Gold nanoprobe, preparation method and viable bacteria visual detection method

Abstract

The application belongs to the field of nanobiological sensing microorganism rapid detection technology, and discloses a gold nanoprobe, a preparation method and a live bacteria visual detection method. The gold nanoprobe comprises gold nanoparticles, pH response conformational switch peptides and LPS specific targeting short peptides which are co-modified on the surface of the gold nanoparticles. The amino acid sequence of the pH response conformational switch peptide is C-GLLHLLHHLLHH-NH2, and the amino acid sequence of the LPS specific targeting short peptide is C-KVRVRVK. The application constructs a targeting anchor and live bacteria microenvironment pH conformational double-molecule lock cooperative sensing system. When only complete metabolic activity gram-negative live bacteria are contained, the double locks are opened at the same time, gold nanoparticles are induced to orderly aggregate, the solution changes from wine red to blue purple, and naked eyes can directly judge. Dead bacteria, bacterial fragments and high protein matrix impurities cannot trigger aggregation, and false positives are completely eliminated. The detection is rapid and convenient, and is resistant to complex matrix interference.

Description

Technical Field

[0001] This invention belongs to the field of nanobiosensing microbial rapid detection technology, specifically involving a gold nanoprobe and its preparation method, and a method for visual detection of live bacteria. It is applicable to rapid visual qualitative screening of Gram-negative live bacteria in scenarios such as drinking water, fresh food homogenates, wound exudate, medical sewage, and disinfection effect verification. Background Technology

[0002] Foodborne Gram-negative pathogens (such as Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Klebsiella pneumoniae) are major sources of risk for food safety, clinical wound infections, and microbial contamination of water bodies. Traditional microbial testing relies on plate culture, which has a testing cycle of 18-48 hours, making it unsuitable for on-site, immediate screening. PCR and enzyme-linked immunosorbent assays (ELISA) require sophisticated instruments and professional operators, resulting in high costs and making them unsuitable for large-scale testing in grassroots or field settings.

[0003] The current naked-eye visible colorimetric detection technology for gold nanoparticles (AuNPs) is the mainstream rapid detection method. Its principle is that monodisperse AuNPs solutions are wine-red; when particles aggregate, the surface plasmon resonance peaks red-shift, and the solution turns blue-purple, which can be directly interpreted with the naked eye. However, existing publicly available technologies have some drawbacks: First, it cannot distinguish between live and dead bacteria. Existing modified ligands, including antibodies, common aptamers, mannose, and single cationic antimicrobial peptides, target bacterial cell membrane fixation structures (lipopolysaccharide LPS, membrane proteins, and nucleic acids). Dead bacteria and bacterial lysate fragments after high-temperature inactivation or antibiotic killing can still bind to the probe, inducing AuNP aggregation and color change, resulting in a high false positive detection rate. It is completely unsuitable for disinfection effect verification and water quality live bacteria counting scenarios.

[0004] Second, complex matrices have poor resistance to interference. Free proteins, oils, and colloidal impurities in blood, wound exudate, high-protein meat homogenates, and turbid sewage can easily cause non-specific electrostatic aggregation of AuNPs. When there are no target bacteria, the solution spontaneously turns blue-purple, which seriously interferes with the naked-eye interpretation results.

[0005] Third, the sensing mechanism is singular, relying solely on a single affinity force to trigger aggregation, lacking a dual screening threshold, thus failing to balance specificity and sensitivity. Increasing ligand specificity leads to a significant decrease in detection sensitivity; conversely, increasing sensitivity results in a substantial increase in matrix interference. Summary of the Invention

[0006] To address the industry bottlenecks of existing gold nanoparticle bacterial visualization detection technologies, such as the inability to distinguish between live and dead bacteria, high false positive rates in complex matrices, and the difficulty in balancing specificity and sensitivity, this invention provides a gold nanoparticle probe and its preparation method, as well as a method for visually detecting live bacteria.

[0007] The first aspect of the present invention provides a gold nanoprobe, comprising gold nanoparticles and a pH-responsive conformational switching peptide and an LPS-specific targeting short peptide co-modified on the surface of the gold nanoparticles. The amino acid sequence of the pH-responsive conformation switch peptide is shown in SEQ ID No. 1 of the amino acid sequence listing: C-GLLHLLHHLLHH-NH2, with a free cysteine ​​thiol group at the N-terminus and an amidation capping at the C-terminus. The amino acid sequence of the LPS-specific targeting short peptide is shown in SEQ ID No. 2 of the amino acid sequence listing, C-KVRVRVK, with a free cysteine ​​thiol group at the N-terminus.

[0008] This invention provides a gold nanoprobe co-modified with a dithiol peptide. By constructing a dual molecular lock sensing mechanism, only intact metabolically active live bacteria can trigger the aggregation and color change of AuNPs, while dead bacteria, bacterial fragments, and matrix impurities do not respond. It combines multiple technical effects such as high specificity, strong anti-interference, and naked-eye visualization.

[0009] pH-responsive conformation switch peptide, complete amino acid sequence: C-GLLHLLHHLLHH-NH2; N-terminal cysteine ​​provides free thiol groups for binding gold nanoparticles, and C-terminal amidation eliminates the negative charge of free carboxyl groups; the polypeptide chain contains multiple histidine residues and hydrophobic leucine residues; under neutral pH conditions, the peptide chain unfolds and becomes hydrophilic, while under the weakly acidic microenvironment (pH 5.0-6.2) around the viable bacterial membrane, histidine imidazole is protonated, and the peptide chain undergoes random coiling and α-helix conformational flipping, exposing hydrophobic residues and providing the driving force for hydrophobic aggregation between particles, thus promoting the aggregation of nanoparticles.

[0010] A short peptide specifically targeting Gram-negative bacteria using LPS, with a complete amino acid sequence: C-KVRVRVK; gold nanoparticles covalently modified with N-terminal cysteine ​​free thiol groups; the peptide is rich in lysine and arginine cationic residues, which can specifically electrostatically bind to the negatively charged phosphate groups of LPS in the outer membrane of viable Gram-negative bacteria, enabling the probe to be anchored at specific sites on the surface of viable bacteria; protein impurities without LPS structure cannot bind to the LPS-specific targeting peptide; although dead bacterial fragments contain a small amount of LPS, the LPS spatial conformation is incomplete, making it difficult to stably bind to the LPS-specific targeting peptide and thus not producing significant interference with the detection signal. This constructs the first line of defense against false positives. LPS is a lipopolysaccharide.

[0011] Free histidine-enriched pH-responsive peptides and free LPS-bound cationic short peptides have only been reported for in vitro antibacterial and sepsis neutralization experiments. In this invention, two functional peptides are co-modified in a fixed molar ratio to construct a bimolecular switch structure of "target recognition lock + live bacteria microenvironment pH-responsive lock" and applied to the AuNPs system for visual sensing of live bacteria in gold nanoparticles.

[0012] Preferably, the molar ratio of the pH-responsive conformational switching peptide to the LPS-specific targeting short peptide is 2:1 to 5:1.

[0013] Preferably, the molar ratio of the pH-responsive conformational switching peptide to the LPS-specific targeting short peptide is 3:1.

[0014] A high proportion of pH-responsive conformation-switching peptides ensures conformation response sensitivity, while a low proportion of LPS-specific targeting peptides achieves precise anchoring, synergistically forming a bimolecular triggering system of "targeting anchoring first lock + pH conformation flipping second lock"; only when both locks are activated simultaneously do gold nanoparticles undergo orderly cross-linking and aggregation.

[0015] Preferably, the gold nanoparticles are monodisperse gold nanoparticles reduced with sodium citrate.

[0016] A second aspect of this invention provides a method for preparing the above-mentioned gold nanoprobe, comprising the following steps: The mixed polypeptide stock solution was mixed with gold nanoparticle sol and incubated to obtain the gold nanoprobe; the mixed polypeptide stock solution was obtained by mixing the pH-responsive conformational switching peptide and the LPS-specific targeting short peptide.

[0017] Preferably, the mixed polypeptide stock solution is obtained by dissolving the pH-responsive conformation switch peptide and the LPS-specific targeting short peptide in HEPES buffer at pH 7.2-7.5.

[0018] HEPES's chemical name is 4-hydroxyethylpiperazine ethanesulfonic acid.

[0019] Preferably, the pH of the HEPES buffer is 7.4.

[0020] pH-responsive conformation switch peptides unfold hydrophilically in neutral environments, and undergo random coiling to α-helix conformational flipping in the weakly acidic microenvironment of pH 5.0-6.2 around the cell membrane of live bacteria, exposing hydrophobic leucine residues and generating the driving force for hydrophobic crosslinking between gold nanoparticles.

[0021] Preferably, the incubation time is 12 hours.

[0022] Preferably, the pH-responsive conformational switching peptide and the LPS-specific targeting short peptide are prepared by solid-phase polypeptide synthesis.

[0023] And / or, after the incubation step, the following steps are also included: salt aging treatment of the incubated product, purification, to obtain the gold nanoprobe.

[0024] And / or, the preparation method of the gold nanoparticles includes the following steps: boil ultrapure water, add 1% sodium citrate aqueous solution and mix evenly, add chloroauric acid aqueous solution all at once, boil and stir for 15 min, cool, and obtain the gold nanoparticles.

[0025] A third aspect of this invention provides a method for visually detecting live bacteria, comprising the following steps: S1. Take the supernatant of the sample to be tested, add the above-mentioned stock solution of gold nanoprobe, mix well, and obtain a mixture. S2. Incubate the mixture at room temperature in the dark for 20-30 minutes. S3. If the incubated mixture remains wine-red, it is negative; if the incubated mixture turns blue-purple or gray-blue, it is positive.

[0026] The live bacteria visualization detection method can distinguish between live bacteria and high-temperature inactivated dead bacteria. Dead bacteria do not have proton transmembrane metabolic activities, and there is no local weakly acidified microenvironment around the cell membrane, so they cannot trigger peptide conformation flipping and will not induce gold nanoparticle aggregation and discoloration.

[0027] Naked-eye interpretation of results: A wine-red color indicates a negative result, meaning the sample contains no live bacteria, only dead bacteria, or impurities; a blue-purple / gray-blue color indicates a positive result, meaning the sample contains Gram-negative live bacteria with intact metabolic activity.

[0028] Preferably, the volume ratio of the sample supernatant to the gold nanoprobe stock solution is 1:(2-4).

[0029] Preferably, the volume ratio of the sample supernatant to the gold nanoprobe stock solution is 1:2.

[0030] The fourth aspect of this invention provides the application of the gold nanoprobes described above in the rapid, naked-eye visual detection of Gram-negative live bacteria.

[0031] It can be applied to the naked-eye visual rapid detection of Gram-negative live bacteria in drinking water, fresh food, wound exudate, and medical wastewater.

[0032] Beneficial effects of this invention: 1. The gold nanoprobe provided by this invention possesses an innovative bimolecular lock-based synergistic sensing mechanism, enabling precise naked-eye differentiation between live and dead bacteria. Existing single-ligand modification systems only recognize fixed bacterial structures, and dead bacterial fragments can trigger color changes. This invention requires simultaneous fulfillment of two conditions: "LPS targeted anchoring + localized weak acidification induced by live bacterial metabolism to induce peptide conformational inversion." Dead bacteria do not undergo proton transmembrane metabolism or localized pH decreases, and the peptides maintain a hydrophilic and relaxed state, unable to form aggregation-driving forces. This completely eliminates false positives caused by dead bacteria and can be directly used in scenarios that traditional technologies cannot cover, such as disinfection effect verification and live bacteria counting in water bodies.

[0033] 2. The gold nanoparticle probe provided by this invention is resistant to interference from complex matrices and is suitable for high-protein, high-oil, and turbid liquid samples. A dual screening threshold forms a two-stage filtration: the first stage involves LPS-specific targeting short peptides that bind only to viable bacteria containing intact LPS, preventing the adsorption of free proteins and colloidal impurities; the second stage involves pH-responsive conformation-switching peptides that can only be activated by the microenvironment of viable bacteria. Even if a small amount of probe non-specifically adsorbs impurities, there is no conformational inversion driving force, preventing aggregation. No spontaneous blue discoloration is observed in blood, meat homogenates, or wastewater samples.

[0034] 3. The live bacteria visualization detection method provided by this invention features fast detection speed, simple operation, and no need for large instruments, making it suitable for on-site screening at the grassroots level. Results are available visually in just 20-30 minutes, eliminating the need for PCR instruments and incubators. The reagent kit is portable, allowing a single person to complete batch sample testing, and the detection cost is far lower than that of immunoassay and nucleic acid testing methods. Attached Figure Description

[0035] Figure 1 TEM image of the gold nanoparticles prepared in Example 1; Figure 2 The image shows the color change and UV-Vis spectrum of the gold nanoprobe before and after reacting with live bacteria in Example 2. Detailed Implementation

[0036] The following will provide a clear and complete description of the concept, specific structure, and technical effects of the present invention in conjunction with embodiments and accompanying drawings, so as to fully understand the purpose, solution, and effects of the present invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0037] Example 1 A gold nanoprobe Preparation method: Mix the mixed peptide stock solution with gold nanoparticles and incubate to obtain gold nanoparticle probes; the mixed peptide stock solution is obtained by mixing pH-responsive conformational switching peptides and LPS-specific targeting short peptides.

[0038] The specific preparation method is as follows: 1. Synthesis of nanoparticle sol Measure 100 mL of ultrapure water into a round-bottom flask and heat to a continuous boil; add 4 mL of 1% sodium citrate aqueous solution and stir magnetically to mix; quickly add 2 mL of 10 mM chloroauric acid aqueous solution at once and keep boiling and stirring for 15 min; the solution changes from light gray to clear wine red, stop heating, and cool naturally to room temperature to obtain AuNPs sol with a particle size of 13 nm - 15 nm, and store at 4 °C protected from light.

[0039] TEM image of the prepared gold nanoparticles is shown below. Figure 1 .

[0040] 2. Description of peptide raw materials The two thiol-modified peptides used in the experiment: Peptide 1 (pH-responsive conformation switch peptide) sequence: C-GLLHLLHHLLHH-NH2; Modification requirements: N-terminal free cysteine ​​thiol group, C-terminal amidation capping; Peptide 2 (LPS-specific targeting short peptide) sequence: C-KVRVRVK; Modification requirement: N-terminal free cysteine ​​thiol group; The peptide was prepared using a solid-phase peptide synthesis method. The synthesized product was desalted with acetate and freeze-dried to obtain a white peptide powder. Peptide synthesis technicians in this field can independently complete the preparation based on this instruction.

[0041] 30 nmol of peptide 1 and 10 nmol of peptide 2 were accurately weighed and dissolved together in 1 mL of sterile pH 7.4 HEPES buffer to prepare a mixed peptide stock solution.

[0042] 3. Double peptide co-modification and aging purification Take 10 mL of cooled AuNPs sol and add 1 mL of peptide stock solution dropwise under magnetic low-speed stirring. After the addition is complete, incubate at room temperature in the dark with continuous stirring for 12 h. The modified layer was stabilized by salt gradient aging: 1 M NaCl solution was added in three small amounts at 30 min intervals, and the final NaCl concentration of the system reached 50 mM, thus completing the stable covalent binding of peptides and gold nanoparticles. Centrifuge the modified mixture at 12000 r / min for 20 min and discard the clear supernatant containing free peptides. Resuspend the precipitate twice in pH 7.4 HEPES buffer to remove unbound free peptides. Finally, resuspend the precipitate in 10 mL of sterile HEPES buffer to obtain the target gold nanoprobe stock solution, and store it in a sealed container at 4°C protected from light.

[0043] Example 2 A Visual Detection Method for Live Bacteria Experimental strain: Escherichia coli ATCC25922 (Gram-negative pattern live bacteria); Inactivation of dead bacteria: Escherichia coli was completely inactivated by boiling water bath at 100℃ for 10 min; Blank substrates: pork homogenate supernatant, simulated wound exudate, and domestic sewage supernatant.

[0044] 1. Live bacteria positive detection system Take 50 μL of a concentration of 10 3CFU / mL of live Escherichia coli solution was added to 100 μL of the gold nanoprobe stock solution prepared in Example 1. The mixture was inverted and mixed thoroughly, and then allowed to stand at room temperature for 20 min. The solution was visibly changed from an initial wine-red color to a uniform blue-purple color, indicating a positive result.

[0045] To verify the pH-responsive aggregation and colorimetric mechanism of the probe of this invention, the plasmon resonance absorption spectra of two groups of samples before and after the reaction were characterized using a UV-Vis spectrophotometer. The instrument scanning wavelength range was set to 400-800 nm. After baseline calibration, the UV-Vis absorption spectra of the two groups of samples were measured. The test results showed that the unreacted dispersed gold nanoprobe had a characteristic absorption peak at 520 nm, corresponding to a wine-red transparent state of the solution. After the probe anchored to live bacteria and underwent a pH-induced conformational change, the gold nanoparticles aggregated hydrophobically, the intensity of the 520 nm characteristic peak decreased significantly, the absorption peak shifted significantly to red, and the macroscopic color of the system changed from wine-red to blue-purple. The color change of the gold nanoprobe before and after the reaction with live bacteria and the UV-Vis spectra are shown in the figure. Figure 2 .

[0046] 2. Dead bacteria negative control system Take 50 μL of Escherichia coli bacterial suspension of the same concentration at high temperature, add an equal volume of probe solution, mix well and let stand for 20 min; the solution remains clear wine red without any blue change, proving that dead bacteria cannot trigger the double-lock aggregation mechanism and there are no false positives.

[0047] 3. Complex matrix interference resistance control Take 50 μL of pork homogenate supernatant, simulated wound exudate, and domestic sewage supernatant (without live bacteria), respectively, add probe and incubate for 20 min. All systems remain wine red and do not spontaneously aggregate or change color, verifying the strong resistance of the present invention to matrix interference.

[0048] 4. Validation of the lowest detection limit Take a series of live Escherichia coli bacterial suspensions with concentration gradients, the concentrations of the various Escherichia coli bacterial suspensions falling between 10 CFU / mL and 10 CFU / mL. 4 Within the CFU / mL range; after 20 min of incubation, the blue staining of a 10 CFU / mL live bacteria system can be stably identified by the naked eye, with a detection limit of 10 CFU / mL, and the sensitivity is superior to existing single peptide-modified AuNPs detection systems.

[0049] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A gold nanoprobe, characterized in that, It includes gold nanoparticles and pH-responsive conformation-switching peptides and LPS-specific targeting short peptides co-modified on the surface of the gold nanoparticles. The amino acid sequence of the pH-responsive conformation switch peptide is C-GLLHLLHHLLHH-NH2, with a free cysteine ​​thiol group at the N-terminus and an amidation-capped C-terminus. The amino acid sequence of the LPS-specific targeting short peptide is C-KVRVRVK, with a free cysteine ​​thiol group at the N-terminus.

2. The gold nanoprobe according to claim 1, characterized in that, The molar ratio of the pH-responsive conformational switching peptide to the LPS-specific targeting short peptide is 2:1 to 5:

1.

3. The gold nanoprobe according to claim 2, characterized in that, The molar ratio of the pH-responsive conformational switching peptide to the LPS-specific targeting short peptide is 3:

1.

4. The gold nanoprobe according to claim 1, characterized in that, The gold nanoparticles are monodisperse gold nanoparticles reduced with sodium citrate.

5. A method for preparing a gold nanoprobe as described in any one of claims 1-4, characterized in that, It includes the following steps: The mixed polypeptide stock solution was mixed with gold nanoparticles and incubated to obtain the gold nanoprobe; the mixed polypeptide stock solution was obtained by mixing the pH-responsive conformational switching peptide and the LPS-specific targeting short peptide.

6. The method for preparing gold nanoprobes according to claim 5, characterized in that, The mixed polypeptide stock solution was obtained by dissolving the pH-responsive conformation switch peptide and the LPS-specific targeting short peptide in HEPES buffer at pH 7.2-7.

5.

7. The method for preparing gold nanoprobes according to claim 6, characterized in that, The pH of the HEPES buffer solution is 7.

4.

8. The method for preparing gold nanoprobes according to claim 5, characterized in that, The pH-responsive conformational switching peptide and the LPS-specific targeting short peptide were prepared by solid-phase polypeptide synthesis, respectively. And / or, after the incubation step, the method further includes the following steps: subjecting the incubated product to salt aging treatment and purification to obtain the gold nanoprobe; And / or, the preparation method of the gold nanoparticles includes the following steps: boil ultrapure water, add 1% sodium citrate aqueous solution and mix evenly, add chloroauric acid aqueous solution all at once, boil and stir for 15 min, cool, and obtain the gold nanoparticles.

9. A method for visually detecting live bacteria, characterized in that, The operation steps are as follows: S1. Take the supernatant of the sample to be tested, add the stock solution of the gold nanoprobe as described in any one of claims 1-4, mix well, and obtain a mixture. S2. Incubate the mixture at room temperature in the dark for 20-30 minutes. S3. If the incubated mixture remains wine-red, it is negative; if the incubated mixture turns blue-purple or gray-blue, it is positive.

10. The application of the gold nanoprobe as described in any one of claims 1-4 in the rapid, naked-eye visual detection of Gram-negative live bacteria.

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