WO2018014719A1 - 检测电极结构及检测孔板与预制检测孔板 - Google Patents

检测电极结构及检测孔板与预制检测孔板 Download PDF

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WO2018014719A1
WO2018014719A1 PCT/CN2017/091435 CN2017091435W WO2018014719A1 WO 2018014719 A1 WO2018014719 A1 WO 2018014719A1 CN 2017091435 W CN2017091435 W CN 2017091435W WO 2018014719 A1 WO2018014719 A1 WO 2018014719A1
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detection
detecting
linear portion
electrode
electrode structure
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PCT/CN2017/091435
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English (en)
French (fr)
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廖玮
郭才华
张晨光
莫亚勤
钟华
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广州易活生物科技有限公司
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Publication of WO2018014719A1 publication Critical patent/WO2018014719A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements

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  • the invention relates to consumable technology of electrochemical detection technology, in particular to a detection electrode structure and a detection orifice plate and a prefabrication detection orifice plate.
  • Liquid biopsy refers to the assessment of the physiological or pathological state of a tissue by genetic or epigenetic information using human body fluids such as plasma, saliva, cerebrospinal fluid, urine, tears, and breast milk.
  • Orifices are a type of container commonly used in culture, storage, measurement or analysis in the biological field. Orifices typically include 96 microwells and are therefore also referred to as 96 well plates. Orifices are usually made of a transparent material for easy viewing and measurement. Orifice plates can be used for liquid biopsy. However, the use of conventional orifice plates for liquid biopsy has a longer reaction time, higher cost, lower sensitivity and higher environmental requirements for implementation. Therefore, the application range is narrow and cannot be satisfied. Market demand.
  • the invention provides a detecting electrode structure and a detecting orifice plate.
  • the detecting electrode structure can apply a voltage through the working electrode to form an electric field, thereby moving and enriching the charged target substance, and obtaining a detection signal such as a concentration of the target substance through the opposite electrode and outputting the detection signal, thereby realizing Fast and accurate detection of target materials.
  • a detection signal such as a concentration of the target substance through the opposite electrode and outputting the detection signal
  • At least one embodiment of the present invention provides a detecting electrode structure including: a base substrate including at least one detecting area; a working electrode disposed on the base substrate and configured to be applied a voltage to form an electric field; and an opposite electrode disposed on the base substrate and configured to acquire a detection signal and output the detection signal, the working electrode including at least one first line having a uniform width
  • the opposing electrode includes at least one second linear portion having a uniform width, and the first linear portion and the second linear portion are disposed in the detection region and alternately spaced apart from each other.
  • the working electrode in the detecting electrode structure, includes an arc-shaped first body portion and the plurality of first and second parallels extending from the first body portion a linear portion
  • the opposite electrode includes an arc-shaped second body portion and the plurality of second linear portions extending parallel to the second body portion, the first body portion and the first portion
  • the two main body portions are oppositely disposed, and the plurality of first linear portions are alternately spaced from the plurality of second linear portions.
  • the working electrode in the detecting electrode structure, includes the first linear portion arranged in a spiral shape, and the opposite electrode includes the second linear portion arranged in a spiral shape
  • the first linear portion and the second linear portion are arranged equidistantly at equal intervals.
  • the first linear portion and the second linear portion have a width ranging from 3 to 20 mils.
  • a distance between the first linear portion and the second linear portion is 3-20 mils.
  • the detecting electrode structure further includes: a reference electrode disposed at an edge of the detecting area.
  • the material of the working electrode and the opposite electrode includes gold.
  • the working electrode and the opposite electrode are disposed in the same plane.
  • At least one embodiment of the present invention provides a detection orifice plate comprising: a cartridge body including a plurality of through holes; and a plurality of detection electrode structures disposed at a bottom of the cartridge body,
  • the detecting electrode structure is the detecting electrode structure according to any one of the above, and the detecting area is provided at a bottom of the through hole and seals a bottom of the through hole.
  • the through holes are cylindrical through holes and are arranged in a matrix in the casing, the number of the through holes being a multiple of four, four Four of the working electrodes in the corresponding detecting electrode structures of the adjacent through holes are electrically connected.
  • the detection aperture plate further includes a circuit board, and the circuit board is electrically connected to the detection electrode structure.
  • the present invention further provides a preformed detection orifice for electrochemically detecting a target molecule, characterized in that a target for binding or capturing the target is fixed in a detection zone of any of the detection orifices described above. Molecular capture.
  • the trap is mixed with a conductive polymer and an ionic compound to form a mixed solution, and then added to the detection area, and the trapped object is fixed by the square wave electric field by the working electrode.
  • the parameters of the square wave electric field are: voltage A: -200 to -500 mV, 1-5 s; voltage B: 800 to 1500 mV, 1 s; 3 to 10 cycles.
  • the electrically conductive polymer is selected from the group consisting of pyrrole, aniline, and thiophene.
  • 885 ⁇ L of ultrapure water is contained per 1 mL of the mixed solution, and the ionization is performed at 3 mol/L. 100 ⁇ L of the compound, 5 ⁇ L of a conductive polymer, and 10 ⁇ L of a 100 ⁇ M capture.
  • the capture is a nucleic acid probe.
  • the capture may also be an antibody.
  • the invention also provides a preparation method of the above prefabricated detection orifice plate, which comprises the following steps and parameters:
  • the parameters of the first square wave electric field are: voltage A: -200-500 mV, 1-5 s; voltage B: 800-1500 mV, 1 s; 3 to 10 cycles.
  • the parameters of the first square wave electric field are set in a software program, and the first square wave electric field is implemented by controlling the working electrode by a software program.
  • the invention also provides a kit for detecting a target molecule, characterized in that it comprises any of the above-mentioned prefabricated detection orifice plates. Further, other conventional reagents for detecting target molecules are also included.
  • FIG. 1 is a schematic plan view showing a structure of a detecting electrode according to an embodiment of the present invention
  • FIG. 2 is a schematic plan view showing another structure of a detecting electrode according to an embodiment of the present invention.
  • FIG. 3 is a schematic plan view showing another structure of a detecting electrode according to an embodiment of the present invention.
  • FIG. 4 is a schematic plan view showing another structure of a detecting electrode according to an embodiment of the present invention.
  • FIG. 5a is a perspective view of a detection orifice plate according to an embodiment of the invention.
  • FIG. 5b is a schematic plan view of a detecting orifice plate according to an embodiment of the invention.
  • FIG. 6 is a partial perspective view of a detecting orifice plate according to an embodiment of the invention.
  • FIG. 7 is a partial perspective view of another detecting orifice plate according to an embodiment of the invention.
  • FIG. 8 is a partial perspective view of another detecting orifice plate according to an embodiment of the invention.
  • FIG. 9 is a partial side elevational view of a detection orifice plate according to an embodiment of the invention.
  • liquid biopsy In the field of liquid biopsy, liquid biopsy has a narrow range of applications due to its long reaction time, high cost, low sensitivity, and the like. Moreover, since liquid biopsy usually needs to be performed in a cleanroom, it is difficult for liquid biopsy to enter the consumer market.
  • the inventors of the present application have thought of using an electric field to generate an electric field to move and concentrate a target substance, and then using an electrical signal to analyze information such as a concentration of a target substance, and using a precise electrode pattern design, thereby enabling liquid activity to be performed quickly and accurately. Detection.
  • Embodiments of the present invention provide a detecting electrode structure and a detecting aperture plate.
  • the detecting electrode structure includes a substrate including at least one detecting region, a working electrode disposed on the substrate, the working electrode configured to apply a voltage to form an electric field, and an opposite surface disposed on the substrate An electrode configured to acquire a detection signal and output the detection signal.
  • the working electrode includes at least one first linear portion having a uniform width
  • the opposite electrode includes at least one second linear portion having a uniform width, the first linear portion and the second linear portion are disposed in the detection region and alternately and equidistantly spaced .
  • the detecting electrode structure can apply a voltage through the working electrode to form an electric field, thereby moving and enriching the charged target substance, and obtaining a detection signal such as a concentration of the target substance through the opposite electrode and outputting the detection signal, thereby realizing Fast and accurate detection of target materials.
  • a detection signal such as a concentration of the target substance through the opposite electrode and outputting the detection signal, thereby realizing Fast and accurate detection of target materials.
  • the working electrode and the opposite electrode to a linear structure having a uniform width, and forming the first linear portion and the second linear portion alternately and equally spaced apart in the detection region, a stable and uniform electric field can be formed, and A fine current is detected, which improves the detection accuracy.
  • FIG. 1 is a schematic plan view showing a structure of a detecting electrode.
  • the detecting electrode structure includes a base substrate 101 including at least one detecting area 102, and a working electrode 103.
  • the electrode 103 is disposed on the base substrate 101 and configured to apply a voltage to form an electric field; and the opposite electrode 104, the opposite electrode 104 is disposed on the base substrate 101 and configured to acquire a detection signal and output the detection signal.
  • the electrode 103 and the opposite electrode 104 are both disposed on the same surface of the base substrate, and therefore, the working electrode 103 and the opposite electrode 104 may be in the same plane. As shown in FIG.
  • the working electrode 103 includes at least one first linear portion 1031 having a uniform width; the opposite electrode 104 includes at least one second linear portion 1041 of uniform width; the first linear portion 1031 and the second linear portion 1041 They are disposed within the detection zone 102 and are alternately spaced apart from one another.
  • the electrode 104 located on the right side of FIG. 1 can be configured as a working electrode to apply a voltage to form an electric field; the electrode 103 located on the left side of FIG. 1 can be The counter electrode is configured to acquire a detection signal and output the detection signal, and the disclosure is not limited herein.
  • the detecting electrode structure can perform a plurality of sets of detection by providing a plurality of detecting areas 102, and performing comparison to further improve the detection accuracy.
  • the working electrode 103 can apply a voltage to generate an electric field to move and concentrate the target substance.
  • the working electrode 103 can apply a square wave alternating voltage to first include the target substance in the liquid to be detected.
  • the charged substance moves to the working electrode 103 to be enriched, so that the target substance can be combined with the probe on the working electrode 103 (a substance that can bind to the target substance, such as a DNA polymer molecule), and then the polarity of the voltage is converted to make the charged substance
  • the other substance not combined with the probe is away from the working electrode 103 (the force of the electric field on the target substance is set to be smaller than the binding force of the target substance and the probe); then, the opposite electrode 104 can acquire the detection signal about the target substance and Outputting a detection signal, for example, a target substance bound to the probe reacts with a specific reagent to generate a current, and thus the opposite electrode 104 can acquire a detection signal about the target substance by detecting the current and output the detection signal; By analyzing the output detection signal, information about the target substance (for example, the concentration of the target substance) can be obtained, thereby Quickly and accurately detected.
  • the target substance for example, the concentration of the target substance
  • first linear portion 1031 and the second linear portion 1041 are linear structures having a uniform width, and within the detection region 102, the first linear portion 1031 and the second linear portion 1041 are alternately equidistantly spaced within the detection region 102. Therefore, the first linear portion 1031 of the working electrode 103 can generate a uniform electric field in the detection region 102, and the second linear portion 1041 of the opposite electrode 104 can detect a minute current in the detection region 102, thereby improving The accuracy of the detection.
  • the density and uniformity of the probe formed on the working electrode 103 can be controlled, and the probe is not overly dense, thereby giving The binding of the target substance to the probe provides space to increase the efficiency of binding of the target substance to the probe, thereby improving the reaction speed of the liquid biopsy and further improving the accuracy of the detection.
  • the detection electrode structure provided in this embodiment can detect not only nucleic acids but also biomolecules such as proteins. Therefore, the detection electrode structure provided in this embodiment can be applied to a detection kit for biomarkers such as ctDNA, genomic DNA, RNA (including non-coding RNAs such as microRNA, lncRNA, circRNA, etc.), SNPs, exosomes, and the like. Meet the different needs of the clinic.
  • the detection electrode structure provided in this embodiment can also be applied to liquid biopsy (ctDNA detection) and pathogenic microbial nucleic acid detection as well as non-coding RNA (microRNA), exosome detection, drug metabolism enzymes, and drug target genes. Detection aspect.
  • the shape of the detecting area 102 is a circle.
  • the working electrode 103 may include an arcuate first body portion 1030 and a plurality of first linear portions 1031 extending from the first body portion 1030.
  • the counter electrode 104 includes an arc-shaped second body portion 1040 and a plurality of second linear portions 1041 that are parallel to each other and extend from the second body portion 1040.
  • the first body portion 1030 is disposed opposite to the second body portion 1040, and the plurality of first linear portions 1031 and the plurality of second linear portions 1041 are disposed in the detection region 102 and are alternately spaced and equidistantly disposed.
  • the working electrode 103 and the opposite electrode 104 have a comb-like structure, and the working electrode 103 and the opposite electrode 104 cross each other to form an interdigitated structure.
  • the range of the detection area may include a plurality of first linear portions and a plurality of second linear portions, and may further include a first main body portion and a second main body portion, which are not limited herein.
  • the working electrode in this embodiment may not be provided with a fixing portion that is in direct contact with the probe, so that the first linear portion may be formed into a line having a uniform width, thereby providing a more uniform electric field, so that the arrangement of the probe is more regular. Thereby improving the efficiency and accuracy of the detection.
  • FIG. 2 shows a schematic plan view of another detection electrode structure in which the shape of the detection region 102 is circular, the working electrode 103 includes a first linear portion 1031 which is spirally arranged, and the opposite electrode 104 includes a spiral arrangement.
  • the second linear portion 1041, the first linear portion 1031 and the second linear portion 1041 are disposed in the detection region 102 and are alternately spaced and equidistantly disposed.
  • the width of the first linear portion is the same as the width of the second linear portion, so that the accuracy of detection can be improved; in addition, the first linear portion and the second linear portion
  • the width can range from 3-20 mils (thousandths of an inch).
  • the pitch of the first linear portion and the second linear portion may range from 3 to 20 mils (thousandths of an inch).
  • the width of the first linear portion and the width of the second linear portion are equal to the spacing between the first linear portion and the second linear portion.
  • FIG. 3 is a schematic plan view of another detecting electrode structure
  • FIG. 4 is a schematic plan view of another detecting electrode structure.
  • the detecting electrode structure provided in an example of the embodiment further includes setting.
  • the reference electrode at the edge of the detection zone since the reference electrode is disposed at the edge of the detection zone, the outer side of the first linear portion and the second linear portion, the reference electrode can provide a contrast in the process of acquiring the detection signal about the target substance. The polarity error of the working electrode is eliminated, thereby further improving the accuracy of the detection.
  • the material of the working electrode and the opposing electrode includes gold. Since the chemical nature of the gold element is stable and does not react with the liquid to be detected and has a lower impedance, the accuracy of the detection can be further improved.
  • the embodiments of the present invention include but are not limited thereto, and other conductive materials such as platinum or indium tin oxide may also be used.
  • the embodiment provides a detection aperture plate, and the detection aperture plate comprises: a box body 200 and a detection electrode structure 100.
  • the casing 200 includes a plurality of through holes 211.
  • the size of the through hole can be referred to the design of a usual 96-well plate.
  • the present disclosure includes but is not limited thereto, and the size of the through hole can be designed according to the concentration and kind of the liquid to be detected.
  • the detecting electrode structure 100 is disposed at the bottom of the casing 200, the detecting electrode structure 100 may be the detecting electrode structure of any of the above-described first embodiment, and the detecting region 102 is disposed at the bottom of the through hole 211.
  • the bottom of the through hole 211 is sealed.
  • the base substrate 101 of the detecting electrode structure 100 and the casing 200 can be made of the same material.
  • the liquid to be detected can be contained in the accommodating space composed of the detection area 102 and the through hole 211, thereby detecting the liquid to be detected.
  • the working electrode 103 may apply a square wave alternating voltage to form a vertical electric field perpendicular to the bottom surface of the through hole 211, first causing the charged substance including the target substance in the liquid to be detected to pass from the respective positions of the through hole 211 to the through hole 211.
  • the bottom moves and moves to the working electrode 103 to enrich, so that the target substance can be combined with the probe on the working electrode 103 (a substance that can bind to the target substance, such as a DNA polymer molecule), and then the polarity of the voltage is converted to make it perpendicular to the pass.
  • the direction of the vertical electric field on the bottom surface of the hole 211 is reversed, so that other substances in the charged substance in the liquid to be detected that are not combined with the probe move from the bottom of the through hole 211 to the upper portion of the through hole 211, thereby causing other substances in the charged substance.
  • the substance not bound to the probe is away from the working electrode 103 (the force of the electric field on the target substance is set to be smaller than the binding force of the target substance and the probe); then, the opposite electrode 104 can acquire the detection signal about the target substance and detect
  • the signal output for example, the target substance bound to the probe reacts with a specific reagent to generate a current, so the opposite electrode 104 can detect the current Acquiring a detection signal about the target substance and outputting the detection signal; then, by analyzing the output detection signal, information about the target substance (for example, the concentration of the target substance) can be obtained.
  • the detecting orifice plate provided in this embodiment includes the detecting electrode structure of any of the above-mentioned first embodiment, it also has a technical effect corresponding to the detecting electrode structure, and the repeated portions are not described herein again.
  • a plurality of through holes 211 are arranged in a matrix in the casing 200.
  • the plurality of through holes 211 are cylindrical through holes.
  • the embodiments of the present invention include but are not limited thereto, and the shape of the plurality of through holes 211 may also be a square cylinder, a triangular cylinder or other cylinders.
  • the number of the plurality of through holes 211 is a multiple of four, as shown in FIG. 8 , four working holes in the detecting electrode structure corresponding to the four adjacent through holes 211 103 is electrically connected.
  • the substrate 111 is formed with a wire 111 and a wire 112.
  • the wire 111 electrically connects the four working electrodes 103.
  • the wires 112 are electrically connected to the four opposite electrodes 104 to extract the electrical signals of the opposing electrode 104. .
  • the four adjacent through holes can be used as a detection group.
  • the embodiments of the present invention include, but are not limited to, any number of working electrodes in the detecting electrode structures corresponding to any number of through holes are electrically connected to each other to provide a uniform voltage.
  • the detection aperture plate provided in this embodiment further includes: a circuit board 110, and the circuit board is electrically connected to the detection electrode structure.
  • An amplification circuit may be disposed on the circuit board 110 to amplify the electrical signal outputted by the opposite electrode or the reference electrode to improve detection accuracy; a voltage stabilization circuit may also be disposed on the circuit board 110 to provide a stable voltage to the working electrode to improve detection accuracy.
  • the present disclosure includes but is not limited thereto, and an overcurrent, overvoltage protection circuit, or the like may be disposed on the circuit board 110.
  • the circuit board 110 may be disposed under the base substrate 101 to more utilize the space.
  • the circuit board 110 may be disposed at other locations, and the disclosure is not limited herein.
  • This embodiment provides a prefabricated detection orifice for detecting a specific target, and the capture is fixed in the detection zone of the detection orifice of the second embodiment.
  • the following examples are used to explain the preparation steps of the prefabricated detection orifice.
  • Step 1 Prepare a mixture of pyrrole and capture probe: take a 1.5mL centrifuge tube, add 885 ⁇ l of ultrapure water, 100 ⁇ l of 3M KCl, vortex and mix, centrifuge; add 5 ⁇ l pyrrole, vortex mixing Uniformly, centrifuge; add 10 ⁇ l of 100 ⁇ M CP (first capture probe or second capture probe); vortex and mix, centrifuge, and obtain a mixture of the first capture probe mixture and the second capture probe, respectively. spare.
  • Step 2 On the detection well plate, add 30 ⁇ l of the prepared mixture of pyrrole and capture probe to each well, and test the reaction well of the 16 subtype group and add the mixture of the first capture probe to detect the 18 subtype. The reaction wells of the group were added to the mixture of the second capture probes.
  • the base sequence of the first capture probe is:
  • the base sequence of the second capture probe is:
  • the corresponding column for the experiment was selected on the EFIRM software.
  • the first square wave electric field parameters were set to: voltage A: 350 mV, 1 s; voltage B: 950 mV, 1 s; 9 cycles were performed. After the electric field treatment is completed, take it out immediately and clean the E-plate plate to obtain a pre-made detection orifice plate.
  • the instruments and software of the EFIRM technology are disclosed in "Electrochemical Sensor for Multiplex Biomarkers Detection, Clin Cancer Res. 2009 Jul 1; 15(13): 4446 - 4452.
  • Kit packaging including the above-mentioned prefabricated detection orifice plate, and
  • a first primer pair and/or a second primer pair are provided.
  • a first nucleic acid combination for detecting HPV 16 subtypes and a second nucleic acid combination for detecting HPV 18 subtypes are included:
  • the first nucleic acid combination comprises a first primer pair and a first capture probe.
  • the first primer pair is used to detect HPV 16 subtypes, including:
  • the upstream primer has a base sequence of: 5'-GAGCCCATTACAATATTGTA-3' (SEQ ID NO. 1);
  • the downstream primer has a biotin label at its 5' end, and the base sequence is: Biotin-5'-GTCTTCCAAAGTACGAATGTCTACGTGTGTGCT-3' (SEQ ID NO. 2).
  • the first capture probe is inversely complementary to the target region (5'-TTGTACGCACAACCGAAGCG-3') of the first target nucleic acid fragment (SEQ ID NO. 7) amplified by the first primer pair.
  • the second primer pair includes:
  • the upstream primer has a base sequence of 5'-AACATTTACCAGCCCGACGA-3' (SEQ ID NO. 4);
  • the downstream primer has a biotin label at the 5' end, and its base sequence is:
  • Biotin-5'-GGAACTGTCTGCTGAGCTTTCTACTACTAGCTCAATTCT-3' SEQ ID NO. 5
  • the second capture probe is inversely complementary to the target region (5'-GGCTTCACACTTACAACAC-3') of the second target nucleic acid fragment (SEQ ID NO. 8) amplified by the second primer pair.

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Abstract

一种检测电极结构及检测孔板与预制检测孔板，包括衬底基板（101），衬底基板（101）包括至少一个检测区域（102）；工作电极（103），工作电极（103）设置在所述衬底基板（101）上并配置为施加电压以形成电场；以及对置电极（104），对置电极（104）设置在所述衬底基板（101）上并配置为获取检测信号并输出所述检测信号；工作电极（103）包括至少一个宽度均匀的第一线状部（1031），对置电极（104）包括至少一个宽度均匀的第二线状部（1041），第一线状部（1031）和第二线状部（1041）设置在检测区域（102）并且相互交替等距间隔设置。

Description

检测电极结构及检测孔板与预制检测孔板 技术领域
本发明涉及电化学检测技术的耗材技术，特别是一种检测电极结构及检测孔板与预制检测孔板。
背景技术
液体活检是指使用人体体液，例如血浆、唾液、脑脊液、尿液、眼泪以及母乳等，通过遗传或表观遗传的信息来评估组织的生理或病理状态。
孔板是生物领域一种常用用于培养、存储、测量或分析的容器。孔板通常包括96个微孔，因此也被称为96孔板。孔板通常采用透明的材料制成，以便于观察和测量。孔板可应用于液体活检，然而，使用通常的孔板进行液体活检的反应时间较长、成本较高、灵敏度较低并且对实施的环境要求较高，因此其应用的范围较窄，无法满足市场的需求。
发明内容
本发明提供一种检测电极结构以及检测孔板。该检测电极结构可通过工作电极施加电压以形成电场，从而可使带电标靶物质移动、富集，通过对置电极可获取如标靶物质的浓度等检测信号并输出该检测信号，从而可实现对标靶物质的快速、准确检测。另外，通过将工作电极和对置电极设置为宽度均匀的线状结构，并将第一线状部和第二线状部在检测区相互交替且等距间隔设置可形成稳定均一的电场，并且可检测到细微的电流，从而可提高检测精度。
本发明至少一个实施例提供一种检测电极结构，其包括：衬底基板，所述衬底基板包括至少一个检测区；工作电极，所述工作电极设置在所述衬底基板上并配置为施加电压以形成电场；以及对置电极，所述对置电极设置在所述衬底基板上并配置为获取检测信号并输出所述检测信号，所述工作电极包括至少一个宽度均匀的第一线状部，所述对置电极包括至少一个宽度均匀的第二线状部，所述第一线状部和所述第二线状部设置在所述检测区并且相互交替等距间隔设置。
例如，在本发明一实施例中，在该检测电极结构中，所述工作电极包括弧状的第一主体部以及与从所述第一主体部延伸而出的多个相互平行的所述第一线状部，所述对置电极包括弧状的第二主体部以及与从所述第二主体部延伸出的多个相互平行的所述第二线状部，所述第一主体部与所述第二主体部相对设置，所述多个第一线状部与所述多个第二线状部交替间隔等距设置。
例如，在本发明一实施例中，在该检测电极结构中，所述工作电极包括螺旋状排列的所述第一线状部，所述对置电极包括螺旋状排列的所述第二线状部，所述第一线状部与所述第二线状部同心交替间隔等距设置。
例如，在本发明一实施例中，在该检测电极结构中，所述第一线状部和所述第二线状部的宽度范围为3-20mil。
例如，在本发明一实施例中，在该检测电极结构中，所述第一线状部和所述第二线状部的间距为3-20mil。
例如，在本发明一实施例中，在该检测电极结构中，还包括：参考电极，所述参考电极设置在所述检测区边缘。
例如，在本发明一实施例中，在该检测电极结构中，所述工作电极和所述对置电极的材料包括金。
例如，在本发明一实施例中，在该检测电极结构中，所述工作电极和所述对置电极设置在同一平面内。
本发明至少一个实施例提供一种检测孔板，其包括：盒体，所述盒体包括多个通孔；以及多个检测电极结构，所述检测电极结构设置在所述盒体的底部，所述检测电极结构为上述任一项所述的检测电极结构，并且，所述检测区设置在所述通孔的底部并将所述通孔的底部密封。
例如，在本发明一实施例中，在该检测孔板中，所述通孔为圆柱形通孔并且成矩阵排列在所述盒体中，所述通孔的数量为四的倍数，四个相邻的所述通孔对应的检测电极结构中的四个所述工作电极电性相连。
例如，在本发明一实施例中，该检测孔板还包括电路板，所述电路板与所述检测电极结构电性相连。
在本发明进一步提供一种用于电化学方法检测靶标分子的预制检测孔板，其特征在于，在权利要去上述任一所述的检测孔板的检测区固定有用于结合或捕获所述靶标分子的捕捉物。
在在本发明一实施例中，所述捕捉物是与导电聚合物和离子化合物混合成混合液后加到所述检测区，通过所述工作电极施加方波电场将所述捕捉物固定在检测区制成；
所述方波电场的参数为：电压A：-200～-500mV，1-5s；电压B:800～1500mV，1s；3～10个循环。
在本发明至少一个实施例中，所述导电聚合物选自吡咯、苯胺和噻吩组成的组。
在本发明至少一个实施例中，每1mL所述混合液中含超纯水885μL,3mol/L所述离子化 合物100μL,导电聚合物5μL,100μM的捕捉物10μL。
在本发明至少一个实施例中，所述捕捉物为核酸探针。本领域技术人员可以根据靶标分子的种类选择或设计合适的捕捉物，例如捕捉物还可以是抗体。
本发明还提供上述预制检测孔板的制备方法，其特征在于包括以下步骤和参数：
(1)预备捕捉物与导电聚合物和离子化合物混合成的混合液；
(2)将所述混合液加入到上述任一所述的检测孔板的检测区中，通过所述工作电极产生的第一方波电场将所述探针固定在反应孔内底部表面制成；
所述第一方波电场的参数为：电压A：-200-500mV，1-5s；电压B:800～1500mV，1s；3～10个循环。
在一些实施例中，所述第一方波电场的参数设置在软件程序中，通过软件程序控制所述工作电极实现所述第一方波电场。
本发明还提供一种用于检测靶标分子的试剂盒，其特征在于，包含上述任一预制检测孔板。进一步地，还包含用于检测靶标分子的其它常规试剂。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例的附图作简单地介绍，显而易见地，下面描述中的附图仅仅涉及本公开的一些实施例，而非对本公开的限制。
图1.为本发明一实施例提供的一种检测电极结构的平面示意图；
图2.为本发明一实施例提供的另一种检测电极结构的平面示意图；
图3.为本发明一实施例提供的另一种检测电极结构的平面示意图；
图4.为本发明一实施例提供的另一种检测电极结构的平面示意图；
图5a.为本发明一实施例提供的一种检测孔板的立体示意图；
图5b.为本发明一实施例提供的一种检测孔板的平面示意图；
图6.为本发明一实施例提供的一种检测孔板的局部立体示意图；
图7.为本发明一实施例提供的另一种检测孔板的局部立体示意图；
图8.为本发明一实施例提供的另一种检测孔板的局部立体示意图；
图9.为本发明一实施例提供的一种检测孔板的局部侧视示意图。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将结合本公开实施例的附图， 对本公开实施例的技术方案进行清楚、完整地描述。显然，所描述的实施例是本公开的一部分实施例，而不是全部的实施例。基于所描述的本公开的实施例，本领域普通技术人员在无需创造性劳动的前提下所获得的所有其他实施例，都属于本公开保护的范围。
除非另外定义，本公开使用的技术术语或者科学术语应当为本公开所属领域内具有一般技能的人士所理解的通常意义。本公开中使用的“第一”、“第二”以及类似的词语并不表示任何顺序、数量或者重要性，而只是用来区分不同的组成部分。“包括”或者“包含”等类似的词语意指出现该词前面的元件或者物件涵盖出现在该词后面列举的元件或者物件及其等同，而不排除其他元件或者物件。“连接”或者“相连”等类似的词语并非限定于物理的或者机械的连接，而是可以包括电性的连接，不管是直接的还是间接的。
在液体活检领域中，液体活检因为捕获靶标分子的反应时间较长、成本较高、灵敏度较低等原因，其应用的范围较窄。并且，由于进行液体活检通常需要在洁净室(cleanroom)内进行，使得液体活检很难进入消费市场。本申请的发明人想到利用电极产生电场来使标靶物质移动并聚集，然后利用电信号来分析标靶物质的浓度等信息，并且利用精密的电极图案设计，从而可迅速、准确地进行液体活检测。
本发明实施例提供一种检测电极结构以及检测孔板。该检测电极结构包括衬底基板，该衬底基板包括至少一个检测区；设置在衬底基板上的工作电极，该工作电极配置为施加电压以形成电场；以及设置在衬底基板上的对置电极，该对置电极配置为获取检测信号并输出所述检测信号。工作电极包括至少一个宽度均匀的第一线状部，对置电极包括至少一个宽度均匀的第二线状部，第一线状部和第二线状部设置在检测区并且相互交替且等距间隔设置。该检测电极结构可通过工作电极施加电压以形成电场，从而可使带电标靶物质移动、富集，通过对置电极可获取如标靶物质的浓度等检测信号并输出该检测信号，从而可实现对标靶物质的快速、准确检测。另外，通过将工作电极和对置电极设置为宽度均匀的线状结构，并将第一线状部和第二线状部在检测区相互交替且等距间隔设置可形成稳定均一的电场，并且可检测到细微的电流，从而可提高检测精度。
下面结合附图对本发明实施例提供的检测电极结构和检测孔板进行说明。
实施例一
本实施例提供一种检测电极结构，图1示出一种检测电极结构的平面示意图，该检测电极结构包括：衬底基板101，衬底基板101包括至少一个检测区域102；工作电极103，工作电极103设置在衬底基板101上并配置为施加电压以形成电场；以及对置电极104，对置电极104设置在衬底基板101上并配置为获取检测信号并输出所述检测信号。例如，这里的工 作电极103和对置电极104均设置在衬底基板的同一表面上，因此，工作电极103和对置电极104可以是位于同一平面内。如图1所示，工作电极103包括至少一个宽度均匀的第一线状部1031；对置电极104包括至少一个宽度均匀的第二线状部1041；第一线状部1031和第二线状部1041设置在检测区102内并且相互交替等距间隔设置。需要说明的是，上述的工作电极103和对置电极104的结构相同，因此，位于图1右侧的电极104可配置为施加电压以形成电场的工作电极；位于图1左侧的电极103可配置为获取检测信号并输出所述检测信号的对置电极，本公开在此不作限定。另外，该检测电极结构可通过设置多个检测区域102以进行多组检测，进行对照以进一步提高检测精度。
在本实施例提供的检测电极结构中，工作电极103可施加电压产生电场来使标靶物质移动并聚集，例如，工作电极103可施加方波交变电压，先使得待检测液体中包括靶标物质的带电的物质向工作电极103移动富集，使得靶标物质可与工作电极103上探针(可与靶标物质结合的物质，例如DNA聚合分子)结合，然后转变电压的极性，使得带电的物质中其他没有与探针结合的物质远离工作电极103(电场对靶标物质的作用力设置为小于靶标物质与探针的结合力)；然后，对置电极104可获取关于标靶物质的检测信号并将检测信号输出，例如，与探针结合的靶标物质会与特定的试剂发生反应而产生电流，因此对置电极104可通过检测电流来获取关于标靶物质的检测信号并将检测信号输出；然后，通过分析输出的检测信号可得出关于标靶物质的信息(例如靶标物质的浓度)，从而可迅速、准确地进行检测。由于第一线状部1031和第二线状部1041是宽度均匀的线状结构，并且在检测区102内，第一线状部1031和第二线状部1041在检测区102内相互交替等距间隔设置，由此，工作电极103的第一线状部1031可在检测区102产生均匀的电场，对置电极104的第二线状部1041可在检测区102可检测到细微的电流，因此可提高检测的精度。另外，由于工作电极103的第一线状部1031与第二线状部1041交替间隔设置，从而可控制形成在工作电极103上探针的密度和均匀度，不会使得探针过密，从而给靶标物质与探针的结合提供空间，提高靶标物质与探针的结合的效率，从而既可提高液体活检的反应速度，又可进一步提高检测的精度。
需要说明的是，本实施例提供的检测电极结构不仅可以检测核酸，还可以检测蛋白质等生物分子。因此，本实施例提供的检测电极结构可以适用于针对ctDNA、基因组DNA、RNA(包含非编码RNA，如microRNA、lncRNA、circRNA等)、SNP、外泌体等生物标记物的检测试剂盒，以满足临床的不同需求。另外，本实施例提供的检测电极结构还可应用于液体活检(ctDNA检测)以及病原微生物核酸检测以及非编码RNA(microRNA)、外泌体检测及药物代谢酶和药物作用靶点基因等的生物检测方面。
例如，在本实施例一示例提供的检测电极结构中，如图1所示，检测区102的形状为圆 形，工作电极103可包括弧状的第一主体部1030以及与从第一主体部1030延伸而出的多个相互平行的第一线状部1031。对置电极104包括弧状的第二主体部1040以及与从第二主体部1040延伸出的多个相互平行的第二线状部1041。第一主体部1030与第二主体部1040相对设置，多个第一线状部1031与多个第二线状部1041设置在检测区102内并交替间隔等距设置。也就是说，工作电极103和对置电极104呈梳齿状结构，并且工作电极103和对置电极104互相交叉以形成插指状结构。需要说明的是，检测区的范围可包括多个第一线状部与多个第二线状部，还可包括第一主体部和第二主体部，本公开在此不作限制。另外，本实施例中的工作电极可不设置与探针直接接触的固定部，因此第一线状部可做成宽度均匀的线状，从而提供更均匀的电场，使得探针的排列更规则，从而提高检测的效率和精度。
例如，图2示出了另一种检测电极结构的平面示意图，检测区102的形状为圆形，工作电极103包括螺旋状排列的第一线状部1031，对置电极104包括螺旋状排列的第二线状部1041，第一线状部1031与第二线状部1041设置在检测区102内并且交替间隔等距设置。
例如，在本实施例一示例提供的检测电极结构中，第一线状部的宽度与第二线状部的宽度相同，从而可提高检测的精度；另外，第一线状部和第二线状部的宽度范围可为3-20mil(千分之一英寸)。
例如，在本实施例一示例提供的检测电极结构中，第一线状部和第二线状部的间距范围可为3-20mil(千分之一英寸)。
例如，在本实施例一示例提供的检测电极结构中，第一线状部的宽度与第二线状部的宽度等于第一线状部和第二线状部的间距。由此，从而可进一步提高电场的均匀度以及检测的均匀度，从而可提高检测的精度。
例如，图3是另一种检测电极结构的平面示意图，图4是另一种检测电极结构的平面示意图；如图3或图4所示，本实施例一示例提供的检测电极结构还包括设置在检测区边缘的参考电极，由于参考电极设置在检测区边缘，第一线状部和第二线状部的外侧，因此参考电极在获取关于标靶物质的检测信号的过程中可提供对照，可消除工作电极的极性误差，从而进一步提高检测的精度。
例如，在本实施例一示例提供的检测电极结构中，工作电极和对置电极的材料包括金。由于金元素化学性质稳定不与待检测液体反应并且具有较低的阻抗，从而可进一步提高检测的精度。当然，本发明实施例包括但不限于此，也可采用其他导电物质，例如铂或氧化铟锡等。
实施例二
本实施例提供一种检测孔板，该检测孔板包括：盒体200以及检测电极结构100。如图5a和图5b所示，盒体200包括多个通孔211。通孔的尺寸可参照通常的96孔板的设计，当然，本公开包括但不限于此，通孔的尺寸可根据待检测液体的浓度和种类进行设计。如图6和7所示，检测电极结构100设置在盒体200的底部，检测电极结构100可为上述实施例一中任一的检测电极结构，并且，检测区102设置在通孔211的底部并将通孔211的底部密封。另外，检测电极结构100的衬底基板101与盒体200可采用同样的材料制作。由此，可在由检测区102和通孔211组成的容置空间中盛放待检测液体，从而对待检测液体进行检测。例如，工作电极103可施加方波交变电压，以形成垂直于通孔211底面的垂直电场，先使得待检测液体中包括靶标物质的带电的物质从通孔211的各个位置向通孔211的底部运动，并向工作电极103移动富集，使得靶标物质可与工作电极103上探针(可与靶标物质结合的物质，例如DNA聚合分子)结合，然后转变电压的极性，使垂直于通孔211底面的垂直电场的方向反转，使得待检测液体中的带电的物质中其他没有与探针结合的物质从通孔211的底部向通孔211的上部运动，从而使得带电的物质中其他没有与探针结合的物质远离工作电极103(电场对靶标物质的作用力设置为小于靶标物质与探针的结合力)；然后，对置电极104可获取关于标靶物质的检测信号并将检测信号输出，例如，与探针结合的靶标物质会与特定的试剂发生反应而产生电流，因此对置电极104可通过检测电流来获取关于标靶物质的检测信号并将检测信号输出；然后，通过分析输出的检测信号可得出关于标靶物质的信息(例如靶标物质的浓度)。因为本实施例提供的检测孔板包括上述实施例一中任一的检测电极结构，因此也具有与该检测电极结构对应的技术效果，重复之处在此不再赘述。
例如，在本实施例提供的检测孔板中，如图5b所示，多个通孔211成矩阵排列在盒体200中。如图6或图7所示，多个通孔211为圆柱形通孔。需要说明的是，本发明实施例包括但不限于此，多个通孔211的形状还可为方形柱体、三角形柱体或其他柱体。
例如，在本实施例提供的检测孔板中，多个通孔211的数量为四的倍数，如图8所示，四个相邻的通孔211对应的检测电极结构中的四个工作电极103电性相连。例如，衬底基板101上形成有导线111以及导线112，导线111将四个工作电极103电性相连；导线112分别与四个对置电极104电性相连以将对置电极104的电信号引出。由此，这四个相邻的通孔可作为一个检测组，在该检测组内，由于四个工作电极电性相连，施加在四个工作电极上的电压一致，四个相邻的通孔可更好地进行对照实验，因此可进一步提高检测精度。当然，本发明实施例包括但不限于此，还可将任意个数的通孔对应的检测电极结构中的任意个数的工作电极电性相连，以提供一致的电压。
例如，如图9所示，本实施例提供的检测孔板还包括：电路板110，电路板与检测电极结构电性相连。电路板110上可设置放大电路以放大对置电极或参考电极输出的电信号，以提高检测精度；电路板110上也可设置稳压电路以向工作电极提供稳定的电压，以提高检测精度。当然，本公开包括但不限于此，电路板110上还可设置过流、过压保护电路等。
例如，如图9所示，电路板110可设置在衬底基板101下，从而更合理地利用空间，当然，电路板110也可设置在其他位置，本公开在此不做限制。
实施例三预制检测孔板及其试剂盒
本实施例提供一种用于检测特定靶标物的预制检测孔板，在实施例二的检测孔板的检测区固定捕捉物。以下示例用于解释预制检测孔板的制备步骤。
示例.用于检测HPV 16亚型和HPV 18亚型的预制检测孔板及试剂盒
步骤1配制吡咯(pyrrole)与捕获探针的混合液：取1个1.5mL离心管，依次加入超纯水885μl，100μl 3M KCl，涡旋震荡混匀，离心；加入5μl pyrrole，涡旋震荡混匀，离心；加入10μl 100μM CP(第一捕获探针或第二捕获探针)；涡旋震荡混匀后离心，分别得到第一捕获探针的混合液和第二捕获探针的混合液，备用。
步骤2.在检测孔板上，每个孔加入30μl的已配制好的pyrrole与捕获探针的混合液，检测16亚型组的反应孔加入第一捕获探针的混合液，检测18亚型组的反应孔加入第二捕获探针的混合液。
第一捕获探针的碱基序列为：
5’-CGCTTCGGTTGTGCGTACAA-3’(SEQ ID NO.3)
第二捕获探针的碱基序列为：
5’-TGTGTTGTAAGTGTGAAGCC-3’(SEQ ID NO.6)。
步骤3.EFIRM电场处理
在EFIRM软件上选择进行实验的对应列，第一方波电场参数设置为：电压A：350mV，1s；电压B:950mV，1s；进行9个循环。电场处理完毕，立刻取出，清洗E-plate板得到预制检测孔板。EFIRM技术的仪器及软件采用“Electrochemical Sensor for Multiplex Biomarkers Detection,Clin Cancer Res.2009 Jul 1；15(13):4446–4452.中公开的。
试剂盒包装：包括上述预制检测孔板，和
第一引物对和/或第二引物对。
包括用于检测HPV 16亚型的第一核酸组合和用于检测HPV 18亚型的第二核酸组合：
其中，第一核酸组合包括第一引物对和第一捕获探针。
第一引物对用于检测HPV 16亚型，包括：
上游引物，其碱基序列为：5’-GAGCCCATTACAATATTGTA-3’(SEQ ID NO.1)；
下游引物，其5’端带有生物素标记，碱基序列为：Biotin-5’-GTCTTCCAAAGTACGAATGTCTACGTGTGTGCT-3’(SEQ ID NO.2)。
第一捕获探针与第一引物对所扩增出的第一靶核酸片段(SEQ ID NO.7)的靶区域(5’-TTGTACGCACAACCGAAGCG-3’)反向互补。
第二引物对包括：
上游引物，其碱基序列为：5’-AACATTTACCAGCCCGACGA-3’(SEQ ID NO.4)；
下游引物，其5’端带有生物素(Biotin)标记，其碱基序列为：
Biotin-5’-GGAACTGTCTGCTGAGCTTTCTACTACTAGCTCAATTCT-3’(SEQ ID NO.5)。
第二捕获探针与第二引物对所扩增出的第二靶核酸片段(SEQ ID NO.8)的靶区域(5’-GGCTTCACACTTACAACACA-3’)反向互补。
有以下几点需要说明：
(1)本发明实施例附图中，只涉及到与本发明实施例涉及到的结构，其他结构可参考通常设计。
(2)为了清晰起见，在用于描述本发明的实施例的附图中，层或微结构的厚度和尺寸被放大。可以理解，当诸如层、膜、区域或基板之类的元件被称作位于另一元件“上”或“下”时，该元件可以“直接”位于另一元件“上”或“下”，或者可以存在中间元件。
(3)在不冲突的情况下，本发明同一实施例及不同实施例中的特征可以相互组合。
以上所述，仅为本公开的具体实施方式，但本公开的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本公开揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本公开的保护范围之内。因此，本公开的保护范围应以所述权利要求的保护范围为准。

Claims (20)

  1. 一种检测电极结构，其特征在于，包括：
    衬底基板，所述衬底基板包括至少一个检测区；
    工作电极，所述工作电极设置在所述衬底基板上并配置为施加电压以形成电场；以及对置电极，所述对置电极设置在所述衬底基板上并配置为获取检测信号并输出所述检测信号，
    所述工作电极包括至少一个宽度均匀的第一线状部，所述对置电极包括至少一个宽度均匀的第二线状部，所述第一线状部和所述第二线状部设置在所述检测区并且相互交替等距间隔设置。
  2. 根据权利要求1所述的检测电极结构，其特征在于，所述工作电极包括弧状的第一主体部以及与从所述第一主体部延伸而出的多个相互平行的所述第一线状部，所述对置电极包括弧状的第二主体部以及与从所述第二主体部延伸出的多个相互平行的所述第二线状部，所述第一主体部与所述第二主体部相对设置，所述多个第一线状部与所述多个第二线状部交替间隔等距设置。
  3. 根据权利要求1所述的检测电极结构，其特征在于，所述工作电极包括螺旋状排列的所述第一线状部，所述对置电极包括螺旋状排列的所述第二线状部，所述第一线状部与所述第二线状部同心交替间隔等距设置。
  4. 根据权利要求1-3任一项所述的检测电极结构，其特征在于，所述第一线状部和所述第二线状部的宽度范围为3-20mil。
  5. 根据权利要求1-3任一项所述的检测电极结构，其特征在于，所述第一线状部和所述第二线状部的间距为3-20mil。
  6. 根据权利要求1-3任一项所述的检测电极结构，其特征在于，还包括：参考电极，所述参考电极设置在所述检测区边缘。
  7. 根据权利要求1-3任一项所述的检测电极结构，其特征在于，所述工作电极和所述对置电极的材料包括金。
  8. 根据权利要求1-3任一项所述的检测电极结构，其特征在于，所述工作电极和所述对置电极设置在同一平面内。
  9. 一种检测孔板，其特征在于，包括：盒体，所述盒体包括多个通孔；以及检测电极结构，所述检测电极结构设置在所述盒体的底部，所述检测电极结构为上述权利要求1-8任一项所述的检测电极结构，并且，所述检测区设置在所述通孔的底部并将所述通孔的底部密封。
  10. 根据权利要求9所述的检测孔板，其特征在于，所述通孔为圆柱形通孔并且成矩阵排列在所述盒体中，所述通孔的数量为四的倍数，四个相邻的所述通孔对应的检测电极结构中的 四个所述工作电极电性相连。
  11. 根据权利要求9所述的检测孔板，其特征在于，还包括：电路板，所述电路板与所述检测电极结构电性相连。
  12. 一种用于电化学方法检测靶标分子的预制检测孔板，其特征在于，在权利要求9-11任一所述的检测孔板的检测区固定有用于结合或捕获所述靶标分子的捕捉物。
  13. 根据权利要求12所述的预制检测孔板，其特征在于：所述捕捉物是与导电聚合物和离子化合物混合成混合液后加到所述检测区，通过所述工作电极施加方波电场将所述捕捉物固定在检测区制成；
    所述方波电场的参数为：电压A：-200～-500mV，1-5s；电压B:800～1500mV，1s；3～10个循环。
  14. 根据权利要求13所述的预制检测孔板，其特征在于：所述导电聚合物选自吡咯、苯胺和噻吩组成的组。
  15. 根据权利要求13或14所述的预制检测孔板，其特征在于：每1mL所述混合液中含超纯水885μL,3mol/L所述离子化合物100μL,导电聚合物5μL,100μM的捕捉物10μL。
  16. 根据权利要求15所述的预制检测孔板，其特征在于：所述捕捉物为核酸探针。
  17. 权利要求12-16任一所述的预制检测孔板的制备方法，其特征在于包括以下步骤和参数：
    (1)预备捕捉物与导电聚合物和离子化合物混合成的混合液；
    (2)将所述混合液加入到权利要求9-11任一所述的检测孔板的检测区中，通过所述工作电极产生的第一方波电场将所述探针固定在反应孔内底部表面制成；
    所述第一方波电场的参数为：电压A：-200-500mV，1-5s；电压B:800～1500mV，1s；3～10个循环。
  18. 根据权利要求17所述的制备方法，其特征在于：所述第一方波电场的参数设置在软件程序中，通过软件程序控制所述工作电极实现所述第一方波电场。
  19. 一种用于检测靶标分子的试剂盒，其特征在于，包含权利要求12-16任一所述的预制检测孔板。
  20. 根据权利要求19所述的试剂盒，其特征在于，还包含用于检测靶标分子的其它常规试剂。
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