TWI385384B - Detecting strip and the detecting method using the same - Google Patents

Description

Fluid testing test piece and fluid detecting method

The present invention relates to a fluid test strip, and more particularly to a fluid test strip for quantitative detection.

In the conventional technique of quantification of biological samples, the specificity of the specificity of the immune molecule is often used for measurement, such as a conventional enzyme linked immunosorbent assay (ELISA), which is mostly in a 96-well plate ( The 96-well plate is operated and the concentration of the substance to be tested is pushed back by detecting the signal from the final enzyme reaction. However, due to this test, in order to avoid non-specific binding of immune molecules to other impurities contained in the analyte, it is necessary to add a washing step at each reaction stage so as not to combine with the analyte or other immune molecules. The reagents were removed from the 96-well plate to avoid false positive signals caused by excessive enzymes remaining in the wells, resulting in a test failure.

Due to the development of electromechanical technology, the test strips with micro-channel structure have been used for immunodetection, simplifying the cumbersome process of cleaning in each of the above-mentioned conventional test methods. However, the currently known test strips still need to add the reagents required for the reaction reagent or reaction to the test piece in an additional manner when detecting. The reagent or the substrate is separated from the test piece and stored in the detection process because of the required reagent or substrate, which cannot be stored for a long time at room temperature and needs to be stored in a specific environment. Medium (such as refrigerated or protected from light) to avoid test errors caused by deterioration of the reactants. Therefore, such test strips are still inconvenient in operation and storage.

In addition, it is known that a fluid detecting test piece having a flow path or a micro flow path structure is not a water absorbing material around the flow path, and the fluid to be tested is a composition having a high viscosity such as protein or sugar, so it is to be treated. After the fluid is measured, it will remain on the flow channel, so that the fluid to be tested cannot be completely reacted. As a result, not only the waste of the fluid to be tested is caused, but also the error of the final test result is more likely.

In addition, in the fluid transfer test piece, the fluid detecting test piece having the micro-channel structure is a capillary phenomenon generated by the micro-channel structure, and the fluid is passively transmitted to the reaction detecting area through the flow path; the other way is When the fluid to be tested is injected, a driving force is given to the fluid by means of pressurization or the like, so that the fluid can actively pass through the flow path to reach the reaction detecting area. However, in either of the above manners, after the fluid to be tested is injected into the flow channel, bubbles of different sizes are often generated to block the flow path, causing an error in actual measurement, and even causing the test to fail.

In order to overcome the above disadvantages, the present invention provides a fluid detecting test piece comprising a substrate having a flow path. The flow path is provided with a first fluid zone, a second fluid zone and a third fluid zone which are sequentially connected, wherein the first fluid zone is for fluid injection. Further, the flow path has a first antibody, a saccharide material, a peroxide oxime, a second antibody, a luminescent agent, and a substrate reaction reagent. The first antibody is located in the first fluid zone for identifying the substance to be tested. The saccharide material and the peroxide enthalpy are located in the first fluid zone or the second fluid zone. The second antibody is immobilized in the second fluid zone and also identifies the same test substance, and the second antibody and the first antibody system recognize distinct epitopes. The luminescent agent and the substrate reaction reagent are co-located in the third fluid zone, and the host reagent comprises a saccharide cerium oxide. Thereby, after the fluid containing the substance to be tested is injected into the flow channel, the first antibody, the saccharide material and the peroxide lanthanum flow with the fluid, and some of the peroxide oxime is combined with the first antibody, the test substance and The second antibody binds and remains in the second fluid zone, and the unbound peroxide oxime flows with the fluid to the third fluid zone, and the saccharide ruthenium catalyzes the oxidation of the saccharide material to the third fluid zone for oxidation reaction. And hydrogen peroxide (H ₂ O ₂ ) is generated, and the luminescent agent is reacted with hydrogen peroxide by the peroxide ruthenium flowing to the third fluid region to generate an optical signal.

Therefore, the main object of the present invention is to provide a fluid detecting test piece. Since all the reagents and materials required for the reaction are provided, the final reaction signal can be directly measured for quantitative detection without cumbersome operation steps.

Another object of the present invention is to provide a fluid detecting test piece in which the reagents and materials required for the reaction are retained in the test piece in a dry manner before the reaction, so that the test can be stored for a long period of time without causing test errors due to deterioration of the reagent. .

The invention also provides a fluid detection method, which mainly comprises the following steps. (1) Providing a fluid having a substance to be tested. (2) providing a substrate, the substrate comprising at least a flow channel, and the flow channel comprises a first fluid zone, a second fluid zone and a third fluid zone sequentially connected, wherein the first fluid zone is for fluid injection. The substrate further comprises a first antibody located in the first fluid region for identifying the substance to be tested in the fluid; a saccharide material located in the first fluid region or the second fluid region; In the first fluid zone or the second fluid zone; the second antibody is fixed in the second fluid zone and also identifies the same substance to be tested, and the second antibody and the first antibody system recognize different antigenic determining positions; and illuminate The reagent and the reagent for the reaction are located in the third fluid zone, and the reagent for the reaction contains glucosinolate. (3) A fluid is applied to the first fluid zone of the flow channel such that the first antibody, the saccharide material, and the peroxide enthalpy flow with the fluid. (4) combining the test substance with the first antibody, the second antibody and a part of the peroxide oxime and leaving it in the second fluid zone, the fluid carrying the saccharide material, the unbound primary antibody and the peroxidation The enthalpy flows to the third fluid zone, so that the saccharide material is oxidized by the saccharide ruthenium ruthenium, and hydrogen peroxide is generated, and the illuminant is catalyzed by the ruthenium hydride catalyzed by the third fluid region. The reaction is carried out and an optical signal is generated. (5) Detecting the generated optical signal.

Therefore, another object of the present invention is to provide a fluid detecting method which can directly measure the final reaction signal for quantitative detection without cumbersome steps by having all the reagents and materials required for the reaction. Still another object of the present invention is to provide a fluid detecting method in which a reagent and a material required for the reaction are retained in a test piece in a dry manner before the reaction, so that it can be stored for a long period of time without causing a test error due to deterioration of the reagent.

Since the present invention discloses a fluid detecting test piece and a test method thereof, the chemical principle and the biological detecting technology utilized by the present invention are well known to those skilled in the relevant art, and therefore, the description below will not be completely described. At the same time, the drawings in the following texts are indicative of the features related to the features of the present invention, and are not required to be completely drawn according to the actual situation.

Please refer to FIG. 1A, which is a schematic diagram of a fluid detecting test piece according to a first preferred embodiment of the present invention. The fluid detecting test piece 1 includes a substrate 10 including a flow path 11. The flow path 11 is provided with a first fluid zone 111, a second fluid zone 112 and a third fluid zone 113 which are sequentially connected, wherein the first fluid zone 111 is for fluid injection.

Please refer to FIG. 1B for a schematic diagram of the distribution of reactive materials in the flow path of the fluid detecting test piece according to the first preferred embodiment of the present invention. The first fluid region 111 has a first antibody 1111, a saccharide material 1112, and a peroxide 脢 1113. The second fluid region 112 has a second antibody 1121 therein, and the second antibody 1121 is immobilized in the second fluid region 112. Further, the first antibody 1111 and the second antibody 1121 are both the same test substance 1101 in the identification fluid, but the first antibody 1111 and the second antibody 1121 are each different in determining the antigen-determining position on the test substance 1101. Therefore, in the selection of the first antibody 1111 and the second antibody 1121, both of them can be monoclonal antibodies (mAbs) or multiple antibodies (pAbs), and only the first antibody 1111 and the second antibody 1121 are required to be a pair of antibodies. (antibody pair), in the case of antigen recognition, it is not necessary to interfere with each other. The third fluid region 113 has a luminescent agent 1131 and a dye-reactive reagent 1132, and the host-reactive reagent 1132 contains glucosinolate 1133. The peroxide 脢 1113 may be HRP (Horseradish Peroxidase), Ascorbate Peroxidase (AP), or Hydrogen Peroxidase (hydrogen peroxidase). In a preferred embodiment, the illuminant 1131 is 3-aminohydrophthalic acid (5-Amino-2,3-dihydro-1,4-phthalazinedione), and the saccharide material 1112 is glucose. At the same time, the sugar bismuth oxide 1133 is selected from glucose cerium oxide.

Please refer to FIG. 1C to FIG. 1E as a schematic diagram showing the distribution of the reaction materials in the flow paths of the fluid detecting test piece in different reaction stages according to the first preferred embodiment of the present invention. First, referring to FIG. 1C, after the fluid containing the substance to be tested 1101 is injected into the flow path 11, the first antibody 1111 recognizes and combines with the substance 1101 to be tested. In addition, since the first antibody 1111, the saccharide material 1112, and the peroxide 脢1113 are not fixed in the first fluid region, the first antibody 1111 and the first antibody 1111 are combined with the test substance 1101 and peroxidized. The substance 1113, and the saccharide material 1112, continue to flow along the fluid to the second fluid zone 112.

Referring to FIG. 1D, after the fluid flows to the second reaction zone 112, the second antibody 1121 will bind to the substance to be tested 1101. At this time, since the second antibody 1121 is fixed in the second fluid region 112, the first antibody 1111 which has been bound to the test substance 1101 and the peroxide 脢 1113 above the first antibody 1111 are combined. The saccharide material 1112 flowing to the second fluid region 112 at this time will be followed by the first antibody 1111 and the peroxide 脢1113 which are not combined with the substance to be tested 1101. The fluid continues to flow to the third fluid zone 113.

Continuing to refer to FIG. 1E, the saccharide material 1112 and the peroxide 脢 1113 not in contact with the first antibody 1111, the second antibody 1112, and the test substance 1101 in the second fluid region 112 flow to the third fluid. District 113. At this time, the saccharide material 1112 is oxidized with the glucoside ruthenium 1133 already present in the third fluid region 113 to generate hydrogen peroxide, and the generated hydrogen peroxide and the illuminant 1131 are subjected to the flow. The peroxide 脢 1113 of the three reaction zone 113 is catalyzed to produce an optical signal. At this time, by detecting the intensity of the optical signal, the concentration of the enzyme involved in the reaction can be estimated, and the amount of the peroxide 脢 1113 flowing to the third fluid region 113 can be estimated by detecting the intensity of the optical signal, and Since the content of the peroxide 脢 1113 to be added is a known fixed value at the time of preparation of the fluid test strip 1, the concentration of the peroxide 脢 1113 remaining in the second fluid region 112 can also be borrowed. It is estimated by simple subtraction between the two. Finally, the concentration of the test substance 1101 contained in the fluid can be further calculated by using the known concentration of the peroxide 脢 1113 remaining in the second fluid region 112 for the purpose of quantitative detection.

Before the unreacted, the binding form of the first antibody 1111 and the peroxide 脢1113 may be direct formation of conjugation; or the first antibody 1111 is conjugated with biotin (Biotin), and The peroxide 脢1113 is conjugated to avidin (Avidin), and the biotin and avidin are strongly combined to form a complex of AB complex, so that when the reaction is carried out, when the first antibody 1111 is left In the second fluid zone 112, a portion of the peroxide crucible 1113 is also retained in the second fluid zone 112. The avidin described above may be Avidin, Streptavidin, or NeutrAvidin.

Please refer to FIG. 1F to FIG. 1H for a schematic diagram showing other modes of distribution of reactive materials in the flow path of the fluid detecting test piece according to the first preferred embodiment of the present invention. For the purpose of quantitative detection of the present invention, the distribution of the saccharide material 1112 and the peroxide 脢1113 in the fluid detecting test piece 1 in the flow path 11 is present in the first flow path 111 except as shown in FIG. 1B. In addition to the distribution mode, it may also be distributed in the second flow path 112.

First, referring to FIG. 1F, the first antibody 1111 and the ruthenium peroxide 1113 are located in the first fluid region 111, and the saccharide material 1112 is located in the second fluid region 112. At this time, the first antibody 1111 and the peroxide The binding mode of 脢1113 is directly conjugated to the above, or the combination of biotin and avidin is formed into a complex of AB complex, and the first antibody 1111 and the peroxide 脢1113 are respectively It exists in a way that combines biotin and avidin. The distribution pattern of the remaining reaction material second antibody 1121, illuminant 1131, substrate reaction reagent 1132, and glucoside ruthenium oxide 1133 in the flow channel 11 is the same as in the first panel. With this distributed state of arrangement, after the fluid flows through the second fluid zone 112, the sugar material 1112 will flow with the fluid to the third fluid zone 113, so the flow distribution of the reactive material in each subsequent reaction stage. And the reactions performed are shown in FIGS. 1C to 1E, and the description thereof will not be repeated here.

Referring again to FIG. 1G, in such a distribution, the first antibody 1111 is located in the first fluid zone 111, and the ruthenium peroxide 1113 and the saccharide material 1112 are located in the second fluid zone 112. In the distribution pattern represented by the 1H diagram, the first antibody 1111 and the saccharide material 1112 are located in the first fluid region 111, and the ruthenium peroxide 1113 is located in the second fluid region 112. The remaining reaction materials of the second antibody 1121, the illuminant 1131, the substrate reaction reagent 1132, and the glucosinolate 1133 in the flow channel 11 are distributed in the same manner as in FIG. 1B, and the reaction materials in the subsequent reaction stages are The flow distribution situation and the reaction performed are shown in FIGS. 1C to 1E, and the description thereof will not be repeated here.

Please continue to refer to Figure 11 for a cross-sectional view of Figure 1A along AA. As shown, a fibrous layer 1110 is included at the bottom of the first fluid zone 111, and a first antibody (1111, FIG. 1B) is formed in the fibrous layer 1110 such that after the fluid is injected into the first fluid zone 111 The first antibody (1111, Figure 1B) will flow with the fluid to the second fluid zone 112. The second nitrocellulose layer 1120 and the third nitrocellulose layer 1130 are each provided at the bottom of the second fluid zone 112 and the third fluid zone 113. The second antibody (1121, Fig. 1B) is fixed in the second nitrocellulose layer 1120, and the host reaction reagent (1132, Fig. 1B) is formed in the third nitrocellulose layer 1130.

The second nitrocellulose layer 1120 and the third nitrocellulose layer 1130 may be laid in the form of a layer of nitrocellulose membrane (NC membrane) at the bottom of the second fluid zone 112 and the third fluid zone 113.

In another preferred form, the nitrocellulose solution can be cast in the second fluid zone 112 and the bottom of the third fluid zone 113 in a casting manner, followed by air drying or freeze drying. Each of the second nitrocellulose layer 1120 and the third nitrocellulose layer 1130 having a hollow network configuration is formed. In addition, in order to reduce the influence of capillary action between the flow path and the fluid, such a flow path made by casting is not a so-called micro flow path of the prior art, and the second fluid zone 112 and the third fluid zone The minimum width of 113 is preferably 0.3 mm, while the substrate 10 can be made of a biocompatible material. For better casting, the surface roughness of the flow path 11 is preferably in the range of 3 to 50 microns, and the average thickness of the second nitrocellulose layer 1120 is equal to the thickness of the third nitrocellulose layer 1130. Further, the flow path 11 may further include a fourth fluid zone (not shown), and a bottom of the fourth fluid zone is also formed with a layer of nitrocellulose for storage of excess fluid, and the nitrocellulose layer has a hollow network configuration.

The above nitrocellulose solution is prepared by mixing nitrocellulose powder with a solvent containing an ester and a ketone, and a preferred volume ratio of the nitrocellulose powder to the ester and the ketone solvent is 1:9. Further, in the state in which the nitrocellulose layer is formed by casting, the second nitrocellulose layer 1120 and the third nitrocellulose layer 1130 are dried, and the second antibody 1121 is injected into the second nitrocellulose layer 1120 as a solution. Then, the second antibody 1121 is retained in the second nitrocellulose layer 1120 in a powder form by air drying or freeze drying.

In addition to the above manner of forming the nitrocellulose layer at the bottom of the fluid region and then forming the second antibody 1121 and the substrate reaction reagent therein, the second antibody 1121 may be injected as a solution. The prepared nitrocellulose solution is firstly mixed, and then poured into the bottom of the second fluid zone 112, and then air-dried or freeze-dried, and the nitrocellulose solution is formed into the second nitrocellulose layer 1120 and the second antibody 1121 as a powder. The way is retained.

In addition, the manner in which the dye-reactive reagent 1132 is formed in the third nitrocellulose layer 1130 is also substantially the same as the manner in which the second antibody 1121 is formed on the second nitrocellulose layer 1120, and the third nitrocellulose layer 1130 may be formed first. The dyed reagent is injected and dried, or mixed with the nitrocellulose solution and injected into the bottom of the third fluid zone 113 to form a dry form. Therefore, the description thereof will not be repeated here.

In addition to the first preferred embodiment described above, the present invention also provides a fluid detecting method, as described in the second preferred embodiment, wherein the detecting test piece used in the detecting method has the structural features and the first The preferred embodiments are substantially the same, so the description will not be repeated, and the number of the test strip components mentioned therein can be referred to FIGS. 1B to 1H.

Please refer to FIG. 2, which is a schematic flow chart of a fluid detecting method according to a second preferred embodiment of the present invention. The fluid detection method 2 comprises the following steps:

Step 21: Provide a fluid containing the substance to be tested 1101, please refer to Figure 1B.

Step 22: Providing a substrate 10 comprising at least a flow channel 11. The flow path 11 includes a first fluid zone 111, a second fluid zone 112 and a third fluid zone 113 which are sequentially connected, and the first fluid zone 111 is for fluid injection. The first fluid region 111 has a first antibody 1111 therein. The second antibody region 112 has a second antibody 1121 therein, and the second antibody 1121 is immobilized in the second antibody region 112. Further, the first antibody 1111 and the second antibody 1121 are both the same test substance 1101 in the identification fluid, but the first antibody 1111 and the second antibody 1121 are each different in determining the antigen-determining position on the test substance 1101. Therefore, in the selection of the first antibody 1111 and the second antibody 1121, both of them can be single antibody or multiple antibodies, and only the first antibody 1111 and the second antibody 1121 are a pair of antibodies, and when antigen identification is performed. It is not necessary to interfere with each other. The third fluid region 113 has a luminescent agent 1131 and a dye-reactive reagent 1132, and the host-reactive reagent 1132 contains glucosinolate 1133. Among them, the peroxide 脢 1113 may be horseradish peroxide 脢, ascorbyl peroxide or guanidine hydroperoxide. In a preferred embodiment, the illuminant 1131 is 3-aminophthalic acid, and the saccharide material 1112 is glucose. Meanwhile, the glucoside ruthenium 1133 is glucose ruthenium oxide. In addition, the various distributions of the saccharide material 1112 and the peroxide 脢1113 in the flow channel 11 are as described in the first preferred embodiment (please refer to FIG. 1B, FIG. 1F, FIG. 1G, and FIG. 1H). ).

Step 23: Adding fluid to the first fluid zone 111 of the flow channel 11. The fluid will sequentially flow from the first fluid zone 111 to the third fluid zone 112 along the flow path 11 to the third fluid zone 113, and the first antibody 1111 and the saccharide material 1112 present in the flow channel 11 And the peroxide 脢 1113 flows with the fluid.

Step 24: The test substance 1101 will flow with the fluid to the second fluid region 112, and the test substance 1101 is combined with the first antibody 1111, the second antibody 1121 and a portion of the peroxide 脢 1113 and left together in the second fluid. In area 112. The fluid continues to flow to the third fluid zone 113 with the saccharide material 1112, the unbound primary antibody 1111, and the peroxide 脢 1113. In the third fluid zone, the saccharide material 1112 is oxidized by the saccharide ruthenium hydride 1133 and produces hydrogen peroxide. The illuminant 1131 is catalyzed by the peroxide 脢 1113 flowing to the third fluid region 113 to react with hydrogen peroxide and generate an optical signal.

Step 25: Detect the optical signal generated by step 24. At this time, by detecting the intensity of the optical signal, the concentration of the enzyme involved in the reaction can be estimated, and the amount of the peroxide 脢 1113 flowing to the third fluid region 113 can be estimated by detecting the intensity of the optical signal, and Since the content of the peroxide 脢 1113 to be added is a known fixed value at the time of preparation of the fluid test strip 1, the concentration of the peroxide 脢 1113 remaining in the second fluid region 112 can also be borrowed. It is estimated by simple subtraction between the two. Finally, the concentration of the test substance 1101 contained in the fluid can be further calculated by using the known concentration of the peroxide 脢 1113 remaining in the second fluid region 112 for the purpose of quantitative detection.

Further, according to the fluid detecting method of the present invention, the combination of the first antibody 1111 and the peroxide 脢1113 and the preferred type, the structural composition of each fluid region, the configuration of the nitrocellulose layer, the formation mode, and the nitrification used The composition and the preferred ratio of the fiber solution, the composition and the formation manner of each of the reaction materials are the same as those of the first preferred embodiment described above, and the detailed description thereof will not be repeated here.

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the present invention. The above description is to be understood by those skilled in the art, and thus the other embodiments are not disclosed. Equivalent changes or modifications made in the spirit of the invention are to be included in the scope of the claims below.

1. . . Fluid test strip

10. . . Substrate

11. . . Runner

1101. . . Substance to be tested

111. . . First fluid zone

1110. . . Fiber layer

1111. . . Primary antibody

1112. . . Sugar material

1113. . . Peroxide

112. . . Second fluid zone

1120. . . Second nitrocellulose layer

1121. . . Secondary antibody

113. . . Third fluid zone

1130. . . Third nitrocellulose layer

1131. . . Luminescent agent

1132. . . Receptor reagent

1133. . . Glycogen

21, 22, 23, 24, 25. . . step

Fig. 1A is a schematic view showing a fluid detecting test piece according to a first preferred embodiment of the present invention.

Fig. 1B is a schematic view showing the distribution of reactive materials in the flow path of the fluid detecting test piece according to the first preferred embodiment of the present invention.

1C to 1E are schematic views showing the distribution of reactant materials in different reaction stages in the flow path of the fluid detecting test piece according to the first preferred embodiment of the present invention.

1F to 1H are schematic views showing other modes of distribution of reactive materials in the flow path of the fluid detecting test piece according to the first preferred embodiment of the present invention.

Fig. 1I is a schematic cross-sectional view showing a fluid detecting test piece according to a first preferred embodiment of the present invention.

Fig. 2 is a flow chart showing a fluid detecting method according to a second preferred embodiment of the present invention.

11‧‧‧ flow path

1101‧‧‧Substance to be tested

111‧‧‧First Fluid Zone

1111‧‧‧First antibody

1112‧‧‧Sugar materials

1113‧‧‧ peroxide 脢

112‧‧‧Second fluid zone

1121‧‧‧Second antibody

113‧‧‧ Third fluid zone

1131‧‧‧Lighting agent

1132‧‧‧Reagents

1133‧‧‧Glycogen

Claims (32)

A fluid detecting test piece mainly comprises a substrate, the substrate comprising at least a first channel, the flow channel comprising a first fluid zone, a second fluid zone and a third fluid zone connected in sequence, the first fluid zone being for fluid Injecting, characterized in that: a first antibody is located in the first fluid region for identifying a substance to be tested; a sugar material is located in the first fluid region or the second fluid region; a first fluid region or a second fluid region; a second antibody fixed in the second fluid region, the second antibody also identifying the test substance, and the second antibody and the first anti-system are different in identification An antigen determining position; and a luminescent agent and a host reaction reagent, located in the third fluid region, the matrix reaction reagent comprising a sugar cerium oxide; thereby, when a fluid containing the substance to be tested is injected into the flow channel The first antibody, the saccharide material, and the peroxide oxime system flow with the fluid, and a portion of the peroxide oxime is combined with the first antibody, the test substance, and the second antibody and left In the second fluid zone, unbound The peroxide enthalpy flows with the fluid to the third fluid zone, and the saccharide cerium oxide catalyzes the oxidation reaction of the saccharide material flowing to the third fluid zone to generate a hydrogen peroxide, and the luminescent agent The peroxide ruthenium, which is passed to the third fluid zone, is catalyzed by the reaction with the hydrogen peroxide and produces an optical signal. The fluid test strip of claim 1, wherein the first antibody and the peroxide oxime are simultaneously located in the first fluid zone and form a conjugate bond. The fluid detecting test piece according to claim 1, wherein the first antibody further forms a conjugate bond with a biotin, and the peroxide lanthanide forms a conjugate bond with the avidin, and the avidin system One of the groups consisting of the following units, including avidin, streptavidin, and neutral streptavidin. The fluid test strip according to claim 1, wherein the first anti-system is a monoclonal antibody or a plurality of antibodies, and the second anti-system is a monoclonal antibody or a plurality of antibodies. The fluid test strip of claim 1, wherein the peroxide is selected from the group consisting of horseradish peroxide and ascorbate peroxide.脢 and hydrogen peroxide. The fluid detecting test piece according to claim 1, wherein the luminescent agent is 3-aminophthalic acid. The fluid detecting test piece according to claim 1, wherein the saccharide material is glucose, and the saccharide cerium oxide is glucosinolate. The fluid test strip of claim 1, wherein the bottom of the first fluid zone comprises a fibrous layer, and the first anti-system is formed in the fibrous layer. The fluid detecting test piece according to claim 1 further includes a nitrocellulose layer formed at a bottom of the second fluid region and the third fluid region, respectively, and the second anti-system is fixed to the nitrocellulose of the second fluid region. The layer, the host reaction reagent is formed in the nitrocellulose layer of the third fluid zone. The fluid test strip of claim 9, wherein the nitrocellulose layer is a nitrocellulose membrane. The fluid detecting test piece according to claim 9 , wherein the nitrocellulose layer is formed by casting a nitrocellulose solution into the bottom of the second fluid zone and the third fluid zone and drying, and the nitrocellulose layer comprises There is a hollow mesh configuration. The fluid test piece according to claim 11, wherein the nitrocellulose solution is formed by mixing a nitrocellulose powder ester and a ketone solvent. A fluid detecting test piece according to claim 12, wherein a preferred ratio of the nitrocellulose powder to the ester and the ketone solvent is 1:9. The fluid test strip of claim 11, wherein the substrate is a biocompatible material. The fluid test strip of claim 11, wherein the average thickness of the nitrocellulose layer of the second fluid zone is equal to the thickness of the nitrocellulose layer of the third fluid zone. The fluid detecting test piece of claim 15, wherein the flow path further comprises a fourth fluid A nitrocellulose layer is also formed at the bottom of the fourth fluid zone, and the nitrocellulose layer comprises a hollow network configuration for storage of excess fluid. A fluid detecting method comprising the steps of: providing a fluid comprising a substance to be tested; providing a substrate comprising at least a first channel, the flow channel comprising a first fluid zone and a second fluid zone sequentially connected And the third fluid region, the first fluid region is for the injection of the fluid, the substrate further comprises: a first antibody, located in the first fluid region, for identifying the substance to be tested in the fluid; The material is located in the first fluid zone or the second fluid zone; a peroxide buffer is located in the first fluid zone or the second fluid zone; a second antibody is fixed in the second fluid zone, and the second antibody is also Identifying the test substance, and the second antibody and the first anti-system identify different antigen-determining positions; and a luminescent agent and a host reaction reagent are located in the third fluid region, and the host reaction reagent comprises a saccharide oxide; adding the fluid to the first fluid zone of the flow channel to cause the first antibody, the saccharide material, and the peroxide raft to flow with the fluid; and the test substance and the first antibody Second antibody a portion of the peroxide oxime is bound and left in the second fluid zone, such that the fluid carries the saccharide material, the unbound primary antibody, and the peroxide to the third fluid zone to cause the sugar The material is oxidized by the saccharide cerium oxide and generates hydrogen peroxide, and the luminescent agent is catalyzed by the peroxide ruthenium flowing to the third fluid region to react with the hydrogen peroxide, and a Optical signal; and detecting the optical signal, estimating the content of the peroxide enthalpy flowing to the third fluid region by the intensity of the optical signal, and fixing the peroxide enthalpy to one of the additives The content is simply subtracted from the content of the peroxide turbulent flow to the third fluid zone to obtain the content of the peroxide oxime remaining in the second fluid zone, and then left in the second fluid zone. The content of the peroxide enthalpy deduces the concentration of the peroxide enthalpy remaining in the second fluid zone, and finally remains in the second stream The concentration of the peroxide oxime in the body region is derived from the concentration of the test substance contained in the fluid. The fluid detecting method of claim 17, wherein the first antibody and the peroxide oxime are simultaneously located in the first fluid region and form a conjugate bond. The fluid detecting method of claim 17, wherein the first antibody further forms a conjugate bond with a biotin, and the peroxide lanthanide forms a conjugate bond with the avidin, and the avidin is selected One of the groups consisting of the following units, including avidin, streptavidin, and neutral streptavidin. The fluid detecting method according to claim 17, wherein the first anti-system is a monoclonal antibody or a plurality of antibodies, and the second anti-system is a monoclonal antibody or a plurality of antibodies. The fluid detecting method according to claim 17, wherein the peroxide oxime is selected from the group consisting of the following units, the group comprising horseradish peroxide 抗, ascorbyl peroxide 脢And hydrazine hydroperoxide. The fluid detecting method according to claim 17, wherein the luminescent agent is 3-aminophthalic acid. The fluid detecting method according to claim 17, wherein the saccharide material is glucose, and the saccharide cerium oxide is glucosinolate. The fluid detecting method of claim 17, wherein the bottom of the first fluid zone comprises a fibrous layer, and the first resistant system is formed in the fibrous layer. The fluid detecting method of claim 17, further comprising a nitrocellulose layer formed at a bottom of the second fluid zone and the third fluid zone, respectively, and the second anti-system is fixed to the nitrocellulose layer of the second fluid zone. The host reaction reagent is formed in the nitrocellulose layer of the third fluid zone. The fluid detecting method of claim 25, wherein the nitrocellulose layer is a nitrocellulose membrane. The fluid detecting method of claim 25, wherein the nitrocellulose layer is formed by pouring a nitrocellulose solution into the bottom of the second fluid zone and the third fluid zone and drying, and the nitrate is formed. The chemical fiber layer contains a hollow network configuration. The fluid detecting method according to claim 27, wherein the nitrocellulose solution is formed by mixing a nitrocellulose powder ester and a ketone solvent. The fluid detecting method according to claim 28, wherein a preferred ratio of the nitrocellulose powder to the ester and the ketone solvent is 1:9. The fluid detecting method of claim 25, wherein the substrate is a biocompatible material. The fluid detecting method of claim 27, wherein the average thickness of the nitrocellulose layer of the second fluid zone is equal to the thickness of the nitrocellulose layer of the third fluid zone. The fluid detecting method of claim 31, wherein the flow path further comprises a fourth fluid zone, and a bottom of the fourth fluid zone is also formed with a nitrocellulose layer, the nitrocellulose layer comprising a hollow mesh structure for Storage of excess fluid.