Detection system for rapidly diagnosing keratitis

Abstract

The invention relates to the technical field of keratitis detection systems, in particular to a detection system for rapidly diagnosing keratitis. The invention provides a detection system for rapidly diagnosing keratitis, which comprises a kit and an excitation light source, the kit comprises three independently packaged reagent single tubes; the three reagent single tubes comprise a first reagent single tube, a second reagent single tube and a third reagent single tube which are arranged in sequence according to a medication sequence; a reagent in the third reagent single tube comprises 6PD-DPAN; reagents in the first reagent single tube and the second reagent single tube are independently DO-DPAS or M1-DPAN, and the reagents in the first reagent single tube and the second reagent single tube are different; and the lambda ex of the excitation light source is equal to 200 to 420 nm. The detection system can simply, conveniently, quickly and accurately detect the infection type of keratitis.

Description

Technical Field

[0001] This invention relates to the field of keratitis detection system technology, and more particularly to a rapid keratitis detection system. Background Technology

[0002] Bacterial and fungal keratitis are common infectious keratitis and a frequent cause of corneal blindness. Rapid and accurate diagnosis is a prerequisite for improving treatment outcomes, thus becoming a key focus and hot topic in ophthalmology. Currently, various methods exist for diagnosing bacterial and fungal keratitis clinically, mainly including clinical empirical diagnosis, microbiological examination, molecular biological diagnosis (high-throughput sequencing technology, polymerase chain reaction assay), corneal confocal microscopy, and artificial intelligence diagnosis.

[0003] Clinical empirical diagnosis is a commonly used and fundamental diagnostic method for infectious keratitis, where clinicians make a diagnosis based on a comprehensive review of the patient's medical history and clinical signs. This method is quick and simple, requiring no special equipment, but it has a long learning curve and a high rate of misdiagnosis.

[0004] Microbiological examination of the pathogenic lesions includes culturing in culture media after sampling, microscopic staining after sampling, and corneal biopsy. This method is considered the gold standard for diagnosing infectious keratitis due to its high heterogeneity. However, it is an invasive procedure, microbial culture is time-consuming, and its accuracy is easily affected by sampling techniques, often resulting in a high negative rate. For example, microbial culture typically takes 48 hours or longer, with a negative rate as high as 32% to 53%. Microscopic staining usually uses Gram staining and Giemsa staining, with a detection rate of approximately 60% to 75%. Corneal biopsy improves the positive rate by obtaining samples from deep corneal tissue, but the negative rate is still as high as 18% to 60%, and in cases where corneal thinning, melting, and necrosis are present, corneal biopsy carries the risk of corneal perforation.

[0005] Molecular biological diagnostics include next-generation sequencing (NGS) and polymerase chain reaction (PCR). NGS can sequence DNA in clinical samples and match the resulting fragments with DNA databases. However, NGS lacks standardization and cannot be used as the sole evidence for diagnosis; it can only be used as an auxiliary method. PCR has the advantages of speed and high sensitivity, and can quickly distinguish between bacterial and fungal keratitis. However, this method also has significant drawbacks, including susceptibility to interference from commensal contaminants, a high false-positive rate from dead bacteria, low specificity, and the need to narrow down the pathogen for the use of specific primers.

[0006] In vivo confocal microscopy (IVCM) is a valuable imaging diagnostic method that allows for non-invasive, repeatable, and high-resolution diagnosis and evaluation of corneal structure and pathology at the cellular and subcellular levels. However, IVCM is primarily used to diagnose fungal keratitis and Acanthamoeba keratitis because its axial resolution is currently limited to 5-7 μm, insufficient to distinguish bacteria (usually <5 μm), and it is also affected by corneal transparency.

[0007] In recent years, artificial intelligence (AI) technology, especially deep learning models, has seen significant advancements in its application in ophthalmic diagnosis. Several deep learning-based convolutional neural network models have been developed for diagnosing infectious keratitis. Studies have shown that these AI models can effectively identify and classify different types of infectious keratitis by analyzing slit-lamp images, handheld camera images, and in vivo confocal microscopy (IVCM) images. Despite the significant potential of AI technology in the diagnosis of infectious keratitis, several challenges remain, including a strong dependence on high-quality, large-scale datasets, variability in image annotation quality, the risk of model overfitting, and the variability of clinical settings. These issues limit the generalization ability of AI models under different imaging conditions and may lead to inaccurate diagnoses when faced with rare or novel infections. Therefore, further optimization and validation are needed to ensure their effectiveness and reliability in clinical settings.

[0008] In summary, although various methods for diagnosing bacterial and fungal keratitis are currently available in clinical practice, each method has its own limitations. Therefore, how to quickly, sensitively, and accurately diagnose and differentiate between bacterial and fungal keratitis remains a hot research topic. Summary of the Invention

[0009] In view of this, the purpose of the present invention is to provide a detection system for rapid diagnosis of keratitis, which can easily, quickly and accurately detect the type of keratitis infection.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0011] This invention provides a rapid diagnostic system for keratitis, comprising a reagent kit and an excitation light source, wherein the reagent kit comprises three individually packaged reagent tubes;

[0012] The three reagent tubes, arranged in the order of medication administration, include a first reagent tube, a second reagent tube, and a third reagent tube set sequentially.

[0013] The reagent in the third reagent tube includes 6PD-DPAN;

[0014] The reagents in the first reagent tube and the second reagent tube are independently either DO-DPAS or M1-DPAN, and the reagents in the first reagent tube and the second reagent tube are different.

[0015] The structure of the M1-DPAN is shown in Formula 1: The structure of the DO-DPAS is shown in Equation 2: The structure of the 6PD-DPAN is shown in Equation 3:

[0016]

[0017] The excitation source λ ex =200~420nm.

[0018] Preferably, the λ of the excitation light source ex =391~395nm.

[0019] Preferably, the excitation source is provided by a slit-lamp microscope or a live confocal microscope.

[0020] Preferably, the reagent in the first reagent tube is DO-DPAS.

[0021] Preferably, the DO-DPAS exists in the form of a DO-DPAS solution;

[0022] The concentration of DO-DPAS in the DO-DPAS solution is 5–20 μmol / L.

[0023] Preferably, the amount of reagent added in the first reagent tube is 5 to 20 μL.

[0024] Preferably, the reagent in the second reagent tube is M1-DPAN.

[0025] Preferably, the M1-DPAN exists in the form of an M1-DPAN solution;

[0026] The concentration of M1-DPAN in the M1-DPAN solution is 5–20 μmol / L.

[0027] Preferably, the amount of reagent added in the second reagent tube is 5–20 μL.

[0028] Preferably, the 6PD-DPAN exists in the form of a 6PD-DPAN solution;

[0029] The concentration of 6PD-DPAN in the 6PD-DPAN solution is 5–20 μmol / L;

[0030] The amount of reagent added in the third reagent tube is 5–20 μL.

[0031] This invention provides a rapid diagnostic system for keratitis, comprising a reagent kit and an excitation light source, wherein the reagent kit comprises three individually packaged reagent tubes;

[0032] The three reagent tubes, arranged in the order of medication administration, include a first reagent tube, a second reagent tube, and a third reagent tube set sequentially.

[0033] The reagent in the third reagent tube includes 6PD-DPAN;

[0034] The reagents in the first reagent tube and the second reagent tube are independently either DO-DPAS or M1-DPAN, and the reagents in the first reagent tube and the second reagent tube are different.

[0035] The structure of the M1-DPAN is shown in Formula 1:

[0036] The structure of the DO-DPAS is shown in Equation 2:

[0037] The structure of the 6PD-DPAN is shown in Equation 3:

[0038]

[0039] The excitation source λ ex =200~420nm.

[0040] The DO-DPAS, M1-DPAN, and 6PD-DPAN in the detection system of this invention exhibit specific fluorescence characteristics after binding with different pathogen grades. DO-DOAS can rapidly and specifically bind to fungi, but not to bacteria, and emits yellow-green fluorescence (λ). em =525nm); M1-DPAN can rapidly and specifically bind to Gram-positive bacteria (Gram-positive bacteria) without binding to Gram-negative bacteria (Gram-negative bacteria) or fungi, and emits the same yellow-green fluorescence (λ). em =525nm); 6PD-DPAN can rapidly bind to bacteria (including Gram- and Gram-negative) and fungi, and also excites yellow-green fluorescence (λ). em =525nm); By placing reagent tubes containing the above three reagents in the order of administration and irradiating them with a slit lamp, specific fluorescence signals can be captured within 5 to 10 minutes, which can quickly and accurately identify and distinguish different types of infectious keratitis.

[0041] Compared with the prior art, the detection system of the present invention has the following advantages:

[0042] 1) High specificity: The DO-DPAS, M1-DPAN and 6PD-DPAN all have high selectivity for specific pathogens. DO-DPAS can specifically recognize fungi, M1-DPAN mainly recognizes Gram-positive bacteria, while 6PD-DPAN shows spectral recognition ability for a variety of pathogens (including Gram-positive, Gram-negative bacteria and fungi). By setting the DO-DPAS, M1-DPAN and 6PD-DPAN, the detection system can accurately identify the types of keratitis infections.

[0043] 2) Fast detection speed: Compared with traditional microbial culture which takes 48 hours or more, the detection system can complete the process in just 10 minutes;

[0044] 3) Good biocompatibility: The DO-DPAS, M1-DPAN and 6PD-DPAN have no obvious toxicity to human corneal tissue cells within the safe concentration range, showing good biocompatibility.

[0045] In summary, the detection system described in this invention has the advantages of high sensitivity, high specificity, rapid response, and good biocompatibility. Attached Figure Description

[0046] Figure 1 The fluorescence signal (excitation wavelength λ) obtained by confocal laser scanning after DO-DPAS treatment of Candida albicans, Gram-positive bacteria (Staphylococcus aureus), and Gram-negative bacteria (Pseudomonas aeruginosa) for 10 min. ex =405nm, emission wavelength λ em =525nm);

[0047] Figure 2 The fluorescence signal (excitation wavelength λ) obtained by confocal laser scanning after treating Candida albicans, Gram-positive bacteria (Staphylococcus aureus), and Gram-negative bacteria (Pseudomonas aeruginosa) with M1-DPAN for 10 min. ex =405nm, emission wavelength λ em =525nm);

[0048] Figure 3 The fluorescence signal (excitation wavelength λ) obtained by confocal laser scanning after 10 min of treatment with 6PD-DPAN for Candida albicans, Gram-positive bacteria (Staphylococcus aureus), and Gram-negative bacteria (Pseudomonas aeruginosa) ex =405nm, emission wavelength λ em=525nm);

[0049] Figure 4 Cell viability of human corneal epithelial cells (AC corresponding to M1-DPAN, 6PD-DPAN, and DO-DPAS, respectively) and THP-1 cells (DF corresponding to M1-DPAN, 6PD-DPAN, and DO-DPAS, respectively) after 30 min of treatment with different concentrations of M1-DPAN, 6PD-DPAN, and DO-DPAS solutions (in the figure, ns: no significant difference, p value: *p<0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, n=5);

[0050] Figure 5 Slit-lamp photography and microbial culture of mice with infectious keratitis. A shows representative slit-lamp images of mice infected with Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa from the control group; B shows representative images of corneal fluorescein-stained corneas of mice infected with Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa from the control group; and C shows microbial culture images of corneal lesion scrapings from mice infected with Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa on Columbia blood agar plates (n=10).

[0051] Figure 6 The images show the histopathological examination of corneal tissue from mice with infectious keratitis. A represents hematoxylin-eosin staining of corneal tissue from the control group, mice infected with *Candida albicans*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa*. B represents Masson staining of corneal tissue from the control group, mice infected with *Candida albicans*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa*. C represents Gram staining of corneal tissue from the control group, mice infected with *Candida albicans*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa*. D represents PAS staining of corneal tissue from the control group, mice infected with *Candida albicans*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa* (n=10).

[0052] Figure 7 Representative confocal laser fluorescence images (λ) of corneal tissue from control, Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa groups were obtained 10 min after treatment with 10 μM M1-DPAN. ex =405nm, λ em =525nm), n=10;

[0053] Figure 8 Representative confocal laser fluorescence images (λ) of corneal tissue from mice treated with 10 μM DO-DPAS for 10 min after infection with Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa were obtained. ex =405nm, λem =525nm), n=10;

[0054] Figure 9 Representative confocal laser fluorescence images (λ) of corneal tissue from control, Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa groups were obtained 10 min after treatment with 10 μM 6PD-DPAN. ex =405nm, λ em =525nm), n=10;

[0055] Figure 10 To determine the optimal treatment method, 10 μL of a 10 μM DO-DPAS solution was added to the corneal surface of normal mice and mice infected with Candida albicans, respectively. After 10 min, the mice in each group were subjected to excitation from a slit lamp under an excitation source λ. ex Representative images of the anterior segment under illumination of 395 nm (n=3). Detailed Implementation

[0056] This invention provides a rapid diagnostic system for keratitis, comprising a reagent kit and an excitation light source, wherein the reagent kit comprises three individually packaged reagent tubes;

[0057] The three reagent tubes, arranged in the order of medication administration, include a first reagent tube, a second reagent tube, and a third reagent tube set sequentially.

[0058] The reagent in the third reagent tube includes 6PD-DPAN;

[0059] The reagents in the first reagent tube and the second reagent tube are independently either DO-DPAS or M1-DPAN, and the reagents in the first reagent tube and the second reagent tube are different.

[0060] The structure of the M1-DPAN is shown in Formula 1:

[0061] The structure of the DO-DPAS is shown in Equation 2:

[0062] The structure of the 6PD-DPAN is shown in Equation 3:

[0063]

[0064] The excitation source λ ex =200~420nm.

[0065] In this invention, the DO-DPAS is a bis(5-pyridine)-based molecule containing one hydroxyl group and two benzene rings. The hydroxyl group enables it to form hydrogen bonds, binding to chitin and dextran in the fungal cell wall. The hydroxyl group serves as the primary active group to enhance heterogeneous binding to the fungal cell wall. The M1-DPAN molecule contains a bis(5-pyridine) structure with a morpholine group and a phenyl group. The benzene ring structure containing the morpholine group provides specific binding to lipoteichoic acid through electrostatic interactions and hydrogen bonds. The 6PD-DPAN is a broad-spectrum binder based on bis(5-pyridine) and benzodiazepine. The derivatives have a structure that allows them to bind to various bacterial and fungal cell wall components. They possess multiple amino and hydroxyl substituents, enabling broad-spectrum binding through hydrogen bonding and electrostatic interactions in diverse cell wall environments. They can bind to various pathogens and emit specific fluorescence. Furthermore, all three substances exhibit yellow-green fluorescence (λ) upon contact with their respective pathogens. ex =525nm), meeting the needs for rapid and sensitive diagnosis of infectious keratitis.

[0066] In this invention, the λ of the excitation light source ex Preferably, the wavelength is 391–395 nm, and more preferably 391 nm.

[0067] In this invention, the excitation light source is preferably provided by a slit-lamp microscope or a live confocal microscope.

[0068] In this invention, the reagent in the first reagent tube is preferably DO-DPAS, and the DO-DPAS is preferably present in the form of a DO-DPAS solution. The concentration of DO-DPAS in the DO-DPAS solution is preferably 5-20 μmol / L, more preferably 10 μmol / L. In this invention, the amount of reagent added in the first reagent tube is preferably 5-20 μL, more preferably 10 μL.

[0069] In this invention, the reagent in the second reagent tube is preferably M1-DPAN, which is preferably in the form of an M1-DPAN solution. The concentration of M1-DPAN in the M1-DPAN solution is preferably 5-20 μmol / L, more preferably 10 μmol / L. In this invention, the amount of reagent added in the second reagent tube is preferably 5-20 μL, more preferably 10 μL.

[0070] In this invention, the reagent in the third reagent tube is preferably 6PD-DPAN, which is preferably present in the form of a 6PD-DPAN solution. The concentration of 6PD-DPAN in the 6PD-DPAN solution is preferably 5–20 μmol / L, more preferably 10 μmol / L. In this invention, the amount of reagent added in the third reagent tube is preferably 5–20 μL, more preferably 10 μL.

[0071] In this invention, the preferred method of using the detection system is as follows:

[0072] Before adding the liquid, adjust the excitation source of the slit lamp to λ. ex =405nm, first add the reagent from the first reagent tube. If a fluorescence signal is detected, stop the detection. If no fluorescence signal is detected, continue adding the reagent from the second reagent tube. If no fluorescence signal is detected, continue adding the reagent from the third reagent tube to detect Gram-negative bacteria or undetermined mixed infections.

[0073] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0074] Verification Example

[0075] The specific binding and colorimetric effects of DO-DPAS, M1-DPAN, and 6PD-DPAN on Gram-positive bacteria, Gram-negative bacteria, and fungi are described below.

[0076] After completing the pathogen culture (the pathogens were Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa, all at a concentration of 1 OD (1*10⁻⁶)), the pathogens were cultured (the pathogens were Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa, all at a concentration of 1 OD (1*10⁻⁶)). 8 10 μL of DO-DPAS, M1-DPAN, and 6PD-DPAN solutions with a concentration of 5 μM were added to the above pathogens, respectively, and incubated at 37°C for 20 min. After incubation, the fluorescence signals of each group were observed using a confocal microscope to analyze the specific recognition effects of the above DO-DPAS, M1-DPAN, and 6PD-DPAN solutions on different pathogens and cells.

[0077] in, Figure 1The fluorescence signal (excitation wavelength λ) obtained by confocal laser scanning after DO-DPAS treatment of Candida albicans, Gram-positive bacteria (Staphylococcus aureus), and Gram-negative bacteria (Pseudomonas aeruginosa) for 10 min. ex =405nm, emission wavelength λ em =525nm)(the scale bar in the lower right corner of the figure is 5μm); where, A is a representative confocal laser scanning fluorescence image after DO-DPAS treatment of Candida albicans, A' is a single-channel green fluorescence image after DO-DPAS treatment of Candida albicans, and A” is a bright-field channel image after DO-DPAS treatment of Candida albicans; B is a single-channel green fluorescence image after DO-DPAS treatment of Staphylococcus aureus, B' is a single-channel green fluorescence image after DO-DPAS treatment of Staphylococcus aureus, and B” is a bright-field channel image after DO-DPAS treatment of Staphylococcus aureus; C is a single-channel green fluorescence image after DO-DPAS treatment of Pseudomonas aeruginosa, C' is a single-channel green fluorescence image after DO-DPAS treatment of Pseudomonas aeruginosa, and C” is a bright-field channel image after DO-DPAS treatment of Pseudomonas aeruginosa; from Figure 1 It is known that DO-DPAS can specifically bind to Candida albicans and emit yellow-green fluorescence, but it failed to bind to Staphylococcus aureus and Pseudomonas aeruginosa.

[0078] Figure 2 The fluorescence signal (excitation wavelength λ) obtained by confocal laser scanning after treating Candida albicans, Gram-positive bacteria (Staphylococcus aureus), and Gram-negative bacteria (Pseudomonas aeruginosa) with M1-DPAN for 10 min. ex =405nm, emission wavelength λ em =525nm)(the scale bar in the lower right corner of the figure is 5μm); where, A is a representative confocal laser scanning fluorescence image after M1-DPAN treatment of Candida albicans, A' is a single-channel green fluorescence image after M1-DPAN treatment of Candida albicans, and A” is a bright-field channel image after M1-DPAN treatment of Candida albicans; B is a representative fluorescence image after M1-DPAN treatment of Staphylococcus aureus, B' is a single-channel green fluorescence image after M1-DPAN treatment of Staphylococcus aureus, and B” is a bright-field channel image after M1-DPAN treatment of Staphylococcus aureus; C is a representative fluorescence image after M1-DPAN treatment of Pseudomonas aeruginosa, C' is a single-channel green fluorescence image after M1-DPAN treatment of Pseudomonas aeruginosa, and C” is a bright-field channel image after M1-DPAN treatment of Pseudomonas aeruginosa; from Figure 2It is known that M1-DPAN can specifically bind to Staphylococcus aureus and emit yellow-green fluorescence, but it failed to bind to Candida albicans and Pseudomonas aeruginosa.

[0079] Figure 3 The fluorescence signal (excitation wavelength λ) obtained by confocal laser scanning after 10 min of treatment with 6PD-DPAN for Candida albicans, Gram-positive bacteria (Staphylococcus aureus), and Gram-negative bacteria (Pseudomonas aeruginosa) ex =405nm, emission wavelength λ em =525nm)(the scale bar in the lower right corner of the figure is 5μm); where, A is a representative confocal laser scanning fluorescence image after 6PD-DPAN treatment of Candida albicans, A' is a single-channel green fluorescence image after 6PD-DPAN treatment of Candida albicans, and A” is a bright-field channel image after 6PD-DPAN treatment of Candida albicans; B is a single-channel green fluorescence image after 6PD-DPAN treatment of Staphylococcus aureus, B' is a single-channel green fluorescence image after 6PD-DPAN treatment of Staphylococcus aureus, and B” is a bright-field channel image after 6PD-DPAN treatment of Staphylococcus aureus; C is a single-channel green fluorescence image after 6PD-DPAN treatment of Pseudomonas aeruginosa, C' is a single-channel green fluorescence image after 6PD-DPAN treatment of Pseudomonas aeruginosa, and C” is a bright-field channel image after 6PD-DPAN treatment of Pseudomonas aeruginosa; from Figure 3 It is known that 6PD-DPAN can bind to Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa and excite specific fluorescence;

[0080] Biocompatibility assessment of DO-DPAS, M1-DPAN, and 6PD-DPAN against different ocular surface cells:

[0081] HCE-T (human corneal epithelial cells) and THP-1 (human monocytes) were seeded into 96-well plates, with 5000 cells per well, and cultured at 37°C and 5 vol% CO2 until 80% confluence. For THP-1 cells, differentiation into macrophages was induced by adding 100 ng / mL phorbol ester (PMA) to the complete culture medium (after 24 h of culture, the cells changed from suspension growth to adherent growth, from round to irregular shape, with further increase in volume, loose cytoplasm, significantly enlarged nuclei, and a few protrusions around the cell membrane, indicating successful induction).

[0082] (2) Prepare solutions of M1-DPAN, 6PD-DPAN, and DO-DPAS at different concentration gradients, with concentration ranges of 0.5 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM. Add each concentration of AIE material solution to the cultured cells, with five replicates per group, and set up a blank control group without AIE material. After incubating the cells for 30 minutes, add CCK-8 reagent to each well and incubate for 4 hours.

[0083] (3) The absorbance (OD450) of each well was measured at a wavelength of 450 nm using an ELISA reader to assess cell viability. Cell viability was calculated using the formula (cell viability = [(experimental wells - blank wells) / (control wells - blank wells)] × 100%) to evaluate the cytotoxic effects of the three aggregation-induced luminescent materials on human corneal epithelial cells (HCE-T cell line) and to determine the safe concentration range.

[0084] The preparation of complete culture medium for human corneal epithelial cells is shown in Table 1:

[0085] Table 1. Preparation of human corneal epithelial cell culture medium

[0086] Element Final concentration DMEM / F12 basal medium / Premium fetal bovine serum 15wt% P / S Penicillin-Streptomycin 1wt% insulin 5μg / mL Recombinant human epidermal growth factor 5ng / mL

[0087] The preparation of complete culture medium for THP-1 cells is shown in Table 2:

[0088] Table 2. Preparation of complete culture medium for THP-1 cells.

[0089] Element Final concentration 1640 basal culture medium / Premium fetal bovine serum 10wt% P / S Penicillin-Streptomycin 1wt% β-Mercaptoethanol 0.05mM HEPES 25mM

[0090] in Figure 4 Human corneal epithelial cells (AC corresponding to M1-DPAN, 6PD-DPAN, and DO-DPAS, respectively) and THP-1 cells (DF corresponding to M1-DPAN, 6PD-DPAN, and DO-DPAS, respectively) were treated with different concentrations of M1-DPAN, 6PD-DPAN, and DO-DPAS solutions for 30 min to determine cell viability (in the figure, ns: no significant difference, p value: *p<0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001, n=5). Figure 4 It can be seen that M1-DPAN, 6PD-DPAN and DO-DPAS have no significant effect on the activity of human corneal epithelial cells and macrophages in the concentration range of 0 to 20 μM, indicating that M1-DPAN, 6PD-DPAN and DO-DPAS have good biocompatibility.

[0091] Construction of a mouse model of infectious keratitis:

[0092] 1) Preparation of bacterial culture:

[0093] Inoculation and incubation: Pick single colonies of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans from solid agar plates. Using a sterile inoculation loop or toothpick, gently touch the colony to ensure that only a single colony is picked. Inoculate the picked colonies into 10 ml of liquid nutrient broth (Nutrient Broth, NB, formula as shown in Table 3), Luria broth (LB, formula as shown in Table 4), and YPD medium (formula as shown in Table 5), respectively. Place the medium in a constant temperature shaker at 37°C and set the shaker speed to 220 rpm, and incubate overnight (12-16 h).

[0094] Table 3 NB Formula (Requires autoclaving at 121℃ for 15 min)

[0095] Element Dosage (based on the amount needed to prepare 1000 mL of culture medium) peptone 10.0g Beef Powder 3.0g Sodium chloride 5.0g pH value 7.2±0.2 temperature 25℃

[0096] Table 4 LB formulation (requires autoclaving at 121℃ for 15 min)

[0097] Element Dosage (based on the amount needed to prepare 1000 mL of culture medium) trypsin 10.0g Yeast extract 5.0g Sodium chloride 10.0g pH value 7.2±0.2 temperature 25℃

[0098] Table 5 YPD culture medium formulation (requires autoclaving at 121℃ for 15 min)

[0099]

[0100]

[0101] Centrifugation and washing: After incubation, transfer the obtained culture to a sterile centrifuge tube and centrifuge at 3000 rpm for 3 min to precipitate the bacteria. Carefully discard the supernatant, retain the bacterial precipitate, add an equal volume of sterile physiological saline, gently mix to resuspend the bacteria, centrifuge again at 3000 rpm for 3 min, discard the supernatant, and repeat the washing process twice.

[0102] Resuspension and Measurement: After washing, the bacterial precipitate was resuspended in an appropriate amount of sterile physiological saline. The optical density (OD600) of the bacterial suspension was measured at a wavelength of 600 nm using a spectrophotometer. The suspension concentration was adjusted according to the measurement results (1.0 OD = 10). 8 (CFU / mL);

[0103] Dilution: Dilute the resuspended bacterial solution with sterile physiological saline to the required concentration (5OD for Staphylococcus aureus, 5OD for Pseudomonas aeruginosa, and 1OD for Candida albicans).

[0104] 2) Animal modeling of infectious keratitis:

[0105] Modeling method: C57BL / 6 mice were anesthetized by intraperitoneal injection of afodin (tribromoethanol, 20μL / g) at 6-8 weeks of age. A crisscross incision was made on the corneal surface of the mice, cutting through the corneal epithelium to the corneal stroma but not exceeding the corneal endothelium. 10μL of bacterial suspension was dripped into the corneal incision area, and the mice were allowed to recover naturally after standing.

[0106] Model evaluation: a: Slit-lamp photography: Fully open the eyelids to expose the eyeball; adjust the illumination band to the maximum width of the diffused illumination state, focus on the mouse eyeball, and take a picture under white light. Instill 1 g / mL sodium fluorescein staining agent under the mouse conjunctival sac, and take a picture under cobalt blue light after 1 min;

[0107] b: Microbial detection: Corneal lesion tissue was sampled with a sterile scalpel blade, inoculated onto Columbia blood agar plates, and cultured in a biochemical incubator for 24 hours. Colony growth was then observed.

[0108] c: Pathological analysis: After obtaining mouse eyeballs, the samples were immediately placed in 4 vol% paraformaldehyde solution and fixed at 4℃ for 24 h. After fixation, they were progressively dehydrated with different concentration gradients of ethanol (70 vol%, 80 vol%, 90 vol%, 95 vol%, and 100 vol%), each concentration for 30 min. After dehydration, the samples were cleared with xylene, and then embedded in paraffin. After embedding, the embedded eyeball tissue was cut into 5 μm thick sections using a microtome, and the sections were laid flat on glass slides and dried in an oven at 37℃ to fix the tissue sections.

[0109] Pathological staining is divided into hematoxylin-eosin (HE) staining, Masson staining, Gram staining, and periodic acid-Schiff (PAS) staining.

[0110] HE staining: After dewaxing, the sections were dehydrated with a series of ethanol solutions, then immersed in hematoxylin staining solution for 5-10 min, rinsed with tap water, differentiated with 1 vol% hydrochloric acid-ethanol, stained with eosin for 2 min, and finally mounted after dehydration with a series of ethanol solutions and clearing with xylene.

[0111] Masson staining: The slides are stained sequentially in iron hematoxylin, acid fuchsin, and azurite, and then dehydrated with graded ethanol before being cleared and mounted.

[0112] Gram staining: performed using crystal violet and fuchsin solutions.

[0113] PAS staining: After treatment with 0.5 vol% periodic acid for 5 min, the sample was stained in a Schiff instrument and reverse stained with hematoxylin.

[0114] After staining, graded ethanol dehydration and xylene clearing, all sections were mounted. Finally, the sections were observed under a microscope to record the verification reaction, fibrosis, and pathogen distribution.

[0115] Figure 5 Slit-lamp photography and microbial culture of mice with infectious keratitis. A shows representative slit-lamp images of mice infected with *Candida albicans*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa* from the control group; B shows representative corneal fluorescein-stained images of mice infected with *Candida albicans*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa* from the control group; C shows microbial culture images of corneal lesion scrapings from mice infected with *Candida albicans*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa* on Columbia blood agar plates (n=10). Figure 5 It can be seen that, by establishing a mouse model of infectious keratitis using the corneal stromal scratch method, typical infectious lesions and corneal epithelial damage appeared in the cornea 24 hours after mice were infected with Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa (AB); microbial culture of corneal lesion tissue showed characteristic colonies of Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa, which proved the successful construction of the mouse model of infectious keratitis at the etiological level (C);

[0116] Figure 6 This document presents histopathological examinations of corneal tissue from mice with infectious keratitis. Image A shows representative hematoxylin-eosin staining images of corneal tissue from control mice, mice infected with *Candida albicans*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa*. Image B shows representative Masson staining images of corneal tissue from control mice, mice infected with *Candida albicans*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa*. Image C shows representative Gram staining images of corneal tissue from control mice, mice infected with *Candida albicans*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa*. Image D shows representative PAS staining images of corneal tissue from control mice, mice infected with *Candida albicans*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa* (n=10). Figure 6 It was found that, compared with the control group corneal tissue, the inflammatory cell infiltration in the stroma of the corneal tissue of mice infected with Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa was significantly increased, and the tissue structure was significantly disordered (AB). Gram staining revealed obvious Gram-positive spherical bacteria in the corneal tissue of mice infected with Staphylococcus aureus (C). Periodic acid-Schiff staining revealed obvious periodic acid-Schiff-positive hyphae in the corneal tissue of mice infected with Candida albicans (D). In conclusion, the mouse model of infectious keratitis was successfully established.

[0117] In vivo experiments validated the specificity of DO-DPAS, M1-DPAN, and 6PD-DPAN in recognizing different pathogens:

[0118] Experimental Methods: 10 μM solutions of DO-DPAS, M1-DPAN, and 6PD-DPAN were accurately delivered to the corneal surface of infected mouse models using a sterile micropipette. Each mouse received approximately 10 μL of the aforementioned AIE solution (DO-DPAS, M1-DPAN, or 6PD-DPAN solution), ensuring coverage of the entire corneal area. To ensure uniform distribution and effective binding of the material, the mouse's eyelids were gently closed for several seconds to promote penetration and full contact of the AIE material (DO-DPAS, M1-DPAN, or 6PD-DPAN) with the corneal surface. After incubation for 10 minutes, the device was covered with a moisturizing gauze over the mouse's head to prevent dry eyes and ensure the material's activity was not affected. During incubation, mice were kept in a stable state to avoid excessive eye movement that could dry out the uniform distribution of AIE. After incubation, the mice's eyes were aseptically removed and immediately rinsed in cold saline to remove any unbound AIE material. The corneal tissue was then carefully dissected using scissors to separate the material from the eye, avoiding damage and interference with fluorescence signals. The dissected corneal tissue was immediately fixed onto a microscope slide, and fluorescence signals were observed and recorded using a confocal microscope. Under the confocal microscope, the AIE material was excited at an appropriate excitation wavelength (405 nm), and the fluorescence emission wavelengths of DO-DPAS, M1-DPAN, and 6PD-DPAN were detected in different pathogen infection models. The fluorescence signal intensity and distribution of each sample were recorded by imaging. By comparing the fluorescence signal intensity and distribution characteristics of Gram-positive bacterial keratitis, Gram-negative bacterial keratitis, and fungal keratitis, the specificity of the AIE material could be evaluated.

[0119] Figure 7 Representative confocal laser fluorescence images (λ) of corneal tissue from control, Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa groups were obtained 10 min after treatment with 10 μM M1-DPAN. ex =405nm, λ em =525nm), n=10, where, in top-to-bottom order, the first row is the fused image, the second row is the bright-field image, and the third row is the fluorescence image; from Figure 7 It can be seen that after treating the control group, Candida albicans, Staphylococcus aureus and Pseudomonas aeruginosa mice with 10 μM M1-DPAN for 10 min, the corneal tissue of the control group mice was not excited to fluoresce, while the corneal tissue of the Staphylococcus aureus-infected mice was specifically excited to fluoresce, indicating the specific diagnostic potential of M1-DPAN for Gram-positive bacterial keratitis.

[0120] Figure 8Representative confocal laser fluorescence images (λ) of corneal tissue from mice treated with 10 μM DO-DPAS for 10 min after infection with Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa were obtained. ex =405nm, λ em =525nm), n=10, where, in top-to-bottom order, the first row is the fused image, the second row is the bright-field image, and the third row is the fluorescence image; from Figure 8 It can be seen that after treating the control group, mice infected with Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa with 10 μM DO-DPAS for 10 min, the corneal tissue of the control group mice was not excited to fluoresce, while the corneal tissue of the Candida albicans-infected mice was specifically excited to fluoresce, indicating the specific diagnostic potential of DO-DPAS for fungal keratitis.

[0121] Figure 9 Representative confocal laser fluorescence images (λ) of corneal tissue from control, Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa groups were obtained 10 min after treatment with 10 μM 6PD-DPAN. ex =405nm, λ em =525nm), n=10, where, in top-to-bottom order, the first row is the fused image, the second row is the bright-field image, and the third row is the fluorescence image; from Figure 9 It can be seen that after treating the control group and mice infected with Candida albicans, Staphylococcus aureus and Pseudomonas aeruginosa with 10 μM 6PD-DPAN for 10 min, the corneal tissue of the control group mice was not excited to fluoresce, while the corneal tissue of mice infected with Candida albicans, Staphylococcus aureus and Pseudomonas aeruginosa were all excited to fluoresce.

[0122] In a slit lamp (excitation source λ) ex Rapid diagnosis of infectious keratitis using DO-DPAS, M1-DPAN, and 6PD-DPAN under 395nm irradiation:

[0123] Experimental methods: 6-8 week old C57BL / 6 mice were randomly divided into normal group and model group. The above corneal scratching method was used to construct a mouse model of Candida albicans infection.

[0124] Mice were anesthetized with intraperitoneal injection of afodin (tribromoethanol, 20 μL / g). 10 μL of a 10 μM DO-DPAS solution was then dropped onto the corneal surface of both normal mice and mice infected with Candida albicans. The eyelids were gently closed for a few seconds, followed by incubation for 10 minutes. After incubation, the conjunctival sac was flushed with 0.9 wt% sodium chloride solution to remove excess DO-DPAS solution. The eyelids were then fully opened to expose the eyeball. The illumination band was adjusted to its maximum width and diffused illumination, focused onto the mouse eyeball, and placed under white light and primary ultraviolet light (slit lamp excitation source λ). ex =395nm) Photo recording;

[0125] Figure 10 To determine the optimal treatment method, 10 μL of a 10 μM DO-DPAS solution was added to the corneal surface of normal mice and mice infected with Candida albicans, respectively. After 10 min, the mice in each group were subjected to excitation from a slit lamp under an excitation source λ. ex Representative anterior segment photographic images (n=3) under 395nm illumination, from Figure 10 It was found that when 10 μL of 10 μM DO-DPAS solution was added to the corneal surface of normal mice and Candida albicans-infected mice, respectively, after 10 min, the control group mice did not exhibit fluorescence, while the corneas of Candida albicans-infected mice exhibited green fluorescence.

[0126] Example

[0127] 6-8 week old C57BL / 6 mice were randomly divided into a normal group and a model group. The Staphylococcus aureus infection mouse model was constructed using the corneal scratching method described above.

[0128] Mice were anesthetized with intraperitoneal injection of afodin (tribromoethanol, 20 μL / g). 10 μL of a 10 μM DO-DPAS solution was then dropped onto the corneal surface of both normal mice and mice infected with Candida albicans. The eyelids were gently closed for a few seconds, followed by incubation for 10 minutes. After incubation, the conjunctival sac was flushed with 0.9 wt% sodium chloride solution to remove excess DO-DPAS solution. The eyelids were then fully opened to expose the eyeball. The illumination band was adjusted to its maximum width and diffused illumination, focused onto the mouse eyeball, and placed under white light and primary ultraviolet light (slit lamp excitation source λ). ex=395nm) Photograph recording; (Result: Staphylococcus aureus-infected mice did not emit light). Next, 10μL of 10μM M1-DPAN solution was added to the corneal surface of normal mice and Candida albicans-infected mice, respectively. After gently closing the mouse's eyelids for a few seconds, the mice were incubated for 10 minutes. After incubation, the conjunctival sac was rinsed with 0.9wt% sodium chloride solution to remove excess M1-DPAN solution. The mouse's eyelids were then fully opened to expose the eyeball. The irradiation band was adjusted to the maximum width of the diffused light and focused on the mouse's eyeball. The irradiation was performed under white light and first-level ultraviolet light (slit lamp excitation source λ). ex =395nm) Photo recording; Recording results: luminescence in mice infected with Staphylococcus aureus.

[0129] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A rapid diagnostic system for keratitis, comprising a reagent kit and an excitation light source, characterized in that, The kit consists of three individually packaged reagent tubes; The three reagent tubes, arranged in the order of medication administration, include a first reagent tube, a second reagent tube, and a third reagent tube set sequentially. The reagent in the third reagent tube includes 6PD-DPAN; The reagents in the first reagent tube and the second reagent tube are independently either DO-DPAS or M1-DPAN, and the reagents in the first reagent tube and the second reagent tube are different. The structure of the M1-DPAN is shown in Formula 1: The structure of the DO-DPAS is shown in Equation 2: The structure of the 6PD-DPAN is shown in Equation 3: The excitation source λ ex =200~420nm.

2. The detection system as described in claim 1, characterized in that, The excitation source λ ex =391~395nm.

3. The detection system as described in claim 2, characterized in that, The excitation source is provided by a slit-lamp microscope or a live confocal microscope.

4. The detection system as described in claim 1, characterized in that, The reagent in the first reagent tube is DO-DPAS.

5. The detection system as described in claim 4, characterized in that, The DO-DPAS exists in the form of a DO-DPAS solution; The concentration of DO-DPAS in the DO-DPAS solution is 5–20 μmol / L.

6. The detection system as described in claim 5, characterized in that, The amount of reagent added in the first reagent tube is 5–20 μL.

7. The detection system as described in claim 1 or 3, characterized in that, The reagent in the second reagent tube is M1-DPAN.

8. The detection system as described in claim 7, characterized in that, The M1-DPAN exists in the form of an M1-DPAN solution; The concentration of M1-DPAN in the M1-DPAN solution is 5–20 μmol / L.

9. The detection system as described in claim 8, characterized in that, The amount of reagent added in the second reagent tube is 5–20 μL.

10. The detection system as described in claim 1, characterized in that, The 6PD-DPAN exists in the form of a 6PD-DPAN solution; The concentration of 6PD-DPAN in the 6PD-DPAN solution is 5–20 μmol / L; The amount of reagent added in the third reagent tube is 5–20 μL.

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