3 mL of LB liquid medium. Incubate on a shaker (37℃, 220 rpm) for 8 h to expand the culture. Add 3 mL of the expanded culture to 600 mL of LB liquid medium and incubate overnight on a shaker (37℃, 220 rpm). Collect the culture and centrifuge at 3000 rpm for 15 min at 4℃. Discard the precipitate and collect the supernatant. Filter the supernatant through a 0.45 μm filter membrane and collect the liquid phase in a sterile new centrifuge tube. Pour the culture into a dedicated ultracentrifuge tube, balance, and centrifuge at 100000g for 2 h 10 min at 4℃ to collect the OMV. After centrifugation, discard the supernatant and resuspend 6 tubes of OMV in 1 mL of sterile DPBS. The collected OMV was passed through a 0.22 μm sieve and stored at 4 °C. 20 μL of the filtered OMV was used for concentration determination. The OMV concentration was determined using the BCA protein concentration method. The preparation method for the protein standard curve included the following steps: Prepare protein standards with a maximum concentration of 5 mg / mL. Prepare BCA working solution by mixing 50 volumes of BCA reagent A with 1 volume of BCA reagent B (50:1) and mixing thoroughly. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard to the wells of a 96-well plate, and add standard diluent to bring the total to 20 μL, corresponding to standard concentrations of 0, 0.25, 0.5, 1, 2, 3, 4, and 5 mg / mL, respectively. Measure the absorbance of A562 using a microplate reader to obtain the protein standard curve. The sample assay method involved adding 20 μL of filtered OMV to each well of a 96-well plate, followed by 200 μL of BCA working solution. The plate was incubated at 37°C for 30 minutes, and the absorbance of A562 was measured using a microplate reader. The concentration of OMV was calculated based on the standard curve and the sample absorbance.

[0075] (2) Extraction and processing of primary peritoneal macrophages from WT mice

[0076] Mice (purchased from Hunan Slack Jingda) were injected intraperitoneally with 3 mL of sterile broth containing 3% thioglycolate to induce peritoneal macrophage aggregation. After 72 hours, mice were euthanized, and the peritoneum was surgically exposed. Primary peritoneal macrophages were collected using a syringe with 10 mL of pre-cooled serum-free 1640 cell culture medium connected to an indwelling needle. The cells were passed through a 40-mesh sieve and homogenized with 1640 complete culture medium (10% fetal bovine serum, 1% penicillin and antibiotics) at a concentration of 1 × 10⁶ cells / mL. 6Macrophages were seeded at a density of 100 μL / mL in 96-well plates and incubated overnight at 37°C with 5% CO2. After overnight cell adhesion, the culture medium was discarded, and the cells were washed three times with PBS. Macrophages were then treated with OMV diluted 10 μg / mL in calcium-free 1640 medium for 12 hours, with the medium-only group serving as a negative control. 1 nM DNA-silver nanoclusters were added, and after incubation at 37°C for 5 min, fluorescence intensity was detected under 488 nm laser excitation.

[0077] II. Experimental Results

[0078] like Figure 5 As shown, OMV-treated cells exhibited a significant fluorescence reaction after the addition of DNA-silver nanoclusters. Based on the confirmed OMV-mediated PS eversion signaling via Caspase-11, this indicates that DNA-silver nanoclusters can detect cell PS eversion.

[0079] Example 4: Validation of the effect of DNA-silver nanoclusters in detecting PS using an animal model

[0080] I. Experimental Procedure

[0081] (1) Mouse modeling

[0082] CLP modeling: C57 / 6J mice were anesthetized with 2% isoflurane (Piramal Critical Care). The abdomen was incised along the longitudinal midline at 1.5 cm to expose the cecum. 75% of the cecum was ligated, and a small amount of feces was expelled through an 18-gauge needle puncture to establish a severe microbial sepsis model. The cecum was repositioned, and the abdomen was sutured. Mice were subcutaneously injected with pre-warmed saline (37°C; 5 mL per 100g body weight) to allow them to recover from anesthesia. A sham-operated CLP control group was established. Blood was collected from the heart 24 hours later and anticoagulated with heparin.

[0083] Endotoxemia modeling: LPS was injected intraperitoneally into C57 / 6J mice at a dose of 20 mg / kg. Blood was collected from the heart 16 hours later and anticoagulated with heparin.

[0084] Sepsis modeling: E. coli was diluted with PBS to a concentration of 10⁹ CFU / mL and injected 100 μL into C57 / 6J mice via the tail vein. Blood was collected from the heart 16 hours later and anticoagulated with heparin.

[0085] (2) Fluorescence detection

[0086] Blood samples from mice with 10 μL CLP model, endotoxemia model, sepsis model, and sham-operated control group were taken, diluted to 1 mL with PBS, mixed well, and 100 μL was taken. 1 nM DNA-silver nanoclusters were added, and the mixture was incubated at 37 degrees for 5 min. The fluorescence intensity was then detected under 488 nm laser excitation.

[0087] II. Experimental Results

[0088] like Figure 6 As shown, the blood of mice with CLP sepsis, endotoxemia, and septicemia models showed a significant fluorescent reaction after the addition of DNA-silver nanoclusters, confirming that DNA-silver nanoclusters can detect PS eversion of blood cells.

[0089] Example 5: Verification of the effectiveness of DNA-silver nanoclusters in detecting PS based on actual clinical samples.

[0090] I. Experimental Procedure

[0091] Blood samples (heparin anticoagulated) were collected from newly diagnosed sepsis patients upon admission. 10 μL of blood sample was taken, diluted to 1 mL with PBS, mixed well, and 100 μL was taken. 1 nM DNA-silver nanoclusters were added, and the mixture was incubated at 37 degrees for 5 min. The fluorescence intensity was then detected under 488 nm laser excitation.

[0092] II. Experimental Results

[0093] like Figure 7 As shown, in patients with sepsis, compared with patients without DIC and those diagnosed with DIC, the fluorescence intensity of blood samples from DIC patients was significantly upregulated after the addition of DNA-silver nanoclusters, indicating that DNA-silver nanoclusters have the potential to predict sepsis-related DIC by detecting PS eversion.

[0094] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. An oligostranded DNA fragment, characterized in that, The nucleotide sequence of the oligo-stranded DNA fragment is shown in SEQ ID NO.

1.

2. A method for preparing DNA-silver nanoclusters, characterized in that, Includes the following steps; The aqueous solution of the oligo-stranded DNA fragment according to claim 1 is mixed with AgNO3 solution, and then NaBH4 solution is added and reacted to obtain the final product.

3. The preparation method according to claim 2, characterized in that, The molar ratio of the oligo-stranded DNA fragment, AgNO3, and NaBH4 is 1:1~1.5:0.2~0.

4.

4. The preparation method according to claim 2, characterized in that, The mixing time is 5-15 minutes.

5. The preparation method according to claim 2, characterized in that, The reaction time is 5-15 minutes.

6. The DNA-silver nanoclusters prepared by the preparation method according to any one of claims 2 to 5.

7. The DNA-silver nanocluster according to claim 6, characterized in that, The DNA-silver nanoclusters have a particle size of 1~3nm.

8. The DNA-silver nanocluster according to claim 6, characterized in that, Excitation with a wavelength of 488 nm yielded the maximum fluorescence emission intensity at 620–640 nm.

9. The use of the oligo-stranded DNA fragment as described in claim 1 or the DNA-silver nanoclusters as described in any one of claims 6 to 8 in the preparation of a reagent for detecting phosphatidylserine, a marker of disseminated intravascular coagulation in sepsis.

10. The application according to claim 9, characterized in that, The method of using the reagent includes: adding the sample to be tested to the DNA-silver nanocluster cluster according to any one of claims 6-8 and then performing detection.

11. The application according to claim 10, characterized in that, The sample is blood cells, blood, serum, or plasma.

12. The application according to claim 9, characterized in that, The sepsis mentioned refers to sepsis caused by bacterial infection.

13. The application according to claim 12, characterized in that, The bacteria include Gram-negative bacteria.

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