Fluorescent sensing composition, fluorescent sensing film and application thereof

Abstract

The invention relates to the field of medical detection, in particular to a fluorescent sensing composition, a fluorescent sensing film and application thereof. According to the invention, by screening a plurality of fluorescent indicators and combinations thereof, an indicator formula with simple components and low cost is finally obtained. Based on the formula, by optimizing the preparation process, the high-efficiency fluorescent sensing film resistant to damp-heat sterilization is developed. After a microorganism culture bottle prepared on the basis of the membrane is inoculated with Gram-negative bacilli, positive cocci and yeast fungi representative quality control strains, experiments of full-automatic microorganism culture system fluorescence curve determination, Gram staining, microorganism mass spectrum identification and the like prove that the fluorescence sensing membrane is excellent in detection performance. The fluorescent sensing film provided by the research is applied to clinical microorganism detection, can provide technical support for blood flow infection diagnosis, and is of great significance in suppressing antibiotic abuse and improving prognosis of blood flow infection patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical detection, in particular to a fluorescent sensing composition, a fluorescent sensing film and application thereof. BACKGROUND

[0002] Blood culture is the simplest, most accurate and commonly used method for diagnosing bloodstream infections, and is the gold standard for identifying pathogenic microorganisms. Developing a fluorescent sensing film with readily available raw materials, simple process and stable performance is crucial to alleviate or even solve this dilemma.

[0003] The current global blood culture market size is about 50 billion yuan, of which the Chinese market accounts for about 10 billion yuan. According to expert consensus, the sampling scheme has been upgraded from the traditional "single-limb single-set" (one bottle of aerobic and one bottle of anaerobic culture bottle collected from one limb) to "double-limb double-set" (one bottle of aerobic and one bottle of anaerobic culture bottle collected from each limb) to improve detection sensitivity. Although this change has improved the detection rate, it has also increased the cost of detection. Under this background, the industry urgently needs to develop more cost-effective solutions, one of which is to optimize the process performance and preparation cost of the sensing film at the bottom of the culture bottle, which has become one of the key technical paths to improve detection efficiency and reduce overall cost.

[0004] Blood culture bottles are a kind of clinical microbiological test products specially designed for detecting whether there are pathogenic microorganisms (such as bacteria and fungi) in blood. After blood is taken from a patient, it is injected into a blood culture bottle preloaded with culture medium, and the blood culture bottle is placed in a blood culture instrument for incubation. The bacteria in the bloodstream of a bloodstream infection patient will grow in the blood culture bottle. The growth of the bacteria will release carbon dioxide, and when the carbon dioxide content reaches a threshold value, the pH at the bottom of the blood culture bottle will change, causing the pre-embedded pH probe to change color or fluorescence to indicate whether there are pathogenic bacteria in the patient's blood. When the blood culture instrument detects the presence of pathogenic bacteria in the blood culture bottle, it will automatically alarm. At this time, the tester needs to record the alarm time and extract the culture for Gram staining. The alarm time and the Gram staining results of the pathogenic bacteria can reflect the initial concentration of the pathogenic bacteria and the type of the pathogenic bacteria. The initial concentration can reflect the severity of bloodstream infection in the patient, and the type of pathogenic bacteria can provide guidance for clinical medication.

[0005] Optimizing the preparation process and performance of key components (such as sensing films) is an important path to promote the technical upgrading of domestic products and reduce the cost of clinical use.

[0006] In blood culture detection, the sensing film is a core functional component of the blood culture detection system, and its performance directly affects the detection sensitivity and reliability of the blood culture bottle. Fluorescence detection uses specific excitation / emission wavelengths, has higher detection sensitivity, and can effectively avoid the interference of other colors of blood samples and reduce the false positive rate. The disclosed sensing film technology is formed by cross-linking and curing the indicator with the film material. The false positive rate and false negative rate in the prior art are as high as 5% or more, of which more than 3% are caused by the quality of the sensing film of the blood culture bottle, which delays the effective treatment of the patient. Secondly, the disclosed technology is complicated to operate and the indicator is not evenly distributed, which is one of the reasons for the poor color development sensitivity. SUMMARY

[0007] Therefore, the present application provides a fluorescent sensing composition, a fluorescent sensing film and applications thereof. The present application improves the problems of complex preparation process, unsatisfactory fluorescent response and high cost of the existing sensing film. Experiments show that the obtained fluorescent sensing film remains stable under moist heat sterilization conditions, can adapt to the currently commonly used imported blood culture instruments, has complete detection functions, and reduces the production cost. It can provide strong support for the localization process of the blood culture market in China, promote the efficiency of clinical diagnosis, and has significant social and economic benefits.

[0008] To achieve the above-mentioned purposes, the present application provides the following technical solutions:

[0009] In a first aspect, the present application provides a fluorescent sensing composition, which comprises the following components in mass parts:

[0010] Silicone A 1000-3000 parts;

[0011] Silicone B 100-350 parts;

[0012] Vulcanization inhibitor 0-20 parts;

[0013] Sodium hydroxide 0.05-1 parts;

[0014] Dispersing agent 10-100 parts;

[0015] Light shielding agent 0.1-10 parts;

[0016] Fluorescent indicator 0.001-80.009 parts;

[0017] The silicone A comprises a functional group-containing polydimethylsiloxane;

[0018] The silicone B comprises a functional group-containing polydimethylsiloxane and platinum gold;

[0019] The functional group comprises one or more of a hydroxyl group, a carboxyl group, an aldehyde group or an amino group;

[0020] The fluorescent indicator includes a combination of two or more of 8-hydroxy-pyrene-1, 3, 6-trisulfonic acid trisodium salt, bromocresol purple, rhodamine B, rhodamine 6G and rhodamine 590 chloride.

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

[0022] In some embodiments of the present application, the vulcanization inhibitor includes ethynylcyclohexanol and polydimethylsiloxane, and the mass ratio of the ethynylcyclohexanol to the polydimethylsiloxane is (0.01-0.05):(9.95-9.99).

[0023] In some embodiments of the present application, the sunscreen 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 embodiments of the present application, the dispersant includes glycerol.

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

[0028] The primer includes C7-C9 isoalkane and titanium tetrabutoxide; the fluorescent sensing composition includes the primer in an amount of 0-90 parts by mass;

[0029] The fluorescent sensing composition includes the solvent in an amount of 50-500 parts by mass; the solvent includes double-distilled water.

[0030] In some embodiments of the present application, the pH value of the fluorescent sensing composition is 7.0-10.0.

[0031] In some embodiments of the present application, the fluorescent sensing composition includes the following components in parts by mass:

[0032] Silicone gel A 245 parts;

[0033] Silicone gel B 273 parts;

[0034] Vulcanization inhibitor 9 parts;

[0035] Sodium hydroxide 0.5 parts;

[0036] Glycerol 82 parts;

[0037] Opacifier 3 parts;

[0038] Fluorescent indicator 0.492 parts;

[0039] Double distilled water 100 parts;

[0040] Primer 27.36 parts;

[0041] The fluorescent indicator comprises:

[0042] 8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt 0.15 parts;

[0043] Bromocresol purple 0.009 parts;

[0044] Rhodamine B 0.009 parts;

[0045] Rhodamine 6G 0.009 parts;

[0046] Rhodamine 590 chloride 0.315 parts;

[0047] or the fluorescent sensing composition comprises:

[0048] Silicone gel A 1000 parts;

[0049] Silicone gel B 100 parts;

[0050] Vulcanization inhibitor 0 parts;

[0051] Sodium hydroxide 0.05 parts;

[0052] Glycerol 10 parts;

[0053] Opacifier 0.1 parts;

[0054] Fluorescent indicator 0.005 parts;

[0055] Double distilled water 100 parts;

[0056] Primer 0 parts;

[0057] The fluorescent indicator comprises:

[0058] 8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt 0.001 parts;

[0059] Bromocresol purple 0.001 parts;

[0060] Rhodamine B 0.001 parts;

[0061] Rhodamine 6G 0.001 parts;

[0062] Rhodamine 590 chloride 0.001 parts;

[0063] or the fluorescent sensing composition comprises:

[0064] Silica gel A 3000 parts;

[0065] Silica gel B 350 parts;

[0066] Vulcanization inhibitor 20 parts;

[0067] Sodium hydroxide 1 part;

[0068] Glycerol 100 parts;

[0069] Light shielding agent 10 parts;

[0070] Fluorescent indicator 80.009 parts;

[0071] Double distilled water 100 parts;

[0072] Primer 90 parts;

[0073] The fluorescent indicator comprises:

[0074] 8-Hydroxy pyrene-1,3,6-trisulfonic acid trisodium salt 20 parts;

[0075] Bromocresol purple 20 parts;

[0076] Rhodamine B 0.009 parts;

[0077] Rhodamine 6G 20 parts;

[0078] Rhodamine 590 chloride 20 parts.

[0079] In a second aspect, the present application further provides the use of the fluorescent sensing composition in the preparation of a fluorescent sensing film.

[0080] In a third aspect, the present application further provides the use of the fluorescent sensing composition in the preparation of a microorganism detection product or a bloodstream infection diagnosis product.

[0081] In a fourth aspect, the present application further provides a fluorescent sensing film comprising the fluorescent sensing composition.

[0082] In a fifth aspect, the present application further provides a preparation method of the fluorescent sensing film, which comprises mixing the fluorescent sensing composition, and curing to obtain the fluorescent sensing film.

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

[0084] In a sixth aspect, the present application further provides the fluorescent sensing film prepared by the preparation method.

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

[0086] In some embodiments of the present application, the blood culture detection device comprises a blood culture bottle.

[0087] In a seventh aspect, the present application further provides use of the fluorescent sensing film in preparation of a blood culture detection device or a blood culture detection system.

[0088] In some embodiments of the present application, the blood culture detection device comprises a blood culture bottle.

[0089] In a seventh aspect, the present application further provides use of the fluorescent sensing film in preparation of a blood culture detection device or a blood culture detection system.

[0090] In a tenth aspect, the present application further provides use of any one of the following in preparation of a microbial detection product or a bloodstream infection diagnosis product:

[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 application further provides a microbial detection product or a bloodstream infection diagnosis product comprising any one of the following:

[0095] (I) the fluorescent sensing composition;

[0096] (II) the fluorescent sensing film;

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

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

[0099] Bloodstream infection is a serious threat to patients' lives. The survival rate of patients can be greatly improved if the diagnosis is made 1 hour earlier, and the abuse of antibiotics can be curbed. As the gold standard for the diagnosis of bloodstream infection, the diagnostic performance, preparation process and cost of blood culture are restricted by the sensing film at the bottom of the blood culture bottle. In this study, a simple and cost-effective indicator formula was finally obtained by screening a variety of fluorescent indicators and their combinations. Based on the formula, a high-efficiency fluorescent sensing film resistant to moist heat sterilization was developed by optimizing the preparation process. The fluorescence curve determination, gram staining and microbial mass spectrometry identification of three representative quality control strains of gram-negative bacilli, positive cocci and yeast fungi confirmed that the fluorescent sensing film not only adapts to mainstream imported blood culture instruments, but also has excellent detection performance. The application of the fluorescent sensing film proposed in this study to the detection of clinical microorganisms can provide technical support for the diagnosis of bloodstream infection, and has important significance for curbing the abuse of antibiotics and improving the prognosis of patients with bloodstream infection. BRIEF DESCRIPTION OF DRAWINGS

[0100] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, a brief introduction will be given below to the drawings needed to be used in the embodiments or prior art description.

[0101] Figure 1 Preparation process and development process of fluorescent sensing film; Note: ELASTOSIL® (Wacker) and Elaplus® silicone gel need to add primer, Liveo® (DuPont) does not need to add primer;

[0102] Figure 2 Fluorescent blood culture bottle and fluorescent sensing film before and after reporting positive; Note: A is the schematic diagram of blood culture bottle; B is the physical diagram of blood culture bottle; C is the fluorescent sensing film before the blood culture bottle is put on the machine; D is the fluorescent sensing film after the blood culture bottle reports positive;

[0103] Figure 3 Fluorescent sensing film and fluorescent curve of comparative example 1; Note: A: fluorescent sensing film before the blood culture bottle is put on the machine for testing; B: fluorescent sensing film after the blood culture bottle reports positive; C: fluorescent curve of the blood culture bottle put on the machine, which is redrawn after extracting data;

[0104] Figure 4 Fluorescent sensing film and fluorescent curve of comparative example 2; Note: A: fluorescent sensing film before the blood culture bottle is put on the machine for testing; B: fluorescent sensing film after the blood culture bottle reports positive; C: fluorescent curve of the blood culture bottle put on the machine, which is redrawn after extracting data;

[0105] Figure 5 Fluorescent sensing film and fluorescent curve of comparative example 3; Note: A: fluorescent sensing film before the blood culture bottle is put on the machine for testing; B: fluorescent sensing film after the blood culture bottle reports positive; C: fluorescent curve of the blood culture bottle put on the machine, which is redrawn after extracting data;

[0106] Figure 6Example 4 sensor film and fluorescence curve; Note: A: fluorescence sensor film before testing on blood culture bottle; B: fluorescence sensor film after blood culture bottle reported positive; C: fluorescence curve of blood culture bottle, redrawn after data extraction;

[0107] Figure 7 Example 5 sensor film and fluorescence curve; Note: A: fluorescence sensor film before testing on blood culture bottle; B: fluorescence curve of blood culture bottle, redrawn after data extraction;

[0108] Figure 8 Example 1 Escherichia coli fluorescence curve and strain identification; Note: A: Escherichia coli fluorescence curve, redrawn after data extraction; B: 24h plate of blood culture bottle reported positive, coated with Columbia blood agar medium; C: Gram staining result of blood culture bottle reported positive; D: Microbial mass spectrum identification result of blood culture bottle reported positive, score is confidence score;

[0109] Figure 9 Example 1 Staphylococcus aureus fluorescence curve and strain identification; Note: A: Staphylococcus aureus fluorescence curve, redrawn after data extraction; B: 24h plate of blood culture bottle reported positive, coated with Columbia blood agar medium; C: Gram staining result of blood culture bottle reported positive; D: Microbial mass spectrum identification result of blood culture bottle reported positive, score is confidence score;

[0110] Figure 10 Example 1 Candida albicans fluorescence curve and strain identification; Note: A: Candida albicans fluorescence curve, redrawn after data extraction; B: 24h plate of blood culture bottle reported positive, coated with Sabouraud agar medium; C: Gram staining result of blood culture bottle reported positive; D: Microbial mass spectrum identification result of blood culture bottle reported positive, score is confidence score;

[0111] Figure 11 Example 2 Escherichia coli fluorescence curve; Note: Redrawn after data extraction;

[0112] Figure 12 Example 3 Escherichia coli fluorescence curve; Note: Redrawn after data extraction. DETAILED DESCRIPTION

[0113] The present application discloses a fluorescence sensing composition, a fluorescence sensing film and its application. Those skilled in the art can refer to the content of the present application and appropriately improve the process parameters. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0114] Terminology:

[0115] Bloodstream infection: septicemia and bacteremia are collectively referred to as bloodstream infection. Septicemia is a blood infection caused by the invasion of blood stream by various pathogenic microorganisms (bacteria or fungi) and toxins; bacteremia is a condition in which bacteria only enter the blood temporarily without clinically apparent symptoms of toxemia (such as blood vessel-related infection).

[0116] Blood culture: blood culture is a method of culturing pathogenic bacteria by inoculating fresh ex vivo blood samples on nutrient medium under certain temperature, humidity, etc. conditions to make the bacteria with higher nutritional requirements grow and reproduce, and to identify them.

[0117] The present application innovatively designs various sensing film preparation schemes, and finally develops a preparation method of high-efficiency fluorescent sensing film (1000) Figure 1 ), the fluorescent sensing film formula and the functions of each component are shown in Table 1, and the physical map and schematic diagram of the fluorescent blood culture bottle are shown in Figure 2 B and Figure 2 A, the color of the fluorescent sensing film will change from purple to purple brown (1000) Figure 2 C and Figure 2 D) after being reported positive, which can be used for naked-eye identification of whether the fluorescent blood culture bottle has been used. The self-made blood culture bottle made based on the method is verified by three standard strains, and the detection curves of the three strains are good, and the preparation process is simpler and the cost is lower.

[0118] Table 1: Fluorescent sensing film formula and function of each component

[0119]

[0120] Table 2: Source of raw materials of fluorescent sensing film

[0121]

[0122]

[0123] The present application provides a fluorescent indicator formula containing a plurality of components: a silica gel A component (1000-3000 mg), a silica gel B component (100-350 mg), a vulcanization inhibitor (0-20 mg), sodium hydroxide (0.05-1 mg), glycerol (10-100 mg), a light shielding agent (0.1-10 mg), a primer (0-90 mg), double distilled water (50-500 mg), and one or more of the following fluorescent indicators: 8-hydroxy pyrene-1, 3, 6-trisulfonic acid trisodium salt, bromocresol purple, rhodamine B, rhodamine 6G, and rhodamine 590 chloride (0.001-20 mg).

[0124] The application also provides a simple fluorescent sensing film preparation process.

[0125] Based on the above research, the application also provides the application of the fluorescent sensing film in a blood culture bottle.

[0126] The application is based on conventional fluorescent reagents, adopts a relatively simple preparation process, and to some extent, improves the problems of complex preparation process, unsatisfactory fluorescent response and high cost of the existing sensing film.

[0127] The application is based on conventional reagents, adopts a relatively simple preparation process, and to some extent, improves the problems of complex preparation process, unsatisfactory signal response and high cost.

[0128] The raw materials and reagents used in the fluorescent sensing composition, the fluorescent sensing film and the application thereof provided by the application can be purchased from the market.

[0129] The application will be further described below in combination with examples:

[0130] Example 1

[0131] ①Preparation of the fluorescent sensing film

[0132] 0.315 mg of rhodamine 590 chloride, 0.15 mg of 8-hydroxy-pyrene-1, 3, 6-trisulfonic acid trisodium salt, 0.009 mg of bromocresol purple, 0.009 mg of rhodamine 6G, 0.009 mg of rhodamine B, 245 mg of ELASTOSIL A glue, 273 mg of ELASTOSIL B glue, 9 mg of vulcanization inhibitor, 82 mg of glycerol, 3 mg of sunscreen, 100 mg of double distilled water, 0.5 mg of sodium hydroxide were stirred and filled into the bottom of a blood culture bottle with 27.36 mg of primer, and then cured at 20 DEG C for 25 h.

[0133] 2. Preparation of working bacterial suspension

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

[0135] 3. Blood culture loading

[0136] The fluorescent blood culture bottle (R1, Example 1) prepared by using the sensing film technology of the present application was subjected to experiments on a BACTEC FX TOP blood culture instrument. Specifically, 5 bottles of each type of blood culture bottle were prepared, and the blood culture bottles were divided into positive group 1, positive group 2, positive group 3 and negative group 1, each group having 5 bottles. The positive group 1 was inoculated with 1 mL (1000 cfu) of working bacterial suspension of Escherichia coli (ATCC25922), the positive group 2 was inoculated with 1 mL (1000 cfu) of working bacterial suspension of Staphylococcus aureus (ATCC25923), and the positive group 3 was inoculated with 1 mL (1000 cfu) of working bacterial suspension of Candida albicans (ATCC18804). After loading, if a positive result was reported, the corresponding plate was inoculated to confirm the growth of bacteria, and then the positive result was recorded. Otherwise, the false positive result was recorded. If no positive result was reported after 96 hours, the blood culture bottle was taken out and observed for no obvious discoloration or turbidity, and then the negative result was recorded. If obvious discoloration or turbidity was observed, the blood culture bottle was inoculated into a blood plate to confirm the presence of bacteria, and then the false negative result was recorded.

[0137] 4. Gram staining and mass spectrometry identification

[0138] The culture solution in the positive blood culture bottle of the positive group 1 was taken out and cultured on Columbia blood agar medium to obtain pure culture single colonies. The Gram staining was performed according to Table 3, and then the single colonies were observed and photographed under an oil immersion lens. Fresh single colonies were picked and subjected to mass spectrometry grouping analysis using an Autof ms 1000 automatic microbial mass spectrometry detection system. The culture solution in the positive blood culture bottle of the positive group 2 was taken out and cultured on Columbia blood agar medium for 24 hours to obtain pure culture single colonies. The Gram staining was performed according to Table 3, and then the single colonies were observed and photographed under an oil immersion lens. Fresh single colonies were picked and subjected to mass spectrometry grouping analysis using an Autof ms 1000 automatic microbial mass spectrometry detection system. The culture solution in the positive blood culture bottle of the positive group 3 was taken out and cultured on Sabouraud agar medium for 24 hours to obtain pure culture single colonies. The Gram staining was performed according to Table 3, and then the single colonies were observed and photographed under an oil immersion lens. Fresh single colonies were picked and subjected to mass spectrometry grouping analysis using an Autof ms 1000 automatic microbial mass spectrometry detection system.

[0139] Table 3 Gram staining

[0140]

[0141] V. Experimental results

[0142] The fluorescence curve and results of E. coli, a gram-negative bacterium, in the self-made blood culture bottle are as follows Figure 8 A and Table 5, the fluorescence curve is stable and good, the positive reporting time is 7-8 h, and the positive reporting fluorescence value is around 0.190, and the performance is excellent, and it grows well in the Columbia blood agar plate Figure 8 B, the gram staining result shows gram-negative bacilli, which is consistent with the expected biological morphology of the strain Figure 8 C, the more accurate microbial mass spectrum identification result shows that the strain in the positive blood culture bottle is E. coli, and the confidence score is as high as 9.587 Figure 8 D, indicating that the fluorescence sensing film of the example is suitable for detection of E. coli, and the positive reporting time is normal and the stability is good. As can be seen from Table 4, compared with the five comparative examples, the positive reporting time of the compound indicator component of Example 1 is about 1 hour faster than that of the fastest positive reporting comparative example 4, and the curve trend is consistent with the imported fluorescence blood culture algorithm logic, i.e. first small decline, then smooth, then large increase, and finally smooth again.

[0143] The fluorescence curve and results of S. aureus, a gram-positive bacterium, in the self-made blood culture bottle are as follows Figure 9 A and Table 5, it can be seen that the fluorescence curve is stable and good, the positive reporting time is 9-10 h, and the positive reporting fluorescence value is around 0.217, and the performance is excellent, and it grows well in the Columbia blood agar plate Figure 9 B, the gram staining result shows gram-positive cocci arranged in grape clusters Figure 9 C, the more accurate microbial mass spectrum identification result shows that the strain in the positive blood culture bottle is S. aureus, and the confidence score is as high as 9.597 Figure 9 D, indicating that the fluorescence sensing film of the example is suitable for S. aureus, and the positive reporting time is normal and the stability is good.

[0144] The fluorescence curve and results of C. albicans, a yeast-type fungus, in the self-made blood culture bottle are as follows Figure 10 A and Table 5, the fluorescence curve is stable and good, the positive reporting time is 13-16 h, and the positive reporting fluorescence value is around 0.212, and the performance is excellent, and it grows well in the Sabouraud agar medium plate Figure 10 B, the gram staining result shows clustered budding yeast-like fungi Figure 10 C, the more accurate microbial mass spectrum identification result shows that the strain in the positive blood culture bottle is C. albicans, and the confidence score is as high as 9.421 Figure 10D), which shows that the fluorescent sensing film of the example is adapted to Candida albicans, the positive reporting time is normal, and the stability is good; the above results show that the fluorescent sensing film of the example is stable for detection of gram-positive bacteria, gram-negative bacteria and yeast fungi, can report positive within a normal time, and is accurate in detection (Table 5), and is well adapted to common imported fluorescent blood culture instruments.

[0145] Example 2

[0146] ①Preparation of fluorescent sensing film

[0147] 0.001 mg of rhodamine 590 chloride, 0.001 mg of 8-hydroxy pyrene-1, 3, 6-trisulfonic acid trisodium salt, 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 sunscreen, 100 mg of distilled water, and 0.05 mg of sodium hydroxide were stirred and filled into the bottom of a blood culture bottle, and then solidified at 70°C for 150 min.

[0148] ②Preparation of working bacterial suspension

[0149] The standard strain Escherichia coli was inoculated into a Columbia blood agar medium, and after being cultured at 35°C for 18 hours, a working bacterial suspension of a required concentration was prepared by dilution with 0.9% sterile sodium chloride solution.

[0150] ③Blood culture on machine

[0151] The fluorescent blood culture bottle prepared by using the sensing film technology of the application was subjected to experiments on a BACTEC FX TOP blood culture instrument. The specific method was as follows: two blood culture bottles were prepared, 1 mL (1000 cfu) of working bacterial suspension of Escherichia coli (ATCC25922) was inoculated, and after being put on the machine, if positive was reported, bacteria growth was confirmed by inoculation on a corresponding plate, and then it was recorded as positive, otherwise, it was recorded as false positive. If no obvious discoloration or turbidity was observed after being taken out at 96 h, it was recorded as negative, and if obvious discoloration or turbidity was observed, bacteria were confirmed by inoculation on a blood plate, and then it was recorded as false negative.

[0152] ④Experimental results

[0153] The fluorescent curve and results of Escherichia coli as gram-negative bacteria in the self-prepared blood culture bottle are shown in Figure 11 The fluorescent curve is stable, and positive is reported at 9 hours and 18 minutes, which is relatively late.

[0154] Example 3

[0155] ①Preparation of fluorescent sensing film

[0156] Mix 20 mg of rhodamine 590 chloride, 20 mg of 8-hydroxy-pyrene-1,3,6-trisulfonic acid trisodium salt, 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 glycerol, 10 mg of sunscreen, 100 mg of double distilled water, and 1 mg of sodium hydroxide, stir, and fill into the bottom of a blood culture bottle with 90 mg of primer, and cure at 110°C for 9 min.

[0157] ②Preparation of working bacterial suspension

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

[0159] ③Blood culture on machine

[0160] The fluorescent blood culture bottle prepared by using the sensing film technology of the application is subjected to experiments on a BACTEC FX TOP blood culture instrument. Specifically, 2 blood culture bottles are prepared, 1 mL (1000 cfu) of working bacterial suspension of Escherichia coli (ATCC25922) is inoculated, if positive is reported after being put on the machine, inoculation into the corresponding plate is needed to confirm bacterial growth, and then it can be recorded as positive, otherwise it is recorded as false positive. If still no positive is reported after 96 hours, it is taken out and observed, and if no obvious discoloration or turbidity is observed, it is recorded as negative, if obvious discoloration or turbidity is observed, inoculation into a blood plate is needed to confirm bacteria, and then it is recorded as false negative.

[0161] ④Experimental results

[0162] The fluorescence curve and results of Escherichia coli as a gram-negative bacterium in the self-prepared blood culture bottle are shown in Figure 12 The fluorescence curve is stable, and the downward trend in the later stage of the curve is obvious, and the positive is reported at 8 hours and 10 minutes, and the positive reporting time is relatively late.

[0163] Comparative Example 1

[0164] ①Preparation of fluorescent sensing film

[0165] Mix 0.03 mg of 8-hydroxy-pyrene-1,3,6-trisulfonic acid trisodium salt, 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, stir, and fill into the bottom of a blood culture bottle with 27.36 mg of primer, and cure at 70°C for 150 min.

[0166] ②Preparation of working bacterial suspension

[0167] Standard strain Escherichia coli was inoculated into Columbia blood agar medium, and cultured at 35°C for 18 hours. Then, the working bacterial suspension was prepared by diluting the bacterial suspension with 0.9% sterile sodium chloride solution to the required concentration.

[0168] ③Blood culture on machine

[0169] The fluorescent blood culture bottle prepared by using the sensing film technology of the present application was subjected to experiments on a BACTEC FX TOP blood culture instrument. Specifically, two blood culture bottles were prepared, and 1 mL (1000 cfu) of working bacterial suspension of Escherichia coli (ATCC25922) was inoculated. If the instrument reported positive after being put on the machine, the corresponding plate was inoculated to confirm the growth of bacteria, so as to be recorded as positive. Otherwise, it was recorded as false positive. If the instrument still reported negative after 96 hours, the bottle was taken out and observed. If there was no obvious discoloration or turbidity, it was recorded as negative. If there was obvious discoloration or turbidity, the bottle was inoculated into a blood plate to confirm the presence of bacteria, and was recorded as false negative.

[0170] ④Experimental results

[0171] The fluorescence curve and results of Escherichia coli in the self-prepared blood culture bottle are shown in Figure 3 . The fluorescence curve did not have a large upward trend in the first 7 hours, and reported positive after 11 hours, which was late.

[0172] Comparative Example 2

[0173] ①Preparation of fluorescent sensing film

[0174] 0.15 mg of rhodamine 6G, 245 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 were stirred and filled into the bottom of a blood culture bottle with 27.36 m primer, and cured at 70°C for 150 min.

[0175] ②Preparation of working bacterial suspension

[0176] Standard strain Escherichia coli was inoculated into Columbia blood agar medium, and cultured at 35°C for 18 hours. Then, the working bacterial suspension was prepared by diluting the bacterial suspension with 0.9% sterile sodium chloride solution to the required concentration.

[0177] ③Blood culture on machine

[0178] The fluorescent blood culture bottle prepared by using the sensing film technology of the present application was subjected to experiments on a BACTEC FX TOP blood culture instrument. Specifically, two blood culture bottles were prepared, and 1 mL (1000 cfu) of working bacterial suspension of Escherichia coli (ATCC25922) was inoculated. If the instrument reported positive after being put on the machine, the corresponding plate was inoculated to confirm the growth of bacteria, so as to be recorded as positive. Otherwise, it was recorded as false positive. If the instrument still reported negative after 96 hours, the bottle was taken out and observed. If there was no obvious discoloration or turbidity, it was recorded as negative. If there was obvious discoloration or turbidity, the bottle was inoculated into a blood plate to confirm the presence of bacteria, and was recorded as false negative.

[0179] IV. Experimental results

[0180] The fluorescence curve and results of E. coli, a gram-negative bacterium, in the self-made blood culture bottle are shown in Figure 4 The fluorescence curve is unstable, and the downward trend in the latter part of the curve is obvious. The positive result is reported at 9 hours and 28 minutes, which is late.

[0181] Comparative Example 3

[0182] I. Preparation of the fluorescence sensing film

[0183] 0.15 mg of rhodamine B, 245.4 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 were stirred and loaded into the bottom of a blood culture bottle with 27.36 mg of primer, and cured at 70°C for 150 min.

[0184] II. Preparation of the working bacterial suspension

[0185] After the standard strain E. coli was inoculated into a Columbia blood agar culture medium and cultured at 35°C for 18 hours, a working bacterial suspension of the required concentration was prepared by dilution with 0.9% sterile sodium chloride solution.

[0186] III. Blood culture

[0187] The fluorescence blood culture bottle prepared by using the sensing film technology of the application was subjected to experiments on a BACTEC FX TOP blood culture instrument. Specifically, two blood culture bottles were prepared, 1 mL (1000 cfu) of the working bacterial suspension of E. coli (ATCC25922) was inoculated, and if a positive result was reported after the instrument was started, the corresponding plate was inoculated to confirm bacterial growth, and then the positive result was recorded. Otherwise, the false positive result was recorded. If the positive result was not reported after 96 hours, the bottle was taken out and observed for no obvious discoloration or turbidity, and the negative result was recorded. If obvious discoloration or turbidity was observed, the bottle was inoculated into a blood plate to confirm the presence of bacteria, and the false negative result was recorded.

[0188] IV. Experimental results

[0189] The fluorescence curve and results of E. coli in the self-made blood culture bottle are shown in Figure 5 The fluorescence curve is unstable, and the downward trend in the latter part of the curve is obvious. The positive result is reported at 10 hours and 24 minutes, which is late.

[0190] Comparative Example 4

[0191] I. Preparation of the fluorescence sensing film

[0192] Mix 0.006 mg of rhodamine 590 chloride, 245 mg of ELASTOSIL® A glue, 273 mg of ELASTOSIL® B glue, 9 mg of vulcanization inhibitor, 82 mg of glycerol, 3 mg of shading agent, 100 mg of double distilled water, 0.5 mg of sodium hydroxide, pour into the bottom of a blood culture bottle with 27.36 mg of primer, and cure at 70°C for 150 min.

[0193] ②Preparation of working bacterial suspension

[0194] Inoculate the standard strain Escherichia coli into Columbia blood agar medium, and incubate at 35°C for 18 hours, then dilute with 0.9% sterile sodium chloride solution to prepare a working bacterial suspension of the desired concentration.

[0195] ③Blood culture on machine

[0196] The fluorescent blood culture bottle prepared using the sensing film technology of the application was tested on a BACTEC FX TOP blood culture instrument. Specifically, 2 blood culture bottles were prepared, and the blood culture bottles were divided into a positive group and a negative group. The positive group was inoculated with 1 mL (1000 cfu) of working bacterial suspension of Escherichia coli (ATCC25922), and the negative group was not inoculated. After being put on the machine, if it reported positive, it needed to be inoculated into the corresponding plate to confirm bacterial growth, and then it could be recorded as positive, otherwise it was recorded as false positive. If it still did not report positive after 96 h, it was taken out and observed for no obvious discoloration or turbidity, and if it was obviously discolored or turbid, it was inoculated into a blood plate to confirm the presence of bacteria and was recorded as false negative.

[0197] ④Experimental results

[0198] The fluorescence curve and results of Escherichia coli, a gram-negative bacterium, in the self-made blood culture bottle are shown in Figure 6 The fluorescence curve is unstable, the downward trend in the latter part of the curve is obvious, the positive reporting time is nearly 9 h, the positive reporting time 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, 245 mg of ELASTOSIL® A glue, 273 mg of ELASTOSIL® B glue, 9 mg of vulcanization inhibitor, 82 mg of glycerol, 3 mg of black shading agent, 100 mg of double distilled water, 0.5 mg of sodium hydroxide, pour into the bottom of a blood culture bottle with 27.36 mg of primer, and cure at 70°C for 150 min.

[0202] ②Preparation of working bacterial suspension

[0203] The standard strain Escherichia coli was inoculated into a Columbia blood agar medium, and was cultured at 35℃ for 18 hours, and then was diluted with 0.9% sterile sodium chloride solution to prepare a working bacterial suspension with a required concentration.

[0204] ③Blood culture on machine

[0205] The fluorescent blood culture bottle prepared by using the sensing film technology of the present application was subjected to experiments on a BACTEC FX TOP blood culture instrument. Specifically, two blood culture bottles were prepared, and the blood culture bottles were divided into a positive group and a negative group. The positive group was inoculated with 1 mL (1000 cfu) of the working bacterial suspension of Escherichia coli (ATCC25922), and the negative group was not inoculated. After being put on the machine, if it was reported positive, it was necessary to inoculate a corresponding plate to confirm the growth of bacteria, and then it could be recorded as positive, otherwise it was recorded as false positive. If it was still not reported positive after 96 hours, it was taken out and observed, and if there was no obvious discoloration or turbidity, it was recorded as negative, and if there was obvious discoloration or turbidity, it was inoculated into a blood plate to confirm the presence of bacteria, and then it was recorded as false negative.

[0206] ④Experimental results

[0207] The fluorescence curve and results of Escherichia coli as a gram-negative bacterium in the self-prepared blood culture bottle are shown in Figure 7 The overall fluorescence curve showed a downward trend, and it was still not reported positive after 4 days of culture.

[0208] Effect example

[0209] Table 4 Summary of Escherichia coli growth test results of five comparative examples and three embodiments

[0210]

[0211] Table 5 Summary of test results of three representative strains of the optimal sensing film (Embodiment 1)

[0212]

[0213] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A fluorescent sensing composition, characterized in that, by mass, comprising: silicone gel A 1000 to 3000 parts; silicone gel B 100 to 350 parts; vulcanization inhibitor 0 to 20 parts; sodium hydroxide 0.05 to 1 part; dispersant 10 to 100 parts; light shielding agent 0.1 to 10 parts; fluorescent indicator 0.001 to 80.009 parts; the silicone gel A includes a functional group-containing polydimethylsiloxane; the silicone gel B includes a functional group-containing polydimethylsiloxane and platinum gold; the functional group includes one or more of a hydroxyl group, a carboxyl group, an aldehyde group, or an amino group; the fluorescent indicator includes a combination of two or more of 8-hydroxypyrene-1, 3, 6-trisulfonic acid trisodium salt, bromocresol purple, rhodamine B, rhodamine 6G, and rhodamine 590 chloride.

2. The fluorescent sensing composition of claim 1, wherein, in the silicone gel B, the mass ratio of the functional group-containing polydimethylsiloxane to the platinum gold is (8 to 9.9):(0.1 to 2).

3. The fluorescent sensing composition of claim 2, wherein the vulcanization inhibitor includes ethynylcyclohexanol and polydimethylsiloxane, and the mass ratio of the ethynylcyclohexanol to the polydimethylsiloxane is (0.01 to 0.05):(9.95 to 9.99).

4. The fluorescent sensing composition of claim 3, wherein the light shielding agent includes polydimethylsiloxane and a pigment; the pigment includes one or more of titanium dioxide, zinc white (ZnO), or calcium carbonate (CaCO3); the mass ratio of the dimethylsiloxane to the pigment is (0.1 to 2):(8 to 9.9).

5. The fluorescent sensing composition of claim 4, wherein, the dispersant includes glycerol.

6. The fluorescent sensing composition of claim 5, wherein, a primer and / or a solvent are further included; the primer includes C7-C9 isoalkane and titanium tetrabutoxide; the fluorescent sensing composition includes the primer 0 to 90 parts by mass; the fluorescent sensing composition includes the solvent 50 to 500 parts by mass; the solvent includes double-distilled water.

7. The fluorescent sensing composition of claim 6, wherein, its pH value includes 7.0~10.

0.

8. The fluorescent sensing composition of claim 7, wherein, by mass, comprising: silicone gel A 2454 parts; silicone gel B 273 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 includes: 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 includes: 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 includes: 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 includes: silicone gel A 3000 parts; silicone gel B 350 parts; vulcanization inhibitor 20 parts; sodium hydroxide 1 part; 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-hydroxy-pyrene-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.

9. Use of the fluorescent sensing composition according to any one of claims 1 to 8 in the preparation of a fluorescent sensing film.

10. Use of the fluorescent sensing composition according to any one of claims 1 to 8 in the preparation of a microbial detection product or a bloodstream infection diagnosis product.

11. A fluorescent sensing film, characterized in that, The fluorescent sensing composition according to any one of claims 1 to 8.

12. The method for preparing the fluorescent sensing film as described in claim 11, characterized in that, The fluorescent sensing film is prepared by mixing the fluorescent sensing composition, and curing.

13. The method of claim 12, wherein the step of preparing is characterized by, The curing temperature is 20-110℃, and the curing time is 9 min-25 h.

14. The fluorescent sensing film prepared by the preparation method according to claim 13.

15. Use of the fluorescent sensing film according to claim 11 in the preparation of a blood culture detection device or a blood culture detection system.

16. The use according to claim 15, wherein The blood culture detection device comprises a blood culture bottle.

17. A blood culture detection device, characterized by The fluorescent sensing film according to claim 11.

18. The blood culture detection device as described in claim 17, characterized in that, The blood culture bottle.

19. A blood culture detection system characterized by, The blood culture detection device according to claim 17.

20. Use of any one of the following in the preparation of a microbial detection product or a bloodstream infection diagnosis product: (I) the fluorescent sensing film according to claim 11; (II) the blood culture detection device according to claim 17; (III) the blood culture detection system according to claim 19.

21. A product for the detection of microorganisms or a product for the diagnosis of blood stream infections, characterized in that, The fluorescent sensing composition according to any one of claims 1 to 8; The fluorescent sensing film according to claim 11; The blood culture detection device according to claim 17; The blood culture detection system according to claim 19. ​

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