CN110726835A - Immune lateral chromatography detection method and device for externally driving biomarker - Google Patents
Immune lateral chromatography detection method and device for externally driving biomarker Download PDFInfo
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Abstract
The invention discloses an immune lateral chromatography detection method and device for externally driving a biomarker, and the device comprises an immune lateral chromatography test paper consisting of a bottom lining 1, a sample pad 2, a combination pad 3, an analysis membrane 4 and a water absorption pad 5, wherein the analysis membrane is provided with a detection belt 6 and a quality control belt 7, and further comprises an upstream driving electrode 8 and a downstream driving electrode 9, the upstream driving electrode is connected with the upstream outer side end of the sample pad, and the downstream driving electrode is connected with the downstream outer side end of the water absorption pad. The method of the invention adopts strong and uniform external driving force generated by the electrode as a 'power source' to externally drive the surging of the biomarker, thereby greatly improving the precision, the accuracy and the detection speed of the existing various immune lateral chromatography technologies, and improving the detection sensitivity by promoting the release of the biomarker on the basis.
Description
The present application is a divisional application entitled "an immuno-lateral chromatography device and method for externally driving a biomarker" on application date 2014, 12/29, application number 201410833035.3.
Technical Field
The invention belongs to the technical field of biological detection in-vitro diagnosis, and relates to an immune lateral chromatography detection method and device for externally driving a biomarker.
Background
Immune lateral chromatography is the most classical field rapid detection method in the technical field of in vitro diagnosis. The physical constitution of the immunity lateral chromatography test paper comprises three types of substances: bioactive molecules, markers, solid phase consumables. The concrete components and functions are as follows:
solid phase consumable: the test paper is made of various microporous materials and can be used as a sample pad, a combination pad, an analysis membrane, a water absorption pad and an adhesive bottom lining of the immune lateral chromatography test paper; on one hand, the solid phase support is provided for the solidification and biological recognition of bioactive molecules, and on the other hand, the capillary siphonage of a microporous structure provides power for the whole chromatography.
Biologically active molecules: the biological macromolecule is a plurality of biological macromolecules with specific biological recognition capability, can be fixed on an analysis membrane as a detection band and a quality control band, is coupled with a marker and is fixed in a combination pad as a biomarker; it is a probe for realizing specific biological recognition in the process of immunodetection.
A marker: particles (including nano, submicron and the like) or chemical macromolecules (including organic, inorganic and the like) with the characteristics of light, electricity, magnetism, color and the like, and can be coupled with bioactive molecules and fixed in the binding pad to serve as a biomarker; the characteristics of light, electricity, magnetism, color and the like of the biological recognition probe can macroscopically display specific biological recognition between the micro-performed bioactive molecules and the detection targets.
As shown in fig. 1, a schematic structural diagram of a prior art immuno lateral chromatography test strip is shown, which comprises a sample pad 2, a conjugate pad 3, an analysis membrane 4 and a bibulous pad 5 stacked from left to right on a substrate 1, wherein a detection zone 6 and a quality control zone 7 are disposed on the analysis membrane 4. The sample pad 2 is used for dropping a sample to be detected, the sample contains a detection target (as shown in A in figure 11), the binding pad 3 is solidified with a biomarker (as shown in B + C in figure 11) coupled by a bioactive molecule (as shown in C in figure 11) and a marker (as shown in B in figure 11), a substance capable of being specifically bound with the detection target A (as shown in A 'in figure 11) is fixed in the detection zone 6, and a substance capable of being specifically bound with the bioactive molecule C (as shown in C' in figure 11) is fixed in the quality control zone 7. The relationship among the bioactive molecule C, the marker B, the detection target A, the substance A 'capable of being specifically combined with the detection target A and the substance C' capable of being specifically combined with the bioactive molecule C in the detection process of the immune lateral chromatography test paper is embodied as follows: as shown in FIG. 11, which shows the reaction process of the prior art immuno-lateral chromatography, panel a in FIG. 11 is a diagram of the state before the liquid sample to be tested is dropped onto the sample pad 11; as shown in the panel B of fig. 11, the liquid sample to be detected dropped into the sample pad 11 permeates into the conjugate pad 3, and the detection target "a" in the sample contacts with "B + C" to perform a specific recognition reaction, so as to form a conjugate "(B + C) -a"; according to the diagram C in fig. 11, the combination "(B + C) -a" gushes into the analysis membrane 4 with the drawing of the liquid by capillary action, and a part of the excess "B + C" fixed on the binding pad 3 also enters into the analysis membrane 4, but due to the trapping and adsorbing effects of the microporous material of the binding pad 3, a part of the combination "(B + C) -a" and "B + C" remains in the binding pad 3; as shown in fig. 11 d, the conjugate "(B + C) -a" entering the analysis membrane 4 flows through the detection zone 6, the substance a 'capable of specifically binding to the detection target a on the detection zone 6 is captured, and a detection complex "[ (B + C) -a' ]" is formed, the detection complex can show a color or a photoelectric signal, the amount of the captured detection complex is shown by the intensity of the color or the photoelectric signal of the detection zone, the larger the amount of the detection complex, the darker the color or the stronger the signal, and the amount of the detection complex is positively correlated with the amount of a in the complex, so that the concentration of the detection target a can be calculated; meanwhile, the 'B + C' entering the analysis membrane crosses the detection zone 6 to reach the quality control zone 7, and is captured by a substance C 'which can be specifically combined with the bioactive molecule C on the quality control zone 7 to form a quality control complex [ (B + C) -C' ], wherein the quality control complex can display color or photoelectric signals, the background of the biomarker 'B + C' is obtained by interpreting the color or the photoelectric signals of the quality control zone, and similarly, the color depth and the signal intensity of the quality control zone are positively correlated with the quantity of the quality control complex captured to the quality control zone. The conjugate "(B + C) -a" and the biomarker "B + C" that are not captured by the detection zone 6 and the quality control zone 7 continue to surge forward into the absorbent pad 5.
It can be seen that, in the detection process of the immuno-lateral chromatography test paper, the relationship among the bioactive molecules, the markers and the solid-phase consumable material is specifically embodied as follows, under the capillary siphon power of the solid-phase consumable material, the biomarker pre-stored in the solid-phase material (the binding pad) is released under the action of the liquid sample, and then flows to the bioactive molecules (the detection zone and the quality control zone) pre-fixed on the solid-phase material (the analysis membrane) along with the sample, and specific biological recognition among the bioactive molecules coupled on the biomarker, the detection target and the bioactive molecules fixed on the detection zone occurs at the position, and the biological recognition can change the amount of the markers at the position through the bioactive molecules in the biomarker, so that the biological recognition can be qualitatively or quantitatively displayed by means of the characteristics of light, electricity, magnetism, color and the like of the markers.
The specific recognition of the bioactive molecules is the fact that the specific recognition exists objectively, and the immune lateral chromatography technology is a carrier for objectively and visually displaying the fact of the specific recognition of the bioactive molecules under the condition that the characteristics (affinity and specificity) of the bioactive molecules are clear. As a display carrier, the immune lateral chromatography technology mainly has two problems in the practical use process, one is the sensitivity and the quantification capability of the identification display, and the other is the precision and the accuracy of the identification display.
In terms of sensitivity and quantification capability, the method is mainly related to the self characteristics of the marker, namely, the legibility and the quantifiability of the optical, electric, magnetic, color and other characteristics of the marker, and various immune lateral chromatography methods in the prior art, such as colloidal gold immune lateral chromatography, fluorescence immune lateral chromatography and paramagnetic particle immune lateral chromatography, are focused on screening and optimizing various tracers, so that the detection sensitivity of the immune lateral chromatography is improved, and the quantification of a detection target is realized.
In terms of precision and accuracy, the method is mainly related to the surging state of the biomarker under the capillary action of the solid phase consumable, namely the microscopic pore size of the solid phase consumable and the uniformity of the capillary siphoning action of the solid phase consumable affect the surging speed and the uniformity of the biomarker. However, the production process of the solid-phase consumable material for the immune lateral chromatography determines that the microscopic pore size and the distribution state of the consumable material are inevitably greatly non-uniform, so that the current chromatography products all face the problem of large variation of detection results; meanwhile, the weak capillary siphon force also enables the lateral chromatography to take relatively long time (about 10-15 minutes).
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the immune lateral chromatography detection method for externally driving the biomarker, which has high precision, good accuracy and high detection speed.
It is a second object of the present invention to provide an immuno-lateral chromatographic device for externally driving a biomarker, which has a simple structure.
The technical scheme of the invention is summarized as follows:
one aspect of the present invention provides a method for exogenously driving an immuno-lateral flow assay for a biomarker, comprising the steps of:
1) immobilizing a biomarker in a binding pad (3) of the immune lateral chromatography test paper, wherein the biomarker is a conjugate of a bioactive molecule and a marker, and the marker has electrical properties, preferably fluorescent particles or paramagnetic particles;
2) an upstream driving electrode (8) is arranged at the upstream of the biological marker fixed in the combining pad (3) of the immune lateral chromatography test paper, and a downstream driving electrode (9) is arranged on the upper surface of the absorbent pad (5) or the outer end part of the absorbent pad;
3) adding a sample to be detected on the sample pad (2), electrifying the upstream driving electrode (8) and the downstream driving electrode (9), and interpreting a detection band (6) and a quality control band (7) on the analysis membrane (4) by naked eyes or an instrument to obtain a qualitative or/and quantitative analysis result of a detection target in the sample to be detected; the detection target is neutral.
In the immune lateral chromatography detection method, the upstream driving electrode (8) is arranged at the upstream outer side end part of the sample pad (2), or the upstream driving electrode (8) is arranged on the upper surface of the sample pad (2) or the upper surface of the binding pad (3) through the lower surface of the upstream driving electrode (8); the downstream driving electrode (9) is arranged at the downstream outer side end of the absorbent pad (5), or the downstream driving electrode (9) is arranged on the upper surface of the absorbent pad (5) through the lower surface of the downstream driving electrode (9).
In the immune lateral chromatography detection method, the electrifying quantity of the upstream driving electrode (8) and the downstream driving electrode (9) is 10V, and the electrifying time is 3 min.
In the immune lateral chromatography detection method, the detection target is methamphetamine.
In the immune lateral chromatography detection method, the marker is fluorescent particles, and the immune lateral chromatography test paper using the fluorescent particles as the marker is quantum dot immune lateral chromatography test paper.
In the immune lateral chromatography detection method, the upstream driving electrode (8) is arranged at the upstream outer side end of the sample pad (2), and the downstream driving electrode (9) is arranged at the downstream outer side end of the absorbent pad (5); or the upstream driving electrode (8) is arranged on the upper surface of the sample pad (2) through the lower surface of the upstream driving electrode, and the downstream driving electrode (9) is arranged at the downstream outer end of the absorbent pad (5); or the upstream driving electrode (8) is arranged on the upper surface of the sample pad (2) through the lower surface thereof, and the downstream driving electrode (9) is arranged on the upper surface of the absorbent pad (5) through the lower surface thereof; or the upstream driving electrode (8) is arranged at the upstream outer end part of the sample pad (2), and the downstream driving electrode (9) is arranged on the upper surface of the absorbent pad (5) through the lower surface of the downstream driving electrode; or the upstream driving electrode (8) is arranged on the upper surface of the bonding pad (3) through the lower surface thereof, and the downstream driving electrode (9) is arranged on the upper surface of the absorbent pad (5) through the lower surface thereof.
In the above-mentioned immune lateral chromatography detection method, the label is paramagnetic particles; the upstream driving electrode (8) is arranged at the upstream outer side end of the sample pad (2), and the downstream driving electrode (9) is arranged at the downstream outer side end of the absorbent pad (5).
The invention also provides an immune lateral chromatography device for externally driving a biomarker, which is used in the immune lateral chromatography method, and comprises an immune lateral chromatography test paper consisting of a bottom lining (1), a sample pad (2), a binding pad (3), an analysis membrane (4) and a water absorption pad (5), wherein the analysis membrane is provided with a detection belt (6) and a quality control belt (7), and further comprises an upstream driving electrode (8) and a downstream driving electrode (9), the upstream driving electrode (8) is connected with the upstream outer end part of the sample pad (2), or the upstream driving electrode (8) is connected with the upper surface of the sample pad (2) or the upper surface of the binding pad (3) through the lower surface of the upstream driving electrode (8); the downstream driving electrode (9) is connected with the downstream outer side end of the water absorption pad (5), or the downstream driving electrode (9) is connected with the upper surface of the water absorption pad (5) through the lower surface of the downstream driving electrode (9);
the biological marker is immobilized in the binding pad (3) as a conjugate of a bioactive molecule and a marker, and the marker has an electrical property, preferably a fluorescent particle or a paramagnetic particle.
The marker is fluorescent particles, and the immune lateral chromatography test paper using the fluorescent particles as the biomarker is quantum dot immune lateral chromatography test paper; the upstream driving electrode (8) is connected with the upstream outer end of the sample pad (2), and the downstream driving electrode (9) is connected with the downstream outer end of the absorbent pad (5); or the upstream driving electrode (8) is connected with the upper surface of the sample pad (2) through the lower surface thereof, and the downstream driving electrode (9) is connected with the downstream outer end of the absorbent pad (5); or the upstream driving electrode (8) is connected with the upper surface of the sample pad (2) through the lower surface thereof, and the downstream driving electrode (9) is connected with the upper surface of the absorbent pad (5) through the lower surface thereof; or the upstream driving electrode (8) is connected with the upstream outer end part of the sample pad (2), and the downstream driving electrode (9) is connected with the upper surface of the water absorption pad (5) through the lower surface of the downstream driving electrode; or the upstream driving electrode (8) is connected with the upper surface of the bonding pad (3) through the lower surface thereof, and the downstream driving electrode (9) is connected with the upper surface of the absorbent pad (5) through the lower surface thereof.
The markers are paramagnetic particles, the upstream driving electrode (8) is connected with the upstream outer side end of the sample pad (2), and the downstream driving electrode (9) is connected with the downstream outer side end of the absorbent pad (5).
The invention has the advantages that:
the method of the invention overcomes the problems of poor precision of detection results, low detection speed and the like caused by uneven micropore structure of solid phase consumables and uneven surging of biomarkers driven by a power source and low speed due to weak capillary siphon suction when the solid phase consumables of the immune lateral chromatography are simultaneously used as a carrier and the power source for biological identification in the prior art. Based on the invention, the function of the immunochromatographic solid-phase consumable is only positioned as a 'carrier' for biological identification, and stronger and uniform external driving force generated by the electrode is used as a 'power source' to externally drive the surging of the biomarker, so that the precision, the accuracy and the detection speed of various existing immune lateral chromatography technologies are greatly improved, and the detection sensitivity is improved by promoting the release of the biomarker on the basis. Finally, on one hand, the requirement of a user on better technical performance of the immune lateral chromatography test paper is met, and on the other hand, the difficulty of quality control of a production enterprise is reduced.
Drawings
FIG. 1 is a prior art immunolateral flow strip;
FIG. 2 is one embodiment of the present invention of an immuno-lateral flow device for the external actuation of a biomarker.
FIG. 3 shows a second immuno-lateral flow device for externally driving a biomarker according to the present invention.
FIG. 4 shows a third immuno-lateral flow device for externally driving a biomarker according to the present invention.
FIG. 5 is a fourth embodiment of the present invention of an immuno-lateral flow device for the external actuation of biomarkers.
FIG. 6 shows a fifth embodiment of the present invention of an immuno-lateral flow device for externally driving a biomarker.
FIG. 7 shows a sixth embodiment of the present invention of an immuno-lateral flow device for externally driving a biomarker.
FIG. 8 shows a seventh aspect of the present invention for an immuno-lateral flow device for externally driving a biomarker.
FIG. 9 shows an eighth immuno-lateral flow device for externally driving a biomarker according to the present invention.
FIG. 10 shows a ninth embodiment of the present invention of an immuno-lateral flow device for externally driving a biomarker.
FIG. 11 is a graph showing the dynamic changes in the use of the prior art immunolateral chromatography test strip; wherein, the panel a in FIG. 11 is a state diagram when a sample to be detected containing a detection target is dripped into a sample pad; FIG. 11, panel b shows the specific binding of the detection target to the biomarker in the conjugate pad; FIG. 11, panel c, shows the binding of the detection target and the biomarker and the movement of the biomarker on the analysis membrane; in FIG. 11, the d-piece shows that the combination of the detection target and the biomarker is specifically bound on the detection band, and the biomarker is specifically bound on the quality control band.
Detailed Description
The invention is further described with reference to the following figures and specific embodiments.
Example 1
An immune lateral chromatography device for externally driving a biomarker comprises an immune lateral chromatography test paper consisting of a bottom lining 1, a sample pad 2, a combination pad 3, an analysis membrane 4 and a water absorption pad 5, wherein the immune lateral chromatography test paper belongs to colloidal gold immune lateral chromatography test paper in the immune lateral chromatography test paper taking colored particles as markers, the analysis membrane is provided with a detection band 6 and a quality control band 7, and the immune lateral chromatography test paper further comprises an upstream driving electrode 8 and a downstream driving electrode 9, the upstream driving electrode is connected with the upstream outer side end of the sample pad, and the downstream driving electrode is connected with the downstream outer side end of the water absorption pad. As shown in fig. 2.
Urine methamphetamine was detected using an immuno-lateral chromatography device of this example that externally actuated the biomarker:
and adding a sample to be detected on the sample pad 2, electrifying the upstream driving electrode 8 and the downstream driving electrode 9 respectively, and carrying out result interpretation on the detection belt 6 and the quality control belt 7 on the analysis membrane 4 by naked eyes.
Example 2
An immune lateral chromatography device for externally driving a biomarker comprises an immune lateral chromatography test paper consisting of a bottom lining 1, a sample pad 2, a combination pad 3, an analysis membrane 4 and a water absorption pad 5, wherein the immune lateral chromatography test paper belongs to quantum dot immune lateral chromatography test paper in the immune lateral chromatography test paper taking fluorescent particles as the marker, a detection belt 6 and a quality control belt 7 are arranged on the analysis membrane, the immune lateral chromatography test paper further comprises an upstream driving electrode 8 and a downstream driving electrode 9, the upstream driving electrode is connected with the upstream outer side end of the sample pad, and the downstream driving electrode is connected with the downstream outer side end of the water absorption pad. As shown in fig. 2.
Urine methamphetamine was detected using an immuno-lateral chromatography device of this example that externally actuated the biomarker:
and adding a sample to be detected on the sample pad 2, electrifying the upstream driving electrode 8 and the downstream driving electrode 9 respectively, and interpreting the results of the detection belt 6 and the quality control belt 7 on the analysis membrane 4 by adopting an optical analysis instrument.
Example 3
An immune lateral chromatography device for externally driving a biomarker comprises an immune lateral chromatography test paper consisting of a bottom lining 1, a sample pad 2, a combination pad 3, an analysis membrane 4 and a water absorption pad 5, wherein the immune lateral chromatography test paper belongs to Cy5 fluorescent immune lateral chromatography test paper in the immune lateral chromatography test paper taking fluorescent macromolecules as markers, the analysis membrane is provided with a detection belt 6 and a quality control belt 7, the immune lateral chromatography test paper further comprises an upstream driving electrode 8 and a downstream driving electrode 9, the upstream driving electrode is connected with the upstream outer end of the sample pad, and the downstream driving electrode is connected with the downstream outer end of the water absorption pad. As shown in fig. 2.
Urine methamphetamine was detected using an immuno-lateral chromatography device of this example that externally actuated the biomarker:
and adding a sample to be detected on the sample pad 2, electrifying the upstream driving electrode 8 and the downstream driving electrode 9 respectively, and interpreting the results of the detection belt 6 and the quality control belt 7 on the analysis membrane 4 by adopting an optical analysis instrument.
Example 4
An immune lateral chromatography device for externally driving a biomarker comprises immune lateral chromatography test paper consisting of a bottom lining 1, a sample pad 2, a combination pad 3, an analysis membrane 4 and a water absorption pad 5, wherein the immune lateral chromatography test paper belongs to paramagnetic particle immune lateral chromatography test paper in the immune lateral chromatography test paper taking paramagnetic particles as the marker, a detection belt 6 and a quality control belt 7 are arranged on the analysis membrane, the immune lateral chromatography test paper further comprises an upstream driving electrode 8 and a downstream driving electrode 9, the upstream driving electrode is connected with the upstream outer side end of the sample pad, and the downstream driving electrode is connected with the downstream outer side end of the water absorption pad. As shown in fig. 2.
Urine methamphetamine was detected using an immuno-lateral chromatography device of this example that externally actuated the biomarker:
a sample to be detected is added on the sample pad 2, the upstream driving electrode 8 and the downstream driving electrode 9 are respectively electrified, and a magnetic analysis instrument is adopted to carry out result interpretation on the detection belt 6 and the quality control belt 7 on the analysis membrane 4.
Example 5
An immune lateral chromatography device for externally driving a biomarker comprises immune lateral chromatography test paper consisting of a bottom lining 1, a sample pad 2, a combination pad 3, an analysis membrane 4 and a water absorption pad 5, wherein the immune lateral chromatography test paper belongs to quantum dot immune lateral chromatography test paper in the immune lateral chromatography test paper taking fluorescent particles as the marker, a detection belt 6 and a quality control belt 7 are arranged on the analysis membrane, the immune lateral chromatography test paper further comprises an upstream driving electrode 8 and a downstream driving electrode 9, and the upstream driving electrode is connected with the upper surface of the sample pad through the lower surface of the upstream driving electrode; the downstream driving electrode is connected with the downstream outer end of the absorbent pad. As shown in fig. 3.
Urine methamphetamine was detected using an immuno-lateral chromatography device of this example that externally actuated the biomarker:
and adding a sample to be detected on the sample pad 2, electrifying the upstream driving electrode 8 and the downstream driving electrode 9 respectively, and interpreting the results of the detection belt 6 and the quality control belt 7 on the analysis membrane 4 by adopting an optical analysis instrument.
Example 6
An immune lateral chromatography device for externally driving a biomarker comprises immune lateral chromatography test paper consisting of a bottom lining 1, a sample pad 2, a combination pad 3, an analysis membrane 4 and a water absorption pad 5, wherein the immune lateral chromatography test paper belongs to quantum dot immune lateral chromatography test paper in the immune lateral chromatography test paper taking fluorescent particles as the marker, a detection belt 6 and a quality control belt 7 are arranged on the analysis membrane, the immune lateral chromatography test paper further comprises an upstream driving electrode 8 and a downstream driving electrode 9, and the upstream driving electrode is connected with the upper surface of the sample pad through the lower surface of the upstream driving electrode; the downstream driving electrode is connected with the upper surface of the water absorption pad through the lower surface of the downstream driving electrode. As shown in fig. 4.
Urine methamphetamine was detected using an immuno-lateral chromatography device of this example that externally actuated the biomarker:
and adding a sample to be detected on the sample pad 2, electrifying the upstream driving electrode 8 and the downstream driving electrode 9 respectively, and interpreting the results of the detection belt 6 and the quality control belt 7 on the analysis membrane 4 by adopting an optical analysis instrument.
Example 7
An immune lateral chromatography device for externally driving a biomarker comprises immune lateral chromatography test paper consisting of a bottom lining 1, a sample pad 2, a combination pad 3, an analysis membrane 4 and a water absorption pad 5, wherein the immune lateral chromatography test paper belongs to quantum dot immune lateral chromatography test paper in the immune lateral chromatography test paper taking fluorescent particles as the marker, a detection belt 6 and a quality control belt 7 are arranged on the analysis membrane, the immune lateral chromatography test paper further comprises an upstream driving electrode 8 and a downstream driving electrode 9, and the upstream driving electrode is connected with the upper surface of the sample pad through the lower surface of the upstream driving electrode; the downstream driving electrode is connected with the upper surface of the analysis membrane at the downstream of the quality control band through the lower surface of the downstream driving electrode. As shown in fig. 5.
Urine methamphetamine was detected using an immuno-lateral chromatography device of this example that externally actuated the biomarker:
and adding a sample to be detected on the sample pad 2, electrifying the upstream driving electrode 8 and the downstream driving electrode 9 respectively, and interpreting the results of the detection belt 6 and the quality control belt 7 on the analysis membrane 4 by adopting an optical analysis instrument.
Example 8
An immune lateral chromatography device for externally driving a biomarker comprises immune lateral chromatography test paper consisting of a bottom lining 1, a sample pad 2, a combination pad 3, an analysis membrane 4 and a water absorption pad 5, wherein the immune lateral chromatography test paper belongs to quantum dot immune lateral chromatography test paper in the immune lateral chromatography test paper taking fluorescent particles as the marker, a detection belt 6 and a quality control belt 7 are arranged on the analysis membrane, the immune lateral chromatography test paper further comprises an upstream driving electrode 8 and a downstream driving electrode 9, and the upstream driving electrode is connected with the upper surface of the sample pad through the lower surface of the upstream driving electrode; the downstream driving electrode is connected with the analysis membrane and the upper surface of the absorbent pad at the downstream of the quality control belt through the lower surface of the downstream driving electrode. As shown in fig. 6.
Urine methamphetamine was detected using an immuno-lateral chromatography device of this example that externally actuated the biomarker:
and adding a sample to be detected on the sample pad 2, electrifying the upstream driving electrode 8 and the downstream driving electrode 9 respectively, and interpreting the results of the detection belt 6 and the quality control belt 7 on the analysis membrane 4 by adopting an optical analysis instrument.
Example 9
An immune lateral chromatography device for externally driving a biomarker comprises immune lateral chromatography test paper consisting of a bottom lining 1, a sample pad 2, a combination pad 3, an analysis membrane 4 and a water absorption pad 5, wherein the immune lateral chromatography test paper belongs to quantum dot immune lateral chromatography test paper in the immune lateral chromatography test paper taking fluorescent particles as markers, the analysis membrane is provided with a detection zone 6 and a quality control zone 7, and the immune lateral chromatography test paper further comprises an upstream driving electrode 8 and a downstream driving electrode 9, and the upstream driving electrode is connected with the upstream outer side end part of the sample pad; the downstream driving electrode is connected with the upper surface of the water absorption pad through the lower surface of the downstream driving electrode. As shown in fig. 7.
Urine methamphetamine was detected using an immuno-lateral chromatography device of this example that externally actuated the biomarker:
and adding a sample to be detected on the sample pad 2, electrifying the upstream driving electrode 8 and the downstream driving electrode 9 respectively, and interpreting the results of the detection belt 6 and the quality control belt 7 on the analysis membrane 4 by adopting an optical analysis instrument.
Example 10
An immune lateral chromatography device for externally driving a biomarker comprises immune lateral chromatography test paper consisting of a bottom lining 1, a sample pad 2, a combination pad 3, an analysis membrane 4 and a water absorption pad 5, wherein the immune lateral chromatography test paper belongs to quantum dot immune lateral chromatography test paper in the immune lateral chromatography test paper taking fluorescent particles as the marker, a detection belt 6 and a quality control belt 7 are arranged on the analysis membrane, the immune lateral chromatography test paper further comprises an upstream driving electrode 8 and a downstream driving electrode 9, and the upstream driving electrode is connected with the upper surface of the combination pad through the lower surface of the upstream driving electrode; the downstream driving electrode is connected with the upper surface of the water absorption pad through the lower surface of the downstream driving electrode. As shown in fig. 8.
Urine methamphetamine was detected using an immuno-lateral chromatography device of this example that externally actuated the biomarker:
and adding a sample to be detected on the sample pad 2, electrifying the upstream driving electrode 8 and the downstream driving electrode 9 respectively, and interpreting the results of the detection belt 6 and the quality control belt 7 on the analysis membrane 4 by adopting an optical analysis instrument.
Example 11
An immune lateral chromatography device for externally driving a biomarker comprises immune lateral chromatography test paper consisting of a bottom lining 1, a sample pad 2, a combination pad 3, an analysis membrane 4 and a water absorption pad 5, wherein the immune lateral chromatography test paper belongs to quantum dot immune lateral chromatography test paper in the immune lateral chromatography test paper taking fluorescent particles as markers, a detection belt 6 and a quality control belt 7 are arranged on the analysis membrane, the immune lateral chromatography test paper further comprises an upstream driving electrode 8 and a downstream driving electrode 9, and the upstream driving electrode is connected with the upper surface of the analysis membrane at the upstream of the detection belt through the lower surface of the upstream driving electrode; the downstream driving electrode is connected with the upper surface of the water absorption pad through the lower surface of the downstream driving electrode. As shown in fig. 9.
Urine methamphetamine was detected using an immuno-lateral chromatography device of this example that externally actuated the biomarker:
and adding a sample to be detected on the sample pad 2, electrifying the upstream driving electrode 8 and the downstream driving electrode 9 respectively, and interpreting the results of the detection belt 6 and the quality control belt 7 on the analysis membrane 4 by adopting an optical analysis instrument.
Example 12
An immune lateral chromatography device for externally driving a biomarker comprises immune lateral chromatography test paper consisting of a bottom lining 1, a sample pad 2, a combination pad 3, an analysis membrane 4 and a water absorption pad 5, wherein the immune lateral chromatography test paper belongs to quantum dot immune lateral chromatography test paper in the immune lateral chromatography test paper taking fluorescent particles as the marker, a detection belt 6 and a quality control belt 7 are arranged on the analysis membrane, the immune lateral chromatography test paper further comprises an upstream driving electrode 8 and a downstream driving electrode 9, and the upstream driving electrode is connected with the sample pad, the combination pad and the upper surface of the analysis membrane at the upstream of the detection belt through the lower surface of the upstream driving electrode; the downstream driving electrode is connected with the upper surface of the water absorption pad through the lower surface of the downstream driving electrode. As shown in fig. 10.
Urine methamphetamine was detected using an immuno-lateral chromatography device of this example that externally actuated the biomarker:
and adding a sample to be detected on the sample pad 2, electrifying the upstream driving electrode 8 and the downstream driving electrode 9 respectively, and interpreting the results of the detection belt 6 and the quality control belt 7 on the analysis membrane 4 by adopting an optical analysis instrument.
Experimental example 1: comparison of the Effect of external Driving of biomarkers from different test strips
[ Experimental procedures ]:
1. comparison of the driving effect of the colloidal gold immunochromatographic strip and the device of example 1:
colloidal gold immune lateral chromatography test paper: adding 100 mul/strip of urine containing 1500ng/ml, 1000ng/ml, 500ng/ml, 100ng/ml, 50ng/ml and 0ng/ml to a test paper sample pad, and standing for observation; each concentration was tested 3 times;
apparatus of example 1: adding 100 mul/strip of urine containing 1500ng/ml, 1000ng/ml, 500ng/ml, 100ng/ml, 50ng/ml and 0ng/ml on the test paper sample pad 2, respectively electrifying the upstream driving electrode 8 and the downstream driving electrode 9 (the electrifying amount is 10V), and standing for observation; each concentration was tested 3 times; see table 1.
2. The quantum dot immune lateral chromatography test paper and the device of the embodiment 2 have a driving effect comparison experiment:
quantum dot immune lateral chromatography test paper: adding 100 mul/strip of urine containing methamphetamine at 100ng/ml, 50ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1ng/ml and 0ng/ml on a test paper sample pad, standing, and analyzing the result by an optical analyzer; each concentration was tested 3 times;
apparatus of example 2: adding 100 mul/strip of urine containing methamphetamine at 100ng/ml, 50ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1ng/ml and 0ng/ml on the test paper sample pad 2, respectively electrifying the upstream driving electrode 8 and the downstream driving electrode 9 (the electrifying quantity is 10V), standing, and analyzing the result by an optical analyzer; each concentration was tested 3 times; see table 1.
Cy5 fluorescence immune lateral chromatography test paper drive effect comparison experiment:
cy5 fluorescence immune lateral chromatography test paper: adding 100 mul/strip of urine containing methamphetamine at 100ng/ml, 50ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1ng/ml and 0ng/ml on a test paper sample pad, standing, and analyzing the result by an optical analyzer; each concentration was tested 3 times;
apparatus of example 3: adding 100 mul/strip of urine containing methamphetamine at 100ng/ml, 50ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1ng/ml and 0ng/ml on the test paper sample pad 2, respectively electrifying the upstream driving electrode 8 and the downstream driving electrode 9 (the electrifying quantity is 10V), standing, and analyzing the result by an optical analyzer; each concentration was tested 3 times; see table 1.
Comparison experiment of driving effect of paramagnetic particle immune lateral chromatography test paper:
paramagnetic particle immune lateral chromatography test paper: adding 100 mul/strip of urine containing 150ng/ml, 100ng/ml, 50ng/ml, 10ng/ml, 5ng/ml and 0ng/ml of methamphetamine on a test paper sample pad, standing, and analyzing the result by a magnetic analyzer; each concentration was tested 3 times;
apparatus of example 4: adding 100 mul/strip of urine containing 150ng/ml, 100ng/ml, 50ng/ml, 10ng/ml, 5ng/ml, 0ng/ml of methamphetamine on the test paper sample pad 2, respectively electrifying the upstream driving electrode 8 and the downstream driving electrode 9 (the electrifying amount is 10V), standing, and analyzing the result by a magnetic analyzer; each concentration was tested 3 times; see table 1.
And (4) analyzing results:
detection time: the time required from the start of the addition of the sample to the appearance of a positive result;
sensitivity: the lowest concentration at which a positive result can be clearly shown is the detection limit, which represents the detection sensitivity;
precision: the detection limit corresponds to the concentration of the sample, and the coefficient of variation (CV ═ SD/Mean) of the three measurement results represents the precision;
the accuracy is as follows: the percentage of the measurement result to the theoretical configured concentration (recovery rate ═ measured concentration/theoretical concentration) for the sample with the detection limit corresponding to the concentration represents the accuracy;
[ results & analysis ]:
TABLE 1
Detection time: after the electrode external drive is added, the detection time of any test paper is shortened from 15min to 3 min;
sensitivity: after the electrode external drive is added, the detection sensitivity of any test paper is obviously improved, and the improvement amplitude is at least 10 times;
precision: after the electrode external drive is added, the detection precision of the quantitative detection test paper is obviously improved;
the accuracy is as follows: after the external drive of the electrodes is added, the detection accuracy of the quantitative detection test paper is obviously improved;
experimental example 2: comparison of effects of quantum dot immune lateral chromatography test paper (fluorescent particle immune lateral chromatography test paper) with electrodes arranged at different positions
[ Experimental procedures ]:
separately, 100. mu.l/strip of urine containing methamphetamine at 10ng/ml, 5ng/ml, 2.5ng/ml, 1ng/ml, 0ng/ml was added to the sample pad 2 of the device of one of examples 5 to 12; energizing the upstream driving electrode 8 and the downstream driving electrode 9 respectively (the energization amount is 10V), standing, and the analysis result of the optical analyzer is shown in Table 2; each concentration was tested 3 times;
and (4) analyzing results:
detection time: the time required from the start of the addition of the sample to the appearance of a positive result;
sensitivity: the lowest concentration at which a positive result can be clearly shown is the detection limit, which represents the detection sensitivity;
precision: the detection limit corresponds to the concentration of the sample, and the coefficient of variation (CV ═ SD/Mean) of the three measurement results represents the precision;
the accuracy is as follows: the percentage of the measurement result to the theoretical configured concentration (recovery rate ═ measured concentration/theoretical concentration) for the sample with the detection limit corresponding to the concentration represents the accuracy;
[ results & analysis ]: see Table 2
TABLE 2
| Examples | Time of detection | Detection limit (sensitivity) | Coefficient of variation (precision) | Recovery rate (accuracy) |
| 5 | 3min | 2.5ng/ml | 3.2％ | 104％ |
| 6 | 3min | 2.5ng/ml | 3.3％ | 102％ |
| 7 | 3min | 2.5ng/ml | 3.5％ | 104％ |
| 8 | 3min | 2.5ng/ml | 3.7％ | 102％ |
| 9 | 3min | 2.5ng/ml | 3.1％ | 102％ |
| 10 | 3min | 2.5ng/ml | 3.2％ | 105％ |
| 11 | 3min | 2.5ng/ml | 3.1％ | 104％ |
| 12 | 3min | 2.5ng/ml | 3.7％ | 102％ |
Detection time: the detection can be completed within 3min no matter where the electrode is arranged;
sensitivity: the detection sensitivity can reach 2.5ng/ml no matter where the electrode is arranged;
precision: no significant difference in precision is made no matter where the electrode is mounted;
the accuracy is as follows: no significant difference in accuracy is made no matter where the electrode is mounted;
the above examples and experimental examples are only specific illustrations of the present invention, and are not intended to limit the technical contents of the present invention. The positions of the upstream driving electrode and the downstream driving electrode can be combined in any way, and are not limited to the way listed above. For the magnetic particle immune lateral chromatography test paper, the driving electrode can be replaced by a magnet to drive the magnetic particle biomarkers.
In practical application, a liquid sample containing a target to be detected is dripped on the sample pad 2, the sample permeates into the combined pad 3 and carries out the solidified biomarkers in the combined pad; furthermore, the mixture of the liquid sample and the biomarker surmounts on the analysis membrane 4 under the driving of the capillary siphon "power source" of the solid phase material, and the specific recognition reaction among the "bioactive molecules in the biomarker", "the target to be detected", and the "bioactive molecules on the detection zone" occurs when the mixture passes through the detection zone 6 and the quality control zone 7, so that the biomarker is fixed on the detection zone 6 and the quality control zone 7, and the biomarker in the fixed biomarker can qualitatively or quantitatively display the occurrence of the biological recognition through the unique characteristics of light, points, magnetism, and the like.
According to the invention, the upstream driving electrode 8 and the downstream driving electrode 9 are respectively connected with the upstream and the downstream of the immune lateral chromatography test paper, so that the surge of the biomarker in the test paper (especially on an analysis membrane) is more uniform and rapid, and the accuracy and precision of detection are improved; the effect of the exogenic force also allows more of the biomarker to be released from the conjugate pad, thereby increasing the sensitivity of the assay.
Claims (10)
1. A method for exogenously driving an immune lateral flow assay for a biomarker comprising the steps of:
1) immobilizing a biomarker in a binding pad (3) of the immune lateral chromatography test paper, wherein the biomarker is a conjugate of a bioactive molecule and a marker, and the marker has electrical properties, preferably fluorescent particles or paramagnetic particles;
2) an upstream driving electrode (8) is arranged at the upstream of the biological marker fixed in the combining pad (3) of the immune lateral chromatography test paper, and a downstream driving electrode (9) is arranged on the upper surface of the absorbent pad (5) or the outer end part of the absorbent pad;
3) adding a sample to be detected on the sample pad (2), electrifying the upstream driving electrode (8) and the downstream driving electrode (9), and interpreting a detection band (6) and a quality control band (7) on the analysis membrane (4) by naked eyes or an instrument to obtain a qualitative or/and quantitative detection result of a detection target in the sample to be detected; the detection target is neutral.
2. The immunochromatographic assay method according to claim 1, wherein the upstream drive electrode (8) is provided at an upstream outer end of the sample pad (2), or the upstream drive electrode (8) is provided on an upper surface of the sample pad (2) or an upper surface of the conjugate pad (3) through a lower surface of the upstream drive electrode (8); the downstream driving electrode (9) is arranged at the downstream outer side end of the absorbent pad (5), or the downstream driving electrode (9) is arranged on the upper surface of the absorbent pad (5) through the lower surface of the downstream driving electrode (9).
3. The immunochromatographic assay method according to claim 1 or 2, wherein the upstream drive electrode (8) and the downstream drive electrode (9) are energized at 10V for 3 min.
4. The method for detecting according to any one of claims 1 to 3, wherein the detection target is methamphetamine.
5. The immuno lateral chromatographic assay according to any one of claims 1 to 4 wherein the label is a fluorescent particle and the immuno lateral chromatographic strip using the fluorescent particle as the label is a quantum dot immuno lateral chromatographic strip.
6. The immunochromatographic assay method according to claim 5, wherein the upstream drive electrode (8) is provided at an upstream outer end of the sample pad (2), and the downstream drive electrode (9) is provided at a downstream outer end of the absorbent pad (5); or
The upstream driving electrode (8) is arranged on the upper surface of the sample pad (2) through the lower surface of the upstream driving electrode, and the downstream driving electrode (9) is arranged at the downstream outer end of the absorbent pad (5); or
The upstream driving electrode (8) is arranged on the upper surface of the sample pad (2) through the lower surface of the upstream driving electrode, and the downstream driving electrode (9) is arranged on the upper surface of the absorbent pad (5) through the lower surface of the downstream driving electrode; or
The upstream driving electrode (8) is arranged at the upstream outer end of the sample pad (2), and the downstream driving electrode (9) is arranged on the upper surface of the absorbent pad (5) through the lower surface of the downstream driving electrode; or
The upstream driving electrode (8) is arranged on the upper surface of the bonding pad (3) through the lower surface of the upstream driving electrode, and the downstream driving electrode (9) is arranged on the upper surface of the water absorption pad (5) through the lower surface of the downstream driving electrode.
7. The method of any one of claims 1-4, wherein the label is a paramagnetic particle; the upstream driving electrode (8) is arranged at the upstream outer side end of the sample pad (2), and the downstream driving electrode (9) is arranged at the downstream outer side end of the absorbent pad (5).
8. An outer driving immune lateral chromatographic device for a biological marker, which is used in the immune lateral chromatographic detection method of any one of claims 1 to 7, and comprises an immune lateral chromatographic test paper consisting of a bottom lining (1), a sample pad (2), a binding pad (3), an analysis membrane (4) and a water absorption pad (5), wherein the analysis membrane is provided with a detection belt (6) and a quality control belt (7), and further comprises an upstream driving electrode (8) and a downstream driving electrode (9), the upstream driving electrode (8) is connected with the upstream outer end part of the sample pad (2), or the upstream driving electrode (8) is connected with the upper surface of the sample pad (2) or the upper surface of the binding pad (3) through the lower surface of the upstream driving electrode (8); the downstream driving electrode (9) is connected with the downstream outer side end of the water absorption pad (5), or the downstream driving electrode (9) is connected with the upper surface of the water absorption pad (5) through the lower surface of the downstream driving electrode (9);
the biological marker is immobilized in the binding pad (3) as a conjugate of a bioactive molecule and a marker, and the marker has an electrical property, preferably a fluorescent particle or a paramagnetic particle.
9. The immuno lateral flow device of claim 8, wherein the label is a fluorescent particle, and the immuno lateral flow test strip using the fluorescent particle as a biomarker is a quantum dot immuno lateral flow test strip;
the upstream driving electrode (8) is connected with the upstream outer end of the sample pad (2), and the downstream driving electrode (9) is connected with the downstream outer end of the absorbent pad (5); or
The upstream driving electrode (8) is connected with the upper surface of the sample pad (2) through the lower surface of the upstream driving electrode, and the downstream driving electrode (9) is connected with the downstream outer end of the absorbent pad (5); or
The upstream driving electrode (8) is connected with the upper surface of the sample pad (2) through the lower surface of the upstream driving electrode, and the downstream driving electrode (9) is connected with the upper surface of the absorbent pad (5) through the lower surface of the downstream driving electrode; or
The upstream driving electrode (8) is connected with the upstream outer end of the sample pad (2), and the downstream driving electrode (9) is connected with the upper surface of the water absorption pad (5) through the lower surface of the downstream driving electrode; or
The upstream driving electrode (8) is connected with the upper surface of the bonding pad (3) through the lower surface of the upstream driving electrode, and the downstream driving electrode (9) is connected with the upper surface of the water absorption pad (5) through the lower surface of the downstream driving electrode.
10. The immuno lateral chromatographic device according to claim 8, wherein the label is a paramagnetic particle, the upstream drive electrode (8) is connected to the upstream outer end of the sample pad (2), and the downstream drive electrode (9) is connected to the downstream outer end of the bibulous pad (5).
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