Synchronous quantitative detection method for cholate-resistant gram-negative bacteria in different active states

Abstract

The invention provides a synchronous quantitative detection method of cholate-resistant gram-negative bacteria in different active states, which comprises the following steps: (1) adding a capture probe into a bacteria solution to be detected, carrying out illumination culture to obtain a compound solution, and centrifuging the compound solution to obtain a supernatant and a precipitate resuspension; (2) adding a signal probe into the resuspension, and carrying out illumination inactivation and incubation to obtain a sandwich compound solution; (3) calculating to obtain the total bacterial count and the number of bile salt-resistant gram-negative bacteria in the living body; (4) calculating to obtain the quantity of bile salt-resistant gram-negative dead bacteria; and (5) calculating to obtain the quantity of the cholate-resistant gram-negative sublethal bacteria. The synchronous quantitative detection of the bile salt-resistant gram-negative bacteria in different active states adopts a Raman and bioluminescence dual-signal detection method, and can realize rapid and sensitive detection of living, dead and sub-lethal bile salt-resistant gram-negative bacteria.

Description

Technical Field

[0001] This invention belongs to the field of bacterial detection, and in particular relates to a method for simultaneous quantitative detection of bile salt-resistant Gram-negative bacteria in different active states. Background Technology

[0002] Bile salt-resistant Gram-negative bacteria are a class of Gram-negative bacteria that can survive and multiply in bile acids. They mainly include *Pseudomonas*, *Enterobacter*, and *Aeromonas*. *Pseudomonas*, primarily including *Pseudomonas aeruginosa* and *Pseudomonas fluorescens*, can cause infections in multiple organs and systemic complications, and is highly susceptible to drug resistance during treatment. *Escherichia coli* is a typical *Enterobacter* bacterium; infection in humans can cause diarrhea, cholecystitis, cystitis, and other diseases. Especially in infants and young children, it can cause acute diarrhea and meningitis, which can be life-threatening in severe cases. *Aeromonas* is a widely distributed opportunistic pathogen in aquatic environments, surviving under both anaerobic and aerobic conditions, and can cause enteritis, sepsis, and other diseases. The broad classification and large number of species of bile salt-resistant Gram-negative bacteria pose a significant challenge to the tracing and analysis of microbial contamination sources and risk control. Therefore, developing accurate, sensitive, and efficient detection methods for bile salt-resistant Gram-negative bacteria is an urgent need for safety and quality control in the food, pharmaceutical, and environmental sectors.

[0003] Meanwhile, the detection of different active bile-resistant Gram-negative bacteria is even more important. Because active bile-resistant Gram-negative bacteria have strong reproductive capabilities and environmental resistance, they can cause large-scale food contamination in a short period, leading to infectious diseases. Therefore, current detection and counting methods focus more on the detection and sterilization of live bacteria, neglecting bacteria in different active states, especially dead and sublethal bile-resistant Gram-negative bacteria. In fact, most bile-resistant Gram-negative bacteria release a secondary metabolite—endotoxin—after death, which poses a greater threat to humans. Endotoxin is mainly composed of phospholipid polysaccharides and proteins, and has strong heat resistance and chemical stability, making it difficult to inactivate and remove using conventional methods. Once endotoxin enters the human bloodstream, it causes a strong pyrogenic reaction, mainly manifested as fever, microcirculatory disturbances, endotoxin shock, and disseminated intravascular coagulation. At the same time, sublethal bile-resistant Gram-negative bacteria should not be ignored. Sublethal bile salt-resistant Gram-negative bacteria refer to bacteria that cannot grow in selective culture media but still possess pathogenicity or potential pathogenicity. When encountering suitable growth conditions, they can reactivate and pose a renewed threat to the surrounding environment and human safety. A typical form of existence is the "live but unculturable" state, and these bile salt-resistant Gram-negative bacteria are frequently found during sterilization processes. Therefore, strengthening our understanding of the active state of bile salt-resistant Gram-negative bacteria and strictly preventing secondary hazards caused by dead and sublethal bile salt-resistant Gram-negative bacteria, while simultaneously detecting live, dead, and sublethal bile salt-resistant Gram-negative bacteria, is highly beneficial for improving the quality of soil, water, air, traditional Chinese medicine materials and processed medicinal slices, and protecting public health.

[0004] Currently, conventional pathogen detection methods such as plate counting, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) have excellent accuracy, but they still have significant limitations in detecting bile salt-resistant Gram-negative bacteria with different activities. For example, culture methods can only count live bacteria and cannot detect dead and sub-lethal bacteria or the risk of residual endotoxins. In addition, antigenic or nucleic acid information of bile salt-resistant Gram-negative bacteria can remain for a long time after death, and ELISA and PCR methods still cannot accurately distinguish bacterial activity and sense the live / dead state. ATP bioluminescence technology is an effective analytical method for sensitively counting live bacteria based on fluorescent enzyme reactions, but it is powerless against dead and sub-lethal bacteria. Surface-enhanced Raman spectroscopy (SERS) is a molecular vibrational spectroscopy that can achieve "fingerprint recognition." With its advantages of high sensitivity, excellent selectivity, and rapid non-destructive detection, it has been used for quantitative detection of bacteria in different active states and rapid differentiation of different bacterial species. However, no research has yet applied this method to the simultaneous quantitative detection of live, sub-lethal, and dead bacteria. Summary of the Invention

[0005] In view of this, the present invention aims to overcome the deficiencies in the prior art and propose a method for simultaneous quantitative detection of bile salt-resistant Gram-negative bacteria in different active states.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0007] This invention provides a method for simultaneous quantitative detection of bile salt-resistant Gram-negative bacteria in different activity states, comprising the following steps:

[0008] (1) Add the capture probe to the bacterial culture to be tested, and after culturing under light, a complex solution is obtained. After centrifuging the complex solution, a supernatant and a precipitate are obtained. The precipitate is resuspended to obtain a resuspension.

[0009] (2) Add a signal probe to the resuspended solution, and obtain a sandwich complex solution after photoinactivation and incubation;

[0010] (3) The Raman spectral signal and relative luminescence intensity of the sandwich complex solution were measured to obtain the relationship between the ratio of the sum of the number of bile salt-resistant Gram-negative dead bacteria and the number of bile salt-resistant Gram-negative sublethal bacteria to the total number of bacteria and the Raman spectral signal intensity, as well as the relationship between the number of bile salt-resistant Gram-negative live bacteria and the relative luminescence intensity. The total number of bacteria and the sum of the number of bile salt-resistant Gram-negative dead bacteria and the number of bile salt-resistant Gram-negative sublethal bacteria were calculated.

[0011] (4) Add the endotoxin targeting probe to the supernatant, and after the reaction, obtain a magnetic dispersion containing endotoxin. Spray the magnetic dispersion containing endotoxin onto the SERS array chip and perform Raman spectroscopy scanning to establish a quantitative relationship between the Raman spectral signal of the characteristic peak of endotoxin and the concentration of endotoxin particles, and calculate the number of bile salt-resistant Gram-negative dead bacteria.

[0012] (5) Calculate the sum of the number of bile salt-resistant Gram-negative dead bacteria and the number of bile salt-resistant Gram-negative sublethal bacteria in step (3) and the difference between the number of bile salt-resistant Gram-negative dead bacteria in step (4) to obtain the number of bile salt-resistant Gram-negative sublethal bacteria.

[0013] The total bacterial count is the sum of the number of bile salt-resistant Gram-negative live bacteria, bile salt-resistant Gram-negative sublethal bacteria, and bile salt-resistant Gram-negative dead bacteria.

[0014] Furthermore, in step (3), when the ratio X1 of the sum of the number of bile salt-resistant Gram-negative sublethal bacteria and bile salt-resistant Gram-negative dead bacteria to the total number of bacteria is 0.01-50%, the relationship between the Raman spectral signal intensity Y1 and the ratio X1 is Y1 = a1 + b1X1, where the value of a1 is 13362.1-14398.3 and the value of b1 is -148.2--108.3; when the ratio X2 of the sum of the number of bile salt-resistant Gram-negative sublethal bacteria and bile salt-resistant Gram-negative dead bacteria to the total number of bacteria is 51-100%, the relationship between the Raman spectral signal intensity Y2 and the ratio X2 is Y2 = a2 + b2X2, where the value of a2 is 12280.6-13945.4 and the value of b2 is -99.5--79.1.

[0015] Furthermore, in step (3), when the concentration Z1 of bile salt-resistant Gram-negative viable bacteria is 1.18-1.18×10⁻⁶... 4 At CFU / mL, the bioluminescence intensity Y BL1 The relationship between Y and the viable bacteria concentration Z1 is: BL1 = aZ1 + b, where a is 80–120 and b is 9–13; when the concentration of bile salt-resistant Gram-negative viable bacteria Z2 is 1.19 × 10⁻⁶. 4 -1.18×10 7 At CFU / mL, the bioluminescence intensity Y BL2 The relationship between Y and the viable bacteria concentration Z2 is as follows: BL2 = aZ2+b, where the value of a is 1600~2100, and the value of b is -6100~-8920;

[0016] In step (4), the quantitative relationship between the Raman spectral signal Y of the characteristic peak of endotoxin and the concentration X of endotoxin particles is Y = a + bX, where the value of a is -900 to -1030, the value of b is 750 to 785, and the concentration of X is 0.01 -8 μg / mL.

[0017] Furthermore, the bile salt-resistant Gram-negative bacteria are one of Escherichia coli, Pseudomonas aeruginosa, Salmonella, or Shigella; the different active states of the bile salt-resistant Gram-negative bacteria include live bile salt-resistant Gram-negative bacteria, dead bile salt-resistant Gram-negative bacteria, and sublethal bile salt-resistant Gram-negative bacteria; the dead bile salt-resistant Gram-negative bacteria are bile salt-resistant Gram-negative bacteria that have died naturally, been inactivated by drugs, or been inactivated by physical conditions; the drug inactivation method is at least one of nutrient deficiency inactivation, antibiotic inactivation, or antimicrobial drug inactivation; the physical inactivation method is at least one of high temperature inactivation, high pressure inactivation, moist heat inactivation, light inactivation, or radiation inactivation; the sublethal bile salt-resistant Gram-negative bacteria are bile salt-resistant Gram-negative sublethal bacteria induced by mild sterilization conditions; the mild sterilization conditions are at least one of low-concentration disinfectant, antibacterial traditional Chinese medicine, or low-concentration antibiotic.

[0018] Furthermore, the capture probe in step (1) is at least one of lectin, bacterial aptamer, bacterial antibody, boric acid-modified metal compound, carbon-based material or black phosphorus nanocomposite.

[0019] Further, the signal probe in step (2) is at least one of a bacterial aptamer or an antibody-modified nanocomposite; the nanocomposite of the bacterial aptamer or antibody-modified nanocomposite is a nanocomposite with a layered structure, the number of layers of the layered structure being greater than or equal to 3; the outermost layer of the nanocomposite is at least one of gold or silver; the nanocomposite contains PB; the nanocomposite is at least one of Ag@PB@Au, Ag@PB@Ag, Au@PB@Au, Au@PB@Ag, Ag@PB@Au@Ag, Ag@PB@Ag@Au, Au@PB@Au@Ag, or Au@PB@Ag@Au.

[0020] Furthermore, in step (1), the light cultivation step is irradiated with LED lamps or near-infrared light for 0.5-12 hours; the culture medium used in step (1) contains 0.3-0.5% bile salts by mass concentration; in step (2), the light inactivation step is irradiated with LED lamps or near-infrared light for 2-30 minutes; in step (2), the incubation time is 30-120 minutes; and the parameters for measuring the Raman spectral signal in step (3) are: excitation wavelength of 785 nm, power of 60-80 mW, acquisition and exposure time of 1 s, and objective lens of 10×.

[0021] Furthermore, the endotoxin targeting probe in step (4) is a nanocomposite modified with bacterial endotoxin aptamers; the nanocomposite is a composite formed by magnetic nanomaterials or metal framework organic materials and gold and / or silver; the nanocomposite is at least one of Fe3O4@Au, Fe3O4@Ag, Fe3O4@Au@Ag, Fe3O4@Ag@Au, PB@Au, PB@Ag, PB@Au@Ag or PB@Ag@Au; the reaction temperature in step (4) is 25-40℃ and the time is 1-5h; the coating material of the SERS array chip in step (4) is gold and / or silver; the substrate of the SERS array chip in step (4) is an aluminum substrate or a silicon substrate; the SERS array chip in step (4) has 1-2000 pores, and the diameter of each pore is 1-1000μm.

[0022] Furthermore, in step (1), the proportion of bile salt-resistant Gram-negative viable bacteria in the test bacterial solution is 0.01-100% of the total number of bacteria, the proportion of bile salt-resistant Gram-negative dead bacteria is 0.01-100% of the total number of bacteria, and the proportion of bile salt-resistant Gram-negative sublethal bacteria is 0.01-100% of the total number of bacteria; the concentration of the capture probe in step (1) is 0.5-3 mg / mL; the concentration of the signal probe in step (2) is 0.5-3 mg / mL; the volume ratio of the signal probe, resuspension, and capture probe in step (1) in step (2) is 1-3:1:1-3; and the volume ratio of the supernatant to the endotoxin targeting probe in step (4) is 1:0.3-1.

[0023] This invention also provides an application of a method for the simultaneous quantitative detection of bile salt-resistant Gram-negative bacteria in different active states. The detection method is used to detect bile salt-resistant Gram-negative bacteria in different active states in water sources, food, traditional Chinese medicine products, or environmental samples.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] The simultaneous quantitative detection method for bile salt-resistant Gram-negative bacteria in different active states described in this invention employs a dual-signal detection method. Compared to the national standard method, this method is time-saving and labor-saving, enabling rapid and sensitive detection of total, live, and dead bile salt-resistant Gram-negative bacteria. Furthermore, this dual-signal detection method integrates dual identification, efficient inactivation, and sensitive detection functions, simplifying the detection process while effectively avoiding secondary contamination. It provides a faster, more accurate, safer, and more systematic solution for food and drug safety monitoring, especially in scenarios where the risks of live bacterial transmission and dead bacterial endotoxins need to be managed simultaneously. Attached Figure Description

[0026] Figure 1 This is a flowchart of the method for simultaneous quantitative detection of bile salt-resistant Gram-negative bacteria in different activity states as described in the embodiments of the present invention;

[0027] Figure 2 The following are transmission electron microscope images of the probe described in the embodiments of the present invention: wherein, image a is Fe3O4@Au modified with lectin in Example 1, and image b is Au@PB@Au@Ag described in Example 2;

[0028] Figure 3 This is the element mapping diagram of Au@PB@Au@Ag described in Embodiment 2 of the present invention;

[0029] Figure 4 This is a transmission electron microscope image of the Fe3O4@Ag-Ap2 endotoxin targeting probe described in Example 5 of the present invention;

[0030] Figure 5 This is the Raman spectrum of the Pseudomonas aeruginosa endotoxin-pyocyanin target probe described in Example 5 of the present invention.

[0031] Figure 6 The following are schematic diagrams of the control group, the "capture probe-bacteria" complex solution, the "signal probe-bacteria" complex solution, and the sandwich complex solution described in Example 7 of the present invention before and after LED irradiation: where, Figure a is a scanning electron microscope image, and Figure b is a confocal fluorescence imaging image;

[0032] Figure 7 The response curves of different concentrations of live Salmonella Typhimurium and Raman spectral intensities described in Example 8 of this invention;

[0033] Figure 8 This is a graph showing the relationship between the sum of the number of bile salt-resistant Gram-negative dead bacteria and the number of bile salt-resistant Gram-negative sublethal bacteria in Example 8 of the present invention and the Raman intensity.

[0034] Figure 9 The following is a linear relationship diagram of bioluminescence of live Salmonella Typhimurium as described in Example 8 of this invention: where, in figure a, 1.18 × 10⁻⁶ 1 –1.18×10 7 The concentration of CFU / mL, as shown in Figure b, is 1.18 × 10⁻⁶. 1 –1.18×10 4 The concentration of CFU / mL, shown in Figure c is 1.19 × 10⁻⁶. 4 –1.18×10 7 CFU / mL concentration;

[0035] Figure 10 The specificity detection described in Example 10 of the present invention is shown in Figure a, where Figure a is the Raman signal and Figure b is the bioluminescent signal. Detailed Implementation

[0036] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0037] The present invention will be described in detail below with reference to the embodiments.

[0038] Example 1: Preparation of a capture probe

[0039] First, 1 mL of 1% HAuCl4 was mixed with 90 mL of ultrapure water and 2 mL of sodium citrate (38.8 mM) and added to a three-necked flask. Then, 1 mL of 1 mg / mL fresh NaBH4 solution was added to the solution and stirred for 5 min to obtain a wine-red Au nanoseed solution.

[0040] Next, 500 mg of Fe3O4 powder was dissolved in a 12.5 mg / mL PEI solution, sonicated for 2.5 h, magnetically separated and washed three times, and then dried to obtain Fe3O4-PEI powder. Then, 30 mg of Fe3O4-PEI was dissolved in 270 mL of Au nanoseed solution, sonicated for 2 h, magnetically separated and washed three times to obtain Fe3O4-PEI-M. seed Solution. Fe3O4-PEI-M seed Dispersed in 150 mL of 0.4 mM HAuCl4 solution, 1.5 mL of 100 mg / mL hydroxylamine hydrochloride solution was added and rapidly sonicated for 5 min. Then, 450 mg of PVP was added and sonicated for 60 min. After magnetic separation three times, the solution was redissolved in ethanol to 3 mL to obtain a 30 mg / mL Fe3O4@Au ethanol solution.

[0041] Then, the Fe3O4@Au ethanol solution was added to 60 mL of anhydrous ethanol, sonicated for 5 min, 12 mL of APTES was added and stirred at room temperature for 6 h, and magnetic separation and washing were performed 3 times to obtain the -NH2 modified Fe3O4@Au solution, which was then redissolved in ethanol to 3 mL.

[0042] Finally, 1 mL of the -NH2-modified Fe3O4@Au solution was dispersed in 10 mL of sterile PBS solution, and 3 mL of 30 mg / mL EDC was added and stirred for 10 min. Then, 2.7 mL of 20 mg / mL NHS was added and stirred for 4 h. After stirring for 3 h, 4 mg of lectin was added and stirred for 3 h. After the end of the process, the solution was washed 3 times with PBS and then reconstituted to 10 mL to obtain the lectin-modified Fe3O4@Au solution.

[0043] Figure 2 Image a shows a transmission electron microscope (TEM) image of Fe3O4@Au modified with lectin. The nanoparticles are spherically distributed with a particle size of approximately 40 nm and exhibit a shell-like distribution, indicating that the lectin ConA is well modified onto the surface of Fe3O4@Au.

[0044] Example 2: Preparation of Salmonella Typhimurium signal probe

[0045] First, 1 mL of 1% HAuCl4 solution was added to a three-necked flask containing 99 mL of ultrapure water. While stirring, the mixture was heated to a gentle boil. Then, 1 mL of 1% sodium citrate solution was quickly added. After stirring for 5 minutes, the power was turned off and stirring was continued for 25 minutes. The mixture was then cooled to room temperature to obtain wine-red Au nanocolloids.

[0046] Next, 10 mL of Au nanocolloid was placed in a 50 mL three-necked flask, 1 mL of 10 mg / mL ascorbic acid was added and stirred for 15 min. Then, 1 mL of 2 mM FeCl2·4H2O solution was added and stirred for 2 min. 1 mL of 2 mM K3[Fe(CN)6] solution was added dropwise over 20 min. After the addition was complete, the mixture was stirred for 10 min. The mixture was then centrifuged at 8000 g and 4 °C for 10 min. The supernatant was removed, and the centrifugation and washing were repeated 3 times. The mixture was then redispersed in 10 mL of sterile PBS to obtain Au@PB nanosolution.

[0047] Then, 1 mL of 20 mg / mL ascorbic acid was added to 10 mL of Au@PB nano solution, and the mixture was stirred for 15 min. Then, 0.15 mL of 1% chloroauric acid solution was added and stirred for 2 min. Next, 1.85 mL of 10 mM AgNO3 solution was added and stirred for 20 min. The mixture was then centrifuged and washed once to obtain Au@PB@Au@Ag nano solution.

[0048] Finally, the activated 2 μM / L Salmonella typhimurium aptamer solution was mixed with Au@PB@Au@Ag nanosolution at a ratio of 1:1 and incubated at 4°C for 8 h. Then, the mixture was centrifuged and washed three times to remove the remaining aptamers in the supernatant, resulting in purified Salmonella typhimurium aptamer-functionalized Au@PB@Au@Ag nanosolution.

[0049] Figure 2 b is a transmission electron microscope image of Au@PB@Au@Ag. It can be seen that the synthesized nanoparticles are spherically distributed, well dispersed and uniform in size, with a particle size of 82 nm.

[0050] Figure 3 The elemental mapping diagram of Au@PB@Au@Ag shows that C, N, Fe, Au, and Ag elements are uniformly distributed on the surface of the Au core, indicating the successful synthesis of Au@PB@Au@Ag nanoparticles. Furthermore, AuAgFe trimetallic doped PB-type metallic framework nanospheres are formed on the Fe, Au, and Ag elements in a ratio of 1:5:62, indicating high iron loading and a moderate level of Au doping.

[0051] Example 3: Preparation of Pseudomonas aeruginosa signal probe

[0052] The only difference from Example 2 is that the aptamer is replaced with 5'-SH-(CH2)6-CCC CCG TTG CTT TCGCTT TTC CTT TCG CTT TTG TTC GTT TCG TCC CTG CTT CCT TTC TTG-3' (SEQ ID NO.1), to obtain the Pseudomonas aeruginosa signal probe.

[0053] Example 4: Preparation of Escherichia coli signal probe

[0054] The only difference from Example 2 is that the aptamer is replaced with 5'-SH-(CH2)6-CAG CTC AGA AGC TTGATC CTA CCA GTA GAC TTT CAA CTT TAC TGC CAT CGT GTG CCC TAA GAC TCG AAG TCGTGC ATC TG-3' (SEQ ID NO.2), thus obtaining the Escherichia coli signal probe.

[0055] Example 5: Preparation of Pseudomonas aeruginosa endotoxin targeting probe

[0056] First, 1 mL of 10 mM AgNO3 was mixed with 90 mL of ultrapure water and 2 mL of sodium citrate (38.8 mM) and added to a three-necked flask to obtain an Ag nano-seed solution. Next, 500 mg of Fe3O4 powder was dissolved in a 12.5 mg / mL PEI solution, sonicated for 2.5 h, magnetically separated and washed three times, and then dried to obtain Fe3O4-PEI powder. Then, 30 mg of Fe3O4-PEI was dissolved in 270 mL of the Ag nano-seed solution, sonicated for 2 h, magnetically separated and washed three times to obtain Fe3O4-PEI-Ag. seed Solution. Fe3O4-PEI-Ag seed The Fe3O4@Ag solution was dispersed in 20 mL of 0.4 mM AgNO3 solution, and 1.5 mL of 80 mg / mL hydroxylamine hydrochloride solution was added. The mixture was rapidly sonicated for 5 min, followed by the addition of 450 mg PVP and sonication for 60 min. After three magnetic separations, the Fe3O4@Ag solution was obtained. The obtained Fe3O4@Ag composite particles were mixed with the Pseudomonas aeruginosa endotoxin aptamer Ap2 at a 1:1 volume ratio in the presence of TCEP and incubated at 4 °C for 2 h. After magnetic separation and washing, the Fe3O4@Ag-Ap2 endotoxin targeting probe was prepared.

[0057] Figure 4 The image shows a transmission electron microscope (TEM) image of the Fe3O4@Ag-Ap2 endotoxin targeting probe. It can be seen that the particles are spherically distributed with an average size of 35 nm. Figure 5 The image shows the Raman spectrum of the *Pseudomonas aeruginosa* endotoxin-targeting probe against the *Pseudomonas aeruginosa* endotoxin-pyocyanin. It can be seen that the Raman signal gradually increases with increasing pyocyanin concentration. This indicates that the prepared endotoxin-targeting probe has a good Raman response to bile salt-resistant Gram-negative bacterial endotoxins.

[0058] Example 6: Preparation of Salmonella Typhimurium endotoxin targeting probe

[0059] The only difference from Example 5 is that the endotoxin aptamer is replaced with the Salmonella typhimurium endotoxin aptamer Ap3, resulting in a Salmonella typhimurium endotoxin targeting probe.

[0060] Example 7: Study on the photothermal sterilization effect of dual-signal biosensor

[0061] First, the ConA-modified Fe3O4@Au capture probe synthesized in Example 1 was mixed with 10 μL of pure water. 7 CFU / mL Salmonella Typhimurium was cultured at 37°C for 60 min, and then separated and washed three times with a magnet to obtain a "capture probe-bacteria" complex solution.

[0062] Secondly, the Au@PB@Au@Ag signal probe, functionalized with the Salmonella typhimurium aptamer synthesized in Example 2, was mixed with 10g of pure water. 7 CFU / mL Salmonella Typhimurium was cultured at 37°C for 60 min, and then separated and washed three times with a magnet to obtain a "signal probe-bacteria" complex solution.

[0063] Next, the ConA-modified Fe3O4@Au capture probe synthesized in Example 1 and the Au@PB@Au@Ag signal probe functionalized with the Salmonella typhimurium aptamer synthesized in Example 2 were sequentially mixed with 10 ml of water diluted with pure water. 7 CFU / mL active Salmonella Typhimurium was incubated at 37℃ for 60 min, and then separated and washed three times with a magnet to obtain a sandwich complex solution of "capture probe-bacteria-signal probe". 10% of this complex was diluted with pure water. 7 CFU / mL active Salmonella Typhimurium was used as the control group.

[0064] Finally, 500 μL of the control group, the "capture probe-bacteria" complex solution, the "signal probe-bacteria" complex solution, and the sandwich complex solution were respectively added to 24-well plates and incubated at 17 W / cm². 2 After irradiation with an LED (5 cm from the well plate) for 10 min, the morphology of the bacteria before and after LED irradiation was observed using SEM, and the survival rate of Salmonella typhimurium was detected using a DMAO / PI double staining kit.

[0065] Figure 6Image a shows scanning electron microscopy (SEM) images of the control group, the "capture probe-bacteria" complex solution, the "signal probe-bacteria" complex solution, and the sandwich-structured complex solution before and after LED irradiation. It can be seen that before LED irradiation, *Salmonella typhimurium* maintained its rod-shaped form, remaining plump, intact, and smooth, with no contents leaking out. Both the capture probe and the signal probe adhered well to the *Salmonella typhimurium*, indicating that their cytotoxicity was negligible. However, after LED irradiation, bacterial fragmentation was clearly observed in the capture probe, signal probe, and sandwich-structured treatment groups, indicating that the bacterial cell structure had collapsed and shrunk. This observation is attributed to the generated high temperature, which disrupted the integrity of the bacterial membrane and led to leakage of cytoplasmic contents. The control group, however, only showed significant shrinkage and indentation, but no rupture. The possible bactericidal mechanism behind this is that the heat generated by the nanotags after LED irradiation causes membrane damage and denaturation of proteins and enzymes, leading to bacterial death. This demonstrates that the capture probe, signal probe, and sandwich-structured complex all have significant photothermal bactericidal effects.

[0066] Figure 6 b shows confocal fluorescence images of the control group, the "capture probe-bacteria" complex solution, the "signal probe-bacteria" complex solution, and the sandwich-filled complex solution before and after LED irradiation. Live *Salmonella typhimurium* and dead *Salmonella typhimurium* showed green and red fluorescence, respectively. It can be seen that before LED irradiation, the fluorescence images of the "capture probe-bacteria" complex solution, the "signal probe-bacteria" complex solution, and the sandwich-filled complex solution were green, further confirming the low cytotoxicity of the capture probe and signal probe against *Salmonella typhimurium*. Furthermore, in the control group, *Salmonella typhimurium* remained viable regardless of laser treatment. This indicates that LED irradiation alone cannot effectively inactivate bacteria, directly confirming the non-invasive effect of LED irradiation on bacteria. However, after LED irradiation, the fluorescence images of the capture probe, signal probe, and sandwich-filled structure treatment groups all changed from green to red, and there was almost no green fluorescence in the sandwich-filled complex solution, indicating a bacterial survival rate of 0, suggesting that this structure has optimal photothermal bactericidal ability.

[0067] Example 8: Simultaneous Detection of Salmonella Typhimurium with Different Active Properties

[0068] First, put 10 3 CFU / mL live Salmonella typhimurium was autoclaved for 20 min to obtain the same concentration of dead Salmonella typhimurium. This was then treated with twice the volume of acidic electrolyzed water containing hypochlorous acid for 10 minutes. 3The same concentration of sublethal Salmonella typhimurium was obtained from live Salmonella typhimurium at CFU / mL. These two types of bacteria were then mixed with freshly cultured live Salmonella typhimurium at the same concentration level in a 1:1:1 ratio to obtain a sample of total Salmonella typhimurium counts at different viable activities. Specifically, the ratio of the sum of the number of bile salt-resistant Gram-negative sublethal bacteria and the number of bile salt-resistant Gram-negative dead bacteria to the total bacterial count was 66.7%.

[0069] Secondly, the capture probe synthesized in Example 1 was combined with the above 10 3 A complex solution was obtained by culturing different active *Salmonella typhimurium* strains at a CFU / mL concentration in a 1:1:1 ratio under near-infrared light for 4 hours. This culturing was performed with the addition of 0.3% bile salts. After culturing and enrichment, the complex solution was centrifuged to obtain a supernatant and a precipitate. The precipitate was then resuspended to obtain a resuspension.

[0070] The signal probe synthesized in Example 2 was added to the resuspension and cultured at 37°C for 60 min. The mixture was then separated and washed three times with a magnet to obtain a sandwich complex solution of "capture probe-mixed total bacteria-signal probe". The solution was then divided into two equal portions.

[0071] Next, a portion of the sandwich composite solution was concentrated 10 times and dropped onto a clean aluminum foil or silicon wafer. After air drying, PB was collected at 2130 cm⁻¹ using a Raman spectrometer. -1 The Raman signal Y2 at the characteristic peak is 12966.5. Based on the relationship between the Raman spectral signal intensity Y2 and the ratio X2 (Y2 = 13020.1 - 80.3X2), the ratio X2 of the sum of the number of bile salt-resistant Gram-negative sublethal bacteria and bile salt-resistant Gram-negative dead bacteria to the total number of bacteria is calculated to be 66.7%, which is consistent with the known ratio, effectively demonstrating the accuracy of the Raman detection. The Raman spectrometer parameters used are as follows: excitation wavelength 785 nm, power 80 mW, acquisition exposure time 1 s, and objective lens 10×.

[0072] Meanwhile, another portion of the sandwich composite solution was taken into a 24-well plate and heated at 17 W / cm². 2 After 10 minutes of irradiation under LED, PB, Au, and Fe3O4 in the complex synergistically exert a photothermal effect, causing localized temperature rise, thereby lysing the cell walls and cell membranes of the contained live bacteria and releasing ATP, yielding a photolyzed lysis buffer. 100 μL of the photolyzed lysis buffer was added to a test tube, along with 100 μL of 3 mg / mL luciferin-luciferase solution, and mixed for 10 seconds. The test tube was then placed in an ATP fluorescence detector for bioluminescence detection, and the bioluminescence signal Y was recorded. BL1 The value is 342.7, according to Y. BL1 Relationship with viable bacteria concentration Z1 Y BL1 =111.26Z1 + 11.27 The viable bacteria concentration Z1 is calculated to be 10.3 The concentration of CFU / mL is consistent with the known concentration added.

[0073] Finally, the *Salmonella typhimurium* endotoxin targeting probe obtained in Example 4 was added to the supernatant. After incubation at 37°C for 2 hours, and magnetic separation and washing, the resulting dispersion containing endotoxin magnetic particles was precisely titrated into each well of the aluminum-core SERS array microchip using a PH-DV200M piezoelectric jet system. Raman spectroscopy was used for point-by-point scanning, and the average Raman intensity value Y of the characteristic peak position of *Salmonella typhimurium* endotoxin was obtained as 101.08. Based on the quantitative relationship between the Raman spectral signal of the characteristic peak position of endotoxin and the concentration of endotoxin particles, Y = -950 + 760X, the endotoxin content of *Salmonella typhimurium* was calculated to be 1.383 μg / mL. Based on the inherent relationship between endotoxin content and bacteria, the number of dead *Salmonella typhimurium* bacteria was estimated to be 10. 3 The CFU / mL concentration is consistent with known concentrations of Salmonella typhimurium in dead mice. Based on the sum of the numbers of bile-resistant Gram-negative dead bacteria and bile-resistant Gram-negative sublethal bacteria, and the difference between the numbers of bile-resistant Gram-negative dead bacteria, the number of bile-resistant Gram-negative sublethal bacteria was determined to be 1.03 × 10⁻⁶. 3 The concentration of CFU / mL is on the same order of magnitude as the sublethal Salmonella typhimurium concentration obtained by known acid treatment, indicating that this method can accurately detect live, dead, and sublethal bile salt-resistant Gram-negative bacteria.

[0074] Figure 7 The response curves of different concentrations of live Salmonella Typhimurium and Raman spectral intensities show that the Raman spectral signal gradually increases with the increase of the concentration of live Salmonella Typhimurium.

[0075] Figure 8 The graph shows the relationship between the ratio of the sum of bile salt-resistant Gram-negative lethal bacteria and bile salt-resistant Gram-negative sublethal bacteria to the total bacterial count and the Raman spectral intensity. It can be seen that the linear relationship of the curve is inconsistent before and after the 50% ratio. Therefore, we divided the curve into two ranges: 0.01-50% and 51%-100%, and examined the linear relationship between the ratio of the sum of bile salt-resistant Gram-negative sublethal bacteria and bile salt-resistant Gram-negative lethal bacteria to the total bacterial count and the Raman spectral intensity.

[0076] Figure 9 AC is 1.18 × 10 1 –1.18×10 7 CFU / mL, 1.18×10 1 –1.18×10 4 CFU / mL and 1.19×10 4 –1.18×10 7Linear relationship of bioluminescence between CFU / mL live Salmonella Typhimurium. It can be seen that the bioluminescence intensity increases with increasing concentration of live Salmonella Typhimurium, but not at 10... 4 At a concentration of CFU / mL, the bioluminescence intensity signal showed a sudden increase. This is because when the concentration of live Salmonella Typhimurium exceeds 1.18 × 10⁻⁶ CFU / mL, the bioluminescence intensity signal increases abruptly. 4 CFU / mL, the surface of live Salmonella typhimurium adsorbs a large number of signal probes, effectively improving the efficiency of targeted photothermal lysis of nanoparticles and releasing more ATP, resulting in a more significant increase in relative luminescence signal intensity. To improve the accuracy of data analysis, we performed segmented analysis of the bioluminescence intensity signal.

[0077] Example 9: Study on the capture and enrichment efficiency of the capture probe

[0078] To investigate the interaction between the capture probe and the bacterial suspension, we designed capture probes of different volumes and examined the bioluminescence intensity of the mixture formed by the capture probe and the live Escherichia coli suspension after different light exposure durations. The higher the bioluminescence intensity, the more bacteria were present, and the more obvious the capture effect and proliferation effect of the capture probe on the bacteria.

[0079] The specific procedures are as follows: Administer 200 μL of 2.9 × 10⁻⁶ solution to four groups respectively. 6 Different volumes of the capture probe described in Example 1 (0, 50 μL, 100 μL, 150 μL) were added to the CFU / mL *Escherichia coli* bacterial suspension. The distance between the LED lamp and the sample was adjusted to maintain a consistent value. The temperature of the well plate after irradiation was measured to be 38°C using an infrared thermal imager. After 1 h, 2 h, and 3 h of light incubation, a complex solution was obtained. The complex solution was centrifuged to obtain the supernatant and precipitate. The precipitate was resuspended to obtain the resuspension. 100 μL of 3 mg / mL luciferin-luciferase solution was added to each of the capture probe groups with different light durations and volumes, and the mixture was stirred for 10 s. The test tubes were then placed in an ATP fluorescence detector for bioluminescence signal detection. The detection results are shown in Table 1.

[0080] Table 1. Bioluminescence detection results

[0081]

[0082] As shown in the table, the bioluminescence intensity increased with prolonged illumination time, indicating that light has a significant impact on promoting bacterial capture and proliferation. Simultaneously, the bioluminescence signal also significantly increased with increasing capture probe concentration. This suggests that the interaction ratio between the capture probe and the test bacterial solution should be adjusted to an appropriate range to facilitate bacterial capture and proliferation.

[0083] Example 10: Study on the specificity of a dual-signal biosensor

[0084] When the number of objects to be detected is 10 7 CFU / mL Bacillus subtilis, 10 7 CFU / mL Staphylococcus aureus, 10 7 CFU / mL Escherichia coli, 10 7 CFU / mL Salmonella Typhimurium and 10 4 A mixed solution of these four bacteria, Pseudomonas aeruginosa, at CFU / mL, yielded the following results: Figure 10 As shown in ab, when the constructed dual-signal sandwich complex detection method was used to detect other pathogenic bacteria standard solutions, even though the concentration of interfering bacteria (Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium) was 1000 times that of Pseudomonas aeruginosa, the Raman signal produced by total Pseudomonas aeruginosa and the bioluminescent signal produced by active Pseudomonas aeruginosa were significantly higher than those of the control bacteria. This indicates that the addition of interfering bacteria does not cause changes in the Raman spectral signal and bioluminescent signal values ​​of the system. Therefore, the constructed dual-signal sandwich complex detection method has high specificity and high selectivity for Pseudomonas aeruginosa. In other words, this dual-signal sandwich complex detection method has one-to-one selectivity for bacterial detection. When detecting different types of bile salt-resistant Gram-negative bacteria, the corresponding capture probe and signal probe are selected to form a sandwich complex, and the resulting detection system has high selectivity for the target bile salt-resistant Gram-negative bacteria. In this embodiment, all bacteria used were live bacteria.

[0085] Example 11: A dual-signal biosensor was used to detect the content of Escherichia coli in different active states in honeysuckle slices.

[0086] As shown in Table 2, different types of Salmonella typhimurium with different activities were added to different categories of honeysuckle slices. The number of added Salmonella typhimurium was then detected using the dual-signal detection method in this scheme, and the recovery rate was obtained.

[0087] Table 2. Detection results of Salmonella Typhimurium in different active states added to honeysuckle decoction pieces.

[0088]

[0089] As shown in Table 2, the recoveries of honeysuckle slices containing live Salmonella typhimurium, dead Salmonella typhimurium, and sublethal Salmonella typhimurium were 93.74-106.01%, 95.70-104.25%, and 98.97-105.0%, respectively, which further demonstrates the high accuracy and reliability of the proposed dual-signal detection method.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for simultaneous quantitative detection of bile salt-resistant Gram-negative bacteria in different active states, characterized in that: Includes the following steps: (1) Add the capture probe to the bacterial culture to be tested, and after culturing under light, a complex solution is obtained. After centrifuging the complex solution, a supernatant and a precipitate are obtained. The precipitate is resuspended to obtain a resuspension. (2) Add a signal probe to the resuspended solution, and obtain a sandwich complex solution after photoinactivation and incubation; (3) The Raman spectral signal and relative luminescence intensity of the sandwich complex solution were measured to obtain the relationship between the ratio of the sum of the number of bile salt-resistant Gram-negative dead bacteria and the number of bile salt-resistant Gram-negative sublethal bacteria to the total number of bacteria and the Raman spectral signal intensity, as well as the relationship between the number of bile salt-resistant Gram-negative live bacteria and the relative luminescence intensity. The total number of bacteria and the sum of the number of bile salt-resistant Gram-negative dead bacteria and the number of bile salt-resistant Gram-negative sublethal bacteria were calculated. (4) Add the endotoxin targeting probe to the supernatant, and after the reaction, obtain a magnetic dispersion containing endotoxin. Spray the magnetic dispersion containing endotoxin onto the SERS array chip and perform Raman spectroscopy scanning to establish a quantitative relationship between the Raman spectral signal of the characteristic peak of endotoxin and the concentration of endotoxin particles, and calculate the number of bile salt-resistant Gram-negative dead bacteria. (5) Calculate the sum of the number of bile salt-resistant Gram-negative dead bacteria and the number of bile salt-resistant Gram-negative sublethal bacteria in step (3) and the difference between the number of bile salt-resistant Gram-negative dead bacteria in step (4) to obtain the number of bile salt-resistant Gram-negative sublethal bacteria. The total bacterial count is the sum of the number of bile salt-resistant Gram-negative live bacteria, bile salt-resistant Gram-negative sublethal bacteria, and bile salt-resistant Gram-negative dead bacteria.

2. The method for simultaneous quantitative detection of bile salt-resistant Gram-negative bacteria in different activity states according to claim 1, characterized in that: In step (3), when the ratio X1 of the sum of the number of bile salt-resistant Gram-negative sublethal bacteria and the number of bile salt-resistant Gram-negative dead bacteria to the total number of bacteria is 0.01-50%, the relationship between the Raman spectral signal intensity Y1 and the ratio X1 is Y1 = a1 + b1X1, where the value of a1 is 13362.1-14398.3 and the value of b1 is -148.2--108.3; when the ratio X2 of the sum of the number of bile salt-resistant Gram-negative sublethal bacteria and the number of bile salt-resistant Gram-negative dead bacteria to the total number of bacteria is 51-100%, the relationship between the Raman spectral signal intensity Y2 and the ratio X2 is Y2 = a2 + b2X2, where the value of a2 is 12280.6-13945.4 and the value of b2 is -99.5--79.

1.

3. The method for simultaneous quantitative detection of bile salt-resistant Gram-negative bacteria in different activity states according to claim 1, characterized in that: In step (3), when the concentration Z1 of bile salt-resistant Gram-negative viable bacteria is 1.18-1.18×10⁻⁶, 4 At CFU / mL, the bioluminescence intensity Y BL1 The relationship between Y and the viable bacteria concentration Z1 is: BL1 = aZ1 + b, where a is 80–120 and b is 9–13; when the concentration of bile salt-resistant Gram-negative viable bacteria Z2 is 1.19 × 10⁻⁶. 4 -1.18×10 7 At CFU / mL, the bioluminescence intensity Y BL2 The relationship between Y and the viable bacteria concentration Z2 is as follows: BL2 = aZ2+b, where the value of a is 1600~2100, and the value of b is -6100~-8920; In step (4), the quantitative relationship between the Raman spectral signal Y of the characteristic peak of endotoxin and the concentration of endotoxin particles X is Y = a + b X, where the value of a is -900 to -1030, the value of b is 750 to 785, and the concentration of X is 0.01-8 μg / mL.

4. The method for simultaneous quantitative detection of bile salt-resistant Gram-negative bacteria in different activity states according to claim 1, characterized in that: The bile salt-resistant Gram-negative bacteria are one of Escherichia coli, Pseudomonas aeruginosa, Salmonella, or Shigella; the different active states of the bile salt-resistant Gram-negative bacteria include live bile salt-resistant Gram-negative bacteria, dead bile salt-resistant Gram-negative bacteria, and sublethal bile salt-resistant Gram-negative bacteria; the dead bile salt-resistant Gram-negative bacteria are bile salt-resistant Gram-negative bacteria that have died naturally, been inactivated by drugs, or been inactivated by physical conditions; the drug inactivation method is at least one of nutrient deficiency inactivation, antibiotic inactivation, or antimicrobial drug inactivation; the physical inactivation method is at least one of high temperature inactivation, high pressure inactivation, moist heat inactivation, light inactivation, or radiation inactivation; the sublethal bile salt-resistant Gram-negative bacteria are bile salt-resistant Gram-negative sublethal bacteria induced by mild sterilization conditions; the mild sterilization conditions are at least one of low-concentration disinfectant, antibacterial traditional Chinese medicine, or low-concentration antibiotic.

5. The method for simultaneous quantitative detection of bile salt-resistant Gram-negative bacteria in different activity states according to claim 1, characterized in that: The capture probe in step (1) is at least one of lectin, bacterial aptamer, bacterial antibody, boric acid-modified metal compound, carbon-based material or black phosphorus nanocomposite.

6. The method for simultaneous quantitative detection of bile salt-resistant Gram-negative bacteria in different activity states according to claim 1, characterized in that: The signal probe in step (2) is at least one of a bacterial aptamer or an antibody-modified nanocomposite; the nanocomposite of the bacterial aptamer or antibody-modified nanocomposite is a nanocomposite with a layered structure, the number of layers of the layered structure being greater than or equal to 3; the outermost layer of the nanocomposite is at least one of gold or silver; the nanocomposite contains PB; the nanocomposite is at least one of Ag@PB@Au, Ag@PB@Ag, Au@PB@Au, Au@PB@Ag, Ag@PB@Au@Ag, Ag@PB@Ag@Au, Au@PB@Au@Ag, or Au@PB@Ag@Au.

7. The method for simultaneous quantitative detection of bile salt-resistant Gram-negative bacteria in different activity states according to claim 1, characterized in that: The light culture step in step (1) uses LED lamps or near-infrared light for irradiation for 0.5-12 hours; the culture medium used in step (1) contains 0.3-0.5% bile salts by mass concentration; the light inactivation step in step (2) uses LED lamps or near-infrared light for irradiation for 2-30 minutes; the incubation step in step (2) lasts for 30-120 minutes; the parameters for measuring the Raman spectral signal in step (3) are: excitation wavelength of 785 nm, power of 60-80 mW, acquisition and exposure time of 1 s, and objective lens of 10×.

8. The method for simultaneous quantitative detection of bile salt-resistant Gram-negative bacteria in different activity states according to claim 1, characterized in that: The endotoxin targeting probe in step (4) is a nanocomposite modified with bacterial endotoxin aptamers; the nanocomposite is a composite formed by magnetic nanomaterials or metal framework organic materials and gold and / or silver; the nanocomposite is at least one of Fe3O4@Au, Fe3O4@Ag, Fe3O4@Au@Ag, Fe3O4@Ag@Au, PB@Au, PB@Ag, PB@Au@Ag or PB@Ag@Au; the reaction temperature in step (4) is 25-40℃ and the time is 1-5h; the coating material of the SERS array chip in step (4) is gold and / or silver; the substrate of the SERS array chip in step (4) is an aluminum substrate or a silicon substrate; the SERS array chip in step (4) has 1-2000 pores, and the diameter of each pore is 1-1000μm.

9. The method for simultaneous quantitative detection of bile salt-resistant Gram-negative bacteria in different activity states according to claim 1, characterized in that: In step (1), the proportion of bile salt-resistant Gram-negative viable bacteria, the proportion of bile salt-resistant Gram-negative dead bacteria, and the proportion of bile salt-resistant Gram-negative sublethal bacteria in the test bacterial solution is 0.01-100% of the total bacterial count; the concentration of the capture probe in step (1) is 0.5-3 mg / mL; the concentration of the signal probe in step (2) is 0.5-3 mg / mL; the volume ratio of the signal probe, resuspension, and capture probe in step (1) in step (2) is 1-3:1:1-3; and the volume ratio of the supernatant to the endotoxin targeting probe in step (4) is 1:0.3-1.

10. The application of the simultaneous quantitative detection method for bile salt-resistant Gram-negative bacteria in different activity states as described in any one of claims 1-9, characterized in that: The detection method described herein is applied to the detection of bile salt-resistant Gram-negative bacteria in different active states in water sources, food, traditional Chinese medicine products, or environmental samples.

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