A single particle nanoprobe and its preparation method and application

Abstract

The present invention provides a single-particle nanoprobe and a preparation method and application thereof, which belongs to the technical field of optical detection of bacterial releases. The single-particle nanoprobe provided by the present invention is composed of gold nanorods (AuNRs) modified with 3-mercaptopropionic acid (MPA) and a black hole quencher BHQ-3. The present invention also provides a preparation method for the above-mentioned single-particle nanoprobe. The single-particle nanoprobe prepared by the present invention can be used for real-time detection or long-distance detection of biological oscillations released by a single bacterial enzyme, overcoming the damage and interference of labeled and contact probes to samples.

Description

Technical Field

[0001] The invention belongs to the technical field of optical detection of bacterial releases, and in particular relates to a single-particle nanoprobe and a preparation method and application thereof. Background Art

[0002] Biological oscillations are ubiquitous in life systems, playing an important role from the gene regulation of single cells to the behavioral level of individual organisms. Therefore, understanding the mechanism of life from biological oscillations has become the main approach. In recent years, people have conducted research on biological oscillations in calcium ions, neurons, proteases, metabolism, genetic genes, and other aspects. Through intuitive periodic, semi-periodic, and indirect oscillation signals, we can further understand the life mechanism of the corresponding organism. How to reveal biological oscillations more accurately has always been the direction of researchers' efforts.

[0003] There are many methods to reveal biological oscillations, such as fluorescent labeling, optogenetics, and population analysis. However, fluorescent labeling cannot avoid the influence of labeled molecules on the molecular dynamics of the analyte, and the photosensitive neurons required for optogenetics have always restricted its development. Population analysis cannot be applied to individual biological samples. Therefore, how to provide a label-free technology that can reveal the method of releasing biological oscillations of bacterial enzymes while avoiding sample damage and overcoming photobleaching is one of the keys to research in this field. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a single-particle nanoprobe that can be used for real-time or remote detection of biological oscillations released by a single bacterial enzyme, thereby overcoming the damage and interference to samples caused by labeled and contact probes.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a single-particle nanoprobe. The nanoprobe consists of a gold nanorod and a black hole quencher BHQ-3. The gold nanorod is modified by 3-mercaptopropionic acid.

[0007] The present invention also provides a method for preparing the above-mentioned single-particle nanoprobe, comprising the following steps:

[0008] 3-Mercaptopropionic acid solution is added to the gold nanorod solution for modification. The modified gold nanorod solution is centrifuged and resuspended in water to obtain a modified gold nanorod aqueous solution. Then a black hole quencher BHQ-3 is added to react to obtain a single particle nanoprobe.

[0009] Preferably, the gold nanorod solution is centrifuged to remove excess CTAB solution, resuspended in water, and then added with 3-mercaptopropionic acid solution for modification.

[0010] Preferably, the concentration of the gold nanorod solution is 0.05-0.15 mg / mL, the concentration of the 3-mercaptopropionic acid solution is 0.05-0.15 mM, and the concentration of the black hole quencher BHQ-3 is 0.5-1.5 μM.

[0011] Preferably, the volume ratio of the gold nanorod aqueous solution to the 3-mercaptopropionic acid solution is 1:90-110.

[0012] Preferably, the modification time is 10 to 14 hours.

[0013] Preferably, the volume ratio of the black hole quencher BHQ-3 to the modified gold nanorod aqueous solution is 1:9-11.

[0014] Preferably, the reaction time is 50 to 70 minutes.

[0015] The present invention also provides the use of the above-mentioned single-particle nanoprobe in real-time detection or long-distance detection of biological oscillations released by a single bacterial enzyme.

[0016] The present invention also provides a method for real-time or long-distance detection of single bacterial enzyme release, comprising the following steps: adding the above-mentioned single particle nanoprobe to a bacterial solution, taking the mixed solution under a dark field microscope to observe the scattering spectrum intensity of the single particle nanoprobe.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The single-particle nanoprobe provided by the present invention realizes real-time optical detection of single bacterial enzyme release, reveals the biological oscillation of single bacterial enzyme release, and also realizes the heterogeneity detection of enzymes at different growth stages of bacteria. By analyzing the enzyme oscillation signal, the growth stage of bacteria can be determined, providing a new solution for accurate drug delivery at different stages of drug-resistant bacteria.

[0019] The single-particle nanoprobe provided by the present invention realizes optical detection of single-bacterial enzyme release by a single particle at a long distance. The detection limit of Gram-negative Escherichia coli is 3μm, and the detection limit of Gram-positive Staphylococcus aureus is 2.5μm, overcoming the damage and interference of labeled and contact probes to samples.

[0020] The method provided by the present invention for real-time detection or long-distance detection of single bacterial enzyme release is a non-invasive optical observation method for biological oscillations of single bacterial enzyme release, which will further promote the development of bacterial biodynamics and provide new ideas for better understanding of bacterial life mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Schematic diagram of the preparation and detection principle of single-particle nanoprobe.

[0022] Figure 2 :Energy suppression and energy recovery of single-particle nanoprobes. a, Real-time changes of energy suppression of nanoprobes; b, Real-time scattering spectrum changes of nanoprobes detecting azoreductase in vitro; c, Real-time scattering spectrum changes of nanoprobes detecting rat liver microsomes in vitro.

[0023] Figure 3 : Single-particle nanoprobe detects the release of a single bacterial enzyme at a long distance. a, dark field images of the nanoprobe for Escherichia coli at different detection distances; b, dark field images of the nanoprobe for Staphylococcus aureus at different detection distances; c, real-time scattering spectrum intensity changes of Escherichia coli at different detection distances; d, changes of scattering spectrum intensity with different detection distances of a single bacterium.

[0024] Figure 4 : Real-time detection of biooscillations of enzyme release from a single bacterium. ad, Detection of biooscillations of enzyme release from a single Escherichia coli at different growth stages: (a) delayed phase, (b) logarithmic phase, (c) stable phase, and (d) decay phase; e, Real-time detection signals of biooscillations of enzyme release from E. coli and Staphylococcus aureus at different growth stages; f, Amplitude changes of E. coli and Staphylococcus aureus at different growth stages; g, Periodic changes of E. coli and Staphylococcus aureus at different growth stages. DETAILED DESCRIPTION

[0025] The invention provides a single-particle nanoprobe. The nanoprobe consists of gold nanorods (AuNRs) and a black hole quencher BHQ-3. The gold nanorods are modified by 3-mercaptopropionic acid (MPA).

[0026] The present invention uses a plasmon resonance energy transfer (PERT) method to prepare the desired nanoprobe. PERT is a phenomenon in which plasmon resonance energy is transferred from metal nanoparticles (as donors) to chemical molecules or conjugated biomolecules (as acceptors). Its triggering mode mainly considers whether the resonance peaks of the donor metal and the acceptor molecule match. The present invention selects gold nanorods and black hole quencher BHQ-3 with matching absorption peaks as donors and acceptors, and modifies the gold nanorods with connecting molecules MPA so that the surface of the gold nanorods has a negative charge, while the conjugated molecule BHQ-3 itself has a positive charge. The two are tightly connected through positive and negative charges to achieve plasmon resonance energy transfer and form a nanoprobe.

[0027] The present invention provides a method for preparing the above-mentioned single-particle nanoprobe, comprising the following steps:

[0028] 3-Mercaptopropionic acid solution is added to the gold nanorod solution for modification. The modified gold nanorod solution is centrifuged and resuspended in water to obtain a modified gold nanorod aqueous solution. Then a black hole quencher BHQ-3 is added to react to obtain a single particle nanoprobe.

[0029] As an optional embodiment, the present invention adds a connecting molecule MPA to commercially purchased AuNRs, shakes and evens out the mixture to obtain AuNRs with a negative charge on the surface; then centrifuges the modified solution at a rate of 5000 to 7000 rap / min for 8 to 12 minutes to remove excess MPA from the solution, and resuspends the centrifuged solution in deionized water; finally, adds BHQ-3 molecules to the aqueous solution of AuNRs modified with the connecting molecule, connects the gold nanorods and the BHQ-3 molecules through positive and negative charges, and obtains the single-particle nanoprobe of the present invention.

[0030] In the present invention, before the gold nanorod solution is added to the 3-mercaptopropionic acid solution for modification, the gold nanorod solution is preferably centrifuged to remove excess CTAB (cetyltrimethylammonium bromide) solution, and then resuspended in water to obtain a gold nanorod aqueous solution. As an optional embodiment, the present invention places commercially purchased AuNRs in a centrifuge with a centrifugal rate of 5000-7000 rap / min and centrifuges 1-2 times, each time for 9-11 minutes, to remove excess CTAB solution in the solution, then adds deionized water and performs ultrasound for 9-11 minutes, so that the gold nanorods are resuspended in the aqueous solution for subsequent modification reactions.

[0031] In the preparation method of the present invention, the concentration of the gold nanorod solution is 0.05-0.15 mg / mL, preferably 0.08-0.1 mg / mL; the concentration of the 3-mercaptopropionic acid solution is 0.05-0.15 mM, preferably 0.08-0.1 mM; the concentration of the black hole quencher BHQ-3 is 0.5-1.5 μM, preferably 0.8-1 μM.

[0032] In the gold nanorod modification process of the present invention, the volume ratio of the gold nanorod aqueous solution to the 3-mercaptopropionic acid solution is 1:90-110, preferably 1:100-105; the modification time is 10-14h, preferably 11-12h.

[0033] In the connection reaction process of the gold nanorods and BHQ-3 of the present invention, the volume ratio of the black hole quencher BHQ-3 to the modified gold nanorod aqueous solution is 1:9-11, preferably 1:10; the reaction time is 50-70 min, preferably 55-60 min.

[0034] The present invention also provides the use of the single-particle nanoprobe in real-time detection or long-distance detection of biological oscillations released by single bacterial enzymes. The single bacteria of the present invention include Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. The enzyme of the present invention is preferably azoreductase.

[0035] The detection principle of the single-particle nanoprobe provided by the present invention in real-time or remote detection of biological oscillations released by a single bacterial enzyme ( Figure 1 ) is as follows: When the gold nanorods with matching absorption peaks are tightly connected to the BHQ-3 molecules, the resonance energy of the AuNRs is transferred to the BHQ-3 molecules, the intensity of the scattering spectrum is significantly reduced, and energy suppression is achieved. When the azoreductase contacts the nanoprobe, the azoreductase cuts off the azo double bond of the BHQ-3 molecule to achieve energy recovery, and the measured scattering spectrum intensity is significantly enhanced. The detection of a single bacterial azoreductase can be achieved based on the change in the measured scattering spectrum intensity. This process can also be reflected in the energy level change. Under the irradiation of white light, the free conduction electrons move from the Fermi level (E F ) rises to a higher energy level (E p ), and collectively oscillate at the resonant optical frequency on the surface of AuNRs. BHQ-3 molecules are conjugated with AuNRs through the connecting molecule MPA, and electrons are transferred from AuNRs to BHQ-3 molecules, and the molecules are transferred from the ground state E g Excited to excited state E2. The scattering spectrum intensity of the nanoprobe decreases significantly, achieving PRET, and the energy is suppressed. When azoreductase is added to the probe of the present invention, the azo double bond breaks, and the electrons will return to AuNRs through the connecting molecules, and the BHQ-3 molecule is reduced from the excited state (E2) to the excited state (E1). In the reaction, because the azo double bonds of the BHQ-3 molecule are not all broken, the BHQ-3 molecule fails to be reduced to the ground state (E g ) is restored to the excited state (E1). At this time, the scattering intensity of the nanoprobe is restored, thereby achieving energy recovery.

[0036] The present invention also provides a method for real-time or long-distance detection of single bacterial enzyme release, comprising the following steps: adding the single-particle nanoprobe of the present invention to a bacterial solution, taking the mixed solution under a dark field microscope to observe the scattering spectrum intensity of the single-particle nanoprobe.

[0037] As an optional embodiment, the present invention takes 0.1-0.2 mL of the prepared single-particle nanoprobe, adds it to 1-2 mL of bacterial solution, shakes slightly to mix the bacteria and the nanoprobe evenly, and draws 10-12 μL of the mixed solution and drops it into the groove of a glass slide with double-sided tape, then covers it with a coverslip, inverts it and observes it under a dark-field microscope, and obtains the relationship between the release rate of azoreductase from a single bacterium and time through the scattering spectrum intensity of the single-particle nanoprobe.

[0038] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0040] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0041] Example 1

[0042] Preparation method of single particle nanoprobe:

[0043] (1) Add 0.1 mM MPA solution to 0.1 mg / mL gold nanorod solution at a volume ratio of 1:100. Shake well and allow to modify for 12 h to make the surface of the gold nanorods negatively charged. After 12 h, centrifuge the modified solution at a rate of 6000 rap / min for 10 min to remove excess MPA molecules in the solution. Resuspend the centrifuged solution in deionized water to obtain a modified gold nanorod aqueous solution (0.1 mg / mL).

[0044] (2) Add 1 μM BHQ-3 molecules to the gold nanorod aqueous solution obtained in step (2) at a volume ratio of 1:10, connect the gold nanorods and BHQ-3 molecules through positive and negative charges, and the reaction time is 55 minutes to form the desired nanoprobe.

[0045] Example 2

[0046] Preparation method of single particle nanoprobe:

[0047] (1) A 0.1 mg / mL gold nanorod solution was placed in a centrifuge at a centrifugal rate of 6000 rap / min and centrifuged twice for 10 min each time to remove excess CTAB solution in the solution. Deionized water was then added and sonicated for 10 min to resuspend the gold nanorods in the aqueous solution (0.1 mg / mL).

[0048] (2) Add 0.1 mM MPA solution to the resuspended gold nanorods at a volume ratio of 1:100. Shake well and allow to modify for 12 hours to make the surface of the gold nanorods negatively charged. After 12 hours, centrifuge the modified solution at a rate of 6000 rap / min for 10 minutes to remove excess MPA molecules in the solution. Resuspend the centrifuged solution in deionized water to obtain a modified gold nanorod aqueous solution (0.1 mg / mL).

[0049] (3) Add 1 μM BHQ-3 molecules to the gold nanorod aqueous solution obtained in step (2) at a volume ratio of 1:10, connect the gold nanorods and BHQ-3 molecules through positive and negative charges, and the reaction time is 55 min to form the desired nanoprobe.

[0050] Example 3

[0051] The difference between this embodiment and embodiment 2 is that in step (1), centrifugation is performed twice in a centrifuge at a centrifugal rate of 6500 rap / min, each time for 9 minutes.

[0052] Example 4

[0053] The difference between this embodiment and embodiment 2 is that the concentration of the gold nanorod solution in step (1) is 0.08 mg / mL.

[0054] Example 5

[0055] The difference between this embodiment and embodiment 2 is that the MPA solution in step (2) is 0.09 mM.

[0056] Example 6

[0057] The difference between this embodiment and embodiment 2 is that in step (2), the volume ratio of the gold nanorod solution to the MPA solution is 1:105.

[0058] Example 7

[0059] The difference between this embodiment and embodiment 2 is that the modification time in step (2) is 11 hours.

[0060] Example 8

[0061] The difference between this embodiment and embodiment 2 is that the rate in step (2) is 6800 rap / min and the time is 9 min.

[0062] Example 9

[0063] The difference between this embodiment and embodiment 2 is that the amount of BHQ-3 molecules added in step (3) is 0.8 μM.

[0064] Example 10

[0065] (1) Energy suppression and energy recovery of single-particle nanoprobes:

[0066] The linker molecule MPA (0.1 mM) was added to AuNRs (0.1 mg / mL) and mixed thoroughly for 12 h at a volume ratio of 1:100 to obtain AuNRs with negative surface charges. Then, 1 μM BHQ-3 molecules were added to the AuNRs solution modified with the linker molecule at a volume ratio of 1:10.

[0067] The energy suppression of the nanoprobe was captured in real time by dark field microscopy and grating spectrometer to explore the energy suppression phenomenon of the nanoprobe under different reaction time conditions. Figure 2 As shown in a, Figure 2The solid line in a corresponds to the scattering spectrum difference (connected to the left axis), the dotted line corresponds to the scattering spectrum difference per unit time (connected to the right axis), and the vertical dotted line in the middle corresponds to the peak value of 674nm.

[0068] Depend on Figure 2 As can be seen from a, the difference in scattering spectra gradually increases with time, indicating that the energy transferred by PRET gradually increases, and the energy suppression becomes more obvious. The difference in scattering spectra per unit time gradually decreases with time, indicating that the energy transferred by PRET per unit time gradually decreases, and the energy suppression gradually tends to saturation. In order to fully suppress the energy of the nanoprobe, the energy suppression time of the nanoprobe used subsequently exceeded 50 minutes.

[0069] (2) To investigate the effectiveness of the constructed nanoprobe in detecting azoreductase released by bacteria:

[0070] Rat liver microsomes contain a variety of reductases and are often used in azo reduction experiments. The performance of the nanoprobe is verified using azo reductase and rat liver microsome solution.

[0071] 0.1 μM azoreductase was added to the nanoprobe prepared in Example 2 at a volume ratio of 1:15. The scattering spectrum intensity of the single-particle nanoprobe was as follows: Figure 2 As shown in b. Figure 2 The curves in b represent 0 min, 5 min, 10 min, 15 min, and 30 min from bottom to top respectively.

[0072] 1 μg / mL rat liver microsomes were added to the nanoprobes prepared in Example 2 at a volume ratio of 1:10. The scattering spectrum intensity of the single-particle nanoprobes was as follows: Figure 2 As shown in c. Figure 2 The curves in c represent 0 min, 5 min, 10 min, 15 min, and 30 min from bottom to top respectively.

[0073] Depend on Figure 2 b. Figure 2 c It can be seen that within 30 minutes, as time goes by, the scattering spectrum intensity of the nanoprobe gradually increases, indicating that the azo double bond in the BHQ-3 molecule is broken and energy recovery is achieved, indicating that the nanoprobe of the present invention has good feasibility for detecting azoreductase.

[0074] Embodiment 11

[0075] Single-particle nanoprobe for remote detection of single bacterial enzyme release:

[0076] The release of azoreductase is carried out through the secretion of outer membrane vesicles, and the single-particle nanoprobe of the present invention can be used for long-distance detection.

[0077] Take 0.1 mL of the nanoprobe obtained in Example 2 and add it to 1 mL of bacteria (Escherichia coli or Staphylococcus aureus) solution. Slightly shake to mix the bacteria and nanoprobe evenly. Then take 10 μL of the mixed solution and drop it into the groove of the slide with double-sided tape. Then cover it with a cover slip and put it upside down under a dark field microscope for observation. The results are shown in Figure 3 .

[0078] Figure 3 a and Figure 3 In b, arrows indicate nanoprobes, scale bar: 2 μm.

[0079] Depend on Figure 3 c It can be observed that the change in scattering intensity gradually increases with time, indicating that the azoreductase released by bacteria is detected by the nanoprobe and increases with the decrease in detection distance. The change in scattering spectrum intensity at a detection distance of 0.5 μm is significantly higher than that at a detection distance of 3 μm.

[0080] Depend on Figure 3 d It can be observed that the detection limit of Escherichia coli is 3 μm, while Staphylococcus aureus is relatively small in size and releases relatively less azoreductase, and the detection limit of Staphylococcus aureus is 2.5 μm.

[0081] Example 12

[0082] Real-time detection of biooscillations in enzyme release from single bacteria:

[0083] The different growth stages of bacteria mainly depend on the culture time of bacteria. It is generally believed that 0 to 4 hours of culture in a shaker at 37°C and a rotation speed of 220 rap / min is the delay period, 6 to 10 hours is the logarithmic period, 10 to 14 hours is the stationary period, and after 20 hours it enters the decay period.

[0084] Take 0.1 mL of the nanoprobe obtained in Example 2 and add it to 1 mL of bacteria (Escherichia coli or Staphylococcus aureus) solution. Slightly shake to mix the bacteria and nanoprobe evenly. Then take 10 μL of the mixed solution and drop it into the groove of the slide with double-sided tape. Then cover it with a cover slip and put it upside down under a dark field microscope for observation. The results are shown in Figure 4 .

[0085] Figure 4 In ad, I is a schematic diagram, II-IV are dark field images of the detection process, and arrows indicate nanoprobes (the inset is an enlarged view). Scale bar: 2 μm.

[0086] Depend on Figure 4ad It can be observed that the nanoprobes are used to detect the four different growth stages of E. coli. The brightness of the nanoprobes (arrows) at different stages gradually increases over time. From the enlarged image of the nanoprobe in the lower right corner, it can be clearly observed that the brightness change is most obvious in the logarithmic period, and the smallest brightness change is in the stable period.

[0087] By calculating the change of scattering spectrum intensity per unit time through the change of scattering spectrum, the relationship between the release rate of azoreductase of Escherichia coli and Staphylococcus aureus and time was obtained, such as Figure 4 As shown in e. Figure 4 It can be observed that the release rate has obvious oscillation behavior, and different periods have different oscillation signals. The strongest amplitude is in the logarithmic period, the weakest is in the stable period, the longest period is in the stable period, and the shortest is in the decay period, such as Figure 4 f and Figure 4 g. This shows that the growth stage of bacteria can be accurately determined based on the obtained oscillation signal, and the life mechanism of bacteria can be further revealed.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A single particle nanoprobe, characterized in that: The nanoprobe is composed of a gold nanorod and a black hole quencher BHQ-3, wherein the gold nanorod is modified with 3-mercaptopropionic acid; and the single particle is a single bacterial enzyme.

2. The method for preparing the single-particle nanoprobe according to claim 1, characterized in that: The following steps are involved: 3-Mercaptopropionic acid solution is added to the gold nanorod solution for modification. The modified gold nanorod solution is centrifuged and resuspended in water to obtain a modified gold nanorod aqueous solution. Then a black hole quencher BHQ-3 is added to react to obtain a single particle nanoprobe.

3. The preparation method according to claim 2, characterized in that: The gold nanorod solution was centrifuged to remove excess CTAB solution, resuspended in water, and then modified by adding 3-mercaptopropionic acid solution.

4. The preparation method according to claim 2, characterized in that: The concentration of the gold nanorod solution is 0.05-0.15 mg / mL, the concentration of the 3-mercaptopropionic acid solution is 0.05-0.15 mM, and the concentration of the black hole quencher BHQ-3 is 0.5-1.5 μM.

5. The preparation method according to claim 2, characterized in that: The volume ratio of the gold nanorod aqueous solution to the 3-mercaptopropionic acid solution is 1:90-110.

6. The preparation method according to claim 2, characterized in that: The modification time is 10 to 14 hours.

7. The preparation method according to claim 2, characterized in that: The volume ratio of the black hole quencher BHQ-3 to the modified gold nanorod aqueous solution is 1:9-11.

8. The preparation method according to claim 2, characterized in that: The reaction time is 50 to 70 minutes.

9. Use of the single particle nanoprobe according to claim 1 in real-time or remote detection of biological oscillations released by a single bacterial enzyme.

10. A method for real-time or remote detection of enzyme release from a single bacterium, characterized in that: The method comprises the following steps: adding the single-particle nanoprobe described in claim 1 into a bacterial solution, and taking the mixed solution to observe the scattering spectrum intensity of the single-particle nanoprobe under a dark field microscope.

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