Method for rapidly detecting heavy metal resistance of bacteria based on QCM-D

Abstract

The invention belongs to the technical field of heavy metal resistance monitoring, and particularly relates to a QCM-D. The method comprises the following steps that a heavy metal solution is injected into a dissipative quartz crystal microbalance, and a heavy metal environment layer is deposited on the surface of a chip of the dissipative quartz crystal microbalance; injecting the bacterial solution into a dissipative quartz crystal microbalance; and injecting the heavy metal solution into the dissipative quartz crystal microbalance to elute non-adhered bacteria, and detecting the change of the resonant frequency of the dissipative quartz crystal microbalance. According to the method, the dissipative quartz crystal microbalance is used for monitoring the change of the resonant frequency generated after the bacteria are in contact with the crystal surface modified by the heavy metal, so that the heavy metal resistance of the bacteria can be accurately detected, and the heavy metal resistance of the bacteria can be rapidly identified only in about 40 minutes.

Description

Technical Field

[0001] The present invention belongs to the technical field of heavy metal resistance monitoring, and more specifically, relates to a method for rapid detection of bacterial heavy metal resistance based on QCM-D. Background Art

[0002] Bacteria can resist and transform heavy metals through complex physiological mechanisms. This resistance mechanism provides an important theoretical basis for bioremediation. Heavy metal-resistant bacteria can resist the toxicity of a variety of heavy metal ions, and this ability may be related to the pathogenicity and environmental adaptability of bacteria. Therefore, rapid and accurate detection of bacterial heavy metal resistance is of great significance for public health, environmental monitoring and related research fields.

[0003] At present, conventional methods for detecting bacterial heavy metal resistance mainly include microbial culture, metagenomic sequencing, high-throughput qPCR technology, DNA microarray, atomic absorption spectroscopy, inductively coupled plasma mass spectrometry or gene chip technology. Although these methods have certain guarantees in terms of accuracy, they have some obvious limitations. For example, microbial culture requires a long culture cycle of several days; atomic absorption spectroscopy and inductively coupled plasma mass spectrometry have relatively complex operation processes and require professional equipment and technicians.

[0004] Quartz Crystal Microbalance with Dissipation (QCM-D) is an emerging quartz crystal microbalance technology that shows great potential in the field of biomolecule detection due to its high sensitivity, real-time monitoring and easy operation. It can provide multiple frequency and dissipation factor data for determining the quality of very thin adsorption layers, and simultaneously provide structural information such as viscoelasticity. This technology can be used to study molecular interactions and molecular, nanoparticle and cell adsorption on many different types of surfaces, and can also detect structural changes in molecules and the dynamic process of adsorption and resolution.

[0005] The study "Effects of electrokinetic phenomena on bacterial deposition monitored by a quartz crystal microbalance (QCM-D) with dissipation monitoring" disclosed: the effect of DC electric field on the deposition of two bacterial strains was quantified using QCM-D technology, and it was found that the QCM-D signal can be used as an indicator of cell attachment rigidity. "Portable computer simulation analysis of real-time measurement of QCM-D system for biomedical applications" introduces the application of QCM-D technology in bacterial detection, which can monitor the interaction between biomolecules and bacteria in real time. Patent CN 107475349A discloses a method for rapid detection of bacterial resistance based on QCM-D, which detects bacterial resistance by measuring the changes in the resonant frequency and dissipation of bacteria adsorbed on the crystal surface after contact with antibiotics by a dissipative quartz crystal microbalance.

[0006] However, there is no case of using dissipative quartz crystal microbalance to detect bacterial heavy metal resistance. Summary of the invention

[0007] Based on the above existing technical problems, the primary purpose of the present invention is to provide a method for rapid detection of bacterial heavy metal resistance based on QCM-D.

[0008] Another object of the present invention is to provide a method for rapid detection of bacterial heavy metal resistance based on QCM-D and its application in detecting bacterial heavy metal resistance.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention claims a method for rapid detection of bacterial heavy metal resistance based on QCM-D, comprising the following steps:

[0011] S1: Injecting heavy metal solution into the dissipative quartz crystal microbalance to deposit a heavy metal environment layer on the chip surface;

[0012] S2: inject the bacterial solution into the dissipative quartz crystal microbalance;

[0013] S3: Inject the heavy metal solution into the dissipative quartz crystal microbalance to wash away the non-adherent bacteria, and detect the change in the resonant frequency of the dissipative quartz crystal microbalance.

[0014] The dissipative quartz crystal microbalance (QCM-D) records the change in chip frequency and the speed of the change from oscillation to complete stillness by intermittently opening / disconnecting the circuit. Based on the characteristic that the dissipative quartz crystal microbalance (QCM-D) can measure the change in quartz crystal frequency (Δf) in real time, QCM-D can be used to measure the change in frequency before and after bacteria adhere to the surface of quartz crystal modified with heavy metals, so as to understand whether the bacteria are resistant or adaptable to the heavy metal.

[0015] If the bacteria are not resistant to such heavy metals, they will move away from the medium surface on the chip surface modified with heavy metals, and the change in the resonant frequency (Δf) will be relatively weak, indicating that they are less deposited on the medium surface. On the contrary, if the bacteria have a certain resistance to heavy metals, they will be more deposited on the chip surface modified with heavy metals, and the resonant frequency (Δf) will drop significantly.

[0016] The method provided by the present invention has a fast detection speed and can realize the rapid identification of bacterial heavy metal resistance in only about 40 minutes.

[0017] Preferably, the type of heavy metal in the heavy metal solution is one or more of silver, cadmium, cobalt, chromium, copper, mercury, nickel, lead, antimony, tellurium, titanium or zinc.

[0018] Preferably, in step S1, the rate of injecting the heavy metal solution into the dissipative quartz crystal microbalance is 1 μL / min to 1 mL / min. More specifically, the heavy metal solution is injected into the dissipative quartz crystal microbalance via a peristaltic pump.

[0019] Preferably, in step S1, the peristaltic pump rate can be adjusted according to the difference in the interaction force between different heavy metal ions and the chip surface.

[0020] Preferably, in step S1, the concentration of the heavy metal solution is 3-8 mM.

[0021] Preferably, the step S1 further includes an adjustment operation, wherein the adjustment operation is: adjusting the baseline of the dissipative quartz crystal microbalance so that its resonant frequency and dissipation value remain unchanged.

[0022] Preferably, in step S2, the rate at which the bacterial solution is injected into the dissipative quartz crystal microbalance is 1 μL / min to 1 mL / min. More specifically, the bacterial solution is injected into the dissipative quartz crystal microbalance via a peristaltic pump.

[0023] Preferably, in step S2, the peristaltic pump rate can be adjusted according to the difference in adhesion of different bacteria.

[0024] Preferably, in the step S2, the bacterial solution is obtained by the following method: activating the bacterial solution, culturing to a logarithmic growth phase, washing by centrifugation, obtaining a supernatant, and adding a buffer solution to obtain the bacterial solution.

[0025] Preferably, the centrifugal rotation speed is 500-10000 rpm.

[0026] Preferably, the centrifugation time is 5-20 min.

[0027] Preferably, the buffer solution is PBS buffer.

[0028] Preferably, after the supernatant is added to the buffer, the bacteria are dispersed by vortexing or ultrasonic vibration.

[0029] Preferably, in step S3, the rate at which the buffer solution is injected into the dissipative quartz crystal microbalance is 1 μL / min to 1 mL / min. More specifically, the buffer solution is injected into the dissipative quartz crystal microbalance via a peristaltic pump.

[0030] Preferably, the peristaltic pump rate in step S3 can be adjusted according to the differences in adaptability of different bacteria to different heavy metal ions.

[0031] Preferably, in step S3, the heavy metal solution is a mixture of heavy metals and deionized water or a buffer solution, wherein the concentration of anions is consistent with the concentration of anions in the background solution where the bacteria are located in step S2.

[0032] Furthermore, the present invention seeks to protect the application of the above method in detecting bacterial heavy metal resistance.

[0033] The present invention has the following beneficial effects:

[0034] The present invention provides a method for detecting bacterial heavy metal resistance. By monitoring the frequency changes generated by the dissipative quartz crystal microbalance when bacteria come into contact with a chip modified with heavy metals, accurate detection of bacterial heavy metal resistance can be achieved. Rapid identification of bacterial heavy metal resistance can be completed in only about 40 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Frequency change of Cd-modified silica chip.

[0036] Figure 2 This is the resonant frequency detection result of Example 1.

[0037] Figure 3 This is the resonant frequency detection result of Example 2.

[0038] Figure 4 This is the resonant frequency detection result of Example 3. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0040] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0041] Analysis of frequency change (Δf3) and calculation of mass change:

[0042]

[0043] Δm is the mass change; C is the mass sensitivity constant; n is the resonant mode number (for Δf3, n=3); Δf is the frequency change.

[0044] Example 1

[0045] In this example, Bacillus subtilis that has not been cultured under heavy metal cadmium (Cd) stress and has no Cd resistance is selected as the bacteria to be examined. The domesticated Bacillus subtilis (Bacillus subtilis bio-168) was purchased from the China General Microbiological Culture Collection Center, with a collection number of CMCC (B) 63501 = A.

[0046] (1) 100uL of Bacillus subtilis culture stored in -80℃ glycerol was taken and added to 5mL LB medium. The culture was activated in a shaker at 35℃ and 170rpm for 24h. 1mL of the activated culture was taken and added to 100mL LB medium. After culturing for 12h, the culture reached the end of the logarithmic growth phase. The culture was centrifuged at 6000rpm for 8min and washed three times. The supernatant was aspirated for the last time and fresh PBS (phosphate buffered saline) was added and mixed thoroughly to obtain a bacterial solution.

[0047] (2) Using 10 mM KCl solution, the baseline of the dissipative quartz crystal microbalance (QCM-D) was leveled and the resonant frequency (Δf) no longer changed.

[0048] (3) A 5 mM CdCl2 heavy metal solution was introduced into the dissipative quartz crystal microbalance for 20 min at a peristaltic pump flow rate of 0.1 mL / min until it reached saturation (e.g. Figure 1 shown).

[0049] (4) After vortexing the bacterial solution prepared in step (1), add it to a 1 mM KCl solution and use a hemocytometer to control the injection volume of the bacterial solution to 3 × 10 7 , added into the dissipative quartz crystal microbalance flow cell through a peristaltic pump, with a peristaltic pump flow rate of 0.1 mL / min.

[0050] (5) After 20 min, 0.5 mM CdCl2 buffer solution dissolved in PBS was added to the dissipative quartz crystal microbalance to elute the non-adherent bacteria and detect the resonant frequency at the same time.

[0051] like Figure 2 As shown, the change in the resonance frequency detected was not obvious, indicating that the Bacillus subtilis of this example was almost not deposited on the interface and had no heavy metal resistance to Cd.

[0052] Example 2

[0053] In this example, a sample containing 50 mg / L Cd 2+ Bacillus subtilis with weak Cd resistance (hereinafter referred to as Group B) cultured in a medium with a stress culture of 10 generations was used as the bacteria to be examined. The bacteria were domesticated Bacillus subtilis bio-168 purchased from the China General Microbiological Culture Collection Center, with a collection number of CMCC (B) 63501 = A.

[0054] (1) 100uL of the group B bacterial solution stored in -80℃ glycerol was taken and added to 5mL LB medium. The solution was activated in a shaker at 35℃ and 170rpm for 24h. 1mL of the activated bacterial solution was taken and added to 100mL LB medium. After culturing for 12h, the solution reached the end of the logarithmic growth phase. The solution was centrifuged at 6000rpm for 8min and washed three times. The supernatant was aspirated for the last time and fresh PBS (phosphate buffered saline) was added and mixed thoroughly to obtain a bacterial solution.

[0055] (2) Using 10 mM KCl solution, the baseline of the dissipative quartz crystal microbalance (QCM-D) was leveled and the resonant frequency (Δf) no longer changed.

[0056] (3) A 5 mM CdCl2 heavy metal solution was introduced into the dissipative quartz crystal microbalance for 20 min at a peristaltic pump flow rate of 0.1 mL / min until it reached saturation.

[0057] (4) After vortexing the bacterial solution prepared in step (1), add it to a 1 mM KCl solution and use a hemocytometer to control the injection volume of the bacterial solution to 3 × 10 7 , added into the dissipative quartz crystal microbalance flow cell through a peristaltic pump, with a peristaltic pump flow rate of 0.1 mL / min.

[0058] (5) After 20 min, 0.5 mM CdCl2 buffer solution dissolved in PBS was added to the dissipative quartz crystal microbalance to elute the non-adherent bacteria and detect the resonant frequency at the same time.

[0059] like Figure 3 As shown, a significant change in the resonance frequency was detected, indicating that the Bacillus subtilis of this embodiment was deposited on the interface and had a certain heavy metal resistance to Cd.

[0060] Example 3

[0061] In this example, a sample containing 100 mg / L Cd 2+The Bacillus subtilis with strong Cd resistance (hereinafter referred to as Group C) which was cultured in a medium with a stress culture of 10 generations was used as the bacteria to be examined. The bacteria were domesticated Bacillus subtilis bio-168 purchased from the China General Microbiological Culture Collection Center, with a collection number of CMCC (B) 63501 = A.

[0062] (1) 100 uL of the bacterial solution of group C stored in glycerol at -80°C was taken and added to 5 mL of LB medium. The solution was activated in a shaker at 35°C and 170 rpm for 24 h. 1 mL of the activated bacterial solution was taken and added to 100 mL of LB medium. After culturing for 12 h, the solution reached the end of the logarithmic growth phase. The solution was centrifuged at 6000 rpm for 8 min and washed three times. The supernatant was aspirated for the last time and fresh PBS (phosphate buffered saline) was added and mixed thoroughly to obtain a bacterial solution.

[0063] (2) Using 10 mM KCl solution, the baseline of the dissipative quartz crystal microbalance (QCM-D) was leveled and the resonant frequency (Δf) no longer changed.

[0064] (3) A 5 mM CdCl2 heavy metal solution was introduced into the dissipative quartz crystal microbalance for 20 min at a peristaltic pump flow rate of 0.1 mL / min until it reached saturation.

[0065] (4) After vortexing the bacterial solution prepared in step (1), add it to a 1 mM KCl solution and use a hemocytometer to control the injection volume of the bacterial solution to 3 × 10 7 , added into the dissipative quartz crystal microbalance flow cell through a peristaltic pump, with a peristaltic pump flow rate of 0.1 mL / min.

[0066] (5) After 20 min, 0.5 mM CdCl2 buffer solution dissolved in PBS was added to the dissipative quartz crystal microbalance to elute the non-adherent bacteria and detect the resonant frequency at the same time.

[0067] like Figure 4 As shown, a significant decrease in the resonance frequency was detected, indicating that the Bacillus subtilis of this example had a strong deposition phenomenon at the interface and had heavy metal resistance and adaptability to Cd.

[0068] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for rapid detection of bacterial heavy metal resistance based on QCM-D, characterized in that: The steps include: S1: Injecting heavy metal solution into the dissipative quartz crystal microbalance to deposit a heavy metal environment layer on the chip surface; S2: inject the bacterial solution into the dissipative quartz crystal microbalance; S3: Inject the heavy metal solution into the dissipative quartz crystal microbalance to wash away the non-adherent bacteria, and detect the change in the resonant frequency of the dissipative quartz crystal microbalance.

2. The method according to claim 1, characterized in that The heavy metals in the heavy metal solution are one or more of silver, cadmium, cobalt, chromium, copper, mercury, nickel, lead, antimony, tellurium, titanium or zinc.

3. The method according to claim 1, characterized in that In the step S1, the rate of injecting the heavy metal solution into the dissipative quartz crystal microbalance is 1 μL / min to 1 mL / min.

4. The method according to claim 1, characterized in that: In the step S1, the concentration of the heavy metal solution is 3-8 mM.

5. The method according to claim 1, characterized in that The step S1 also includes an adjustment operation, which is: adjusting the baseline of the dissipative quartz crystal microbalance so that its resonant frequency and dissipation value remain unchanged.

6. The method according to claim 1, characterized in that In the step S2, the rate at which the bacterial solution is injected into the dissipative quartz crystal microbalance is 1 μL / min to 1 mL / min.

7. The method according to claim 1, characterized in that In the step S2, the bacterial solution is obtained by the following method: activating the bacterial solution, culturing it to a logarithmic growth phase, washing it by centrifugation, obtaining a supernatant, and adding a buffer solution to obtain the bacterial solution.

8. The method according to claim 1, characterized in that In the step S3, the rate at which the buffer solution is injected into the dissipative quartz crystal microbalance is 1 μL / min to 1 mL / min.

9. The method according to claim 1, characterized in that: In step S3, the heavy metal solution is a mixture of heavy metal and deionized water or buffer solution.

10. Use of the method according to any one of claims 1 to 9 in detecting bacterial heavy metal resistance.

Images