CN110736844A - detection method of cardiac troponin - Google Patents
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- CN110736844A CN110736844A CN201911246006.6A CN201911246006A CN110736844A CN 110736844 A CN110736844 A CN 110736844A CN 201911246006 A CN201911246006 A CN 201911246006A CN 110736844 A CN110736844 A CN 110736844A
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- 230000000747 cardiac effect Effects 0.000 title claims abstract description 21
- 238000001514 detection method Methods 0.000 title description 31
- 238000006243 chemical reaction Methods 0.000 claims abstract description 165
- 239000007788 liquid Substances 0.000 claims abstract description 123
- 238000002156 mixing Methods 0.000 claims abstract description 74
- 230000000739 chaotic effect Effects 0.000 claims abstract description 61
- 238000000034 method Methods 0.000 claims abstract description 23
- 238000004140 cleaning Methods 0.000 claims abstract description 22
- 239000000427 antigen Substances 0.000 claims abstract description 10
- 108091007433 antigens Proteins 0.000 claims abstract description 8
- 102000036639 antigens Human genes 0.000 claims abstract description 8
- 238000012546 transfer Methods 0.000 claims abstract description 8
- 238000001035 drying Methods 0.000 claims abstract description 7
- 230000008105 immune reaction Effects 0.000 claims abstract description 6
- 239000012528 membrane Substances 0.000 claims description 33
- 239000002699 waste material Substances 0.000 claims description 31
- 210000002966 serum Anatomy 0.000 claims description 19
- 238000011049 filling Methods 0.000 claims description 14
- 238000001917 fluorescence detection Methods 0.000 claims description 8
- 230000000149 penetrating effect Effects 0.000 claims description 6
- 238000007789 sealing Methods 0.000 claims description 2
- 102000004903 Troponin Human genes 0.000 abstract description 12
- 108090001027 Troponin Proteins 0.000 abstract description 12
- 239000012530 fluid Substances 0.000 abstract description 9
- 238000013461 design Methods 0.000 abstract description 2
- 230000010354 integration Effects 0.000 abstract 1
- 238000010586 diagram Methods 0.000 description 9
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- 238000001215 fluorescent labelling Methods 0.000 description 1
- 230000017525 heat dissipation Effects 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 238000002032 lab-on-a-chip Methods 0.000 description 1
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Abstract
The invention discloses a method for detecting cardiac troponins, which comprises the following steps of (1) adding a quantitative sample from a liquid adding port by a liquid transfer device, (2) enabling the sample to flow into a reaction cavity through a micro flow channel, enabling a micro fluid chip and a chaotic convection mixing device to be in integration design, starting mixing uniformly for 3-15 min, enabling an antigen of the sample to carry out an immune reaction with an antibody in the reaction cavity, stopping the chaotic convection mixing device, (3) pushing air into the liquid adding port by the liquid transfer device to push liquid in the micro fluid chip to move forward to blow the reaction cavity and the flow channel, drying the reaction cavity and the flow channel, 4) adding a cleaning liquid into the liquid adding port to fill the reaction cavity, starting the chaotic convection mixing device again, mixing uniformly for 1-3 min, cleaning, stopping the chaotic convection mixing device, pushing air into the micro fluid chip by the liquid transfer device to blow the micro flow channel and the reaction cavity, 5) repeating the step (4) and cleaning for 3-5.
Description
Technical Field
The invention belongs to the technical field of medical equipment, and particularly relates to a detection method of cardiac troponins.
Background
Microfluidics is a technology applied across various disciplines including engineering, physics, chemistry, microtechnology, and biotechnology. Microfluidics involves the study of minute quantities of fluids and how to manipulate, control and use such small quantities of fluids in various microfluidic systems and devices, such as microfluidic detection chips. For example: microfluidic biochips (known as "lab-on-a-chip") are used in the field of molecular biology to integrate assay operations for purposes such as analyzing enzymes and DNA, detecting biochemical toxins and pathogens, diagnosing diseases, and the like.
The micro-fluidic chip Analysis takes the chip as an operation platform, simultaneously takes analytical chemistry as a basis, relies on micro-electromechanical processing technology, takes a micro-pipeline network as a structural characteristic, takes life science as a main application object at present, and is the key point of the development of the field of the micro-fluidic Analysis system.
However, when the micro-fluid chip is used for detection, how to improve the uniform mixing effect of a sample in a closed narrow reaction cavity of the micro-fluid chip is a problem which needs to be solved urgently, a mode of uniformly mixing magnetic beads is adopted, compared with the traditional separation method, the magnetic beads are used for separating complex components of a biochemical sample, separation and enrichment can be realized simultaneously, the separation speed and the enrichment efficiency are effectively improved, and meanwhile, the sensitivity of analysis and detection is greatly improved.
Therefore, it is necessary to develop methods for detecting cardiac troponin, which has high mixing efficiency, high accuracy of detection results, reduced cost and simple operation.
Disclosure of Invention
The invention aims to solve the technical problem of providing methods for detecting cardiac troponin by adopting a microfluid chip, which have the advantages of high immune reaction mixing efficiency, high result accuracy, reduced cost and simpler and more convenient operation.
In order to solve the technical problems, the technical scheme adopted by the invention is that the method for detecting cardiac troponin by using the microfluid chip specifically comprises the following steps:
(1) adding a quantitative sample plasma/serum from the liquid adding port by using a liquid transfer machine;
(2) the sample plasma/serum sequentially passes through the middle-layer liquid adding through hole, the liquid adding input micro-channel and the reaction cavity input channel to flow into the reaction cavity, the micro-fluidic chip and the chaotic convection blending device are designed in an body mode, the chaotic convection blending device is started to blend the sample plasma/serum in the reaction cavity for 3-15 min, a sample antigen and an antibody in the reaction cavity perform an immune reaction, and the chaotic convection blending device is stopped;
(3) pushing air into the liquid adding opening by adopting the liquid shifter, pushing liquid in the microfluidic chip to move forwards, and drying the reaction cavity and the flow channel;
(4) adding a cleaning solution into the liquid adding port until the reaction cavity is filled, starting the chaotic convection mixing device again, mixing for 1-3 min, cleaning, stopping the chaotic convection mixing device, and pushing air into the microfluidic chip by a liquid shifter to blow dry the microfluidic channel and the reaction cavity of the chip;
(5) repeating the step (4) and cleaning for 3-5 times;
(6) and carrying out fluorescence detection on the microfluidic chip to obtain a reaction fluorescence value.
By adopting the technical scheme, the sample plasma/serum is sucked by a liquid transfer device through manual sample introduction and is added into a liquid adding port, the sample enters a reaction cavity, the mixture is uniformly mixed by a chaotic convection uniformly mixing device, the immunoreaction is carried out, and the fluorescence signal value is detected after the steps of cleaning and the like; the sample reaction fluorescence value of the immune reaction carried out by uniformly mixing the mixture by the chaotic convection uniformly mixing device is high, and the immune reaction of the antibody and the antigen is more sufficient.
The invention aims to solve the technical problem of providing microfluid detection chips, which can be matched with a high-efficiency mixing device for mixing uniformly, and have the advantages of high mixing efficiency, high detection result accuracy, reduced cost and simpler operation.
In order to solve the technical problems, the technical scheme includes that the microfluidic chip used for detecting the cardiac troponin comprises a chip body, the chip body sequentially comprises a lower chip, a middle chip and an upper chip from bottom to top, the chip body comprises a liquid adding port, a reaction cavity and a micro-channel, the middle chip and the lower chip are matched to define a closed micro-channel and a reaction cavity, the liquid adding port is communicated with the reaction cavity through the micro-channel, at least grooves are formed in the reaction cavity, the grooves are formed in the reaction cavity and used for coating antibodies, fluorescence-labeled antibodies are fixed in an area outside the grooves in the reaction cavity, compared with the previous design, the positions of the coated antibodies are more concentrated, fluorescence signals after reaction are more concentrated, the peak position of a fluorescence detection curve is more stable, the CV value of a detection result is smaller, repeatability is good, the detection accuracy is improved, the detection chip enters sample liquid through the liquid adding port manually, the detection chip can be used independently, if the number of the detection chip is smaller, the result can be quickly given out, the detection is suitable for verifying the detection, the detection is more convenient for reducing the reaction cost of a sample liquid and the detection, the detection is more convenient for the concentrated reaction of a fluorescence detection instrument, the detection of a sample, the detection material, the detection is more concentrated, the detection of the sample liquid, the detection of the sample liquid, the sample liquid is more concentrated reaction, the detection of the.
, the bottom of the reaction cavity is provided with a chaotic convection mixing device, a microfluid chip and the chaotic convection mixing device are integrated, wherein the convection mixing device is of a 3-layer structure, the material and the microfluid chip are both made of plastics, and the microfluid chip is bonded or adhered with the microfluid chip after being integrally bonded to form a body for detection, namely, the microfluid chip is provided with the mixing device, no additional matching mixing instrument is needed, and the use is convenient.
As a preferred technical solution of the present invention, the reaction chamber includes an upper reaction chamber disposed on the back surface of the middle chip and a lower reaction chamber disposed through the lower chip, and the position of the upper reaction chamber on the back surface of the middle chip corresponds to the position of the lower reaction chamber on the lower chip; the bottom of the lower reaction chamber is covered on the back surface of the lower chip by an elastic film for sealing, and the groove is arranged in the upper reaction chamber. The fluorescence labeling antibody is fixed in the area outside the groove of the upper reaction chamber or on the elastic film of the lower reaction chamber on the front surface of the lower chip; the groove is arranged in the upper reaction chamber, so that the position of the coated antibody is more concentrated, the fluorescent signal after reaction is more concentrated, the repeatability is good, and the detection accuracy is improved; the bottom of the lower reaction chamber is covered and sealed by an elastic film, so that the transmission of blending energy is facilitated, and the high-efficiency blending device, namely a disturbance column cap of the chaotic convection blending device, is matched with the reaction chamber to enhance the blending effect.
As a preferred technical scheme of the invention, the grooves are inwards recessed circular grooves, the number of the grooves is 3, and the 3 grooves are uniformly distributed in the reaction cavity, the three circular grooves are arranged in the reaction cavity and can be used for coating three antibodies, a chip product is a three-way card, three indexes of the same sample can be detected, the simultaneous detection of multiple indexes of the sample can be realized, compared with the traditional multi-index microfluidic chip, the production cost can be reduced, the experimental operation is simpler and more convenient, and the structure of a matched detection instrument is simpler.
As a preferred technical scheme, the micro flow channel comprises a reaction cavity input flow channel and a reaction cavity output flow channel, the reaction cavity input flow channel and the reaction cavity output flow channel are arranged on the back surface of the middle chip, the reaction cavity input flow channel is communicated with the end of the upper reaction cavity, the reaction cavity output flow channel is communicated with the other end of the upper reaction cavity, the reaction cavity input flow channel is communicated with the liquid filling port, a backflow prevention structure is arranged between the reaction cavity input flow channel and the liquid filling port, the backflow prevention structure can achieve the effect that sample liquid in the reaction cavity cannot flow to the front and back directions of the reaction cavity in the flow channel within the common shaking range of the reaction cavity, the reaction cavity input flow channel and the reaction cavity output flow channel are arranged on the back surface of the middle chip, gas is enabled to be filled with the sample liquid in the reaction, the gas is prevented from possibly occupying the upper part of the reaction cavity when the reaction cavity input flow channel and the reaction cavity output flow channel of the lower chip are arranged on the front surface of the lower chip, and the gas possibly occupies the upper part of the reaction cavity, so that the sample liquid cannot.
As a preferred technical scheme of the invention, the liquid adding port is penetratingly arranged on the upper chip, the middle chip is penetratingly arranged with a middle liquid adding through hole at a corresponding position of the liquid adding port, and the front of the lower chip is provided with a liquid adding input micro-channel at a corresponding position of the liquid adding port; the liquid feeding input micro-channel is communicated with the liquid feeding port through the middle-layer liquid feeding through hole, and the reaction cavity input channel is communicated with the liquid feeding port through the liquid feeding input micro-channel. The filling opening is funnel-shaped on the upper chip, the opening of the filling opening is gradually reduced from the front surface of the upper chip to the back surface of the upper chip, and the sample to be detected is better added from the filling opening.
As a preferable technical scheme, the backflow prevention structure comprises a vertical flow channel , a second vertical flow channel and a backflow prevention connecting flow channel, the backflow prevention connecting flow channel is arranged on the back face of the upper chip, the vertical flow channel and the second vertical flow channel penetrate through the middle chip, and the liquid feeding input micro flow channel sequentially passes through the second vertical flow channel, the backflow prevention connecting flow channel and the vertical flow channel and then is communicated with the reaction cavity input flow channel.
As a preferred technical scheme of the invention, the chip body further comprises a waste liquid cavity, the waste liquid cavity is arranged on the lower chip, and a middle-layer waste liquid cavity through hole is arranged on the middle chip in a penetrating manner at a position corresponding to the waste liquid cavity; the back of the upper chip is provided with a waste liquid cavity cover plate at a position corresponding to the waste liquid cavity, correspondingly, the front of the upper chip is provided with a waste liquid cavity exhaust hole, the front of the middle chip is provided with an auxiliary leakage-proof waste liquid cavity at the upper part of the waste liquid cavity, and the auxiliary leakage-proof waste liquid cavity is provided with a waste liquid cavity middle exhaust hole at a position corresponding to the waste liquid cavity exhaust hole in a penetrating manner.
As a preferable technical solution of the present invention, the reaction chamber is sequentially communicated with the waste liquid chamber through the reaction chamber output flow channel and the waste liquid output micro flow channel, the waste liquid output micro flow channel is disposed on the front surface of the lower chip, a conductive rubber valve is disposed between the waste liquid output micro flow channel and the reaction chamber output flow channel, and the conductive rubber valve includes an upper conductive rubber valve structure disposed on the upper chip and a middle conductive rubber valve structure disposed at a position corresponding to the middle chip.
The method is characterized in that a chaotic convection mixing device is arranged on the back surface of a lower chip and is integrated with a chip body, the chaotic convection mixing device sequentially comprises an upper layer structure, a middle layer structure and a lower layer structure from top to bottom, a disturbance column through hole penetrates through the upper layer structure at a position corresponding to a reaction cavity, an elastic membrane is arranged between the upper layer structure and the middle layer structure, a disturbance column is arranged on the elastic membrane, the disturbance column penetrates through the disturbance column through hole to be in contact with an elastic membrane coated outside the bottom of the reaction cavity, an elastic membrane cavity is matched with the upper layer structure and the middle layer structure at a position corresponding to the reaction cavity, a middle layer air outlet and at least disturbance air inlets are arranged on the elastic membrane cavity, a lower layer air outlet is arranged at a position corresponding to the middle layer air outlet, an air inlet and an air inlet channel are further arranged on the middle layer structure, the air inlet is communicated with the disturbance air inlet through the air inlet channel, the technical scheme is adopted, the microfluidic chip is arranged with the chaotic convection mixing device , wherein the material and the microfluidic chip 57 are both of the chaotic convection mixing device and are in a chaotic convection mixing device, so that the chaotic convection reaction of a chaotic column and a chaotic reaction chip bonding reaction is more convenient and a chaotic reaction is realized, even if the chaotic convection mixing device is used for forming a chaotic reaction is more convenient and a chaotic reaction, the chaotic mixing device, the chaotic convection detection of a chaotic convection sample is more convenient stirring device, the chaotic mixing device, the chaotic convection sample is more convenient stirring device, the chaotic mixing device is more convenient stirring device, the chaotic.
As a preferred technical solution of the present invention, the elastic film cavity includes an upper layer elastic film cavity disposed on the back side of the upper layer structure and a middle layer elastic film cavity disposed on the front side of the middle layer structure; the elastic diaphragm is arranged between the upper layer elastic diaphragm cavity and the middle layer elastic diaphragm cavity; the middle layer air outlet and the disturbance air inlet are arranged on the middle layer elastic membrane cavity. The upper layer structure and the middle layer structure are tightly combined by the arrangement, the elastic membrane is ensured to be air-tight, meanwhile, the elastic membrane can cover a disturbance air inlet on the middle layer elastic membrane cavity, two air flows are convected in the middle layer elastic membrane cavity, two alternative motion modes are formed, chaotic convection is formed, and then a sample liquid in a reaction cavity contacted with a disturbance column is disturbed by the disturbance column on the elastic membrane between the middle layer elastic membrane cavity and the upper layer elastic membrane cavity, so that the liquid in the reaction cavity is uniformly mixed.
As a preferable technical scheme of the invention, the air inlet is arranged on the side surface of the middle layer structure, the air inlet channel is arranged on the back surface of the middle layer structure, the end of the air inlet channel is connected with the air inlet, and the other end of the air inlet channel is communicated with the disturbance air inlet of the middle layer elastic membrane cavity.
As a preferred technical solution of the present invention, the upper layer elastic membrane cavity is provided with an upper layer exhaust passage on the back of the upper layer structure, correspondingly, the middle layer structure is provided with a middle layer exhaust port , the lower layer structure is provided with a lower layer exhaust port at a position corresponding to the middle layer exhaust port , and the end of the upper layer exhaust passage away from the upper layer elastic membrane cavity is communicated with the middle layer exhaust port after the upper layer structure, the middle layer structure and the lower layer structure are connected in a matching manner.
As a preferable technical solution of the present invention, the shape and size of the upper layer elastic film cavity are the same as those of the reaction cavity, the shape of the middle layer elastic film cavity is the same as those of the upper layer elastic film cavity, the size of the middle layer elastic film cavity is larger than that of the upper layer elastic film cavity, and the size of the elastic diaphragm is not smaller than that of the middle layer elastic film cavity.
As a preferred technical solution of the present invention, the elastic diaphragm cavity is shaped like an olive, the number of the perturbation columns is 2, the 2 perturbation columns are disposed on the elastic diaphragm and are uniformly distributed along the central line of the radial direction of the olive, and correspondingly, the number of the perturbation column through holes is 2, and the perturbation column through holes are disposed at the positions of the upper layer structure corresponding to the perturbation columns.
As a preferable embodiment of the present invention, the upper layer exhaust duct extends to both sides in a direction perpendicular to the olive-shaped radial direction, so that the middle layer exhaust port is disposed outside the middle layer elastic membrane cavity.
As a preferable technical solution of the present invention, the number of the disturbance air inlets is 2, and the disturbance air inlets are disposed in the middle layer elastic film cavity and at both ends of the olive shape in the radial direction. Such an arrangement may ensure an alternating perturbation of the flexible diaphragm by the inlet port.
As a preferable technical scheme of the present invention, the number of the air inlets and the number of the air inlet channels are 2, 2 air inlets are respectively arranged on the side surface of the middle layer structure, and the distance between the 2 air inlets is greater than the radial length of the middle layer elastic membrane cavity; the air inlet channel is L-shaped.
As a preferable technical scheme of the invention, the middle layer air outlet penetrates through the middle part in the middle layer elastic membrane cavity. So that the middle exhaust port is centered with respect to the perturbation column also on the elastomeric membrane.
By adopting the technical scheme, air is respectively fed through the two air inlets, so that two air flows are formed in the chaotic convection blending device, two alternate motion modes are formed in the elastic membrane cavity by the two air flows, chaotic convection is formed, and then the reaction cavity which is in contact with the chaotic convection is disturbed through the disturbance column on the elastic membrane, so that high-efficiency blending is realized.
Drawings
The following is a more detailed description of the embodiments , taken in conjunction with the accompanying drawings:
FIG. 1 is a schematic perspective view of a microfluidic chip according to the present invention;
FIG. 2 is a schematic diagram of a three-layer explosion structure of a microfluidic chip of the present invention;
FIG. 3 is a schematic diagram of the front side of the lower chip of the microfluidic chip of the present invention;
FIG. 4 is a schematic diagram of the backside structure of the lower chip of the microfluidic chip of the present invention;
FIG. 5 is a schematic diagram of the front side of a middle layer chip of the microfluidic chip of the present invention;
FIG. 6 is a schematic diagram showing the reverse structure of a middle layer chip of the microfluidic chip of example 1 of the present invention;
FIG. 7 is a schematic diagram showing the reverse structure of a middle layer chip of the microfluidic chip of example 2 of the present invention;
FIG. 8 is a schematic diagram of the structure of the front side of the upper chip of the detection microfluidic chip according to the present invention;
FIG. 9 is a schematic view of the structure of the reverse side of the upper chip of the microfluidic chip of the present invention;
FIG. 10 is a top three-layer explosive structure diagram of the chaotic convection blending device after bonding of the microfluidic chip and the chaotic convection blending device of the present invention;
FIG. 11 is a bottom three-layer explosion structure diagram of the chaotic convection blending device after the microfluidic chip of the present invention is bonded to the chaotic convection blending device;
FIG. 12 is a perspective structure view of the chaotic convection mixing device of the microfluidic chip of the present invention;
FIG. 13 is a top perspective view of a microfluidic chip of the present invention bonded to a chaotic convective blending device;
FIG. 14 is a bottom perspective view of a microfluidic chip of the present invention bonded to a chaotic convective mixer;
the device comprises a substrate.
Detailed Description
The elastic flow channel comprises a chip body, a micro flow channel, a reaction channel, a heat dissipation channel, a thermal dissipation channel.
The micro-fluidic chip comprises a micro-fluidic chip, a reaction chamber, a gas inlet port, a reaction chamber, a gas outlet port, a micro-fluidic chip, a film inlet port, a film inlet port, a film inlet port, a film inlet port, a film inlet port, a film inlet, a film inlet, a film, a film, a film, a film, a film, a film, a film inlet, a film, a film, a film inlet, a film, a film, a film, a film, a film, a film, a film, a film, a film, a film, a film, a film, a film, a film, a film, a film, a film, a film, a film.
The elastic film 7052 comprises an upper layer structure 701, a middle layer structure 702 and a lower layer structure 703 from top to bottom, wherein the upper layer structure 701 penetrates through a disturbing column through hole 704 at a position corresponding to the reaction cavity 5, an elastic film 706 is arranged between the upper layer structure 701 and the middle layer structure 702, the disturbing column 707 is arranged on the elastic film 706, the disturbing column passes through the disturbing column through hole 704 and contacts with an elastic film 5021 at the outer periphery of the bottom of the reaction cavity 5, the upper layer structure 701 and the middle layer structure 702 are provided with an elastic film cavity 705 at a position corresponding to the reaction cavity 5, a middle layer exhaust port 708 and at least air inlet ports 709 are arranged on the elastic film cavity 705, the lower layer structure 703 is provided with an air inlet port 7011 and an air inlet port 7012 at a position corresponding to the middle layer exhaust port 7052 at the middle layer 7052, the air inlet port 7052 and the air inlet port 7012 are arranged on the middle layer structure 7052, the upper layer 7052 and the middle layer 7052, the elastic film 7052 is arranged at a position corresponding to the upper layer 7052, the middle layer 7052, the elastic film 7052 and the elastic film 7052, the upper layer 7052 is arranged at a central line 7012, the upper layer 7052, the elastic film 7052 and the upper layer 7012, the upper layer 7012 are arranged at the middle layer 7012, the upper layer 7052, the upper layer 7012, the elastic film 7052 and the upper layer 7012 are arranged at the upper layer structure, the upper layer 7012, the upper layer 7052, the elastic film 7051 is arranged at the upper layer 7052, the upper layer 7012, the middle layer 7012, the elastic film 7012, the upper layer 7012 is arranged at the upper layer 7012, the elastic film 7012 is arranged at the same length of the middle layer 7012, the elastic film 7052, the elastic film 7012 and the elastic film 7051 is arranged at the same length of the same as the elastic film 7052, the elastic film 7012, the elastic film 7052, the upper layer 7012, the elastic film 7012, the middle layer 7012, the elastic film 7051 and the elastic film 7012, the same length of the elastic film 7012, the elastic film 7012 is arranged at the elastic film 7012, the elastic film 7012 of the elastic film 7012, the elastic film 7052 with the elastic film 7012, the elastic film 7012 is arranged at the same as the elastic film 7052, the same as the elastic film 7012, the elastic film 7052, the elastic film 705, the elastic film 7012, the upper layer 705, the upper layer 7012, the same as the elastic film 7012, the same as the elastic film 7012, the elastic film 7052, the elastic film 7012, the.
Example 4: the method for detecting cardiac troponin by matching the micro-fluidic chip in the embodiment 1 with the chaotic convection blending device in the embodiment 3 specifically comprises the following steps:
(1) adding 200 μ L of sample plasma/serum from the addition port 4 with a pipette;
(2) the sample plasma/serum sequentially passes through the middle-layer liquid adding through hole 401, the liquid adding input micro-channel 603 and the reaction cavity input channel 601 to flow into the reaction cavity 5, the microfluid chip and the chaotic convection blending device are designed in an body mode, the chaotic convection blending device 7 is started to blend the sample plasma/serum in the reaction cavity 5 uniformly for 10min, the sample antigen and the antibody in the reaction cavity 5 perform immunoreaction, and the chaotic convection blending device 7 is stopped;
(3) pushing air into a liquid adding port 4 of the microfluidic chip by using the liquid shifter, pushing liquid in the microfluidic chip to move forwards, and drying a reaction cavity 5 and a micro-channel 6;
(4) adding 200 mu L of cleaning solution into the liquid adding port 4 until the reaction cavity 5 is filled, starting the chaotic convection uniformly-mixing device 7 again, uniformly mixing for 1min, cleaning, stopping the chaotic convection uniformly-mixing device 7, and pushing air into the microfluidic chip by a liquid transfer device to blow dry the microfluidic channel 6 and the reaction cavity 5 of the chip;
(5) repeating the step (4) and cleaning for 3-5 times;
(6) and carrying out fluorescence detection on the microfluidic chip to obtain a reaction fluorescence value, and further detecting the content of the cardiac troponin index.
Example 5 (standing control example): the difference from the embodiment 4 is that in the step (2), a chaotic convection mixing device is not adopted for mixing, and the sample plasma/serum is added and kept standing for 10 min; in particular, the amount of the solvent to be used,
(1) adding 200 μ L of sample plasma/serum from the addition port 4 with a pipette;
(2) the sample plasma/serum sequentially flows into the reaction cavity 5 through the middle-layer liquid adding through hole 401, the liquid adding input micro-channel 603 and the reaction cavity input channel 601, and stands for 10min, so that the sample antigen and the antibody in the reaction cavity 5 perform immunoreaction;
(3) pushing air into a liquid adding port 4 of the microfluidic chip by using the liquid shifter, pushing liquid in the microfluidic chip to move forwards, and drying a reaction cavity 5 and a micro-channel 6;
(4) then adding 200 mu L of cleaning solution into the liquid adding port 4 until the reaction cavity 5 is filled, standing for 1min, cleaning, and pushing an air-blowing micro-channel 6 and the reaction cavity 5 into the microfluidic chip by a liquid shifter;
(5) repeating the step (4) and cleaning for 3-5 times;
(6) and carrying out fluorescence detection on the microfluidic chip to obtain a reaction fluorescence value, and further detecting the content of the cardiac troponin index.
Example 6: the difference from the embodiment 4 is that a rotor is arranged in the reaction cavity in advance, and the sample plasma/serum in the reaction cavity is uniformly mixed through the rotor; specifically, the method comprises the following steps:
(1) adding 200 μ L of sample plasma/serum from the addition port 4 with a pipette;
(2) the sample plasma/serum sequentially passes through the middle-layer liquid adding through hole 401, the liquid adding input micro-channel 603 and the reaction cavity input channel 601 to flow into the reaction cavity 5, the magnetic stirring device is started to uniformly mix the sample plasma/serum in the reaction cavity 5 for 10min, the sample antigen and the antibody in the reaction cavity 5 carry out immunoreaction, and the magnetic stirring device is stopped;
(3) pushing air into a liquid adding port 4 of the microfluidic chip by using the liquid shifter, pushing liquid in the microfluidic chip to move forwards, and drying a reaction cavity 5 and a micro-channel 6;
(4) adding 200 mu L of cleaning solution into the liquid adding opening 4 until the reaction cavity 5 is filled, starting the magnetic stirring device again, mixing uniformly for 1min, cleaning, stopping the magnetic stirring device, and pushing air into the microfluidic chip by a liquid shifter to blow dry the microfluidic channel 6 and the reaction cavity 5 of the chip;
(5) repeating the step (4) and cleaning for 3-5 times;
(6) and carrying out fluorescence detection on the microfluidic chip to obtain a reaction fluorescence value, and further detecting the content of the cardiac troponin index.
Example 7: the difference from the embodiment 4 lies in that the microfluidic chip is placed on a shaking table and the antibody and the sample in the reaction chamber are mixed uniformly by adopting a shaking table mixing mode, specifically:
(1) adding 200 μ L of sample plasma/serum from the addition port 4 with a pipette;
(2) the sample plasma/serum sequentially passes through the middle-layer liquid adding through hole 401, the liquid adding input micro-channel 603 and the reaction cavity input channel 601 to flow into the reaction cavity 5, the micro-fluid chip is placed into a shaking table and uniformly mixed for 10min, the sample antigen and the antibody in the reaction cavity 5 are subjected to immunoreaction, and the micro-fluid chip is taken out of the shaking table;
(3) pushing air into a liquid adding port 4 of the microfluidic chip by using the liquid shifter, pushing liquid in the microfluidic chip to move forwards, and drying a reaction cavity 5 and a micro-channel 6;
(4) adding 200 mu L of cleaning solution into the liquid adding port 4 until the reaction cavity 5 is filled, putting the mixture into a shaking table, uniformly mixing for 1min, cleaning, taking out the micro-liquid chip from the shaking table, and pushing air into the micro-liquid chip by a liquid transfer device to blow dry the micro-flow channel 6 and the reaction cavity 5 of the chip;
(5) repeating the step (4) and cleaning for 3-5 times;
(6) and carrying out fluorescence detection on the microfluidic chip to obtain a reaction fluorescence value, and further detecting the content of the cardiac troponin index.
The fluorescence values of the reaction obtained by mixing different sample concentrations in the methods of examples 4 to 7 are shown in Table 1.
Comparing the reaction fluorescence values of the standing control and the 3 mixing methods, the reaction fluorescence value of the chaotic convection mixing method is the highest, which shows that the mixing effect is better and the antibody-antigen immunoreaction is more sufficient.
TABLE 1 fluorescence values of the reactions of the different homogenization methods
The fluorescence values of the reaction obtained by mixing different sample concentrations in the methods of examples 4 to 7 are shown in Table 1.
Comparing the reaction fluorescence values of the standing control and the 3 mixing methods, the reaction fluorescence value of the chaotic convection mixing method is the highest, which shows that the mixing effect is better and the antibody-antigen immunoreaction is more sufficient.
It will be appreciated by those skilled in the art that the foregoing embodiments are not intended to limit the invention, which is described herein, but are merely illustrative of the principles of the invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention, such as minor changes in the arrangement of the chambers, which fall within the scope of the invention as claimed.
Claims (13)
1, method for detecting cardiac troponin by using a microfluidic chip, which is characterized by comprising the following steps:
(1) adding a quantitative sample plasma/serum from the liquid adding port by using a liquid transfer machine;
(2) the sample plasma/serum sequentially passes through the middle-layer liquid adding through hole, the liquid adding input micro-channel and the reaction cavity input channel to flow into the reaction cavity, the micro-fluidic chip and the chaotic convection blending device are designed in an body mode, the chaotic convection blending device is started to blend the sample plasma/serum in the reaction cavity for 3-15 min, a sample antigen and an antibody in the reaction cavity perform an immune reaction, and the chaotic convection blending device is stopped;
(3) pushing air into the liquid adding opening by adopting the liquid shifter, pushing liquid in the microfluidic chip to move forwards, and drying the reaction cavity and the flow channel;
(4) adding a cleaning solution into the liquid adding port until the reaction cavity is filled, starting the chaotic convection mixing device again, mixing for 1-3 min, cleaning, stopping the chaotic convection mixing device, and pushing air into the microfluidic chip by a liquid shifter to blow dry the microfluidic channel and the reaction cavity of the chip;
(5) repeating the step (4) and cleaning for 3-5 times;
(6) and carrying out fluorescence detection on the microfluidic chip to obtain a reaction fluorescence value.
2. The method for detecting cardiac troponin using a microfluidic chip according to claim 1, wherein the microfluidic chip for detecting cardiac troponin comprises a chip body, the chip body comprises a lower chip, a middle chip and an upper chip in sequence from bottom to top, the chip body comprises a liquid filling port, a reaction chamber and a microchannel, and is characterized in that the middle chip and the upper chip are matched to define a closed microchannel and a reaction chamber, the liquid filling port is communicated with the reaction chamber through the microchannel, and at least grooves are formed in the reaction chamber.
3. The method for detecting cardiac troponin using a microfluidic chip according to claim 2, wherein the bottom of the microfluidic chip is provided with a chaotic convective mixer.
4. The method for detecting cardiac troponin using a microfluidic chip according to claim 3, wherein the reaction chamber comprises an upper reaction chamber disposed on the rear surface of the middle chip and a lower reaction chamber disposed through the lower chip, the upper reaction chamber being disposed at a position on the rear surface of the middle chip corresponding to the position of the lower reaction chamber on the lower chip; the bottom of the lower reaction chamber is covered on the back surface of the lower chip by an elastic film for sealing, and the groove is arranged in the upper reaction chamber.
5. The microfluidic chip according to claim 3, wherein the grooves are inwardly recessed circular grooves, the number of the grooves is 3, and 3 of the grooves are uniformly distributed in the reaction chamber.
6. The method for detecting cardiac troponin using a microfluidic chip according to claim 4 or 5, wherein the microchannel comprises a reaction chamber input channel and a reaction chamber output channel, the reaction chamber input channel and the reaction chamber output channel are both disposed on the back side of the middle layer chip, the reaction chamber input channel communicates with the end of the upper reaction chamber, the reaction chamber output channel communicates with the other end of the upper reaction chamber, the reaction chamber input channel communicates with the filling opening, and an anti-backflow structure is disposed between the reaction chamber input channel and the filling opening.
7. The method for detecting cardiac troponin using a microfluidic chip according to claim 6, wherein the upper chip is provided with the filling opening in a penetrating manner, the middle chip is provided with a middle filling through hole in a penetrating manner at a position corresponding to the filling opening, and the front surface of the lower chip is provided with a filling input microchannel at a position corresponding to the filling opening; the liquid feeding input micro-channel is communicated with the liquid feeding port through the middle-layer liquid feeding through hole, and the reaction cavity input channel is communicated with the liquid feeding port through the liquid feeding input micro-channel.
8. The method for detecting cardiac troponin using a microfluidic chip according to claim 7, wherein the anti-backflow structure comprises a vertical flow channel , a second vertical flow channel and an anti-backflow connection flow channel, the anti-backflow connection flow channel is disposed on the back surface of the upper chip, both the vertical flow channel and the second vertical flow channel are disposed through the middle chip, and the liquid feeding input micro flow channel sequentially passes through the second vertical flow channel, the anti-backflow connection flow channel and the vertical flow channel and then is communicated with the reaction chamber input flow channel.
9. The method of claim 8, wherein the chip body further comprises a waste liquid chamber disposed on the lower chip, and a middle waste liquid chamber through hole is formed in the middle chip at a position corresponding to the waste liquid chamber; the back of the upper chip is provided with a waste liquid cavity cover plate at a position corresponding to the waste liquid cavity, correspondingly, the upper chip is provided with a waste liquid cavity exhaust hole in a penetrating manner, the front of the middle chip is provided with an auxiliary leakage-proof waste liquid cavity at the upper part of the waste liquid cavity, and the auxiliary leakage-proof waste liquid cavity is provided with a waste liquid cavity middle exhaust hole in a penetrating manner at a position corresponding to the waste liquid cavity exhaust hole.
10. The method of claim 9, wherein the reaction chamber is connected to the waste liquid chamber via the reaction chamber output channel and a waste liquid output microchannel in sequence, the waste liquid output microchannel is disposed on the front surface of the lower chip, and a conductive rubber valve is disposed between the waste liquid output microchannel and the reaction chamber output channel, and the conductive rubber valve comprises an upper conductive rubber valve structure disposed on the upper chip and a middle conductive rubber valve structure disposed at a position corresponding to the middle chip.
11. The microfluidic chip according to claim 4, wherein the chaotic convective mixer is disposed on the back of the lower chip and is -shaped with the chip body, the chaotic convective mixer sequentially comprises an upper layer, a middle layer and a lower layer from top to bottom, the upper layer is provided with a disturbing column through hole at a position corresponding to the reaction chamber, an elastic membrane is disposed between the upper layer and the middle layer, the elastic membrane is provided with a disturbing column, the disturbing column passes through the disturbing column through hole and contacts with an elastic membrane coated on the bottom of the reaction chamber, the upper layer and the middle layer are provided with an elastic membrane chamber at a position corresponding to the reaction chamber, the elastic membrane chamber is provided with a middle layer exhaust port and at least disturbing air inlets, the lower layer is provided with a lower layer exhaust port at a position corresponding to the middle layer exhaust port, the middle layer is further provided with an air inlet and an air inlet channel, and the air inlet is communicated with the disturbing air inlet through an air inlet channel.
12. The microfluidic chip according to claim 11, wherein the elastic membrane cavities comprise an upper elastic membrane cavity disposed on a back side of the upper layer structure and a middle elastic membrane cavity disposed on a front side of the middle layer structure; the elastic diaphragm is arranged between the upper layer elastic diaphragm cavity and the middle layer elastic diaphragm cavity; the middle layer air outlet and the disturbance air inlet are arranged on the middle layer elastic membrane cavity.
13. The microfluidic chip according to claim 12, wherein the upper elastic membrane cavity is provided with an upper exhaust channel on a back surface of the upper structure, and correspondingly, the middle structure is provided with a middle exhaust port , the lower structure is provided with a lower exhaust port at a position corresponding to the middle exhaust port , and an end of the upper exhaust channel away from the upper elastic membrane cavity is connected to the middle exhaust port after the upper structure, the middle structure and the lower structure are connected in a matching manner.
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