Fluorescent sensing composition, fluorescent sensing film and use thereof

Abstract

The present application relates to the field of medical detection, in particular to a fluorescence sensing composition, a fluorescence sensing film and application thereof. Through screening a variety of fluorescence indicators and combinations thereof, a simple and cost-effective indicator formula is finally obtained. Based on the formula, through optimizing the preparation process, a high-efficiency fluorescence sensing film resistant to moist heat sterilization is developed. After inoculating gram-negative bacilli, gram-positive cocci and yeast-type fungi representative quality control strains into the microorganism culture bottle prepared based on the film, through fluorescence curve determination of the full-automatic microorganism culture system, gram staining, microorganism mass spectrum identification and other experiments, it is proved that the fluorescence sensing film has excellent detection performance. The fluorescence sensing film proposed in the present application can be applied to the detection of clinical microorganisms, which can provide technical support for the diagnosis of bloodstream infections, and has important significance for curbing the abuse of antibiotics and improving the prognosis of patients with bloodstream infections.

Description

Technical Field

[0001] This invention relates to the field of medical testing, and particularly to fluorescence sensing compositions, fluorescence sensing membranes, and their applications. Background Technology

[0002] Blood culture is the simplest, most accurate, and commonly used method for diagnosing bloodstream infections, and it is also the gold standard for identifying pathogenic microorganisms. Developing fluorescent sensing membranes with readily available raw materials, simple processes, and stable performance is crucial to alleviating or even solving this dilemma.

[0003] The current global blood culture market is approximately 50 billion yuan, with my country accounting for about 10 billion yuan. According to expert consensus, sampling protocols have been upgraded from the traditional "single-sided single-set" (collecting one aerobic and one anaerobic culture bottle from one limb) to "double-sided double-set" (collecting one aerobic and one anaerobic culture bottle from each limb) to improve detection sensitivity. While this change has increased the detection rate, it has also increased testing costs. Against this backdrop, the industry urgently needs to develop more cost-effective solutions, and optimizing the process performance and manufacturing cost of the detection sensor membrane at the bottom of the culture bottle has become one of the key technological paths to improve detection efficiency and reduce overall costs.

[0004] Blood culture bottles are clinical microbiology testing products specifically designed to detect the presence of pathogenic microorganisms (such as bacteria and fungi) in blood. Blood is drawn from a patient and injected into a pre-filled culture bottle. The bottle is then placed in a blood culture analyzer for incubation. Pathogens from the patient's bloodstream will grow in the bottle. This growth releases carbon dioxide. When the carbon dioxide level reaches a threshold, the pH at the bottom of the bottle changes, causing a color or fluorescence change in a pre-embedded pH probe to indicate the presence of pathogens in the patient's blood. The blood culture analyzer will automatically alarm when pathogens are detected in the bottle. At this time, the laboratory personnel need to record the alarm time and extract the culture for Gram staining. The alarm time and the Gram staining results reflect the initial concentration and type of pathogens. The initial concentration reflects the severity of the patient's bloodstream infection, and the pathogen type can provide guidance for clinical medication.

[0005] Optimizing the manufacturing process and performance of key components (such as sensing membranes) is an important way to promote the technological upgrading of domestic products and reduce the cost of clinical use.

[0006] In blood culture testing, the sensor membrane is a core functional component of the blood culture testing system, and its performance directly affects the detection sensitivity and reliability of the blood culture bottle. Fluorescence detection uses specific excitation / emission wavelengths, resulting in higher detection sensitivity and effectively avoiding interference from other colors in the blood sample, thus reducing the false positive rate. The disclosed sensor membrane technology involves cross-linking and curing the indicator with the membrane material. In existing technologies, the false positive and false negative rates are as high as 5% or more, with more than 3% due to quality issues with the blood culture bottle's sensor membrane, delaying effective treatment for patients. Furthermore, the currently disclosed technology is cumbersome to operate, and the uneven distribution of the indicator is also one of the reasons for the insensitive color development. Summary of the Invention

[0007] In view of this, the present invention provides a fluorescent sensing composition, a fluorescent sensing membrane, and their applications. The present invention improves upon the problems of complex preparation processes, less than ideal fluorescence response, and high costs associated with existing sensing membranes. Experiments show that the obtained fluorescent sensing membrane remains stable under moist heat sterilization conditions, is compatible with commonly used imported blood culture instruments in clinical practice, has complete detection functions, and simultaneously reduces production costs. This can provide strong support for the localization of blood culture in my country, improve the efficiency of clinical diagnosis, and has significant social and economic benefits.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a fluorescence sensing composition comprising, by weight, the following components:

[0010] Silicone gel A, 1000-3000 parts;

[0011] Silicone gel B100-350 parts;

[0012] Sulfation inhibitor 0-20 parts;

[0013] Sodium hydroxide 0.05–1 part;

[0014] 10-100 parts of dispersant;

[0015] 0.1 to 10 parts of light-blocking agent;

[0016] Fluorescent indicator: 0.001–80.009 parts;

[0017] The silicone gel A comprises polydimethylsiloxane containing functional groups;

[0018] The silicone gel B comprises polydimethylsiloxane containing functional groups and platinum;

[0019] The functional group includes one or more of hydroxyl, carboxyl, aldehyde or amino groups;

[0020] The fluorescent indicator comprises two or more of the following: trisodium 8-hydroxypyrene-1,3,6-trisulfonate, bromocresol purple, rhodamine B, rhodamine 6G, and rhodamine 590 chloride.

[0021] In some specific embodiments of the present invention, in the silicone gel B, the mass ratio of the functionalized polydimethylsiloxane to the platinum is (8-9.9):(0.1-2).

[0022] In some specific embodiments of the present invention, the sulfurization inhibitor comprises ethynylcyclohexanol and polydimethylsiloxane, wherein the mass ratio of ethynylcyclohexanol to polydimethylsiloxane is (0.01-0.05):(9.95-9.99).

[0023] In some specific embodiments of the present invention, the light-blocking agent includes polydimethylsiloxane and pigment;

[0024] The pigment includes one or more of titanium dioxide, zinc white (ZnO), or calcium carbonate (CaCO3);

[0025] The mass ratio of the dimethylsiloxane to the pigment is (0.1-2): (8-9.9).

[0026] In some specific embodiments of the present invention, the dispersant includes glycerol.

[0027] In some specific embodiments of the present invention, the fluorescent sensing composition further includes a primer and / or a solvent;

[0028] The primer comprises C7-C9 isoalkanes and titanium tetrabutoxide; the fluorescent sensing composition comprises 0 to 90 parts of the primer by weight.

[0029] The fluorescent sensing composition comprises 50 to 500 parts by weight of the solvent; the solvent includes double-distilled water.

[0030] In some specific embodiments of the present invention, the pH value of the fluorescent sensing composition includes 7.0 to 10.0.

[0031] In some specific embodiments of the present invention, the fluorescence sensing composition comprises, by weight, the following components:

[0032] Silicone gel A2454 parts;

[0033] Silicone gel B273 parts;

[0034] Nine parts of sulfurization inhibitor;

[0035] Sodium hydroxide 0.5 parts;

[0036] 82 parts glycerin;

[0037] 3 parts of sunscreen;

[0038] 0.492 parts of fluorescent indicator;

[0039] 100 portions of double-distilled water;

[0040] 27.36 parts of primer;

[0041] The fluorescent indicator includes:

[0042] 0.15 parts of trisodium 8-hydroxypyrene-1,3,6-trisulfonic acid;

[0043] 0.009 parts of bromocresol purple;

[0044] Rhodamine B 0.009 copies;

[0045] Rhodamine 6G 0.009 copies;

[0046] Rhodamine 590 chloride 0.315 parts;

[0047] Or the fluorescence sensing composition may include:

[0048] 1000 parts of silicone gel A;

[0049] 100 parts of silicone gel B;

[0050] 0 parts of sulfurization inhibitor;

[0051] 0.05 parts of sodium hydroxide;

[0052] 10 parts glycerin;

[0053] 0.1 parts of light-blocking agent;

[0054] 0.005 parts of fluorescent indicator;

[0055] 100 portions of double-distilled water;

[0056] 0 parts primer;

[0057] The fluorescent indicator includes:

[0058] 0.001 parts of trisodium 8-hydroxypyrene-1,3,6-trisulfonic acid;

[0059] 0.001 parts of bromocresol purple;

[0060] Rhodamine B 0.001 copies;

[0061] Rhodamine 6G 0.001 copies;

[0062] Rhodamine 590 chloride 0.001 parts;

[0063] Or the fluorescence sensing composition may include:

[0064] 3000 parts of silicone gel A;

[0065] 350 parts of silicone gel B;

[0066] 20 parts of sulfurization inhibitor;

[0067] 1 part sodium hydroxide;

[0068] 100 parts glycerin;

[0069] 10 parts of sunblock agent;

[0070] 80.009 parts of fluorescent indicator;

[0071] 100 portions of double-distilled water;

[0072] 90 parts of primer;

[0073] The fluorescent indicator includes:

[0074] 20 parts of trisodium 8-hydroxypyrene-1,3,6-trisulfonate;

[0075] 20 parts of bromocresol purple;

[0076] Rhodamine B 0.009 copies;

[0077] Rhodamine 6G 20 copies;

[0078] Rhodamine 590 chloride 20 parts.

[0079] Secondly, the present invention also provides the use of the fluorescent sensing composition in the preparation of fluorescent sensing films.

[0080] Thirdly, the present invention also provides the application of the fluorescent sensing composition in the preparation of microbial detection products or bloodstream infection diagnostic products.

[0081] Fourthly, the present invention also provides a fluorescent sensing film comprising the fluorescent sensing composition.

[0082] Fifthly, the present invention also provides a method for preparing the fluorescent sensing membrane, wherein the fluorescent sensing composition is mixed and cured to obtain the fluorescent sensing membrane.

[0083] In some specific embodiments of the present invention, the curing temperature is 70°C and the curing time is 150 min.

[0084] In a sixth aspect, the present invention also provides a fluorescent sensing film prepared by the aforementioned preparation method.

[0085] In a seventh aspect, the present invention also provides the application of the fluorescent sensing membrane in the preparation of a blood culture detection device or a blood culture detection system.

[0086] In some specific embodiments of the present invention, the blood culture detection device includes a blood culture bottle.

[0087] Eighthly, the present invention also provides a blood culture detection device, including the aforementioned fluorescent sensing membrane.

[0088] In some specific embodiments of the present invention, the blood culture detection device includes a blood culture bottle.

[0089] In a ninth aspect, the present invention also provides a blood culture detection system, including the blood culture detection device.

[0090] In a tenth aspect, the present invention also provides the use of any one of the following in the preparation of microbial detection products or bloodstream infection diagnostic products;

[0091] (I) The fluorescent sensing film;

[0092] (II) The blood culture detection device;

[0093] (III) The blood culture detection system.

[0094] In an eleventh aspect, the present invention also provides microbial detection products or bloodstream infection diagnostic products, including any one of the following:

[0095] (I) The fluorescence sensing composition;

[0096] (II) The fluorescent sensing film;

[0097] (III) The blood culture detection device;

[0098] (IV) The blood culture detection system.

[0099] Bloodstream infections pose a serious threat to patients' lives. Early diagnosis (every hour earlier) can significantly improve survival rates and curb antibiotic overuse. Blood culture, as the gold standard for diagnosing bloodstream infections, is limited in its diagnostic performance, preparation process, and cost by the sensing membrane at the bottom of the blood culture bottle. This study screened various fluorescent indicators and their combinations, ultimately obtaining a simple and cost-effective indicator formulation. Based on this formulation, a highly efficient fluorescent sensing membrane resistant to moist heat sterilization was developed through optimized preparation processes. Experiments including fluorescence curve determination, Gram staining, and microbial mass spectrometry identification using representative control strains of three Gram-negative bacilli, Gram-positive cocci, and yeast-type fungi confirmed that the fluorescent sensing membrane is not only compatible with mainstream imported blood culture instruments but also exhibits excellent detection performance. Applying the fluorescent sensing membrane proposed in this study to the detection of clinical microorganisms can provide technical support for the diagnosis of bloodstream infections and is of great significance for curbing antibiotic overuse and improving the prognosis of patients with bloodstream infections. Attached Figure Description

[0100] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0101] Figure 1 This document describes the fabrication process and development flow of the fluorescent sensing film. Note: A primer is required when using ELASTOSIL® (Wacker) and Elaplus® silicone gel, but not when using Liveo® (DuPont).

[0102] Figure 2 The diagram shows a fluorescent blood culture bottle and the fluorescent sensing membrane before and after a positive result is reported. Note: A is a schematic diagram of a blood culture bottle; B is an actual picture of a blood culture bottle; C is the fluorescent sensing membrane of the blood culture bottle before it is loaded into the instrument; D is the fluorescent sensing membrane of the blood culture bottle after a positive result is reported.

[0103] Figure 3 Comparative Example 1 shows the fluorescence sensing membrane and fluorescence curve; Note: A: Fluorescence sensing membrane before blood culture bottle is tested; B: Fluorescence sensing membrane after blood culture bottle reports positive; C: Fluorescence curve of blood culture bottle after data extraction and redrawing.

[0104] Figure 4 Comparative Example 2 shows the fluorescence sensing membrane and fluorescence curve; Note: A: Fluorescence sensing membrane before blood culture bottle is tested; B: Fluorescence sensing membrane after blood culture bottle reports positive; C: Fluorescence curve of blood culture bottle after data extraction and redrawing.

[0105] Figure 5 Comparative Example 3 shows the fluorescence sensing membrane and fluorescence curve; Note: A: Fluorescence sensing membrane before blood culture bottle is tested; B: Fluorescence sensing membrane after blood culture bottle reports positive; C: Fluorescence curve of blood culture bottle after data extraction and redrawing.

[0106] Figure 6Comparative Example 4 shows the sensing membrane and fluorescence curve; Note: A: Fluorescence sensing membrane before blood culture bottle is tested; B: Fluorescence sensing membrane after blood culture bottle reports positive; C: Fluorescence curve of blood culture bottle after data extraction and redrawing.

[0107] Figure 7 Comparative Example 5 shows the sensing membrane and fluorescence curve; Note: A: Fluorescence sensing membrane of blood culture bottle before testing; B: Fluorescence curve of blood culture bottle after data extraction and redrawing.

[0108] Figure 8 Example 1 shows the fluorescence curve and strain identification of Escherichia coli; Note: A: Escherichia coli fluorescence curve, redrawn after data extraction; B: Reported positive blood culture bottle plated on Columbia blood agar medium and incubated for 24 hours; C: Gram staining results of strains from reported positive blood culture bottles; D: Microbial mass spectrometry identification results of strains from reported positive blood culture bottles, with scores representing confidence scores;

[0109] Figure 9 Example 1 shows the fluorescence curve and strain identification of Staphylococcus aureus; Note: A: Staphylococcus aureus fluorescence curve, redrawn after data extraction; B: Reported positive blood culture bottle plated on Columbia blood agar medium and incubated for 24 hours; C: Gram staining results of strains from reported positive blood culture bottles; D: Microbial mass spectrometry identification results of strains from reported positive blood culture bottles, with scores representing confidence scores;

[0110] Figure 10 Example 1 shows the fluorescence curve and strain identification of Candida albicans; Note: A: Fluorescence curve of Candida albicans, redrawn after data extraction; B: Plates of positive blood culture bottles on Sabouraud agar medium after 24 hours of incubation; C: Gram staining results of strains from positive blood culture bottles; D: Microbial mass spectrometry identification results of strains from positive blood culture bottles, with scores representing confidence scores;

[0111] Figure 11 Example 2 shows the fluorescence curve of Escherichia coli; Note: This is a redrawing after data extraction.

[0112] Figure 12 The fluorescence curve of Escherichia coli in Example 3 is shown; Note: This is a redrawing after data extraction. Detailed Implementation

[0113] This invention discloses a fluorescent sensing composition, a fluorescent sensing film, and their applications. Those skilled in the art can refer to the content herein and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0114] Terminology Explanation:

[0115] Bloodstream infection: Septicemia and bacteremia are collectively referred to as bloodstream infection. Septicemia is a bloodstream infection caused by various pathogenic microorganisms (bacteria or fungi) and toxins entering the bloodstream; if bacteria only enter the bloodstream temporarily without clinically obvious toxemia symptoms (such as vascular-related infections), it is called bacteremia.

[0116] Blood culture: Blood culture is a culture method in which fresh, isolated blood samples are inoculated onto a nutrient culture medium and, under certain temperature and humidity conditions, bacteria with high nutritional requirements are allowed to grow and multiply, and then identified to determine the pathogens.

[0117] This invention innovatively designs various sensing membrane preparation schemes, and ultimately develops a method for preparing a high-efficiency fluorescent sensing membrane. Figure 1 The formulation and functions of each component of the fluorescent sensing membrane are shown in Table 1. A photograph and schematic diagram of the fluorescent blood culture bottle are provided. Figure 2 B and Figure 2 A, the color of the fluorescent sensing film will change from purple to purplish-brown after a positive reaction. Figure 2 C and Figure 2 (D) This method can be used to visually identify whether a fluorescent blood culture bottle has been used. The self-made blood culture bottle prepared based on this method was validated by experiments with three standard strains, showing good detection curves for all three strains. Furthermore, the preparation process is simpler and less expensive.

[0118] Table 1. Formulation and Functions of Fluorescent Sensing Membrane

[0119]

[0120] Table 2 Sources of raw materials for fluorescent sensing membranes

[0121]

[0122]

[0123] This invention provides a fluorescent indicator formulation containing multiple components: silicone gel A (1000-3000 mg), silicone gel B (100-350 mg), sulfurization inhibitor (0-20 mg), sodium hydroxide (0.05-1 mg), glycerin (10-100 mg), opacifier (0.1-10 mg), primer (0-90 mg), double-distilled water (50-500 mg), and fluorescent indicator: one or more of the following: 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt, bromocresol purple, rhodamine B, rhodamine 6G, and rhodamine 590 chloride (0.001-20 mg).

[0124] This invention also provides a simple process for preparing a fluorescence sensing membrane. Various raw materials are stirred evenly and added to the bottom of a blood culture bottle coated with a primer; after drying, a fluorescence sensing membrane is obtained.

[0125] Based on the above research, this invention also provides the application of a fluorescent sensing membrane in blood culture bottles. The fluorescent sensing membrane not only has a simple preparation process and controllable cost, but also exhibits good growth of three quality control strains in blood culture bottles, with fluorescence curves meeting requirements and timely reporting of positive results.

[0126] This invention, based on conventional fluorescent reagents and employing a relatively simple preparation process, improves upon the problems of complex preparation processes, less than ideal fluorescence response, and high costs associated with existing sensor membranes. Experiments show that the resulting fluorescent sensor membrane remains stable under moist heat sterilization conditions, is compatible with commonly used imported blood culture instruments in clinical settings, offers comprehensive detection functions, and simultaneously reduces production costs. This invention can provide strong support for the localization of blood culture technology in my country, improve the efficiency of clinical diagnosis, and has significant social and economic benefits.

[0127] This invention utilizes conventional reagents and a relatively simple preparation process, which to some extent improves upon the problems of complex preparation processes, less than ideal signal response, and high costs associated with existing sensor membranes. Experiments show that the obtained fluorescent sensor membrane can withstand moist heat sterilization, and when representative quality control strains are inoculated into culture flasks equipped with the fluorescent sensor membrane, they can be successfully detected and reported as positive. The development of this fluorescent sensor membrane can provide strong support for the localization of blood culture in my country, thereby improving the efficiency of clinical diagnosis and yielding significant social and economic benefits.

[0128] The fluorescent sensing composition, fluorescent sensing membrane, and raw materials and reagents used in the application of the present invention are all commercially available.

[0129] The present invention will be further illustrated below with reference to the embodiments:

[0130] Example 1

[0131] ①Preparation of fluorescent sensing film

[0132] Mix 0.315 mg of Rhodamine 590 chloride, 0.15 mg of trisodium 8-hydroxypyrene-1,3,6-trisulfonic acid, 0.009 mg of bromocresol purple, 0.009 mg of Rhodamine 6G, 0.009 mg of Rhodamine B, 2454 mg of ELASTOSIL® A glue, 273 mg of ELASTOSIL® B glue, 9 mg of vulcanization inhibitor, 82 mg of glycerol, 3 mg of opacifier, 100 mg of double-distilled water, and 0.5 mg of sodium hydroxide. Pour the mixture into the bottom of a blood culture bottle containing 27.36 mg of primer and cure at 20°C for 25 hours.

[0133] ② Preparation of working bacterial suspension

[0134] Standard strains of Escherichia coli and Staphylococcus aureus were inoculated onto Columbia blood agar medium, and Candida albicans was inoculated onto YPD agar medium. After incubation at 35°C for 18 hours, the culture was diluted with 0.9% sterile sodium chloride solution to prepare a working bacterial suspension of the required concentration.

[0135] ③ Blood culture machine

[0136] Fluorescent blood culture bottles (R1, Example 1) prepared using the sensing membrane technology of this invention were tested on a BACTECFX TOP blood culture instrument. Specifically, five bottles of each type were prepared and divided into four groups: positive group 1, positive group 2, positive group 3, and negative group 1, with five bottles in each group. Positive group 1 was inoculated with 1 mL (1000 CFU) of working Escherichia coli (ATCC25922), positive group 2 with 1 mL (1000 CFU) of working Staphylococcus aureus (ATCC25923), and positive group 3 with 1 mL (1000 CFU) of working Candida albicans (ATCC18804). If a positive result was reported after loading the instrument, it was necessary to inoculate onto the corresponding plate to confirm bacterial growth before recording it as positive; otherwise, it was recorded as a false positive. If no positive result was reported after 96 hours, the bottle was removed and observed; if there was no obvious discoloration or turbidity, it was recorded as negative. If there was obvious discoloration or turbidity, it was inoculated onto a blood plate to confirm bacterial growth and was recorded as a false negative.

[0137] ④ Gram staining and mass spectrometry identification

[0138] The culture medium from the positive blood culture bottles of group 1 was extracted and cultured on Columbia blood agar to obtain pure single colonies. Gram staining was performed according to Table 3, and the colonies were observed and photographed under an oil immersion microscope. Fresh single colonies were picked and analyzed by mass spectrometry using the Antu Bio fully automated microbial mass spectrometry detection system (Autof ms 1000). The culture medium from the positive blood culture bottles of group 2 was extracted and cultured on Columbia blood agar for 24 hours to obtain pure single colonies. Gram staining was performed according to Table 3, and the colonies were observed and photographed under an oil immersion microscope. Fresh single colonies were picked and analyzed by mass spectrometry using the Antu Bio fully automated microbial mass spectrometry detection system (Autof ms 1000). The culture medium from the positive blood culture bottles of group 3 was extracted and cultured on Sabouraud agar for 24 hours to obtain pure single colonies. Gram staining was performed according to Table 3, and the colonies were observed and photographed under an oil immersion microscope. Fresh single colonies were picked and analyzed by mass spectrometry using the Antu Bio fully automated microbial mass spectrometry detection system (Autof ms 1000).

[0139] Table 3 Gram staining

[0140]

[0141] ⑤ Experimental Results

[0142] The fluorescence curve and results of *Escherichia coli*, a Gram-negative bacterium, in a self-made blood culture bottle are as follows: Figure 8 As shown in A and Table 5, the fluorescence curves are stable and good, with positive reporting times of 7–8 hours and positive fluorescence values ​​around 0.190, indicating excellent performance. It grows well on Columbia blood agar plates. Figure 8 B), Gram staining results showed it to be a Gram-negative bacillus, consistent with the expected strain's biological morphology. Figure 8 C), the microbial mass spectrometry identification results more accurately showed that the strain in the positive blood culture bottle was Escherichia coli, and the confidence score was as high as 9.587 ( Figure 8 D) indicates that the fluorescent sensing membrane in this example is suitable for the detection of E. coli, with normal positive reporting time and good stability. As can be seen from Table 4, compared with the five comparative examples, Implementation 1, as a component of the composite indicator, reported a positive time about 1 hour faster than Comparative Example 4, which reported the fastest positive time. Moreover, the curve trend conforms to the logic of the imported fluorescent blood culture algorithm, that is, it first decreases slightly, then becomes smooth, then increases significantly, and finally becomes smooth again.

[0143] The fluorescence curve and results of Staphylococcus aureus, a Gram-positive bacterium, in a self-made blood culture flask are as follows: Figure 9 As shown in A and Table 5, its fluorescence curve is stable and good, with positive reporting times of 9–10 h and positive fluorescence values ​​around 0.217, indicating excellent performance. It grows well on Columbia blood agar plates. Figure 9 B), Gram staining results showed Gram-positive cocci arranged in grape-like clusters ( Figure 9 C), the microbial mass spectrometry identification results more accurately showed that the positive blood culture bottle contained Staphylococcus aureus, and the confidence score was as high as 9.597 ( Figure 9 (D) indicates that the fluorescent sensing membrane in this example is compatible with Staphylococcus aureus, has a normal positive reporting time, and good stability.

[0144] The fluorescence curve and results of *Candida albicans*, a yeast-type fungus, in a self-made blood culture bottle are as follows: Figure 10 As shown in Table A and Table 5, its fluorescence curves are stable and good, with positive reporting times ranging from 13 to 16 hours and positive fluorescence values ​​around 0.212, indicating excellent performance. It grows well on Sabouraud agar plates. Figure 10 B), Gram staining results showed clumps of budding yeast-like fungi ( Figure 10 C), the microbial mass spectrometry identification results more accurately showed that the positive blood culture bottle contained Candida albicans, and the confidence score was as high as 9.421 ( Figure 10D) indicates that the fluorescent sensing membrane in this example is compatible with Candida albicans, with normal positive reporting time and good stability; the above results show that the fluorescent sensing membrane in this example is stable in detecting Gram-positive bacteria, Gram-negative bacteria, and yeast-type fungi, and can report positive within the normal time and with accurate detection (Table 5), and is well compatible with common imported fluorescent blood culture instruments.

[0145] Example 2

[0146] ①Preparation of fluorescent sensing film

[0147] Mix 0.001 mg of Rhodamine 590 chloride, 0.001 mg of trisodium 8-hydroxypyrene-1,3,6-trisulfonic acid, 0.001 mg of bromocresol purple, 0.001 mg of Rhodamine 6G, 0.001 mg of Rhodamine B, 1000 mg of Liveo® A glue, 100 mg of Liveo® B glue, 0 mg of vulcanization inhibitor, 10 mg of glycerol, 0.1 mg of opacifier, 100 mg of double-distilled water, and 0.05 mg of sodium hydroxide, pour the mixture into the bottom of a blood culture flask, and cure at 70°C for 150 min.

[0148] ② Preparation of working bacterial suspension

[0149] Inoculate standard strains of Escherichia coli onto Columbia blood agar medium, incubate at 35°C for 18 hours, and then dilute with 0.9% sterile sodium chloride solution to prepare a working bacterial suspension of the required concentration.

[0150] ③ Blood culture machine

[0151] The fluorescent blood culture bottles prepared using the sensing membrane technology of this invention were tested on a BACTEC FX TOP blood culture instrument. Specifically, two blood culture bottles were prepared, and 1 mL (1000 CFU) of working Escherichia coli (ATCC25922) suspension was inoculated into each bottle. If a positive result was reported after loading the instrument, it was necessary to inoculate the bottle onto the corresponding agar plate to confirm bacterial growth before recording it as positive; otherwise, it was recorded as a false positive. If no positive result was reported after 96 hours, the bottle was removed and observed; if there was no obvious discoloration or turbidity, it was recorded as negative. If there was obvious discoloration or turbidity, it was inoculated onto a blood agar plate to confirm bacterial growth and was recorded as a false negative.

[0152] ④ Experimental Results

[0153] The fluorescence curve and results of *Escherichia coli*, a Gram-negative bacterium, in self-made blood culture flasks are shown below. Figure 11 The fluorescence curve was stable, and a positive result was reported after 9 hours and 18 minutes, which was relatively late.

[0154] Example 3

[0155] ①Preparation of fluorescent sensing film

[0156] Mix 20 mg of Rhodamine 590 chloride, 20 mg of trisodium 8-hydroxypyrene-1,3,6-trisulfonic acid, 20 mg of bromocresol purple, 20 mg of Rhodamine 6G, 0.009 mg of Rhodamine B, 3000 mg of ELASTOSIL® A glue, 350 mg of ELASTOSIL® B glue, 20 mg of vulcanization inhibitor, 100 mg of glycerin, 10 mg of opacifier, 100 mg of double-distilled water, and 1 mg of sodium hydroxide. Pour the mixture into the bottom of a blood culture bottle containing 90 mg of primer and cure at 110°C for 9 minutes.

[0157] ② Preparation of working bacterial suspension

[0158] Inoculate standard strains of Escherichia coli onto Columbia blood agar medium, incubate at 35°C for 18 hours, and then dilute with 0.9% sterile sodium chloride solution to prepare a working bacterial suspension of the required concentration.

[0159] ③ Blood culture machine

[0160] The fluorescent blood culture bottles prepared using the sensing membrane technology of this invention were tested on a BACTEC FX TOP blood culture instrument. Specifically, two blood culture bottles were prepared, and 1 mL (1000 CFU) of working Escherichia coli (ATCC25922) suspension was inoculated into each bottle. If a positive result was reported after loading the instrument, it was necessary to inoculate the bottle onto the corresponding agar plate to confirm bacterial growth before recording it as positive; otherwise, it was recorded as a false positive. If no positive result was reported after 96 hours, the bottle was removed and observed; if there was no obvious discoloration or turbidity, it was recorded as negative. If there was obvious discoloration or turbidity, it was inoculated onto a blood agar plate to confirm bacterial growth and was recorded as a false negative.

[0161] ④ Experimental Results

[0162] The fluorescence curve and results of *Escherichia coli*, a Gram-negative bacterium, in self-made blood culture flasks are shown below. Figure 12 The fluorescence curve was stable, but the downward trend was obvious in the latter part of the curve. It reported a positive result after 8 hours and 10 minutes, which was relatively late.

[0163] Comparative Example 1

[0164] ①Preparation of fluorescent sensing film

[0165] Mix 0.03 mg of trisodium 8-hydroxypyrene-1,3,6-trisulfonic acid, 2454 mg of ELASTOSIL® A glue, 273 mg of ELASTOSIL® B glue, 82 mg of glycerol, 100 mg of double-distilled water, and 0.5 mg of sodium hydroxide, and pour the mixture into the bottom of a blood culture bottle containing 27.36 mg of primer. Cure at 70°C for 150 min.

[0166] ② Preparation of working bacterial suspension

[0167] The standard strain of Escherichia coli was inoculated onto Columbia blood agar medium and incubated at 35°C for 18 hours. Then, it was diluted with 0.9% sterile sodium chloride solution to prepare a working bacterial suspension of the required concentration.

[0168] ③ Blood culture machine

[0169] The fluorescent blood culture bottles prepared using the sensing membrane technology of this invention were tested on a BACTEC FX TOP blood culture instrument. Specifically, two blood culture bottles were prepared, and 1 mL (1000 CFU) of working Escherichia coli (ATCC25922) suspension was inoculated into each bottle. If a positive result was reported after loading the instrument, it was necessary to inoculate the bottle onto the corresponding agar plate to confirm bacterial growth before recording it as positive; otherwise, it was recorded as a false positive. If no positive result was reported after 96 hours, the bottle was removed and observed; if there was no obvious discoloration or turbidity, it was recorded as negative. If there was obvious discoloration or turbidity, it was inoculated onto a blood agar plate to confirm bacterial growth and was recorded as a false negative.

[0170] ④ Experimental Results

[0171] The fluorescence curves and results of Escherichia coli in homemade blood culture flasks are as follows: Figure 3 As shown, the fluorescence curve did not show a significant upward trend in the first 7 hours, but reported a positive result after 11 hours, which was late.

[0172] Comparative Example 2

[0173] ①Preparation of fluorescent sensing film

[0174] Mix 0.15 mg of Rhodamine 6g, 2454 mg of ELASTOSIL® A glue, 273 mg of ELASTOSIL® B glue, 82 mg of glycerin, 100 mg of double-distilled water, and 0.5 mg of sodium hydroxide, and pour the mixture into the bottom of a blood culture bottle with a 27.36 μm primer. Cure at 70°C for 150 min.

[0175] ② Preparation of working bacterial suspension

[0176] The standard strain of Escherichia coli was inoculated onto Columbia blood agar medium and incubated at 35°C for 18 hours. Then, it was diluted with 0.9% sterile sodium chloride solution to prepare a working bacterial suspension of the required concentration.

[0177] ③ Blood culture machine

[0178] The fluorescent blood culture bottles prepared using the sensing membrane technology of this invention were tested on a BACTEC FX TOP blood culture instrument. Specifically, two blood culture bottles were prepared, and 1 mL (1000 CFU) of working Escherichia coli (ATCC25922) suspension was inoculated into each bottle. If a positive result was reported after loading the instrument, it was necessary to inoculate the bottle onto the corresponding agar plate to confirm bacterial growth before recording it as positive; otherwise, it was recorded as a false positive. If no positive result was reported after 96 hours, the bottle was removed and observed; if there was no obvious discoloration or turbidity, it was recorded as negative. If there was obvious discoloration or turbidity, it was inoculated onto a blood agar plate to confirm bacterial growth and was recorded as a false negative.

[0179] ④ Experimental Results

[0180] The fluorescence curve and results of *Escherichia coli*, a Gram-negative bacterium, in self-made blood culture flasks are shown below. Figure 4 The fluorescence curve was unstable, with a clear downward trend in the latter part of the curve. It reported a positive result at 9 hours and 28 minutes, which was late.

[0181] Comparative Example 3

[0182] ①Preparation of fluorescent sensing film

[0183] Mix 0.15 mg of Rhodamine B, 2454 mg of ELASTOSIL® A glue, 273 mg of ELASTOSIL® B glue, 82 mg of glycerin, 100 mg of double-distilled water, and 0.5 mg of sodium hydroxide, and pour the mixture into the bottom of a blood culture bottle containing 27.36 mg of primer. Cure at 70°C for 150 min.

[0184] ② Preparation of working bacterial suspension

[0185] The standard strain of Escherichia coli was inoculated onto Columbia blood agar medium and incubated at 35°C for 18 hours. Then, it was diluted with 0.9% sterile sodium chloride solution to prepare a working bacterial suspension of the required concentration.

[0186] ③ Blood culture machine

[0187] The fluorescent blood culture bottles prepared using the sensing membrane technology of this invention were tested on a BACTEC FX TOP blood culture instrument. Specifically, two blood culture bottles were prepared, and 1 mL (1000 CFU) of working Escherichia coli (ATCC25922) suspension was inoculated into each bottle. If a positive result was reported after loading the instrument, it was necessary to inoculate the bottle onto the corresponding agar plate to confirm bacterial growth before recording it as positive; otherwise, it was recorded as a false positive. If no positive result was reported after 96 hours, the bottle was removed and observed; if there was no obvious discoloration or turbidity, it was recorded as negative. If there was obvious discoloration or turbidity, it was inoculated onto a blood agar plate to confirm bacterial growth and was recorded as a false negative.

[0188] ④ Experimental Results

[0189] The fluorescence curves and results of Escherichia coli in homemade blood culture flasks are as follows: Figure 5 As shown, the fluorescence curve is unstable, with a significant downward trend in the latter part of the curve, and the positive result is reported late, at 10 hours and 24 minutes.

[0190] Comparative Example 4

[0191] ①Preparation of fluorescent sensing film

[0192] Mix 0.006 mg of Rhodamine 590 chloride, 2454 mg of ELASTOSIL® A glue, 273 mg of ELASTOSIL® B glue, 9 mg of vulcanization inhibitor, 82 mg of glycerol, 3 mg of opacifier, 100 mg of double-distilled water, and 0.5 mg of sodium hydroxide, and pour the mixture into the bottom of a blood culture bottle containing 27.36 mg of primer. Cur at 70°C for 150 min.

[0193] ② Preparation of working bacterial suspension

[0194] The standard strain of Escherichia coli was inoculated onto Columbia blood agar medium and incubated at 35°C for 18 hours. Then, it was diluted with 0.9% sterile sodium chloride solution to prepare a working bacterial suspension of the required concentration.

[0195] ③ Blood culture machine

[0196] The fluorescent blood culture bottles prepared using the sensing membrane technology of this invention were tested on a BACTEC FX TOP blood culture instrument. Specifically, two blood culture bottles were prepared and divided into a positive group and a negative group. The positive group was inoculated with 1 mL (1000 CFU) of working Escherichia coli (ATCC25922) suspension, while the negative group was not inoculated. If a positive result was reported after loading the instrument, it was necessary to inoculate the bottle onto the corresponding agar plate to confirm bacterial growth before it could be recorded as positive; otherwise, it was recorded as a false positive. If no positive result was reported after 96 hours, the bottle was removed and observed; if there was no obvious discoloration or turbidity, it was recorded as negative. If there was obvious discoloration or turbidity, it was inoculated onto a blood agar plate to confirm bacterial growth and was recorded as a false negative.

[0197] ④ Experimental Results

[0198] The fluorescence curve and results of *Escherichia coli*, a Gram-negative bacterium, in a self-made blood culture bottle are as follows: Figure 6 As shown, the fluorescence curve is unstable, with a significant downward trend in the latter part of the curve. The positive reporting time is nearly 9 hours, which is late, and the positive fluorescence value is 0.378.

[0199] Comparative Example 5

[0200] ①Preparation of fluorescent sensing film

[0201] Mix 0.144 mg of bromocresol purple, 2454 mg of ELASTOSIL® A glue, 273 mg of ELASTOSIL® B glue, 9 mg of vulcanization inhibitor, 82 mg of glycerin, 3 mg of black opacifier, 100 mg of double-distilled water, and 0.5 mg of sodium hydroxide. Pour the mixture into the bottom of a blood culture bottle containing 27.36 mg of primer and cure at 70°C for 150 min.

[0202] ② Preparation of working bacterial suspension

[0203] The standard strain of Escherichia coli was inoculated onto Columbia blood agar medium and incubated at 35°C for 18 hours. Then, it was diluted with 0.9% sterile sodium chloride solution to prepare a working bacterial suspension of the required concentration.

[0204] ③ Blood culture machine

[0205] The fluorescent blood culture bottles prepared using the sensing membrane technology of this invention were tested on a BACTEC FX TOP blood culture instrument. Specifically, two blood culture bottles were prepared and divided into a positive group and a negative group. The positive group was inoculated with 1 mL (1000 CFU) of working Escherichia coli (ATCC25922) suspension, while the negative group was not inoculated. If a positive result was reported after loading the instrument, it was necessary to inoculate the bottle onto the corresponding agar plate to confirm bacterial growth before it could be recorded as positive; otherwise, it was recorded as a false positive. If no positive result was reported after 96 hours, the bottle was removed and observed; if there was no obvious discoloration or turbidity, it was recorded as negative. If there was obvious discoloration or turbidity, it was inoculated onto a blood agar plate to confirm bacterial growth and was recorded as a false negative.

[0206] ④ Experimental Results

[0207] The fluorescence curve and results of *Escherichia coli*, a Gram-negative bacterium, in a self-made blood culture bottle are as follows: Figure 7 As shown, the fluorescence curve showed an overall downward trend, and no positive result was reported after 4 days of incubation.

[0208] Example of effect

[0209] Table 4. Summary of E. coli growth test results for five comparative examples and three specific examples.

[0210]

[0211] Table 5 Summary of test results for three representative strains of the optimal sensing membrane (Example 1)

[0212]

[0213] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle 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 fluorescent sensing composition, characterized in that, by mass, comprising: silicone gel A 4.54 parts; silicone gel B 2.73 parts; vulcanization inhibitor 9 parts; sodium hydroxide 0.5 parts; glycerol 82 parts; light shielding agent 3 parts; fluorescent indicator 0.492 parts; double distilled water 100 parts; primer 27.36 parts; the fluorescent indicator comprises: 8-hydroxypyrene-1, 3, 6-trisulfonic acid trisodium salt 0.15 parts; bromocresol purple 0.009 parts; rhodamine B 0.009 parts; rhodamine 6G 0.009 parts; rhodamine 590 chloride 0.315 parts; or the fluorescent sensing composition comprises: silicone gel A 1000 parts; silicone gel B 100 parts; vulcanization inhibitor 0 parts; sodium hydroxide 0.05 parts; glycerol 10 parts; light shielding agent 0.1 parts; fluorescent indicator 0.005 parts; double distilled water 100 parts; primer 0 parts; the fluorescent indicator comprises: 8-hydroxypyrene-1, 3, 6-trisulfonic acid trisodium salt 0.001 parts; bromocresol purple 0.001 parts; rhodamine B 0.001 parts; rhodamine 6G 0.001 parts; rhodamine 590 chloride 0.001 parts; or the fluorescent sensing composition comprises: silicone gel A 3000 parts; silicone gel B 350 parts; vulcanization inhibitor 20 parts; sodium hydroxide 1 parts; glycerol 100 parts; light shielding agent 10 parts; fluorescent indicator 80.009 parts; double distilled water 100 parts; primer 90 parts; the fluorescent indicator comprises: 8-hydroxypyrene-1, 3, 6-trisulfonic acid trisodium salt 20 parts; bromocresol purple 20 parts; rhodamine B 0.009 parts; rhodamine 6G 20 parts; rhodamine 590 chloride 20 parts.

2. Use of the fluorescent sensing composition according to claim 1 in the preparation of a fluorescent sensing film.

3. Use of the fluorescent sensing composition according to claim 1 in the preparation of a microorganism detection product or a bloodstream infection diagnosis product.

4. A fluorescent sensing film, characterized by comprising the fluorescent sensing composition according to claim 1.

5. The method for preparing the fluorescent sensing film as described in claim 4, characterized in that, mixing the fluorescent sensing composition, curing to obtain the fluorescent sensing film.

6. The method of claim 5, wherein the step of preparing is characterized by, the temperature of the curing is 20-110℃, and the time of the curing is 9 min-25 h.

7. The fluorescent sensing film prepared by the preparation method according to claim 6.

8. Use of the fluorescent sensing film according to claim 4 in the preparation of a blood culture detection device or a blood culture detection system.

9. The use according to claim 8, wherein the compound is ###0002### the blood culture detection device comprises a blood culture bottle.

10. A blood culture detection device, characterized by comprising the fluorescent sensing film according to claim 4.

11. The blood culture detection device as described in claim 10, characterized in that, comprising a blood culture bottle.

12. A blood culture detection system characterized in that, comprising the blood culture detection device according to claim 10.

13. Use of any of the following in the preparation of a microorganism detection product or a bloodstream infection diagnosis product: (I) the fluorescent sensing film according to claim 4; (II) the blood culture detection device according to claim 10; (III) the blood culture detection system according to claim 12.

14. A product for the detection of microorganisms or a product for the diagnosis of blood stream infections, characterized in that, comprising any of the following: (I) the fluorescent sensing composition according to claim 1; (II) the fluorescent sensing film according to claim 4; (III) the blood culture detection device according to claim 10; (IV) the blood culture detection system according to claim 12.

Images