CN116586132A - Microfluidic chip of coupling phage modified biosensor and method for quantitatively detecting bacteria by using microfluidic chip - Google Patents
Microfluidic chip of coupling phage modified biosensor and method for quantitatively detecting bacteria by using microfluidic chip Download PDFInfo
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Abstract
The invention provides a microfluidic chip of a coupling phage modified biosensor and a method for quantitatively detecting bacteria, belonging to the technical field of microorganism detection. The microfluidic chip of the coupling phage modified biosensor provided by the invention comprises a bottom layer and a top layer; the top layer comprises a sample injection area, a detection area and a sample outlet area; the detection zone has a plurality of linear channels, with different phage-modified biosensors coupled in different channels. The microfluidic chip can simultaneously realize the rapid and accurate detection of the bacterial content of different pathogenic bacteria, and has the advantages of high sensitivity, high accuracy, strong specificity, short test time and simple and convenient operation.
Description
Technical Field
The invention relates to the technical field of microorganism detection, in particular to a microfluidic chip of a coupling phage modified biosensor and a method for quantitatively detecting bacteria by using the microfluidic chip.
Background
There are several methods for detecting bacteria, such as gold standard method, enzyme-linked immunosorbent assay (ELISA), polymerase Chain Reaction (PCR) technique, etc. These methods have excellent accuracy, but also have the disadvantages of time consuming, cumbersome to operate, expensive large instruments, high professional quality of operators and easy generation of false positive results. Although the above-mentioned techniques provide standards for detecting different types of pathogenic bacteria in laboratories, most of them need to be developed in laboratories, and they cannot meet different requirements of detection and analysis such as on-site detection and clinical diagnosis when dealing with public emergencies in the fields of foods and biomedicine.
Microfluidic technology is the forefront technical field of the hottest detection technology at present. Compared with the traditional experiment platform, the detection device is applied to bacteria detection, can greatly shorten detection time, reduce consumable, reduce cost, and has high integration level, high sensitivity, and small and convenient detection equipment. In addition, the advantages of flexible combination of various unit operations, overall controllability and the like of the micro-flow control enable the micro-flow control to be combined with a research thought of bacteria detection, and the micro-flow control becomes an important technical platform in the field of bacteria detection research.
Compared with the traditional bacterial detection method, the bacterial detection in the micro-flow control has the unique advantage. The relatively closed environment formed by the microfluidic channels and the two-dimensional flow of fluid in the channels enable more complex physicochemical reactions to be performed on bacteria. How to realize integrated and portable detection by utilizing a microfluidic chip technology is a current research trend. In addition, since the existing sample preparation technology is very limited, particularly when the concentration of pathogenic bacteria is very low, the introduction of efficient bacteria separation and enrichment strategies on the chip is of great importance for the rapid and sensitive detection of bacteria. In addition, due to the complexity of the matrix in the bacterial detection process, the target biomolecules may generate non-specific adsorption on the microfluidic chip to cause false negative results, so that some biological probes are generally required to be introduced to perform appropriate surface modification on the microfluidic channel to reduce the non-specific adsorption, so as to ensure the sensitivity and repeatability of the detection system. Meanwhile, in order to realize simultaneous detection of multiple bacteria, designing multiple microcavities and multiple fluid channel devices on a single chip is a future development direction for realizing multiple detection. Therefore, according to practical requirements, developing a low-cost, user-friendly, portable, high-sensitivity and accurate multi-channel detection microfluidic chip method is an important research point for detecting food-borne pathogenic bacteria.
A bacteriophage is a virus that can selectively bind to specific cell surface receptors in vitro. It also has strong selectivity and binding affinity for bacterial strains, and a certain phage can only target a certain specific bacterium. The phage probe prepared by the method has high specificity, sensitivity and stability, and is easy to prepare and store. Different from the antibody and the aptamer, the phage not only can distinguish the types of bacteria, but also can identify different strains of the same bacteria, and has wide application prospect in pathogenic bacteria detection.
ATP is present only in living bacteria, in oxygen and Mg 2+ In the situation, the Biological Luminous (BL) reaction catalyzed by luciferase can be caused, the biological luminous signal is in direct proportion to the ATP amount released by the bacteria to be lysed, and the high-sensitivity and high-efficiency detection of the living bacteria can be realized.
Therefore, developing a technology that can be combined with phage probes to achieve integrated, portable, rapid and sensitive detection of bacteria is an objective of current detection technology development.
Disclosure of Invention
The invention aims to provide a microfluidic chip of a coupling phage modified biosensor and a quantitative detection method thereof, and by adopting the microfluidic chip, the rapid and accurate detection of the bacterial content of different pathogenic bacteria can be realized simultaneously, and the microfluidic chip has the advantages of high sensitivity, high accuracy, strong specificity, short test time and simple and convenient operation.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a microfluidic chip of a coupling phage modified biosensor, which comprises a bottom layer and a top layer;
the top layer comprises a sample injection area, a detection area and a sample outlet area;
the detection zone is provided with a plurality of linear channels, and different bacteriophage-modified biosensors are coupled in different channels.
Preferably, the bottom layer is a glass slide;
the sample injection area of the top layer is divided into a sample injection hole of a sample to be detected, a buffer solution injection hole, a cell lysate/D-fluorescein/luciferase injection hole;
the sample injection area is connected with the detection area through a single serpentine channel, and the detection area is positioned between the sample injection area and the sample outlet area;
the length of the linear channel of the detection area is 3-4 cm, and the width is 70-90 mu m.
The invention also provides a method for quantitatively detecting bacteria by the microfluidic chip of the coupling phage modified biosensor, which comprises the following steps:
(1) Adding PBS buffer solution into a buffer solution sample adding hole of the sample adding area to clean a detection area of the microfluidic chip;
(2) Adding a sample liquid into a sample adding hole of a sample to be detected in a sample injection area, combining bacteria in the sample liquid with phage in a linear channel of a detection area, and then adding PBS buffer solution into the sample adding hole from the buffer solution to clean unbound bacteria;
(3) Sequentially adding the cell lysate, the D-luciferin and the luciferase into the cell lysate/the D-luciferin/the luciferase sample adding hole to perform luminescence reaction, and generating fluorescence for detection;
(4) And calculating to obtain the bacterial concentration according to the detected fluorescence intensity and the standard curve of the bacteria to be detected.
Preferably, the temperature is maintained at 35-37 ℃ throughout the detection process.
Preferably, the time for the bacteria to bind to phage in step (2) is from 5 to 10 minutes.
Preferably, step (3) is followed by addition of D-luciferin and luciferase after 10-15 s of Tris-HCl cell lysate.
Preferably, the wavelength of fluorescence detected in step (3) is 540 to 600nm.
Compared with the prior art, the technical scheme has the following beneficial effects:
(1) Compared with the traditional analysis by using a large-scale instrument, the method for quantitatively detecting the bacterial content shortens the detection time, simplifies the operation steps, is convenient to carry and is suitable for on-site analysis.
(2) The microfluidic chip can simultaneously realize quantitative detection of various bacteria, namely, under the state that various bacteria are uniformly mixed, the quantity of each bacteria entering three channels is basically equal, and only the quantity of the bacteria in one channel is calculated and then multiplied by the number of the channels, so that the method is more rapid and effective compared with the traditional method or other microfluidic bacteria detection methods.
(3) The micro-fluidic chip of the invention is coupled with the biosensor modified with phage, and has strong specificity and high sensitivity.
(4) The phage-modified biosensor coupling microfluidic chip has the advantages of simple preparation process, mild reaction conditions, low cost and high safety coefficient.
Drawings
Fig. 1 is a schematic structural diagram of a PDMS template of a microfluidic chip prepared in example 1;
1-waste liquid hole, 2-detection area, 3-phage modified biosensor, 4-D-fluorescein and luciferase/cell lysate sample hole, 5-sample hole to be detected, 6-buffer solution sample hole, 7-sample area, 8-sample outlet area and 9-snake channel;
FIG. 2 is a schematic diagram of the portable fluorescence detector in example 2;
FIG. 3 is a standard graph of Salmonella plotted in example 2;
FIG. 4 shows the bioluminescence intensity signal (4A) and its corresponding standard curve (4B) at different S.T concentrations in example 3.
Detailed Description
The invention provides a microfluidic chip of a coupling phage modified biosensor and a method for quantitatively detecting bacteria, which have the advantages of high sensitivity, high accuracy, strong specificity, short test time and simple operation.
The invention provides a microfluidic chip of a coupling phage modified biosensor, which comprises a bottom layer and a top layer;
the top layer comprises a sample injection area, a detection area and a sample outlet area;
the detection zone is provided with a plurality of linear channels, and different bacteriophage-modified biosensors are coupled in different channels.
In the invention, the bottom layer is a glass slide;
the sample injection area of the top layer is divided into a sample injection hole of a sample to be detected, a buffer solution injection hole, a cell lysate/D-fluorescein/luciferase injection hole;
the sample injection area is connected with the detection area through a single serpentine channel, and the detection area is positioned between the sample injection area and the sample outlet area;
the length of the linear channel of the detection zone is 3-4 cm, preferably 3.5cm, and the width is 70-90 μm, preferably 80 μm.
In the invention, the detection area is connected with the sample outlet area through a branch bend or a straight channel.
In the invention, the sample outlet area is provided with a waste sample hole for collecting waste liquid.
In the present invention, the channel width of the sample outlet region is preferably 150 to 200. Mu.m, more preferably 180. Mu.m.
The invention also provides a method for quantitatively detecting bacteria by coupling the microfluidic chip of the phage-modified biosensor, which comprises the following steps:
(1) Adding PBS buffer solution into a buffer solution sample adding hole of the sample adding area to clean a detection area of the microfluidic chip;
(2) Adding a sample liquid into a sample adding hole of a sample to be detected in a sample injection area, combining bacteria in the sample liquid with phage in a linear channel of a detection area, and then adding PBS buffer solution into the sample adding hole from the buffer solution to clean unbound bacteria;
(3) Sequentially adding the cell lysate, the D-luciferin and the luciferase into the cell lysate/the D-luciferin/the luciferase sample adding hole to perform luminescence reaction, and generating fluorescence for detection;
(4) And calculating to obtain the bacterial concentration according to the detected fluorescence intensity and the standard curve of the bacteria to be detected.
In the present invention, the temperature of the whole detection process is maintained at 35 to 37 ℃, preferably 37 ℃.
In the present invention, the time for the bacteria to bind to phage in step (2) is 5 to 10min, preferably 8min.
In the present invention, the step (3) is performed after adding Tris-HCl cell lysate for 10 to 15s, and D-luciferin and luciferase are added, preferably for 12s.
In the present invention, the wavelength of fluorescence detected in step (3) is 540 to 600nm, preferably 560nm.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation method of microfluidic chip
Firstly, manufacturing a chip mould by using an AI drawing and 3D printing technology, carrying out hydrophobic treatment on the chip mould by using an analytically pure octadecyl trichlorosilane toluene solution with the mass concentration of 2 percent for 30min, then washing the chip mould by using analytically pure toluene for 3 times, airing the chip mould at room temperature to obtain the chip mould subjected to hydrophobic treatment, pouring PDMS (polydimethylsiloxane) into the chip mould, and curing the PDMS main agent (A) and the PDMS (B) at the temperature of 60 ℃ for 2 hours to obtain the PDMS template.
The structure of which is schematically shown in figure 1.
The PDMS layer includes three regions: sample injection area 7, detection area 2 and play appearance district 8, and detection area 2 is located between sample injection area 7 and play appearance district 8. The sample injection area 7 comprises a single serpentine channel 9 which is connected with the detection area 2 in a straight channel, the detection area 2 is provided with three channels, the length of each channel is the same, the channels are 3.5cm, and the channel width is 80 mu m. The sample injection area 7 comprises a sample injection hole 5 of a sample to be detected, a buffer solution injection hole 6 and a D-fluorescein and luciferase/cell lysate injection hole 4. The channel width of the sample injection area is 180 μm. The sample outlet area contains waste sample holes 1, and the diameter of each waste sample hole is 1.5mm.
The phage biosensor is snapped into channel 3 of the detection zone.
And (3) placing the PDMS template and the glass sheet with the supporting function into a plasma cleaning machine, carrying out plasma treatment at 25 ℃ for 0.5min, and then sealing in a 90 ℃ oven for 2h to obtain the microfluidic chip. The sealed whole chip is stored in a refrigerator at 4 ℃ and irradiated by an ultraviolet lamp for 30min before the magnetic bead@phage is added.
Preparation of phage biosensors
Preparation of magnetic beads: first, 3.6 g of anhydrous sodium acetate and 1.3 g of finely ground FeCl were sonicated 3 .6H 2 O was dissolved in 40 ml of ethylene glycol to give a yellow viscous solution. The resulting viscous solution was sealed in an autoclave (100 ml volume) and heated at 200 ℃ for 6 hours. After cooling to room temperature (25 ℃) the magnetic nanoparticles (MB) were obtained by three separate washes with ethanol and water, and finally dried under vacuum at 50℃for 12 hours for further use.
Preparation of MB-Phage probe: 10mg of MB was dissolved in 1.0mL of H 2 O and sonicated for 10 minutes to give a MB solution at a concentration of 10 mg/ml. Then, 1% (wt%) of a solution of PDDA (polydiallyl dimethyl ammonium chloride) in water was added to MB (Fe) 3 O 4 Solution) and reacted at 2200rpm for 1 hour to give magnetic bead coupled PDDA (MB@PDDA). Thereafter, using H 2 O repeatedly washed the resulting MB@PDDA three times, 200. Mu.L of PBS solution was added, and 300. Mu.L of 10 at 2200rpm 13 PFU/mL Salmonella typhimurium phage solution was added thereto and coupled for 1 hour (phage will be immobilized on the surface of MB using electrostatic adsorption due to the negative charge of phage head). The coupled product, salmonella typhimurium phage modified biosensor (M-P1 probe), was isolated with a magnet. Phage coupling process requiresThe addition of the salmonella typhimurium phage solution was repeated 4 times, each for 1 hour. Thereafter, using H 2 O the resulting M-P1 was repeatedly washed three times, and 1% (wt%) BSA was added thereto, and reacted at 2200rpm for 1 hour to avoid nonspecific adsorption. Finally, H is used for the M-P1 probe 2 O was washed 3 times and stored in a refrigerator at 4℃for further use. Before use, M-P1 is pumped into the detection area 3 by the sample adding hole 5 by a micro injection pump at a flow rate of 10 mu l min < -1 > under positive pressure, magnetic materials and magnetic beads are mutually attracted and combined, and then PBS is used for washing.
Example 2
Quantitative detection of Salmonella content Using the microfluidic chip of the coupled phage-modified biosensor prepared in example 1
Firstly, 5ml of PBS buffer solution is introduced into a buffer solution sample-adding hole, then 1ml of sample solution is introduced into the sample-adding hole of a sample to be detected of the microfluidic chip, the sample solution is continuously introduced by a syringe pump, salmonella in the sample solution is combined with salmonella phage on a biological sensor in a detection area channel and adsorbed on the channel, after 8min, PBS buffer solution is introduced into the buffer solution sample-adding hole to flush out superfluous bacteria, 50mmol/LTri-HCl lysate with the volume ratio of 0.1:1 of the buffer solution to the bacteria solution is added, after 12s, 5ml of mixed solution of 15mg/ml of D-fluorescein working solution and 1ml of 1mg/L luciferase is added into the D-fluorescein sample-adding hole, and fluorescence is generated on the channel after reaction. At this time, the portable fluorescence detector and the smart phone (shown in fig. 2) are combined, and the total length of the channel capable of generating fluorescence in the channel of the detection area of the microfluidic chip is measured.
Simultaneously drawing a standard curve of salmonella: salmonella was configured to a concentration of 10 9 cfu/ml,10 8 cfu/ml,10 7 cfu/ml,10 6 cfu/ml,10 5 cfu/ml,10 4 cfu/ml,10 3 cfu/ml,10 2 cfu/ml and bacterial liquid with concentration of 10 cfu/ml. And (3) measuring fluorescence values of salmonella solutions with different concentrations instead of the sample solution. A standard curve is plotted as shown in fig. 3. And calculating the concentration of salmonella in the sample liquid according to a standard curve.
The whole step is carried out in a portable incubator at 37 ℃.
The portable fluorescence detector (fig. 2) used in this embodiment is mainly composed of a black box and an LED light source. The overall dimensions of the black box were 15cm×8cm×5cm (length×width×height), and the observation window was 6cm×4cm (length×width). The detection window is arranged at the top of the black box and is covered by the mobile phone. A7 cm by 0.5cm by 1cm (length by width by height) fixed support is designed near the detection window, and the power of the light source is 3W.
Example 3
Different milk and water samples were used as actual samples, and the bacterial concentration of salmonella (s.t.) was measured using the microfluidic chip prepared in example 1 and the detection method provided in example 2. Recovery of the four samples was calculated using standard addition methods. The results are shown in Table 1.
For milk sample 1, three groups were separated, and 0CFU/mL and 10 were added respectively 3 CFU/mL、10 6 The salmonella standard solution of CFU/mL is then used for detecting the bacterial concentration of salmonella (S.T.) by using the microfluidic chip prepared in the example 1 and the detection method provided in the example 2, and the result shows that the S.T. content is not detected by the labeled 0CFU/mL group; label 10 3 CFU/mL group assay S.T. content was (0.966.+ -. 0.035). Times.10 3 CFU/mL, recovery rate of 96.5% and RSD of 3.5; label 10 6 CFU/mL group detection S.T. content was (0.948.+ -. 0.071). Times.10 6 CFU/mL, recovery was 94.8% and RSD was 7.1.
For milk sample 2, three groups of 0CFU/mL and 10 were added 3 CFU/mL、10 6 The salmonella standard solution of CFU/mL is then used for detecting the bacterial concentration of salmonella (S.T.) by using the microfluidic chip prepared in the example 1 and the detection method provided in the example 2, and the result shows that the S.T. content is (43+/-6) CFU/mL detected by the labeled 0CFU/mL group; label 10 3 CFU/mL group assay S.T. content was (0.974.+ -. 0.023). Times.10 3 CFU/mL, recovery rate of 97.4% and RSD of 2.3; label 10 6 CFU/mL group detection S.T. content was (0.983.+ -. 0.076). Times.10 6 CFU/mL, recovery was 98.3% and RSD was 7.6.
For water sample 1, three groups were separated and added with 0CFU/mL and 10 3 CFU/mL、10 6 CFU/mL salmonella standard solution, then the micro-fluidic chip prepared in the example 1 and the detection method provided in the example 2 are used for detecting the concentration of bacteria of salmonella (S.T.), and the result shows that the S.T. content is not detected by the labeled 0CFU/mL group; label 10 3 CFU/mL group detection S.T. content was (0.992.+ -. 0.077). Times.10 3 CFU/mL, recovery of 99.2% and RSD of 7.7; label 10 6 CFU/mL group assay S.T. content was (1.014.+ -. 0.052). Times.10 6 CFU/mL, recovery was 101.4% and RSD was 5.2.
For water sample 2, three groups were separated and added with 0CFU/mL and 10 3 CFU/mL、10 6 The salmonella standard solution of CFU/mL is then used for detecting the bacterial concentration of salmonella (S.T.) by using the microfluidic chip prepared in the example 1 and the detection method provided in the example 2, and the result shows that the S.T. content is not detected in the labeled 0CFU/mL group; label 10 3 CFU/mL group assay S.T. content was (1.027.+ -. 0.059). Times.10 3 CFU/mL, recovery rate 102.7%, RSD 5.9; label 10 6 CFU/mL group detection S.T. content was (0.988.+ -. 0.038). Times.10 6 CFU/mL, recovery was 98.8% and RSD was 3.8.
TABLE 1 bioluminescence assay to detect S.T concentration in real samples
The recovery rate of S.T in two milk and two water samples is over 94%, and the RSD value is less than 8%.
The lower detection limit was calculated by the 3 delta method (which is repeated three times to find its standard deviation at low concentrations, from 3 times the standard deviation, divided by its slope, which is its lower detection limit). The results are shown in FIG. 4.
From fig. 4A, different concentrations of S.T were subjected to bioluminescence detection with increasing bioluminescence intensity as S.T concentration increased. And at 10 1 To 10 7 Concentration range of CFU/mLThe interior shows a good linear relationship, and the linear regression equation is as follows: logbl=0.161logcs.t+1.951, r 2 0.984 (fig. 4B). The calculation showed that S.T had a lower limit of detection (LOD) of 9CFU/mL.
Compared with other existing bacteria detection methods, the detection method disclosed by the invention has the advantages that the detection range is wider and the sensitivity is higher, and the results are shown in Table 2.
TABLE 2 detection limits and detection ranges for detecting bacterial content by comparing different methods
Detection method | Detection limit (CFU.mL) -1 ) | Detection Range (CFU.mL) -1 ) |
Multimode spot filtration type immunoassay system | 10 | 10 1 ~10 6 |
UCNPsPCR fluorescence sensor | 11 | 11~1.14×10 9 |
Electrochemical immunoassay | 3 | 10~10 7 |
Colorimetric method | 7 | 10~10 7 |
The method of the invention | 9 | 10~10 9 |
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
1. A microfluidic chip of a coupled phage-modified biosensor, wherein the microfluidic chip comprises a bottom layer and a top layer;
the top layer comprises a sample injection area, a detection area and a sample outlet area;
the detection zone is provided with a plurality of linear channels, and different bacteriophage-modified biosensors are coupled in different channels.
2. The microfluidic chip of claim 1, wherein the bottom layer is a glass slide;
the sample injection area of the top layer is divided into a sample injection hole of a sample to be detected, a buffer solution injection hole, a cell lysate/D-fluorescein/luciferase injection hole;
the sample injection area is connected with the detection area through a single serpentine channel, and the detection area is positioned between the sample injection area and the sample outlet area;
the length of the linear channel of the detection area is 3-4 cm, and the width is 70-90 mu m.
3. A method for quantitatively detecting bacteria using the microfluidic chip of the coupled phage-modified biosensor of claim 1 or 2, comprising the steps of:
(1) Adding PBS buffer solution into a buffer solution sample adding hole of the sample adding area to clean a detection area of the microfluidic chip;
(2) Adding a sample liquid into a sample adding hole of a sample to be detected in a sample injection area, combining bacteria in the sample liquid with phage in a linear channel of a detection area, and then adding PBS buffer solution into the sample adding hole from the buffer solution to clean unbound bacteria;
(3) Sequentially adding the cell lysate, the D-luciferin and the luciferase into the cell lysate/the D-luciferin/the luciferase sample adding hole to perform luminescence reaction, and generating fluorescence for detection;
(4) And calculating to obtain the bacterial concentration according to the detected fluorescence intensity and the standard curve of the bacteria to be detected.
4. A method for quantitatively detecting bacteria as claimed in claim 3, wherein the temperature is maintained at 35 to 37 ℃ throughout the detection process.
5. The method for quantitatively detecting bacteria of claim 4, wherein the time for the bacteria to bind to phage in the step (2) is 5 to 10min.
6. The method for quantitatively detecting bacteria according to claim 5, wherein the step (3) is performed by adding D-luciferin and luciferase 10 to 15 seconds after the Tris-HCl cell lysate is added.
7. The method for quantitatively detecting bacteria according to claim 6, wherein the wavelength of the fluorescence detected in the step (3) is 540 to 600nm.
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