CN112011446A - Micro-fluidic chip for bacterial lysis, bacterial lysis device and application thereof - Google Patents
Micro-fluidic chip for bacterial lysis, bacterial lysis device and application thereof Download PDFInfo
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- CN112011446A CN112011446A CN202010893915.5A CN202010893915A CN112011446A CN 112011446 A CN112011446 A CN 112011446A CN 202010893915 A CN202010893915 A CN 202010893915A CN 112011446 A CN112011446 A CN 112011446A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/06—Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
- C12N1/066—Lysis of microorganisms by physical methods
Abstract
The invention provides a micro-fluidic chip for bacterial lysis, a bacterial lysis device and application thereof, belonging to the technical field of cell biology; the chip is of a three-layer laminated structure, wherein the first layer is an acrylic cover plate 1, the second layer is a 3M double-sided adhesive tape 2 and the third layer is a glass slide 4 from top to bottom; and an interdigital electrode 3 is fixed on the glass slide 4. Compared with the prior widely used chemical method (such as an alkaline lysis method), the method does not need to add any chemical reagent, avoids the damage of the chemical reagent to the content of the bacterial cells and the subsequent operation of removing the chemical reagent, and is more convenient. The micro-fluidic chip has simple manufacturing process and low cost. The chip of the invention avoids the defects of high price and the like caused by using materials such as Polydimethylsiloxane (PDMS) and the like, has small product volume and convenient carrying, and can completely realize bacterial electrofusion functionally.
Description
Technical Field
The invention relates to the technical field of cell biology, in particular to a micro-fluidic chip for bacterial lysis, a bacterial lysis device and application thereof.
Background
Microfluidics (Microfluidics), which refers to the science and technology involved in systems using microchannels (tens to hundreds of microns in size) to process or manipulate tiny fluids (nanoliters to attoliters in volume), is an emerging interdiscipline of chemistry, fluid physics, microelectronics, new materials, biology and biomedical engineering.
Currently, there are studies on the application of microfluidic technology to the electrolytic bacteria. Poudineh et al used a three-dimensional tip electrode (3DSTE) to lyse E.coli and achieve release of biomarker RNA while determining and optimizing the conditions for bacterial lysis under this electrode (Poudineh M, Mohamadi RM, Sage A, Mahmoudian L, Sargent EH, Kelley SO. three-dimensional, share-targeted electrodes applied fields to enable direct electric release of interaction biomarkers from cells [ J ] Lab on a chip 2014,14(10): 1785-1790.). Abdossmaad Talebour et al achieved the electrolysis of gram-negative and gram-positive bacteria including E.coli in a Surface-Enhanced high-impedance electrode Lysis chamber and demonstrated that the applied electric field acted on bacterial Lysis simultaneously with the high temperature environment (Talebour A, Maaskant R, Khine AA, Alavie T.Using of Surface Enhanced Blocking (SEB) Electrodes for Microbial Cell Lysis in Flow-Through Devices [ J ]. PLoS one.2014,9 (7)). In the fast Detection of low-level Escherichia coli, in order to overcome the problems of slow Detection time caused by low concentration of intracellular mRNA, Besant et al use an electrochemical method to crack Escherichia coli on a chip, obtain mRNA and make the mRNA contact with a sensor in a short distance, thereby greatly improving the performance of low-level Bacterial Detection (Justin D.Besant, Jagotamoy Das, Edward H.Sargent.Proximal Bacterial and Detection in Nanoliter Wells Using Electrochemistry [ J ]. NAACS no,2013,7(9): 8183-. However, the current research on related electro-cracking still has the limitation of complex structure of the cracking device.
Disclosure of Invention
The invention aims to provide a micro-fluidic chip for bacteria lysis, a bacteria lysis device and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a micro-fluidic chip for bacterial lysis, which is of a three-layer laminated structure, wherein the first layer is an acrylic cover plate 1, the second layer is a 3M double-sided adhesive tape 2 and the third layer is a glass slide 4 from top to bottom; the acrylic cover plate 1 is provided with a sample inlet 1-1 and a sample outlet 1-2; the sample inlet and the sample outlet are communicated with the acrylic cover plate 1; the 3M double-sided adhesive tape 2 is provided with a micro-channel 2-1, a micro-channel inlet 2-2 and a micro-channel outlet 2-3 at two ends of the micro-channel; the micro-channel 2-1 penetrates through the 3M double-sided adhesive tape 2; the micro-channel inlet 2-2 corresponds to the sample inlet 1-1 up and down; the micro-channel outlet 2-3 corresponds to the sample outlet 1-2 up and down; an interdigital electrode 3 is fixed on the glass slide 4; the micro-channel 2-1 is adhered to the interdigital electrode 3.
Preferably, the length of the micro-channel 2-1 is 4-5 mm, the width is 2-2.2 mm, and the depth is 75-85 μm.
Preferably, the diameter of the micro-channel inlet 2-2 is 0.8-1.2 mm; the diameter of the micro-channel outlet 2-3 is 1.8-2 mm.
The invention also provides a bacteria cracking device, which comprises the micro-fluidic chip 6, the bacteria liquid injection assembly 7, the lysate collection assembly 8 and the arbitrary function generator 9; the bacterial liquid injection assembly 7 is communicated with a sample inlet 1-1 of the micro-fluidic chip 6 through a pipeline; the lysate collecting component 8 is communicated with a sample outlet 1-2 of the microfluidic chip 6 through a pipeline; the interdigital electrode 3 is connected to an arbitrary function generator 9.
The invention also provides application of the microfluidic chip or the bacteria lysis device in bacteria lysis.
Preferably, the bacteria comprise escherichia coli.
Preferably, the application comprises the following steps:
1) injecting bacterial liquid containing bacteria from a sample inlet 1-1, and entering a micro-channel 2-1 through a micro-channel inlet 2-2;
2) alternating current is introduced into the interdigital electrode 3 to electrically crack bacteria in the micro-channel 2-1 to obtain a cracking solution;
3) and the lysate after the electric cracking is output from the sample outlet 1-2 through the micro-channel outlet 2-3 and collected.
Preferably, the injection flow rate of the bacterial liquid in the step 1) is 0.5-2 muL/min.
Preferably, the voltage of the alternating current in the step 2) is 5-10VP-PThe frequency is 1 to 1000 kHz.
Preferably, the interdigital electrode 3 and the arbitrary function generator 9 are electrically connected in the step 2); the waveform of the arbitrary function generator is square wave, and the phase difference is 180 degrees.
The invention has the beneficial effects that: the invention provides a micro-fluidic chip for bacterial lysis, which is of a three-layer laminated structure, wherein the first layer is an acrylic cover plate 1, the second layer is a 3M double-sided adhesive tape 2 and the third layer is a glass slide 4 from top to bottom; the acrylic cover plate 1 is provided with a sample inlet 1-1 and a sample outlet 1-2; the sample inlet and the sample outlet are communicated with the acrylic cover plate 1; the 3M double-sided adhesive tape 2 is provided with a micro-channel 2-1, a micro-channel inlet 2-2 and a micro-channel outlet 2-3 at two ends of the micro-channel; the micro-channel 2-1 penetrates through the 3M double-sided adhesive tape 2; the micro-channel inlet 2-2 corresponds to the sample inlet 1-1 up and down; the micro-channel outlet 2-3 corresponds to the sample outlet 1-2 up and down; an interdigital electrode 3 is fixed on the glass slide 4; the micro-channel 2-1 is adhered to the interdigital electrode 3.
Compared with the prior widely used chemical method (such as an alkaline lysis method), the method does not need to add any chemical reagent, avoids the damage of the chemical reagent to the content of the bacterial cells and the subsequent operation of removing the chemical reagent, and is more convenient. The micro-fluidic chip has simple manufacturing process and low cost. The chip of the invention avoids the defects of high price and the like caused by using materials such as Polydimethylsiloxane (PDMS) and the like, has small product volume and convenient carrying, and can completely realize bacterial electrofusion functionally.
The invention also provides a bacteria cracking device, which comprises the micro-fluidic chip 6, the bacteria liquid injection assembly 7, the lysate collection assembly 8 and the arbitrary function generator 9; the bacterial liquid injection assembly 7 is communicated with a sample inlet 1-1 of the micro-fluidic chip 6 through a pipeline; the lysate collecting component 8 is communicated with a sample outlet 1-2 of the microfluidic chip 6 through a pipeline; the arbitrary function generator 9 is electrically connected with the interdigital electrode 3 of the microfluidic chip 6.
According to the invention, the bacterial liquid injection assembly 7 is used for controlling the flow rate of the bacterial liquid, the bacterial liquid is injected into the micro-channel 2-1 through the micro-channel inlet 2-2 for cracking, the lysate flows into the lysate collection assembly 8 for collection through the micro-channel outlet 2-3, manual operation is not required in the cracking process, parameters can be adjusted at any time or continuation or suspension of the cracking process is determined, the automation degree is high, and the cracking process is continuously controllable.
The bacteria cracking device can realize high-efficiency bacteria cracking, and the cracking efficiency can reach more than 90%.
Drawings
FIG. 1 is a structural diagram of a microfluidic chip according to the present invention, wherein an acrylic cover plate 1, a 3M double-sided adhesive tape 2, an interdigital electrode 3, a glass slide 4, a sample inlet 1-1, a sample outlet 1-2, a microchannel 2-1, a microchannel inlet 2-2, and a microchannel outlet 2-3;
FIG. 2 is a structural diagram of an interdigital electrode in accordance with the present invention;
FIG. 3 is a structural diagram of the bacteria lysis apparatus of the present invention, wherein the micro-fluidic chip 6, the bacteria liquid injection assembly 7, the lysate collection assembly 8, and the optional function generator 9 are arranged;
FIG. 4 shows the results of the electrolysis of the micro-fluidic bacteria, wherein a is the results of the plate coating with the bacteria liquid without lysis, and b is the results of the plate coating with the bacteria liquid at a frequency of 500kHz and a flow rate of 2 μ L/min and a voltage of 4Vp-pC is a voltage of 8V at a frequency of 500kHz and a flow rate of 2. mu.L/minp-pThe result of bacterial lysis of (a);
FIG. 5 shows the lysis efficiency of bacteria when different voltages are applied;
FIG. 6 shows the bacterial lysis efficiency when different alternating current frequencies are applied;
FIG. 7 shows the lysis efficiency of bacteria at different flow rates.
Detailed Description
The invention provides a micro-fluidic chip for bacterial lysis, which is of a three-layer laminated structure, wherein the first layer is an acrylic cover plate 1, the second layer is a 3M double-sided adhesive tape 2 and the third layer is a glass slide 4 from top to bottom; the acrylic cover plate 1 is provided with a sample inlet 1-1 and a sample outlet 1-2; the sample inlet and the sample outlet are vertically communicated with the acrylic cover plate 1; the 3M double-sided adhesive tape 2 is provided with a micro-channel 2-1, a micro-channel inlet 2-2 and a micro-channel outlet 2-3 at two ends of the micro-channel; the micro-channel 2-1 penetrates through the 3M double-sided adhesive tape 2 from top to bottom; the micro-channel inlet 2-2 corresponds to the sample inlet 1-1 up and down; the micro-channel outlet 2-3 corresponds to the sample outlet 1-2 up and down; an interdigital electrode 3 is fixed on the glass slide 4; the micro-channel 2-1 is adhered to the interdigital electrode 3.
The structure diagram of the microfluidic chip of the invention is shown in figure 1, wherein an acrylic cover plate 1, a 3M double-sided adhesive tape 2, an interdigital electrode 3, a glass slide 4, a sample inlet 1-1, a sample outlet 1-2, a microchannel 2-1, a microchannel inlet 2-2 and a microchannel outlet 2-3.
In the present invention, the interdigital electrode 3 is derived from a conventional commercially available one; the distance between the interdigital electrodes is preferably 100 μm, and the width of each electrode is preferably 100 μm; the structure of the interdigital electrode is shown in fig. 2.
In the invention, the acrylic cover plate tightly covers the upper layer of the micro-channel and is used for keeping the micro-fluidic chip in a sealed environment; the length of the acrylic cover plate is preferably 20mm, the width of the acrylic cover plate is preferably 20mm, and the thickness of the acrylic cover plate is preferably 1 mm.
In the invention, the shapes of the sample inlet 1-1 and the sample outlet 1-2 are preferably circular respectively, and in the specific implementation process of the invention, the sample inlet 1-1 and the sample outlet 1-2 are obtained by drilling holes through a drill; the diameter of the sample inlet 1-1 is preferably consistent with that of the microchannel inlet 2-2; the diameter of the sample outlet 1-2 is preferably the same as the diameter of the microchannel outlet 2-3.
In the invention, the length of the micro-channel 2-1 is preferably 4-5 mm, more preferably 4.5mm, the width is preferably 2-2.2 mm, and the depth is preferably 75-85 μm, more preferably 80 μm; the micro-channel 2-1 is used for introducing bacterial suspension.
In the invention, the diameter of the micro-channel inlet 2-2 is preferably 0.8-1.2 mm, and more preferably 1 mm; the diameter of the micro-channel outlet 2-3 is preferably 1.8-2 mm.
In the present invention, the interdigital electrode 3 is fixed on the glass slide 4 preferably by tightly adhering and fixing with 3M double-sided adhesive tape; the interdigital electrode 3 is used for providing an electric field and cracking bacteria.
In the invention, the glass slide is preferably sprayed with an ethanol water solution with the volume concentration of 70-80% before use, and is wiped clean by using a lens wiping paper.
The invention also provides a bacteria cracking device, which comprises the micro-fluidic chip 6, the bacteria liquid injection assembly 7, the lysate collection assembly 8 and the arbitrary function generator 9; the bacterial liquid injection assembly 7 is communicated with a sample inlet 1-1 of the micro-fluidic chip 6 through a pipeline; the lysate collecting component 8 is communicated with a sample outlet 1-2 of the microfluidic chip 6 through a pipeline; the arbitrary function generator 9 is electrically connected with the interdigital electrode 3 of the microfluidic chip 6. In the invention, the bacteria liquid injection assembly 7 is preferably communicated with a sample inlet 1-1 of the microfluidic chip 6 through a hose; the lysate collecting component 8 is preferably communicated with the sample outlet 1-2 of the microfluidic chip 6 through a hose.
The structure diagram of the bacteria cracking device of the invention is shown in figure 3, wherein a microfluidic chip 6, a bacteria liquid injection assembly 7, a cracking liquid collection assembly 8 and an arbitrary function generator 9 are arranged in the device.
In the present invention, the bacteria liquid injection assembly 7 preferably comprises a syringe; the injector is preferably connected with a syringe pump; the syringe pump is preferably a syringe pump available from langer pump (hargerpump) (Harvard PHD 2000); the injection pump is used for fixing the injector and adjusting the flow rate of the bacterial liquid; before use, the bacteria liquid injection assembly 7 is preferably sterilized, and more preferably sterilized by ethanol water solution with a volume concentration of 75%.
In the present invention, the lysate collection assembly 8 preferably comprises an EP tube.
In the present invention, the arbitrary function generator 9 is preferably a model AFG3102 available from Tektronix, Inc.; the arbitrary function generator 9 is used to provide an electrical signal.
The invention also provides the application of the micro-fluidic chip or the bacteria lysis device in bacteria lysis; the bacterium preferably includes Escherichia coli, and more preferably Escherichia coli JM 109.
In the present invention, the application preferably comprises the steps of:
1) injecting bacterial liquid containing bacteria from a sample inlet 1-1, and entering a micro-channel 2-1 through a micro-channel inlet 2-2;
2) alternating current is introduced into the interdigital electrode 3 to electrically crack bacteria in the micro-channel 2-1 to obtain a cracking solution;
3) and the lysate after the electric cracking is output from the sample outlet 1-2 through the micro-channel outlet 2-3 and collected.
Firstly, injecting bacterial liquid containing bacteria from a sample inlet 1-1, and entering a micro-channel 2-1 through a micro-channel inlet 2-2; the injection flow rate of the bacterial liquid is preferably 0.5-2 mu L/min;
in the invention, the bacterial liquid is preferably prepared by a method comprising the following steps:
s1, activating and culturing bacteria to be lysed to obtain seed liquid;
s2, inoculating the seed liquid to an LB culture medium, carrying out amplification culture, centrifuging the culture liquid, taking a precipitate, re-suspending the precipitate and diluting by 10-5～10-6And doubling to obtain a bacterial liquid.
Firstly, carrying out activated culture on bacteria to be lysed to obtain seed liquid; the OD value of the seed liquid is preferably 0.8-1.2, and more preferably 1; the conditions of the activation culture depend on the bacteria to be cultured; when the bacteria are escherichia coli, the temperature of the activation culture is preferably 37 ℃, the time is preferably 16-20 h, more preferably 18h, and the mode of the activation culture is preferably shake culture; the rotation speed of the shaking table culture is preferably 180-200 rpm; the storage temperature of the seed liquid is preferably-80 ℃.
After the seed liquid is obtained, the seed liquid is inoculated to an LB culture medium for amplification culture, centrifugation, sediment taking, heavy suspension and sediment dilution are carried out for 10-5～10-6Multiplying to obtain a bacterial liquid; the proportion of the seed liquid to the LB culture medium is preferably 50 mu L: 5 mL; the conditions of the expanded culture depend on the bacteria to be cultured; when the bacteria are escherichia coli, the temperature of the amplification culture is preferably 37 ℃, the time is preferably 10-14 h, more preferably 12h, and the mode of the amplification culture is preferably shake culture; the rotation speed of the shaking table culture is preferably 180-200 rpm; the reagent used for resuspending the pellet is preferably ultrapure water.
After the bacteria liquid is injected into the micro-channel 2-1, alternating current is introduced into the interdigital electrode 3 to electrically crack bacteria in the micro-channel 2-1 to obtain a cracking liquid; the voltage of the alternating current is preferably 5-10VP-PThe frequency is preferably 1 to 1000kHz, more preferably 50 to 300kHz, and most preferably 100 to 200 kHz.
In the invention, the arbitrary function generator 9 provides an electric signal to the interdigital electrode 3 through a copper wire; the waveform of the arbitrary function generator is preferably square wave, and the phase difference is preferably 180 degrees; 5-10VP-PRefers to the single channel voltage of any function generator 9; the output voltage of each channel is the same, and the total output voltage of the channels is two.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1A method for the lysis of E.coli
Step 1: design and manufacture of micro-fluidic chip
The schematic structure of the microfluidic chip is shown in fig. 1.
From top to bottom:
the first layer is an acrylic cover plate 1, holes are drilled on the acrylic cover plate through a drilling machine and respectively comprise a sample inlet 1-1 and a sample outlet 1-2, and the distance between the sample inlet 1-1 and the sample outlet 1-2 is the length of a micro channel 2-1. The micro-channel cover closely covers the upper layer of the micro-channel 2-1, and the positions of the sample inlet 1-1 and the sample outlet 1-2 correspond to the micro-channel inlet 2-2 and the micro-channel outlet 2-3 one by one.
The second layer uses 3M double sided tape 2 etched micro-channels 2-1. And (3) taking the other glass slide, spraying and wiping the other glass slide with alcohol, sticking a 3M double-sided adhesive tape with a certain length on the glass slide, and carving a straight micro-channel with the length of 4-5 mm and the width of 2mm on the 3M double-sided adhesive tape by using a scalpel, wherein the straight micro-channel comprises an inlet (the diameter of 1mm) and an outlet (the diameter of 1.8 mm). And (3) placing the etched micro-channel 2-1 under a microscope for observation to ensure that the micro-channel 2-1 is flat. The micro-channel 2-1 is adhered to the interdigital electrode 3 so as to be tightly adhered.
The third layer is a glass slide 4, and the glass slide 4 is sprayed with a self-prepared ethanol water solution with the volume concentration of 75% before use and is wiped clean by a piece of lens wiping paper. The interdigital electrode 3 is tightly fixed on the glass slide 4 by using 3M double-sided adhesive tape.
Step 2: escherichia coli culturing and processing method
The strain used is Escherichia coli JM109, and the culture medium is LB medium. The existing bacterial liquid is plated on an LB solid culture medium for streaking, and is cultured in an incubator at 37 ℃ for overnight at constant temperature, and a single clone is selected to be cultured in 5mL of LB liquid culture medium for 18h under shaking, and the constant temperature shaking table is set at 37 ℃ and is cultured at 200rpm until the OD value is about 1.0. And (5) placing the bacterial liquid in a refrigerator at-80 ℃ for freezing storage. Inoculating 50 μ L of the bacterial liquid into 5mL LB culture medium before experiment, shaking at constant temperature for 12h under the same conditions, centrifuging at 13000r/min for 1min, discarding supernatant, resuspending with 1mL of ultrapure water, and diluting 10%-5～10-6Double, ready for electrolysis.
And step 3: connection and use of bacteria lysis device
A schematic of the bacterial lysis apparatus is shown in FIG. 3. Injecting the diluted bacteria liquid into an injector which is disinfected by ethanol aqueous solution with volume concentration of 75% in advance and then washed and cleaned by ultrapure water, connecting an arbitrary function generator (Tektronix model AFG3102) with the interdigital electrode micro-fluidic chip, connecting the injector with the inlet of the micro-channel through a hose, and connecting the outlet of the micro-channel with an EP (ethylene propylene) tube through a hose. The syringe was secured with a syringe pump (Harvard PHD 2000).
And 4, step 4: bacterial lysis and bacterial lysis efficiency testing
The method comprises the steps of sterilizing a micro-channel, a used syringe and a used hose by using alcohol and washing the micro-channel, the used syringe and the used hose by using ultrapure water, sucking diluted bacteria liquid into the syringe, connecting an experimental device, adjusting an arbitrary function generator to be square waves, adjusting the phase difference to be 180 degrees and adjusting the phase difference to be 0-10Vp-pChanging alternating current voltage within the range of (single-channel voltage, each channel has the same output voltage, and the total number of the channels is two), changing alternating current frequency within the range of 1 kHz-50 MHz, and changing the flow rate of the injection pump within the range of 0.5-10 muL/min. The collected lysate and the original bacterial liquid which is not lysed are respectively coated on a plate with 100 mu L, and then are compared, and the colony number on the agar plate is counted, so that the lysis effect is judged.
The results of lysis are shown in FIG. 4 (results of electrolysis of micro-fluidic bacteria), where a is the results of plating with bacteria solution without lysis, and b is the results of plating with 4V at a frequency of 500kHz and a flow rate of 2. mu.L/minp-pC is a voltage of 8V at a frequency of 500kHz and a flow rate of 2. mu.L/minp-pThe result of bacterial lysis of (1). When an electric field is applied to the chip, the number of colibacillus colonies on the agar plate is obviously reduced, and the method can effectively crack the colibacillus and has the voltage of 8Vp-pWhen the number of colonies on the agar plate was significantly reduced, the voltage was 8Vp-pThe lysis effect of bacteria can be improved.
The bacterial lysis efficiency in this project is defined as follows:
η＝(n0-n)/n0×100％ (1)
wherein n is the colony number of colibacillus in lysate, and n is0The number of colonies in the original bacterial liquid was 100. mu.L, and eta was n0-n and n0The ratio is the bacteria lysis efficiency.
Results of bacterial lysis efficiency calculations are shown in FIGS. 5 and 6, whichFIG. 5 shows the lysis efficiency of bacteria when different voltages are applied; FIG. 6 shows the lysis efficiency of bacteria when different AC frequencies were applied. As can be seen from FIG. 5, the voltage is 10Vp-pThe bacteria lysis efficiency is highest; as can be seen from FIG. 6, the bacterial lysis efficiency is high within the range of 1-1000 kHz.
The Escherichia coli cracking efficiency is high under the condition of a small flow rate (0.5-2 mu L/min). See in particular fig. 7.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A micro-fluidic chip for bacteria lysis is of a three-layer laminated structure, wherein the chip comprises a first acrylic cover plate (1), a second 3M double-faced adhesive tape (2) and a third glass slide (4) from top to bottom;
the acrylic cover plate (1) is provided with a sample inlet (1-1) and a sample outlet (1-2); the sample inlet and the sample outlet are vertically communicated with the acrylic cover plate (1);
a micro-channel (2-1) is formed in the 3M double-sided adhesive tape (2), and a micro-channel inlet (2-2) and a micro-channel outlet (2-3) are formed in two ends of the micro-channel; the micro-channel (2-1) penetrates through the 3M double-sided adhesive tape (2) from top to bottom; the micro-channel inlet (2-2) corresponds to the sample inlet (1-1) up and down; the micro-channel outlet (2-3) corresponds to the sample outlet (1-2) up and down;
an interdigital electrode (3) is fixed on the glass slide (4); the micro-channel (2-1) is adhered to the interdigital electrode (3).
2. The microfluidic chip according to claim 1, wherein the microchannel (2-1) has a rectangular shape; the length of the micro-channel (2-1) is 4-5 mm, the width is 2-2.2 mm, and the depth is 75-85 μm.
3. The microfluidic chip according to claim 1, wherein the diameter of the microchannel inlet (2-2) is 0.8-1.2 mm; the diameter of the micro-channel outlet (2-3) is 1.8-2 mm.
4. A bacteria lysis device, comprising the microfluidic chip (6) of claim 1, a bacteria liquid injection assembly (7), a lysate collection assembly (8) and an arbitrary function generator (9); the bacterial liquid injection assembly (7) is communicated with a sample inlet (1-1) of the micro-fluidic chip (6) through a pipeline; the lysate collecting component (8) is communicated with a sample outlet (1-2) of the microfluidic chip (6) through a pipeline; the arbitrary function generator (9) is connected with the interdigital electrode (3) of the microfluidic chip (6) through a copper wire.
5. Use of the microfluidic chip according to any one of claims 1 to 3 or the bacterial lysis device according to claim 4 for bacterial lysis.
6. The use of claim 5, wherein the bacteria comprise E.
7. The application according to claim 5 or 6, characterized in that it comprises the following steps:
1) injecting bacterial liquid containing bacteria from a sample inlet (1-1) and entering a micro-channel (2-1) through a micro-channel inlet (2-2);
2) alternating current is introduced into the interdigital electrode (3) to carry out electric lysis on bacteria in the micro-channel (2-1);
3) and the lysate after the electric cracking is output from the sample outlet (1-2) through the micro-channel outlet (2-3) and is collected.
8. The use of claim 7, wherein the injection flow rate of the bacterial liquid in the step 1) is 0.5-2 μ L/min.
9. The use according to claim 7, wherein the voltage of the alternating current in step 2) is 5-10VP-PThe frequency is 1 to 1000 kHz.
10. Use according to claim 7, characterized in that in step 2) the interdigital electrodes (3) are connected to an arbitrary function generator (9); the waveform of the arbitrary function generator is square wave, and the phase difference is 180 degrees.
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