Paper folding type microfluidic paper chip analysis device for detecting adenosine triphosphate and application

Abstract

The invention relates to the technical field of micro-fluidic chips and biosensors, in particular to a paper folding type micro-fluidic paper chip analysis device for detecting ATP (adenosine triphosphate) based on a difunctional DNA tetrahedron and application of the paper folding type micro-fluidic paper chip analysis device. The analysis device comprises a detection layer and a washing layer, and is characterized in that a difunctional DNA tetrahedron system containing detection ATP is added into a reaction zone; wherein the bifunctional DNA tetrahedron comprises a capture DNA tetrahedron (B-TDN) and a detection DNA tetrahedron (D-TDN), and a single-stranded DNA sequence extending from any vertex of the tetrahedron formed by the B-TDN and a single-stranded DNA sequence extending from any vertex of the tetrahedron formed by the D-TDN can be complementary. The paper folding type micro-fluidic paper chip analysis device based on the difunctional DNA tetrahedron has the advantages of being small in size, low in scientific research cost, biodegradable, high in sensitivity, good in selectivity and the like, a new method is provided for rapid ATP detection in the medical field, and the paper folding type micro-fluidic paper chip analysis device is suitable for remote areas and economic outdated areas.

Description

Technical Field

[0001] The invention relates to the field of microfluidic chip technology and biosensor technology, in particular to an origami-type microfluidic paper chip analysis device for detecting adenosine triphosphate (ATP) based on a bifunctional DNA tetrahedron and its application. Background Art

[0002] Adenosine triphosphate (ATP) is an essential high-energy compound in living organisms. It is the main energy carrier of cells in the body and plays a vital role in signal transduction and cell metabolism. Studies have shown that abnormal ATP content can cause a variety of diseases, such as hypoglycemia, Parkinson's disease, malignant tumors, cardiovascular disease, Alzheimer's disease, etc. To date, common methods for ATP analysis include high-performance liquid chromatography, capillary electrophoresis, chemiluminescence, and mass spectrometry. Although these techniques can effectively detect ATP, there are still obstacles such as time-consuming sample preparation, high reagent consumption, and expensive instruments. Therefore, it is very necessary to develop a fast and economical ATP detection technology.

[0003] Microfluidic analytical devices based on cellulose paper have developed into a widely used analytical sensing platform in recent years due to their advantages such as portability, low price, easy preparation and small sample volume. However, traditional microfluidic paper chip analytical devices can only provide qualitative measurements. In terms of quantitative analysis, paper-based analytical devices have been integrated with a variety of analytical methods, such as colorimetry, electrochemistry, surface-enhanced Raman spectroscopy and fluorescence. Among them, the fluorescence analysis platform based on microfluidic paper chips has attracted much attention due to its advantages such as simple operation and fast detection speed. However, there is still room for improvement in its sensitivity. Based on this, we introduced a bifunctional DNA tetrahedron, in which one tetrahedron is used as a capture substrate to improve the efficiency of target recognition, and the other tetrahedron is combined with a fluorescent dye as a strong fluorescent indicator. Combining it with a microfluidic paper chip can significantly improve the sensitivity of ATP detection. Summary of the invention

[0004] The purpose of the present invention is to provide an origami-type microfluidic paper chip analysis device for detecting adenosine triphosphate (ATP) based on a bifunctional DNA tetrahedron and its application.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] An origami-type microfluidic paper chip analysis device for detecting adenosine triphosphate, the analysis device comprising a detection layer and a washing layer, characterized in that: a bifunctional DNA tetrahedron system containing detection ATP is added into a reaction zone; wherein the bifunctional DNA tetrahedron comprises a capture DNA tetrahedron (B-TDN) and a detection DNA tetrahedron (D-TDN), a single-stranded DNA sequence extending from any vertex of the tetrahedron formed by the B-TDN can be complementary to a single-stranded DNA sequence extending from any vertex of the tetrahedron formed by the D-TDN; and a single-stranded DNA extending from any vertex of the tetrahedron formed by the D-TDN can bind to the adenosine triphosphate to be detected.

[0007] The capture DNA tetrahedron (B-TDN) and the detection DNA tetrahedron (D-TDN) are respectively self-assembled using four different single-stranded DNAs; wherein, the B-TDN is composed of three single-stranded DNAs modified with biotin and one unmodified biotin intertwined to form a tetrahedron, and the single-stranded DNA unmodified with biotin in the regular tetrahedron extends an arm chain from any vertex of the tetrahedron; the D-TDN is composed of four single-stranded DNAs unmodified with biotin intertwined to form a tetrahedron, and an arm chain extends from any vertex of the tetrahedron.

[0008] The single-stranded DNA in the capture DNA tetrahedron (B-TDN) and the detection DNA tetrahedron (D-TDN) were mixed in equal moles, incubated in TM buffer at 95° C. for 7 minutes, then cooled to 4° C. and incubated for another 25 minutes to self-assemble into corresponding DNA tetrahedrons.

[0009] The bifunctional DNA tetrahedron system containing ATP detection is prepared by adding the assembled B-TDN to the reaction zone, and then adding a mixture of the assembled detection DNA tetrahedron (D-TDN), a fluorescent dye and a sample to be detected therein; wherein the volume ratio of (B-TDN) to (fluorescently modified D-TDN) is 5:1.

[0010] The final concentrations of the two tetrahedrons mentioned above are roughly the same, generally 10 -6 M.

[0011] The single-stranded DNA in the assembled B-TDN are respectively S1-S4 sequences, and the single-stranded DNA in the assembled D-TDN are respectively D1-D4 sequences;

[0012]

[0013] The origami-type microfluidic paper chip analysis device is an origami-type microfluidic paper chip that is formed by folding a cellulose material in half to form a detection layer having a hydrophilic reaction zone and a washing layer having a hydrophilic washing channel; wherein the detection layer contains a reaction zone, and the washing layer contains a corresponding washing channel, wherein the reaction zone and the washing channel are hydrophilic zones, and the remaining portion is a hydrophobic barrier zone for preventing diffusion of reagents.

[0014] The above-mentioned origami-type microfluidic paper chip analysis device consists of two functional components: a detection layer with a hydrophilic reaction zone and a washing layer with a hydrophilic washing channel. Folding the paper layer along the axis can connect the reaction zone and the washing channel to achieve the washing function; wherein, the detection layer contains a reaction zone with a diameter of 9 mm; the washing layer contains a corresponding washing channel.

[0015] The white areas of the device (reaction areas and wash channels) are hydrophilic areas, and the remaining black or gray areas of each layer are hydrophobic barriers to prevent diffusion of reagents.

[0016] The detection layer and the washing layer are folded along the axis to form a paper-based analysis device that overlaps up and down; during detection, the reaction area of ​​the detection layer is separated from the washing channel of the washing layer; during washing, the two overlap to form a fluid channel.

[0017] The detection layer and the washing layer are Whatman No.1 filter paper.

[0018] A method for preparing an origami-type microfluidic paper chip analysis device,

[0019] 1) The required chip pattern was made using Adobe Illustrator software and directly wax-printed onto Whatman No.1 filter paper using a wax inkjet printer. After printing, the wax-printed filter paper was heated in a 140°C drying oven for 60 seconds to form a hydrophobic barrier. After cutting and folding, an origami device with a reaction area and a washing channel was obtained;

[0020] 2) When the reaction zone of the detection layer was separated from the washing channel of the washing layer, chitosan (5.0 μL, 2.5×10 -4 g / mL), glutaraldehyde (5.0 μL, 2.5%), streptavidin (5.0 μL, 5×10 -5 g / mL), bovine serum albumin (5.0 μL, 0.5%), B-TDN (5.0 μL, 10 -6 M), biotin (0.1%, 5.0 μL), react at room temperature and wash with PBST washing solution.

[0021] In the above 2), when the reaction zone of the detection layer is separated from the washing channel of the washing layer, chitosan (5.0 μL, 2.5×10 -4g / mL). Glutaraldehyde (5.0 μL, 2.5%) was added after 10 min and reacted at room temperature for 2 h. During washing, the detection layer and the washing layer were folded along the axis so that the two overlapped up and down to form a fluid channel, and washed with PBST washing solution with pH = 7.4. After washing, the detection layer and the washing layer were separated, and streptavidin (5.0 μL, 5×10 -5 g / mL) was added to the reaction zone and reacted for 15 min. Subsequently, bovine serum albumin (5.0 μL, 0.5%) was introduced to block nonspecific sites and washed with PBST to remove unbound or physically adsorbed molecules. The washing steps were as above. B-TDN (5.0 μL, 10 -6 M) and incubated for 15 min. After washing with PBST buffer, 0.1% biotin (5.0 μL) was added to block the remaining non-specific sites of streptavidin in the reaction area. Finally, the obtained B-TDN functionalized microfluidic paper chip analysis device was washed with PBST buffer to remove unbound biotin and dried for later use.

[0022] An application of the device, wherein the device is used in qualitative and / or quantitative detection of adenosine triphosphate in a sample.

[0023] A method for detecting adenosine triphosphate, using the origami-type microfluidic paper chip analysis device, dripping a solution containing self-assembled capture DNA tetrahedrons (B-TDN) into a reaction zone of a detection layer, then mixing detection DNA tetrahedrons (D-TDN) with fluorescent dyes for reaction, continuously adding a sample to be detected and incubating, then dripping it into the reaction zone, and using fluorescent signals to qualitatively and / or quantitatively detect adenosine triphosphate.

[0024] The DNA tetrahedron (D-TDN) and fluorescent dye mixed reaction is to mix D-TDN and SG I evenly and react for 15 minutes to generate SG-D-TDN for standby use.

[0025] After the sample to be detected is added dropwise to the reaction zone, if the sample to be detected does not contain ATP, the D-TDN combined with SGI can specifically bind to B-TDN through base pair hybridization, thereby showing a strong fluorescence signal. If the sample to be detected contains ATP, ATP will preferentially bind to the extended single strand of D-TDN. In this case, the D-TDN combined with SGI cannot bind to B-TDN, resulting in a decrease in the fluorescence signal. Therefore, ATP analysis is achieved by detecting the fluorescence intensity of the reaction zone.

[0026] The principle is as follows: the present invention uses B-TDN as a capture probe and grafts the detection probe in a direct direction and with a clear spacing. SGI is used to couple D-TDN as a detection probe to generate an enhanced fluorescence signal. This is because the double-stranded DNA structure in D-TDN can adsorb a large amount of fluorescent molecules SGI. In the absence of ATP, the detection probe specifically binds to the capture probe and displays a strong fluorescence signal. When ATP is present, ATP will preferentially bind to the detection probe. In this case, the detection probe cannot bind to the capture probe, resulting in a decrease in the fluorescence signal.

[0027] The present invention has the following advantages:

[0028] 1) Due to its strong rigid structure, the B-DNA in the present invention can accurately regulate the orientation and distribution density of the ligand when used as a surface-immobilized substrate, thereby improving the recognition efficiency of the target. At the same time, we introduce a strong fluorescent signal indicator SGI functionalized D-TDN (SG-D-TDN) to improve the detection sensitivity.

[0029] 2) The analytical device designed in the present invention uses cellulose paper as an immobilization matrix, which effectively reduces scientific research expenses and promotes the portability of the device.

[0030] 3) The device of the present invention has the advantages of small size, no need for professional operation, biodegradability, high sensitivity and good selectivity, etc. It provides a new method for rapid detection of ATP in the medical field and is suitable for remote areas and economically backward areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A schematic diagram of a chip pattern of an origami-type microfluidic paper chip analysis device provided in an embodiment of the present invention, wherein the white area is the hydrophilic area, and the black and gray areas are the hydrophobic areas.

[0032] Figure 2 A schematic diagram of a self-assembled DNA tetrahedron provided in an embodiment of the present invention.

[0033] Figure 3 Electrophoresis diagram of self-assembled DNA tetrahedron provided by an embodiment of the present invention. Wherein, A: B-TDN, channel M: Marker, lane 1: S1, lane 2: S2, lane 3: S1+S2, lane 4: S1+S2+S3, lane 5: B-TDN (S1+S2+S3+S4); B: D-TDN, Lane M: Marker, lane 1: D1, lane 2: D2, lane 3: D1+D2, lane 4: D1+D2+D3, lane 5: D-TDN (D1+D2+D3+D4)

[0034] Figure 4 The actual object and working principle diagram of the origami-type microfluidic paper chip analysis device provided in an embodiment of the present invention.

[0035] Figure 5 The invention provides a fluorescence signal response and a standard curve for measuring different concentrations of ATP using a device. DETAILED DESCRIPTION

[0036] The following embodiments will further explain the present invention in detail with reference to the accompanying drawings, but the present invention is not limited thereto. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Example 1

[0038] like Figure 1 As shown, the origami-type microfluidic paper chip analysis device is formed by folding cellulose paper (Whatman No.1 filter paper) in half, and the two parts divided by the folding serve as two functional components, namely, a detection layer with a hydrophilic reaction area and a washing layer with a hydrophilic washing channel; wherein, after folding, the reaction area and the washing channel can be connected to realize the washing function; wherein, the detection layer of this embodiment contains a reaction area, and the reaction area is a circle with a diameter of 9 mm; and the washing layer contains a washing channel corresponding thereto.

[0039] One end of the washing channel corresponds to the reaction area of ​​the detection layer, and the other end is a liquid storage tank for storing waste liquid.

[0040] The white areas of the device (reaction areas and wash channels) are hydrophilic areas, and the remaining black or gray areas of each layer are hydrophobic barriers to prevent diffusion of reagents.

[0041] Origami-type microfluidic paper chip analysis device: during detection, the reaction area of ​​the detection layer is separated from the washing channel of the washing layer; during washing, the two overlap to form a fluid channel.

[0042] The reaction area is used as a sample addition detection area to drop a bifunctional DNA tetrahedron for detecting ATP. The bifunctional DNA tetrahedron includes a capture DNA tetrahedron (B-TDN) and a detection DNA tetrahedron (D-TDN), which are self-assembled using four different single-stranded DNAs in a suitable temperature and buffer; wherein B-TDN is composed of three single-stranded DNAs modified with biotin and one unmodified biotin; D-TDN is composed of four single-stranded DNAs unmodified with biotin; the single-stranded DNA sequence extending from the vertex of B-TDN can bind to the single-stranded DNA sequence extending from the vertex of D-TDN through complementary bases ( Figure 2 ).

[0043] The single-stranded DNAs in the capture DNA tetrahedron (B-TDN) and the detection DNA tetrahedron (D-TDN) are respectively recorded in the following table, that is, S1-S4 are interwoven to form B-TDN, and D1-D4 are interwoven to form D-TDN.

[0044]

[0045]

[0046] Among them, the four single-stranded DNAs S1-S4 were mixed in equal molar proportions, incubated in TM buffer at 95°C for 7 minutes, and then cooled to 4°C and incubated for another 25 minutes to self-assemble into a capture DNA tetrahedron (B-TDN).

[0047] The four single-stranded DNAs D1-D4 were mixed in equal molar proportions, incubated in TM buffer at 95°C for 7 min, then cooled to 4°C and incubated for a further 25 min to self-assemble into a detection DNA tetrahedron (D-TDN).

[0048] The different tetrahedrons obtained above were characterized by agarose gel electrophoresis, see Figure 3 .

[0049] Depend on Figure 3 It can be seen that due to the increase in the number of DNA chains involved in self-assembly, the movement speed of channel 5 is the slowest, indicating that we have successfully synthesized B-TDN ( Figure 3 5 in A) and D-TDN( Figure 3 (5 questions in B).

[0050] Example 2

[0051] like Figure 4 As shown, the preparation method of the origami-type microfluidic paper chip analysis device for detecting ATP based on bifunctional DNA tetrahedron is specifically as follows:

[0052] 1) Use Adobe Illustrator drawing software to make the required rectangular chip pattern, including the detection layer and washing layer patterns, and wax print them directly onto Whatman No.1 filter paper using a wax inkjet printer. After printing, heat the wax-printed filter paper in a 140°C drying oven for 60 seconds to form a hydrophobic barrier. Cut the paper into rectangles (85mm×40mm), then fold the prepared rectangular paper in half, that is, form two folded parts as the detection layer with a hydrophilic reaction area and the washing layer with a hydrophilic washing channel, and the size of the two layers is 40mm×40mm respectively;

[0053] The reaction area contains a circle with a diameter of 9 mm, and the washing layer contains a corresponding washing channel; one end of the washing channel corresponds to the reaction area of ​​the detection layer, and the other end is a reservoir for storing waste liquid. The washing channel is used in the washing process. The white area of ​​the device (reaction area and washing channel) is a hydrophilic area, and the remaining black or gray area of ​​each layer is a hydrophobic barrier to prevent the diffusion of reagents.

[0054] 2) When the reaction zone of the detection layer was separated from the washing channel of the washing layer, chitosan (5.0 μL, 2.5×10 -4 g / mL). Glutaraldehyde (5.0 μL, 2.5%) was added after 10 min and reacted at room temperature for 2 h. During washing, the detection layer and the washing layer were folded along the axis so that the two overlapped up and down to form a fluid channel, and washed with PBST washing solution with pH = 7.4.

[0055] After washing, the detection layer and the washing layer were separated, and streptavidin (5.0 μL, 5 × 10 -5 g / mL) was added to the reaction zone and reacted for 15 min. Subsequently, bovine serum albumin (5.0 μL, 0.5%) was introduced to block nonspecific sites and washed with PBST to remove unbound or physically adsorbed molecules. The washing steps were as above. B-TDN (5.0 μL, 10 -6 M) and incubated for 15 min. After washing with PBST buffer, 0.1% biotin (5.0 μL) was added to block the remaining non-specific sites of streptavidin in the reaction area. Finally, the obtained B-TDN functionalized microfluidic paper chip analysis device was washed with PBST buffer to remove unbound biotin and dried for later use.

[0056] Then, in the detection layer reaction area, the detection DNA tetrahedron D-TDN and SG I were mixed evenly and reacted for 15 minutes to generate SG-D-TDN (wherein the final concentration of D-TDN was 10 -6 M, the final concentration of SGI is 0.6 mM) and then used for detection.

[0057] Example 3

[0058] The qualitative and / or quantitative detection of ATP using the origami-type microfluidic paper chip analysis device obtained in Examples 1 and 2 is performed as follows:

[0059] Using the origami-type microfluidic paper chip analysis device obtained in Examples 1 and 2, that is, before assembling the surface of the B-TDN functionalized paper chip device, D-TDN and SG I were mixed evenly and reacted for 15 min to generate SG-D-TDN (wherein the final concentration of D-TDN was 10 -6 Then, different concentrations of ATP (0, 10-12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 and 10 -7 M) was mixed with SG-D-TDN and incubated for 10 min. Finally, 5.0 μL of the mixture (the mixture was ATP and SG-D-TDN with a volume ratio of 4:1) was added to the reaction area of ​​the detection layer and reacted at room temperature for 15 min. After washing, the chip device was placed on the stage of the fluorescence spectrophotometer for signal detection. Figure 5 It can be seen that within a certain concentration range, the fluorescence signal decreases with the increase of ATP concentration, and at 10 -12 ~10 -7 Within the range of M, the change in fluorescence signal intensity (ΔF = F0–F) was linearly correlated with the ATP concentration.

[0060] Example 4

[0061] The origami-type microfluidic paper chip analysis device obtained in Examples 1 and 2 is used to qualitatively and / or quantitatively detect ATP in human serum samples, and the steps are as follows:

[0062] Using the origami-type microfluidic paper chip analysis device obtained in Examples 1 and 2, that is, before assembling the surface of the B-TDN functionalized paper chip device, D-TDN and SG I were mixed evenly and reacted for 15 min to generate SG-D-TDN (wherein the final concentration of D-TDN was 10 -6 M, the final concentration of SGI was 0.6 mM). Subsequently, different concentrations of ATP (10 -12 , 10 -11 , 10 -10 M) was mixed with SG-D-TDN and incubated for 10 minutes (the volume ratio of SG-D-TDN to ATP diluted in human serum was 1:4). Finally, 5.0 μL of the mixture was added to the reaction area of ​​the detection layer and reacted at room temperature for 15 minutes. After washing, the chip device was placed on the stage of the fluorescence spectrophotometer for signal detection. As shown in Table 1, the spike recovery rate of ATP in human serum samples was in the range of 91.2% to 109.6%, and the RSD did not exceed 5%. This shows that the analysis results of the designed origami device are reliable, and there is broad room for development in detecting the ATP content in human serum samples.

[0063]

Claims

1. An origami-type microfluidic paper chip analysis device for detecting adenosine triphosphate, the analysis device comprising a detection layer and a washing layer, characterized in that: A bifunctional DNA tetrahedron system containing detection ATP is added into the reaction zone; wherein the bifunctional DNA tetrahedron includes a capture DNA tetrahedron (B-TDN) and a detection DNA tetrahedron (D-TDN), and a single-stranded DNA sequence extending from any vertex of the tetrahedron formed by the B-TDN can be complementary to a single-stranded DNA sequence extending from any vertex of the tetrahedron formed by the D-TDN.

2. The origami-type microfluidic paper chip analysis device for detecting adenosine triphosphate according to claim 1, characterized in that: The capture DNA tetrahedron (B-TDN) and the detection DNA tetrahedron (D-TDN) are respectively self-assembled using four different single-stranded DNAs; wherein, the B-TDN is composed of three single-stranded DNAs modified with biotin and one unmodified biotin intertwined to form a tetrahedron, and the single-stranded DNA unmodified with biotin in the regular tetrahedron extends an arm chain from any vertex of the tetrahedron; the D-TDN is composed of four single-stranded DNAs unmodified with biotin intertwined to form a tetrahedron, and an arm chain extends from any vertex of the tetrahedron.

3. The origami-type microfluidic paper chip analysis device for detecting adenosine triphosphate according to claim 2, characterized in that: The single-stranded DNA in the capture DNA tetrahedron (B-TDN) and the detection DNA tetrahedron (D-TDN) were mixed in equal moles, incubated in TM buffer at 95° C. for 7 minutes, then cooled to 4° C. and incubated for another 25 minutes to self-assemble into corresponding DNA tetrahedrons.

4. The origami-type microfluidic paper chip analysis device for detecting adenosine triphosphate according to claim 3, characterized in that: The bifunctional DNA tetrahedron system containing ATP detection is prepared by adding the assembled B-TDN to the reaction zone, and then adding a mixture of the assembled detection DNA tetrahedron (D-TDN), a fluorescent dye and a sample to be detected therein; wherein the volume ratio of (B-TDN) to (fluorescently modified D-TDN) is 5:

1.

5. The origami-type microfluidic paper chip analysis device for detecting adenosine triphosphate according to any one of claims 1 to 4, characterized in that: The single-stranded DNA in the assembled B-TDN are sequences S1-S4, and the single-stranded DNA in the assembled D-TDN are sequences D1-D4; 6. The origami-type microfluidic paper chip analysis device for detecting adenosine triphosphate according to claim 1, characterized in that: The origami-type microfluidic paper chip analysis device is an origami-type microfluidic paper chip that is formed by folding a cellulose material in half to form a detection layer having a hydrophilic reaction zone and a washing layer having a hydrophilic washing channel; wherein the detection layer contains a reaction zone, and the washing layer contains a corresponding washing channel, wherein the reaction zone and the washing channel are hydrophilic zones, and the remaining portion is a hydrophobic barrier zone for preventing diffusion of reagents.

7. An application of the device according to claim 1, characterized in that: The device is used in qualitative and / or quantitative detection of adenosine triphosphate in a sample.

8. A method for detecting adenosine triphosphate, characterized in that: Using the origami-type microfluidic paper chip analysis device described in claim 1, a solution containing self-assembled capture DNA tetrahedrons (B-TDN) is dripped into the reaction area of ​​the detection layer, and then the detection DNA tetrahedrons (D-TDN) and the fluorescent dye are mixed and reacted, and the sample to be tested is continuously added and incubated and then dripped into the reaction area, and adenosine triphosphate is qualitatively and / or quantitatively detected using the fluorescent signal.

9. The method for detecting adenosine triphosphate according to claim 8, characterized in that: After the sample to be detected is added dropwise to the reaction zone, if the sample to be detected contains ATP, it can bind to the extended single-stranded DNA in D-TDN, so that D-TDN and B-TDN cannot bind, thereby reducing the fluorescence signal; if the sample to be detected does not contain ATP, D-TDN combined with SGI specifically binds to B-TDN, thereby showing a strong fluorescence signal.

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