CN113176310B - Automatic phage panning platform based on digital microfluidic technology - Google Patents
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Abstract
The invention discloses an automatic phage panning platform based on a digital microfluidic technology, which comprises a digital microfluidic platform, wherein the digital microfluidic platform consists of a microfluidic chip, an integrated circuit, a temperature control module and a magnetic separation module, the microfluidic chip and the integrated circuit are used for fluid manipulation, the temperature control module is used for providing proper temperature for bacteria and phage amplification, and the magnetic separation module is used for realizing integrated magnetic bead washing. The automated panning protocol can automatically complete three rounds of biopanning within 16 hours, greatly freeing up labor as compared to the cumbersome manual operation of the traditional method for nearly one week. In addition, the reagent dosage of the scheme is only 3% of that of the conventional method, and the reagent cost is greatly reduced. In summary, the present invention will further facilitate affinity polypeptide screening and its use in diagnostic, imaging, delivery, and therapeutic fields.
Description
Technical Field
The invention relates to an automatic panning platform and panning implementation scheme based on phage display, and belongs to the technical field of digital microfluidics.
Background
Molecular recognition is the molecular basis for physiological and pathological activities, with most complex physiological and pathological activities being dependent on molecular recognition. Phage display technology is one of the effective methods of studying molecular recognition. Phage display technology involves inserting exogenous genes into the phage genome by genetic engineering to fusion express exogenous peptides or antibodies on the phage surface (angelw.chem.int.ed.2019, 58, 14428-14437). The display peptide or antibody on the surface of phage can be directly subjected to affinity screening aiming at different targets, and the sequence of the display peptide or antibody can be easily obtained through phage gene sequencing. To date, a large number of affinity ligands have been identified by phage display technology, with a broad range of targets including small molecules, DNA, proteins, cells, bacteria, tissue sections, even nanomaterials, etc., and are widely used in the fields of biosensing, vaccine development, in vitro and in vivo imaging, targeted delivery and therapy (biotechnol.j.2016, 11, 732-745).
In order to obtain affinity ligands, phage display technology typically enriches the corresponding phage round by affinity screening methods (also known as biopanning), which involves a cyclic process of multiple rounds of reverse screening, forward screening, washing, elution and amplification. However, multiple rounds of biopanning have problems of high reproducibility, long time consumption, labor intensive operations, large reagent consumption, etc., limiting the development process of novel ligands.
Therefore, there is a need to develop a technique suitable for ligand development.
Disclosure of Invention
The invention provides an automatic phage panning platform which is based on a digital microfluidic technology and has the advantages of automation, integration, labor relief, less reagent consumption and the like aiming at the problems of high repeatability, long time consumption, intensive operation, large reagent consumption and the like in the affinity screening of the traditional phage display technology.
One of the technical schemes of the invention is as follows:
an automatic phage panning platform based on digital microfluidic technology is characterized in that: the device comprises a digital micro-fluidic chip, wherein the digital micro-fluidic chip comprises a driving electrode array, transition electrodes, a washing liquid storage tank, a culture medium storage tank, a washing liquid storage tank, a positive sieve magnetic bead storage tank, a neutralization liquid storage tank and a reverse sieve magnetic bead storage tank, and auxiliary electrodes for controlling the on and off of the driving electrode array, each storage tank and each transition electrode; the transition electrode comprises a first transition electrode and a second transition electrode, each liquid storage tank is connected with the driving electrode array through the first transition electrode and at least one second transition electrode respectively, and liquid drops are generated from each liquid storage tank to the driving electrode array through the power-off and the power-on of the first transition electrode, the second transition electrode and the driving electrode;
the magnetic beads are arranged in the driving electrode array, and the driving electrodes are connected with one another in a parallel manner;
and comprises an electric heating plate for heating the microfluidic chip.
Preferably, the digital micro-fluidic chip comprises an upper plate and a lower plate, and is fixed by a clamp at intervals of four layers of double faced adhesive tapes and connected with a control system for use. The upper plate was a Teflon AF1600 hydrophobized conductive Indium Tin Oxide (ITO) glass. The lower electrode plate takes quartz glass as a substrate, a chromium conductive layer of about 300nm is sputtered on the lower electrode plate, a SU-8 1040 medium layer of about 11.7 mu m is photoetched, and a Teflon AF1600 hydrophobic layer of about 200nm is finally coated.
Preferably, the integrated circuit is a digital microfluidic circuit control system. The software operation is transmitted to the circuit board through the digital-analog conversion unit, and then after being processed by the sine wave generator, the power amplifier and the high-voltage alternating current switch matrix, signals are transmitted to the chip contact electrode through the PCB spring thimble.
Preferably, the magnet is a cone-shaped magnet.
Preferably, the drive electrode array comprises 27 drive electrodes, each area 4mm x 4mm; the reservoirs were contact electrodes, each contact electrode having an area of 6mm x 8mm. Each first transition electrode area is 3mm×2 mm), each transition electrode area is 2mm×2mm.
Preferably, the drive electrode spacing is 40 μm.
Preferably, the electrode leads in the driving electrode array are in a serpentine shape, the wiring is designed in a line-in-line mode, and the width of the electrode leads is 60 mu m.
Preferably, the culture medium liquid storage tank, the washing liquid storage tank and the pickling liquid storage tank are respectively connected with the driving electrode array through a first transition electrode and a second transition electrode so as to respectively generate large liquid drops; the positive sieve magnetic bead liquid storage tank, the neutralization liquid storage tank and the reverse sieve magnetic bead liquid storage tank are respectively connected with the driving electrode array through the first transition electrode and the two second transition electrodes so as to respectively generate small liquid drops. Preferably, the large droplet is 8. Mu.L and the small droplet is 2. Mu.L.
Preferably, the incubation of 15-17 μl droplets is handled by uninterrupted droplet looping movements with 2×2 four drive electrodes as one droplet incubation unit.
Preferably, the chip can be operated simultaneously with three of the above droplet incubation units.
Preferably, any three driving electrodes and magnets in the driving electrode array are connected in series to form a magnetic separation module, and the selective power-on/power-off strategy of each driving electrode is utilized to realize the non-uniform splitting of liquid drops, so that most magnetic beads are retained in the small liquid drops, and the magnetic separation with high magnetic bead retention rate and high washing efficiency is realized.
Preferably, the magnetic separation process of the present invention is as follows: the cone magnet is placed above the chip so that the edge of the magnet is tangential to the edge of the electrode and the edge of the droplet. Dragging the bead droplet back and forth causes the bead everywhere in the droplet to be attracted to the edge of the droplet at the interface of E2 and E3. Then E2 and E1 are sequentially turned on to ensure that the beads are still at the edge of the droplet. E3 is opened, E2 is rapidly closed, two inhomogeneous droplets are obtained after the droplet is split, a small droplet containing magnetic beads is left on E3, and a large supernatant droplet is left on E1, so that efficient magnetic separation is realized. Wherein the E2 driving electrode is a driving electrode positioned in the middle.
Preferably, magnetic beads are used to modify the target proteins and the counter-screen proteins, thereby realizing an integrated screening procedure.
Preferably, 0.2M glycine-HCl, 1mg/mL BSA, pH 2.2 was used as the pickling solution, and the four-electrode cycle was incubated for 10min as the elution condition.
Preferably, 0.1M tris-hydrochloric acid, pH 9.0 is used as the neutralization solution, and the elution and neutralization solution volumes are 5:1 or 4:1, so that the elution system can be more completely neutralized.
Preferably, the screening parameters are flexibly adjustable. With the increase of the number of selection rounds, the input amount of the positive screening protein and the incubation time are gradually reduced, and the washing times, the input amount of the negative screening protein and the incubation time are gradually increased.
The invention also provides an automated biopanning application as described above, for use in screening for affinity polypeptides or affinity antibodies. The method comprises the following steps:
step A: introducing the diluted phage library into a driving electrode array of a microfluidic chip;
and (B) step (B): respectively loading each reagent into each liquid storage tank of the digital microfluidic chip;
step C: the on-off control of the electrodes is carried out according to a certain sequence, and the counter-sieve magnetic bead liquid storage tank generates counter-sieve magnetic bead liquid drops; incubating the phage library with reverse-screening magnetic beads, and after incubation, removing the magnetic beads by magnetic separation, and retaining unbound phage droplets;
step D: the positive sieve magnetic bead liquid storage tank generates positive sieve magnetic bead liquid drops. The remaining phage droplets were incubated with positive screen magnetic beads. After incubation, the magnetic beads were retained by magnetic separation.
Step E: the wash liquid reservoir generates wash liquid droplets. After thoroughly mixing with the magnetic beads, the magnetic separation retains the magnetic beads and removes the cleaning liquid. This is a wash.
Step F: and E, washing for multiple times according to the washing method in the step E, and finally, reserving the magnetic beads for the last time and removing washing liquid.
Step G: the pickle liquor liquid storage tank generates pickle liquor liquid drops. The beads were resuspended in the pickling solution and incubated for 10min. Subsequently, the beads were rapidly magnetically separated and removed, and the supernatant was retained.
Step H: the neutralizing liquid reservoir generates neutralizing liquid droplets. The supernatant was rapidly mixed with the neutralisation buffer.
Step I: the medium reservoir produces droplets of medium. The droplets were mixed with the medium and incubated for 5 hours.
And (3) repeating the step B-the step I to finish the second round of screening, and repeating the step B-the step H to finish the third round of screening.
Preferably, as the number of selection rounds increases, the positive incubation time gradually decreases and the number of washes gradually decreases; the negative incubation time gradually increased.
Compared with the prior art, the invention has the advantages that:
(1) By means of digital microfluidic technology, the invention can realize more accurate space-time control of liquid, and the automatic and integrated flow replaces the tedious manual operation of the traditional method, liberates labor force, and can be expanded to multi-target parallel screening.
(2) Depending on digital microfluidic technology, the invention can automatically complete 3 rounds of biopanning within 16 hours, whereas the traditional method requires nearly one week of manual operation.
(3) Because digital microfluidics can accurately manipulate small volumes of liquid, the reagent consumption of the invention is only 3% of that of the traditional method, and the cost is saved.
(4) The magnetic beads of the integrated magnetic separation component have high retention rate, so that most of binding sequences are retained, and the success rate of automatic screening is ensured.
Drawings
FIG. 1 is a schematic diagram showing a cross-sectional structure of a digital microfluidic chip according to the present invention
Fig. 2 is a top view of the overall structure of the chip of the present invention.
Fig. 3 is an enlarged view of an electrode edge lead of the chip of the present invention (corresponding to a in fig. 2).
Fig. 4 is a partial view of a droplet generation unit of the chip of the present invention.
FIG. 5 is a diagram of droplet splitting in operation of the magnetic separation module of the present invention.
Fig. 6 is a schematic operation diagram of the temperature control module according to the present invention.
FIG. 7 shows the washing efficiency of the magnetic separation device of the present invention.
FIG. 8 shows the magnetic bead retention of the magnetic separation device of the present invention.
FIG. 9 is a diagram of the screening results of an embodiment of the present invention.
Reference numerals
1 glass substrate 2 ITO 3 hydrophobic layer 4 four layers double faced adhesive tape 5 hydrophobic layer 6 medium layer 7 electrode layer 8 glass substrate 9 auxiliary electrode 10 culture medium reservoir 11 washing liquid reservoir 12 pickling liquid reservoir 13 positive screen magnetic bead reservoir 14 neutralization liquid reservoir 15 reverse screen magnetic bead reservoir 16 reservoir 17 first transition electrode 18 second transition electrode 19 drive electrode 20 power-on electrode 21 conical magnet 22 magnetic bead 23 upper electrode plate 24 lower electrode plate 25 electric heating sheet
Detailed Description
The invention will be further described in detail below with reference to the accompanying drawings using an automated polypeptide panning platform as an example.
The automatic panning platform consists of four parts, including a digital micro-fluidic chip, an integrated circuit, a magnetic separation module and a temperature control module. Referring to fig. 1 and 2, the digital microfluidic chip includes, in order from top to bottom, a glass substrate 1, an ITO (indium tin oxide) layer 2, a hydrophobic layer 3, a spacer layer (four-layer double sided tape) 4, a hydrophobic layer 5, a dielectric layer 6, an electrode layer 7, and a glass substrate 8.
Wherein, referring to fig. 2, the electrode layer 7 comprises a driving electrode array composed of 27 driving electrodes 19 positioned in the middle, and the 27 driving electrodes 19 are arranged in three rows, 9 in each row. Above the array, a washing liquid reservoir 11, a culture medium reservoir 10 and a pickling liquid reservoir 12 are provided in this order from left to right. Under the array, a positive sieve magnetic bead liquid storage tank 13, a neutralization liquid storage tank 14 and a reverse sieve magnetic bead liquid storage tank 15 are arranged at intervals from left to right. In this embodiment, each reservoir (10-15) is actually an electrode. By moving the upper polar plate, part of the contact electrode is exposed, liquid is directly dripped on the contact electrode, and the liquid can stay on the electrode to serve as a liquid storage tank due to the change of the contact angle after the liquid is electrified.
Referring also to fig. 4 i, each upper row of contact electrodes 16 (representing the culture medium reservoir 10, the washing liquid reservoir 11, and the washing liquid reservoir 12) is provided with a first transition electrode 17 downward, the transition electrode 17 is connected downward to the driving electrode array through a second transition electrode 18, and the connection positions of the washing liquid reservoir 11, the culture medium reservoir 10, and the pickling liquid reservoir 12 are sequentially the leftmost, middle, and rightmost driving electrodes 19 of the upper row of the array.
Referring also to fig. 4 ii, each lower row of contact electrodes 16 (representing the positive sieve bead reservoir 13, the neutral liquid reservoir 14, and the negative sieve bead reservoir 15) is provided with a first transition electrode 17 upward, and the first transition electrode 17 is connected to the drive electrode array upward through two second transition electrodes 18 connected in series up and down. The positions of connection of the positive sieve magnetic bead liquid storage tank 13, the neutral liquid storage tank 14 and the reverse sieve magnetic bead liquid storage tank 15 are sequentially provided with a leftmost driving electrode 19, a middle position and a rightmost driving electrode 19 of the lower row of the array.
The drive electrodes 19 are provided with 27, nine in each row, three in rows, sufficient to perform complex droplet manipulations of forward screening, reverse screening, washing, elution, and incubation.
The auxiliary electrode array is arranged above the washing liquid storage tank 11, the culture medium storage tank 10 and the pickling liquid storage tank 12, the auxiliary electrode array comprises 48 auxiliary electrodes 9, the auxiliary electrodes are divided into 3 rows, 16 auxiliary electrodes are arranged in each row, each storage tank (10 to 15), each first transition electrode 17, each second transition electrode 18 and each driving electrode 19 are respectively controlled by an auxiliary electrode through a conductive circuit.
In performing droplet processing, each reservoir (10 to 15) and the transition electrode (first transition electrode 17 and second transition electrode 18) are used only as droplet generation units, and the remaining droplet manipulations, including droplet mixing, magnetic separation, and the like of the forward screen, reverse screen, washing and elution, are performed on 27 drive electrodes 19 of the drive electrode array.
Referring to fig. 3, the electrode is routed in a serpentine pattern with a serpentine lead L1 and a wire-to-wire L2. All electrode edges adopt serpentine leads L1, which can increase the smoothness of droplet movement. The electrode surrounded by the electrode adopts a line center line L2, so that the electrode which is not exposed can be connected with a lead.
Referring to fig. 4, each driving electrode 19 has an area of 4mm×4mm, each first transition electrode has an area of 3mm×2mm, each second transition electrode 18 has an area of 2mm×2mm, and each reservoir 16 has an area of 6mm×8mm. With the selective power on/off strategy, two droplets of 2 μl/unit and 8 μl/unit can be generated by one set of droplet generation units. Taking fig. 4 ii as an example, the power on/off strategy for generating 2 μl droplets is divided into two steps: first, the electrodes 16, 17 and 18 below are energized, and the 18 above is deenergized, so that the droplets are located at the electrodes 16, 17 and 18 below; second, the holding electrode 16 and the lower 18 are energized, the electrode 17 is de-energized, and the upper 18 and electrode 19 are rapidly energized, causing the droplet to break at the electrode 17, leaving a small droplet on the electrode 18, approximately 2 μl in volume. The power on/off strategy for generating 8 μl droplets is divided into two steps: first, the electrode 17, the two 18 and 19 are energized, so that the droplet is located at the electrode 17, the two 18 and 19; second, the hold 19 is energized, rapidly energizing electrode 16 and de-energizing electrode 17 and both 18, causing the droplet to break at electrodes 17, 18, leaving a large droplet on electrode 19, with a volume of about 8 μl.
FIG. 4I is used primarily to generate 8. Mu.L droplets. First, electrodes 16, 17, 18 and 19 are energized and second, electrodes 16 and 19 are kept energized and electrodes 17 and 18 are de-energized so that the droplets break at electrodes 17, 18, leaving large droplets on electrode 19 in a volume of about 8 μl.
Referring to fig. 5, the magnetic separation unit includes three electrodes E1, E2 and E3 connected in sequence, and the electrodes E1, E2 and E3 may be any three driving electrodes 19 connected in the driving electrode array. The workflow is as follows: a conical magnet 21 is placed over the chip with drops on E1 and E2, and magnetic beads 22 are mixed in the drops so that the edges of the magnet are tangential to the edges of the adjacent electrodes E2-E3 and the edges of the drops. Dragging the bead droplet back and forth causes the bead everywhere in the droplet to be attracted to the edge of the droplet. Then E2 and E1 are sequentially turned on to ensure that the beads are still at the edge of the droplet. E3 is turned on and E2 is turned off rapidly, two non-uniform droplets are obtained after droplet splitting, leaving a small droplet containing magnetic beads on E3 and a large supernatant droplet on E1.
Referring to fig. 6, the temperature control module includes an upper plate 23, a lower plate 24, and an electric heating plate 25 located below the lower plate. Wherein the upper plate 23 comprises a glass substrate 1, an ITO (indium tin oxide) layer 2 and a hydrophobic layer 3, and the lower plate 24 comprises a hydrophobic layer 5, a dielectric layer 6, an electrode layer 7 and a glass substrate 8. The upper polar plate and the lower polar plate are fixed by a clamp and are connected with a control system for use by adopting four layers of double faced adhesive tape 4 at intervals. The workflow of the temperature control module is as follows: the electric heating plate 25 with temperature control is closely attached to the lower part of the lower polar plate 24 of the chip, the thermocouple is used for temperature measurement and calibration, and the required temperature is regulated by the temperature controller.
As a preferred embodiment of the invention, the specific procedures of the invention for targeting an automated ephrin A receptor 2 (EphA 2) polypeptide panning are:
1) The positive and negative magnetic beads adopt toluenesulfonyl magnetic beads modified by EphA2 and HSA respectively. As wash solutions, 0.1% w/vTween-20 and 0.5% w/v Tween 20 in Tris buffer were used.
Phage library 10. Mu.L phD12 phage library ten-fold diluted with wash was used in a total volume of 100. Mu.L.
2) The diluted phage library is introduced into the drive electrode array of the chip, and can be added through any liquid storage tank. All reagents were loaded into the corresponding reservoirs. Referring to FIGS. 2 and 4 II, droplets of the medium, droplets of the washing liquid and droplets of the pickling liquid each of 8. Mu.L can be produced by the medium reservoir 10, the washing liquid reservoir 11 and the pickling liquid reservoir 12, respectively. Referring to fig. 2 and 4 i, the droplets of the positive sieve magnetic beads, the droplets of the neutralization solution, and the droplets of the reverse sieve magnetic beads, each 2 μl, can be generated by the positive sieve magnetic bead reservoir 13, the neutralization solution reservoir 14, and the reverse sieve magnetic bead reservoir 15, respectively.
3) The counter-sieve magnetic bead reservoir 15 generates 2 μl of counter-sieve magnetic bead droplets. And mixed with phage libraries. Incubating the phage library with the counter-screened magnetic beads for 20min, removing the magnetic beads by magnetic separation, discarding the magnetic beads, and retaining unbound phage droplets.
4) The positive sieve magnetic bead reservoir 13 generates 2 μl of positive sieve magnetic bead droplets. The remaining phage droplets were incubated with 2. Mu.L of positive sieve beads for 60min. After incubation, the magnetic separation retains the magnetic beads, which remain at the incubation of the primary drive electrode 19. The magnetic separation is shown in FIG. 5.
5) The washing liquid reservoir 11 generates 16. Mu.L of washing liquid droplets, which are thoroughly mixed with the magnetic beads on the drive electrode 19, and then the magnetic beads are magnetically separated and retained, thereby removing the washing liquid. This is a wash.
6) Washing for 6-8 times, retaining magnetic beads for the last time, and removing washing liquid.
7) The pickle liquor reservoir 12 produces 8 μl pickle liquor droplets. The beads were resuspended in the pickling solution and incubated for 10min. Subsequently, the beads were rapidly magnetically separated and removed, and the supernatant was retained.
8) The neutralization liquid reservoir 14 generates 2 μl of neutralization liquid droplets. The supernatant was rapidly mixed with 2. Mu.L of neutralization buffer.
9) The medium reservoir 10 generates 20. Mu.L of medium droplets. The droplets were mixed with the medium and incubated for 5 hours.
10 Repeating steps 2) -8) to complete the second round of screening, and repeating steps 2) -7) to complete the third round of screening. As the number of selection rounds was increased, the positive incubation time was reduced from 60 minutes to 30 minutes and the wash time was gradually increased from 6 to 8. The negative incubation time was gradually increased from 20 minutes to 30 minutes.
As shown in figures 7-8, after three times of washing, 99% of nonspecific adsorption can be removed by adopting the magnetic separation mode of the invention, which is equivalent to the efficiency of the traditional washing mode outside the chip. After eight washes, the retention rate of the magnetic beads is about 91.8%, which is equivalent to that of the magnetic beads in the traditional off-chip magnetic separation mode (95.2%).
As shown in FIG. 9, an enriched library of EphA2 was obtained using the automated phage panning platform of the present invention, any 30 of which were characterized, all of which exhibited a higher affinity for EphA2, indicating that the platform successfully enriched for EphA2 binding sequences.
The applicant states that the detailed features and detailed methods of the present invention are described by way of the above examples, but the present invention is not limited to the detailed features and detailed methods described above, i.e., it is not meant that the present invention must rely on the detailed features and detailed methods to practice the present invention. It will be apparent to those skilled in the art that any modifications of the invention, equivalent substitutions of selected components of the invention, selection of specific modes, etc., are within the scope of the invention and the disclosure.
Claims (9)
1. An automatic phage panning platform based on digital microfluidic technology is characterized in that: the device comprises a digital micro-fluidic chip, wherein the digital micro-fluidic chip comprises a driving electrode array, transition electrodes, a washing liquid storage tank, a culture medium storage tank, a washing liquid storage tank, a positive sieve magnetic bead storage tank, a neutralization liquid storage tank and a reverse sieve magnetic bead storage tank, and auxiliary electrodes for controlling the on and off of the driving electrode array, each storage tank and each transition electrode; the transition electrode comprises a first transition electrode and a second transition electrode, each liquid storage tank is connected with the driving electrode array through the first transition electrode and at least one second transition electrode respectively, and liquid drops are generated from each liquid storage tank to the driving electrode array through the power-off and the power-on of the first transition electrode, the second transition electrode and the driving electrode;
the magnetic beads are arranged in the driving electrode array, and the driving electrodes are connected with one another in a parallel manner;
comprising an electrical heating plate for heating the microfluidic chip;
the electrode leads in the driving electrode array are in a serpentine shape, and the wiring is designed in a line-in-line mode.
2. The automated phage panning platform based on digital microfluidic technology of claim 1, wherein the digital microfluidic chip comprises an upper plate and a lower plate, the lower plate comprises a substrate, an electrode layer, a dielectric layer and a hydrophobic layer from bottom to top, and the upper plate comprises a substrate and a hydrophobic layer from top to bottom.
3. The automated phage panning platform based on digital microfluidic technology of claim 1, wherein: the culture medium liquid storage tank, the washing liquid storage tank and the pickling liquid storage tank are respectively connected with the driving electrode array through a first transition electrode and a second transition electrode so as to respectively generate large liquid drops; the positive sieve magnetic bead liquid storage tank, the neutralization liquid storage tank and the reverse sieve magnetic bead liquid storage tank are respectively connected with the driving electrode array through the first transition electrode and the two second transition electrodes so as to respectively generate small liquid drops.
4. The automated phage panning platform based on digital microfluidic technology of claim 1, wherein: any three driving electrodes and magnets in the driving electrode array are connected in series to form a magnetic separation module, and the selective power-on/power-off strategy of each driving electrode is utilized to realize the non-uniform splitting of liquid drops, so that most magnetic beads are retained in small liquid drops, and the magnetic separation with high magnetic bead retention rate and high washing efficiency is realized.
5. The automated phage panning platform based on digital microfluidic technology according to claim 4 wherein: the position of the magnet is tangential to the edge of the electrode and the edge of the liquid drop; by dragging the magnetic liquid drop back and forth, the magnetic beads at all positions in the liquid drop are attracted to the edge of the liquid drop close to the junction of the electrodes E2 and E3; the E2, E1, E3 drive electrodes are then sequentially turned on, with the E2 drive electrode being the centrally located drive electrode, and the E2 is turned off rapidly, causing the droplet to split unevenly, leaving a small droplet containing magnetic beads on E3 and a large supernatant droplet on E1.
6. Use of an automated phage panning platform based on digital microfluidics according to any one of claims 1 to 5, wherein: for phage display library-based panning of polypeptides.
7. Use of an automated phage panning platform based on digital microfluidics according to any one of claims 1 to 5, wherein: antibody panning for phage display library-based.
8. A fully automated phage panning method using the digital microfluidic technology-based automated phage panning platform of any one of claims 1-5, comprising the steps of:
step A: introducing the diluted phage library into a driving electrode array of a microfluidic chip;
and (B) step (B): respectively loading each reagent into each liquid storage tank of the digital microfluidic chip;
step C: the on-off control of the electrodes is carried out according to a certain sequence, and the counter-sieve magnetic bead liquid storage tank generates counter-sieve magnetic bead liquid drops; incubating the phage library with reverse-screening magnetic beads, and after incubation, removing the magnetic beads by magnetic separation, and retaining unbound phage droplets;
step D: the positive sieve magnetic bead liquid storage tank generates positive sieve magnetic bead liquid drops; incubating the remaining phage droplets with positive screen magnetic beads; after incubation, magnetic separation retains the magnetic beads;
step E: the washing liquid storage tank generates washing liquid drops; after thoroughly mixing with the magnetic beads, magnetically separating the retained magnetic beads and removing the cleaning liquid; this is a wash;
step F: e, washing for multiple times according to the washing method in the step E, reserving magnetic beads for the last time, and removing washing liquid;
step G: the pickling solution liquid storage tank generates pickling solution liquid drops; re-suspending the magnetic beads in an acid washing solution, and incubating; then, rapidly magnetically separating and removing the magnetic beads, and retaining the supernatant;
step H: the neutralization liquid storage tank generates neutralization liquid drops; rapidly mixing the supernatant with a neutralization buffer;
step I: the culture medium liquid storage tank generates culture medium liquid drops; mixing the liquid drops with a culture medium, and culturing;
and C, repeating the step C-the step I to finish the second round of screening, and repeating the step C-the step H to finish the third round of screening.
9. A fully automated phage panning method as claimed in claim 8, wherein: with the increase of the number of selection rounds, the positive incubation time is gradually reduced, and the washing times are gradually increased; the negative incubation time gradually increased.
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| TWI542879B (en) * | 2015-03-16 | 2016-07-21 | 國立交通大學 | Magnetic bead-based digital microfluidic immunoanalysis device and method thereof |
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| EP1333096A1 (en) * | 2000-09-01 | 2003-08-06 | Yale University | Method to identify gene function using phage display screening |
| CN101059515A (en) * | 2007-05-14 | 2007-10-24 | 西安交通大学 | Use of phage chip as protein chip or gene chip |
| CN105063758A (en) * | 2015-08-17 | 2015-11-18 | 上海交通大学 | Preparation method of high-flux specific antibody library |
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