CN115895859A - Microarray chip for detecting biological sample and detection method - Google Patents

Abstract

A biological sample detection microarray chip and a detection method are provided, wherein the biological sample detection microarray chip comprises a first substrate structure, a second substrate structure, a reaction area positioned between the first substrate structure and the second substrate structure, and a detection module. The first substrate structure comprises more than one electric regulation and control module, the second substrate structure comprises more than one temperature control module, and the detection module comprises a light sensing element. The microarray chip is used for detecting biological samples, the whole process is automatically executed by equipment under the control of external signals, the error of manual operation is reduced, the quantity of the required biological samples is less, the consumption of biological reagents can be greatly reduced, and the detection efficiency is improved.

Description

Microarray chip for detecting biological sample and detection method

Technical Field

The present invention relates to microfluidic technology, and is especially one kind of microarray chip for detecting biological sample and its usage.

Background

Direct detection is difficult because of the low concentration of the molecules to be detected in the biological sample and the very weak biological signal. Commonly used methods, for example, detection of nucleic acid molecules, typically involve cyclic amplification of the nucleic acid molecule such that the biological signal is enhanced to a detectable level, and detection of the biological signal using a particular detection device. These detection devices usually require complex heating and temperature control devices, and the overall device is bulky. The detection process also involves the use of a large amount of samples and reagents, and has the disadvantages of complex technical operation, large sample and time requirement, easy generation of manual errors, inaccurate test results of different batches, poor repeatability and the like.

The microfluidic technology is a technology capable of controlling fluid at a micrometer scale, and can integrate basic operation units such as sample preparation, reaction, separation, detection and the like on a chip at a centimeter scale. The technology greatly reduces the sample cost and improves the detection efficiency. In recent years, the method has attracted much attention and is applicable to fields such as biology, chemistry, and medical treatment.

Disclosure of Invention

Based on the above-mentioned related technical background and problems, the present application provides a biological sample detection microarray chip and a method for applying the same to biological sample detection. And provides a microarray chip based on the above.

In one aspect of the present application, there is provided a biological sample detection microarray chip comprising:

a first substrate structure having a first surface and a second surface,

the first substrate structure comprises a first substrate, and more than one electrical conditioning module on the first substrate, the electrical conditioning module comprising one or more working electrodes;

a second substrate structure having a first substrate and a second substrate,

the second substrate structure comprises a second substrate and at least one temperature control module positioned on the second substrate, the temperature control module comprising a temperature change member;

a reaction region between the first substrate structure and the second substrate structure; and one or more detection modules comprising a light sensing element;

the surface of the electric regulation module facing the reaction area is coated with a functional membrane, and under the regulation and control of an electric field, the hydrophilic or hydrophobic state of the functional membrane can be changed.

In another aspect, the present application also provides a method for detecting a biological sample, comprising the steps of:

a) Injecting a biological sample into the reaction region of a biological sample detection microarray chip provided herein to form microdroplets;

b) Controlling the temperature of the temperature control module so that at least one temperature-stable temperature zone is formed in the reaction area;

c) Under the action of the electric regulation and control module, the micro-droplets move to a specified temperature zone or stay in the specified temperature zone as required;

d) Under the action of the electric regulation and control module, the micro liquid drops move to the position corresponding to the detection module;

e) And receiving the signal detected by the detection module.

In another aspect, the present application also provides use of the microarray chip of the present application for at least one of molecular diagnostics, biological sample analysis, chemical sample analysis, food analysis, and environmental analysis.

The module and the detection module required by temperature control of the biological sample are integrated on one chip, so that the volume of the device can be greatly reduced. Because a plurality of temperature control modules, electric regulation modules and detection modules can be designed on the chip, a plurality of channels can be designed for detecting biological samples, the whole process is automatically executed by equipment under the control of external signals, the error of manual operation is reduced, and the accuracy and the repeatability of test results are improved;

the chip has little biological sample amount required for detection, and can greatly reduce the consumption of biological reagents, thereby having great advantage on the detection cost;

the temperature control module, the electric regulation and control module and the detection module of the chip are distributed on different substrates. Since the functional modules are distributed on the substrates at both sides of the reaction chamber, mutual interference between the modules can be avoided. On the other hand, because the functional modules on the single substrate are reduced, the metal interconnection layer does not need to be increased, and the standard production process of the existing thin film transistor can be adapted, so that the development period can be greatly shortened, and the robustness can be greatly improved.

Additional features and advantages of the present application will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the present application. Other advantages of the present application may be realized and attained by the instrumentalities and combinations particularly pointed out in the specification and the drawings.

Drawings

The accompanying drawings are included to provide an understanding of the present disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the principles of the disclosure and not to limit the disclosure.

FIG. 1A is a side view of a biological sample detection microarray chip in one embodiment of the present application;

FIG. 1B is a diagram of a cartridge of a biological sample detection microarray chip in one embodiment of the present application;

FIG. 2 is a side view of an electrical control module and a detection module of a biological sample detection microarray chip according to an embodiment of the present application, wherein the electrical control module and the detection module are fabricated by a thin film transistor process;

FIG. 3 is a side view of an electrical conditioning module of a biological sample detection microarray chip according to another embodiment of the present application, wherein the electrical conditioning module is fabricated by a printed circuit board process;

FIG. 4 is a schematic diagram illustrating the structure and detection of a microarray chip for biological sample detection according to one embodiment of the present application, wherein the microarray chip is fabricated by a printed circuit board process;

fig. 5 is a side view of a temperature control module of a biological sample detection microarray chip in one embodiment of the present application.

Detailed Description

Polymerase Chain Reaction (PCR) and real-time fluorescent quantitation (q-PCR) techniques are commonly used techniques for amplifying DNA fragments. The basic principle of PCR technology is similar to the natural replication process of DNA, and its specificity depends on oligonucleotide primers complementary to both ends of the target sequence. PCR consists of three basic reaction steps of denaturation-annealing-extension: denaturation of template DNA: heating the template DNA to about 95 ℃ for a certain time, dissociating the double-stranded template DNA or the double-stranded DNA formed by PCR amplification to form a single strand so that the single strand can be combined with the primer to prepare for the next reaction; annealing (annealing) of template DNA to primer: heating and denaturing the template DNA into single strands, cooling to about 55 ℃, and pairing and combining the primers and the complementary sequences of the template DNA single strands; extension of the primer: the DNA template-primer combination is used for synthesizing a new semi-reserved replication chain which is complementary with a template DNA chain by taking dNTP as a reaction raw material and a target sequence as a template according to the base complementary pairing and semi-reserved replication principles under the action of DNA polymerase (such as Taq DNA polymerase) at 72 ℃. Repeating the three processes of denaturation, annealing and extension, more 'semi-reserved replication chains' can be obtained, and the new chains can become templates of the next cycle. The amplification of the target gene to be amplified can be amplified by millions of times within 2 to 3 hours after each cycle is completed. A critical part of PCR technology is the need for precise temperature control.

As a basic technology of modern molecular biology, PCR technology is widely used in analysis of biological samples, infectious disease screening, food analysis, and chemical substance analysis. These application scenarios often involve multi-sample detection, but PCR is inefficient and costly to perform multiple sample screens. Therefore, it is necessary to develop a system capable of performing multi-sample detection screening in real time and high throughput to meet the detection screening requirements in practical application scenarios. This system cannot be a simple linear superposition of PCR devices to increase the detection throughput, which would otherwise result in a large volume and high cost.

In order to miniaturize the device, the heating temperature control module, the biological sample control module and the detection module can be integrated on one chip, so that the volume of the device can be greatly reduced, and high-throughput biological sample detection can be performed. One design approach may be to integrate all modules on the same substrate to improve production efficiency and integration. However, since the operating voltage and the operating current required by each module are not very same, there may be a case where the modules interfere with each other, resulting in poor chip robustness. On the other hand, since all modules are integrated on one substrate, an additional metal interconnection layer is inevitably added, which results in a longer production cycle of the chip. And the production process route of the existing thin film transistor is relatively fixed, and the excessive metal interconnection layers can cause that the existing production line cannot be matched with the chip production, so that the chip processing is difficult.

Based on the above situation, the present invention provides a method for researching a biological sample with low cost, in which a heating module and a sample manipulation module are separated and respectively manufactured on substrates at two sides of a reaction chamber. Since the functional modules are distributed on the substrates on both sides of the reaction region, mutual interference between the modules can be avoided. On the other hand, because the functional modules on the single substrate are reduced, the metal interconnection layer does not need to be increased, and the standard production process of the existing thin film transistor can be adapted, so that the development period can be greatly shortened, and the robustness can be improved. On the other hand, the flux number can be greatly improved due to the adoption of the semiconductor process, multi-component detection can be carried out at the same time, and the detection efficiency is improved.

In addition to applications to detection of nucleic acid samples by PCR, the biological sample detection microarray chip of the present application can also be applied to more biological samples (including proteins, cells, etc.) or biological processes that are detected by monitoring changes in optical properties.

In one aspect of the present application, there is provided a biological sample detection microarray chip comprising:

a first substrate structure having a first surface and a second surface,

the first substrate structure comprises a first substrate and more than one electric regulation and control module positioned on the first substrate, wherein the electric regulation and control module comprises one or more working electrodes;

a second substrate structure having a first substrate and a second substrate,

the second substrate structure comprises a second substrate and at least one temperature control module positioned on the second substrate, wherein the temperature control module comprises a temperature change element;

a reaction region between the first substrate structure and the second substrate structure; and

one or more detection modules including a light sensing element;

the surface of the electric regulation module facing the reaction area is coated with a functional membrane, and under the regulation and control of an electric field, the hydrophilic or hydrophobic state of the functional membrane can be changed.

In some embodiments, the biological sample detection microarray chip further comprises one or more channels for providing fluid communication between the biological sample detection microarray chip and the outside. In some embodiments, the material of the channels comprises a wicking material, such as capillaries or fibers. In some embodiments, the channels may be of other materials, such as glass, high molecular weight polymers, and the like.

In some embodiments, at least one of the first substrate and the second substrate is made of a transparent material. In some embodiments, the first substrate and the second substrate are the same or different in material, including glass, polyimide (PI), polyethylene naphthalate (PEN), polyethylene terephthalate (PET), and Printed Circuit Board (PCB) board substrates. In some embodiments, the electrical conditioning module and the detection module can be fabricated on a first substrate, and the temperature control module can be fabricated on a second substrate, where the first substrate is made of a transparent material. In some embodiments, the detection module may also be disposed on the second substrate, and the second substrate is made of a transparent material.

In some embodiments, at least a portion of a surface of the second substrate structure facing the reaction region is coated with a functional film. In some embodiments, at least a portion of a surface of the second substrate structure facing the reaction region is coated with a functional film. The functional film is in a hydrophobic state when no electric field is applied, and becomes a hydrophilic state when an electric field exceeding a certain strength is applied. In some embodiments, the functional membrane is a membrane comprised of a fluoropolymer, preferably polytetrafluoroethylene. In some embodiments, the material constituting the functional membrane includes, but is not limited to, silicone oil, cytop, teflon, and the like.

In some embodiments, the reaction area is used for amplification, biochemical reactions, and movement of a biological sample.

In some embodiments, the electrical regulation module further comprises a thin film transistor or a metal trace. In some embodiments, the electrical tuning module may be either an active design (including a thin film transistor) or a passive design (not including a thin film transistor), and includes at least one conductive working electrode for applying an electric field to the functional membrane to adjust the hydrophilic or hydrophobic state of the functional membrane.

In some embodiments, the thin film transistor, the working electrode and the functional film layer may be stacked in sequence, from bottom to top, in sequence as a thin film transistor-working electrode-functional film layer. Each small area (pixel) is composed of a thin film transistor, and can apply voltage (current) signals to the working electrode of the upper layer and perform timing control on the signals; the working electrode is used for controlling and giving no voltage (current) signal and carrying out hydrophobic-hydrophilic state switching control on a small area (pixel) of an upper functional film layer; the uppermost part is a functional film layer, and the film layer is in a hydrophobic state when no voltage (current) is loaded below the film layer; the membrane layer is switched to a hydrophilic state after a voltage (current) is applied to the membrane layer.

In some embodiments, when a target droplet of a certain size is located on the surface of the working electrode and the functional film layer, the droplet can be located right above at least one electrode in two dimensions on the surface of the electric control module at any time. When no voltage (or current) signal is applied to the electrode where the liquid drop is positioned by the current signal routing, the surface (interface) of the film layer above the electrode is in a hydrophobic state; the next electrode in the direction of droplet travel applies a voltage (or current) signal to the electrode through the signal trace, and the membrane layer surface (interface) above the electrode is switched to be hydrophilic. The droplet is thus "attracted" to the hydrophilic electrode side along the hydrophobic electrode, achieving the most basic "step" movement. By analogy, the continuous one-dimensional motion of the liquid drops along the preset path in a step mode can be realized by sequentially applying working signals to the electrodes passing through the liquid drop moving path. In some embodiments, the control circuitry applies a time-varying voltage via the input-output circuitry to the set of working electrodes through respective electronic switches (which may be thin film transistors or MOS circuits in the substrate or off-chip) to generate an electric field across the droplets to move the droplets along the path.

In some embodiments, under the control of the working electrode and the functional film of the electrical control module, a variety of actions of the microdroplets may be achieved, including but not limited to: movement of droplets, in-situ oscillation, coalescence between droplets, tearing of large droplets to small droplets, and the like. In some embodiments, according to the specific route of the biological experiment, different reagents (or samples) added externally can be matched to achieve the corresponding biological function requirement.

In some embodiments, the working electrode is controlled by a thin film transistor or by metal traces connecting other external components. In some embodiments, the working electrode is a light transmissive conductive electrode comprising Indium Tin Oxide (ITO), indium Zinc Oxide (IZO), and silver nanowires (AgNW).

In some embodiments, the electrical control module further includes an insulating layer, which is typically SiOx (silicon oxide) or SiNx (silicon nitride), or a stacked structure of the two. In some embodiments, in an electrical modulation module fabricated using a thin film transistor process, if the semiconductor layer is an organic semiconductor, the second insulating layer and the third insulating layer may be organic insulating layers. In some embodiments, the electrical modulation control module manufactured by the PCB process has a metal material of Cu or Au, and the insulating layer is a resin material.

In some embodiments, the temperature change member in the temperature control module is a metal member that can be heated by applying an electric current, and the material of the metal member includes: mo, ti, mo-Al alloys, ti-Al alloys, au and Ag. In some embodiments, the temperature change element in the temperature control module can be other heating elements whose temperature can be controlled. In some embodiments, the temperature control module may further comprise a temperature sensing element for detecting the temperature of the local region.

In some embodiments, the temperature change member in the temperature control module is formed of a metal film formed by a thin film deposition process, and the region of the metal film can be heated by applying a current to the metal film. Since the resistance value of the metal film and the temperature show a linear relationship, the temperature value of the area can be obtained by monitoring the resistance of the metal film.

In some embodiments, the temperature change element in the temperature control module is formed from a thin metal film layer, and upon application of an electric current to the metal, joule heat is generated in the area of the metal film layer, thereby achieving a localized temperature increase. On the other hand, the resistance of the thin film metal changes with the change of the temperature, so that the current temperature value can be obtained by monitoring the resistance of the thin film metal. The stable temperature zone can be obtained by PID (Proportional Integral Derivative) control. In some embodiments, another thin-film metal layer located near the thin-film metal layer of the temperature change element can be added as the temperature-sensing element, and the current temperature value can be obtained by monitoring the resistance of the temperature-sensing element. The film metal layer of the temperature change element and the film metal layer of the temperature sensing element can be the same layer of metal or different layers of metal films. In some embodiments, the material of the temperature-sensitive element includes: mo, ti, mo-Al alloy, ti-Al alloy, au and Ag.

In some embodiments, when light is sensed, the electrical characteristics of the light sensing elements in the detection module may change, including current, voltage, resistance, and capacitance.

In some embodiments, the light sensing element in the detection module may be a photosensitive device fabricated by a thin film transistor process, such as amorphous silicon, polysilicon, oxide, organic semiconductor, PN junction, etc., or may be a photodiode fabricated by a Complementary Metal Oxide Semiconductor (CMOS) process, a photomultiplier tube, etc. of an integrated circuit.

In some embodiments, the light sensing element is a semiconductor element, and this property can be used to achieve light detection because light can change the energy band of the semiconductor, causing a change in current. In some embodiments, other light sensing elements of similar design and construction are included.

In some embodiments, the detection module of the present application may also be removed and the optical change observable may be detected using external light detection means.

In another aspect, the present application also provides a method for detecting a biological sample, comprising the steps of:

a) Injecting a biological sample into a reaction area of a biological sample detection microarray chip provided by the present application to form micro droplets;

b) Controlling the temperature of the temperature control module so that at least one temperature-stable temperature zone is formed in the reaction area;

c) Under the action of the electric regulation and control module, the micro-droplets move to a specified temperature zone or stay in the specified temperature zone as required;

d) Under the action of the electric regulation module, the micro liquid drops move to the position corresponding to the detection module;

e) And receiving the signal detected by the detection module.

In some embodiments, the biological sample comprises DNA, RNA, PNA (peptide nucleic acid), LNA (locked nucleic acid), ANA (arabinonucleic acid), HNA (hexose nucleic acid) oligonucleotides, or the like. In some embodiments, the biological sample may also include a protein sample. In some embodiments, the biological sample may also include a cell sample, including but not limited to bacteria, fungi, animal cells, plant cells, archaea. In some embodiments, the biological sample may also include a biological tissue sample consisting of multiple cells.

In some embodiments, the biological sample injected in step a) may be a single sample, and other reagents for processing the biological sample may be provided step by step in step c) at different times during the assay as desired. In some embodiments, the biological sample injected in step a) may comprise all reagents required for subsequent reactions. These reagents include: various reagents used in biological experiments, such as lipids, saccharides, nucleic acids, enzymes, primers, buffers, antibodies, fluorescein, stains, magnetic beads, inorganic salts, various organic reagents, and the like.

In some embodiments, in step a), the reaction region of the biological sample detection microarray chip of the present application may be a cavity, and the biological sample is provided in the form of a droplet encapsulated in a liquid. Or the reaction area is filled with oily substances, and a water-in-oil structure is formed after the biological sample is injected into the reaction area.

In some embodiments, step c) further comprises adding additional reagents or additional samples externally as needed at any time during step c).

In some embodiments, a complete PCR reaction can be achieved in the biological sample detection microarray chip of the present application through control of an external signal. By generating a local constant temperature zone for denaturation, annealing and extension in the reaction zone, and matching with an electric regulation module, the amplification of DNA can be realized in the reaction zone. After amplification is complete, detection can be performed to determine whether the biological sample contains the target DNA fragment sequence. If the analyte in the biological sample contains a sequence of a target DNA fragment, it will bind to the target DNA and the label will undergo an observable optical change (e.g., fluorescence, or color change). As amplification proceeds, the change in the observable optical properties will become apparent. In some embodiments, the biological sample detection microarray chip of the present application may further include a laser capable of emitting laser light of a specific wavelength for exciting the fluorescent label added to the sample to be detected.

In some embodiments, the methods of the present application can also be used to detect protein and/or cell samples by stepwise addition of different reagents in step c), subjecting the protein and/or cell samples to multiple treatments, which then produce an optical change that can be detected by the detection module. In some embodiments, the methods of the present application can also be used in conjunction with optical indicators to detect a variety of biological properties and/or processes in a biological sample.

In another aspect, the present application also provides a microarray chip, wherein one or more of the biological sample detection microarray chips of the present application are integrated on the chip.

In some embodiments, the manufacturing process of the electric control module is a thin film transistor process or a printed circuit board process. In some embodiments, the process for fabricating the electrical regulation module and the detection module is a thin film transistor process, and the electrical regulation module and the detection module are located on the same substrate.

In some embodiments, the manufacturing process of the electrical modulation control module is a printed circuit board process; the detection module is located the chip outside, and the manufacturing technology is integrated circuit CMOS technology to the detection module includes: photodiodes, avalanche diodes, photomultiplier tubes.

In another aspect, the present application also provides use of the microarray chip of the present application for molecular diagnostics, biological sample analysis, chemical sample analysis, food analysis, and environmental analysis.

On the other hand, the microarray chip of the present application can be manufactured by a standard semiconductor process such as "film formation-photolithography-development-etching-peeling" and the like, and the design and manufacture of the microarray chip can be integrally realized on a glass, silicon wafer, metal or plastic rigid (flexible) substrate. In some embodiments, the microarray chip of the present application may be chip designed using thin film transistors and finished on a production line of the thin film transistors.

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

The present application describes embodiments, but the description is illustrative rather than limiting and it will be apparent to those of ordinary skill in the art that many more embodiments and implementations are possible within the scope of the embodiments described herein. Although many possible combinations of features are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are possible. Any feature or element of any embodiment may be used in combination with, or instead of, any other feature or element of any other embodiment, unless expressly limited otherwise.

The present application includes and contemplates combinations of features and elements known to those of ordinary skill in the art. The embodiments, features and elements disclosed in this application may also be combined with any conventional features or elements to form a unique inventive concept as defined by the claims. Any feature or element of any embodiment may also be combined with features or elements from other inventive aspects to form yet another unique inventive aspect, as defined by the claims. Thus, it should be understood that any of the features shown and/or discussed in this application may be implemented individually or in any suitable combination. Accordingly, the embodiments are not limited except as by the appended claims and their equivalents. Furthermore, various modifications and changes may be made within the scope of the appended claims.

Further, in describing representative embodiments, the specification may have presented the method and/or process as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps herein, the method or process should not be limited to the particular sequence of steps. Other orders of steps are possible as will be understood by those of ordinary skill in the art. Therefore, the particular order of the steps set forth in the specification should not be construed as limitations on the claims. Further, the claims directed to the method and/or process should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the embodiments of the present application.

All directional indicators in the embodiments of the present invention (such as upper, lower, left, right, front, and rear … …) are used only to explain the relative positional relationship between the components, the movement, etc. in a particular attitude (as shown in the drawings), and if the particular attitude changes, the directional indicator changes accordingly. In the present invention, unless otherwise expressly stated or limited, the terms "connected," "secured," and the like are to be construed broadly, and for example, "secured" may be a fixed connection, a removable connection, or an integral part; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations. In addition, the descriptions related to "first", "second", etc. in the present invention are only for descriptive purposes and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature.

Fig. 1A is a side view of a biological sample detection microarray chip in one embodiment of the present application. The biological sample detection microarray chip includes: a first substrate structure composed of a first substrate 001, a functional layer 002 and a functional film 003, a second substrate structure composed of a second substrate 010, a temperature-controlling layer 005 and a functional film 003, and a reaction region 004 between the second substrate structure and the first substrate structure. Wherein, detection module 009 and electricity accuse module 008 among the functional layer 002 are fixed on first base plate 001, and electricity accuse module 008 can exert the electric field to functional membrane 003, changes the hydrophilic or hydrophobic characteristic of functional membrane 003, and then adjusts and controls the action of little liquid drop 007 in reaction zone 004. The cyclic path with the arrow in the middle means that the micro-droplets 007 can move cyclically between different regions.

In this embodiment, the detection module 009 is located at a lower layer of the electrical regulation and control module 008 and fixed on the first substrate 001, and the micro droplets 007 can move above the detection module 009 under the action of the upper electrical regulation and control module 008, and in this embodiment, the working electrodes in the electrical regulation and control module 008 are light-permeable conductive electrodes, so that the change of the optical characteristics in the micro droplets 007 can be detected by the detection module 009 through the electrical regulation and control module 008. In other embodiments, the detection module 009 may have other different configurations, such as the configuration shown in fig. 4.

At least one temperature control module 006 is disposed in the temperature control layer 005 of the second substrate structure for receiving an external signal and maintaining a local region corresponding thereto in the reaction region 004 at a constant temperature. The functional film 003 in the second substrate structure is optional, and may not be present in other embodiments.

In the whole process of the microarray chip, the temperature zones (for example, temperature zone a, temperature zone B, temperature zone C, and temperature zone D in fig. 1A) with different temperatures are formed in the reaction region 004 under the control of the electric control module 008, then the micro droplet 007 containing the biological sample and the reaction reagent moves back and forth between the different temperature zones, or is kept in one temperature zone for a period of time, and after the reaction is completed, the information in the biological sample is known through the change of the optical signal received by the detection module 009. During the reaction, other reagents or other samples may be added externally as needed. Other reagents include reagents that can perform a series of biological processes including modification, denaturation, depolymerization, catalysis, charging, staining, washing, etc. on a biological sample of interest. In some embodiments, the other reagents include one or more of lipids, carbohydrates, nucleic acids, enzymes, antibodies, magnetic beads, stains, and the like.

For example, in the detection of a target nucleic acid in a sample using PCR, the micro-droplet 007 contains a biological sample, an enzyme and buffer required for amplification, and a label required for detection. The micro-droplets 007 move cyclically between different temperature zones according to the steps required by PCR, and the whole amplification reaction is completed. If the nucleic acid fragments of the biological sample comprise the target nucleic acid fragment sequence, the tag will bind to the target nucleic acid fragment. The label undergoes a change in an optical characteristic that is observable. As amplification proceeds, the observable change in optical properties will become apparent. By detecting a change in the observable optical properties of the tag, it can be determined whether the reaction mass comprises the sequence of the target nucleic acid fragment. To detect changes in the optical properties of the label, a laser may optionally be configured in some embodiments. In some embodiments, the detection module of the present application may also be removed and the optical change observable may be detected using external light detection means.

FIG. 1B is a diagram of a cartridge of a biological sample detection microarray chip in one embodiment of the present application. From bottom to top are a first substrate 101, a functional layer 102, a functional film 103, a micro-droplet 104, a functional film 103, a temperature control layer 105, and a second substrate 106, respectively.

Fig. 2 is a side view of an electrical modulation module and a detection module of a biological sample detection microarray chip according to an embodiment of the present application, in which the electrical modulation module and the detection module are manufactured by a thin film transistor process.

In the figure, an electric conditioning module 211 and a detection module 210 are disposed on a substrate 201. The electric regulation module 211 is a first metal layer 202, a first insulating layer 203, a semiconductor layer 204, a second metal layer 205, a second insulating layer 206, a working electrode 207, and a third insulating layer 208, respectively, from bottom to top. The detection module 210 is a first metal layer 202, a first insulating layer 203, a semiconductor layer 204, a second metal layer 205, and a second insulating layer 206, respectively, from bottom to top. The material of the first metal layer is usually Mo, ti, or a composite metal layer composed of Mo-Al, ti-Al, and may also be metal such as Au, ag, etc. The material of the second metal layer is usually Mo, ti, or a composite metal layer composed of Mo-Al, ti-Al, and can also be metal such as Au, ag, etc. The working electrode is typically a light-transmissive conductive electrode, such as ITO, IZO, agNW (silver nanowires). The first insulating layer is usually SiOx (silicon oxide) or SiNx (silicon nitride), and may have a stacked-layer structure of the two. For the semiconductor layer being an organic semiconductor, the first insulating layer is an organic insulating layer. The second insulating layer and the third insulating layer are typically SiOx (silicon oxide) or SiNx (silicon nitride), and may have a stacked-layer structure. For the semiconductor layer to be an organic semiconductor, the second insulating layer and the third insulating layer may be organic insulating layers.

The first metal layer 202 is typically a gate metal layer for adjusting an energy band of the semiconductor layer 204 to change an on/off state of the semiconductor. The first insulating layer 203 is typically a gate insulating layer. The first metal layer 202, the first insulating layer 203, and the semiconductor layer 204 form a metal-insulator-semiconductor (MIS) structure, which is a core unit of the thin film transistor device. Second metal layer 205 is typically a source/drain metal layer for conducting signals to the working electrode. The working electrode 207 is used to apply an electric field to the functional membrane 209 to change the hydrophilic or hydrophobic properties of the functional membrane. When the hydrophilic and hydrophobic characteristics of the functional film of the connected pixels are different, the micro-droplets move, and then the action of the surface micro-droplets can be controlled, wherein the action of the surface micro-droplets comprises the movement of the droplets, in-situ oscillation, combination of the micro-droplets, tearing from large droplets to small droplets and the like. The biological sample control mode can be divided into an active mode (at least comprising one thin film transistor unit) and a passive mode (not comprising the thin film transistor unit) according to whether the electric control module comprises the thin film transistor or not. Fig. 2 shows a design of an active electrical control module, in which the thin film transistor type is an amorphous silicon thin film transistor, and other types of thin film transistors, such as oxide transistor, organic transistor, and low temperature polysilicon transistor, can also achieve similar functions. For the design of a passive electric regulation module, the voltage can be applied to the working electrode only through metal wiring. When the working electrode is applied with voltage, the hydrophilic or hydrophobic property of the functional membrane above the working electrode is affected, so that the moving operation of the biological sample is realized. The way in which the voltage of the working electrode is not controlled by the thin film transistor is also included in the embodiment of the electrical regulation module.

In the detection module 210, since the energy band of the semiconductor can be changed by the light energy, the current changes, and thus the change of the current passing through the semiconductor layer 204 can be detected by using the characteristic, so as to realize the detection of the optical characteristic change in the micro-droplet. In other embodiments, the optical property change can be detected by other designs and structures capable of achieving similar functions, such as a photosensitive device manufactured by a thin film transistor process, such as amorphous silicon, polysilicon, oxide, an organic semiconductor, a PN junction, and the like.

Fig. 3 is a side view of an electrical regulatory module of a biological sample detection microarray chip according to another embodiment of the present application, in which a manufacturing process of the electrical regulatory module is shown as a printed circuit board process.

In the figure, a first metal layer 302, a first insulating layer 303, a second metal layer 304 (working electrode), a second insulating layer 305, and a functional film 306 are arranged on a substrate 301 in this order. The first metal layer 302 passes the control voltage passed via the external metal trace to the second metal layer 304, i.e., the working electrode. The second metal layer 304 (working electrode) applies an electric field to the functional membrane 306 to change the hydrophilic or hydrophobic characteristics of the functional membrane. Thereby controlling the action of the surface micro-droplets.

Fig. 4 is a schematic view illustrating the structure and detection of a biological sample detection microarray chip according to an embodiment of the present application, in which the microarray chip is fabricated by a printed circuit board process.

The configuration in the figure is similar to that in fig. 1A, and comprises a first substrate structure consisting of a first substrate 401, a functional layer 402 and a functional film 403, a second substrate structure consisting of a second substrate 410, a temperature control layer 405 and a functional film 403, a reaction region 404 between the second substrate structure and the first substrate structure, and an off-chip detection module 409. The electrical control module 408 in the functional layer 402 is fixed on the first substrate 401, and the electrical control module 408 can apply an electric field to the functional film 403 to change the hydrophilic or hydrophobic characteristics of the functional film 003, thereby controlling the movement of the micro-droplets 407 in the reaction region 404. The circular path with the arrows in the middle means that the micro-droplets 407 can move cyclically between different zones. In this embodiment, the second substrate 410 of the integrated temperature control module 405 is made of a transparent material, such as glass, polymer (PI, PEN, PET), and the like. The electrical tuning control module 408 manufactured by using the PCB process is opaque, so that the detection module 409 is located at one side of the temperature control module 405 to detect whether the observable optical characteristics in the micro-droplets 407 change. The movement, amplification, and the like of the micro-droplets 407 in the reaction region 404 are the same as those in FIG. 1A.

Fig. 5 is a side view of a temperature control module of a biological sample detection microarray chip in one embodiment of the present application.

In the figure, a first metal layer 502, a first insulating layer 503, a second metal layer 504, a second insulating layer 505, a third metal layer 506 (common electrode), a third insulating layer 507, and a functional film 508 are integrated in this order on a substrate 501.

Wherein the third metal layer 506 (common electrode), similar to the working electrode in fig. 2, may be used to provide an electric field to the functional film 508 to change its hydrophilic or hydrophobic properties. The first metal layer 502 and the second metal layer 504 are used for local area heating and temperature monitoring of the reaction area. The metal film layer for heating may be a first metal layer or a second metal layer. By applying an electric current to the metal, a local area where the metal is located can be heated. The temperature monitoring can be carried out in two ways, one is to monitor the resistance value of the metal layer for heating, and the temperature of a local area can be obtained through conversion; another way is to monitor the resistance of another metal layer directly above or below the metal layer used for heating to obtain the temperature of the heated region. The current is adjusted in accordance with the monitored temperature so that the area where the metal is located for heating obtains a constant temperature. Each temperature control module can independently control the temperature of a local area at the temperature control module. In some embodiments, a constant temperature in a localized area may also be achieved by other designs and structures that perform similar functions.

Claims (24)

1. A biological sample detection microarray chip comprising:

a first substrate structure having a first surface and a second surface,

the first substrate structure comprises a first substrate, and more than one electrical conditioning module on the first substrate, the electrical conditioning module comprising one or more working electrodes;

a second substrate structure having a first substrate and a second substrate,

the second substrate structure comprises a second substrate and at least one temperature control module located on the second substrate, wherein the temperature control module comprises a temperature change element;

a reaction region between the first substrate structure and the second substrate structure; and

one or more detection modules comprising a light sensing element;

the surface of the electric regulation and control module facing the reaction area is coated with a functional membrane, and under the regulation and control of an electric field, the hydrophilic or hydrophobic state of the functional membrane can be changed.

2. The biological sample detection microarray chip of claim 1, further comprising one or more channels providing fluid communication between the biological sample detection microarray chip and the outside.

3. The microarray chip for detecting biological samples according to claim 1, wherein at least one of the first substrate and the second substrate is made of a transparent material.

4. The microarray chip for detecting biological samples according to claim 3, wherein the first substrate and the second substrate are made of the same or different materials, and the materials are selected from one or more of glass, PI, PEN, PET and PCB plate substrates.

5. The biological sample detection microarray chip of claim 1, wherein a surface of the second substrate structure facing the reaction region is coated with the functional film.

6. The biological sample detection microarray chip of any one of claims 1 to 5, wherein the electrical regulation module further comprises a thin film transistor or a metal trace.

7. The biological sample detection microarray chip of claim 6, wherein the working electrode is controlled by the thin film transistor or by the metal trace connecting other external elements.

8. The microarray chip for detecting biological samples according to claim 1, wherein the working electrode is a light-transmissible conductive electrode comprising ITO, IZO and AgNW.

9. The microarray chip for detecting biological samples according to claim 1, wherein the material of said functional membrane comprises silicone oil, cytop, teflon.

10. The microarray chip for detecting biological samples according to claim 1, wherein said temperature changing member is a first metal member that can be heated after application of electric current, and the temperature of a local area can be converted by monitoring the change in resistance of said first metal member.

11. The microarray chip for testing biological samples according to claim 10, wherein said temperature control module further comprises a temperature sensing element for detecting the temperature of a local area.

12. The microarray chip for detecting biological samples according to claim 11, wherein the temperature-sensing element is a second metal element, and the temperature of the local area is obtained by monitoring the change in the resistance of the second metal element.

13. The microarray chip for detecting biological samples according to claim 12, wherein the material of the first metal member and the second metal member comprises: mo, ti, mo-Al alloy, ti-Al alloy, au and Ag.

14. The biological sample detection microarray chip of claim 1, wherein the optical sensing element has a change in electrical characteristics when light is sensed, the electrical characteristics including current, voltage, resistance, and capacitance.

15. The biological sample detection microarray chip of claim 14, wherein the light sensing element is a semiconductor element.

16. The biological sample detection microarray chip of any one of claims 1 to 5 and 8 to 15, wherein the biological sample detection microarray chip further comprises a laser capable of emitting laser light of a specific wavelength.

17. The microarray chip for biological sample detection according to claim 1, wherein the electrical control module is fabricated by a thin film transistor process or a printed circuit board process.

18. The microarray chip for biological sample detection according to claim 1, wherein the electrical manipulation module and the detection module are fabricated by a thin film transistor process, and are located on the same substrate.

19. The biological sample detection microarray chip of any one of claim 1, wherein the electrical regulation and control module is fabricated by a printed circuit board process;

the detection module is positioned at the outer side of the chip, the manufacturing process is an integrated circuit CMOS process, and the detection module comprises: photodiodes, avalanche diodes, photomultiplier tubes.

20. A method of detecting a biological sample, comprising the steps of:

a) Injecting a biological sample into the reaction region of the biological sample detection microarray chip of any one of claims 1 to 19 to form microdroplets;

b) Controlling the temperature of the temperature control module so that more than one stable temperature zones with different temperatures are formed in the reaction area;

c) Under the action of the electric regulation and control module, the micro-droplets move to a specified temperature zone or stay in the specified temperature zone as required;

d) The micro liquid drops are moved to the position corresponding to the detection module under the action of the electric regulation module;

e) And receiving the signal detected by the detection module.

21. The method for detecting a biological sample according to claim 20, wherein the biological sample comprises DNA, RNA, PNA, LNA, ANA and HNA.

22. The method for testing a biological sample according to claim 20, wherein in step a), the reaction region of the biological sample testing microarray chip of any one of claims 1 to 19 is filled with an oily substance, and the biological sample is injected into the reaction region to form a water-in-oil structure.

23. The method for detecting a biological sample according to claim 20, wherein the step c) further comprises adding other reagents or other samples from the outside as required at any time of the step c).

24. Use of the biological sample detection microarray chip of claims 1-23 for at least one of molecular diagnostics, biological sample analysis, chemical sample analysis, food analysis, and environmental analysis.

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