Colorimetric sensing composition, colorimetric sensing film and application of colorimetric sensing film

Abstract

The invention relates to the field of medical detection, in particular to a colorimetric sensing composition, a colorimetric sensing film and application of the colorimetric sensing film. According to the invention, a domestic indicator is adopted, and the colorimetric sensing film resistant to damp-heat sterilization is developed through a simple preparation process. The sensing membrane has the characteristics of simple preparation process, low cost, stable performance and the like, and can effectively meet clinical detection requirements. The preparation method provided by the research is simple, convenient and effective, is expected to provide technical support for increasing the market proportion of domestic blood culture products and improving the blood flow infection diagnosis efficiency, and has positive significance for promoting reasonable medication and improving the treatment success rate.

Description

TECHNICAL FIELD

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

[0002] Bloodstream infection is a major public health challenge worldwide, causing millions of deaths each year. As the gold standard for clinical diagnosis, blood culture technology plays a key role in the diagnosis and treatment system of infectious diseases.

[0003] 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 detection rate, it has also increased detection cost. This situation highlights the urgency of blood culture product localization. By optimizing the preparation process of key materials such as detection sensing film, reducing production cost, and building a self-controllable industrial chain, it will become an important breakthrough to promote the development of blood culture technology in China. Achieving this goal not only helps to reduce the medical burden, but also contributes to enhancing the self-innovation capability of China's high-end medical equipment.

[0004] Blood culture bottle is a clinical microbiological examination product specially designed for detecting pathogenic microorganisms (such as bacteria, fungi) in blood. Its working principle is to inject patient blood samples into a culture bottle preloaded with culture medium, and then place it in a blood culture instrument for incubation. When the patient has bloodstream infection, the pathogenic bacteria in the blood will proliferate in the culture bottle, and the carbon dioxide released during the metabolic process will cause the pH value at the bottom of the culture bottle to change, thereby activating the pre-embedded pH probe, indicating the presence of pathogenic bacteria through color or fluorescence change. When the blood culture instrument detects that the carbon dioxide concentration reaches the threshold value, it will automatically alarm, at which time the laboratory personnel need to record the alarm time and perform gram staining on the culture. The alarm time can reflect the initial concentration of pathogenic bacteria, indicating the severity of infection; while the gram staining result can quickly identify the type of pathogenic bacteria, providing important guidance for clinical medication.

[0005] The research and development of blood culture bottles in China started relatively late, and the industrial foundation is relatively weak. Currently, domestic blood culture bottles still have room for improvement in core technology. Against this background, promoting the technological innovation and industrialization development of domestic blood culture bottles, and realizing the self-controllable of key materials, are of great significance to perfecting China's medical equipment supply chain and reducing medical costs. Through optimizing production process, improving product stability and other ways, it will help to promote the application of domestic blood culture bottles and provide more quality choices for clinical.

[0006] In blood culture bottles, the bottom pH sensor (fluorescent or colorimetric) is one of the decisive factors in the bottle's performance. Existing sensor membrane technologies involve cross-linking and curing the indicator with the membrane material. However, these technologies suffer from false positive and false negative rates exceeding 5%, with over 3% attributed to quality issues with the blood culture bottle's sensor membrane, delaying effective patient treatment. Furthermore, the cumbersome operation and uneven indicator distribution of current technologies also contribute to insensitive color development. Our company uses common chemical reagents such as thymol blue, bromothymol blue, methyl red, xylenol blue, and phenolphthalein as colorimetric indicators, eliminating the need for additional chemically synthesized indicators. Simply mixing these with silica gel AB glue ensures uniform indicator distribution. After adding the membrane to the bottom of the blood bottle and drying the substrate to solidify, the colorimetric sensor membrane is ready. Additionally, the prepared colorimetric sensor membrane is highly stable and can withstand temperatures up to 115°C, allowing for high-temperature and high-pressure sterilization.

[0007] Currently, the preparation process of culture media for domestically produced blood culture bottles of the same type is complex and costly. Therefore, providing colorimetric sensing membranes, their preparation methods and applications, as well as blood culture bottles containing such colorimetric sensing membranes, is of significant practical importance. Summary of the Invention

[0008] In view of this, the present invention provides a colorimetric sensing composition, a colorimetric sensing membrane, and their applications. The colorimetric sensing membrane provided by the present invention uses inexpensive raw materials, is simple to prepare (requiring no complex chemical synthesis), exhibits excellent performance, can withstand moist heat sterilization, and is compatible with common imported blood culture instruments based on colorimetric principles. Blood culture bottles prepared using this method can reduce the cost of preparing the sensing membrane while improving the performance of our company's blood culture bottles.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] In a first aspect, the present invention provides a colorimetric sensing composition comprising, by parts by weight, the following components:

[0011] Silicone gel A, 2500-3000 parts;

[0012] Silicone gel B250-350 parts;

[0013] Sulfation inhibitor 1-5 parts;

[0014] Sodium hydroxide 0.1–0.3 parts;

[0015] 50-150 parts of dispersant;

[0016] 2-6 parts of light-blocking agent;

[0017] Indicator 0.4–1.2 parts;

[0018] The silicone gel A comprises polydimethylsiloxane;

[0019] The silicone gel B comprises polydimethylsiloxane containing functional groups and platinum;

[0020] The functional group includes one or more of hydroxyl, carboxyl, aldehyde or amino groups.

[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 indicator includes one or more of thymol blue, bromothymol blue, methyl red, xylenol blue, and phenolphthalein;

[0027] The dispersant includes glycerol.

[0028] In some specific embodiments of the present invention, the colorimetric sensing composition further includes a primer and / or a solvent;

[0029] The primer comprises C7-C9 isoalkanes and titanium tetrabutoxide; by weight, the colorimetric sensing composition comprises 5 to 20 parts of the primer;

[0030] The colorimetric sensing composition comprises 100-300 parts by weight of the solvent; the solvent includes double-distilled water. Preferably, the colorimetric sensing composition comprises 200 parts by weight of the solvent.

[0031] In some specific embodiments of the present invention, the primer includes functionalized polydimethylsiloxane, C7-C9 isoalkanes, tetrabutoxide titanium, and tetraethyl silicate.

[0032] The mass ratio of the functionalized polydimethylsiloxane, the C7-C9 isoalkane, the titanium tetrabutoxide, and the tetraethyl silicate is (0.05-0.2):(0.5-0.9):(0.01-0.1):(0.005-0.05).

[0033] Preferably, the ratio of the functionalized polydimethylsiloxane, the C7-C9 isoalkane, the tetrabutoxide titanium, and the tetraethyl silicate is 0.108:0.826:0.066:(0.005~0.05).

[0034] or

[0035] The primer comprises tetrabutyl titanate and heptane; the ratio of tetrabutyl titanate to heptane is (0.1-0.4):(0.6-0.9).

[0036] In some specific embodiments of the present invention, the pH value of the colorimetric sensing composition includes 7.0 to 10.0.

[0037] In some specific embodiments of the present invention, the colorimetric sensing composition comprises, by weight, the following components:

[0038] Silicone gel A, 2500-3000 parts;

[0039] Silicone gel B250~350 parts;

[0040] Five parts of sulfurization inhibitor;

[0041] Sodium hydroxide 0.1~0.3 parts;

[0042] 50-150 parts glycerin;

[0043] 2-6 parts of sunblock agent;

[0044] Indicator 0.4~1.2 parts;

[0045] 100-300 portions of double-distilled water;

[0046] 12.36 parts of primer.

[0047] In some specific embodiments of the present invention, the colorimetric sensing composition comprises, by weight, the following components:

[0048] 3000 parts of silicone gel A;

[0049] 350 parts of silicone gel B;

[0050] Five parts of sulfurization inhibitor;

[0051] 0.3 parts sodium hydroxide;

[0052] 150 parts glycerin;

[0053] 6 parts of sunblock agent;

[0054] Indicator 1.2 parts;

[0055] 300 portions of double-distilled water;

[0056] 12.36 parts primer;

[0057] Or the colorimetric sensing composition may include:

[0058] 2500 parts of silicone gel A;

[0059] 250 parts of silicone gel B;

[0060] Five parts of sulfurization inhibitor;

[0061] Sodium hydroxide 0.1 parts;

[0062] 50 parts glycerin;

[0063] Two parts of sunscreen;

[0064] 0.4 parts of indicator;

[0065] 100 portions of double-distilled water;

[0066] 12.36 parts primer;

[0067] Or the colorimetric sensing composition may include:

[0068] 2700 parts of silicone gel A;

[0069] 300 parts of silicone gel B;

[0070] Five parts of sulfurization inhibitor;

[0071] Sodium hydroxide 0.2 parts;

[0072] 80 parts glycerin;

[0073] 5 parts of sunscreen;

[0074] 0.8 parts of indicator;

[0075] 200 portions of double-distilled water;

[0076] 12.36 parts of primer.

[0077] Secondly, the present invention also provides the application of the colorimetric sensing composition in the preparation of colorimetric sensing membranes.

[0078] Thirdly, the present invention also provides the application of the colorimetric sensing composition in the preparation of microbial detection products or bloodstream infection diagnostic products.

[0079] Fourthly, the present invention also provides a colorimetric sensing membrane comprising the colorimetric sensing composition.

[0080] Fifthly, the present invention also provides a method for preparing the colorimetric sensing membrane, wherein the colorimetric sensing composition is mixed and cured to obtain the colorimetric sensing membrane.

[0081] In some specific embodiments of the present invention, the mixing time is 25 min; the curing temperature is 50~90℃; and the curing time is 60~240 min.

[0082] In a sixth aspect, the present invention also provides a colorimetric sensing membrane prepared by the aforementioned preparation method.

[0083] In a seventh aspect, the present invention also provides the application of the colorimetric sensing membrane in the preparation of a blood culture detection device or a blood culture detection system.

[0084] In some specific embodiments of the present invention, the blood culture detection device includes a blood culture bottle.

[0085] Eighthly, the present invention also provides a blood culture detection device, including the colorimetric sensing membrane.

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

[0087] In a ninth aspect, the present invention also provides a blood culture detection system, including the blood culture detection device.

[0088] 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;

[0089] (I) The colorimetric sensing film;

[0090] (II) The blood culture detection device;

[0091] (III) The blood culture detection system.

[0092] In an eleventh aspect, the present invention also provides microbial detection products or bloodstream infection diagnostic products, including any one of the following:

[0093] (I) The colorimetric sensing composition;

[0094] (II) The colorimetric sensing membrane;

[0095] (III) The blood culture detection device;

[0096] (IV) The blood culture detection system.

[0097] Bloodstream infections pose a serious threat to patients' lives, and timely diagnosis is crucial for improving prognosis. Blood culture, as the gold standard for diagnosis, faces a major challenge in clinical practice due to its insufficient detection speed. Optimizing sensor membrane performance is a key step in improving the efficiency of blood culture. This study developed a colorimetric sensor membrane resistant to moist heat sterilization using domestically produced indicators and a simplified preparation process. This sensor membrane features simple preparation, low cost, and stable performance, effectively meeting clinical testing needs. The preparation method proposed in this study is simple and effective, and is expected to provide technical support for increasing the market share of domestically produced blood culture products and improving the diagnostic efficiency of bloodstream infections, thus having a positive impact on promoting rational drug use and improving treatment success rates. Attached Figure Description

[0098] 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.

[0099] Figure 1 The diagram shows the structure of the blood culture bottle and the changes in the sensing membrane after a positive result is reported. A shows a schematic diagram of the blood culture bottle structure; B shows an actual image of the blood culture bottle; C shows the state of the bottom sensing membrane before a positive result is reported; and D shows the color change of the bottom sensing membrane after a positive result is reported.

[0100] Figure 2 This demonstrates the development process and strategy; Note: A primer is required when using ELASTOSIL® (Wacker) and Elaplus® silicone gel, but not when using Liveo® (DuPont);

[0101] Figure 3 The color reaction characteristics of different indicator concentrations under different pH conditions are shown; where A shows the absorbance changes of different concentrations of bromothymol blue at various pH values; B shows the absorbance changes of different concentrations of xylenol blue at various pH values.

[0102] Figure 4 The experimental results of inoculating the blood culture bottle with the self-made sensor membrane blood culture bottle with the standard strain of Escherichia coli in Example 1 are shown; where A shows the growth curve on the fully automated blood culture instrument, which is redrawn after data extraction; B shows the blood culture bottle with positive results coated with Columbia blood agar medium and cultured for 24 hours.

[0103] Figure 5 The experimental results of inoculating the blood culture bottle with the self-made sensor membrane of Example 1 with the standard strain of Staphylococcus aureus are shown; where A shows the growth curve on the fully automated blood culture instrument, which is redrawn after data extraction; B shows the blood culture bottle with positive results coated with Columbia blood agar medium and cultured for 24 hours.

[0104] Figure 6The experimental results of inoculating the blood culture bottle with the self-made sensor membrane in Example 1 with the standard strain of Candida albicans are shown; where A shows the growth curve on the fully automated blood culture instrument, which is redrawn after data extraction; B shows the blood culture bottle with positive results coated with Sabouraud agar medium and cultured for 24 hours.

[0105] Figure 7 The experiment results of inoculating the blood culture bottle with the self-made sensor membrane and the standard strain of Escherichia coli in Example 2 are shown; A shows the growth curve on the fully automated blood culture instrument, which was redrawn after data extraction; B shows the blood culture bottle with positive results coated with Columbia blood agar medium and cultured for 24 hours.

[0106] Figure 8 The experimental results of inoculating the blood culture bottle with the self-made sensor membrane and Staphylococcus aureus standard strain in Example 2 are shown; where A shows the growth curve on the fully automated blood culture instrument, which was redrawn after data extraction; B shows the blood culture bottle with positive results coated with Columbia blood agar medium and cultured for 24 hours.

[0107] Figure 9 The experimental results of inoculating the blood culture bottle with the self-made sensor membrane and the standard strain of Candida albicans in Example 2 are shown; where A shows the growth curve on the fully automated blood culture instrument, which is redrawn after data extraction; B shows the blood culture bottle with positive results coated with Sabouraud agar medium and cultured for 24 hours.

[0108] Figure 10 The following are the experimental results of inoculating blood culture bottles with the self-made sensor membrane blood culture bottle with standard strain of Escherichia coli in Example 1; where A shows the growth curve on the fully automated blood culture instrument, which was redrawn after data extraction; B shows the blood culture bottle with positive blood culture medium coated with Columbia blood agar and cultured for 24 h; C shows the Gram staining photograph of the blood culture bottle with positive blood culture; D shows the MALDI-TOF MS spectrum and identification results.

[0109] Figure 11 The following are the experimental results of inoculating blood culture bottles with the self-made sensor membrane and Staphylococcus aureus standard strain in Example 1. Among them, A shows the growth curve on the fully automated blood culture instrument, which was redrawn after data extraction; B shows the blood culture bottle with positive blood culture medium coated with Columbia blood agar and cultured for 24 hours; C shows the Gram staining photograph of the blood culture bottle with positive blood culture; D shows the MALDI-TOF MS spectrum and identification results.

[0110] Figure 12 The experiment results of inoculating the blood culture bottle with the self-made sensor membrane and the standard strain of Candida albicans in Example 1 are shown. Among them, A shows the growth curve on the fully automated blood culture instrument, which is redrawn after data extraction; B shows the blood culture bottle with positive results coated with Sabor agar medium and cultured for 24 hours; C shows the Gram staining photograph of the blood culture bottle with positive results; D shows the MALDI-TOF MS spectrum and identification results. Detailed Implementation

[0111] This invention discloses a colorimetric sensing composition, a colorimetric sensing membrane, and their applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note 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 will clearly be able to 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.

[0112] Terminology Explanation:

[0113] 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.

[0114] 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.

[0115] Table 1. Functions of each component in a colorimetric sensor membrane

[0116]

[0117]

[0118]

[0119] This invention designs various methods for preparing sensor membranes, ultimately resulting in a method for preparing a colorimetric sensor membrane. Its core technology achieves real-time, highly sensitive pathogen detection by detecting carbon dioxide (CO2) produced by microbial metabolism. When pathogens proliferate in blood culture bottles, they metabolize and produce large amounts of CO2 gas, which dissolves to form carbonic acid (H2CO3), leading to a significant decrease in the pH of the culture environment. To accurately detect this change while avoiding interference from the color and turbidity of the blood sample itself, this invention employs a composite sensor membrane structure. This sensor membrane immobilizes a pH-sensitive indicator within a specially formulated gas-permeable polymer matrix. This matrix allows CO2 to pass freely but completely blocks liquids and solids such as blood and culture medium. In the early stages of culture or when no microbial growth is observed, the sensor membrane remains blue in a neutral or weakly alkaline environment (e.g., ...). Figure 1(As shown in B and C); when CO2 produced by microbial proliferation permeates into the membrane layer, it creates a localized acidic environment. When the pH drops below the indicator's color change threshold, the sensor membrane's color irreversibly changes from blue to yellow (as shown in Figure B and C). Figure 1 (As shown in D). The optical detector of the fully automated culture system can monitor this color change in real time and trigger a positive alarm. This design physically isolates the indicator from the sample, which not only avoids the problem of interference in traditional colorimetric methods and improves the specificity and signal-to-noise ratio of the detection, but also ensures the stability and low cost of the sensing system, providing a high-performance, low-cost, and reliable technical solution for the localization of blood culture bottles.

[0120] This invention addresses, to some extent, the problems of complex preparation processes, less than ideal signal response, and high costs associated with existing sensor membranes. Experiments show that the resulting colorimetric sensor membrane can withstand moist heat sterilization, and when representative quality control strains are inoculated into culture flasks equipped with the colorimetric sensor membrane, they can be successfully detected and reported as positive. The development of this colorimetric 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.

[0121] The colorimetric sensing composition, colorimetric sensing membrane, and raw materials and reagents used in the application of the present invention are all commercially available.

[0122] The present invention will be further illustrated below with reference to the embodiments:

[0123] Preliminary test case

[0124] This study focuses on the effects of key factors such as colorimetric substrate, system water content, temperature, sodium hydroxide addition, opacifier addition, and glycerol addition on various aspects of the sensor membrane's performance by fixing the proportions of the remaining components, in order to determine the key processes and sensor membrane formulation.

[0125] (1) Colorimetric characterization of substrates in response to pH changes

[0126] In the development of colorimetric sensing membranes, pH value is a key factor affecting the color change of indicators. To develop a colorimetric sensing membrane suitable for blood culture bottles, this study screened bromothymol blue and p-xylenol blue as candidate indicators from commonly used colorimetric pH indicators (Table 2). These two indicators exhibit significant color responses within this pH range and theoretically can respond well to pH changes caused by carbon dioxide produced during microbial growth. Colorimetric response tests were conducted using bromothymol blue and p-xylenol blue as candidate indicators. The preparation method for the bromothymol blue stock solution was as follows: 10 mg of bromothymol blue was dissolved in 8 mL of 0.02 mol / L NaOH solution, and the pH was adjusted to 7.00 ± 0.01 after measurement. The solution was then diluted to 20 mL with deionized water to obtain a stock solution with a concentration of 500 mg / L, which was dark green. A 320 mg / L working solution was prepared by diluting the solution proportionally and mixing it in 1.5 mL or 2 mL EP tubes for later use. The preparation method for the xylenol blue stock solution is as follows: Weigh 10 mg of xylenol blue and dissolve it in 8 mL of 0.02 mol / L NaOH solution. After measuring the pH, adjust it to 7.00 ± 0.01, then bring the volume to 20 mL with purified water to obtain a 500 mg / L stock solution. Subsequently, measure and adjust the pH to 9.6; the solution should be deep blue. Dilute proportionally to obtain a 320 mg / L working solution, mix it in 1.5 mL or 2 mL EP tubes, and set aside. Bromothymol blue and xylenol blue working solutions at different pH conditions were added to transparent 96-well plates, with each well containing 0.15 mL. The color reaction and absorbance changes of the two candidate indicators at different concentrations were characterized using a TECAN SPARK microplate reader. Figure 3 ).

[0127] Table 2. Common colorimetric pH indicators

[0128]

[0129] Experimental results show that bromothymol blue exhibits the most significant color difference and exhibits high sensitivity to absorbance changes within the pH range of 6–7.5 at a concentration of 480 mg / L, meeting the requirements for detecting pH changes during microbial growth. Higher concentrations of the indicator would result in material waste. Experimental results recommend bromothymol blue as the indicator for colorimetric sensing membranes, with an optimal concentration of 480 mg / L.

[0130] (2) Effect of moisture content on uniform dispersion of substrate in AB glue

[0131] To simplify the preparation process of colorimetric sensing membranes, this study used water as the indicator solvent and systematically investigated the effect of water content on the uniform dispersion of the substrate. In the experiment, a fixed concentration of colorimetric indicator working solution was used as the raw material, and different water content gradients (approximately 27.3%, 13.6%, 6.8%, and 3.4%) were added to component A. After mixing, an equal amount of component B was added, and the mixture was stirred for 10 min before being allowed to cure at 90℃. The results showed that when the water content was higher than 13.6%, the strong hydrophilicity of water was incompatible with the hydrophobicity of the silicone matrix of the AB adhesive, easily leading to phase separation or local aggregation of the indicator, resulting in uneven color spots on the membrane surface. Simultaneously, excessive water would compete with the crosslinking agent and curing agent in the AB adhesive, prolonging the curing time, causing pores or agglomeration in the membrane structure, and reducing performance consistency. When the water content was controlled at 6.8%, the indicator dispersion was the most uniform, with good solubility, a stable curing process, and a smooth and uniform surface and color of the resulting sensing membrane. Further reducing the water content would result in insufficient solvent volume, incomplete dissolution of the indicator, and precipitation. Considering both the indicator's solubility and the system's homogeneity, the water content was controlled at approximately 6.8%, which ensured sufficient substrate dispersion while avoiding phase separation and solidification interference.

[0132] Table 3. Effect of moisture content on uniform dispersion of substrate in AB glue

[0133]

[0134] (3) The effect of temperature on colorimetric sensing film

[0135] The silicone rubber matrix used in this study requires a relatively high temperature environment to achieve full cross-linking and solidification, forming a structurally stable membrane. Furthermore, to simplify the overall production and application process, the sensing membrane must be able to withstand high-temperature sterilization conditions to avoid membrane failure due to the sterilization process. This study, while maintaining a constant system ratio, set different curing temperature gradients (50℃, 70℃, 90℃, 110℃) to evaluate the solidification effect and the color stability of the formed film. The uniformly mixed sensing membrane was prepared according to a predetermined ratio, injected into empty blood culture bottles, and placed in a constant temperature oven for curing at the set temperature for 150 min. After curing, samples were taken and cooled to room temperature, and color changes were recorded. The smoothness, adhesion, and color consistency of the membrane at different temperatures were compared. Experimental results showed that the membrane could be successfully cured within the temperature range of 50–110℃, with no obvious structural differences or color shifts observed, indicating that the curing effect was not significantly different within this temperature range, and the color was relatively stable before and after solidification. However, after high-temperature sterilization, the sensor membrane generally turned yellow, and this phenomenon was more pronounced at 121℃ for 20 minutes than at 115℃ for 30 minutes. Considering the importance of color stability to the performance of the sensor membrane, 115℃ will be selected as the sterilization condition for subsequent experiments. In addition, to mitigate the heat-induced color background change, experiments with the addition of sodium hydroxide will be conducted to optimize the chemical stability of the membrane.

[0136] Table 4. Effect of temperature on colorimetric sensing film

[0137]

[0138] (4) Effect of sodium hydroxide addition on colorimetric sensor membrane

[0139] Sodium hydroxide, a common alkaline regulator, primarily affects the chemical stability and color change of indicators by adjusting the acid-base environment of the sensing membrane system. In colorimetric sensing membranes, the addition of sodium hydroxide can effectively mitigate color shifts caused by high-temperature sterilization, helping to improve the color stability of the sensing membrane after high-temperature treatment and ensuring that the optical performance of the membrane remains consistent under different conditions. With other components fixed, three experimental groups were set up with 0.1 ml (experimental group 1), 0.2 ml (experimental group 2), and 0.3 ml (experimental group 3) of 0.035M sodium hydroxide solution. After stirring and mixing for 25 minutes, the mixture was poured into the bottom of blood culture bottles containing 12.36 mg of primer and cured at 90℃ for 150 minutes, resulting in four sets of experimental sensing membranes. Nutrient solution was then added to obtain three sets of self-made blood culture bottles. Each blood culture bottle was divided into a positive group and a negative group, with two bottles in each group. The positive group was inoculated with 1 mL of working Escherichia coli (ATCC 25922, purchased from Microbiologics®) suspension (1000 CFU), and the results were statistically analyzed after 12 hours of incubation. The results showed that experimental group 1, with the addition of 0.2 mL of sodium hydroxide, exhibited more stable performance, and the difference between the positive and negative groups was more significant, indicating that this amount of added sodium hydroxide helps optimize the stability and detection sensitivity of the sensing membrane.

[0140] Table 5. Effect of sodium hydroxide addition on colorimetric sensor membrane

[0141]

[0142] (5) Effect of the amount of opaque agent added on colorimetric sensing film

[0143] As a crucial factor affecting the performance of colorimetric sensor membranes, the opacifier's role is to regulate the membrane's optical properties, thereby controlling its sensitivity and color change under light. With other components kept constant, four experimental groups were prepared with opacifier additions of 0 mg (experimental group 1), 2 mg (experimental group 2), 4 mg (experimental group 3), and 6 mg (experimental group 4). The mixture was stirred for 25 minutes and then poured into the bottom of blood culture bottles containing 12.36 mg of primer. The mixture was cured at 90°C for 150 minutes to prepare the four experimental sensor membranes. Nutrient solution was then added to obtain four sets of self-made blood culture bottles. Each blood culture bottle was divided into a positive group and a negative group, one bottle per group. The positive group was inoculated with 1 mL (1000 CFU) of working Escherichia coli (ATCC25922, purchased from Microbiologics®). After 12 hours of incubation, the results were statistically analyzed. Experimental results showed that when the dosage was too high (60g), the overall color of the sensing membrane became lighter and white and cloudy, which weakened the color reaction difference during the detection process, resulting in insufficient differentiation between the positive and negative groups and a significant decrease in detection sensitivity. When the dosage was too low (e.g., 0mg, 2mg), the transparency of the sensing membrane was too high, making it susceptible to interference from external light, resulting in insufficient performance stability and responsiveness. Taking all factors into consideration, 4mg was determined to be the optimal dosage. This dosage can effectively block external light interference to ensure stability, while also preserving the color reaction intensity and detection sensitivity of the sensing membrane, meeting the needs of practical applications.

[0144] Table 6. Effect of Opacity Addition on Colorimetric Sensing Film

[0145]

[0146] (6) Effect of glycerol addition on colorimetric sensor membrane

[0147] In the preparation of colorimetric sensing membranes, glycerol, as one of the solvents, mainly plays a role in helping the indicator to disperse uniformly. Due to its high hydrophilicity and good solubility, glycerol can effectively reduce the tendency of the indicator to aggregate in the matrix, thereby promoting the uniform dispersion of the indicator into small droplets. By optimizing the amount of glycerol added, the dispersibility of the indicator in the matrix can be improved, ensuring that the sensing membrane has consistent performance and response speed under different conditions. Keeping the other components constant, three experimental groups were set up with glycerol addition amounts of 0 mg (experimental group 1), 80 mg (experimental group 2), and 160 mg (experimental group 3). After stirring and mixing for 25 minutes, the mixture was poured into the bottom of a blood culture bottle containing 12.36 mg of primer and cured at 90℃ for 150 minutes to prepare the three sets of experimental sensing membranes. Nutrient solution was then added to obtain three sets of self-made blood culture bottles. Each blood culture bottle was divided into a positive group and a negative group, with one bottle in each group. The positive group was inoculated with 1 mL (1000 CFU) of working Escherichia coli (ATCC25922, purchased from Microbiologics®). Results were analyzed after 12 hours of incubation. The results showed that the amount of glycerol added significantly affected the performance of the sensor membrane. When the amount of glycerol added was 80 mg, experimental group 2 exhibited the best dispersibility and responsiveness, significantly superior to the other experimental groups. With too little glycerol (0 mg) or too much (160 mg), the detection sensitivity of the sensor membrane was low and the color reaction differences were small. In conclusion, 80 mg was determined to be the optimal amount of glycerol added, which effectively optimizes the dispersibility and detection performance of the sensor membrane.

[0148] Table 7. Effect of Glycerin Addition Amount on Colorimetric Sensing Membrane

[0149]

[0150] Example 1: Three engineered bacterial strains were simultaneously tested on a blood culture bottle using a self-made colorimetric sensing membrane.

[0151] (1) Preparation of colorimetric sensing membrane

[0152] Mix 1.2 mg bromothymol blue, 3000 mg ELASTOSIL® A glue, 350 mg ELASTOSIL® B glue, 6 mg opacifier, 5 mg inhibitor, 150 mg glycerol, and 0.3 ml 0.035 M sodium hydroxide solution to a weakly alkaline state. Stir for 25 minutes and pour the mixture into the bottom of a blood culture bottle containing 12.36 mg primer. Cure at 90°C for 60 minutes to obtain the desired sensing membrane.

[0153] (2) Preparation of working bacterial suspension

[0154] 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.

[0155] (3) Blood culture experiment

[0156] The colorimetric blood culture bottles prepared using the sensing membrane technology of this embodiment were used in a comparative experiment on a fully automated microbial culture system. Specifically, eight bottles of each type of blood culture bottle were prepared and divided into four groups: positive group 1, positive group 2, positive group 3, and negative group, with two bottles in each group. Positive group 1 was inoculated with 1 mL (1000 CFU) of working bacterial suspension of *Escherichia coli* (ATCC25922, purchased from Microbiologics®), positive group 2 was inoculated with 1 mL (1000 CFU) of working bacterial suspension of *Staphylococcus aureus* (ATCC25923, Microbiologics®), and positive group 3 was inoculated with 1 mL (1000 CFU) of working bacterial suspension of *Candida albicans* (ATCC18804, purchased from ATCC®). After 48 hours of incubation, the results were analyzed. If a positive result was reported, the culture had to be inoculated onto the corresponding plate to confirm bacterial growth before being recorded as positive; otherwise, it was recorded as negative.

[0157] (4) Experimental results

[0158] For Escherichia coli, the reflectance detection curve is as follows: Figure 4 As shown in Figure A, the positive result is reported within 9-10 hours; verification can be performed using Columbia blood agar plate culture (e.g., ...). Figure 4 (As shown in B)

[0159] For Staphylococcus aureus, the reflectance detection curve is as follows: Figure 5 As shown in A, the positive reporting time is 17-19 hours; verification via Columbia blood agar plate culture (e.g.) Figure 5 (As shown in B)

[0160] For Candida albicans, the reflectance detection curve is as follows: Figure 6 As shown in Figure A, the positive reporting time is 17-18 hours; verification via Sabouraud agar plate culture (e.g.) Figure 6 (As shown in B)

[0161] The experimental data above show that the colorimetric sensing membrane described in this invention can accurately detect Gram-negative bacteria, Gram-positive bacteria, and yeast-type fungi. The positive reporting time for each strain meets clinical testing requirements, the detection results are accurate and reliable, and it exhibits good compatibility with existing mainstream blood culture instruments. The results demonstrate that this product performs excellently in all performance indicators and meets the requirements for blood culture.

[0162] Table 8. Experimental Results of Example 1

[0163]

[0164] Example 2: Three engineered bacterial strains were simultaneously tested on a blood culture bottle using a self-made colorimetric sensing membrane.

[0165] (1) Preparation of colorimetric sensing membrane

[0166] Mix 0.4 mg bromothymol blue, 2500 mg ELASTOSIL® A glue, 250 mg ELASTOSIL® B glue, 2 mg opacifier, 5 mg inhibitor, 50 mg glycerol, and 0.1 ml 0.035 M sodium hydroxide solution until weakly alkaline. Stir and mix for 25 minutes, then pour the mixture into the bottom of a blood culture bottle containing 12.36 mg primer. Cure at 50°C for 240 minutes to obtain the desired sensing membrane.

[0167] (2) Preparation of working bacterial suspension

[0168] 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.

[0169] (3) Blood culture experiment

[0170] The colorimetric blood culture bottles prepared using the sensing membrane technology of this embodiment were used in a comparative experiment on a fully automated microbial culture system. Specifically, eight bottles of each type of blood culture bottle were prepared and divided into four groups: positive group 1, positive group 2, positive group 3, and negative group, with two bottles in each group. Positive group 1 was inoculated with 1 mL (1000 CFU) of working bacterial suspension of *Escherichia coli* (ATCC25922, purchased from Microbiologics®), positive group 2 was inoculated with 1 mL (1000 CFU) of working bacterial suspension of *Staphylococcus aureus* (ATCC25923, Microbiologics®), and positive group 3 was inoculated with 1 mL (1000 CFU) of working bacterial suspension of *Candida albicans* (ATCC18804, purchased from ATCC®). After 48 hours of incubation, the results were analyzed. If a positive result was reported, the culture had to be inoculated onto the corresponding plate to confirm bacterial growth before being recorded as positive; otherwise, it was recorded as negative.

[0171] (4) Experimental results

[0172] For Escherichia coli, the reflectance detection curve is as follows: Figure 7 As shown in Figure A, the positive result is reported within 8-9 hours; verification can be performed using Columbia blood agar plate culture (e.g., Figure 7 (As shown in B)

[0173] For Staphylococcus aureus, the reflectance detection curve is as follows: Figure 8 As shown in Figure A, the positive result was reported within 18-19 hours; verification was performed using Columbia blood agar plate culture (as shown in Figure A). Figure 8 (As shown in B)

[0174] For Candida albicans, the reflectance detection curve is as follows: Figure 9 As shown in Figure A, the positive reporting time is 16-17 hours; verification via Sabouraud agar plate culture (e.g.) Figure 9 (As shown in B)

[0175] The experimental data above show that the colorimetric sensing membrane described in this invention can accurately detect Gram-negative bacteria, Gram-positive bacteria, and yeast-type fungi. The positive reporting time for each strain meets clinical testing requirements, the detection results are accurate and reliable, and it exhibits good compatibility with existing mainstream blood culture instruments. The results demonstrate that this product performs excellently in all performance indicators and meets the requirements for blood culture.

[0176] Table 9. Experimental Results of Example 2

[0177]

[0178] Example 3: Three engineered bacterial strains were simultaneously tested on a blood culture bottle using a self-made colorimetric sensing membrane.

[0179] (1) Preparation of colorimetric sensing membrane

[0180] Mix 0.8 mg bromothymol blue, 2700 mg ELASTOSIL® A glue, 300 mg ELASTOSIL® B glue, 5 mg opacifier, 5 mg inhibitor, 80 mg glycerol, and 0.2 ml 0.035 M sodium hydroxide solution until weakly alkaline. Stir for 25 minutes and pour into the bottom of a blood culture bottle containing 12.36 mg primer. Cure at 70°C for 150 minutes to obtain the desired sensing membrane.

[0181] (2) Preparation of working bacterial suspension

[0182] 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.

[0183] (3) Blood culture experiment

[0184] The colorimetric blood culture bottles prepared using the sensing membrane technology of this embodiment were used in a comparative experiment on a fully automated microbial culture system. Specifically, eight bottles of each type of blood culture bottle were prepared and divided into four groups: positive group 1, positive group 2, positive group 3, and negative group, with two bottles in each group. Positive group 1 was inoculated with 1 mL (1000 CFU) of working bacterial suspension of *Escherichia coli* (ATCC25922, purchased from Microbiologics®), positive group 2 was inoculated with 1 mL (1000 CFU) of working bacterial suspension of *Staphylococcus aureus* (ATCC25923, Microbiologics®), and positive group 3 was inoculated with 1 mL (1000 CFU) of working bacterial suspension of *Candida albicans* (ATCC18804, purchased from ATCC®). After 48 hours of incubation, the results were analyzed. If a positive result was reported, the culture had to be inoculated onto the corresponding plate to confirm bacterial growth before being recorded as positive; otherwise, it was recorded as negative.

[0185] (4) Gram staining and mass spectrometry identification

[0186] All blood culture bottles reporting positive results underwent subsequent microbiological verification. A small amount of culture medium was aseptically aspirated and directly smeared. Gram staining was performed according to Table 10, followed by observation under an oil immersion microscope to preliminarily determine the morphological characteristics and staining properties of the microorganisms, which were then photographed and recorded. Simultaneously, the culture medium from the bottle was transferred and streaked onto blood agar plates, incubated at 35°C for 24 hours to obtain pure single colonies. After colony formation, their morphological characteristics were further observed and recorded. Fresh single colonies were then selected and subjected to matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS, model Antu Autof ms1000) for final species identification to ensure that the identification results were completely consistent with the inoculated strain.

[0187] Table 10. Gram staining procedure

[0188]

[0189] (4) Experimental results

[0190] For Escherichia coli, the reflectance detection curve is as follows: Figure 10 As shown in Figure A, the positive reporting time is 8-9 hours (Table 11); this was verified by culture on Columbia blood agar plates (e.g., Figure 10 As shown in Figure B), Gram staining and microscopic examination revealed Gram-negative short bacilli (such as...). Figure 10 As shown in C), the MALDI-TOF MS microbial mass spectrometry identification score was 9.421 (as shown in C). Figure 10 (As shown in D), the identification result was Escherichia coli;

[0191] For Staphylococcus aureus, the reflectance detection curve is as follows: Figure 11As shown in Figure A, the positive reporting time is 17-19 hours (Table 11); this was verified by culture on Columbia blood agar plates (e.g., Figure 11 As shown in Figure B), Gram staining and microscopic examination revealed Gram-positive cocci (e.g., ...). Figure 11 As shown in C), the VITEK MS microbial mass spectrometry identification score was 9.625 (as shown in C). Figure 11 As shown in Figure D), the identification result was Staphylococcus aureus;

[0192] For Candida albicans, the reflectance detection curve is as follows: Figure 12 As shown in Figure A, the positive reporting time is 16-17 hours (Table 11); this was verified by culture on Sabouraud agar plates (e.g., Figure 12 As shown in Figure B), Gram staining and microscopic examination revealed Gram-positive yeast-like fungi (such as...). Figure 12 As shown in C), the VITEK MS microbial mass spectrometry identification score was 9.538 (as shown in C). Figure 12 As shown in D), the identification result was Candida albicans;

[0193] The experimental data above show that the colorimetric sensing membrane described in this invention can accurately detect Gram-negative bacteria, Gram-positive bacteria, and yeast-type fungi. The positive reporting time for each strain meets clinical testing requirements, the detection results are accurate and reliable, and it exhibits good compatibility with existing mainstream blood culture instruments. The results demonstrate that this product performs excellently in all performance indicators and meets the requirements for blood culture.

[0194] Table 11. Experimental Results of Example 3

[0195]

[0196] 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 colorimetric sensing composition characterized in that, by mass, comprising: silicone gel A 2500~3000 parts; silicone gel B 250~350 parts; vulcanization inhibitor 1~5 parts; sodium hydroxide 0.1~0.3 parts; dispersant 50~150 parts; opacifier 2~6 parts; indicator 0.4~1.2 parts; the silicone gel A comprises polydimethylsiloxane; the silicone gel B comprises functional group-containing polydimethylsiloxane and platinum gold; the functional group comprises one or more of hydroxyl, carboxyl, aldehyde or amino.

2. The colorimetric 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 (8~9.9):(0.1~2).

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

4. The colorimetric sensing composition of claim 3, wherein the opacifier comprises polydimethylsiloxane and pigment; the pigment comprises one or more of titanium dioxide, zinc white (ZnO) or calcium carbonate (CaCO3); the mass ratio of the dimethylsiloxane to the pigment is (0.1~2):(8~9.9).

5. The colorimetric sensing composition of claim 4, wherein the indicator comprises one or more of thymol blue, bromothymol blue, methyl red, p-dimethylphenyl blue, phenolphthalein; the dispersant comprises glycerol.

6. The colorimetric sensing composition of claim 5, wherein a primer and / or a solvent are further included; the primer comprises C7-C9 isoalkane and titanium tetrabutoxide; the colorimetric sensing composition comprises the primer 5~20 parts by mass; the colorimetric sensing composition comprises the solvent 100~300 parts by mass; the solvent comprises double distilled water.

7. The colorimetric sensing composition of claim 6, wherein the primer comprises functional group-containing polydimethylsiloxane, C7-C9 isoalkane, titanium tetrabutoxide and tetraethyl silicate; the mass ratio of the functional group-containing polydimethylsiloxane, the C7-C9 isoalkane, the titanium tetrabutoxide and the tetraethyl silicate is (0.05~0.2):(0.5~0.9):(0.01~0.1):(0.005~0.05); or the primer comprises titanium tetrabutoxide and heptane; the ratio of the titanium tetrabutoxide to the heptane is (0.1~0.4):(0.6:~0.9).

8. The colorimetric sensing composition of claim 7, wherein, its pH value comprises 7.0~10.

0.

9. The colorimetric sensing composition of claim 8, wherein, by mass, comprising: silicone gel A 2500~3000 parts; silicone gel B 250~350 parts; vulcanization inhibitor 5 parts; sodium hydroxide 0.1~0.3 parts; glycerol 50~150 parts; opacifier 2~6 parts; indicator 0.4~1.2 parts; double distilled water 100~300 parts; primer 12.36 parts.

10. The colorimetric sensing composition of claim 9, wherein by mass, comprising: silicone gel A 3000 parts; silicone gel B 350 parts; vulcanization inhibitor 5 parts; sodium hydroxide 0.3 parts; glycerol 3 parts; opacifier 6 parts; indicator 1.2 parts; double distilled water 300 parts; primer 12.36 parts; or the colorimetric sensing composition comprises: silicone gel A 2500 parts; silicone gel B 250 parts; vulcanization inhibitor 5 parts; sodium hydroxide 0.1 parts; glycerol 50 parts; opacifier 2 parts; indicator 0.4 parts; double distilled water 100 parts; primer 12.36 parts; or the colorimetric sensing composition comprises: silicone gel A 2700 parts; silicone gel B 300 parts; vulcanization inhibitor 5 parts; sodium hydroxide 0.2 parts; glycerol 80 parts; sunscreen 5 parts; indicator 0.8 parts; double distilled water 200 parts; primer 12.36 parts.

11. Use of the colorimetric sensing composition according to any one of claims 1 to 10 in the preparation of a colorimetric sensing film.

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

13. A colorimetric sensing membrane characterized by, comprising the colorimetric sensing composition according to any one of claims 1 to 10.

14. The method for preparing the colorimetric sensing membrane as described in claim 13, characterized in that, mixing, curing the colorimetric sensing composition according to any one of claims 1 to 10 to obtain the colorimetric sensing film.

15. The method of claim 14, wherein the step of preparing is characterized by, The mixing time is 25 min; the curing temperature is 50-90℃, and the curing time is 60-240 min.

16. The colorimetric sensing film prepared according to the preparation method of claim 15.

17. Use of the colorimetric sensing film according to claim 13 or claim 16 in the preparation of a blood culture detection device or a blood culture detection system.

18. The use according to claim 17, wherein The blood culture detection device comprises a blood culture bottle.

19. A blood culture detection device, characterized by comprising the colorimetric sensing film according to claim 13 or claim 16.

20. The blood culture detection device as described in claim 19, characterized in that, comprising a blood culture bottle.

21. A blood culture detection system characterized in that, comprising the blood culture detection device according to claim 19 or claim 20.

22. Use of any one of: (I) the colorimetric sensing film according to claim 13 or claim 16; (II) the blood culture detection device according to claim 19 or claim 20; (III) the blood culture detection system according to claim 21.

23. A product for the detection of microorganisms or a product for the diagnosis of blood stream infections, characterized in that, comprising any one of: (I) the colorimetric sensing composition according to any one of claims 1 to 10; (II) the colorimetric sensing film according to claim 13 or claim 16; (III) the blood culture detection device according to claim 19 or claim 20; (IV) the blood culture detection system according to claim 21.

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