CN117420297A - Method for realizing efficient enzyme-linked immunosorbent assay based on magnetic field driven micro-nano robot - Google Patents
Method for realizing efficient enzyme-linked immunosorbent assay based on magnetic field driven micro-nano robot Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
Abstract
The invention belongs to the technical field of instant detection, provides a method for realizing high-efficiency enzyme-linked immunosorbent assay based on a magnetic field driven micro-nano robot, and relates to a preparation and detection application method of MNRS-Ab1 of the micro-nano robot. The technology can solve the problem that the traditional ELISA detects in the same hole to cause mutual interference between different steps. The detection process can be spatially separated, the detection efficiency is improved, and the instant ELISA detection is developed in the future. Meanwhile, the method can also be applied to Polymerase Chain Reaction (PCR) to improve the detection efficiency.
Description
Technical Field
The invention belongs to the technical field of instant detection, and provides a method for realizing high-efficiency enzyme-linked immunosorbent assay based on a magnetic field driven micro-nano robot.
Background
Enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used labeled immunoassay techniques. It is based on an enzyme-labeled antibody capable of detecting an antigen immobilized on a 96-well or 384-well elisa plate, and the added substrate can produce a color change or light signal that correlates with the amount of antigen present in the original sample. This is a simple and rapid technique for detecting antibodies or antigens attached to a solid surface. ELISA is commercially valuable in laboratory research, disease biomarker diagnosis, and quality control in various industries as one of the most sensitive immunoassay methods. Wherein, the sandwich method ELISA requires the processes of antibody coating, antigen-antibody combination, plate washing for multiple times, ELISA signal detection and the like.
The main defects existing in the prior art are as follows:
1. the capture antibody of enzyme-linked immunosorbent assay commonly used in the market is mainly coated on the hole wall of a porous plate, the contact area of an object to be detected and the capture antibody is small, long time is required to reach the combination balance, and the detection efficiency is low.
2. The ELISA requires multiple steps of reaction, all the reactions are completed in the same hole at present, and in order to avoid the influence of residual reagents in different steps, multiple times of cleaning are needed, and the process is complicated.
3. At present, the ELISA detection needs to be completed by personnel with expert knowledge, and operation errors are easy to introduce.
Disclosure of Invention
In view of the problems existing in the prior art, the invention provides a method for realizing high-efficiency enzyme-linked immunosorbent assay (ELISA) based on a magnetic field driven micro-nano robot, which can be applied to the field of point of care testing (POCT) in the future. The technology can solve the problem that the traditional ELISA detects in the same hole to cause mutual interference between different steps. The detection process can be spatially separated, the detection efficiency is improved, and the instant ELISA detection is developed in the future. Meanwhile, the method can also be applied to Polymerase Chain Reaction (PCR) to improve the detection efficiency.
Specifically, the invention is realized by the following technical scheme:
firstly, the invention provides a preparation method of a movable enzyme-linked immunosorbent assay probe, which comprises the following steps:
(1) Fe is added to 3 O 4 @SiO 2 The nanorods were dispersed in ethylene glycol and APTE was added to react for several hours at room temperature, the product was collected by centrifugation and washed clean with solvent; dispersing the washed product in DMF, adding SAA and TEA to the above solution, stirring at room temperature for several hours, centrifuging, washing with solvent, and collecting silica-coated Fe with carboxyl groups 3 O 4 A nanorod;
(2) Coating silicon dioxide with carboxyl group on Fe 3 O 4 The nanorods were dispersed in MES, then EDC and NHS were added and mixed for a certain time, and then the reaction product was washed with PBS and dispersed in PBS; adding capture antibody into the above solution and reacting for several hours, then washing the reaction product with PBS and dispersing it in PBS, adding a certain amount of BSA into the mixture and mixing for a certain time to obtain Fe 3 O 4 @SiO 2 Wash Ab1 with PBS, i.e., MNRs-Ab1.
As a preferable technical scheme of the invention, fe 3 O 4 @SiO 2 The ratio of the nanorods to the APTE is Fe 3 O 4 @SiO 2 (4mg)：APTE(200μL)。
As a preferable technical scheme of the invention, fe 3 O 4 @SiO 2 The ratio of the nanorods to SAA and TEA is as follows: fe (Fe) 3 O 4 @SiO 2 (4 mg), SAA (50 mg) and TEA (52.1. Mu.L).
As a preferred embodiment of the present invention, the silica-coated Fe having carboxyl groups 3 O 4 The ratio of the nanorods to EDC to NHS is as follows: fe (Fe) 3 O 4 @SiO 2 (1 mg) nanorods bearing carboxyl groups, 1.5g EDC and 1.6g NHS.
As a preferred embodiment of the present invention, the washing solvent is EtOH.
As a preferable embodiment of the present invention, the reaction time and stirring time in the step (1) are 24 hours, respectively.
As a preferable technical scheme of the invention, the mixing time in the step (2) is respectively 1 hour.
As a preferred embodiment of the present invention, the BSA concentration by mass is 1% and the solvent is water.
As a preferred embodiment of the present invention, fe is obtained 3 O 4 @SiO 2 Ab1 was washed with PBS and stored in 1mL Tris (1% bovine serum albumin, mass concentration, solvent water, tris buffer salt Tris buffer salt) at 4℃for future use.
As a preferred embodiment of the present invention, ab1 and Ab2 are usedELISA kit, ancillary reagent kit 1.Catalog No.DY007 Lot P234196.
The invention further provides an automatic enzyme-linked immunosorbent assay method based on the magnetic field micro-nano robot, which comprises the following steps:
adding MNRS-Ab1, an analyte (PCT), a detection antibody Ab2 and streptavidin HRP prepared by the method into a reaction hole for reacting for a certain time, driving the MNRS-Ab 1-analyte-Ab 2-HRP with a sandwich structure into a washing hole filled with a washing buffer solution through a micro-channel, and rotating for a certain time to wash out conjugated MNRs;
MNR-Ab 1-analyte-Ab 2-HRP was then driven into the TMB-filled detection wells for a spin reaction for a period of time, and back into the wash wells, 50 μl of stop solution was added to stop the enzyme reaction, and the assay units were placed into the microplate reader for detection.
Wherein, MNR: micro-nano robot
MNRs-Ab1: micro-nano motor with surface modified antibody 1
ELISA: ELISA detection
And (2) PCR: polymerase chain reaction
APTE: 3-aminopropyl triethoxysilane
DMF: n, N-dimethylformamide
SAA: succinic anhydride
TEA: triethylamine
MES: 4-morpholinoethanesulfonic acid
EDC: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride
NHS: n-hydroxysuccinimide
PBS: phosphate buffer
BSA: acetamide compound
PCT: procalcitonin
HRP: horseradish peroxidase
TMB:3,3', 5' -tetramethylbenzidine
Ab2: antibody 2
Tris: trimethylolaminomethane
The excellent effects of the present invention over the prior art include:
(1) The technology can solve the problem that the traditional ELISA detects in the same hole to cause mutual interference between different steps.
(2) The detection process can be spatially separated, the detection efficiency is improved, and the instant ELISA detection is developed in the future. Meanwhile, the method can also be applied to Polymerase Chain Reaction (PCR) to improve the detection efficiency.
Drawings
FIG. 1 is a three-view of a detection unit of an ELISA detection unit;
FIG. 2 is a schematic diagram of the magnetic field driven micro-nano robot rotary motion enhancement motion characterization, wherein in the diagram, a is a screenshot of R6G distribution in a solution at different moments under the action of MNR rotation; b MNR rotation frequency is 4Hz, and different rotation times R6G are mixed with coefficient distribution. c under the condition of the same MNR rotation time, the mixing coefficient change distribution is carried out under different rotation frequencies; d MNR rotation (4 Hz) of the surrounding flow velocity distribution; e MNR distribution of surrounding streamlines as it rotates (4 Hz).
FIG. 3 is a schematic diagram of a magnetic field control system and a detection unit in the drawing, wherein the magnetic field drives a micro-nano robot to combine with an autonomous design detection unit groove to detect results; b, isolating the different functional grooves through micro-channels, and respectively carrying out pictures on the solutions in the different functional grooves before and after 1 hour; c MNR realizes shuttling between different functional grooves under the action of a magnetic field; optical density at 450nm after enzymatic reaction with (d) different rotation times (rotation frequency of 6 hz) and (e) different rotation frequencies (rotation time of 30 min). f optical densities at 450nm in detection wells of standard and test samples of PCT of different concentrations dispersed in PBS (blue dotted line is a standard sample linear fit, red dot is serum measurement).
Detailed Description
The invention is further described below with reference to the accompanying drawings and the detailed description, but the invention is not limited thereto.
Example 1
Preparation of a mobile enzyme-linked immunosorbent assay probe:
fe is added to 3 O 4 @SiO 2 (4 mg) nanorods were dispersed in ethylene glycol (20 mL), and APTE (200. Mu.L) was added to react at room temperature for 24 hours. The product was collected by centrifugation (9000 r/min,5 min) and washed 3 times with EtOH. The washed product (2 mg) was dispersed in DMF (10 mL). Then, SAA (50 mg) and TEA (52.1. Mu.L) were added to the above solution, and stirred at room temperature for 24 hours. The silica-coated Fe with carboxyl groups was collected by centrifugation (8000 r/min,5 min) 3 O 4 The nanorods were washed 3 times with EtOH.
Fe 3 O 4 @SiO 2 (1 mg) of the nanorods bearing carboxyl groups were dispersed in 1ml MES, and then 1.5g of EDC and 1.6g of NHS were added and mixed for 1h. The reaction product was then washed 3 times with PBS at 4℃and dispersed in 1ml PBS. To the above solution, 0.25mg of the capture antibody was added and reacted for 4 hours. The reaction product was then washed 3 times with PBS and dispersed in 1mL of PBS. Mu.l BSA (1%) was added to the mixture for 1 hour. Obtaining Fe 3 O 4 @SiO 2 Ab1 was washed with PBS and stored in 1mL Tris (1% bovine serum albumin) at 4 ℃ for future use.
Example 2
Automatic enzyme-linked immunosorbent assay detection flow is realized based on a magnetic field micro-nano robot:
mu.L of MNRS-Ab1 prepared in example 1, 30. Mu.L of analyte (PCT), 30. Mu.L of detection antibody Ab2 and 30. Mu.L of streptavidin HRP were added to the reaction wells and reacted for a certain period of time. Then, as shown in FIG. 1, a sandwich structure (MNR-Ab 1-analyte-Ab 2-HRP) was driven into a wash well filled with 100. Mu.L of wash buffer through a microchannel and spun for 10 minutes to wash out conjugated MNR. MNR-Ab 1-analyte-Ab 2-HRP was driven into a detection well filled with 100. Mu.L TMB for spin reaction (10 min) and back into the wash well. Add 50 μl of stop solution to stop the enzyme reaction, we put the assay unit into the microplate reader to detect OD values.
In the detection process, a detection groove is arrangedIn the magnetic field control system, ab1 and Ab2 are usedELISA kit, ancillary reagent kit 1.Catalog No.DY007 Lot P234196. The test results are shown in fig. 2 and 3, respectively.
To demonstrate the mixing properties of MNR, in the experiment, a solution (10. Mu.L, 10) of a fluorescent dye (rhodamine 6G (R6G)) was injected into a PEG solution (. About.30cst) (10. Mu.L, 0.2 mg/mL) containing dispersed MNR -4 M). The MNR is activated and rotated by application of an external magnetic field. Then, we record fluorescence images at different time intervals. Since MNRs cause local interference, the R6G dye will mix rapidly with PEG solution. In control experiments without any active mix of MNRs, the distribution of R6G dye (red region) remained almost unchanged and the interface between R6G and PEG remained clear. The mixing coefficient (MI) was introduced in the experiment, and when mixed more uniformly, MI would be closer to 0. Without active mixing, MI fluctuates between 0.37 and 0.4 (grey line in fig. 2 b). The MI was significantly reduced when active mixing was turned on and reduced to near 0 in about 2 minutes, experiments demonstrated that active rotation could significantly enhance molecular species migration to form a homogeneous solution. The rotation frequency of the MNR also plays an important role in the mixing effect. In the same rotation time of 120s, MI drops from 0.37+ -0.03 to 0.007+ -0.004 when the rotation frequency is increased from 0 to 4 Hz. However, if the rotation frequency is increased from 6Hz to 12Hz, MI is increased again to 0.32±0.04 (fig. 2 c) due to the step-out phenomenon.
The simulated velocity profile (FIG. 2 d) and streamlines (FIG. 2 e) show the fluid flow on X-Y when the MNR rotates at 4 Hz. The maximum observed velocity of the MNR surface near the MNR outermost tip (15.08 μm/s). Furthermore, the maximum local velocity appears to be linearly dependent on the rotation frequency of the MNR. The streamlines show the circulation pattern formed around the MNR, which increases the disturbance to the enhanced mixing. Furthermore, the MNR endpoint pressure was much higher (0.1 Pa) compared to the other zones. Such a significant pressure gradient may cause additional mass transfer within the fluid to further enhance the rotation-induced mixing.
A test cell containing three functional wells was designed and manufactured in the experiments. The reaction tank (a-1 in FIG. 3 a) was used to incubate MNR-Ab1s with analyte (PCT), detection antibody (Ab 2 s) and streptavidin HRP. The wash tank (a-2) and the detection tank (a-3 in FIG. 3 a) were pre-filled with wash buffer and 3,3', 5' -Tetramethylbenzidine (TMB) substrate, respectively. These functional channels are connected by micro-channels filled with paraffin oil (cyan) to form a water-oil interface, separating the different functional channels (fig. 3 b). Under the action of rotation, the detection probe is quickly combined with the object to be detected. After a period of time, under a gradient magnetic field, the conjugate enters different functional grooves. The optical density values at different times at the same rotation frequency and at different rotation speeds at the same time were tested in experiments, respectively (fig. 3d and 3 e). PCT values in simulated blood samples were also tested using the developed system, and PCT concentrations detected in the experiments were very consistent with the PCT amounts added to serum samples (fig. 3 f). This demonstrates that the developed nR-ELISA system provides sufficient resolution to distinguish healthy from inflamed subjects, indicating the potential of use of our system in clinical testing applications.
The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.
Claims (10)
1. A preparation method of a movable enzyme-linked immunosorbent assay probe is characterized by comprising the following steps of:
(1) Fe is added to 3 O 4 @SiO 2 The nanorods were dispersed in ethylene glycol and APTE was added to react for several hours at room temperature, the product was collected by centrifugation and washed clean with solvent; dispersing the washed product in DMF, adding SAA and TEA to the above solution, stirring at room temperature for several hours, centrifuging, washing with solvent, and collecting silica-coated Fe with carboxyl groups 3 O 4 A nanorod;
(2) Silica having carboxyl groupsCoated Fe 3 O 4 The nanorods were dispersed in MES, then EDC and NHS were added and mixed for a certain time, and then the reaction product was washed with PBS and dispersed in PBS; adding capture antibody into the above solution and reacting for several hours, then washing the reaction product with PBS and dispersing it in PBS, adding a certain amount of BSA into the mixture and mixing for a certain time to obtain Fe 3 O 4 @SiO 2 Wash Ab1 with PBS, i.e., MNRs-Ab1.
2. The method for preparing the mobile enzyme-linked immunosorbent assay probe according to claim 1, wherein the method comprises the following steps: fe (Fe) 3 O 4 @SiO 2 The ratio of the nanorods to the APTE is Fe 3 O 4 @SiO 2 (4mg)：APTE(200μL)。
3. The method for preparing the mobile enzyme-linked immunosorbent assay probe according to claim 1, wherein the method comprises the following steps: fe (Fe) 3 O 4 @SiO 2 The ratio of the nanorods to SAA and TEA is as follows: fe (Fe) 3 O 4 @SiO 2 (4 mg), SAA (50 mg) and TEA (52.1. Mu.L).
4. The method for preparing the mobile enzyme-linked immunosorbent assay probe according to claim 1, wherein the method comprises the following steps: silica coated Fe with carboxyl groups 3 O 4 The ratio of the nanorods to EDC to NHS is as follows: fe (Fe) 3 O 4 @SiO 2 (1 mg) nanorods bearing carboxyl groups, 1.5g EDC and 1.6g NHS.
5. The method for preparing the mobile enzyme-linked immunosorbent assay probe according to claim 1, wherein the method comprises the following steps: the washing solvent is EtOH; the reaction time and stirring time in the step (1) were 24 hours, respectively.
6. The method for preparing the mobile enzyme-linked immunosorbent assay probe according to claim 1, wherein the method comprises the following steps: in the step (2), the mixing time is 1 hour respectively.
7. The method for preparing the mobile enzyme-linked immunosorbent assay probe according to claim 1, wherein the method comprises the following steps: the mass concentration of BSA was 1% and the solvent was water.
8. The method for preparing the mobile enzyme-linked immunosorbent assay probe according to claim 1, wherein the method comprises the following steps: fe obtained 3 O 4 @SiO 2 Ab1 was washed with PBS and stored in 1mL Tris (1% bovine serum albumin, mass concentration, solvent water, tris buffer salt Tris buffer salt) at 4℃for future use.
9. The method for preparing the mobile enzyme-linked immunosorbent assay probe according to claim 1, wherein the method comprises the following steps: ab1 and Ab2 areELISA kit, ancillary reagent kit 1.
10. The method for realizing automatic enzyme-linked immunosorbent assay based on the magnetic field micro-nano robot comprises the following steps:
adding MNRs-Ab1, an analyte (PCT), a detection antibody Ab2 and streptavidin HRP prepared by the preparation method of any one of claims 1-9 into a reaction hole for reacting for a certain time, driving the MNRs-Ab 1-analyte-Ab 2-HRP with a sandwich structure into a washing hole filled with washing buffer through a micro-channel, and rotating for a certain time to wash out conjugated MNRs;
MNR-Ab 1-analyte-Ab 2-HRP was then driven into the TMB-filled detection wells for a spin reaction for a period of time, and back into the wash wells, 50 μl of stop solution was added to stop the enzyme reaction, and the assay units were placed into the microplate reader for detection.
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| CN202311155602.XA CN117420297A (en) | 2023-09-08 | 2023-09-08 | Method for realizing efficient enzyme-linked immunosorbent assay based on magnetic field driven micro-nano robot |
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| CN202311155602.XA CN117420297A (en) | 2023-09-08 | 2023-09-08 | Method for realizing efficient enzyme-linked immunosorbent assay based on magnetic field driven micro-nano robot |
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