CN209841886U - AME micro-fluidic detection chip and detection system based on immunomagnetic separation - Google Patents
AME micro-fluidic detection chip and detection system based on immunomagnetic separation Download PDFInfo
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Abstract
The utility model provides a micro-fluidic detection chip of chain gessol monomethyl ether micro-fluidic detection chip based on immunity magnetic separation. The microfluidic detection chip comprises three layers of structures which are sequentially stacked together and sealed with each other, wherein the three layers are a cover plate layer, a middle layer and a substrate layer from top to bottom; the middle layer is provided with a plurality of parallel independent fluid channels, each fluid channel is provided with a sample inlet and a sample outlet, and the sample inlet and the sample outlet respectively correspond to the sample inlet and the liquid outlet on the cover plate layer; the fluid channel is sequentially provided with a reaction chamber A, a micro-mixing area and a reaction chamber B; GNP-mAbs coupling pad is placed in reaction chamber A, and magnetic bead-BSA-AME complex is contained in reaction chamber B. The chip can be used for rapidly, simply, highly sensitively and quantitatively detecting the AME in the sample at high flux, has strong detection specificity and simple operation, and can be widely applied to the quantitative detection of the AME in the fruit and vegetable sample.
Description
Technical Field
The utility model relates to a food safety monitoring field, specifically speaking relates to a chain gessol monomethyl ether micro-fluidic detection chip and detecting system based on immunity magnetic separation.
Background
Alternariol Monomethyl Ether (AME) is one of the main secondary metabolites of Alternaria alternata, and has carcinogenicity, teratogenicity and mutagenicity. Currently, the methods for detecting alternaria alternate AME mainly rely on the traditional detection method of liquid chromatography-mass spectrometry. However, these conventional detection methods typically require large, expensive instrumentation and specialized technicians.
Currently, a few reports on quantitative detection of AME by immunoassay methods, such as colloidal gold immunochromatographic test strips, competitive enzyme-linked immunosorbent assay (ELISA), etc., have appeared. These detection methods have low detection sensitivity and cannot achieve rapid high-throughput detection of AME.
Microfluidic chips, also known as "lab-on-a-chips", have been listed as the most important leading technology row and column in the 21 st century, and as a potential analysis platform, it can integrate biological or chemical experimental operations such as preparation, separation, reaction, detection, etc. to reduce the sample consumption, shorten the analysis time, improve the detection sensitivity, and finally realize the integrated, miniaturized, high-throughput and automated detection of the sample. In addition, the combination of microfluidic chips with immunoassay methods is considered to be one of the promising analytical platforms for high-throughput, sensitive, automated detection.
SUMMERY OF THE UTILITY MODEL
The utility model aims at providing an alternariol monomethyl ether micro-fluidic detection chip based on immune magnetism separation.
In order to realize the purpose of the utility model, in the first aspect, the utility model provides an Alternaria phenol monomethyl ether micro-fluidic detection chip (micro-fluidic chip) based on immunomagnetic separation, which comprises a three-layer structure that is stacked together in sequence and sealed with each other, and is a cover plate layer, an intermediate layer and a substrate layer from top to bottom respectively;
the cover plate layer is provided with a plurality of sample inlets and liquid outlets, and the sample inlets and the liquid outlets respectively and independently penetrate through the cover plate layer and the middle layer;
the middle layer is provided with a plurality of parallel independent fluid channels, each fluid channel is provided with a sample inlet and a sample outlet, the sample inlet corresponds to a sample inlet on the cover plate layer, and the sample outlet corresponds to a liquid outlet on the cover plate layer; the fluid channel is sequentially provided with a reaction chamber A, a micro-mixing area and a reaction chamber B along the direction from the sample inlet to the sample outlet; a colloidal gold-AME monoclonal antibody compound coupling pad (GNP-mAbs coupling pad) is placed in the reaction chamber A, and a magnetic bead (MNP) -BSA-AME compound is added in the reaction chamber B.
In the present invention, the preparation method of the colloidal gold-AME monoclonal antibody compound is described in CN201610539564.1 specification, examples 1 and 2.
Preferably, the cover sheet layer and the intermediate layer are made of NOA81(Norland Optical Adhesive 81) Optical cement, PDMS or the like.
Preferably, the substrate layer is a glass substrate or a PDMS substrate or the like.
Preferably, the coupling pad is made of a glass cellulose membrane.
Preferably, the micro-mixing zone in the intermediate layer is a zigzag microfluidic mixing channel.
Preferably, the coupling pad is coated with 6 μ L of 6.7-fold concentrated colloidal gold-AME monoclonal antibody complex, and 15 μ L of 3mg/mL magnetic bead-BSA-AME complex is added into the reaction chamber B.
Optionally, the reaction zone chamber is circular and the capture zone chamber is elliptical.
Preferably, the length of the microfluidic chip is 65mm, and the width of the microfluidic chip is 43 mm.
The thickness of the cover plate layer and the middle layer of the microfluidic chip is about 0.5 mm.
The depth, length and width of the fluid channel of the middle layer of the microfluidic chip are respectively about 0.5mm, about 43mm and about 150 mu m.
In a second aspect, the utility model provides an alternariol monomethyl ether micro-fluidic detection system based on immunomagnetic separation, including but not limited to above-mentioned micro-fluidic chip, magnet, feed liquor pipe, drain pipe, ELIASA and peristaltic pump.
The utility model provides a micro-fluidic chip and detecting system is applicable in fruit and vegetable AME's quantitative determination.
Borrow by above-mentioned technical scheme, the utility model discloses following advantage and beneficial effect have at least:
the utility model provides an alternariol monomethyl ether micro-fluidic detection chip based on immune magnetic separation. The chip comprises a cover plate layer, an intermediate layer and a substrate layer from top to bottom; the middle layer is provided with a plurality of parallel independent fluid channels, and each fluid channel is sequentially provided with a reaction chamber A, a micro-mixing area and a reaction chamber B; a colloidal gold-monoclonal antibodies (GNP-mAbs) coupling pad is placed in the reaction chamber A, and a magnetic bead-BSA-AME compound is contained in the reaction chamber B. The chip takes magnetic bead-BSA-AME as a capture probe and GNP-mAbs as a detection probe. Meanwhile, the magnetism of the magnetic beads is utilized to carry out rapid separation and purification. The micro-fluidic chip can rapidly (complete detection within 15 minutes), simply, highly sensitively and quantitatively detect the AME in the sample at high flux, has the lowest detection limit of 12.5pg/mL, has strong detection specificity and simple operation, and can be widely applied to the quantitative detection of the AME in the fruit and vegetable sample.
Drawings
Fig. 1 is a schematic diagram (top perspective view) of the whole structure of the microfluidic immune chip of the present invention. The device comprises a substrate layer (bottom layer), a 2-middle layer, a 3-cover plate layer (upper layer), a 4-sample inlet/sample inlet, a 5-liquid outlet/sample outlet, a 6-reaction chamber A, a 7-Z-shaped micro-mixing area and a 8-reaction chamber B.
FIG. 2 is a schematic diagram of the detection of the microfluidic immune chip of the present invention.
Fig. 3 is an exploded view of a microfluidic chip according to the present invention. The device comprises a sample inlet, a sample outlet, a 3-reaction chamber A, a 4-reaction chamber B, a 5-Z-shaped micro-mixing area, a 6-cover plate layer (upper layer), a 7-middle layer and an 8-substrate layer (bottom layer).
Fig. 4 is a cross-sectional view of the microfluidic chip of the present invention, showing the specific structures of the substrate, the sample fluid channel, the reaction chamber a, the reaction chamber B, the sample inlet, and the liquid outlet in the chip. The device comprises a substrate layer 1, a middle layer 2, a cover plate layer 3, a sample inlet 4, a sample outlet 5, a sample outlet 6, a reaction chamber A and a reaction chamber B8.
Fig. 5 shows the structure of the intermediate layer of the microfluidic chip according to the present invention. The device comprises a sample inlet, a sample outlet, a 6-reaction chamber A, a 7-Z-shaped micro-mixing area and a 8-reaction chamber B, wherein the sample inlet is arranged at the bottom of the reaction chamber A, the sample outlet is arranged at the bottom of the reaction chamber A, and the sample outlet is arranged at the bottom of the reaction chamber B.
Fig. 6 is a schematic diagram of a process for constructing a microfluidic chip according to the present invention. Wherein, A: preparation processes of the chip intermediate layer and the chip upper layer, B: nano material embedding and chip bonding process, C: mask structure and channel dimension structure chart.
FIGS. 7a to 7f are graphs showing the optimization results of the reaction reagents and reaction conditions in example 2 of the present invention. Wherein, fig. 7 a: optimization of CTAB concentration, fig. 7 b: results of AA concentration optimization, fig. 7 c: HAuCl4Results of solution concentration optimization, fig. 7 d: optimization of the amount of GNP-mAbs, FIG. 7 e: effect of reaction time on immunogold amplification, fig. 7 f: na (Na)2S2O3The effect of addition of (a) on immuno-gold amplification.
FIG. 8 is the electron micrograph of GNP (a) and the UV characterization of the GNP-mAbs complex described in example 2 of the present invention.
FIG. 9 is a diagram showing the specificity of the microfluidic immunoassay method described in example 3 of the present invention.
FIG. 10 is a diagram showing the quantitative detection of AME by the microfluidic immunoassay method in example 4 of the present invention.
Detailed Description
In the description of the present invention, unless otherwise specified, the terms "upper", "lower", "inside", "outside", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience in describing the present invention and simplifying the description, but do not indicate or imply that the device or element referred to must have a specific orientation, be constructed in a specific orientation, and be operated, and thus should not be construed as limiting the present invention.
Example 1 preparation of Alternaria phenol monomethyl ether microfluidic detection chip based on immunomagnetic separation
The embodiment provides an alternariol monomethyl ether microfluidic detection chip based on immunomagnetic separation, which uses magnetic beads (MNP) -BSA-AME as capture probes and colloidal gold-monoclonal antibodies (GNP-mAbs) as detection probes. Meanwhile, the magnetism of the magnetic beads is utilized to carry out rapid separation and purification, and signal amplification is carried out based on the autocatalysis characteristic of the immune gold. The change in absorbance of the immuno-gold is proportional to the amount of AME in the sample. The preparation method of the microfluidic chip comprises the following specific steps:
1. preparation of micro-fluidic chip channel structure layer (intermediate layer)
Thoroughly cleaning the glass substrate by using water and absolute ethyl alcohol, drying the glass substrate by using nitrogen, and fixing the mask frame on the glass substrate; dripping 0.4mL of NOA81 optical cement into the mask frame, and then slightly covering the mask with the channel structure with the NOA81 optical cement; after UV exposure, the mask and mask frame were gently torn off, the uncured NOA81 photoresist was rinsed with a mixture of acetone and alcohol (4:1, v/v), and blown dry with cold air.
2. Preparation of cover sheet layer (upper layer) structure of microfluidic chip
Fixing the mask frame on a chromium plate, dripping 0.3mL of NOA81 photoresist, and slightly covering the upper mask on the NOA81 photoresist; after ultraviolet exposure, the mask frame and the NOA81 photoresist layer are slightly torn off from the chromium plate; and cleaning the uncured photoresist, and removing the mask frame.
3. Nanomaterial embedding and die bonding
The magnetic bead-BSA-AME complex and the GNP-mAbs coupling pad prepared in advance were added to the reaction chamber separately. And fixing the optical cement structure on the upper mask and the optical cement in the middle layer by using a UV bonding technology, and slightly tearing off the upper mask. And obtaining the assembled microfluidic chip.
The microfluidic chip comprises a three-layer structure which is stacked together in sequence and sealed mutually, and a cover plate layer (upper layer), a middle layer and a substrate layer (bottom layer) from top to bottom.
Wherein, be equipped with 6 introduction port and 6 liquid outlets on the cover plate layer respectively, introduction port, liquid outlet link up cover plate layer and intermediate level independently respectively.
The middle layer is provided with 6 parallel independent fluid channels, the fluid channels are provided with sample inlets and sample outlets, the sample inlets correspond to sample inlets on the cover plate layer, and the sample outlets correspond to liquid outlets on the cover plate layer; the fluid channel is sequentially provided with a reaction chamber A, Z-shaped micro-mixing area (micro-mixing channel) and a reaction chamber B along the direction from the sample inlet to the sample outlet. A colloidal gold-AME monoclonal antibody compound coupling pad (GNP-mAbs coupling pad) is placed in the reaction chamber A, and a magnetic bead (MNP) -BSA-AME compound is added in the reaction chamber B.
The micro-mixing area of the middle layer is a Z-shaped micro-fluid channel.
The length of the micro-fluidic chip is 65mm, and the width is 43 mm. The total thickness of the microfluidic chip is about 2 mm.
The thickness of the cover plate layer and the middle layer of the microfluidic chip is about 0.5 mm.
The depth, length and width of the fluid channel of the middle layer of the microfluidic chip are respectively about 0.5mm, about 43mm and about 150 mu m.
The overall structure schematic diagram of the microfluidic immune chip is shown in figure 1. The detection principle is shown in figure 2. The structure is shown in an exploded view in fig. 3. The sectional view is shown in fig. 4. The structure of the intermediate layer is shown in fig. 5. The preparation process of the microfluidic chip is shown in fig. 6.
EXAMPLE 2 optimization of reagents and reaction conditions
1. Optimization of CTAB concentration
5mL of water was added to each 15mL centrifuge tube. Adding 22.8-409mg of CTAB into each centrifuge tube according to the table 1; then, 12.5. mu.L of 0.1M HAuCl was added to each tube4Solutions ofIncubating at 37 ℃ until the color of the mixed solution becomes transparent and light yellow; after the subsequent addition of 65. mu.L of 100mM AA, the color of the mixture quickly changed to colorless. Adding 100 μ L of CTAB mixture with different concentrations into 96-well plate, adding 6 μ L of re-suspended GNP-mAbs, and adding 10mM Na into each well after 10min2S2O3Solution 10. mu.L. The optimization results are shown in fig. 7 a.
TABLE 1 respective reagent amounts for CTAB concentration optimization experiments
2. Optimization of AA (ascorbic acid) concentration
1mL of 100mM CTAB was added to a series of 1.5mL centrifuge tubes, respectively; 0.1M HAuCl was added as in Table 24The solution was 2.5. mu.L, incubated at 37 ℃ until the color of the mixture became clear pale yellow; 100mM AA 10-26. mu.L each was added. To 96 wells, 100. mu.L of the mixture was added, followed by 6. mu.L of GNP-mAbs. After reaction for 10min, 10mM Na was added to each well separately2S2O3Solution 10. mu.L. The optimization results are shown in FIG. 7 b.
Table 2 amounts of reagents used for AA concentration optimization experiments
3、HAuCl4Optimization of solution concentration
Adding 2mL of 100mM CTAB solution into a series of 4mL centrifuge tubes respectively; 0.1M HAuCl was added separately as shown in Table 34Incubating the solution at 37 deg.C to 0.5-9 μ L until the color of the mixture turns into transparent light yellow; mu.L of 100mM AA was added sequentially. To 96 wells, 100. mu.L of the mixture was added, followed by 6. mu.L of GNP-mAbs. After reaction for 10min, 10mM Na was added to each well separately2S2O3Solution 10. mu.L. The optimization results are shown in fig. 7 c.
TABLE 3 for HAuCl4Respective reagent dosage of concentration optimization experiment
4. Preparation of optimal homogeneous immunogold growth liquid
According to the optimization results of the above 1, 2 and 3 (fig. 7 a-7 c), the optimal formulation of homogeneous immunogold growth solution is obtained as follows: 100mM CTAB +1.5mM AA +0.25mM HAuCl4。
The preparation method comprises the following steps: 182.2mg CTAB and 12.5. mu.L of 0.1M HAuCl4The solution was added to 5mL of purified water and incubated at 37 ℃ until the mixture became clear and pale yellow. Then 75. mu.L of 100mM AA was added and the color of the mixture rapidly changed to colorless. Cooling the mixed solution to room temperature to obtain the homogeneous immunogold increasing solution.
5. Optimization of the amount of GNP-mAbs
Growth of the optimal homogeneous immunogold (100mM CTAB +1.5mM AA +0.25mM HAuCl)4) Respectively adding 1-10 mu L of the resuspended GNP-mAbs; after reaction for 10min, 10mM Na was added to each well2S2O3Solution 10. mu.L. The optimization results are shown in FIG. 7 d. By optimization, the optimal GNP-mAbs dosage is 6. mu.L.
6. Reaction time and Na2S2O3Effect on immunogold amplification
100 mu L of immune gold growth solution is added into 96 wells, 6 mu L of GNP-mAbs is added, and the ultraviolet absorption value (400-800nm) is measured once per minute by a microplate reader. At 7 min, 10mM Na was added2S2O3Solution 10. mu.L. The optimization results are shown in fig. 7e and 7 f. The optimal growth time of the immune gold is 10min through optimization; na (Na)2S2O3Can effectively stop the growth of the immunogold. In this example, GNP electron microscopy is shown in FIG. 8 (a) and the UV characterization of GNP-mAbs complexes is shown in FIG. 8 (b).
Example 3 establishment of competitive fluorescent immunosensing assay based on magnetic beads
AME standards were diluted with Tri-HCl (10mM, pH 7.4) to standard solutions at concentrations of 600,400,200,150, 100,50,25,12.5 and 0pg/mL, respectively. In peristaltic pumpsUsing, 50 μ L of AME standard solutions of different concentrations were injected into the microfluidic chip prepared in example 1; firstly, in a micro-mixing channel of a GNP-mAbs coupling pad, AME in a sample is fully incubated and combined with the GNP-mAbs; the remaining GNP-mAbs and the formed GNP-mAbs-AME complex flow into the MNP-BSA-AME reaction chamber, and the remaining GNP-mAbs will be captured by MNP-BSA-AME; after magnetic separation, 30. mu.L of the supernatant was added to 100. mu.L of the optimized homogeneous immuno-gold growth medium (100mM CTAB +1.5mM AA +0.25mM HAuCl)4) After 10min, 10. mu.L of 10mM Na was added2S2O3And performing ultraviolet detection by using a microplate reader (400-.
As a result, it was found that as the concentration of AME increases, the intensity of the absorbance change also increases; and the change in absorbance intensity was linear with the concentration of AME in the range of 12.5-200 pg/mL (FIG. 10). Δ Abs ═ 0.001CAME-0.0021，R20.9975. The minimum detection limit was 12.5 pg/mL.
Example 4 specificity experiment for competitive fluorescent immunosensing assay based on magnetic beads
Two structural analogues of AME (e.g., Alternariol (AOH), alternarionic acid (TeA)) and mixtures thereof were selected for quantitative detection using the microfluidic immunoassay method described in example 2, and the sample solutions determined were: a is 200pg/mL AME solution; b is 1ng/mL AOH; c is 1ng/mL TeA; d is a mixture containing 200pg/mL AME and 1ng/mL AOH; e is a mixture containing 200pg/mL AME and 1ng/mL TeA; f is a mixture containing 1ng/mL AOH and 1ng/mL TeA. The results are shown in FIG. 9. As can be seen from the figure, negligible changes in the absorbance intensity can only be found in samples containing AOH or TeA. Whereas a significant change in fluorescence intensity was only found in samples containing AME. Therefore, the prepared microfluidic immunoassay method has better detection specificity.
Example 5 application of microfluidic immunoassay method to AME detection in fruits (spiking experiment)
1. Sample pretreatment: cutting 1kg of cherries, oranges and apples into small pieces and grinding into pulp; respectively weighing 5g of homogenized cherry, orange and apple, and adding water to 5 mL; to each of cherry, orange and apple was added 10. mu.L, 5. mu.L, 2.5. mu.L and 1.25. mu.L of 0.1mg/mL AME standard and 20mL of acetonitrile solution containing 100mM citric acid, and after incubation with stirring at room temperature for 30min, 2g of sodium chloride was added. Centrifuging at 10000rpm for 5min, and purifying the supernatant with a solid phase extraction column; after 4mL of the extract was dried under nitrogen, 0.1mL of acetonitrile was used for reconstitution. Finally, the recombinant AME solutions were diluted with 10mM Tris-HClbuffer (pH 7.4) to concentrations of 150, 75, 37.5 and 18.75 pg/mL for assay.
2. And (3) quantitative detection: AME was simultaneously detected by a microfluidic immunoassay method and HPLC-MS, and the results are shown in Table 4. The recovery rate of this detection method was 88.35-94.15% (fig. 10). The result shows that the method has reliability and practicability in quantitative detection of AME in fruit actual samples.
TABLE 4 spiking detection of AME in fruit samples
Although the invention has been described in detail with respect to the general description and the specific embodiments, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Therefore, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (9)
1. The Alternaria phenol monomethyl ether microfluidic detection chip based on immunomagnetic separation is characterized by comprising a three-layer structure which is stacked together in sequence and sealed with each other, wherein the three-layer structure comprises a cover plate layer, a middle layer and a substrate layer from top to bottom;
the cover plate layer is provided with a plurality of sample inlets and liquid outlets, and the sample inlets and the liquid outlets respectively and independently penetrate through the cover plate layer and the middle layer;
the middle layer is provided with a plurality of parallel independent fluid channels, each fluid channel is provided with a sample inlet and a sample outlet, the sample inlet corresponds to a sample inlet on the cover plate layer, and the sample outlet corresponds to a liquid outlet on the cover plate layer; the fluid channel is sequentially provided with a reaction chamber A, a micro-mixing area and a reaction chamber B along the direction from the sample inlet to the sample outlet; a colloidal gold-AME monoclonal antibody compound coupling pad is placed in the reaction chamber A, and a magnetic bead-BSA-AME compound is added in the reaction chamber B.
2. The microfluidic detection chip according to claim 1, wherein the cover sheet layer and the middle layer are made of NOA81 optical cement or PDMS.
3. The microfluidic detection chip according to claim 1, wherein the substrate layer is a glass substrate or a PDMS substrate.
4. The microfluidic detection chip according to claim 1, wherein the coupling pad is made of a glass cellulose membrane.
5. The microfluidic detection chip according to claim 1, wherein the length of the chip is 65mm and the width thereof is 43 mm.
6. The microfluidic detection chip according to claim 1, wherein the thickness of the cover sheet layer and the middle layer is 0.5 mm.
7. The microfluidic detection chip of claim 1, wherein the depth, length and width of the fluid channel are 0.5mm, 43mm and 150 μm, respectively.
8. The microfluidic detection chip according to any one of claims 1 to 7, wherein the micro-mixing region in the intermediate layer is a zigzag microfluidic mixing channel.
9. The Alternaria phenol monomethyl ether microfluidic detection system based on immunomagnetic separation is characterized by comprising the microfluidic detection chip, a magnet, a liquid inlet pipe, a liquid outlet pipe, an enzyme labeling instrument and a peristaltic pump according to any one of claims 1 to 8.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109900899A (en) * | 2019-04-08 | 2019-06-18 | 北京农业质量标准与检测技术研究中心 | Rod method phenol monomethyl ether micro-fluidic detection chip and detection method based on immunomagnetic isolation |
| CN112505029A (en) * | 2020-11-26 | 2021-03-16 | 北京农业质量标准与检测技术研究中心 | Colorimetric sensing detection method for pathogenic bacteria and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109900899A (en) * | 2019-04-08 | 2019-06-18 | 北京农业质量标准与检测技术研究中心 | Rod method phenol monomethyl ether micro-fluidic detection chip and detection method based on immunomagnetic isolation |
| CN112505029A (en) * | 2020-11-26 | 2021-03-16 | 北京农业质量标准与检测技术研究中心 | Colorimetric sensing detection method for pathogenic bacteria and application thereof |
| CN112505029B (en) * | 2020-11-26 | 2022-12-09 | 北京市农林科学院 | Colorimetric sensing detection method for pathogenic bacteria and application thereof |
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