CN113832030A - Integrated electrochemical micro-column platform and application thereof - Google Patents
Integrated electrochemical micro-column platform and application thereof Download PDFInfo
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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- G—PHYSICS
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Abstract
The application belongs to the technical field of biological detection, and particularly relates to an integrated cloud electrochemical micro-column platform and application thereof, wherein the integrated cloud electrochemical micro-column platform comprises a cell micro-column culture unit, a micro-droplet detection unit, an integrated cloud electrochemical working unit and a data receiving and analyzing unit, and the cell micro-column culture unit comprises a plurality of micro-columns; the micro-droplet detection unit is provided with a plurality of monitoring pieces, and each micro-column is provided with a monitoring piece; the integrated cloud electrochemical working unit is used for collecting target electrochemical signals of the micro-droplet detection unit and transmitting the target electrochemical signals to the data receiving and analyzing unit; and the data receiving and analyzing unit is used for receiving the information transmitted by the integrated cloud electrochemical working unit and analyzing and judging the information. The integrated cloud electrochemical microcolumn platform is used for cell analysis, drug screening and disease multi-marker detection, and has the advantages of parallelization, high flux, high sensitivity and good accuracy.
Description
Technical Field
The application belongs to the technical field of biological detection, and particularly relates to an integrated electrochemical micro-column platform and application thereof.
Background
The real-time cell culture monitoring can further understand cell proliferation and information interaction mechanism, and is also helpful for further research on disease origin, drug screening and the like. Currently, some companies develop and develop monitoring equipment for cell real-time culture: the CytoSMART system developed by Aichi Intelligent company is used for living cell imaging and observation, a mini microscope is used for observing cells in a culture dish, and due to the limitation of the field of view and the magnification of the microscope, only one batch of cell growth can be observed and only ten times of cell growth can be amplified each time, so that the CytoSMART system is not beneficial to deep observation and research of cells; the CGQ system produced by Aquila Biolabs is a device for monitoring biomass in a shake flask in real time on line, the shake flask does not need to be taken out of a shaking table during measurement, and the operation of the shaking table does not need to be stopped, the device can realize batch signal reading, but the read signal is only limited to the concentration of cells in the flask to analyze the growth dynamics of microorganisms; the ImageXpress Pico system combines CellReporterXpress imaging acquisition and analysis software to detect the cells, and the imaging device system is provided with an environmental control room for controlling and monitoring temperature and CO2And O2Concentration and humidity, while the system provides five fluorescence channels and colorimetric imaging, using invasive fluorescent dyes to monitor cell proliferation, differentiation, compound toxicity and various other cell-based assays; the IncuCyte ZOOM of the Essen company is a long-time, real-time, dynamic and non-injurious living cell imaging analysis platform, an instrument is divided into a microscopic imager and a controller, the microscopic imager can be placed in an incubator, standard consumables with various specifications and sizes are placed in the middle, a microscopic photographic device is arranged below the microscopic imager, cultured cells are continuously monitored through automatic focusing and automatic shooting, and a dynamic growth curve based on cell coverage and cell number is generated through a networked computer. These devices mainly suffer from several problems: 1) inability to high throughput cellular detection; 2) the monitoring cost is high; 3) invasive cell signal reading; 4) insufficient signal sensitivity; 5) the monitoring equipment is separated from the culture equipment, so that the real-time culture monitoring cannot be thoroughly realized.
Disclosure of Invention
The application aims to provide an integrated electrochemical micro-column platform and application thereof, and aims to solve the problems of low cell culture cost, high efficiency, low cell monitoring sensitivity and poor accuracy to a certain extent.
In order to achieve the purpose of the application, the technical scheme adopted by the application is as follows:
in a first aspect, the present application provides an integrated cloud electrochemical micro-column platform, comprising a cell micro-column culture unit, a micro-droplet detection unit, an integrated cloud electrochemical working unit, and a data receiving and analyzing unit,
the cell microcolumn culture unit comprises a plurality of microcolumns for loading micro liquid drops;
the micro-droplet detection unit is provided with a plurality of monitoring pieces, and each micro-column is provided with a monitoring piece for monitoring the change of a target electrochemical signal in the micro-droplet;
the integrated cloud electrochemical working unit is used for collecting target electrochemical signals of the micro-droplet detection unit and transmitting the target electrochemical signals to the data receiving and analyzing unit;
and the data receiving and analyzing unit is used for receiving the information transmitted by the integrated cloud electrochemical working unit and analyzing and judging the information.
Furthermore, the surface of the micro-column is modified with a hydrophobic and hydrophobic organic layer, and the hydrophobic and hydrophobic organic layer comprises a surfactant and/or an adhesive.
Further, the surfactant comprises one or more of perfluorodecyl trichlorosilane, perfluorobutyric acid, fluorochloropentanoic acid, fluorine-containing alkyl sodium sulfonate and perfluorooxaamido quaternary ammonium salt.
Further, the adhesive comprises one or more of cyanoacrylate, acryl based adhesive, phenolic resin adhesive, polyurethane adhesive.
Further, the monitoring part adopts a three-electrode system.
Furthermore, the electrode material of the three-electrode system is one or more of conductive polymer, carbon material fiber, metal wire and nano composite material; and/or
Three electrodes contained in the three-electrode system are arranged in the micro-column, one ends of the three electrodes extend out of the top end of the micro-column and are in contact with micro-droplets loaded on the top end of the micro-column, and the other ends of the three electrodes are connected with the integrated cloud electrochemical working unit.
Furthermore, the surface of the electrode of the three-electrode system is modified with one or more of nanogold, nanosilver, two-dimensional material graphene and quantum dots.
In a second aspect, the application provides an application of an integrated cloud electrochemical micro-column platform, including the integrated cloud electrochemical micro-column platform and the application of the integrated cloud electrochemical micro-column platform prepared by the method in the aspects of high-throughput cell analysis, drug screening and disease multi-marker detection.
In a third aspect, the application provides a method for monitoring and culturing high-throughput cells in real time, which comprises culturing and monitoring cells in real time by using the integrated cloud electrochemical microcolumn platform.
Further, the method for monitoring the high-throughput cell culture in real time comprises the following steps:
dropping a culture medium containing cells on the micro-column of the cell micro-column culture unit for culture treatment;
dripping an electrochemical signal agent into the culture medium, switching on a power supply, and collecting an electrochemical signal of the trace liquid drop detection unit by the integrated cloud electrochemical working unit and transmitting the electrochemical signal to the data receiving and analyzing unit;
and (4) analyzing the data by selecting a current time method or a cyclic voltammetry method on a data receiving and analyzing unit.
The integrated form high in clouds electrochemistry microcolumn platform that this application first aspect provided can realize parallelization, high flux cultivation to the cell with the cell microcolumn culture unit, simultaneously, adopts trace drop detecting element and integrated form high in clouds electrochemistry work cell to carry out non-invasive monitoring to the cell, has improved the degree of accuracy and the sensitivity of cell monitoring greatly, with data receiving analysis unit wireless connection, and transmission information realizes the high in the clouds real-time supervision to the cell.
The application of the integrated form high in the clouds electrochemistry micro-column platform that this application second aspect provided adopts foretell integrated form high in the clouds electrochemistry micro-column platform to carry out cell analysis, drug screening, the many markers of disease detect, have parallelization, high flux, sensitivity height, advantage that the accuracy is good.
The method for monitoring the high-throughput cell real-time culture adopts the integrated cloud electrochemical microcolumn platform to monitor the cell culture, realizes non-invasive monitoring on the cell by using an electrochemical detection technology, greatly improves the sensitivity and accuracy of cell monitoring, adopts the cell microcolumn culture unit to culture the cell, has high throughput, parallelization and intellectualization, and greatly improves the efficiency of cell culture and monitoring.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
Fig. 1 is a schematic diagram of an integrated cloud-based electrochemical microcolumn platform and high-throughput signal transmission according to an embodiment of the present disclosure;
fig. 2 is a schematic diagram illustrating a positional relationship between a three-electrode system and a cell microcolumn culture unit of the integrated cloud-based electrochemical microcolumn platform according to an embodiment of the present disclosure;
fig. 3 is a graph of cell culture versus current time signal variation for the integrated cloud electrochemical micro-column platform provided in the embodiments of the present application;
fig. 4 is a current-voltage diagram of the screening of anticancer drugs by the integrated cloud electrochemical micro-column platform provided in the embodiment of the present application.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present application more clearly apparent, the present application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
In this application, the term "and/or" describes an association relationship of associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a is present alone, A and B are present simultaneously, and B is present alone. Wherein A and B can be singular or plural. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
In the present application, "at least one" means one or more, "a plurality" means two or more. "at least one of the following" or similar expressions refer to any combination of these items, including any combination of the singular or plural items. For example, "at least one (one) of a, b, or c," or "at least one (one) of a, b, and c," may each represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, and c may be single or plural, respectively.
It should be understood that, in various embodiments of the present application, the sequence numbers of the above-mentioned processes do not mean the execution sequence, some or all of the steps may be executed in parallel or executed sequentially, and the execution sequence of each process should be determined by its function and inherent logic, and should not constitute any limitation to the implementation process of the embodiments of the present application.
The terminology used in the embodiments of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in the examples of this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The weight of the related components mentioned in the description of the embodiments of the present application may not only refer to the specific content of each component, but also represent the proportional relationship of the weight among the components, and therefore, the content of the related components is scaled up or down within the scope disclosed in the description of the embodiments of the present application as long as it is scaled up or down according to the description of the embodiments of the present application. Specifically, the mass in the description of the embodiments of the present application may be in units of mass known in the chemical industry, such as μ g, mg, g, and kg.
The terms "first" and "second" are used for descriptive purposes only and are used for distinguishing purposes such as substances from one another, and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. For example, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX, without departing from the scope of embodiments of the present application. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature.
The term "PDMS" is an abbreviation for "polydimethysiloxane", representing Polydimethylsiloxane; the term "PMMA" is an abbreviation for "poly methyl methacrylate", which means polymethyl methacrylate; the term "PLA" denotes polylactic acid; "MCF-7 cells" means breast cancer cells.
In a first aspect, the present application provides an integrated cloud electrochemical micro-column platform, comprising a cell micro-column culture unit, a micro-droplet detection unit, an integrated cloud electrochemical working unit, and a data receiving and analyzing unit,
the cell microcolumn culture unit comprises a plurality of microcolumns for loading micro liquid drops;
the micro-droplet detection unit is provided with a plurality of monitoring pieces, and each micro-column is provided with a monitoring piece for monitoring the change of a target electrochemical signal in the micro-droplet;
the integrated cloud electrochemical working unit is used for collecting target electrochemical signals of the micro-droplet detection unit and transmitting the target electrochemical signals to the data receiving and analyzing unit;
and the data receiving and analyzing unit is used for receiving the information transmitted by the integrated cloud electrochemical working unit and analyzing and judging the information.
The integrated cloud electrochemical micro-column platform provided by the first aspect of the embodiment of the invention has the following advantages:
cell culture parallelization, high-throughput: the cell microcolumn culture unit comprises a plurality of microcolumns, each microcolumn can gather a micro-drop respectively, so each micro-drop can be used as an independent cell culture dish, thereby parallelizing and culturing a large number of cells in a high-throughput manner, the cost is low, the time is short, each microcolumn platform only needs 10-100 mu L of culture medium, the culture medium required by a batch of cell culture of the microcolumn platform is only equivalent to the amount of cells cultured in one culture dish at present, and the cell research cost and time are greatly reduced;
non-invasive signal acquisition of cells: according to the embodiment of the invention, the integrated cloud electrochemical working unit and the micro-droplet detection unit are adopted to collect the electric signals in the cell culture process, so that the traditional observation of the cells by adding fluorescent dye, the resistance signal fluctuation caused by collecting the cell self-redox signals or cell adherent proliferation and the like is avoided, the influence of external factors on the cell signals is greatly avoided, and the accuracy and the authenticity of monitoring signals are improved;
the sensitivity of monitoring the real-time cell culture is improved: the traditional cell observation usually utilizes optical signal acquisition, such as fluorescence, colorimetry or direct microscopic visual observation, the signal acquisition means has low sensitivity and can only locally observe the cell number and the cell death and activity.
The remote real-time culture monitoring is really realized: the method comprises the steps of combining an integrated cloud electrochemical working unit and hundreds of monitoring units of cell microcolumn culture units to construct a multi-channel intelligent test system, wherein the cell real-time monitoring data of each microcolumn can realize cloud monitoring and analysis, the research and development iteration period and the closed-loop time are greatly shortened, and the cell uninterrupted real-time culture monitoring is really realized.
In the embodiment of the present application, the cell microcolumn culture unit included in the integrated cloud-based electrochemical microcolumn platform includes a substrate and a plurality of microcolumns extending from the surface of the substrate, as shown in fig. 1, the cell microcolumn culture unit is peeled off from the mold, embedding a three-electrode system in each micro-column, connecting the three-electrode system with the integrated cloud electrochemical working unit, adhering the integrated cloud electrochemical working unit to the surface of the substrate away from the micro-column, as shown in fig. 2, each microcolumn 2 is disposed perpendicular to the substrate 1, the electrodes 3 in the three-electrode system are disposed in parallel inside the microcolumn 2, and the top of each electrode 3 is flush with the top of the microcolumn 2, when the micro-droplet 4 is loaded on the top of the micro-column 2, each electrode 3 of the three-electrode system can be contacted with the micro-droplet 4, so that a target electrochemical signal in the micro-droplet 4 can be monitored.
In the embodiment of the present application, the prepolymer for preparing the micro-column may be variously selected according to the acid-base environment, the temperature, the solution polarity, etc. used in the micro-column, including but not limited to Polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polylactic acid (PLA), the cell micro-column culture unit platform has excellent flexibility, the micro-column droplet will not fall off under stretching, bending and twisting, and in addition, the adhesion of the micro-column surface to the droplet, the flexibility and hardness of the micro-column, etc. may be achieved by adjusting the ratio of the prepolymer to the curing agent.
In the embodiment of the application, the diameter of the microcolumn is 0.5-5mm, the height is 0.5-5mm, the microcolumn in the range is easier to polymerize droplets, and the droplets are too close to each other due to too large diameter of the microcolumn, so that the interference and stirring disturbance among the droplet microreactors are easily caused.
In the embodiments of the present application, the cell microcolumn culture unit may be a square array of 2 × 2, 3 × 3, 9 × 9, 10 × 10, etc., or may be a rectangular array of 2 × 4, 3 × 7, 10 × 10, etc., or may be an array in which a plurality of microcolumns are arranged in other shapes, including but not limited to a polygon such as a triangle, a diamond, a hexagon, etc., wherein the number of the microcolumns in the cell microcolumn culture unit is not limited and is set according to the size of the cell microcolumn culture unit, in the specific example of the application, the number of the micro-columns in the cell micro-column culture unit can be 96, 384 or 1000, 96, 384 or 1000 high-throughput cell cultures and signal real-time culture monitoring can be simultaneously carried out, in one embodiment of the present application, 96, i.e., 8 × 12 rectangular cell microcolumn culture units are preferred.
In the embodiment of the application, the surface of the micro-column is further modified with a hydrophobic and hydrophobic organic layer, and the hydrophobic and hydrophobic organic layer comprises a surfactant and/or a binder, so that stable storage and operation of a complex liquid system such as a culture medium and the like can be realized.
In the embodiment of the application, the surfactant comprises one or more of perfluorodecyl trichlorosilane, perfluorobutyric acid, fluorochloropentanic acid, sodium fluoroalkyl sulfonate and perfluorooxaamido quaternary ammonium salt, and the surfactant has lower surface energy and has the performance of a hydrophobic and hydrophobic organic reagent, so that the micro-column has the characteristics of water resistance, pollution prevention, adhesion resistance, oxidation resistance, corrosion resistance and self-cleaning, and simultaneously has the performance of preventing current conduction, and the phenomenon of short circuit caused by the contact of the micro-column and a monitoring unit of a micro-droplet detection unit is prevented.
In the embodiment of the application, the adhesive comprises one or more of cyanoacrylate, acryl-based adhesive, phenolic resin adhesive and polyurethane adhesive, and the adhesive has the characteristics of low VOC content, low or no environmental pollution, non-combustion, simple bonding process, excellent low temperature resistance, excellent stability and the like.
In the embodiment of the present application, the micro droplet detecting unit adopts a two-electrode system or a three-electrode system, preferably a three-electrode system, and the three-electrode system introduces a reference electrode for stabilizing the working electrode on the basis of a conventional two-electrode system (working electrode and auxiliary electrode), so that a large error of the electrode potential caused by the polarization current can be eliminated.
In an embodiment of the present application, the electrode material of the micro droplet detection unit is one or more of a conductive polymer, a carbon fiber, a metal wire, and a nanocomposite material, and in a specific embodiment of the present application, the electrode material of the micro droplet detection unit is a gold-silver-platinum metal wire, wherein the working electrode is a gold wire, and does not affect cells during a cell culture process; the reference electrode adopts silver wires, has good reproducibility and low temperature coefficient and is a reversible electrode; the auxiliary electrode adopts a platinum wire, so that the property is stable and the resistance is small.
In the embodiment of the application, in order to meet the requirements of monitoring of special monitored objects and high-sensitivity analysis at the same time, on the basis that the gold-silver-platinum wires are used as a trace liquid drop detection unit, multifunctional modification is carried out on the electrodes so as to improve the sensitivity and the practicability of the small-sized microcolumn sensor, specifically, one or more of nano-gold, nano-silver, two-dimensional material graphene and quantum dots are adopted to modify the electrode surfaces of the three electrodes, so that the whole electrode can be modified, and only the monitoring unit can be modified.
In an embodiment of the present application, a method for preparing an integrated cloud electrochemical micro-column platform includes the following steps: preparing a cell microcolumn culture unit, axially penetrating a micro-droplet detection unit, namely a three-electrode system through a microcolumn of the cell microcolumn culture unit, enabling the detection end of the micro-droplet detection unit to be flush with or slightly higher than the upper end of the microcolumn and be used for contacting with micro-droplets loaded by the microcolumn so as to collect electrochemical signals in the micro-droplets, and enabling the other end of the micro-droplet detection unit to be connected with an integrated cloud electrochemical working unit.
In an embodiment of the application, the data receiving and analyzing unit includes, but is not limited to, a mobile phone, a computer, and a tablet, and software or a program wirelessly connected to the integrated cloud electrochemical working unit may be installed, so as to perform real-time monitoring on cells in micro droplets on the micro-column platform by selecting a monitoring mode on the data receiving and analyzing unit.
In the embodiment of the present application, the method for preparing the cell microcolumn culture unit may be selected from one of a 3D printing method, a template stripping method, and a milling machine processing method, and the specific steps are as follows: and (3) mixing the prepolymer and the curing agent according to the ratio of 10:1, pouring the mixture on a template with arrayed holes, drying the mixture in an oven at 80 ℃ for 3 hours, and stripping the solidified mixture from the template to obtain the arrayed micro-column platform.
The application provides an application of integrated form high in clouds electrochemistry micro-column platform in the second aspect, including the application in the aspect of foretell integrated form high in clouds electrochemistry micro-column platform high flux cell analysis, drug screening, the many markers of disease detect, have parallelization, high flux, sensitivity height, advantage that the accuracy is good.
The third aspect of the application provides a method for monitoring the real-time culture of high-throughput cells, which comprises the step of monitoring the culture of the cells by using the integrated cloud electrochemical microcolumn platform.
In an embodiment of the present application, the method for monitoring the real-time culture of the high-throughput cells includes the following steps:
dropping a culture medium containing cells on the micro-column of the cell micro-column culture unit for culture treatment;
dripping an electrochemical signal agent into the culture medium, switching on a power supply, and collecting an electrochemical signal of the trace liquid drop detection unit by the integrated cloud electrochemical working unit and transmitting the electrochemical signal to the data receiving and analyzing unit;
and (4) analyzing the data by selecting a current time method or a cyclic voltammetry method on a data receiving and analyzing unit.
The method of high flux cell real-time cultivation control that this application second aspect provided adopts foretell integrated form high in clouds electrochemistry microcolumn platform to cultivate the control to the cell, utilizes electrochemical detection technique to realize the non-invasive monitoring to the cell, has improved the sensitivity and the degree of accuracy of cell monitoring greatly, adopts cell microcolumn culture unit to cultivate the cell simultaneously, and high flux, parallelization, intellectuality have improved the efficiency of cell cultivation and monitoring greatly.
In the embodiment of the present application, the number of the cell microcolumn culture units includes, but is not limited to, 64, 96, 360, 640, 1000, each microcolumn can be used as an independent cell culture dish, and high-throughput, parallelized cell culture and real-time monitoring can be realized.
In the embodiment of the application, the electrochemical signaling agent adopts organic salt with redox property, and nonspecific phosphatase on the cell membrane catalyzes the organic salt to generate redox decomposition reaction, so that the electrochemical signal in the trace liquid drop is enhanced, the observation of the cell by adding fluorescent dye in the prior art is avoided, the influence of external factors on the cell signal is greatly avoided, and the accuracy and the authenticity of the monitoring signal are improved.
The current time method is that a constant voltage is applied to an electrode in the experimental process, the result of the current changing along with the time is collected, the correlation condition of the current and the cell differentiation in the micro liquid drop is analyzed, and the increase and decrease condition of the cell number in the micro liquid drop is obtained.
The cyclic voltammetry controls the potential of an electrode at different rates in the experimental process, and repeatedly scans the electrode once or for multiple times in a triangular waveform along with time, the potential range enables different reduction and oxidation reactions to alternately occur on the electrode, a current-potential curve is recorded, and the reversibility of the electrode reaction, the possibility of adsorption or new phase formation of intermediates and phase boundaries, the property of coupling chemical reaction and the like can be judged according to the curve shape.
In order to make the above implementation details and operations of the present application clearly understood by those skilled in the art, and to make the progress of the integrated cloud electrochemical micro-column platform and the preparation method thereof significantly apparent, the above technical solutions are illustrated by a plurality of examples below.
Example 1: preparation of integrated cloud electrochemical micro-column platform
Preparing a micro-column platform: as shown in fig. 1, a cell micro-column culture unit of Polydimethylsiloxane (PDMS) with an array number of 12 × 8 is prepared by a template stripping method, a prepolymer and a curing agent are mixed and poured on a template with array holes according to a ratio of 10:1, the template is dried in an oven at 80 ℃ for 3 hours, the prepolymer and the curing agent are stripped off the template after curing to obtain a micro-column platform with an array, a surfactant such as perfluorodecyl trichlorosilane and an adhesive are mixed and then coated on the surface of the micro-column, and the surface has certain hydrophobic and hydrophobic properties after drying, and can be used for fixing a culture medium for cell culture and real-time culture monitoring;
integrating the microcolumns with an integrated workstation: embedding a micro-droplet detection unit in each microcolumn, guiding an electrode wire to axially penetrate through the microcolumn from the bottom of a substrate of a cell microcolumn culture unit by using a needle, and shearing the electrode wire to enable the electrode wire to be flush with the upper end of the microcolumn, wherein a gold wire is used as a working electrode, a silver wire is used as a reference electrode, a platinum wire is used as an auxiliary electrode, and the electrode wire at the bottom of the substrate is welded with an integrated cloud electrochemical working unit;
the data receiving and analyzing unit adopts a mobile phone and can be in wireless connection with the integrated cloud electrochemical working unit to receive information transmitted by the integrated cloud electrochemical working unit and analyze and process the information.
Example 2: application of integrated cloud electrochemical micro-column platform
High throughput cell real-time culture monitoring (current time method):
dropping a culture medium containing cells on a cell microcolumn culture unit of a 12 x 8 array microcolumn to form micro liquid drops, culturing the cells, adding 4-aminophenyl sodium phosphate as an electrochemical signal agent, switching on a power supply, wirelessly connecting an integrated cloud electrochemical working unit with a data receiving and analyzing unit, converting the electrochemical signals detected by the micro liquid drop detection unit into wireless signals by the integrated cloud electrochemical working unit, transmitting the wireless signals to the data receiving and analyzing unit, namely a mobile phone or a computer, analyzing the received information by selecting a current time method on the mobile phone or the computer, because nonspecific phosphatase of cell membranes can oxidize the 4-aminophenyl sodium phosphate into 4-aminophenol, the 4-aminophenol can be subjected to redox under the electrochemical action, and the cells can proliferate and differentiate along with the increase of time, the 4-aminophenylphosphate sodium salt is decomposed to promote the enhancement of electrochemical signals, as shown in figure 3, the electrochemical signals are continuously enhanced along with the prolonging of the cell culture time, which shows that the number of cells in a culture medium on a cell microcolumn culture unit is more and more, the culture and detection mode does not need to take out the cells in the culture process for detection, does not need to add reagents in the subsequent process, has sensitive signals, and can realize the processes of culturing, monitoring the differentiation, canceration and the like of the cells in real time.
Anti-cancer drug screening (cyclic voltammetry):
dropping a culture medium containing MCF-7 cells (breast cancer cells) on a cell microcolumn culture unit of a 12 x 8 array microcolumn to form micro liquid drops, carrying out cell culture, adding 4-aminophenylphosphate sodium salt as an electrochemical signal agent, switching on a power supply, wirelessly connecting an integrated cloud electrochemical working unit with a data receiving and analyzing unit, converting the electrochemical signals detected by a micro liquid drop detection unit into wireless signals by the integrated cloud electrochemical working unit, transmitting the wireless signals to the data receiving and analyzing unit, namely a mobile phone or a computer, selecting a cyclic voltammetry scanning on the mobile phone or the computer, increasing the resistance when the cells are adhered to the surface of an electrode along with the proliferation of the cells, reducing the electrochemical signals, realizing the screening of the anti-cancer drugs on a microcolumn platform by utilizing the point, adding the MCF-7 cells (breast cancer cells) on the microcolumn and respectively adding diclofenac, vast manganese zinc, okadaic acid, ascorbic acid and phosphate buffer are used as blanks, electrochemical signal detection is carried out by using a differential pulse method after 24-hour cell culture, as shown in figure 4, the electrochemical signals of four drugs are large, which shows that the drugs can kill cancer cells, the blank group of cells proliferate fast, the electrode resistance is large, so that the signals are small, the integrated cloud micro electrochemical micro-column platform high-flux cell monitoring advantage is utilized, the parallel analysis of multiple groups of drugs and the simultaneous analysis of multiple drugs can be simultaneously completed, the evaluation of new drugs is greatly shortened while the accuracy of the drug treatment effect is ensured, and a new idea is provided for the screening of anti-cancer drugs.
The present invention is not intended to be limited to the particular embodiments shown and described, but is to be accorded the widest scope consistent with the principles and novel features herein disclosed.
Claims (10)
1. An integrated cloud electrochemical micro-column platform is characterized by comprising a cell micro-column culture unit, a micro-droplet detection unit, an integrated cloud electrochemical working unit and a data receiving and analyzing unit,
the cell microcolumn culture unit comprises a plurality of microcolumns for loading micro liquid drops;
the micro-droplet detection unit is provided with a plurality of monitoring pieces, and each micro-column is provided with the monitoring piece and is used for monitoring the change of a target electrochemical signal in the micro-droplet;
the integrated cloud electrochemical working unit is used for collecting the target electrochemical signals of the micro-droplet detection unit and transmitting the target electrochemical signals to the data receiving and analyzing unit;
and the data receiving and analyzing unit is used for receiving the information transmitted by the integrated cloud electrochemical working unit and analyzing and judging the information.
2. The integrated cloud-based electrochemical micro-column platform according to claim 1, wherein the surface of the micro-column is further modified with a hydrophobic organic layer, and the hydrophobic organic layer comprises a surfactant and/or a binder.
3. The integrated cloud-based electrochemical microcolumn platform of claim 2, wherein the surfactant comprises one or more of perfluorodecyltrichlorosilane, perfluorobutyric acid, fluorochloropentanoic acid, sodium fluoroalkylsulfonate, and perfluorooxanamide quaternary ammonium salt.
4. The integrated cloud-based electrochemical microcolumn platform of claim 2, wherein the adhesive comprises one or more of cyanoacrylate, an acryl-based adhesive, a phenolic adhesive, a polyurethane adhesive.
5. The integrated cloud electrochemical microcolumn platform of claim 1, wherein the monitoring member is a three-electrode system.
6. The integrated cloud-based electrochemical micro-column platform according to claim 5, wherein the electrode material of the three-electrode system is selected from one or more of conductive polymer, carbon fiber, metal wire, and nanocomposite; and/or
And three electrodes contained in the three-electrode system are arranged in the micro-column, one end of each electrode extends out of the top end of the micro-column and is in contact with the micro-droplets loaded on the top end of the micro-column, and the other end of each electrode is connected with the integrated cloud electrochemical working unit.
7. The integrated cloud-based electrochemical micro-column platform according to claim 5 or 6, wherein the electrode surface of the three-electrode system is modified with one or more of nanogold, nanosilver, two-dimensional material graphene, and quantum dots.
8. An application of the integrated cloud electrochemical micro-column platform of any one of claims 1 to 7 in high-throughput cell analysis, drug screening, and multi-marker detection of diseases.
9. A method for high-throughput real-time cell culture monitoring, which is characterized in that the integrated cloud electrochemical micro-column platform of any one of claims 1 to 7 is used for carrying out culture and real-time monitoring treatment on cells.
10. The method for high throughput real-time cell culture monitoring according to claim 9, comprising the steps of:
dropping a culture medium containing cells on the micro-column of the cell micro-column culture unit for culture treatment;
dripping an electrochemical signal agent into the culture medium, switching on a power supply, and collecting an electrochemical signal of the trace liquid drop detection unit by the integrated cloud electrochemical working unit and transmitting the electrochemical signal to the data receiving and analyzing unit;
and (4) analyzing the data by selecting a current time method or a cyclic voltammetry method on a data receiving and analyzing unit.
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