TWI572330B - Device for blood typing - Google Patents

Description

Blood type detecting device

The invention relates to a blood type detecting device, which can quickly and accurately obtain blood type analysis results at low cost.

On red blood cells, 328 different antigens have been identified and divided into 30 different blood group. Among them, ABO blood group and RH blood type are the most common categories, and based on the mechanism of anti-hemoglobulin, blood group detection is to observe the agglutination reaction between the antibody on the surface of red blood cells and the corresponding antigen, and the antibody and antigen interaction are triggered. Red blood cells are tightly bound.

The importance of accurately identifying blood types is high, and the Blood Bank or Medical Center provides donors and recipients with safe blood transfusions and metastases, while transfusion of blood types can lead to fatal hemolytic consequences. In addition, blood types are also used in other clinical applications, such as the treatment of diseases and prognosis. Traditional blood type detection methods, such as tube test, tube column agglutination, slide method, solid phase test method to detect agglutinated red blood cells, the process must be performed by medical personnel or inspectors, while collecting more blood (blood needs) Amount >1mL), generally use a syringe to draw blood to collect the required amount of blood, the overall time is long (tens of minutes), and external inspection equipment (such as centrifuge, microscope, etc.) is needed for analysis. Increasing the cost of testing reagents and equipment is more limited in environments with limited resources, such as battlefields or developing countries.

In order to solve the above problems, today's clinical detection-based concepts have developed platforms for cell types that require fewer blood samples and do not require the use of larger external pump systems and processing power supplies. Most of these platforms use paper detection. However, in order to obtain better and clear results, it is necessary to wash the reaction zone with a buffer solution; and, the immune interaction of red blood cells and antibodies on paper, under limited diffusion and reaction time, It may be detrimental to the detection of weak interactions, such as blood group of group A3, blood type of group B3, and blood type of patients with hematologic malignancy or thalassemia.

In view of this, there is an urgent need for a detection system that is fast, inexpensive, and low in blood demand, which can greatly improve the speed and quality of medical diagnosis, and can be used in related fields to greatly shorten the detection time.

An object of the present invention is to provide a blood type detecting device, comprising: a first substrate having a first through hole and a second through hole; and a second substrate disposed opposite to the first substrate, the second substrate Facing one surface of the first substrate, the plurality of grooves have a plurality of grooves, and the grooves are connected to each other and include a filter zone and optionally a first-class track zone and a mixing zone, wherein the filter zone comprises a coloring portion and a narrow And a third substrate disposed between the first substrate and the second substrate, the third substrate having a third through hole and a plurality of additional recesses facing a surface of the first substrate a groove, the additional grooves are connected to each other and include the flow channel region and the mixing region; wherein the first through hole is connected to the flow channel region, the flow channel region is connected to the mixing zone, and the mixing zone is connected to the filter zone The coloring portion, the coloring portion of the filtering region is connected to the slit portion of the filtering region, and the slit portion of the filtering region is connected to the second through hole to form a flow path.

The first through hole may be a blood antibody and a blood sample input to the blood sample detecting device. The type of the first through hole is not limited, and may be, for example, a screw hole. When the first through hole is a screw hole, the first through hole may have a diameter of 300 to 5,000 micrometers, preferably 1000. Up to 3000 microns.

Wherein, the second through hole may be an outlet for outputting the remaining liquid, and the second through hole may have a diameter of 100 to 2,000 micrometers, preferably 500 to 1200 micrometers. Due to the miniature size of the outlet end, blood samples do not easily flow out to the outside environment, reducing the possibility of contamination.

Wherein, the flow channel region may be a buffer before the blood antibody and the blood sample are input into the mixing zone, and the total length of the grooves or the additional grooves in the flow channel region may be 500 to 300,000 micrometers. Preferably, the grooves or the additional grooves in the flow channel region have a depth of 5 to 1,000 micrometers, preferably 50 to 500 micrometers, which facilitates the smooth passage of red blood cells and is not squeezed. Pressure cracking; however, the invention is not limited thereto.

Wherein, the mixing zone may be an area where the blood antibody and the blood sample are contacted and reacted. For the purpose of uniform mixing, the mixing zone preferably has a distinct difference in depth and a sufficient reaction distance, and therefore, in the mixing zone Preferably, the grooves or the additional grooves have two or more depths, and each groove has a depth difference from the adjacent grooves, and the difference in depth is preferably between 5 and 2,000 microns, preferably. It is from 100 to 900 microns; and the total length of the grooves or the additional grooves in the mixing zone may be from 500 to 300,000 microns, preferably from 10,000 to 30,000 microns.

The filter zone may include the coloring portion and the slit portion, and the coloring portion is a place for reading the detection result. In order to facilitate the observer to directly observe the detection result by the naked eye, the grooves in the color forming portion preferably constitute a pattern, which may be curved, curved, or V-shaped, or the color portion The grooves have a wide width of 50 to 5,000 microns, for example, 500 to 2,000 microns; however, the invention is not limited thereto. The slit portion is a region for screening whether red blood cells in the blood sample bind to blood antibodies, and the grooves in the slit portion may have a depth of 0.1 to 3 μm, for example, 2 μm. In detail, when the red blood cells in the blood sample are combined with the blood antibodies, the combined volume increases and the slit cannot be entered. Therefore, the successfully bound red blood cells and antibodies accumulate in the coloring portion, and the unbound blood antibody and blood sample can be output from the second through hole.

The blood type detecting device of the present invention may further include: a fourth substrate on the opposite side of the second substrate on which the third substrate is disposed, and the fourth substrate includes a recess; wherein the second substrate further has a a fourth through hole, and the groove of the fourth substrate is connected to the fourth through hole. In this case, the first substrate may further have an opening, the second substrate may further have a fifth through hole, the third substrate may further have a sixth through hole, and the opening is connected to the sixth through hole The sixth through hole is connected to the fifth through hole, and the fifth through hole is connected to the groove of the fourth substrate.

Further, the blood type detecting device of the present invention can simultaneously detect a plurality of blood types. Under this requirement, for example, a plurality of second through holes may be disposed on the first substrate, and a plurality of flow path regions, a plurality of mixing regions, and a plurality of filter regions may be disposed on the second substrate. Accordingly, the first through hole can simultaneously connect a plurality of flow channel regions, and each flow channel region sequentially connects the mixing regions, the filter regions, and each of the second through holes to form a plurality of flow paths.

The present invention further provides a method for performing blood detection using the blood detecting device, comprising the steps of: (A) taking a blood antibody, and injecting the first through hole into the mixing region of the blood type detecting device; (B) taking one a blood sample injected from the first through hole into the mixing zone of the blood type detecting device to uniformly mix the blood sample with the blood antibody to form a mixture; (C) entering the mixture into the filtration zone; and (D) It is observed whether the coloring portion in the filtration zone is colored.

In the above step (A), when the blood antibody is injected from the first through hole, a pressure is applied to drive the blood antibody to flow in the flow path, for example, by screwing the first through hole downward. The screw applies pressure, or the syringe is used to connect the second through hole with a needle tip, and the syringe is pushed or withdrawn to drive the blood antibody to move, so that the blood antibody reaches the mixing zone.

In the above step (B), when the blood sample is injected from the first through hole, the blood sample can be driven into the mixing region by using the pressure (step) in the above step (A) to make the blood sample and the blood antibody. Mix evenly.

In the above step (C), in the mixture formed by uniformly mixing the blood sample and the blood antibody, if the red blood cells in the blood sample are combined with the blood antibody, the combination will stay in the coloring portion, the unbound red blood cells and Blood antibodies enter the slit. Finally, in the above step (D), the observer can directly observe the detection result of the appearance of the coloring portion with the naked eye.

Through the blood type detecting device of the present invention, a blood sample can be collected by using a relatively invasive blood collecting needle method for micro blood sampling (such as finger puncture) without using a syringe for blood drawing, and only 1 microliter (μL) is required. The amount of blood and the detection time of 1 minute. The blood type detecting device of the present invention can detect the blood type of normal red blood cells, and is also suitable for judging other weak interactions, for example, the subtype A blood type-A3 blood type, the B subtype blood type-B3 blood type, and the hematological malignant tumor. The blood type of patients with (hematologic malignancy) or thalassemia can be detected. Therefore, the blood type detecting device of the present invention performs blood type detection, and the detection result can be obtained quickly and efficiently. The detection method is simple, the sensitivity is high, and the device cost is low, so that it can be implemented under various environments and conditions.

10,20‧‧‧ blood type detection device

1‧‧‧First substrate

2‧‧‧second substrate

11‧‧‧ first through hole

12‧‧‧Second through hole

13‧‧‧ openings

21‧‧‧ surface

22‧‧‧ sixth through hole

23‧‧‧ third through hole

211,212,213,214,215‧‧‧ grooves

216‧‧‧Multiple grooves

31‧‧‧Runner Area

32‧‧‧ mixed area

33‧‧‧Filter zone

331‧‧‧Coloring Department

332‧‧‧Slits

4‧‧‧ blood collection device

5‧‧‧ screws

6‧‧‧ Third substrate

61‧‧‧Linear groove

62‧‧‧V-shaped groove

63‧‧‧fourth through hole

64‧‧‧5th through hole

7‧‧‧4th substrate

71‧‧‧Linear groove

Fig. 1A is a schematic cross-sectional view showing a blood type detecting device according to a first embodiment of the present invention.

Fig. 1B is a perspective view of a blood type detecting device according to a first embodiment of the present invention.

Fig. 2 is a graph showing the results of a withstand voltage test according to a second embodiment of the present invention.

3A and 3B are diagrams showing the result of the groove test of the mixing zone in the fourth embodiment of the present invention.

4 is a schematic view of a blood detecting method according to Embodiment 5 of the present invention.

Fig. 5 (a) to (e) are diagrams showing blood test results of Example 5 of the present invention.

6(a) to 6(d) are exploded views of the blood type detecting device of the sixth embodiment of the present invention.

Figure 7 is a top plan view of a blood type detecting device according to a sixth embodiment of the present invention.

The embodiments of the present invention are described by way of specific examples, and those skilled in the art can readily appreciate the other advantages and advantages of the present invention. The present invention may be embodied or applied in various other specific embodiments. The details of the present invention can be variously modified and changed without departing from the spirit and scope of the invention.

[Example 1] Blood type detecting device

1A and 1B, FIG. 1A is a cross-sectional view of the blood type detecting device 10 of the present embodiment, FIG. 1B is a perspective view, and FIG. 1A is a cross-sectional view taken along line L-L' of FIG. 1B. The blood type detecting device 10 of the present embodiment includes a first substrate 1 having a first through hole 11 and a second through hole 12, and a second substrate 2 disposed opposite to the first substrate 1 and arranged in parallel. The surface of the second substrate 2 facing the first substrate 1 has a plurality of grooves 211, 212, 213, 214, 215 interconnected by 211, 212, 213, 214, 215 and divided into a first-class track area 31, a mixing area 32, and a filter area. 33, and the filtering area 33 includes a coloring portion 331 and a slit portion 332, wherein the first through hole 11 is connected to the flow channel region 31, and the flow channel region 31 is connected to the mixing region 32, and the mixing region 32 is connected The coloring portion 331 of the filtering region 33, the coloring portion 331 of the filtering region 33 is connected to the slit portion 332 of the filtering region 33, and the slit portion 332 of the filtering region 33 is connected to the second through hole 12, Form a circulation path.

In this embodiment, a cyclo olefin polymer (COP) is used as the material of the first substrate and the second substrate. However, those skilled in the art can consider cost, weight, process, transparency, and blood. Factors such as biocompatibility are easily replaced with other materials. The dimensions used in this embodiment are 3 cm x 3 cm x 4 mm (first substrate) and 3 cm x 3 cm, respectively. Made of COP substrate (ZEONOR 1020R, Zeon Corporation, Tokyo, Japan) of m x 2 mm (second substrate). As shown in FIG. 1B, three second through holes 12 having a diameter of 650 micrometers are first drilled on the first substrate, and the first through holes 11 are drilled at the center point of the first substrate by using a drill having a diameter of 1.5 mm. Then, a commercially available stainless steel screw having a diameter of 2 mm was transferred into the first through hole 11 to make a thread on the inner side wall thereof. Next, a plurality of grooves are formed on the second substrate to form a flow channel region 31, a mixing region 32, and a filtration zone 33, wherein the slit portion (refer to FIG. 1A, reference numeral 332) is an ultraviolet ozone (UVO; Novascan, Ames, IA), after surface modification, is formed by swelling of cyclohexane vapor. Finally, the two COP substrates were treated by a hot press at a pressure of 3.45 MP for 1 minute, and sealed into a wafer to complete the blood type detecting device 10 of the present embodiment. According to this, the blood type detecting device 10 can simultaneously detect a plurality of blood types, and the first through holes can simultaneously connect three flow channel regions, and each flow channel region is sequentially connected to each mixing region, each filtering region, and each second through hole. Three flow paths are formed.

The red blood cells are generally red blood cells showing a double concave disk shape with a thickness of 2.5 μm and a diameter of 7.5 μm. The red blood cells are elastic and will appear like a slipper shape and a parachute shape when flowing in the human blood circulation system, indicating that the red blood cells will be deformed during the flow, resulting in dimensional shrinkage. According to this principle, referring to FIG. 1A, the depth D4 of the groove 215 of the slit portion 332 is 2 μm, which can block the agglomerated red blood cells (red blood cells combined with blood antibodies), and the uncondensed red blood cells (not combined with blood antibodies). The red blood cells can pass through the slit portion 332.

In the embodiment, the first through hole 11 can be a screw hole, which facilitates the application of pressure by screwing in a screw (a commercially available stainless steel screw). The first through hole has a diameter of 2,000 microns. In addition, the second through hole may be a pinhole that pushes or draws the injection syringe to drive the blood sample or the blood antibody to move. The second through hole has a diameter of 700 μm.

In this embodiment, referring to FIG. 1A, the total length of the grooves 211 in the flow path region 31 may be 20,000 micrometers, and the depth D1 of the grooves 211 in the flow channel region 31 is 100 micrometers. The grooves 212, 213 in the mixing zone 32 have two depths D2 (100 microns), D3 (200 microns), and each concave The groove 212 has a depth difference D5 between the adjacent grooves 213, and the depth difference is 100 μm; and the total length of the grooves in the mixing zone is 20,000 μm.

In the present embodiment, the flow path area 31, the mixing area 32, and the filtration area 33 all constitute a curved pattern, however, the pattern is only used to facilitate the observer to observe with the naked eye. Alternatively, referring to FIG. 1A, the width W1 of the grooves 214 in the color-developing portion 331 can be designed to be wider than other regions, for example, 1,000 micrometers, and can also be conveniently observed by an observer.

[Example 2] Withstand voltage test

The blood type detecting device prepared in Example 1 was fixed on a micro-carving machine stage, and drills having dimensions of 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, and 0.5 mm were respectively used, and the rotational speed was set to 15000 rpm, Z. The axial feed rate is 2mm/sec, the through hole is formed on the first substrate, and the burrs generated by the drilling are removed by using a deburring knife, and the threaded stainless steel is used for tapping the 0.5 mm through hole to generate the needle. Type interface (smooth surface), other sizes of through holes use a general 2mm tapping to create a thread inside the slot. Next, the liquid chromatography was used to test the pump, and the experimental results are shown in Fig. 2. Through the penetration pressure of the screw pump generated by the 2mm tapping, it was found that the through hole formed by the drill bit with the size of 1.45 to 1.65mm can withstand the joint pressure of more than 20MPa and the size of 1.5mm. The through hole formed by the drill bit, the average withstand pressure of the screw pump can exceed 20 MPa. Therefore, it is made of a 1.5 mm drill bit, which has better pressure resistance stability and can withstand a pressure value greater than 20 MPa.

[Example 3] Optimal load volume test

Using the blood type detecting device prepared in Example 1, the test was carried out, using 5 different volume ratios of pure water, using a micropipette to take 4 μL - 8 μL of pure water droplets to the first through hole (a screw hole made of a 1.5 mm drill), and then Use stainless steel screws to turn to the bottom of the unit. First measure the weight of the paper towel before the water absorption, then place the paper towel next to the screw to absorb the excess liquid, and measure the weight of the paper towel after the water absorption, then the two weights The value of the difference is the amount of liquid wasted. The results of the experiment are reported in the table below, and as a result, it was found that the optimum loading amount of the blood type detecting device was 6 μL.

[Example 4] Mixed zone groove test

Fluorescence is used to analyze the groove design of the mixing zone. The fluorescent reagent and deionized water are mixed first, and then injected into the groove with the mixed mixing zone downstream to obtain the blood type prepared in Example 1 respectively. The mixing process diagram of the mixing zone of the detecting device and the drawing process of Figure 3B (the same depth groove, the groove depth is 100 micrometers) are divided into (i) according to the distance from the injection end in the groove. The mixing result of 0 cm (at the injection end) after the flow path, (ii) 1.2 cm after entering the flow path, and (iii) 2.5 cm after entering the flow path, the X-axis of FIGS. 3A and 3B indicates the width direction of the groove. The location on the top. The flow rate of the injected fluorescent reagent and deionized water used in the test was 5 μL/min.

For the experimental results, please refer to FIG. 3A and FIG. 3B. FIG. 3A shows that the fluorescein and the deionized water are mixed and passed through grooves having different depths (that is, the grooves of the mixing region of the blood type detecting device prepared in Embodiment 1), and FIG. 3B is The fluorescein and deionized water are mixed and passed through the same depth groove. It can be seen from the experimental results that if the grooves having different depths are not used, the red blood cells and the blood antibodies cannot fully function, and thus, the antibody does not completely bind to the antigen on the surface of the red blood cells in a short time, and it is difficult to obtain. Fast and accurate test results.

[Example 5] Blood type test

The blood type detecting device 10 prepared in Example 1 was used for the test. Referring to FIGS. 1A and 1B, 0.5 μL of antibody A, antibody B, and antibody RH are respectively dropped into a first through hole 11 using a micropipette, a rubber tube is connected to the second through hole 12, and a rubber tube is connected with a needle tip. Thereafter, the syringe is pushed or withdrawn to drive the blood antibody to the mixing zone 32. If the test is not performed immediately, the first through hole and the second through hole may be sealed with a tape, and after the antibody is dried, it is stored in a refrigerator at 4 degrees C.

The blood samples used in this embodiment are men and women aged 20 to 40, and there are no major diseases. There are 30 blood samples, and the blood type includes type A, type B, type AB, type O, and thalassemia type O blood. All experiments were performed in accordance with relevant laws and regulations, and samples of blood samples were approved by CGM Hospital. Referring to FIG. 4, the detection procedure is as follows: 1 μL of blood is placed in the first through hole 11 of the blood type detecting device 10 by using a blood collection device 4, and then a screw 2 of 2 mm is screwed into the first through hole 11 to drive blood flow. Referring to FIG. 1A together, the blood sample is reacted (evenly mixed) with the blood antibody previously injected into the mixing zone 32, and the red blood cells bound to the antibody are agglomerated and are blocked in front of the narrow area 332, accumulating in The coloring portion 331 was observed, and the coloring result was observed with the naked eye as shown in FIG. In Fig. 5, (a) shows the results of type A blood, (b) shows the results of type B blood, (c) shows the results of type AB blood, and (d) shows the detection of type O blood. As a result, and (e) the results of the detection of thalassemia type O blood; the number of red blood cells in the Mediterranean anemia is smaller than that of the average person, and the size is smaller than that of the average person, and the apparent coloration range is smaller than that of the general type O blood. From this, it is understood that the blood type detecting device of the present invention has 100% sensitivity and specificity.

[Embodiment 6] Another blood type detecting device

However, the blood type detecting device of the present invention is not limited to the embodiment 1, and those skilled in the art can easily understand the spirit of the present invention and thereby simply adjust the device structure. For example, please refer to FIGS. 6( a ) to ( d ) and FIG. 7 , and FIGS. 6( a ) to ( d ) are a first substrate, a third substrate, a second substrate, and a fourth substrate of the blood type detecting device 20, respectively. The blood type detecting device of the present invention is in addition to the first substrate 1 and In addition to the second substrate 2, the third substrate 6 and the fourth substrate 7 may be further included. The first substrate 1 and the second substrate 2 are disposed opposite to each other, and the third substrate 6 is interposed between the first substrate 1 and the second substrate 2, The fourth substrate 7 is located on the opposite side of the second substrate 2 on which the third substrate 6 is disposed. The first substrate 1 includes a first through hole 11 and a second through hole 12, and further includes an opening 13; the second substrate 2 The linear substrate 61 includes a V-shaped groove 62, a fourth through hole 63, and a fifth through hole 64. The third substrate 6 includes a plurality of grooves 216, and further includes a sixth through hole 22 and a third through hole 23; The substrate 7 includes a linear groove 71. In this embodiment, the same portions as those in Embodiment 1 will not be described again.

The first substrate to the fourth substrate are press-sealed into a single wafer by using a hot press, that is, the blood type detecting device 20 of the present embodiment is completed (as shown in FIG. 7). Accordingly, the first through hole 11 is connected to the plurality of grooves 216, and the plurality of grooves 216 are sequentially connected to the second through hole 12 and the third through hole 23, respectively, and the third through hole 23 is connected to the V-shaped groove 62 and the linear groove 61. Forming a first circulation path. It should be noted that the opening 13 is connected to the sixth through hole 22, the sixth through hole 22 is connected to the fifth through hole 64, the fifth through hole 64 is connected to the linear groove 71, and the linear groove 71 is connected to the fourth through hole 63, fourth The through hole 63 connects the linear groove 61 and the V-shaped groove 62 via the slit portion (provided between the linear groove 61 and the fourth through hole 63) to form a second flow path. As shown in FIG. 7 , the first through hole 11 and the fourth through hole 63 overlap but are not directly connected, and the second through hole 12 and the third through hole 23 do not overlap, and the opening 13 and the sixth through hole 22 and the fifth through hole are not overlapped. 64 overlaps, and the diameter of the opening 13 is significantly larger than the sixth through hole 22 and the fifth through hole 64.

In this embodiment, the blood antibody can be injected from the second through hole 12, the rubber tube is externally connected to the opening 13, the rubber tube is connected with the needle tip, and the injection syringe is pushed or extracted to drive the blood antibody to move into the plurality of grooves 216. a mixing zone (not shown), a blood sample is added from the first through hole 11, a syringe is pushed or withdrawn to drive the blood sample to mix with the blood antibody, and the syringe is pushed or extracted to drive the blood sample and blood. The mixture of antibodies reaches the V-shaped groove 62 through the third through hole 23, and a slit portion (not shown) is disposed between the linear groove 61 and the fourth through hole 63, and the condensed red blood cells and antibodies accumulate in the V shape. At the groove 62, the detection result can be visually recognized.

In addition, the blood type detecting device of the present invention can also be applied to other fluid detecting methods, for example, using the principle of binding of an antigen to an antibody, and can be further extended to the detection of various immune mechanisms. And, this hair In the blood type detecting device of the Ming, the flow path is formed by the through holes and the grooves. Therefore, the red blood cells may also flow to other regions such as the mixing region or the flow channel region after being combined with the antibody, so that the region to be read is not limited to the coloring portion. The condensed red blood cells accumulate, making the circulation path appear red, indicating the presence of a corresponding blood antigen.

The blood type detecting device of the invention can achieve the characteristics of rapid detection and high sensitivity, and the amount of blood required for 1 μL can be obtained by using a less invasive finger puncture method, and the ABO blood type and the Rh blood type can be detected, and In the minute, the results of blood agglutination can be correctly observed, and the blood types such as A and B subtypes and thalassemia can be judged, so that the blood type can be quickly re-verified before the clinical operation. The amount of blood used in the test is small, the amount of antibody is small, the number of manual steps is small, the reaction time is short, and it is safer to use non-use glass, and there is no risk of infection due to exposure of blood waste liquid.

The above-mentioned embodiments are merely examples for convenience of description, and the scope of the claims is intended to be limited to the above embodiments.

1‧‧‧First substrate

2‧‧‧second substrate

21‧‧‧ surface

11‧‧‧ first through hole

12‧‧‧Second through hole

211,212,213,214,215‧‧‧ grooves

31‧‧‧Runner Area

32‧‧‧ mixed area

33‧‧‧Filter zone

331‧‧‧Coloring Department

332‧‧‧Slits

Claims (13)

A blood type detecting device includes: a first substrate having a first through hole and a second through hole; a second substrate disposed opposite to the first substrate, the second substrate facing the first substrate One of the surfaces has a plurality of grooves interconnected and including a filter zone and optionally a first-order zone and a mixing zone, the filter zone comprising a coloring portion and a slit portion; and the optional a third substrate is disposed between the first substrate and the second substrate, and a surface of the third substrate facing the first substrate has a third through hole and a plurality of additional grooves, and the additional holes are The groove is interconnected and includes the flow channel region and the mixing region; wherein the first through hole is connected to the flow channel region, the flow channel region is connected to the mixing zone, and the mixing zone is connected to the coloring portion of the filter zone, The coloring portion of the filtering region is connected to the slit portion of the filtering region, and the slit portion of the filtering region is connected to the second through hole to form a flow path; wherein the grooves or the holes in the mixing region The extra grooves have more than two depths. The blood type detecting device of claim 1, wherein each of the grooves or the additional grooves of the mixing zone has a depth difference between the groove and the adjacent groove. The depth difference is between 100 and 900 microns. The blood type detecting device of claim 1, wherein the total length of the grooves or the additional grooves in the mixing zone is 500 to 300,000 micrometers. The blood type detecting device according to claim 1, wherein the grooves in the coloring portion have a width of 50 to 5,000 μm. The blood type detecting device according to claim 1, wherein the grooves in the color forming portion constitute a pattern which is curved, curved, or V-shaped. The blood type detecting device according to claim 1, wherein the grooves in the slit portion have a depth of 0.1 to 3 μm. The blood type detecting device according to claim 1, wherein the total length of the grooves or the additional grooves in the flow path region is 500 to 300,000 μm. The blood type detecting device according to claim 1, wherein the grooves or the additional grooves in the flow path region have a depth of 5 to 1,000 μm. The blood type detecting device according to claim 1, wherein the first through hole is a screw hole. The blood type detecting device according to claim 1, wherein the first through hole has a diameter of 300 to 5,000 μm. The blood type detecting device according to claim 1, wherein the second through hole has a diameter of 100 to 2,000 μm. The blood type detecting device of claim 1, further comprising: a fourth substrate disposed on a reverse side of the second substrate on which the third substrate is disposed, wherein the fourth substrate comprises a recess; The second substrate further has a fourth through hole, and the groove of the fourth substrate is connected to the fourth through hole. The blood type detecting device of claim 12, wherein the first substrate further has an opening, the second substrate further has a fifth through hole, and the third substrate further has a sixth through hole, and The opening is connected to the sixth through hole, the sixth through hole is connected to the fifth through hole, and the fifth through hole is connected to the groove of the fourth substrate.