CN113399007B - Surface open type microfluidic chip, immunosensor and method for detecting rhodamine B and sudan red pigment - Google Patents
Surface open type microfluidic chip, immunosensor and method for detecting rhodamine B and sudan red pigment Download PDFInfo
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- CN113399007B CN113399007B CN202110510858.2A CN202110510858A CN113399007B CN 113399007 B CN113399007 B CN 113399007B CN 202110510858 A CN202110510858 A CN 202110510858A CN 113399007 B CN113399007 B CN 113399007B
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Abstract
The invention discloses a surface open type microfluidic chip, an immunosensor and a method for detecting rhodamine B and sudan red pigment. The surface-open microfluidic chip comprises a chip substrate and a plurality of parallel channels positioned on the chip substrate, wherein each parallel channel is provided with 5 reaction chambers and 1 tail chamber which are sequentially connected, and each chamber is provided with a concave microstructure and is mutually connected through a microfluidic flow channel; the first reaction chamber is used for loading a sample to be detected, a magnetic bead antigen and an enzyme-labeled antibody mixed solution, the second reaction chamber is loaded with PBS, and the third reaction chamber is loaded with a substrate Amplex Red/H 2 O 2 The fourth reaction chamber was loaded with PBS and the fifth reaction chamber was loaded with Gluocose/Amplex Red/HRP substrate. The method can detect the contents of rhodamine B and Sudan red in a plurality of samples at one time, has high sensitivity and simple and convenient operation, and is very suitable for the field detection of prohibited pigments in food.
Description
Technical Field
The invention relates to the technical field of food safety detection, in particular to a surface open type microfluidic chip, an immunosensor and a method for detecting rhodamine B and Sudan red pigment.
Background
The pepper condiment has a large share in the global condiment market and is an important raw material for increasing the flavor, color and preservation time of food. However, some merchants illegally add rhodamine B, sudan red and other industrial dyes into the pepper seasoning in order to make the color of the product more gorgeous and thus earn high profits and do not consider the health problems of consumers. Researches show that the dyes have great harm to human health, wherein rhodamine B has specific potential toxicity, carcinogenicity and mutagenicity. The list of non-edible substances that may be illegally added to food and food additive varieties that are easily abused (first lot) in Ministry of health was listed as prohibited pigments; sudan red (Sudan I, Sudan II, Sudan III and Sudan IV) is classified as three carcinogens by the international cancer organization, has carcinogenic effect on animals, and has liver as the main target organ. Sudan red is prohibited as a food additive in countries such as European Union in 1995, and is prohibited in Ming dynasty in food additive health Standard in 1996 in China. At present, the detection method for the prohibited pigments such as rhodamine B, Sudan red and the like in food mainly comprises an instrumental method, including a high performance liquid chromatography, a high performance liquid chromatography-tandem mass spectrometry method, a gas chromatography-tandem mass spectrometry method and the like, and an immunoassay method, such as an enzyme-linked immunosorbent assay, an immunoaffinity chromatography and the like. Although the instrumental method has high accuracy and precision, the pretreatment of the sample is complex and expensive instruments and equipment are required; the existing immunoassay method has the defects of single detection object, need of a large-scale enzyme-labeling instrument and the like although the operation is simple and convenient and the sensitivity is high, and the application of the immunoassay method in food safety field screening is limited. The patent CN103439514A discloses a method for detecting multiple residues of an agricultural and veterinary drug based on a microarray detection chip, which comprises the steps of coupling a micromolecular agricultural and veterinary drug hapten and bovine serum albumin to prepare an immune antigen, immunizing a mouse with the immune antigen to prepare a corresponding agricultural and veterinary drug monoclonal antibody, and spotting a capture monoclonal antibody probe on an agarose-modified slide solid-phase carrier by using a biochip spotting instrument to prepare a multiple-repetition capture monoclonal antibody probe microarray. The hapten of the veterinary drug and the ovalbumin are coupled to prepare a 'hapten-OVA' coupling body, and then the coupling body is marked by a fluorescent molecule Cy 3. Mixing a sample solution to be detected and a 'marked detection antigen' according to a certain concentration, incubating with a chip, directly and competitively combining the antigen to be detected and the corresponding 'marked detection antigen' in the sample with a corresponding 'capture monoclonal antibody probe' fixed on the chip, eluting under a certain condition, and detecting a result by using a biochip scanner; although the biochip is used for detection, the microarray detection chip is still too cumbersome to use, subsequent matched detection needs to depend on laboratory instruments and equipment, and the detection target which is simple, efficient and quick on site cannot be achieved.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a surface open type microfluidic chip for detecting rhodamine B and Sudan red pigment.
The second purpose of the invention is to provide a biosensor for detecting rhodamine B and Sudan red pigment.
The third purpose of the invention is to provide the method for detecting rhodamine B and the Sudan red pigment by using the biosensor.
The above object of the present invention is achieved by the following technical solutions:
a surface open type microfluidic chip for detecting rhodamine B and Sudan red pigment comprises a chip substrate and a plurality of parallel channels positioned on the chip substrate, wherein each parallel channel is provided with 5 reaction chambers and 1 tail chamber which are sequentially connected, and each chamber is provided with a sunken microstructure and is mutually connected through a microfluidic flow channel; the first reaction chamber is used for loading a sample to be detected, a magnetic bead antigen and an enzyme-labeled antibody mixed solution, the second reaction chamber is loaded with PBS liquid drops, and the third reaction chamber is loaded with a substrate Amplex Red/H 2 O 2 A droplet, the fourth reaction chamber is loaded with a PBS droplet, and the fifth reaction chamber is loaded with a Gluocose/Amplex Red/HRP substrate droplet; the magnetic bead antigen is a rhodamine B antigen-magnetic bead conjugate and a Sudan red antigen-magnetic bead conjugate; the enzyme-labeled antibody is a horseradish peroxidase-labeled rhodamine B antibody and a glucose oxidase-labeled sudan red antibody.
The surface-open type microfluidic chip takes a rhodamine B antigen-magnetic bead conjugate (RhB-Ag-Mb) and a Sudan red antigen-magnetic bead conjugate (SuD-Ag-Mb) as sensor substrates, and takes a horse radish peroxidase-labeled rhodamine B antibody (RhB-Ab-HRP) and a glucose oxidase-labeled Sudan red antibody (SuD-Ab-GOx) as signal probes. A mixed solution containing magnetic bead antigens and enzyme-labeled antibodies is added into a first reaction chamber of a microfluidic chip channel filled with mineral oil, and water-in-oil droplets are formed to serve as a micro reaction area. After the immune recognition is finished, the magnetic beads combined with the enzyme-labeled antibody can be pulled into a second reaction chamber (containing PBS) for cleaning through the action of an additional magnet. After washing, the beads were again pulled into the third chamber (containing the substrate Amplex Red/H) 2 O 2 A droplet of (a). Wherein, RhB-Ab-HRP catalytic substrate Amplex Red/H 2 O 2 Generation of fluorescent substance Resorufin, the signal intensity is proportional to the RhB content in the system; following washing of the immunomagnetic beads in the fourth reaction chamber (PBS-containing droplet), the beads were drawn into a fifth reaction chamber containing a Gluocose/Amplex Red/HRP substrate droplet, and SuD-Ab-GOx first catalyzes Glucose to generate H 2 O 2 And then further catalyzing the Amplex Red/HRP to generate a fluorescent substance Resorufin, wherein the signal intensity is in proportional relation with the SuD content in the system, and the concentration of rhodamine B and Sudan Red in the sample to be detected is calculated through a linear relation curve of standard concentration and fluorescence intensity. The content of rhodamine B and Sudan red in a plurality of samples can be detected once and simultaneously.
Preferably, the number of parallel channels is 16.
Preferably, the reaction chamber is 12.76mm long, 3.2mm wide and 3.7mm high; the tail cavity is 3.2mm long, 3.2mm wide and 3.7mm high.
A biosensor for detecting rhodamine B and Sudan red pigment comprises a biosensor shell 1, a power module 2, an incubation module 3, an immunoreaction module 4, a fluorescence excitation module 5 and a signal acquisition module 6, wherein the power module 2, the incubation module 3, the immunoreaction module 4 and the fluorescence excitation module 5 are arranged in the biosensor shell 1; the power supply module 2 is arranged at the bottom of the biosensor shell 1 and is respectively connected with the incubation module 3 and the fluorescence excitation module 5; the incubation module 3 is arranged below the immunoreaction module 4; the fluorescence excitation module 5 is arranged on the inner side of the top of the biosensor shell 1; the immune reaction module 4 comprises a microfluidic chip tray 41 and any one of the open-surface microfluidic chips 42.
The biosensor can realize the simultaneous detection of the contents of rhodamine B and Sudan red in a plurality of samples at a time by using the surface-open microfluidic chip; the power module provides a stable power supply for the incubation module and the fluorescence excitation module, the incubation module can provide a constant-temperature heating environment for the immunoreaction module, the fluorescence excitation module is used for fluorescence excitation of a substrate, the signal acquisition module ensures the consistency of the positions of acquired fluorescence signals, the batch difference is reduced, and the detection accuracy is improved. The biosensor can be used for real-time, on-site and high-precision rapid detection of rhodamine B and Sudan red.
Preferably, the power module 2 is a recyclable battery box, and a 12V battery box can be adopted for supplying power to the power modules of the whole biosensor.
Preferably, the incubation module 3 is composed of an intelligent temperature controller 31 and a constant temperature heater 32 which are connected.
More preferably, the constant temperature heater 32 is a 12V micro-heating plate for incubating the immunoreaction module.
Preferably, one side of the biosensor housing 1 is provided with a temperature display screen 7, and the temperature display screen 7 is connected with the intelligent temperature controller 31.
Preferably, the chip tray 41 is a drawer tray, and can be used for conveying the open-surface microfluidic chip 42, and the design of the handle also takes human mechanics into consideration, so as to improve the comfort of the user.
Preferably, the fluorescence excitation module 5 is composed of an LED array, an excitation filter and an emission filter.
More preferably, the LED array is at an angle of 45 ° to the top of the biosensor housing 1, the LED array is a 60 × 15mm, 2W green LED array, and the excitation wavelength is 525 nm.
Preferably, the signal acquisition module 6 is composed of a signal acquisition hole 61, a smart phone 62 and a fixing support 63, the signal acquisition hole 61 is arranged at the top of the biosensor housing 1 and is matched with a camera of the smart phone 62, and the fixing support 63 is arranged at the outer side of the top of the biosensor housing 1 and is used for fixing the smart phone 62.
Preferably, the mobile phone comprises an image information acquisition module, a colorimetric module and a concentration display module, and is used for acquisition and processing of detection signals and display of results, and the Android version is required to be 4.3 or more.
Preferably, the biosensor housing 1 is further provided with a first switch 8 for controlling the incubation module 3 and a second switch 9 for controlling the fluorescence excitation module 5 on the top. In particular, the first switch is used to control the thermostatic heater 32 and the second switch is used to control the LED array.
The invention also provides a method for detecting rhodamine B and Sudan red pigment, which comprises the following steps:
s1, measuring the fluorescence intensity generated by the biosensor under the concentration of the standard series rhodamine B and the concentration of Sudan red by adopting any one of the biosensors, and drawing a linear relation curve of the concentration and the fluorescence intensity;
s2, detecting the rhodamine B and the Sudan red pigment in the sample to be detected by using any one of the biosensors, and calculating the concentration of the rhodamine B and the Sudan red pigment in the sample by using the linear relation curve obtained in the step S1.
Compared with the prior art, the invention has the following beneficial effects:
the surface-open microfluidic chip disclosed by the invention is simple in structure, convenient to operate, free from pump driving and capable of realizing high-flux detection; the biosensor can be used for simultaneously detecting the content of rhodamine B and Sudan red in a plurality of samples at one time, has high sensitivity (the detection limits of the rhodamine B, the Sudan red No. 1 and No. 2 are respectively 0.0072ng/mL, 0.0040ng/mL and 0.026ng/mL), is simple and convenient to operate, is very suitable for field detection of prohibited pigments in food, and has a wide application prospect.
Drawings
Fig. 1 is a schematic structural diagram of a surface-open microfluidic chip and a chip mold design according to embodiment 1 of the present invention. A is a chip parameter diagram, and B is a die parameter diagram.
FIG. 2 is a schematic diagram of the overall structure separation of a portable biosensor for detecting rhodamine B and Sudan red in embodiment 2 of the invention.
FIG. 3 is a schematic diagram of a portable biosensor for detecting rhodamine B and Sudan red according to embodiment 2 of the invention.
FIG. 4 shows the detection results of antigen-magnetic bead conjugates of rhodamine B and Sudan red pigment, and HRP-labeled rhodamine B antibody and GOx-labeled Sudan red antibody. A is an SDS gel electrophoresis detection result of the antigen-magnetic bead conjugate based on the rhodamine B and the Sudan red pigment of the nanometer magnetic bead; b is a rhodamine B antibody, HRP and RhB-Ab-HRP ultraviolet (230-600 nm) scanning identification result; c is the color change result of the Sudan red antibody Ab, GOx enzyme and enzyme labeled antibody Ab-GOx mixed and incubated with the same amount of TMB/HRP solution.
Drawing notes: 1-a biosensor housing; 2-a power supply module; 3-an incubation module; 4-an immunoreaction module; 5-a fluorescence excitation module; 6-a signal acquisition module; 7-temperature display screen; 8-a first switch; 9-a second switch; 11-a biosensor front housing; 12-biosensor rear housing; 21-a battery case; 22-battery box base; 31-intelligent temperature controller; 32-constant temperature heater; 41-chip tray; 42-surface open microfluidic chip; 51-a first excitation light fixture; 52-a second excitation light fixture; 61-signal acquisition wells; 62-mobile phone; 63-fixing the bracket.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Embodiment 1a surface open microfluidic chip for detecting rhodamine B and Sudan red
1. Preparation of surface-open microfluidic chip
As shown in fig. 1A, a surface-open microfluidic chip (90 × 80 × 0.5cm) is provided, which includes a surface-open microfluidic chip, and includes a chip substrate and 16 parallel channels located on the chip substrate, each channel is provided with 5 reaction chambers and 1 tail chamber connected in sequence, each chamber has a recessed microstructure, and is connected to each other through a microfluidic flow channel, the reaction chambers have a length of 12.76mm, a width of 3.2mm, and a height of 3.7mm, and the tail chambers have a length of 3.2mm, a width of 3.2mm, and a height of 3.7 mm; the first reaction chamber is used for loading a sample to be detected, a magnetic bead antigen and an enzyme-labeled antibody mixed solution, the second reaction chamber is loaded with PBS liquid drops, and the third reaction chamber is loaded with a substrate Amplex Red/H 2 O 2 A droplet, a fourth reaction chamber loaded with a droplet of PBS and a fifth reaction chamber loaded with a droplet of glocose/Amplex Red/HRP substrate.
The surface open type micro-fluidic chip uses rhodamine B antigen-magnetic bead conjugate (RhB-Ag-Mb) and a sudan red antigen-magnetic bead conjugate (SuD-Ag-Mb) are used as sensor substrates, and horseradish peroxidase labeled rhodamine B antibody (RhB-Ab-HRP) and glucose oxidase labeled sudan red antibody (SuD-Ab-GOx) are used as signal probes. A mixed solution containing magnetic bead antigens and enzyme-labeled antibodies is added into a first reaction chamber of a microfluidic chip channel filled with mineral oil, and water-in-oil droplets are formed to serve as a micro reaction area. After the immune recognition is finished, the magnetic beads combined with the enzyme-labeled antibodies can be drawn into a second reaction chamber (containing PBS) for cleaning through the action of an additional magnet. After washing, the beads were again pulled into the third chamber (containing the substrate Amplex Red/H) 2 O 2 A droplet of (a). Wherein, RhB-Ab-HRP catalyzes a substrate Amplex Red/H2O2 to generate fluorescent substance Resorufin, and the signal intensity is in a proportional relation with the RhB content in the system; following washing of the immunomagnetic beads in the fourth reaction chamber (PBS-containing droplet), the beads were drawn into a fifth reaction chamber containing a Gluocose/Amplex Red/HRP substrate droplet, and SuD-Ab-GOx first catalyzes Glucose to generate H 2 O 2 And then further catalyzing Amplex Red/HRP to generate fluorescent substance Resorufin, wherein the signal intensity is in proportion to the SuD content in the system.
The micro-fluidic chip is obtained by a reverse mold method, and the specific preparation method comprises the following steps:
first, a mold is prepared, and a mold design is shown in fig. 1B and is obtained by machining. Then the chip is prepared. 30 g of glue A and 3 g of glue B of PDMS polydimethylsiloxane (Dow Corning 184SYLGARD184) as a pouring sealant are weighed, stirred and mixed, and then vacuumized. And (3) performing reverse molding on the mixed glue on a mold, vacuumizing again, removing bubbles, placing the mold in a 90-DEG C oven, heating for 30 minutes, and curing and molding. And (5) after the mixture is placed at room temperature, peeling off the die to obtain the chip.
2. Preparing antigen-magnetic bead conjugates of rhodamine B and Sudan red pigment based on nano magnetic beads respectively:
(1) and (4) activating the magnetic beads. Putting 100 mu L of magnetic beads into 1.5mL of EP tubes respectively, putting the tubes in a magnetic separation rack, enriching the magnetic beads, and removing supernatant; adding 200 μ L of 1mM hydrochloric acid diluted solution precooled at 4 deg.C, vortex shaking for 15s, placing on a magnetic separation rack to enrich magnetic beads, and removing supernatant.
(2) Antigen coupling. mu.L of each antigen solution (RhB-Ag at a concentration of 100. mu.g/mL and SuD-Ag at a concentration of 100. mu.g/mL) was added thereto, and vortexed for 30 seconds to mix them uniformly. And (3) placing the EP pipe on a vortex oscillator, adjusting to the lowest gear, and oscillating for 1.5h in an automatic mode.
(3) And separating supernatant. Magnetic beads are enriched by adopting a magnetic separation rack, and supernatant is preserved.
(4) And (5) cleaning. Add 200. mu.L of blocking solution (3M ethanolamine, pH 9.0), vortex for 15s, discard the supernatant and repeat this 4 times.
(5) And (5) sealing. Add 200. mu.L of blocking solution and mix with shaking for 2 h.
(6) And (5) separating. Magnetic beads were enriched using a magnetic separation rack and the supernatant was discarded.
(7) And (5) secondary sealing. 200 μ L of 1% BSA solution was added, vortexed for 15s, and the supernatant was discarded, and this operation was repeated 2 times. Then 200. mu.L of 1% BSA solution was added and mixed by shaking for 1 hour.
(8) And (5) cleaning. Add 200. mu.L of ultrapure water, vortex for 15s, and discard the supernatant.
(9) And (5) storing. Add 200. mu.L of storage solution (0.05% sodium azide in PBS), vortex for 15s, and discard the supernatant. This operation was repeated twice. Then 100. mu.L of preservation solution is added, mixed fully and preserved at 4 ℃ for standby.
(9) And (5) identifying. Performing SDS gel electrophoresis, respectively taking RhB-Ag with the concentration of 100 μ g/mL and SuD-Ag with the concentration of 100 μ g/mL and the supernatant in the step (3), adding the supernatant into different lanes, and finally evaluating the coupling efficiency by comparing the color shades of the lanes. The results are shown in fig. 4A, the protein gel color development of rhodamine antigen and sudan red antigen in the supernatant after coupling is much lighter than that before coupling, which indicates that the protein concentration of the supernatant after coupling is reduced, i.e. most of the antigen is coupled to the surface of the magnetic bead.
3. Preparing an HRP-labeled rhodamine B antibody and a GOx-labeled Sudan red antibody respectively:
(1) 5mg of HRP and 5mg of GOx were weighed out and dissolved in 500. mu.L of CH3COONa-CH, respectively 3 To the COOH (0.2mol/L, pH 5.6) buffer, 100. mu.L of 0.1mol/L NaIO was added 4 The solution was stirred at 4 ℃ for 30min in the absence of light.
(2) 500. mu.L of 2.5% ethylene glycol was added dropwise, and the reaction was carried out at room temperature for 30 min.
(3) mu.L of rhodamine B antibody (1mg/mL) was added dropwise to HRP, 100. mu.L of Sudan Red antibody (1mg/mL) was added dropwise to GOx, and the mixture was stirred at 4 ℃ for 30min and then NaHCO was used 3 -Na 2 CO 3 (0.05mol/L, pH 9.6) the buffer solution was dialyzed overnight.
(4) The following day the antibodies were removed and 100. mu.L of 5mg/mL NaBH was added separately 4 The solution was stirred at 4 ℃ for 2 h.
(5) Adding equivalent saturated ammonium sulfate solution, reacting at 4 deg.C for 30min, and standing for 1 hr.
(6) Centrifuging at 4000r/min at 4 ℃ for 20min, discarding the supernatant, and draining. The precipitate was dissolved in a small amount of PBS (0.01mol/L, pH 7.4), packed in a dialysis bag, and dialyzed with PBS (0.01mol/L, pH 7.4) overnight.
(7) Taking out the supernatant of the dialysate on the third day, collecting the supernatant of the dialysate, namely enzyme-labeled antibodies RhB-Ab-HRP and SuD-Ab-GOx, mixing uniformly, subpackaging, and storing at-20 ℃ for later use.
(8) And (5) ultraviolet scanning identification. And (3) performing ultraviolet (230-600 nm) scanning identification on the rhodamine B antibody, the HRP and the RhB-Ab-HRP respectively, wherein the HRP has a characteristic absorption peak at 400nm, and the highest absorption values of the substances before and after labeling are compared, so that characteristic peaks of the HRP and the HRP labeled antibody at 400nm (shown in a figure 4B) are found, and the antibody has no characteristic peak at the position, so that the reaction product is a compound of the HRP and the antibody, and the labeling is successful. Production of H from glucose using GOx enzyme catalysis 2 O 2 And further, mixing and incubating Sudan red antibody Ab, GOx enzyme and enzyme labeled antibody Ab-GOx with the same amount of TMB/HRP solution for 30 minutes according to the principle of changing the color of the TMB/HRP solution. The results (FIG. 4C) show that the antibody group did not discolor TMB, while both the GOx group and the GOx-Ab group turned TMB blue, indicating that GOx-Ab coupling was successful.
4. Assembly of immunosensor for simultaneously detecting rhodamine B and Sudan red pigment
Based on the surface-open microfluidic chip obtained in the step 1, the first reaction chamber is used for loading a sample to be detected, a mixed solution of magnetic bead antigens (RhB-Ag-Mb, SuD-Ag-Mb) and enzyme-labeled antibodies (RhB-Ab-HRP, SuD-Ab-GOx) to serve as a micro-reactionA zone; the second reaction chamber was loaded with PBS droplets and the third reaction chamber was loaded with the substrate Amplex Red/H 2 O 2 A droplet, a fourth reaction chamber loaded with a droplet of PBS and a fifth reaction chamber loaded with a droplet of glocose/Amplex Red/HRP substrate. After the immune recognition is finished, the magnetic beads combined with the enzyme-labeled antibody can be pulled into a second reaction chamber (containing PBS) for cleaning through the action of an additional magnet. After washing, the beads are pulled into a third chamber (containing the substrate Amplex Red/H) 2 O 2 A droplet of (a). Wherein, RhB-Ab-HRP catalytic substrate Amplex Red/H 2 O 2 Generating fluorescent substance Resorufin, wherein the signal intensity is in proportion to the content of RhB in the system; following washing of the immunomagnetic beads in the fourth reaction chamber (PBS-containing droplet), the beads were drawn into a fifth reaction chamber containing a Gluocose/Amplex Red/HRP substrate droplet, and SuD-Ab-GOx first catalyzes Glucose to generate H 2 O 2 And then further catalyzing Amplex Red/HRP to generate fluorescent substance Resorufin, wherein the signal intensity is in proportion to the SuD content in the system. And calculating the concentrations of rhodamine B and Sudan red in the sample to be detected through a linear relation curve of the standard concentration and the fluorescence intensity.
Example 2
As shown in fig. 2 and 3, the portable biosensor for detecting rhodamine B and sudan red comprises a biosensor shell 1, a power module 2, an incubation module 3, an immunoreaction module 4, a fluorescence excitation module 5 and a signal acquisition module 6, wherein the power module 2, the incubation module 3, the immunoreaction module 4 and the fluorescence excitation module 5 are arranged in the biosensor shell 1; the power supply module 2 is arranged at the bottom of the biosensor shell 1 and is respectively connected with the incubation module 3 and the fluorescence excitation module 5; the incubation module 3 is arranged below the immunoreaction module 4 and is formed by connecting an intelligent temperature controller 31 and a constant temperature heater 32; a temperature display screen 7 is arranged on one side of the biosensor shell 1, and the temperature display screen 7 is connected with an intelligent temperature controller 31; the immunoreaction module 4 consists of a chip tray 41 and an open-surface microfluidic chip 42 which is arranged on the chip tray 41 and is described in the embodiment 1; the fluorescence excitation module 5 is arranged on the inner side of the top of the biosensor shell 1 and consists of an LED array, an excitation filter and an emission filter, and the LED array and the top of the biosensor shell 1 form an included angle of 45 degrees; the signal acquisition module 6 consists of a signal acquisition hole 61, a mobile phone 62 and a fixing support 63, wherein the signal acquisition hole 61 is arranged at the top of the biosensor shell 1 and is matched with a camera of the mobile phone 62, and the fixing support 63 is arranged at the outer side of the top of the biosensor shell 1 and is used for fixing the mobile phone 62; the top of the biosensor housing 1 is further provided with a first switch 8 for controlling the incubation module 3 and a second switch 9 for controlling the fluorescence excitation module 5.
The biosensor housing 1 includes a biosensor front housing 11 and a biosensor rear housing 12. The power module 2 is a recyclable battery box, and comprises a battery box body 21 and a battery box base 22, and a 12V battery box can be adopted for supplying power to each power module of the whole biosensor. The constant temperature heater 32 is a 12V micro-heating plate for incubating the immunoreaction module 4. The incubation temperature is displayed in real time through the temperature display screen 7, so that the constant temperature heater 32 can be conveniently controlled. A surface-open microfluidic chip 42 with 16 channels is used as an immunoreaction site, and immunoreactions are carried out in parallel in each channel. Rhodamine B antigen-magnetic bead conjugate (RhB-Ag-Mb) and Sudan red antigen-magnetic bead conjugate (SuD-Ag-Mb) are used as sensor substrates, horseradish peroxidase labeled rhodamine B antibody (RhB-Ab-HRP) and glucose oxidase labeled Sudan red antibody (SuD-Ab-GOx) are used as signal probes, and the signals can flexibly pass through each reaction cell in a channel of the microfluidic chip 42 under the traction of an external magnet. The chip tray 41 is a drawer-type tray, and can be used for conveying the microfluidic chip 42, and the design of the handle of the tray also considers human mechanics factors, so that the comfort of a user can be improved. The fluorescence excitation module 5 comprises a first excitation light holder 51 and a second excitation light holder 52, and is used for fixing the fluorescence excitation module 5, the LED array is a green LED array with 60 × 15mm and 2W, and the excitation wavelength is 525 nm. The mobile phone 62 includes an image information acquisition module, a colorimetric module, and a concentration display module, and is used for acquisition and processing of detection signals and display of results, which require Android versions 4.3 and above. The first switch 8 is used to control the thermostatic heater 32 and the second switch 9 is used to control the LED array.
The biosensor that this embodiment provided includes power module 2, incubate module 3, immunoreaction module 4, fluorescence arouses module 5 and signal acquisition module 6, through power module 2 for incubating module 3 and fluorescence arouses module 5 and provide stable power, it can provide constant temperature heating environment for immunoreaction module 4 to incubate module 3, fluorescence arouses module 5 is used for the fluorescence excitation of substrate, signal acquisition module 6 guarantees to gather the uniformity of fluorescence signal position, reduce the lot difference, improve the accuracy that detects. The biosensor can be used for real-time, real-time and high-precision rapid detection of rhodamine B and Sudan red.
Example 4A method for detecting rhodamine B concentration and Sudan Red
1. The biosensor in the embodiment 3 is adopted to measure the fluorescence intensity generated by the biosensor under the concentration of standard series of rhodamine B and Sudan red, and linear relation curves of the concentration of the rhodamine B and the Sudan red and the fluorescence intensity are respectively drawn; the method specifically comprises the following steps:
(1) mixing rhodamine B solution and Sudan red solution with the RhB-Ab-HRP and SuD-Ab-GOx solutions respectively, extracting 30 mu L of mixed solution, injecting the mixed solution into a first liquid pool of the chip, adding 1 mu L of immunomagnetic beads RhB-Ag-Mb, and incubating for 20 minutes at 37 ℃.
(2) And (4) utilizing a magnet to pull the magnetic beads to the washing liquid pool, and washing the magnetic beads in a reciprocating manner.
(3) The beads were further pulled to a volume containing 40. mu.L of Amplex Red/H 2 O 2 And incubating again at 37 ℃ for 5 minutes.
(4) Turning off the constant temperature heater and turning on the LED light source; fluorescent signal acquisition by utilizing mobile phone to detect APP
(5) And (3) utilizing a magnet to pull the magnetic beads to the washing liquid pool 2, and washing the magnetic beads in a reciprocating way.
(6) The beads were further pulled to a substrate pool containing 30. mu.L of Glucose (4mM) and Amplex Red/HRP and incubated at 37 ℃ for 30 min.
(7) And opening the mobile phone detection app to acquire the image photo. The specific operation is as follows. Clicking 'New test' to enter a detection interface; clicking 'Take Picture' to acquire an image, clicking 'V' to convert the image into a gray value image; after the sample points are framed, click on "analysis" to calculate the gray value of each point.
Inputting the analyte concentration corresponding to each point, clicking 'Fitting', and generating a fitted standard curve; clicking 'Testing with the curve', setting the standard curve as a rule, and reappearing an image shooting interface; clicking 'Take Picture' to obtain a sample image, clicking 'V' to convert the image into a gray value image; after the sample points are framed, clicking analysis to calculate the gray value click Testing of each sample with unknown concentration, obtaining the actual concentration of the sample points according to the gray value and the established standard curve, and naming a file to store records.
2. And (3) detecting rhodamine B and Sudan red pigment in the pepper condiment by using the biosensor in the embodiment 3 according to the method in the step (1), and calculating the concentration of the rhodamine B and the Sudan red pigment in the sample by using the linear relation curve drawn in the step (1). The method can accurately, quickly and conveniently detect the concentrations of rhodamine B and the sudan red in the sample to be detected.
Claims (1)
1. A method for detecting rhodamine B and Sudan red pigment is characterized by comprising the following steps:
s1, measuring the fluorescence intensity generated by a biosensor under the concentration of standard series rhodamine B and the concentration of Sudan red by adopting the biosensor, and drawing a linear relation curve of the concentration and the fluorescence intensity;
s2, detecting rhodamine B and Sudan red pigment in the sample to be detected by using a biosensor, and calculating the concentrations of the rhodamine B and the Sudan red pigment in the sample by using the linear relation curve obtained in the step S1;
the biosensor comprises a biosensor shell (1), a power supply module (2), an incubation module (3), an immunoreaction module (4), a fluorescence excitation module (5) and a signal acquisition module (6), wherein the power supply module, the incubation module (3), the immunoreaction module (4) and the fluorescence excitation module are arranged in the biosensor shell (1); the power supply module (2) is arranged at the bottom of the biosensor shell (1) and is respectively connected with the incubation module (3) and the fluorescence excitation module (5); the incubation module (3) is arranged below the immunoreaction module (4); the fluorescence excitation module (5) is arranged on the inner side of the top of the biosensor shell (1); the immune reaction module (4) comprises a micro-fluidic chip tray (41) and a micro-fluidic chip (42) with an open surface;
the incubation module (3) is formed by connecting an intelligent temperature controller (31) and a constant temperature heater (32);
a temperature display screen (7) is arranged on one side of the biosensor shell (1), and the temperature display screen (7) is connected with an intelligent temperature controller (31);
the fluorescence excitation module (5) consists of an LED array, an excitation optical filter and an emission optical filter;
the signal acquisition module (6) consists of a signal acquisition hole (61), a smart phone (62) and a fixing support (63), the signal acquisition hole (61) is formed in the top of the biosensor shell (1) and matched with a camera of the smart phone (62), and the fixing support (63) is formed in the outer side of the top of the biosensor shell (1) and used for fixing the smart phone (62);
the top of the biosensor shell (1) is also provided with a first switch (8) for controlling the incubation module (3) and a second switch (9) for controlling the fluorescence excitation module (5);
the surface-open microfluidic chip (42) comprises a chip substrate and a plurality of parallel channels positioned on the chip substrate, wherein each parallel channel is provided with 5 reaction chambers and 1 tail chamber which are sequentially connected, and each chamber is provided with a concave microstructure and is mutually connected through a microfluidic flow channel; the first reaction chamber is used for loading a sample to be detected, a magnetic bead antigen and an enzyme-labeled antibody mixed solution, the second reaction chamber is loaded with PBS liquid drops, and the third reaction chamber is loaded with a substrate Amplex Red/H 2 O 2 A droplet, the fourth reaction chamber is loaded with a PBS droplet, and the fifth reaction chamber is loaded with a Gluocose/Amplex Red/HRP substrate droplet; the magnetic bead antigen is a rhodamine B antigen-magnetic bead conjugate and a Sudan red antigen-magnetic bead conjugate; the enzyme-labeled antibody is a horseradish peroxidase-labeled rhodamine B antibody and a glucose oxidase-labeled sudan red antibody;
16 parallel channels are provided;
the reaction chamber is 12.76mm long, 3.2mm wide and 3.7mm high; the tail chamber is 3.2mm long, 3.2mm wide and 3.7mm high.
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