CN109001269B - Bacterial chip integrating DEP separation, magnetic microsphere selective enrichment and EIS in-situ detection and detection method thereof - Google Patents
Bacterial chip integrating DEP separation, magnetic microsphere selective enrichment and EIS in-situ detection and detection method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biochemical analysis, and particularly relates to a bacterial chip integrating DEP separation, selective enrichment of glycosylated magnetic microspheres and EIS in-situ detection and a detection method thereof. The bacterium chip integrates three functions of DEP separation, glycosylated magnetic microsphere enrichment and EIS in-situ detection, can rapidly and efficiently determine the concentration of the salmonella, and has the detection limit of 100 CFU/mL; the provided detection method has selectivity, wherein the magnetic microspheres modified by the mannose derivatives can selectively mark salmonella, escherichia coli, vibrio cholerae or klebsiella pneumoniae; DEP can enrich marked salmonella, colibacillus, vibrio cholerae or klebsiella pneumoniae in suspension, and other unmarked bacteria and redundant magnetic microspheres are discharged by a sample outlet channel along with the solution, thereby ensuring the selectivity of the detection method.
Description
Technical Field
The invention belongs to the technical field of biochemical analysis, and particularly relates to a bacterial chip integrating DEP separation, selective enrichment of glycosylated magnetic microspheres and EIS in-situ detection and a detection method thereof.
Background
With the development of social economy and the improvement of living standard of people, the requirements of people on environmental safety, food safety, rapid diagnosis and treatment and the like are higher and higher, and the rapid detection technology aiming at specific pathogenic bacteria becomes a hot point of attention and research at home and abroad. The current detection methods commonly used for pathogenic bacteria comprise: plate colony counting method, PCR, enzyme linked immunosorbent assay, etc. but they all have the disadvantages of long time consumption, complicated procedure, etc. The microfluidic chip (microfluidic chip) becomes a new platform for rapid detection of bacteria due to its advantages of being easy to be rapid, accurate, high in sensitivity, easy to be integrated, portable and the like.
A micro-fluidic bacteria detection chip integrated with a functional micro-channel and a micro-electrode array is a research and development field which is concerned about. Patent CN 101788515 a reports a microfluidic chip and method for detecting bacteria based on Electrochemical Impedance (EIS), which does not relate to experimental methods for operability such as pretreatment of samples, although the detection limit is 103CFU/mL, which cannot be matched with requirements that cannot be detected in national standards; patent CN 101694476 a reports a bacterial impedance detection method, which adopts a simplified chip preparation process, but lacks the separation and enrichment treatment of bacteria, is difficult to be used for effective detection of a small amount of pathogenic bacteria in an actual sample, and does not provide important bacterial test indexes such as bacterial detection limit. It can be seen that, because the actual sample contains a small amount of pathogenic bacteria and often contains a plurality of bacteria, one of the difficulties in the rapid, sensitive and specific detection of pathogenic bacteria is the pretreatment of the sample. The magnetic enrichment technology based on the magnetic microspheres or the magnetic nanoparticles is widely applied due to the advantages of high enrichment efficiency and simple method. The Recognition receptor (Recognition receptor) is an essential element for the magnetic microsphere to selectively capture pathogenic bacteria, and the currently reported Recognition receptor mainly comprises a nucleic acid aptamer and an antibody. Patent 106053805A reports a method for capturing salmonella by using aptamers, and patent CN 106702016A reports a method for capturing salmonella by using immunomagnetic beads, but aptamers and antibodies have the defects of long screening time, complex steps and high labor and financial cost. Therefore, the reported technology and method for detecting the microfluidic bacterial chip are difficult to meet the requirements of selective detection of pathogenic bacteria in the application fields of environmental monitoring, food safety monitoring, clinical rapid diagnosis and treatment and the like.
Disclosure of Invention
In order to solve the problems, the invention provides a bacterial chip for DEP (dielectrophoresis) separation, selective enrichment of glycosylated magnetic microspheres and EIS in-situ detection, which integrates three functions of DEP separation, selective enrichment and EIS in-situ detection, can realize rapid and sensitive detection of salmonella, and has the detection limit as low as 100 CFU/mL.
A micro-fluidic bacterial chip integrating DEP separation, magnetic microsphere selective enrichment and EIS in-situ detection is formed by bonding a glass substrate containing a microelectrode array and a PDMS (polydimethylsiloxane) cover plate containing a micro-channel. The device is characterized in that the micro-channel is provided with a sample introduction channel, a mixing channel, a detection zone and a sample outlet channel which are sequentially communicated; the two sample introduction channels are communicated with the mixing channel to form a Y shape, the included angle of the two sample introduction channels is 60-180 degrees, sample introduction ports are respectively arranged at the initial ends of the sample introduction channels, the length of each sample introduction channel is 10-25 mm, the width of each sample introduction channel is 300-1000 micrometers, and the depth of each sample introduction channel is 50-100 micrometers; the mixing channel is integrated with 10-100 micro units in Tesla configuration, and the total length is 10-100 mm; the detection area is circular, the diameter of the detection area is 1000-5300 mu m, and the depth of the detection area is the same as that of the sample injection channel; the sampling channel is a straight channel, the length of the sampling channel is 1-10 mm, and the width and the depth of the sampling channel are the same as those of the sampling channel; the tail end of the sample outlet channel is provided with a sample outlet.
The microelectrode array consists of 2-20 groups of interdigital electrodes, each group of interdigital electrodes consists of two arc-shaped interdigital microelectrodes, the configuration and the position of each interdigital microelectrode correspond to the configuration and the position of the detection area, and each interdigital microelectrode can be completely immersed in the detection area; the diameter of the arc-shaped interdigital microelectrode is 100-5000 microns, 10-100 interdigital parts are arranged, the length of the interdigital part is 200-1000 microns, the width of the interdigital part is 10-30 microns, the thickness of the interdigital part is 50-200 nm, and the distance between every two adjacent interdigital parts in each group of interdigital electrodes is 10-30 microns.
Preferably, the preparation method of the PDMS cover sheet comprises: preparing an SU8 positive membrane containing a microchannel by using an MEMS (micro electro mechanical systems) processing technology, pouring a mixture of Polydimethylsiloxane (PDMS) and a curing agent onto the SU8 positive membrane by using a thermoplastic method, curing for 20-60 min at 60-100 ℃ after bubbles are removed, and stripping the solidified PDMS from the positive membrane to obtain the PDMS cover plate with the microchannel.
Preferably, the material of the substrate is glass, quartz, silicon or polymer.
Preferably, the interdigital electrode is made of gold, platinum, copper, aluminum or palladium, and is prepared by adopting a magnetron sputtering technology of MEMS processing.
The invention also provides a detection method using the bacterial chip, which specifically comprises the following steps:
(1) sampling 10-1000 uL of bacterial liquid from one side of a Y-shaped channel of a sampling channel, simultaneously sampling 10-1000 uL0.1-1 mg/mL of aqueous solution of glycosylated magnetic microspheres from the other side, wherein the sampling mode is peristaltic pump injection sampling or negative pressure sampling, and the bacterial liquid and the aqueous solution of the glycosylated magnetic microspheres flow into a mixing channel to be fully mixed, so as to complete the marking of the glycosylated magnetic microspheres on bacteria and obtain suspension of the glycosylated magnetic microspheres marked bacteria and redundant glycosylated magnetic microspheres;
(2) when the suspension obtained in the step (1) enters a detection area, DEP (dielectrophoresis) is started, the voltage is 2-10V, the frequency is 400-600 kHz, the DEP time is 1-30 min, the peristaltic pump is stopped, DEP is continued until the flow rate of a mixing channel is 0, the DEP is stopped, and the bacteria marked by the glycosyl magnetic microspheres are enriched between interdigital electrodes in the detection cell;
(3) immediately switching to an EIS detection mode after DEP is stopped, wherein an alternating current disturbance signal of the EIS is 50-500 mV, the frequency is 10-50 kHz, and a signal value Z is recorded;
(4) repeating the steps (1) to (3) by taking bacterial liquid with different concentrations and the glycosylated magnetic microsphere solution, recording the signal value Zn of the suspension with different bacterial concentrations, wherein n is more than or equal to 5, and subtracting the blank signal value to obtain delta Zn. Is Δ ZnOrdinate, Δ Z- [ C ] plotted against the concentration of bacteria in suspension as abscissa]；
(5) Taking a sample X to be detected, and determining a signal to Z according to the steps (1) to (3)xAnd according to the relation Δ Z- [ C]Obtaining the bacterial concentration of the suspension of the sample to be detected, and then calculating the bacterial concentration in the sample X to be detected according to the sample introduction volume of the sample to be detected and the sample introduction volume of the glycosylated magnetic microsphere aqueous solution;
the glycosylated magnetic microsphere is a mannose derivative modified magnetic microsphere.
Preferably, the preparation method of the glycosylated magnetic microsphere comprises the following steps: taking 60-6000 mu L of carboxylated magnetic microspheres subjected to ultrasonic treatment at 15-30 ℃, enriching the carboxylated magnetic microspheres by using a magnet, removing supernatant, adding 300-3000 mu L of 100-300 mmol/L EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 300-3000 mu L of 100-300 mmol/L NHS (N-hydroxysuccinimide), shaking in the dark for 30-60 min, adding 300-3000 mu L of 0.2-2 mg/mL new mannose derivatives, shaking in the dark for 40-120 min, adding 300-3000 mu L of 1-5% bovine serum albumin solution, shaking in the dark for 10-120 min, stopping the reaction, enriching the magnet, removing supernatant, and washing with a buffer solution to obtain the glycosylated magnetic microspheres.
The mannose derivative is a compound having mannose as a main structure, such as 4-aminophenyl- α -mannopyranoside, mannobiose, mannotriose, and mannose dendrimer.
The buffer is Phosphate Buffered Saline (PBS).
The DEP (dielectrophoresis) separation, glycosylation magnetic microsphere selective enrichment and EIS in-situ detection bacterial chip and/or the detection method using the chip are/is applied to the detection of salmonella typhimurium, escherichia coli expressing type 1 pilus, solitary cholera or/and klebsiella pneumoniae.
The invention has the beneficial effects that:
1. the bacterial chip provided by the invention integrates three functions of DEP separation, glycosylation magnetic microsphere enrichment and EIS in-situ detection, can rapidly and efficiently determine the concentration of salmonella, and has the detection limit as low as 100 CFU/mL;
2. the detection method provided by the invention has good selectivity, wherein the magnetic microspheres modified by the mannose derivatives can selectively mark salmonella, escherichia coli, vibrio cholerae or klebsiella pneumoniae, the marked salmonella, escherichia coli, vibrio cholerae or klebsiella pneumoniae in the suspension can be enriched through DEP, and redundant magnetic microspheres are discharged along with the solution outlet channel, so that the high-efficiency detection of target bacteria is ensured.
Drawings
FIG. 1 is a schematic diagram of the substrate structure (left) and the array fork structure (right) of the bacterial chip of the present invention;
FIG. 2 is a schematic diagram of the structure of a PDMS cover plate (left) and a part of the structure of a mixing channel (right) of the bacterial chip of the present invention;
FIG. 3 is a graph Δ Z- [ C ] obtained in example 3.
In the figure: the device comprises a substrate 1, a microelectrode 2, an interdigital electrode 3, an interdigital microelectrode 4, an interdigital pad 5, a PDMS cover plate 6, a sample injection channel 7, a sample injection port 701, a mixing channel 8, a detection zone 9, a sample outlet channel 10, a sample outlet port 101, a microcell 11 and a microchannel 12.
Detailed Description
The invention will be further explained below with reference to the drawings.
The reagents and equipment used in the present invention are commercially available unless otherwise specified. The term "Tesla configuration" in the present invention refers to a commonly used configuration of micromixers in the Chemical Engineering field, in particular, see the documents "Shakhawat Hossain, et al, analysis and optimization of a micromixer with a modified Tesla structure [ J ], Chemical Engineering Journal 158(2010), 305-.
The following is a bacterial chip structure provided by the invention, and the change of the integration number of the interdigital electrodes and the microcircuit in the invention still belongs to the protection scope of the invention. Also, those skilled in the art can obtain technical solutions (such as modifications to the number of fingers, the inter-finger space, the number of mixed region micro-units, etc.) without creative efforts and still fall within the protection scope of the present invention.
Example 1
The bacterial chip provided by the invention is obtained by bonding a glass substrate 1 integrated with a microelectrode array and a PDMS (polydimethylsiloxane) cover plate 6 containing a microchannel 12, wherein two groups of interdigital electrodes 3 (figure 1) are integrated on the glass substrate 1, each interdigital electrode 3 consists of two arc-shaped interdigital microelectrodes 4, the diameter of each interdigital electrode is 700 mu m, each interdigital electrode contains 10 fingers, the length of each finger is 500-800 mu m, the width of each finger is 20 mu m, the thickness of each finger is 100nm, and the distance between adjacent fingers of each group of interdigital electrodes is 10 mu m. Two groups of micro-channels 12 (figure 2) are integrated on the Polydimethylsiloxane (PDMS) cover plate 6, and a sample introduction channel 7, a mixing channel 8, a detection zone 9 and a sample outlet channel 10 are sequentially arranged on the PDMS cover plate. The sampling channels 7 are two and form a Y-shaped structure with the mixing channel 8, the included angle of the two sampling channels 7 is 60 degrees, the initial ends of the sampling channels are respectively provided with a sampling port, the length of the sampling channel 7 is 10mm, the width is 300 mu m, and the depth is 50 mu m; the mixing channel 8 is integrated with 10 micro-units 11 in Tesla configuration, with a total length of 10mm and a depth of 50 μm; the detection zone 9 is circular in configuration, with a circular area of 1000 μm diameter and 50 μm depth; the sampling channel 10 is a straight channel, the length of the sampling channel is 3mm, and the width and the depth of the sampling channel are the same as those of the sampling channel 7; the end of the sample outlet channel 10 is provided with a sample outlet 101.
Example 2
The preparation method of the glycosylated magnetic microsphere comprises the following steps: 60 μ L of 1mg/mL carboxylated magnetic microspheres (sonicated before use) were taken in a 5mL glass vial at room temperature, the magnetic microspheres were enriched with a magnet, and the supernatant was removed. Add the mixed solution of 300. mu.L EDC (200mM) and 300. mu.L NHS (50mM) to activate the carboxyl group on the magnetic microsphere, and shake the reaction for 30min continuously in the dark. Then 300. mu.L (1mg/ml) of freshly prepared 4-aminophenyl alpha-D-mannopyranoside was added and shaken in the dark for 1 h. Add 300. mu.L of 1% bovine serum albumin solution, shake for 2h away from light, stop the reaction. Enriching the glycosylated magnetic microspheres by a magnet, removing the supernatant and separating the supernatant from free mannose, washing the supernatant for 3 times by using 200 mu L of buffer solution, finally suspending the supernatant to 300 mu L to obtain the glycosylated magnetic microspheres with the concentration of about 0.2mg/mL, and storing the glycosylated magnetic microspheres at 4 ℃. The buffer was Phosphate Buffered Saline (PBS).
Example 3
Salmonella typhimurium (S.typhimurium) was picked with an inoculating loop and cultured in 60mL sterilized lactose broth for 16h at 37 ℃. Taking 1ml, centrifuging at 6000rpm for 3min, resuspending in ultrapure water, repeating for 3 times. Diluting with ultrapure water to obtain 105CFU/mL、5×104CFU/mL、104CFU/mL、5×103CFU/mL、103CFU/mL、102CFU/mL and 50CFU/mL series of concentrations of Salmonella standard solution.
The bacterial chip of example 1 was used to establish a standard curve for salmonella detection using the above standard solution. Firstly, respectively taking 1mL of the standard solution, and injecting a sample from a sample inlet; 1mL of the glycosylated magnetic microsphere prepared in example 2 (0.5mg/mL) was injected from another injection port at a flow rate of 1 uL/min. And after the bacteria and the redundant magnetic microsphere mixed suspension are fully mixed by a mixing channel on the chip, the bacteria marked by the glycosylated magnetic microsphere and the redundant magnetic microsphere mixed suspension are obtained and then enter a detection area. DEP was performed for 20min at the detection zone using a voltage of 10V and a frequency of 500 kHz. At this time, the bacteria marked by the glycosylated magnetic microspheres are captured on the interdigital electrodes, and the redundant glycosylated magnetic microspheres are not captured. And immediately switching to an in-situ impedance detection mode, wherein an alternating current disturbance signal is 100mV, the frequency is 40KHz, a relation graph Delta Z- [ C ] (see FIG. 3) of Delta Z and bacterial concentration is obtained, the obtained linear regression equation is 1583lg [ C ] -15155, the correlation coefficient is 0.9826, and the detection limit can reach 100 CFU/mL. And (3) taking a normal human urine sample, adding 200CFU/mL of salmonella bacterial liquid, and performing an experiment according to the method, wherein the detection result is 189CFU/mL, the recovery rate is 96%, and the relative standard deviation is 6.45(n is 5).
Example 4
The concentration is selected to be 5X 104Five pathogenic bacteria, i.e., S.typhimurium ATCC14028, E.coli JM109, E.coli DH5 alpha, S.aureus, P.aeruginosa, and the like, of CFU/mL were tested in example 3, and the results showed that S.typhimurium ATCC14028 and E.coli JM109 expressing type 1 pili (containing FimH adhesin) had good responses, and Normalized values of impedance change (NIC) reached-124. + -. 18% and-91. + -. 9%, and significant differences from the other three bacteria (NIC values of-8.5. + -. 0.5%, -7.8. + -. 1.8%, -9.0. + -. 1.9%, respectively). The results show that the detection method described in the present invention selectively recognizes bacteria expressing type 1 pili (containing FimH adhesin) and does not recognize other bacteria.
NIC is the normalized value of impedance value Z at 40 KHz; z(Bacreria)The impedance value of the bacterial solution obtained after the experiment according to the embodiment 3 is obtained; z(Control)Impedance values obtained after the experiment in example 3 were measured for ultrapure water containing no bacteria.
Claims (6)
1. The method for detecting the bacteria chip by utilizing integrated DEP separation, magnetic microsphere selective enrichment and EIS in-situ detection is characterized in that the chip is bonded by a substrate (1) integrated with a microelectrode array (2) and a PDMS cover plate (6) integrated with a microchannel (12), and the microchannel (12) is provided with a sample introduction channel (7), a mixing channel (8), a detection zone (9) and a sample outlet channel (10) which are sequentially communicated; the two sample introduction channels (7) are communicated with the mixing channel (8) to form a Y shape, the included angle of the two sample introduction channels (7) is 60-180 degrees, the initial ends of the sample introduction channels (7) are respectively provided with a sample introduction port (701), the length of each sample introduction channel (7) is 10-25 mm, the width of each sample introduction channel is 300-1000 mu m, and the depth of each sample introduction channel is 50-100 mu m; the mixing channel (8) is integrated with 10-100 micro units (11) in Tesla configuration, and the total length is 10-100 mm; the detection area (9) is circular, the diameter is 1000-5300 mu m, and the depth is the same as that of the sample injection channel; the sample outlet channel (10) is a straight channel, the length of the sample outlet channel is 1-10 mm, the width and the depth of the sample outlet channel are the same as those of the sample inlet channel (7), and a sample outlet (101) is arranged at the tail end of the sample outlet channel (10); the microelectrode array (2) consists of 2-20 groups of interdigital electrodes (3), each group of interdigital electrodes (3) consists of arc interdigital microelectrodes (4), the configuration and the position of each interdigital microelectrode array correspond to those of the detection area (9), and the interdigital microelectrodes can be completely immersed in the detection area (9); the diameter of the arc-shaped interdigital microelectrode (4) is 100-5000 microns, 10-100 interdigital parts are arranged, the length of the interdigital part is 200-1000 microns, the width of the interdigital part is 10-30 microns, the thickness of the interdigital part is 50-200 nm, and the distance between every two adjacent interdigital parts in each group of interdigital electrodes is 10-30 microns; preparing an SU8 positive membrane containing a microchannel by using an MEMS (micro electro mechanical systems) processing technology, pouring a mixture of Polydimethylsiloxane (PDMS) and a curing agent onto the SU8 positive membrane by using a thermoplastic method, curing for 20-60 min at 60-100 ℃ after bubbles are removed, and stripping the solidified PDMS from the positive membrane to obtain a PDMS cover plate with the microchannel; the method specifically comprises the following steps:
(1) sampling 10-1000 uL of bacterial liquid from one sample inlet of a sample injection channel, sampling 10-1000 uL of 0.1-1 mg/mL of aqueous solution of the glycosylated magnetic microspheres simultaneously from the other sample inlet, wherein the sample injection mode is peristaltic pump injection sample injection or negative pressure sample injection, the bacterial liquid and the aqueous solution of the glycosylated magnetic microspheres flow into a mixing channel to be fully mixed, and marking the bacteria by the glycosylated magnetic microspheres to obtain a suspension of the glycosylated magnetic microsphere marked bacteria and redundant glycosylated magnetic microspheres;
(2) when the suspension obtained in the step (1) enters a detection area, DEP (dielectrophoresis) is started, the voltage is 2-10V, the frequency is 400-600 kHz, the DEP time is 1-30 min, the peristaltic pump is stopped, DEP is continued until the flow rate of a mixing channel is 0, the DEP is stopped, and bacteria marked by the glycosylated magnetic microspheres are enriched between interdigital electrodes in the detection cell;
(3) immediately switching to an EIS detection mode after DEP is stopped, wherein an alternating current disturbance signal of the EIS is 50-500 mv, the frequency is 10-50 kHz, and an impedance signal value Z is recorded;
(4) repeating the steps (1) - (3) by taking bacterial liquid with different concentrations and the glycosylated magnetic microsphere solution, and recording the signal values Z of suspensions with different bacterial concentrationsnN is more than or equal to 5, the blank signal value is subtracted to obtain annTo be atnTaking the concentration of bacteria in the suspension as the abscissa as the ordinate] ；
(5) Taking a sample X to be measured, and determining a signal value Z according to the steps (1) - (3)xIn relation to Δ Z- [ C]Obtaining the bacterial concentration of the suspension of the sample to be detected, and then calculating the bacterial concentration in the sample X to be detected according to the sample introduction volume of the sample to be detected and the sample introduction volume of the glycosylated magnetic microsphere aqueous solution; the glycosylated magnetic microsphere is a mannose derivative modified magnetic microsphere.
2. The method of claim 1, wherein the substrate is made of glass, quartz, silicon, or a polymer.
3. The method according to claim 1, wherein the material of the interdigital electrode is gold, platinum, copper, aluminum or palladium, and the interdigital electrode is prepared by a magnetron sputtering technique of MEMS processing.
4. The method of claim 1, wherein the glycosylated magnetic microsphere is prepared by the following steps: at 15-30 ℃, taking 60-6000 mu L of carboxylated magnetic microspheres subjected to ultrasonic treatment, enriching the carboxylated magnetic microspheres by using a magnet, removing supernatant, adding 300-3000 mu L of 100-300 mmol/L EDC and 300-3000 mu L of 100-300 mmol/L NHS, shaking in the dark for 30-60 min, then adding 300-3000 mu L of 0.2-2 mg/mL new mannose derivatives, shaking in the dark for 40-120 min, adding 300-3000 mu L of 1-5% bovine serum albumin solution, shaking in the dark for 10-120 min, stopping reaction, enriching the magnet, removing supernatant, and washing by using a buffer solution to obtain the glycosylated magnetic microspheres.
5. The method of claim 1, wherein the mannose derivative is 4-aminophenyl- α -mannopyranoside, mannobiose, mannotriose, or a mannose dendrimer having mannose as a main structure.
6. Use of the method of any one of claims 1 to 5 for the detection of Salmonella typhimurium, Escherichia coli expressing type 1 pili, Arthrobacter choleraesuis, or/and Klebsiella pneumoniae.
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