CN117106578B - Electrochemical detection kit and detection method - Google Patents
Electrochemical detection kit and detection method Download PDFInfo
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- CN117106578B CN117106578B CN202311386208.7A CN202311386208A CN117106578B CN 117106578 B CN117106578 B CN 117106578B CN 202311386208 A CN202311386208 A CN 202311386208A CN 117106578 B CN117106578 B CN 117106578B
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- 238000001514 detection method Methods 0.000 title claims abstract description 121
- 238000000835 electrochemical detection Methods 0.000 title claims abstract description 30
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- 230000003321 amplification Effects 0.000 claims abstract description 38
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- KTWOOEGAPBSYNW-UHFFFAOYSA-N ferrocene Chemical compound [Fe+2].C=1C=C[CH-]C=1.C=1C=C[CH-]C=1 KTWOOEGAPBSYNW-UHFFFAOYSA-N 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention discloses an electrochemical detection kit and a detection method, wherein the electrochemical detection kit comprises a kit main body, a reaction zone and a detection part, the reaction zone comprises a reaction cavity with a detection zone and an amplification zone, the detection zone is provided with an induction electrode, a capture probe is arranged on the induction electrode, after a sample to be detected which is specifically paired with a signal probe enters the reaction cavity, the residual section of the sample to be detected is specifically paired with the capture probe, and the signal probe is triggered, so that the signal probe changes the current or voltage of the induction electrode and transmits a signal to the external detection part, thereby being capable of simultaneously carrying out qualitative detection on the sample to be detected and improving the accuracy of a detection result; meanwhile, the sample to be detected is amplified through the arrangement of the amplification area, the condition that the sample to be detected flows into the detection area to leak detection is avoided, and the accuracy of the detection result can be further improved.
Description
Technical Field
The invention relates to the technical field of molecular diagnosis, in particular to an electrochemical detection kit and a detection method.
Background
The molecular diagnosis technique is a technique for diagnosing a human state and a disease by detecting the presence, defect or abnormal expression of a gene using a molecular biological technique using DNA and RNA as diagnostic materials. The basic principle is to detect whether the structure of DNA or RNA is changed, the quantity of DNA or RNA is more or less and the expression function is abnormal, so as to determine whether the detected person has abnormal change of gene level, and the method has important significance for preventing, predicting, diagnosing, treating and prognosis of diseases. All molecular biology-level-based methodological techniques are colloquially simple and belong to the molecular diagnostic arts, such as polymerase chain reaction techniques, gene sequencing techniques, and the like.
The polymerase chain reaction (Polymerase chain reaction, PCR) is a molecular biological technique for amplifying specific DNA fragments (target genes of interest), i.e. specific in vitro amplification processes of target genes of interest. The basic principle of PCR is that double-stranded DNA is denatured and melted into single-stranded DNA at high temperature, double strands can be renatured after the temperature is reduced, and the denaturation and renaturation of the DNA can be controlled through temperature change; the primer, DNA polymerase, deoxyribonucleoside triphosphate (dNTP) and corresponding buffer solution (collectively referred to as PCR reaction system) are added into the sample to be detected, and the PCR reaction system can complete in vitro copy amplification of the target gene by matching with temperature control (namely heating-cooling circulation treatment).
After the target gene of the sample to be detected is copied, the copied product is detected, the existing detection method is a fluorescence detection method, namely, the optical detection system is utilized to carry out optical irradiation treatment on the copied product and detect a fluorescent signal transmitted by the irradiated copied product, and finally, the qualitative detection is carried out on the sample to be detected according to the fluorescent signal, wherein the qualitative detection refers to judging whether the sample to be detected carries the target gene.
When the fluorescence detection method is used for carrying out fluorescence detection on the replication product, the detection result is inaccurate easily due to the fluorescence crosstalk problem, which is an unavoidable defect problem of the fluorescence detection method.
Therefore, how to improve the accuracy of the detection result of the sample to be detected is a problem to be solved.
Disclosure of Invention
The invention aims to provide an electrochemical detection kit and a detection method, which can improve the accuracy of a detection result of a sample to be detected.
In order to achieve the above purpose, the present invention provides a technical solution:
an electrochemical detection kit comprising:
the kit body is used for containing a sample to be tested;
the reaction zone is connected with the kit main body and comprises a reaction cavity, the reaction cavity is communicated with the kit main body and comprises a detection zone and an amplification zone, the detection zone is provided with a sensing electrode, and the sensing electrode is provided with a capture probe; the amplification region comprises a first temperature control region for heating and a second temperature control region for cooling, a temperature control flow passage is arranged in the amplification region, one part of the temperature control flow passage is positioned in the first temperature control region, the other part of the temperature control flow passage is positioned in the second temperature control region, and the sample to be tested alternately flows between the first temperature control region and the second temperature control region along the temperature control flow passage;
and the detection component is electrically connected with the induction electrode.
Optionally, the reactor further comprises an electrode plate detachably connected in the reaction cavity, and the induction electrode is arranged on the electrode plate.
Optionally, the temperature control runner is coil-type along the direction of height Z of the kit main body, the upper half part of the temperature control runner is located in the first temperature control area, and the lower half part of the temperature control runner is located in the second temperature control area.
Optionally, a heat insulation layer is arranged between the first temperature control area and the second temperature control area.
Optionally, the reaction zone further comprises a reaction plate, the reaction plate is connected with the kit main body, and a sample inlet channel and a sample outlet channel are formed in the reaction plate;
the reaction cavity is arranged on the reaction plate, one side of the reaction cavity is communicated with the sample inlet channel, the other side of the reaction cavity is communicated with the sample outlet channel, and the main body of the kit is communicated with the sample inlet channel and the sample outlet channel at the same time.
Optionally, the temperature control flow channel includes an inlet and an outlet, the inlet is communicated with the sample injection channel, the outlet is communicated with the detection area, and the height of the outlet is higher than that of the sensing electrode.
Optionally, the reaction zone further includes a sealing film, and the sealing film covers the surface of the reaction plate, so that the reaction cavity, the sample inlet channel and the sample outlet channel form a closed space.
Optionally, the kit main body includes a box body and a switching valve, the box body includes a central through hole, a plurality of mutually isolated accommodating cavities are configured at the periphery of the central through hole, and one accommodating cavity is used for accommodating the sample to be tested;
the switching valve comprises a cylinder body inserted in the central through hole and a valve seat positioned at one end of the cylinder body, a plunger is embedded in a cylinder cavity of the cylinder body, an inlet and an outlet communicated with the cylinder cavity are arranged on the valve seat, and when the inlet and the outlet are aligned and communicated with the containing cavity for containing the sample to be tested, the plunger is pushed to enable the sample to be tested to come in and go out of the cylinder cavity; when the inlet and the outlet are aligned and communicated with the sample inlet channel or the sample outlet channel, the plunger is pushed to enable the sample to be detected to enter and exit the reaction zone.
Optionally, a drainage plate is arranged at the inlet of the temperature control flow channel, one end of the drainage plate is communicated with the inlet of the temperature control flow channel, and the other end of the drainage plate is communicated with the sample injection channel;
the height of the drainage plate is higher than that of the temperature control flow passage.
The invention also provides a technical scheme that:
a method of detection comprising:
the plunger controls a sample to be tested to enter the sample injection channel;
the sample to be measured in the sample injection channel is controlled by the plunger and flows into one of the temperature control flow channels, and the sample to be measured at least fills part of the flow channels and stays for 4s-6s;
the plunger controls the sample to be tested to flow again, and the reciprocating cycle is carried out until the sample to be tested completely enters the detection area through the temperature control flow channel;
the capture probe in the detection area is specifically paired with the residual section of the sample to be detected, and the signal probe is triggered, so that the signal probe changes the current or voltage of the induction electrode, and a current signal or voltage signal is transmitted to an external detection component.
Compared with the prior art, the invention has the beneficial effects that:
1. the electrochemical detection kit comprises a kit main body, a reaction zone and a detection part, wherein the reaction zone comprises a reaction cavity, the reaction cavity comprises a detection zone and an amplification zone, the detection zone is provided with an induction electrode, a capture probe is arranged on the induction electrode, the induction electrode is also electrically connected with the detection part, as a part of a sample to be detected is specifically paired with a signal probe before the sample to be detected enters the reaction cavity, after the sample to be detected which is specifically paired with the signal probe enters the reaction cavity, the residual section of the sample to be detected is specifically paired with the capture probe, and the signal probe is triggered, so that the signal probe changes the current or voltage of the induction electrode, and a current signal or a voltage signal is transmitted to an external detection part, thereby being capable of carrying out qualitative detection on the sample to be detected, avoiding the problem of fluorescence crosstalk by the detection method, and improving the accuracy of detection results; the amplification area comprises a first temperature control area for heating and a second temperature control area for cooling, a temperature control flow passage is arranged in the amplification area, one part of the temperature control flow passage is located in the first temperature control area, the other part of the temperature control flow passage is located in the second temperature control area, so that a sample to be detected flows alternately between the first temperature control area and the second temperature control area along the temperature control flow passage, when the sample to be detected flows to the detection area, the sample to be detected is amplified, the amplified sample to be detected is specifically paired with the capture probe, so that detection is completed, and the acquired target gene concentration in the sample to be detected is unknown, so that the condition that the sample to be detected flows into the detection area to have omission is avoided, the sample to be detected enters the detection area for detection after amplification, and the accuracy of a detection result is further improved.
2. The temperature control flow channel of the electrochemical detection kit is arranged in a coil pipe shape, along the height direction Z of the main body of the kit, the upper half part of the coil pipe is positioned in a first temperature control area, DNA in the area is denatured and melted into single-stranded DNA at high temperature, the lower half part of the coil pipe is positioned in a second temperature control area, double chains can be formed in the area again through renaturation, and the denaturation and renaturation of the DNA can be controlled through temperature change, so that amplification is completed; because the temperature control flow channel is in a coil type arrangement, a sample to be detected can be amplified once in one flow channel of the coil, and can be amplified for a plurality of times in a plurality of flow channels in a similar way, so that the target base factor quantity of the sample to be detected flowing into the detection area is increased, the capture probe can better capture the target gene, the condition of missing detection is avoided, and the accuracy of the detection result is improved.
3. The heat insulation layer is arranged between the first temperature control region and the second temperature control region of the electrochemical detection kit, the heat transmission between the first temperature control region and the second temperature control region can be avoided through the arrangement of the heat insulation layer, the first temperature control region is always in a high temperature constant temperature state, the second temperature control region is always in a low temperature constant temperature state, and the denaturation and renaturation of DNA are controlled through temperature change, so that amplification is completed.
4. The temperature control flow channel of the electrochemical detection kit comprises an inlet and an outlet, wherein the inlet is communicated with the sample injection channel, the outlet is communicated with the detection area, and the height of the outlet is higher than that of the sensing electrode, so that a sample to be detected can flow to the detection area conveniently, and the sample can be captured by a capture probe on the sensing electrode more easily.
5. The temperature control flow channel of the electrochemical detection kit comprises a plurality of flow channels, the plunger is controlled to move through the control part, so that the plunger controls a sample to be detected to flow into the temperature control flow channel, at the moment, the plunger firstly controls the sample to be detected to flow into one flow channel, at least one part of the flow channel is filled with the sample to be detected, the sample stays for 4-6 s, the denaturation and renaturation of DNA are controlled, then the plunger moves again, and the sample is reciprocally circulated until all the sample to be detected enters a detection area through the temperature control flow channel for detection, and the stay time of the sample to be detected in the whole amplification area is shorter, so that the detection time of the whole kit is shorter, and meanwhile, the sample to be detected can control the denaturation and renaturation of the DNA in each flow channel, so that the target gene can be amplified for multiple times, the concentration of the target gene can be quickly improved in a shorter time, and the accuracy of a detection result is improved.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a schematic diagram of the structure of an electrochemical detection kit of the present invention;
FIG. 2 is a schematic diagram of the structure of the hidden sealing film of the electrochemical detection kit of the invention;
FIG. 3 is a partial enlarged view of a first view of a reaction zone of an electrochemical detection kit of the present invention;
FIG. 4 is a partial enlarged view of a second view of the reaction zone of the electrochemical detection kit of the present invention;
FIG. 5 is a front view of FIG. 4;
FIG. 6 is a schematic view showing a partial structure of a case body of the electrochemical detection kit of the present invention;
FIG. 7 is a bottom view of FIG. 6;
FIG. 8 is a schematic diagram showing the internal structure of a switching valve of the electrochemical detection kit of the present invention.
In the figure:
1. a kit body; 11. a case body; 111. a liquid passing hole; 112. a central through hole; 12. a top cover; 13. a base; 14. a switching valve; 141. a cylinder; 142. a valve seat; 143. a plunger; 144. a barrel cavity; 145. a first access port; 146. a second inlet and outlet; 147. a treatment zone; 101. a sample chamber; 102. a lysate chamber; 103. a cleaning liquid chamber; 104. a buffer chamber; 105. a waste liquid chamber; 106. a primer probe mixed liquid cavity; 107. a first preformed cavity; 108. a second preformed chamber; 2. a reaction zone; 21. a reaction chamber; 211. a detection zone; 2111. an electrode plate; 2112. an induction electrode; 212. an amplification region; 2121. A first temperature control zone; 2122. a second temperature control zone; 2123. a temperature control flow passage; 2124. an inlet; 2125. an outlet; 2126. a drainage plate; 22. a reaction plate; 23. a sample introduction channel; 24. a sample outlet channel; 25. and (5) sealing a film.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the present invention, unless explicitly specified and limited otherwise, the terms "mounted," "connected," "secured," and the like are to be construed broadly and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific circumstances.
The existing detection method is a fluorescence detection method, namely, an optical detection system is used for carrying out optical irradiation treatment on the replication and amplification product and detecting a fluorescence signal transmitted by the replication and amplification product after irradiation, and finally, qualitative detection is finished on a sample to be detected according to the fluorescence signal. Qualitative refers to determining whether the sample to be tested carries the target gene of interest. This detection mode is called real-time fluorescent quantitative polymerase chain reaction (Quantitative Real-time Polymerase Chain Reaction, qPCR). The qPCR reaction is to add a reporter group to a target gene in a PCR reaction system, when the target gene undergoes one reaction cycle (namely after undergoing one replication), the fluorescent signal intensity emitted by the reporter group is enhanced once, and an optical detection system of a PCR instrument can monitor the change of the reaction product amount by detecting the change of the fluorescent signal intensity of a sample to be detected after each reaction cycle. When the detection method is used for qualitatively judging the sample to be detected, the problem of fluorescence crosstalk can occur, so that the detection result is wrong, and a large amount of amplification is needed for the sample to be detected, so that the time required for completing the detection is longer.
The present company previously filed a patent about the kit, the patent number is "cn202110518972.X", and since the patent cannot solve the problems of fluorescence crosstalk and upper detection limit in multi-index detection, the present application improves on the basis of the above patent, and the present application can solve the above problems after improvement.
Referring to fig. 1-8, the embodiment of the disclosure provides an electrochemical detection kit, which includes a kit main body 1, a reaction zone 2 and a detection component, where the reaction zone 2 includes a reaction chamber 21, the reaction chamber 21 includes a detection zone 211 and an amplification zone 212, the detection zone 211 is provided with an electrode plate 2111, the electrode plate 2111 is provided with an sensing electrode 2112, the sensing electrode 2112 is provided with a capture probe, and the sensing electrode 2112 is further electrically connected with the detection component, because a part of a section of the sample to be detected is already specifically paired with a signal probe before the sample to be detected enters the reaction chamber 21, after the sample to be detected specifically paired with the signal probe enters the reaction chamber 21, the remaining section of the sample to be detected is specifically paired with the capture probe, and triggers the signal probe, so that the signal probe changes the current or voltage of the sensing electrode 2112, and transmits the current signal or the voltage signal to the external detection component, thereby being able to qualitatively detect the sample to be detected, the fluorescence crosstalk problem is avoided by the detection method, and the accuracy of the detection result is improved; the amplification region 212 includes a first temperature control region 2121 for heating and a second temperature control region 2122 for cooling, and the amplification region 212 is internally provided with a temperature control flow channel 2123, a part of the temperature control flow channel 2123 is located in the first temperature control region 2121, and the other part of the temperature control flow channel 2123 is located in the second temperature control region 2122, so that a sample to be detected flows alternately between the first temperature control region 2121 and the second temperature control region 2122 along the temperature control flow channel 2123, when the sample to be detected flows to the detection region 211, the sample to be detected has completed amplification, and the amplified sample to be detected is specifically paired with a capture probe, so that detection is completed.
The capture probe is fixed on the surface of the sensing electrode 2112, because the partial section of the sample to be tested is specifically paired with the signal probe before the sample to be tested enters the reaction cavity 21, after the sample to be tested which is specifically paired with the signal probe enters the reaction cavity 21, the residual section of the sample to be tested is specifically combined with the capture probe, so that the signal probe is close to the surface of the sensing electrode 2112, the signal probe is marked with ferrocene molecules, the ferrocene molecules are close to the sensing electrode 2112, oxidation-reduction reaction occurs to further cause electron transfer to generate current or voltage change, and the detection part detects the current or voltage value, so that the identification and the judgment of the target gene are carried out, and because the identification are not carried out by an illumination method, the fluorescent crosstalk problem is avoided, and the accuracy of the detection result is improved. Meanwhile, the detection method does not need to carry out a large number of copying and amplification in advance through a special temperature control device, so that the detection speed is increased, the temperature control device is omitted, and the production cost of the instrument is saved.
The electrode plate 2111 described above may be detachably connected to the reaction chamber 21, and since the sensing electrode 2112 is provided on the electrode plate 2111, replacement of the sensing electrode 2112 can be facilitated.
The electrode plate 2111 is connected with a display screen, and since each sensing electrode 2112 is electrically connected to the electrode plate 2111, when the sensing electrode 2112 is combined with the target gene, the sensing electrode 2112 will generate a change in current or voltage, and the change will react on the electrode plate 2111 and be displayed through the display screen, so that the user can observe conveniently.
The capture probes can be arranged in one or more ways, when one capture probe is arranged, only one target gene can be detected, and when the capture probes are arranged in a plurality of ways, a plurality of target genes can be detected through one detection experiment, so that multi-index detection is realized, and the detection efficiency is improved.
In some embodiments, the temperature-controlled flow channel 2123 is arranged in a coil-type along the height direction Z of the kit main body 1, the upper half part of the coil is located in a first temperature-controlled region 2121, the DNA is denatured and melted into single-stranded DNA at a high temperature in the region, the lower half part of the coil is located in a second temperature-controlled region 2122, the double-stranded DNA can be renatured in the region, and the denaturation and renaturation of the DNA can be controlled through temperature change, so that amplification is completed; because the temperature control flow channel 2123 is in a coil type arrangement, a sample to be detected can be amplified once in one flow channel of the coil, and can be amplified for a plurality of times in a plurality of flow channels in a similar way to a certain order of magnitude, so that the target base factor in the sample to be detected flowing into the detection area 211 is increased, the capture probe can capture the target gene better, the condition of missing detection is avoided, and the accuracy of the detection result is improved. Of course, the magnitude of amplification can also be controlled, and the amount of amplification can be controlled by controlling the residence time of the sample to be tested inside the temperature-controlled flow channel 2123.
Wherein, a thermal insulation layer is arranged between the first temperature control region 2121 and the second temperature control region 2122, and the temperature transmission between the first temperature control region 2121 and the second temperature control region 2122 can be avoided through the arrangement of the thermal insulation layer, so that the first temperature control region 2121 is always a high temperature constant temperature region, the second temperature control region 2122 is always a low temperature constant temperature region, and the denaturation and renaturation of DNA are controlled through temperature change, thereby completing amplification.
The thermal insulation layer can be arranged when the temperature control flow passage 2123 is processed, namely, the temperature control flow passage 2123 is divided into two parts for processing or is divided after being integrally processed, and then the thermal insulation layer is coated at the joint of the two parts of the temperature control flow passage 2123.
The temperature control flow passage 2123 controls the temperature through the control component, so that the temperature control flow passage 2123 in the first temperature control region 2122 can be controlled to be always in a high temperature constant temperature state, the DNA is denatured and melted into single-stranded DNA at a high temperature, the temperature control flow passage 2123 in the second temperature control region 2122 can be controlled to be always in a low temperature constant temperature state, the DNA is renatured into double-stranded DNA at a low temperature, and the denaturation and renaturation of the DNA are controlled through the change of temperature, so that amplification is completed.
In some embodiments, the sensing electrodes 2112 are configured in a cylindrical structure and are uniformly distributed on the surface of the electrode plate 2111, where the size of each sensing electrode 2112 may be the same or different, the number of capture probes immobilized on each sensing electrode 2112 may be the same or different, and the types of capture probes may be the same or different, depending on the requirements of the application.
Alternatively, the sensing electrode 2112 is configured in a cuboid structure, and a plurality of capture probes are arranged on the sensing electrode 2112 in the cuboid structure, so that the capture probes can capture the target genes of interest; the sensing electrodes 2112 can be provided in plurality, and each sensing electrode 2112 is provided with a type of capture probe, so that the sensing electrodes 2112 can be provided with a plurality of capture probes, and a plurality of target genes can be detected through one detection experiment, so that multi-index detection is realized, and the detection efficiency is improved.
The arrangement direction of the sensing electrodes 2112 with a cuboid structure may be perpendicular to the direction in which the sample to be measured enters the detection area 211, and a plurality of sensing electrodes 2112 may be arranged, and the sensing electrodes 2112 with a plurality of cuboid structures are arranged on the surface of the electrode plate 2111 along the height direction Z of the kit main body 1, which is only one arrangement mode, but may also be other arrangement modes.
It should be appreciated that the sensing electrodes 2112 may be any other shape, such as oval, triangle, quadrangle, pentagon, etc., and the number and type of capture probes provided on each sensing electrode 2112 are not limited, and may be set according to the use requirements.
In some embodiments, the reaction zone 2 further comprises a reaction plate 22, the reaction plate 22 is connected with the kit main body 1, and a sample inlet channel 23 and a sample outlet channel 24 are formed on the reaction plate 22; the reaction chamber 21 is arranged on the reaction plate 22, one side of the reaction chamber 21 is connected with the sample inlet channel 23, the other side is connected with the sample outlet channel 24, and the sample inlet channel 23 and the sample outlet channel 24 are communicated with the inside of the kit main body 1 at the same time. That is, when liquid feeding is needed, the sample feeding channel 23 is communicated with the kit main body 1, the sample discharging channel 24 is blocked, the sample to be detected flows into the reaction cavity 21 through the sample feeding channel 23, and after detection is completed, the sample to be detected flows back to the kit main body 1 through the sample discharging channel 24.
The temperature control flow channel 2123 includes an inlet 2124 and an outlet 2125, the inlet 2124 is communicated with the sample injection channel 23, the outlet 2125 is communicated with the detection zone 211, and the height of the outlet 2125 is higher than that of the sensing electrode 2112, so that a sample to be detected can flow to the detection zone 211 conveniently and is captured by a capture probe on the sensing electrode 2112 more easily.
The reaction zone 2 further includes a sealing film 25, where the sealing film 25 covers the surface of the reaction plate 22, so that the reaction chamber 21, the sample inlet channel 23 and the sample outlet channel 24 form a closed space, which can facilitate the inflow and outflow of the sample to be measured. Here, the sealing film 25 can also cover the temperature control flow passage 2123.
The reaction area 2 and the main body 1 of the kit are integrally formed or welded.
In some embodiments, the kit body 1 includes a case 11 and a switching valve 14, the case 11 includes a central through hole 112, and the outer circumference of the central through hole 112 is configured with a plurality of receiving chambers isolated from each other, one receiving chamber for receiving a sample to be measured; the switching valve 14 comprises a cylinder 141 inserted in the central through hole 112 and a valve seat 142 positioned at one end of the cylinder 141, wherein a plunger 143 is embedded in a cylinder cavity 144 of the cylinder 141, an inlet and an outlet which are communicated with the cylinder cavity 144 are formed in the valve seat 142, when the inlet and the outlet are aligned and communicated with a containing cavity for containing a sample to be tested, the plunger 143 is pushed to enable the sample to be tested to enter and exit the cylinder cavity 144, when the inlet and the outlet are aligned and communicated with the sample feeding channel 23 or the sample discharging channel 24, the plunger 143 is pushed to enable the sample to be tested to enter and exit the reaction zone 2, so that the sample to be tested can be controlled to enter or exit the reaction zone 2 through the movement of the plunger 143, and the operation is convenient. Here, a control member is connected to the plunger 143, and the control member controls the plunger 143 to move in the cylinder.
The temperature control flow channel 2123 includes a plurality of flow channels, the control component controls the plunger 143 to move, so that the plunger 143 controls the sample to be tested to flow into the temperature control flow channel 2123, at this moment, the plunger 143 controls the sample to be tested to flow into one flow channel, and the sample to be tested at least fills up a part of the flow channel, stays for 4s-6s, controls the denaturation and renaturation of DNA, then the plunger 143 moves again, and circulates reciprocally until the sample to be tested all enters the detection area 211 for detection through the temperature control flow channel 2123, and the stay time is in seconds, so that the stay time of the sample to be tested in the whole amplification area 212 is shorter, so that the detection time of the whole kit is shorter, and meanwhile, the sample to be tested can control the denaturation and renaturation of DNA in each flow channel, so that the target gene can be amplified for multiple times, so that the concentration of the target gene can be improved rapidly in a shorter time, and the accuracy of the detection result can be improved.
Here, the temperature control channels 2123 are provided in advance, so that the number and volume of channels in the temperature control channels 2123 are also known, and the volume of the sample to be measured is also known, and the structure inside the cartridge body 1 is also known, so that the control part can control the sample to be measured to flow into the reaction region 2 by controlling the moving distance of the plungers 143. Of course, the volume of the sample to be measured can be filled with half flow channels, one flow channel, two flow channels or a plurality of flow channels, and can be specifically determined according to the volume of the sample to be measured injected into the accommodating cavity.
For example, the volume of the sample to be measured can fill up half of the channels, by pushing the plunger 143 to move downward, the sample to be measured in the accommodating cavity can be pushed to the sample injection channel 23, then flows to one of the channels 2123, and fills up the high temperature constant temperature area of the first channel, after filling up, the control part controls the plunger 143 to move downward again, then gives a pulse to the sample to be measured, makes it enter the low temperature constant temperature area of the first flow tube, after the sample to be measured stays for 4s-6s, the control part controls the plunger 143 to move downward again, makes it enter the high temperature constant temperature area of the second channel, after the sample to be measured stays for 4s-6s, the control part controls the plunger 143 to move downward, makes it enter the low temperature constant temperature area of the second flow channel, and after the sample to be measured stays for 4s-6s, and so on, the sample to be measured flows into the detection area 211 after multiple amplifications, and detects in the detection area 211.
Or, the volume of the sample to be measured can fill up one flow channel, by pushing the plunger 143 to move downwards, the sample to be measured in the accommodating cavity can be pushed to the sample injection channel 23, then a part of the sample to be measured flows to one flow channel in the temperature control flow channel 2123 and fills up the high temperature constant temperature area of the first flow channel, after the part of the sample to be measured stays for 4s-6s, the control part further controls the plunger 143 to move downwards, then a pulse is given to the sample to be measured, so that the part of the sample to be measured enters the low temperature constant temperature area of the first flow channel, and meanwhile, another part of the sample to be measured enters the high temperature constant temperature area of the first flow channel to fill up the same, and stays for 4s-6s, the control part further controls the plunger 143 to move downwards, so that the sample to be measured enters the next flow channel, and then the sample to be measured flows into the detection area 211 after being amplified for multiple times and is detected in the detection area 211.
Or, the volume of the sample to be measured can fill up a plurality of channels, the amplification mode is the same as that when the volume of the sample to be measured can fill up one channel, but after the sample to be measured in the first channel enters the second channel, the first channel is filled up with the sample to be measured in the sample inlet channel 23, and so on, the sample to be measured flows into the detection area 211 after being amplified for a plurality of times, and is detected in the detection area 211.
The flow channel is divided into two parts by the high temperature constant temperature area and the low temperature constant temperature area, the length of the flow channel in the high temperature constant temperature area is the same as that of the flow channel in the low temperature constant temperature area (the two are basically the same, even if a small part of the flow channel is poor, the final result is not influenced), and the moving distance of the sample to be measured is the length of one half flow channel.
Because the runner size is less, so the sample to be measured has adhesive force on the surface of the runner, and the sample to be measured is difficult to flow by itself under the action of the adhesive force, and can move mainly by virtue of the movement of the plunger 143.
Meanwhile, the plunger 143 herein can be fully automatically controlled, and since the parameters are known, it can be fully controlled by the control part; alternatively, the plunger 143 may be semi-automatically controlled, and since the sealing film 25 is transparent, the movement of the plunger 143 may be controlled by manually observing the filling condition in the flow path and then manipulating the control member.
In some embodiments, when the temperature control flow channel 2123 and the sealing film 25 are not completely adhered, the inlet 2124 of the temperature control flow channel 2123 may be provided with a drainage plate 2126, the first end of the drainage plate 2126 is communicated with the inlet 2124 of the temperature control flow channel 2123, the second end of the drainage plate 2126 is communicated with the sample introduction channel 23, the drainage plate 2126 mainly plays the role of drainage and transition, and the height of the drainage plate 2126 may be slightly higher than that of the temperature control flow channel 2123, by setting the drainage plate 2126, the sample to be measured can be introduced into the inlet 2124 of the temperature control flow channel 2123, and then flows along the flow channel of the temperature control flow channel 2123, so as to prevent the sample to be measured from directly flowing to other positions of the amplification region 212, so as to further ensure that the sample to be measured can move according to the preset path of the temperature control flow channel 2123, and avoid loss of control of amplification of the sample to be measured.
Or, when the temperature control flow channel 2123 and the sealing film 25 are attached, the drainage plate 2126 may not be provided, the sample to be measured may directly enter the temperature control flow channel 2123 through the sample injection channel 23, at this time, the sample to be measured may flow into the high temperature constant temperature region of the first flow channel through the low temperature constant temperature region of the first flow channel, and then stay for 4s to 6s, and the subsequent amplification method is consistent with the above, so that no description is repeated here.
As shown in fig. 2, 6-8, in some embodiments, the cartridge body 1 includes a cartridge body 11 and a switching valve 14; the case 11 includes a central through hole 112, wherein a plurality of holding cavities isolated from each other are configured at the periphery of the central through hole 112, one of the holding cavities is configured as a sample cavity 101, the sample cavity 101 is used for holding a sample to be tested, the other holding cavities can be configured as reagent cavities for holding various reagents required for preprocessing the sample to be tested, such as a lysis solution cavity 102, a cleaning solution cavity 103, a buffer solution cavity 104, a waste solution cavity 105, a primer probe mixed solution cavity 106, a first reserved cavity 107 and a second reserved cavity 108, wherein the first reserved cavity 107 and the second reserved cavity 108 serve as standby holding cavities for holding any possible reagents or serve as other functions, and the bottom of each holding cavity is provided with a liquid passing hole 111; the switching valve 14 comprises a cylinder body 141 inserted in the central through hole 112 and a valve seat 142 positioned at one end of the cylinder body 141, wherein a plunger 143 is embedded in a cylinder cavity 144 of the cylinder body 141, a processing area 147 is arranged in the valve seat 142, an inlet and an outlet which are communicated with the processing area 147 are also arranged on the valve seat 142, the inlet and the outlet comprise a first inlet and an outlet 145 and a second inlet and outlet 146 which are arranged at an angle on the same circumference radius, the angle range is 60-180 degrees, preferably 180 degrees, wherein one end of the first inlet and outlet 145 can be communicated with a liquid passing hole 111 at the bottom of the containing cavity in an aligned mode, the other end of the first inlet and outlet 145 is communicated with the processing area 147, one end of the second inlet and outlet 146 can be communicated with the liquid passing hole 111 at the bottom of the containing cavity in an aligned mode, the other end of the second inlet and outlet is communicated with the processing area 147 and the cylinder cavity 144, the switching valve 14 can be driven to rotate around the axis of the central through hole 112 so as to realize that any one of the first inlet and the second inlet and the outlet 146 is communicated with one of a plurality of containing cavities in an aligned mode, and when one of the first inlet and the second inlet and the outlet 146 is communicated with one of the containing cavity and the other containing cavity and is not communicated with all of the containing cavities; the sample inlet channel 23 and the sample outlet channel 24 are respectively communicated with the corresponding liquid through holes 111 of the accommodating cavity.
The following describes the process of the sample to be measured entering the sample introduction channel 23 from the sample chamber 101 and finally flowing to the reaction chamber 21: the switching valve 14 rotates to enable the liquid passing hole 111 of the sample cavity 101 to be aligned and communicated with the second inlet and outlet 146, the plunger 143 moves upwards in the barrel cavity 144, the pressure in the barrel cavity 144 is reduced, the sample to be detected flows from the sample cavity 101 to the liquid passing hole 111, the first inlet and outlet 146 and finally flows to the treatment area 147 and the barrel cavity 144 in sequence, then the switching valve 14 rotates again to enable the liquid passing hole 111 of the sample feeding channel 23 to be aligned and communicated with the first inlet and outlet 145, the plunger 143 descends in the barrel cavity 144, the pressure in the barrel cavity 144 is increased, the sample to be detected flows from the treatment area 147 and the barrel cavity 144 to the first inlet and outlet 145 and the liquid passing hole 111 of the sample feeding channel 23 and finally enters the reaction cavity 21 through the sample feeding channel 23, and similarly, after the sample to be detected is detected, the switching valve 14 rotates again to enable the liquid passing hole 111 of the sample feeding channel 24 to be aligned and communicated with one of the first inlet and outlet 145 or the second inlet and outlet 146, the plunger 143 moves upwards in the barrel cavity 144, the pressure in the sample feeding channel 23 is reduced, and the sample to be detected flows to the treatment area 147 and the barrel cavity 144 through the sample feeding channel 24.
As shown in fig. 2, the kit main body 1 further includes a top cover 12 and a base 13, and the connection mode between the top cover 12 and the kit body 11 may be welding, bonding or other sealing connection modes; the base 13 and the box body 11 can be detachably connected and also can be fixedly connected, when the base 13 and the box body 11 are detachably connected, at least one buckle is arranged on the base 13, a clamping groove is formed in the box body 11 corresponding to the buckle, the buckle is arranged in the clamping groove through the clamping, so that the base 13 is connected with the box body 11, the box body 11 and the base 13 can be firmly connected, the connection is convenient, the box body 11 and the switching valve 14 can be checked through the base 13, and the damaged switching valve 14 or the box body 11 can be replaced conveniently.
In summary, in the electrochemical detection kit provided in this embodiment, since the part of the section of the sample to be detected is already specifically paired with the signal probe before the sample to be detected enters the reaction chamber 21, after the sample to be detected after being specifically paired with the signal probe enters the reaction chamber 21, the remaining section of the sample to be detected is specifically paired with the capture probe, and triggers the signal probe, so that the signal probe changes the current or voltage of the sensing electrode 2112, and transmits the current signal or voltage signal to the external detection component, thereby being capable of carrying out qualitative detection on the sample to be detected, avoiding the fluorescent crosstalk problem by the detection method, and improving the accuracy of the detection result; the amplification region 212 is internally provided with a temperature control flow channel 2123, one part of the temperature control flow channel 2123 is located in a high temperature constant temperature region, the other part of the temperature control flow channel 2123 is located in a low temperature constant temperature region, so that a sample to be detected flows alternately between the high temperature constant temperature region and the low temperature constant temperature region along the temperature control flow channel 2123, when the sample to be detected flows to the detection region 211, the sample to be detected is amplified, the amplified sample to be detected is specifically paired with a capture probe, so that detection is completed, and the acquired target gene concentration in the sample to be detected is unknown, so that the condition that the sample to be detected flows into the detection region 211 and is missed to be detected after the sample to be detected is amplified, and the accuracy of a detection result is further improved.
The embodiment of the disclosure also provides a detection method, which comprises the following steps:
plunger 143 controls the sample to be tested to enter sample injection channel 23;
the sample to be measured in the sample injection channel 23 is controlled by the plunger 143 and flows into one flow channel in the temperature control flow channel 2123, and the sample to be measured at least fills part of the flow channel and stays for 4s-6s;
plunger 143 controls the sample to be tested to flow again, and to cycle back and forth until the sample to be tested all enters detection zone 211 through temperature control flow channel 2123;
the capture probe in the detection zone 211 specifically mates with the remaining section of the sample to be detected and triggers the signaling probe, causing the signaling probe to change the current or voltage of the sensing electrode 2112 and transmit the current signal or voltage signal to an external detection component.
Since the detection method is performed in the above-mentioned electrochemical detection kit and has the same or similar technical effects as described above, and the detection method is also described above, the description of the above-mentioned electrochemical detection kit will not be repeated.
In the description of the present invention, it should be understood that the orientation or positional relationship indicated is based on the orientation or positional relationship shown in the drawings, and is merely for convenience in describing the present invention and simplifying the description, and does not indicate or imply that the apparatus or element referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present invention.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. An electrochemical test kit comprising:
the kit body is used for containing a sample to be tested;
the reaction zone is connected with the kit main body and comprises a reaction cavity, the reaction cavity is communicated with the kit main body and comprises a detection zone and an amplification zone, the detection zone is provided with a sensing electrode, and the sensing electrode is provided with a capture probe; the amplification region comprises a first temperature control region for heating and a second temperature control region for cooling, a temperature control flow passage is arranged in the amplification region, one part of the temperature control flow passage is positioned in the first temperature control region, the other part of the temperature control flow passage is positioned in the second temperature control region, and the sample to be tested alternately flows between the first temperature control region and the second temperature control region along the temperature control flow passage;
a detection member electrically connected to the induction electrode;
the temperature control flow channel is arranged in a coil-type manner along the height direction (Z) of the kit main body, the upper half part of the temperature control flow channel is positioned in the first temperature control area, and the lower half part of the temperature control flow channel is positioned in the second temperature control area.
2. The electrochemical detection kit of claim 1, further comprising an electrode plate removably coupled within the reaction chamber, the sensing electrode disposed on the electrode plate.
3. The electrochemical detection kit of claim 1, wherein a thermal barrier is disposed between the first temperature control zone and the second temperature control zone.
4. The electrochemical detection kit according to any one of claims 1 to 3, wherein the reaction zone further comprises a reaction plate, the reaction plate is connected with the kit main body, and a sample inlet channel and a sample outlet channel are formed on the reaction plate;
the reaction cavity is arranged on the reaction plate, one side of the reaction cavity is communicated with the sample inlet channel, the other side of the reaction cavity is communicated with the sample outlet channel, and the main body of the kit is communicated with the sample inlet channel and the sample outlet channel at the same time.
5. The electrochemical detection kit of claim 4, wherein the temperature-controlled flow channel comprises an inlet and an outlet, the inlet is in communication with the sample introduction channel, the outlet is in communication with the detection zone, and the outlet is higher than the sensing electrode.
6. The electrochemical detection kit according to claim 4, wherein the reaction zone further comprises a sealing film, and the sealing film covers the surface of the reaction plate, so that the reaction chamber, the sample introduction channel and the sample discharge channel form a closed space.
7. The electrochemical detection kit according to claim 4, wherein the kit body comprises a case body and a switching valve, the case body comprises a central through hole, the periphery of the central through hole is configured with a plurality of receiving cavities isolated from each other, and one of the receiving cavities is used for receiving the sample to be detected;
the switching valve comprises a cylinder body inserted in the central through hole and a valve seat positioned at one end of the cylinder body, a plunger is embedded in a cylinder cavity of the cylinder body, an inlet and an outlet communicated with the cylinder cavity are arranged on the valve seat, and when the inlet and the outlet are aligned and communicated with the containing cavity for containing the sample to be tested, the plunger is pushed to enable the sample to be tested to come in and go out of the cylinder cavity; when the inlet and the outlet are aligned and communicated with the sample inlet channel or the sample outlet channel, the plunger is pushed to enable the sample to be detected to enter and exit the reaction zone.
8. The electrochemical detection kit according to claim 7, wherein a drainage plate is arranged at the inlet of the temperature control flow channel, one end of the drainage plate is communicated with the inlet of the temperature control flow channel, and the other end of the drainage plate is communicated with the sample injection channel;
the height of the drainage plate is higher than that of the temperature control flow passage.
9. A detection method based on the electrochemical detection kit according to any one of claims 1 to 8, characterized by comprising:
the plunger controls a sample to be tested to enter the sample injection channel;
the sample to be measured in the sample injection channel is controlled by the plunger and flows into one of the temperature control flow channels, and the sample to be measured at least fills part of the flow channels and stays for 4s-6s;
the plunger controls the sample to be tested to flow again, and the reciprocating cycle is carried out until the sample to be tested completely enters the detection area through the temperature control flow channel;
the capture probe in the detection area is specifically paired with the residual section of the sample to be detected, and the signal probe is triggered, so that the signal probe changes the current or voltage of the induction electrode, and a current signal or voltage signal is transmitted to an external detection component.
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| TW201908729A (en) * | 2017-07-17 | 2019-03-01 | 國立臺灣師範大學 | Portable rapid temperature regulation and control thermal circulation device having characteristics of instant detection and rapid temperature control |
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Address after: Building 1, No. 42 Qibei Road, Changping District, Beijing 102209 Patentee after: Kunpeng Gene (Beijing) Scientific Instrument Co.,Ltd. Country or region after: China Patentee after: Kunpeng (Xuzhou) Scientific Instrument Co.,Ltd. Address before: 2202, 19th Floor, Building 9, No. 8 Ronghua Middle Road, Daxing District, Beijing Patentee before: ROCGENE TECNOLOGY Co. Country or region before: China Patentee before: Kunpeng (Xuzhou) Scientific Instrument Co.,Ltd. |