CN112852739A

CN112852739A - Screening method of drug for accurate treatment of tumor

Info

Publication number: CN112852739A
Application number: CN202110074407.9A
Authority: CN
Inventors: 贾艳伟; 翟蛟; 李浩然; 麦沛然; 马许愿
Original assignee: University of Macau
Current assignee: University of Macau
Priority date: 2021-01-20
Filing date: 2021-01-20
Publication date: 2021-05-28

Abstract

The invention discloses a screening method of a drug for accurate tumor treatment, and relates to the technical field of drug screening. The screening method comprises the following steps of in vitro screening: respectively culturing the primary tumor cells under various different culture conditions; the primary tumor cells are derived from tumor tissues of individuals suffering from tumors; a variety of different culture conditions include: at least one first culture condition in the presence of a drug to be screened and at least one second culture condition in the absence of a drug to be screened; comparing the survival of the primary tumor cells in a plurality of different culture conditions, and selecting the drug to be screened used in the first culture condition, which survives the first culture condition worse than the second culture condition, as the drug for the precise treatment of the tumor. In the application, the primary tumor cells are directly used for screening, the primary tumor cells do not need to be cultured in vitro, and the condition that the deviation exists in the drug screening result due to accidental mutation of the neutron cells in the culture process is effectively avoided.

Description

Screening method of drug for accurate treatment of tumor

Technical Field

The invention relates to the technical field of drug screening, in particular to a screening method of a drug for accurate tumor treatment.

Background

Precise medicine in oncology requires tailoring treatment strategies to individual cancer patients. To date, most targeted drugs are based on genetic information, and some drugs may be prescribed to patients with certain genetic mutations for optimal efficacy; while patients with other gene mutations are not given because of the large side effects. However, clinical data show that more and more genes are involved in tumor response to certain drugs, complicating prognosis based on genetic accurate medicine. Alternatively, direct drug screening of a biopsy sample from a patient may provide direct information on drug sensitivity. However, biopsy samples contain only a limited number of cells, which is troublesome for traditional 96-well plate based drug screening. Although sufficient tumor cell numbers can be obtained by taking multiple biopsy samples, the risk of cancer spreading is also increased. Sufficient cell numbers can be obtained by culturing primary tumor cells in vitro prior to drug screening. However, this may lead to accidental mutation of the daughter cells and mislead implications for drug screening.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a screening method of a medicament for accurately treating tumors.

The invention is realized by the following steps:

in a first aspect, the present invention provides a method for screening a drug for precise treatment of tumors, comprising the steps of in vitro screening:

the in vitro screening step comprises: respectively culturing the primary tumor cells under various different culture conditions; the primary tumor cells are derived from tumor tissue of an individual suffering from a tumor;

a variety of different culture conditions include: at least one first culture condition in the presence of a drug to be screened and at least one second culture condition in the absence of a drug to be screened;

comparing the survival of the primary tumor cells under the first culture condition and the second culture condition, and selecting the drug to be screened used in the first culture condition, in which the survival of the primary tumor cells under the first culture condition is worse than the survival of the primary tumor cells under the second culture condition, as the drug for tumor precision treatment.

In a second aspect, the present application provides an application of a drug selected by the above method for screening a drug for precise treatment of tumors in the preparation of a drug for treating tumors in precise medical treatment.

In a third aspect, the present application provides a digital microfluidic chip for implementing the above tumor-accurate screening method for individualized drugs, which includes a bottom plate, wherein an electrode array group and an output voltage welding spot are formed on the bottom plate; the electrode array group comprises at least two sample injection electrode groups used for respectively injecting samples of primary tumor cells and drugs in a drug group to be screened, a mixing electrode used for mixing the injected primary tumor cells and the drugs in the drug group to be screened to obtain drug-loaded cells, a moving electrode group used for moving the drug-loaded cells and a culture electrode group used for moving the drug-loaded cells to a culture area for culture; wherein the mixing electrode is simultaneously adjacent to the feeding electrode group and the moving electrode group, and the culturing electrode group is adjacent to the moving electrode group.

In a fourth aspect, the present application provides a portable digital microfluidic system, which includes the digital microfluidic chip of any one of the above embodiments.

The invention has the following beneficial effects:

according to the screening method for the medicine for the accurate treatment of the tumor, the primary tumor cells and the medicine to be screened are directly adopted for culture, the primary tumor cells do not need to be cultured in vitro, and the condition that deviation exists in a medicine screening result due to accidental mutation of the neutron cells in the culture process is effectively avoided. The application also establishes a tumor model, and the drug screened by the screening method for the drug for the precise tumor treatment is applied to the tumor model and is consistent with the corresponding individual tumor reaction result, so that the screening method for the drug for the precise tumor treatment provided by the application is used for rapidly determining the effectiveness of the drug with the optimal drug effect and the potential value of personalized tumor treatment. In addition, the digital microfluidic chip provided by the application can realize the culture of primary tumor cells under various different culture conditions, and the portable digital microfluidic system provided by the application is portable in operation and small in size of the workbench, and can quickly determine effective drugs for treating personalized cancer.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

Fig. 1 is a schematic structural diagram of a digital microfluidic chip provided in an embodiment of the present application;

fig. 2 is a schematic structural diagram of a bottom plate of a digital microfluidic chip provided in an embodiment of the present application; wherein V1 to V24 are numbers of output voltage pads, and E1 to E24 are numbers of electrodes;

fig. 3 is a schematic structure of a single electrode array group in a digital microfluidic chip provided in an embodiment of the present application;

fig. 4 is a schematic diagram of a serial structure of electrodes with the same number in a single electrode array group in a digital microfluidic chip provided in an embodiment of the present application;

fig. 5 is a schematic diagram illustrating a principle of a digital microfluidic chip for driving a droplet to move by energizing adjacent electrodes according to an embodiment of the present disclosure;

fig. 6 is a schematic diagram of a mobile mixed culture performed after a drug-loaded sample and a cell sample are loaded on a digital microfluidic chip provided in an embodiment of the present application;

fig. 7 is a schematic diagram of a serial structure of identically numbered electrodes of a single electrode array group in a digital microfluidic chip according to another embodiment of the present application;

FIG. 8 is a schematic diagram of a drug concentration generator for combined drug toxicity testing according to an embodiment of the present application;

fig. 9 is a schematic structural diagram of a portable digital microfluidic system according to an embodiment of the present disclosure;

fig. 10 is a schematic diagram of a design of a portable digital microfluidic system provided in an embodiment of the present application;

fig. 11 is a schematic view of a portable digital microfluidic system for drug screening for transplantation of biopsy samples of a murine breast cancer model according to an embodiment of the present disclosure;

FIG. 12 is a diagram of an experimental process of an experimental method provided in an embodiment of the present application; wherein A represents a roadmap for guiding in vivo treatment by establishing a breast cancer MDA-MB-231 tumor model, obtaining a biopsy sample, and screening on-chip drugs and screening results; b shows the results of the biopsy needle and the biopsy specimen taken from the tumor site; c represents an image of skin adhesion around the tumor site of the mouse before and after biopsy; D-F represents the treatment result of the mouse, D represents the relation between the administration frequency and the tumor volume, and E represents the relation between the administration frequency and the mouse body weight; f represents the photographing result of the tumor size after the drug treatment;

fig. 13 is an experimental process diagram of the influence of ejection parameters of the drug concentration generator on the ejection volume provided in the embodiment of the present application; wherein, (a) is the design and working principle schematic diagram of the drug concentration generator; (b) a correction curve between the concentration of the cisplatin and the fluorescence intensity of the medicine is obtained, (c) the influence of the injection frequency on the injection volume is obtained, (c) the width of an injection electrode strip is fixed at 400 micrometers, the peak value of the injection voltage is fixed at 1020v, (d) the injection voltage is obtained, (e) the influence of the injection time on the injection volume is obtained, and the injection frequency is fixed at 800 Hz; the drug used in this experiment was cisplatin;

FIG. 14 shows the results of on-chip toxicity testing of drug cisplatin on tumor cells dissociated from mouse 2 biopsy samples;

FIG. 15 shows the results of on-chip drug EP toxicity testing of tumor cells dissociated from biopsy samples of rat 2;

FIG. 16 shows the results of on-chip drug wzb toxicity testing of tumor cells dissociated from biopsy samples of murine 2; wherein the red fluorescent cells are dead cells.

Icon: 100-digital microfluidic chip; 110-a base plate; 111-electrode array group; 112-sample introduction electrode group; 112 a-a drug-loaded sample injection unit; 112 b-a cell sample introduction unit; 113-a hybrid electrode; 114-moving the electrode set; 115-culture electrode set; 116-output voltage pad; 120-a gasket; 130-a top plate; 131-a sample addition hole; 200-portable digital microfluidic systems; 210-a circuit control board; 211-a power interface; 212-control buttons; 213-a signal generator; 214-a transformer; 215-an electromagnetic relay; 220-handheld device body.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

The application provides a screening method of a drug for accurate treatment of tumors, which comprises the following in vitro screening steps: respectively culturing the primary tumor cells under various different culture conditions; a variety of different culture conditions include: at least one first culture condition in the presence of a drug to be screened and at least one second culture condition in the absence of a drug to be screened;

comparing the survival of the primary tumor cells under the first culture condition and the second culture condition, and selecting the drug to be screened used in the first culture condition, in which the survival of the primary tumor cells under the first culture condition is inferior to the survival of the primary tumor cells under the second culture condition, as the drug for tumor precision treatment.

Wherein the primary tumor cells are derived from tumor tissues of tumor-affected individuals, and the tumor types of the tumor-affected individuals are any one of breast cancer, lung cancer, kidney cancer, endometrial cancer, esophageal cancer, stomach cancer, pancreatic cancer, liver cancer, glioma, ovarian cancer, prostate cancer, myelogenous leukemia and colon cancer. The individual suffering from the tumor is a mammal; preferably, the mammal is a human; preferably, the mammal is a non-human mammal; preferably, the non-human mammal is selected from any one of mouse, rat, dog, pig, rabbit, cow, horse, sheep, monkey, and ape.

In the application, the primary tumor cells are directly mixed with various different drugs to be screened and cultured, so that the primary tumor cells do not need to be cultured and proliferated in vitro, and accidental mutation in the process of in vitro culture and proliferation can be effectively avoided. In the application, the sensitivity of the primary tumor cells to the drug to be screened is judged through the survival condition of the primary tumor cells under each culture condition, specifically, dead cells and/or live cells are stained by adding a fluorescent agent into the culture condition, and finally, the sensitivity of the primary tumor cells to the drug to be screened is judged according to the number of the fluorescent cells. There are a variety of methods for staining cells or fluorescent agents, including, but not limited to, any of Dead cell stain, Live/Dead stain, protein expression stain, and neutral red stain. It should be understood that other staining methods can be used as long as the number of cells can be counted, and the application is not listed.

There are also various ways of culturing primary tumor cells and the drug to be screened in this application, including but not limited to 96-well plates, digital microfluidic systems, or pipeline microfluidics.

Among them, the number of primary tumor cells required by a 96-well plate is large, and multiple biopsy sampling is required, which brings trouble to traditional drug screening based on the 96-well plate. The microfluidic technology has potential application value in the field of biomedicine by the characteristic of small sample volume requirement, and particularly has the aspect of drug screening based on primary tumor cells. Microfluidic technologies can be divided into two categories according to principles: mechanical force-driven based channel microfluidics and voltage-driven based digital microfluidics. Drug screening has been extensively studied in channel microfluidic chips. In these works, researchers have performed a series of drug sensitivity tests using human primary tumor specimens from different organs. Since the ultimate goal of drug screening trials is to find relatively effective drugs for individual patients to extend their life. However, these documents do not further discuss the in vivo effects of related drugs, and thus the application value of the drugs is greatly reduced. Furthermore, the use of elongated capillaries in channel microfluidics makes the waste of biopsy samples inevitable; the large size of the auxiliary equipment and the mode of operation of the machine also prevents its wider spread.

Unlike channel microfluidics, Digital Microfluidics (DMF) utilizes an electrical drive signal based on the dielectric wetting (EWOD) phenomenon to manipulate individual droplets on an electrode array, facilitating automated analysis of individual samples. Cell culture on DMF chips has been explored by many groups, such as primary cell culture, single cell culture, or drug toxicity testing of commercial tumor cell lines. However, all of these previous studies used commercial cancer cell lines as model cells. The primary tumor sample culture and drug screening work has never been performed on a portable digital microfluidic system. More importantly, it is not clear whether in vitro drug screening can reliably direct in vivo therapy.

Therefore, in the present application, it is preferable to use a digital microfluidic system to culture the primary tumor cells and the drug to be screened.

Specifically, a medicament and primary tumor cells are injected into a digital microfluidic system, and then adjacent electrodes in the digital microfluidic system are electrified to move and mix the injected samples; the digital microfluidic system is internally provided with a plurality of electrodes which are respectively used for moving and mixing liquid drops on the electrodes, and the plurality of electrodes are driven by corresponding voltages; the number of voltages is less than or equal to the number of electrodes, that is, each of the plurality of electrodes in the present application may be controlled by using a single output voltage channel, or some of the plurality of electrodes may be controlled by using a common output voltage channel.

Specifically, in the present application, a digital microfluidic chip in a digital microfluidic system is preferably designed, and only as a typical illustrative example, an electrode array group and an output voltage welding spot are formed on a bottom plate of the digital microfluidic chip in the present application; the electrode array group comprises at least two sample introduction electrode groups used for respectively introducing samples of different samples, a mixing electrode used for mixing the introduced different samples, a movable electrode group used for moving the mixed samples and a culture electrode group used for moving the mixed samples to the culture area for culture; wherein, the mixing electrode is simultaneously adjacent to the sample feeding electrode group and the moving electrode group, and the culture electrode group is adjacent to the moving electrode group.

The sample injection method may be various, for example, the sample injection method may be performed after mixing the pre-prepared drugs with different concentrations of the drugs with the primary tumor cell suspension; or; injecting the prepared medicines with different concentrations of various medicines and the primary tumor cell suspension into the sample injection electrode group and then mixing; or; the method comprises the steps of injecting a medicament to be mixed in a plurality of medicaments into an injection electrode group, and then mixing primary tumor cell suspension with medicaments with different volumes.

Preferably, a sharp pulse wave amplified by a square wave is applied to a medicine concentration generator arranged on the sampling electrode group to generate a medicine liquid with a volume smaller than the volume of the sampling medicine; the drug concentration generator comprises a first electrode strip and a second electrode strip, wherein the width of the second electrode strip is 7-9 times that of the first electrode strip; the width of the first electrode stripes is 10-60 μm, and the width of the second electrode stripes is 300-500 μm.

Next, the specific structure of the digital microfluidic chip 100 will be explained in detail in this application.

Referring to fig. 1, the present application provides a digital microfluidic chip 100, which includes a bottom plate 110, a spacer 120, and a top plate 130. The top plate 130 is connected to the upper side of the bottom plate 110 through the spacer 120, and the bottom plate 110 is formed with an electrode array group 111 and an output voltage pad 116 (fig. 2).

Specifically, referring to fig. 2 and 3, the electrode array set 111 includes a sample electrode set 112, a mixing electrode 113, a moving electrode set 114, and a culture electrode set 115.

The sample injection electrode group 112 is used for injecting samples of different samples respectively.

The number of the sampling electrode groups 112 is at least two, sampling of at least two samples can be realized, each sampling electrode group 112 comprises a plurality of electrodes which are sequentially arranged and have different numbers, the electrode numbers of all the sampling electrode groups 112 are the same, and in the application, the adjacent electrodes are sequentially electrified to promote the movement of the sampled sample liquid drops.

Preferably, in this embodiment, the number of the sampling electrode groups 112 is two, and specifically includes a drug-loaded sample sampling unit 112a and a cell sample sampling unit 112b, which are respectively used for respectively sampling a tumor cell sample and a drug sample with different concentrations. The drug-loaded sample injection unit 112a and the cell sample injection unit 112b are respectively connected to two sides of the mixing electrode 113; the injected drug-loaded sample and cell sample move to the mixing electrode 113 and are mixed at the mixing electrode 113. Specifically, in the present application, the left side of the mixing electrode 113 is a drug-loaded sample injection unit 112a, and the right side is a cell sample injection unit 112 b. It should be understood that the positions of the drug-loaded sample injection unit 112a and the cell sample injection unit 112b can be interchanged, and the number and the shape of the internal electrodes thereof can be adjusted according to the circumstances, which is not specifically limited in this application.

Specifically, the drug-loaded sample introduction unit 112a has a plurality of electrodes arranged in sequence and different in number, and the number of the electrodes in the cell sample introduction unit 112b are the same as those of the electrodes in the drug-loaded sample introduction unit 112 a; the electrodes with the same number in the drug-loaded sample injection unit 112a and the cell sample injection unit 112b are connected in series and are commonly connected to an output voltage pad (see fig. 4). Through advance kind unit 112a and cell sample and advance kind unit 112b and set to the electrode that has the same number and the same serial number with the medicine carrying sample in this application, advance kind the back at medicine carrying sample and cell sample, can realize carrying out charge control to the electrode of the same serial number through controlling same output voltage to the removal of realization to medicine carrying sample and cell sample is controlled simultaneously.

The mixing electrode 113 is used for mixing different samples to be injected.

The mixing electrode 113 is adjacent to the sampling electrode group 112 and the moving electrode group 114, and the mixing electrode 113 can mix the drug-loaded sample and the cell sample which move to the mixing electrode 113, and output the mixed sample.

The moving electrode group 114 is used to move the mixed sample.

Since the moving principle diagram of the present application is shown in fig. 5, in order to avoid an error in the moving direction of the droplet, the numbers of any three adjacent electrodes need to be different, and therefore, the moving electrode group 114 in the present application includes at least three electrodes that are sequentially arranged and have different numbers. This design principle (the number of any three adjacent electrodes needs to be different) is also applicable to the sample electrode set 112 and the culture electrode set 115.

The movable electrode group 114 at least comprises a movable electrode unit, the movable electrode unit at least comprises three electrodes which are adjacent in sequence, one electrode of the movable electrode unit, which is far away from the mixed electrode, shares an output voltage welding point 116 with the mixed electrode 113, and the other electrodes of the movable electrode unit and the electrodes of the sample injection electrode group 112, which are close to the mixed electrode 113, have the same corresponding numbers, and share the same output voltage welding point 116 with the same numbers.

The culture electrode assembly 115 is used for moving the mixed sample to the culture area for culture.

The culture electrode group 115 is adjacent to any one of the moving electrode group 114, and the mixed sample on the moving electrode group 114 can be changed in direction to move to the culture electrode group 115, and the electrodes of the culture electrode group 115 in this application are hydrophilic pretreated electrodes, which can promote cell adhesion.

Specifically, the culture electrode group 115 includes a plurality of groups of the same or different culture units; the culture unit is provided with a plurality of electrodes which are arranged in sequence and have different numbers; any one of the moving electrode sets 114 may be adjacent to multiple different sets of culture units; alternatively, different electrodes in the moving electrode group 114 may be adjacent to a corresponding number of identical sets of culture units;

as can be seen in fig. 3, the set of culture electrodes 115 are two different sets of culture units, which are adjacent to both sides of the electrode of the moving electrode unit that shares one output voltage pad 116 with the mixing electrode 113. The number of culture units per set is the same as the number of moving electrode units.

In the present application, the number of the electrodes in the sample electrode group 112, the number of the mixed electrode 113, the number of the moving electrode group 114, and the number of the culturing electrode group 115 are respectively numbered (E1 to E24), and the number of the output voltage pads 116 are numbered (V1 to V24). The number of the output voltage welding points 116 is the same as the number of the electrodes in the electrode array group 111; the electrodes with different numbers are respectively and correspondingly connected to the output voltage welding points 116; the identically numbered electrodes are connected in series and in common to an output voltage pad 116.

For a clearer explanation of the present application, the plurality of electrodes in the electrode array group 111 are numbered and illustrated one by one in the present application by way of a typical non-limiting example.

In this example, the sample electrode group 112 includes two groups of 10 electrodes, which are a drug-loaded sample injection unit 112a and a cell sample injection unit 112b, the drug-loaded sample injection unit 112a has 5 electrodes, which are numbered as E22, E23, E24, E5, and E3, and similarly, the cell sample injection unit 112b also has 5 electrodes, which are numbered as E22, E23, E24, E5, and E3, and the numbers of which are consistent with the numbers of the drug-loaded sample injection unit 112 a. The hybrid electrode 113 is one, which is numbered E4. The moving electrode group 114 has 3 electrodes with different numbers, which are respectively numbered as E5, E3 and E4, wherein one electrode of the moving electrode unit away from the mixing electrode 113 is numbered as E4 and has the same number as the mixing electrode 113, therefore, the two electrodes share one output voltage pad 116, and the other electrodes of the moving electrode unit have two electrodes (i.e., the electrodes numbered as E5 and E3 in the moving electrode unit) which are correspondingly numbered as 2 electrodes of the sample electrode group 112 close to the mixing electrode 113 (i.e., the electrodes numbered as E5 and E3 in the electrode group 112), and the same number shares the same output voltage pad. The moving electrode group 114 has 3 moving electrode units in total and 9 electrodes in total. The culture electrode group 115 has 6 groups of 12 electrodes in total to realize culture analysis of 6 drug-loaded samples with different concentrations, specifically, each group has 2 electrodes, which are respectively numbered as E1 and E2, or numbered as E6 and E7, specifically, the electrode groups numbered as E1 and E2 have 3 groups, and the electrode groups numbered as E6 and E7 also have 3 groups.

At least two electrodes in the sample electrode group 112 are numbered the same as two electrodes in the moving electrode group 114, i.e. the electrodes numbered E3 and E5 in the sample electrode group 112 and the electrodes numbered E3 and E5 in the moving electrode group 114 have the same number, and the electrode numbered E4 in the moving electrode group 114 is numbered the same as the mixed electrode 113. Because in this application, the electrodes with the same number are connected in series and are connected to one output voltage welding spot 116 in common, so that one electrode array group 111 of this application has 24 electrodes in total, but only needs 8 output voltages can realize moving, mixing and moving the drug-loaded sample and the cell sample to the culture electrode.

The electrode energization sequence in this example is: the drug-loaded sample and the cell sample enter two electrodes E22 from the sample inlet hole respectively, the electrode E22 is electrified simultaneously, then the electrode E23 → E24 → E5 → E3 → E4 mixed electrode 113 is used for mixing the drug-loaded sample and the cell sample, the electrode E5 is electrified again, at the moment, the mixed sample moves along the movable electrode group 114, and when the mixed sample moves to the electrode E4, the electrode E2 or the electrode E7 can be electrified selectively, so that the mixed sample moves to different culture electrodes.

It should be noted that in other embodiments of the present application, the number of the feeding electrode set 112 may be selected (see an example given in fig. 7), as long as at least one electrode is included, and similarly, at least one set of 3 electrodes with different numbers may be included in the moving electrode set 114, and the number may be selected to be different from that of the mixing electrode 113, and the culturing electrode set 115 may have only one electrode. Such electrode setting can realize moving, mixing medicine-carrying sample and cell sample and finally move to the cultivation electrode through letting adjacent electrode circular telegram in proper order.

In the typical example of the present application, the number, number and arrangement sequence of the electrodes can be used to realize continuous sample injection. Specifically, the application aims at that 6 drug-loaded samples with different concentrations and cell samples are mixed and then are cultured simultaneously so as to observe the sensitivity of the cells to the drug-loaded samples with different concentrations, and therefore the optimal drugs can be screened out. After entering an electrode No. E22, the drug-loaded sample and the cell sample can be injected again when moving to an electrode No. E5 of an injection electrode group 112, at this time, the sample for the first injection moves from a droplet on the electrode No. E5 to an electrode No. E3 and then moves to the electrode No. E4 for mixing, the sample for the second injection moves from an electrode No. E22 to an electrode No. E23 and then moves to an electrode No. E24, and when the sample for the first injection passes through the electrode No. E5 of the movable electrode group 114 again, the sample for the first injection moves along the movable electrode group 114 and passes through the electrode No. E3 and the electrode No. E4; and the sample in the second sample introduction moves to the electrode No. E5 of the sample introduction electrode group 112, and then is mixed through the electrode No. E3 and the electrode No. E4. Similarly, when the sample of the second sample introduction moves to electrode E5 of the sample introduction electrode group 112, the sample introduction can be performed again. When the mixed sample of the first sample is moved to the last electrode (i.e. the electrode numbered as E4 in the third group) of the moving electrode group 114, at this time, the mixed sample of the second sample is moved to the electrode numbered as E4 in the second group of the moving electrode group 114, and the mixed sample of the third sample is moved to the electrode numbered as E4 in the first group of the moving electrode group 114, at this time, it can be selected to enter the culturing electrode group 115, that is, it can realize one-time entry of three mixed samples. Continuous feeding can be realized through such setting in this application, and the multiunit sample moves at different position points simultaneously, is favorable to realizing that same voltage controls a plurality of electrodes to maximize reaction point quantity and drug screening flux.

Further, in the present application, the number of the electrode array groups 111 is three and the three electrode array groups are arranged in parallel, wherein the electrodes E22, E23 and E24 of the three electrode array groups 111 can be respectively connected in series to achieve synchronous feeding.

In addition, in the present application, the material of the top plate 130 is ITO glass; the top plate 130 is provided with a plurality of sample injection holes 131 for injecting samples into the sample injection electrode group 112. Barriers for preventing the drug-loaded sample and the cell sample from drifting are further arranged around each electrode of the bottom plate 110; preferably, the material of the fence is SU-8; the thickness of the fence is 50-70 microns. Polytetrafluoroethylene layers with the thickness of 80-120nm are coated on the upper surface of the bottom plate 110 and the lower surface of the top plate 130; the polytetrafluoroethylene layer is advantageous in promoting smooth movement of the droplets. The spacer 120 is used to connect the bottom plate 110 and the top plate 130, specifically, the thickness of the spacer 120 is 80-120 microns, and the material of the spacer 120 includes, but is not limited to, conductive adhesive.

The digital microfluidic chip 100 in the present application is prepared by etching. Specifically, three parallel electrode array groups 111 are designed as masks by using AutoCAD software.

SU-8 as a dielectric layer was coated on the base plate 110 to a thickness of 10 μm, and then patterned on the base plate 110. After development, a second 60 micron thick layer of SU-8 pattern was applied as a barrier to prevent droplet drift. ITO glass having a size of 35mm by 20mm is cut as the top plate 130. A hole having a diameter of 1.5 mm was drilled in the ITO glass as the well 131 using an ZKJ laser cutter (Shanghai ZKJ laser). In order to promote smooth movement of the droplets, polytetrafluoroethylene is coated to a thickness of 80-120nm on the top plate 130 and the bottom plate 110. A spacer 120 of 80-120 microns thickness connects the bottom plate 110 and the top plate 130.

In addition, it should be noted that, in the present application, a drug concentration generator (see fig. 8) is disposed on the sample injection electrode set, the drug concentration generator includes a first electrode strip and a second electrode strip, and a width of the second electrode strip is 7-9 times a width of the first electrode strip; the width of the first electrode stripes is 10-60 μm, and the width of the second electrode stripes is 300-500 μm. Applying a square wave amplified sharp pulse wave to a drug concentration generator to produce a series of small volume drug liquids; this method can produce drug concentration gradients spanning a range of three to four orders of magnitude.

By applying a high voltage pulse signal to a special electrode strip with a drop, the drop will eject a large number of small volume drops. When the cell suspension is mixed with the ejected small volume droplets of drug, a mixture of drug cell suspensions of a certain concentration will be produced. By adjusting the ejection parameters (ejection voltage, ejection electrode strip width and ejection time), the drug concentration generator can easily achieve the generation of different drug concentrations (three to four orders of magnitude).

The drug concentration generator can flexibly generate a series of drug concentrations and can effectively evaluate the drug toxicity. Due to the simplicity of electrode strip design, the flexibility of producing drug concentrations spanning four orders of magnitude, the designed drug concentration generator has great potential application value in optimizing combinatorial drug screening for cancer therapy and other related concentration studies.

In a second aspect, please refer to fig. 9 and 10, the present application further provides a portable digital microfluidic system 200, which includes the digital microfluidic chip 100.

Specifically, the portable digital microfluidic system 200 further includes a handheld device body 220 and a circuit control board 210, and the circuit control board 210 and the digital microfluidic chip 100 are integrated in the handheld device body 220. The portable digital microfluidic system 200 has overall dimensions of 20-25cm x 14-18cm x 3-4 cm. The portable digital microfluidic system 200 has a small volume, occupies a small space for operation, and is portable.

Preferably, the circuit control board 210 comprises a power interface 211, a control button 212 and a signal generator 213 for providing an alternating current driving signal, the signal generator 213 is connected with the control button 212 in a communication way, the power interface 211 is electrically connected with the control button 212, and the control button 212 is electrically connected with the digital microfluidic chip 100 and the electromagnetic relay 215; preferably, the AC drive signal is a sine wave of 0.5-10 Vrms. In this application, the control buttons 212 are aligned with the chip electrodes to control the power on/off of each electrode.

The circuit control board 210 further includes a transformer 214 for amplifying the ac driving signal and charging the electrodes on the digital microfluidic chip 100, the transformer 214 being electrically connected to the control buttons 212 and the electromagnetic relay 215.

The signal generator 213 may provide a drive signal of a certain waveform, and then the sine wave signal provided by the signal generator 213 is amplified by the transformer 214 into a droplet drive signal of 70-120V. In view of the safety of portable applications of the portable digital microfluidic system 200, the present application uses the electromagnetic relay 215 as a relay station in both the dc amplification circuit and the ac amplification circuit to achieve low voltage activation of the push button switch. When the button switch is pressed, a 5v power supply connection power interface 211 provided by the 5v power adapter connects the alternating current amplifying circuit through the button switch and the electromagnetic relay 215 to charge the electrodes on the digital microfluidic chip 100. Since the digital microfluidic chip 100 is provided with three parallel electrode array groups 111 to improve the flux of drug screening, the button switch is also provided with an array group corresponding to the electrode sequence on the chip, thereby simplifying the operation, specifically, the number of the control buttons 212 is multiple and corresponds to the number of the output voltage pads 116 on the digital microfluidic chip 100 to independently control the voltages of the electrodes with different numbers.

Referring to fig. 11, the working process of the portable digital microfluidic system 200 provided by the present application is as follows:

the drug-loaded sample and the cell sample are respectively injected into the sample injection electrode group 112 of the digital microfluidic chip 100 through the two sample injection holes, and then the button switches of the electrodes with the numbers are switched and continuously charged in sequence, so that the drug-loaded sample and the cell sample are moved on the sample injection electrode group 112, after the mixed sample is moved to the position of the mixed electrode 113 and mixed, the mixed sample is charged again according to the electrodes with the numbers, so that the mixed sample continues to move along the movable electrode group 114, and finally moves to the culture electrode group 115. Each electrode array group 111 can sample 6 drug-loaded samples with different concentrations, the 6 drug-loaded samples with different concentrations are respectively mixed with cell samples and then moved to respective culture electrodes, and then the digital microfluidic chip 100 is placed in a cell culture dish for culture, and is observed under a fluorescence microscope after the culture is completed (see fig. 6). The portable digital microfluidic system 200 in the application has 3 parallel electrode array groups 111, so that three different drugs (each drug has 6 concentration gradients) can be screened at the same time, and the flux of drug screening is improved by 3 times compared with the traditional design. The whole working process of primary tumor sample acquisition and chip drug toxicity test can be completed within 36 hours, and the individual drug screening result of the mouse on the chip is consistent with the individual tumor treatment effect, which suggests that the reliability of the optimal effective drug can be rapidly determined based on the animal tumor model of xenotransplantation. This "series circuit" design approach will improve chip utilization. The portable digital microfluidic system 200 provided by the present application has great application potential in personalized cancer therapy.

The reagents referred to in this application are:

SU8 and SU-8 developers were purchased from MicroChem; pluronic F127 was purchased from Sigma Aldrich; silicone oil (1cSt) was purchased from Clearco, usa; phosphate Buffered Saline (PBS) was purchased from Gibco; HBSS is available from Life Technologies, USA; cisplatin (II) dichloro, epirubicin hydrochloride (EP) purchased from Sigma; wzb117 was purchased from Selleckchem; DMEM/F12 medium was purchased from Hyclone; insulin, hydrocortisone, cholera toxin, and hyaluronidase were purchased from Sigma; epidermal Growth Factor (EGF) was purchased from Invitrogen; collagenase III was purchased from woxinton; RBC lysis buffer was purchased from eBioscience, usa; StemMACS iPS Brew XF medium was purchased from Milton Biotech, USA; EthD-1 was purchased from Saimer Feishel technologies.

First, experiment method

(1) Transplant nude mouse model

All animal experiments were performed according to the Australian animal welfare method. The invention makes 100 microliter of human breast cancer MDA-MB-231 cell suspension (2X 10)⁶Individual cells/ml) were injected subcutaneously into the right side of a single female nude mouse. 30 mice were used and labeled in this experiment. Mice were 6 weeks of age. Mice were examined every other day during the study. When the tumor is palpable, the two-dimensional size (length and width) of the tumor is measured with calipers. Tumor Volume (TV) according to the formula a x b²(a and b) calculationRepresenting the longest and shortest diameters, respectively). When the tumor volume of the mice is increased to 0.1-0.3cm³When (note that due to individual differences, the tumor growth rate of each mouse was different, and mice with tumor volumes greater or smaller than most mice were discarded in the experiment), mice were anesthetized with Avermectin (250mg/kg), and primary tumor specimens were removed with a biopsy needle of 16 G.times.9 cm, 1cm sampling slot. A total of 21 mice were biopsied in this experiment. The sample size was about 2X 10 mm. The biopsy specimens were placed into 1.5ml sterile PBS centrifuge tubes and numbered. The skin around the mouse tumor was then adhered with medical adhesive to prevent infection. The obtained biopsy samples were used for primary tumor isolation and on-chip drug screening.

(2) Primary tumor sample dissociation

According to the method, the dissociation operation is carried out on the sample in the experiment according to the conventional dissociation method of the primary tumor specimen, and the dissociation step is greatly simplified. Briefly, the present invention first transfers mouse biopsy samples obtained using a biopsy needle to 24-well plates, respectively, and washes twice with PBS. Then, after pipetting out PBS, 0.5mL of digestion buffer I (DMEM/F12 medium containing 5% FBS, 5. mu.g/mL insulin, 500ng/mL hydrocortisone, 10ng/mL Epidermal Growth Factor (EGF), 20ng/mL cholera toxin, 300U/mL collagenase III and 100U/mL hyaluronidase) was added to each of the dissociated samples in the 24-well plate. The 24-well plate was then placed in an incubator (37 ℃, 5% CO2) and digested with shaking at 100rpm for about 3 h. The solution was gently pipetted every 30 minutes to accelerate the disintegration. The suspensions in the well plates were then individually transferred to 1.5ml sterile centrifuge tubes, spun at 400g for 3 minutes, then the supernatant in the centrifuge tubes was discarded, and 0.5ml of RBC lysis buffer (eBioscience, USA) was added to the centrifuge tubes for red blood cell lysis for 30s, and finally 0.5ml of HBSS (Life Technologies, USA) was added to terminate the lysis reaction. Finally, the present invention counted the dissociated cells with a cytometer and diluted with StemMACS iPS Brew XF medium to a cell density of 1.5 x 10 (milrattan biotechnology, usa)⁶Individual cells/ml for chip drug screening.

(3) On-chip drug screening

With cisplatin(II) dichloro (Cis), Wzb117 (glucose transporter 1 inhibitor, Wzb for short) and epirubicin hydrochloride (EP) are used as drug models, and the toxicity of the drug models to the tumor cells dissociated from the separated primary biopsy tumor samples is monitored within 24 hours. Of these three drugs, Cis is a widely used anti-cancer chemotherapeutic drug. wzb can block glucose transport, promote apoptosis, and inhibit tumor growth. EP has a wide range of antitumor effects, and is particularly effective in the treatment of metastatic breast cancer and small cell lung cancer. The present invention first prepares Cis, wzb and EP series concentrations (0, 1, 10, 20, 40. mu.M) by in vitro serial dilution. Pluronic F127 and EthD-1 were then added separately to mice (1.5X 10) in PCR tubes⁶Individual cells/ml) at final concentrations of 0.01% Pluronic F127 and 2 μ M EthD-1, respectively. Thereafter, the DMF chips were filled with silicone oil (1 cSt). Subsequently, the cell suspension and a series of concentrations (0, 1, 10, 20, 40 μ M) of drug (Cis, wzb, EP) were pipetted into the wells and the mixed droplets were moved to the target culture electrodes by sequentially energizing the adjacent electrodes. The chip was then placed in a cell culture dish, placed in a cell incubator for 24 hours, and finally a picture of the cells in the red fluorescent channel and bright field was taken with an inverted fluorescence microscope (Olympus) at 10-fold magnification. And calculating the drug toxicity result by counting the number of the red fluorescent cells and the total number of the cells on the target cell culture electrode.

(4) Xenograft nude mouse drug therapy

According to the results of the on-chip drug toxicity test of each mouse, the present invention divided the mice into three groups of 7 mice each. One group was injected with relatively effective drug (Cis), another group was injected with relatively ineffective drug (wzb), and the third group was injected with PBS as a non-treatment control. The injection is intraperitoneal injection, and is administered 2 times per week. The injection dose of Cis is 10 mg/kg. To ensure that the injected doses were equal in the three groups, the therapeutic dose was also 10mg/kg in the other two groups. Mouse body weight and tumor volume were measured and calculated before each administration, with a course of treatment of 1 month.

II, specific experimental operation:

(1) on-chip drug screening of primary tumor samples

The procedure for obtaining primary tumor samples is shown in FIGS. 12A-C. Briefly, the present invention injects 30 female nude mice with a human breast cancer MDA-MB-231 cell suspension. When the tumor was visible to the naked eye, its size was measured. When the tumor volume of the mice is increased to 0.1-0.3cm³When required, it was weighed and anesthetized with Avermectin (250 mg/kg). The invention then uses a biopsy needle (16 G.times.9 cm, 1cm sample well) to take primary tumor samples from mice (FIG. 12, B). Wounds around the mouse tumor were adhered with medical adhesive to prevent infection (C in fig. 12). The invention then dissociates the obtained biopsy samples and uses them for on-chip drug screening. Cisplatin (II) dichloro (Cis), Wzb117 (glucose transporter 1 inhibitor, Wzb for short) and epirubicin hydrochloride (EP) were used as drug models herein. The invention takes the primary tumor specimen of 14 mice to carry out on-chip drug screening research, and then takes the same number of mice as the contrast to carry out in vivo treatment research. The present invention uses "-" and "+"/"to describe the level of drug sensitivity. "/" indicates that no drug sensitivity values were obtained because cytotoxicity was not determined due to limited sample numbers. When the value of cell viability at a certain drug concentration is greater than 90% of the cell viability at 0 drug concentration, it is described by "-". The number of "+" positively correlated with drug sensitivity.

As shown in table 1, all three drugs were toxic to primary tumor samples of 14 mice to varying degrees. For Cis treatment, the primary tumor sample from mouse 14 was the least sensitive ("+"), while the primary tumor sample from mouse 2 was the most sensitive ("+ + + + +"). In addition to no toxicity measured in the primary tumor sample of mouse 9, the sensitivity levels of the other samples were also different, being "+ +", "+ + + + +", or "+ + + + + + + + + + +", respectively. For EP treatment, primary tumor samples from

mice

9, 13 and 14 were not sensitive, except that no toxicity was measured for primary tumor samples from

mice

1 and 8. The primary tumor samples from other mice had different sensitivities, either "+", "+ + + + + +" or "+ + + + + + + +". The results of fluorescence microscopy imaging of toxicity assays of the three drugs on primary tumor samples from mouse 2 are shown in fig. 14, 15 and 16. When the cells died, the fluorescent dye EthD-1 entered the cells and emitted red fluorescence. It is clear from the present invention that the number of red fluorescent cells increases with increasing concentration of the drug (Cis and EP). For wzb, there was no significant change in red blood cell number with increasing drug concentration. Overall, primary tumor specimens from mice are more sensitive to Cis treatment than EP treatment. For wzb treatment, none of the 11 mice were sensitive to the drug. Only samples from mice 3 and 13 were sensitive to the drug. Overall, the results in table 1 indicate that different individuals respond differently to treatment with the same drug or different drugs.

TABLE 1 results of on-chip drug toxicity testing of primary tumor samples from nude mice.

Note: "/: cytotoxicity was not measured; -: no obvious cytotoxicity exists; cytotoxicity (+ ++++ > + + >) "

(2) Xenograft nude mouse drug therapy

In order to study whether the screening result of the primary tumor cell drug in vitro is consistent with the in vivo drug treatment result, the invention researches the growth condition of the tumor by injecting an effective drug or ineffective drug screened on a chip or PBS buffer solution as a control into each mouse. Each group had 7 mice. The invention researches the in-vivo treatment effect of the medicament by injecting a certain dose of medicament into the abdominal cavity of the MDA-MB-231 breast cancer cell xenograft mouse. According to the screening result of the chip drugs, the invention divides the mice into three groups, and each group comprises 7 mice. One group injected with relatively potent drugs (Cis); another group injected with relatively low-potency drugs (wzb); the third group was injected with PBS as a control (mean tumor volume was consistent in the three groups of mice). The results in FIG. 12, D, show that the tumor volume in Cis treated group was significantly less than wzb treated group and PBS control group at 1 month of treatment, which matched well with the results of the on-chip drug screening. In addition, the tumor volumes of the three groups of mice varied in trend, suggesting individual differences. Tumor volume measurements in Cis-treated mice 2 were minimal, consistent with on-chip drug screening results, for the same treatment period (primary tumor samples from mice 2 were most sensitive to drug Cis). In vivo drug toxicity evaluation methods typically employ body weight measurements. In FIG. 12E, there was no significant change in body weight in the wzb-treated and control PBS-treated mice. Mice in the Cis-treated group had some weight loss, suggesting potential toxicity of Cis. The photograph of the tumor after treatment is shown as F in fig. 12. It can be seen that the tumor volume of mice in the Cis-treated group is significantly smaller than that of other groups.

All results show that comparison of the best performing drug, the worst performing drug, and the control group, selected by on-chip screening, clearly reveal: the result of the screening of the mouse drugs on the in vitro chip is consistent with the individual treatment effect, suggesting the feasibility of the portable digital microfluidic system 200 for accurate medicine. Considering that the sample for chip drug screening can be as small as a section with the diameter of 1mm and the length of 10mm, the result can be returned within 24 hours, the DMF platform is portable, the size of the workbench is small, and the DMF system can quickly determine the effective drug for personalized cancer treatment. Therefore, on-chip drug screening of the portable digital microfluidic system 200 has certain guiding significance for in vivo drug therapy.

(3) Influence of spray parameters of the concentration generator on the spray volume

In the present application, the injection volume is quantified by fluorescence measurement. Fig. 13 (a) shows the design and operation of the drug concentration generator. The present application investigated the effect of firing parameters (firing frequency, firing electrode strip width and firing voltage) on firing volume (b-e in fig. 13). For common medicines without fluorescence emission characteristics, adding fluorescent dye into the medicines is a convenient method for realizing accurate volume quantification of the sprayed micro liquid drops. The present application utilizes cisplatin as a model for non-fluorescent drugs for jet volume quantification testing. A4 mM cisplatin stock solution (dissolved in 50% DMSO solution) containing 10. mu.M cy3 was used as a spray drop. DMEM medium without fluorescence was used as pickup drops. Prior to the experiment, the fluorescence intensity of different cisplatin concentrations (dissolved in DMEM medium) was measured to obtain a cisplatin standard curve (b in fig. 13). The ejection volume was quantified by measuring the fluorescence intensity of the picked-up drops and calculating from a cisplatin standard curve. The present work first investigated the relationship between injection frequency and injection quantity. As shown in c in fig. 13, the injection amount increases as the injection frequency increases. But as the firing frequency increases, the error between each parallel test also increases. This may be due to the following reasons: when the ejection frequency exceeds 800Hz, the ejection area exceeds the range of the electrode picking up the droplet, resulting in the waste of some minute droplets ejected. Therefore, the present application selects 800Hz as the fixed injection frequency for the following experiments. The present application then investigated the relationship between the ejection electrode stripe width and the ejection volume. In fig. 13 d indicates: regardless of the ejection voltage (556v, 660v, 768v, 864v, 980v, 1020v), the ejection amount is increased when the ejection electrode bar width is wide. For the tested jetting electrode strip widths (50 microns, 100 microns, 200 microns, 300 microns, and 400 microns), the jetting volume has a linear relationship to the jetting voltage. Therefore, it can be concluded that the ejection volume is smaller when the ejection electrode stripe width is narrowed and the ejection voltage is lower, and vice versa. For the tested spray electrode strip width and spray voltage parameters, the results are shown in fig. 13 as d: the minimum injection quantity is 0.015nL, the electrode strip width is 50 microns, and the voltage peak-to-peak value is 556 v; the maximum ejection volume was 0.829nL, the electrode stripe width was 400 μm, and the voltage peak-to-peak value was 1020 v. Thus, by adjusting the firing electrode strip width and firing voltage parameters, the firing volume will cover a range of 0.015nL-0.829nL, with a range of two orders of magnitude. Then, the present application tested the injection time, which is another factor affecting the injection amount. As shown by e in fig. 13, the injection amount is linear with the injection time. For a firing electrode stripe width of 400 microns, firing 2s with a peak 1020v voltage may produce a volume of 0.722nL, while firing 30s may produce a volume of 11.93 nL.

Epirubicin hydrochloride having a red fluorescence emission characteristic was used as another drug model for the ejection quantity quantitative test. A 5mM epirubicin hydrochloride stock solution (DMSO: H2O (volume ratio) ═ 1: 1) was used as ejection droplets. DMEM medium without fluorescence was used as pickup drops. The concentration generator is designed to produce an injection volume in the range of 0.0015nL to 11.39 nL. Traditionally, in drug toxicity testing, drug concentrations range from 2 to 3 orders of magnitude. Therefore, by using the on-chip concentration generator designed by the application, the drug toxicity test can be easily and flexibly completed.

To sum up, the screening method for the drugs for the precise treatment of the tumors provided in the application directly adopts the primary tumor cells and the drugs to be screened to culture, does not need to culture the primary tumor cells in vitro, and effectively avoids the condition that the deviation exists in the drug screening result due to the accidental mutation of the neutron cells in the culture process. The application also establishes a tumor model, and the drug screened by the screening method for the drug for the precise tumor treatment is applied to the tumor model and is consistent with the corresponding individual tumor reaction result, so that the screening method for the drug for the precise tumor treatment provided by the application is used for rapidly determining the effectiveness of the drug with the optimal drug effect and the potential value of personalized tumor treatment. The application provides a digital microfluidic chip 100 for drug screening of biopsy samples of a heterogeneous mouse breast cancer model. Compared with the traditional design mode of the electrodes on the chip, the three parallel electrode array groups 111 are designed on the small-sized digital microfluidic chip 100(3.76cm multiplied by 3.76cm) in a series circuit mode, so that the toxicity of three anti-cancer drugs can be detected simultaneously (in the traditional design, 24 output voltage channels control 24 electrodes; in the design of the application, 24 output voltage channels control 96 electrodes). The whole work flow of obtaining primary tumor samples in mice and drug toxicity tests can be completed within 36 hours, and the drug screening result on a single mouse chip is consistent with the corresponding individual tumor reaction result, which implies that the effectiveness of the drug with the best drug effect is rapidly determined on a DMF chip based on a tumor model of a xenograft animal and the potential value of personalized tumor treatment based on a DMF platform.

The drug susceptibility test is performed by dissociating a biopsy sample (diameter 1mm, length 10mm, about 10000 cells) of a transplanted rat, and loading it on the digital microfluidic chip 100, mixed with each drug at different concentrations. The sensitivity of primary tumor cells to various drugs was evaluated based on the survival rate of cells after 24 hours of culture. Then the most sensitive medicine screened in vitro is injected into the corresponding mouse for treatment, and the potential highest curative effect is achieved.

In order to verify the effectiveness of the portable digital microfluidic system 200 and the method, human breast cancer cells (MDA-MB-231) are transplanted into a nude mouse body, and a human breast cancer model is established. Cisplatin (Cis), Wzb117 (glucose transporter 1 inhibitor, Wzb for short) and epirubicin hydrochloride (EP) are used as model drugs for drug screening and in vivo treatment research. Comparison of the best, the worst, and the control group, screened on-chip, clearly revealed: the result of the mouse drug screening on the in vitro chip is consistent with the individual treatment effect, which suggests the feasibility of the digital microfluidic platform for accurate medicine. Considering that a sample for screening chip drugs can be reduced to a slice with the diameter of 1mm and the length of 10mm, the result can be returned within 24 hours, the portable digital microfluidic system 200 is portable, and the size of the workbench is small, the portable digital microfluidic system 200 provided by the application is a promising platform, and can quickly determine effective drugs for treating personalized cancers.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A screening method of a drug for accurate treatment of tumors is characterized by comprising the following in vitro screening steps:

2. The method of claim 1, wherein the at least one culture condition for the presence of the drug to be screened comprises: a plurality of culture conditions exist for different drugs to be screened.

3. The screening method for a drug for tumor precision treatment according to claim 1 or 2, characterized in that the survival rate of primary tumor cells in each culture condition is reflected by counting the number of fluorescent cells present in the culture condition after staining the cells with a fluorescent agent;

preferably, the fluorescent agent comprises any one of Dead cell stain, Live/Dead stain, protein expression stain and neutral red stain.

4. The screening method for drugs for tumor precision therapy according to claim 1 or 2, characterized in that the tumor type of the individual suffering from tumor is any one of breast cancer, lung cancer, kidney cancer, endometrial cancer, esophageal cancer, stomach cancer, pancreatic cancer, liver cancer, glioma, ovarian cancer, prostate cancer, myelogenous leukemia and colon cancer.

5. The screening method for a drug for the precise treatment of tumor according to claim 1, wherein the individual suffering from tumor is a mammal;

preferably, the mammal is a human;

preferably, the mammal is a non-human mammal;

preferably, the non-human mammal is selected from any one of a mouse, rat, dog, pig, rabbit, cow, horse, sheep, monkey, and ape.

6. The screening method of drugs for tumor precision therapy according to claim 1, characterized in that the culturing of the primary tumor cells under a plurality of different culture conditions is performed on a 96-well plate, a digital microfluidic system or a pipeline microfluidic system;

preferably, the sample is moved and mixed by electrifying adjacent electrodes in the digital microfluidic system;

preferably, the plurality of electrodes are driven by corresponding voltages; the number of the voltages is less than or equal to the number of the electrodes;

preferably, the digital microfluidic system comprises a digital microfluidic chip, and an electrode array group and an output voltage welding spot are formed on a bottom plate of the digital microfluidic chip; the electrode array group comprises at least two sample introduction electrode groups for respectively introducing samples of different samples, a mixing electrode for mixing the introduced different samples, a movable electrode group for moving the mixed samples and a culture electrode group for moving the mixed samples to a culture area for culture; wherein the mixing electrode is simultaneously adjacent to the feeding electrode group and the moving electrode group, and the culturing electrode group is adjacent to the moving electrode group.

7. The screening method of drugs for tumor precise therapy according to claim 6, characterized in that the drug with different concentrations of the pre-prepared drugs is mixed with the primary tumor cells and injected; or;

injecting the prepared medicines with different concentrations of various medicines and the primary tumor cells into the sample injection electrode group and then mixing; or;

injecting the medicines to be mixed in the medicines into the injection electrode group, and then mixing the primary tumor cells with the medicines with different volumes;

preferably, a sharp pulse wave amplified by a square wave is applied to a medicine concentration generator arranged on the sample injection electrode group to generate medicine liquid with a volume smaller than the volume of the sample injection medicine;

preferably, the drug concentration generator comprises a first electrode strip and a second electrode strip, the width of the second electrode strip is 7-9 times the width of the first electrode strip;

preferably, the width of the first electrode stripes is 10-60 μm, and the width of the second electrode stripes is 300-500 μm.

8. Use of the drug selected by the method for screening a drug for the precise treatment of tumor according to any one of claims 1 to 7 for the preparation of a drug for the treatment of tumor in the precise medical treatment.

9. A digital microfluidic chip for implementing the method for screening precise and individualized drugs for tumors according to any one of claims 1 to 7, comprising a base plate on which an electrode array group and output voltage pads are formed;

the electrode array group comprises at least two sample injection electrode groups used for respectively injecting samples of primary tumor cells and drugs in a drug group to be screened, a mixing electrode used for mixing the injected primary tumor cells and the drugs in the drug group to be screened to obtain drug-loaded cells, a moving electrode group used for moving the drug-loaded cells and a culture electrode group used for moving the drug-loaded cells to a culture area for culture; wherein the mixing electrode is simultaneously adjacent to the feeding electrode group and the moving electrode group, and the culturing electrode group is adjacent to the moving electrode group.

10. A portable digital microfluidic system comprising the digital microfluidic chip of claim 9;

preferably, the portable digital microfluidic system further comprises a handheld device body and a circuit control board, wherein the circuit control board and the digital microfluidic chip are integrated in the handheld device body;

preferably, the circuit control board comprises a power supply, a control button and a signal generator for providing an alternating current driving signal, the signal generator is in communication connection with the control button, the power supply is electrically connected with the control button, and the control button is electrically connected with the digital microfluidic chip;

preferably, the alternating current driving signal is a sine wave of 0.5-10 Vrms;

preferably, the number of the control buttons is multiple and corresponds to the number of the output voltage welding points on the digital microfluidic chip so as to respectively and independently control the voltages of the electrodes with different numbers;

preferably, the circuit control board further comprises a transformer for amplifying the alternating current driving signal and charging the electrodes on the digital microfluidic chip, and the transformer is electrically connected with the control button and the digital microfluidic chip.