A paper-based microfluidic chip analysis device for detecting adenosine triphosphate and application thereof

Abstract

The present application relates to the field of microfluidic chip technology and biosensor technology, in particular to a paper-based microfluidic chip analysis device for detecting adenosine triphosphate (ATP) based on bifunctional DNA tetrahedron and application. The analysis device comprises a detection layer and a washing layer, characterized in that: a bifunctional DNA tetrahedron system containing ATP detection is added in the reaction area; wherein the bifunctional DNA tetrahedron comprises a capture DNA tetrahedron (B-TDN) and a detection DNA tetrahedron (D-TDN), and the single-stranded DNA sequence extending from any one vertex of the tetrahedron formed by the B-TDN can be complementary to the single-stranded DNA sequence extending from any one vertex of the tetrahedron formed by the D-TDN. The paper-based microfluidic chip analysis device based on bifunctional DNA tetrahedron provided by the present application has the advantages of small volume, low research cost, biodegradability, high sensitivity and good selectivity, and provides a new method for rapid detection of ATP in the medical field, and is suitable for remote and economically backward areas.

Description

Technical Field

[0001] This invention relates to the fields of microfluidic chip technology and biosensor technology, specifically to an origami-type microfluidic paper chip analysis device and its application based on the detection of adenosine triphosphate (ATP) using a bifunctional DNA tetrahedron. Background Technology

[0002] Adenosine triphosphate (ATP) is an essential high-energy compound in living organisms and the main energy carrier in cells, playing a crucial role in signal transduction and cellular metabolism. Studies have shown that abnormal ATP levels can cause various diseases, such as hypoglycemia, Parkinson's disease, malignant tumors, cardiovascular diseases, and Alzheimer's disease. To date, common methods for ATP analysis include high-performance liquid chromatography (HPLC), capillary electrophoresis, chemiluminescence immunoassay, and mass spectrometry. While these techniques can effectively detect ATP, they still face obstacles such as time-consuming sample preparation, high reagent consumption, and expensive equipment. Therefore, developing a rapid and economical ATP detection technique is essential.

[0003] Cellulose paper-based microfluidic analytical devices have become a widely used analytical sensing platform in recent years due to their advantages such as portability, low cost, ease of preparation, and small sample volume requirements. However, traditional microfluidic paper-based analytical devices can only provide qualitative measurements. In quantitative analysis, paper-based analytical devices have been integrated with various analytical methods, such as colorimetry, electrochemistry, surface-enhanced Raman spectroscopy, and fluorescence. Among them, fluorescence analysis platforms based on microfluidic paper chips have attracted much attention due to their simple operation and fast detection speed. However, there is still room for improvement in their sensitivity. Based on this, we introduced a bifunctional DNA tetrahedron. One tetrahedron serves as a capture substrate to improve target recognition efficiency, while the other tetrahedron combines with a fluorescent dye to act as a strong fluorescent indicator. Coupling this with a microfluidic paper chip can significantly improve the sensitivity of ATP detection. Summary of the Invention

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

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

[0006] A paper-based microfluidic paper chip analyzer for detecting adenosine triphosphate (ATP) is disclosed. The analyzer includes a detection layer and a washing layer. The feature is that a bifunctional DNA tetrahedron system containing ATP for detection is added to the reaction zone. The bifunctional DNA tetrahedron includes a capture DNA tetrahedron (B-TDN) and a detection DNA tetrahedron (D-TDN). The single-stranded DNA sequence extending from any vertex of the B-TDN tetrahedron is complementary to the single-stranded DNA sequence extending from any vertex of the D-TDN tetrahedron. The single-stranded DNA extending from any vertex of the D-TDN tetrahedron can bind to the ATP to be detected.

[0007] The captured DNA tetrahedron (B-TDN) and the detected DNA tetrahedron (D-TDN) are each assembled from four different single-stranded DNA molecules. The B-TDN is formed by the interweaving of three biotin-modified and one unmodified single-stranded DNA molecules, with an arm extending from any vertex of the unmodified single-stranded DNA molecule. The D-TDN is formed by the interweaving of four unmodified single-stranded DNA molecules, with an arm extending from any vertex of the tetrahedron.

[0008] The single-stranded DNA in the captured DNA tetrahedron (B-TDN) and the detected DNA tetrahedron (D-TDN) are mixed in equal molar amounts, and then incubated in TM buffer at 95°C for 7 min, followed by cooling to 4°C and incubation for another 25 min to self-assemble into the corresponding DNA tetrahedron.

[0009] The bifunctional DNA tetrahedral system containing ATP detection involves adding the assembled B-TDN to the reaction region, followed by adding a mixture of the assembled detection DNA tetrahedral (D-TDN), fluorescent dye, and the sample to be tested; wherein the volume ratio of (B-TDN) to (fluorescently modified D-TDN) is 5:1.

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

[0011] The single-stranded DNA in the assembled B-TDN consists of sequences S1-S4, and the single-stranded DNA in the assembled D-TDN consists of sequences D1-D4.

[0012]

[0013] The origami-type microfluidic paper chip analysis device is an origami-type microfluidic paper chip formed by folding cellulose material to create a detection layer with a hydrophilic reaction zone and a washing layer with a hydrophilic washing channel. The detection layer contains a reaction zone, and the washing layer contains a corresponding washing channel. The reaction zone and the washing channel are hydrophilic regions, and the remaining part is a hydrophobic barrier region to prevent reagent diffusion.

[0014] The aforementioned origami-type microfluidic paper chip analyzer 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 connects the reaction zone and the washing channel to achieve the washing function. The detection layer contains a reaction zone with a diameter of 9 mm, and the washing layer contains a corresponding washing channel.

[0015] The white areas (reaction zone and washing channel) of the device are hydrophilic, while the remaining black or gray areas in each layer are hydrophobic barriers to prevent reagent diffusion.

[0016] The detection layer and the washing layer are folded along the axis to form a paper-based analytical device with overlapping layers; during detection, the reaction zone 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 washing layer are Whatman No.1 filter paper.

[0018] Method for fabricating a paper-folding type microfluidic paper chip analysis device

[0019] 1) The required chip pattern was created 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℃ drying oven for 60 seconds to form a hydrophobic barrier. After cutting and folding, a paper-folding device with a reaction zone and washing channels was obtained.

[0020] 2) When the detection layer reaction zone separates from the washing layer washing channel, chitosan (5.0 μL, 2.5 × 10⁻⁶) is sequentially introduced onto the detection layer reaction zone. -4 Glutaraldehyde (5.0 μL, 2.5%), Streptavidin (5.0 μL, 5 × 10 g / mL), glutar ... Streptavidin (5.0 μL, 5 -5 g / mL), bovine serum albumin (5.0 μL, 0.5%), B-TDN (5.0 μL, 10 g / mL), -6 M), biotin (0.1%, 5.0 μL), reacted at room temperature and washed with PBST washing solution.

[0021] In step 2), when the detection layer reaction zone separates from the washing layer washing channel, chitosan (5.0 μL, 2.5 × 10⁻⁶) is introduced into the detection layer reaction zone. -4After 10 min, add glutaraldehyde (5.0 μL, 2.5%) and react at room temperature for 2 h. During washing, fold the detection layer and wash layer along the axis so that they overlap to form a fluid channel, and wash with PBST washing solution at pH 7.4. After washing, separate the detection layer and wash layer, dry them, and then add streptavidin (5.0 μL, 5 × 10 g / mL). -5 Add g / mL) to the reaction zone and react for 15 min. Then, introduce bovine serum albumin (5.0 μL, 0.5%) to block nonspecific sites, and wash with PBST to remove unbound or physically adsorbed molecules, following the washing steps above. Add B-TDN (5.0 μL, 10 g / mL) to the reaction zone. -6 Incubate for 15 min (M). After washing with PBST buffer, add 0.1% biotin (5.0 μL) to block the remaining non-specific sites of streptavidin in the reaction zone. Finally, wash the obtained B-TDN-functionalized microfluidic paper chip analyzer with PBST buffer to remove unbound biotin, and dry it for later use.

[0022] An application of the device described herein, specifically its use in the qualitative and / or quantitative detection of adenosine triphosphate (ATP) in a sample.

[0023] A method for detecting adenosine triphosphate (ATP) involves using the origami-type microfluidic paper chip analysis device to add a solution containing self-assembled capture DNA tetrahedrons (B-TDN) to the reaction area of ​​the detection layer, then mixing and reacting the detection DNA tetrahedrons (D-TDN) with a fluorescent dye, adding the sample to be tested for incubation, and then adding it to the reaction area. ATP is then qualitatively and / or quantitatively detected using the fluorescence signal.

[0024] The DNA tetrahedron (D-TDN) and fluorescent dye mixing reaction is carried out by mixing D-TDN with SG I and reacting for 15 min to generate SG-D-TDN, which is then ready for use.

[0025] After the sample to be tested is added to the reaction zone, if the sample does not contain ATP, the D-TDN bound with SG I can specifically bind to B-TDN through base pair hybridization, thus exhibiting a strong fluorescent signal. If the sample contains ATP, ATP will preferentially bind to the extended single strand of D-TDN. In this case, the D-TDN bound with SG I cannot bind to B-TDN, resulting in a decreased fluorescent signal. Therefore, ATP analysis is achieved by detecting the fluorescence intensity in the reaction zone.

[0026] The principle is as follows: This invention uses B-TDN as a capture probe and grafts a detection probe with a direct direction and defined spacing. SGI-coupled D-TDN is used as the detection probe, generating an enhanced fluorescence signal. This is because the double-stranded DNA structure in D-TDN can adsorb a large amount of the fluorescent molecule SGI. In the absence of ATP, the detection probe specifically binds to the capture probe, exhibiting a strong fluorescence signal. However, when ATP is present, it preferentially binds to the detection probe. In this case, the detection probe cannot bind to the capture probe, resulting in a decreased fluorescence signal.

[0027] The present invention has the following advantages:

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

[0029] 2) The analytical device designed in this invention uses cellulose paper as the immobilization substrate, which effectively reduces research expenditures and promotes the portability of the device.

[0030] 3) The device of the present invention has advantages such as small size, no need for professional personnel to operate, biodegradability, high sensitivity and good selectivity, providing a new method for rapid detection of ATP in the medical field, and is suitable for remote and economically underdeveloped areas. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the chip pattern of the 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 This is a schematic diagram of a self-assembled DNA tetrahedron provided in an embodiment of the present invention.

[0033] Figure 3 This is a tetrahedral electrophoresis diagram of self-assembled DNA provided in 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 diagram shows the physical object and working principle of the origami-type microfluidic paper chip analysis device provided in the embodiment of the present invention.

[0035] Figure 5 The fluorescence signal response and standard curves of ATP at different concentrations are obtained using the device provided in the embodiments of the present invention. Detailed Implementation

[0036] The following embodiments will further illustrate the present invention with reference to the accompanying drawings, but are not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort 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. The two parts formed by the fold serve as two functional components: a detection layer with a hydrophilic reaction zone and a washing layer with a hydrophilic washing channel. After folding, the reaction zone and the washing channel can be connected to realize the washing function. In this embodiment, the detection layer contains a reaction zone, which is a circle with a diameter of 9 mm. The washing layer contains a corresponding washing channel.

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

[0040] The white areas (reaction zone and washing channel) of the device are hydrophilic, while the remaining black or gray areas in each layer are hydrophobic barriers to prevent reagent diffusion.

[0041] Origami-type microfluidic paper chip analysis device: During detection, the reaction zone of the detection layer and the washing channel of the washing layer are separated; during washing, the two overlap to form a fluid channel.

[0042] The reaction zone, serving as the sample loading and detection zone, allows for the addition of bifunctional DNA tetrahedra for ATP detection. These bifunctional DNA tetrahedra comprise a capture DNA tetrahedron (B-TDN) and a detection DNA tetrahedron (D-TDN), each self-assembled from four different single-stranded DNA strands at suitable temperatures and buffer solutions. The B-TDN consists of three biotin-modified single-stranded DNA strands and one unmodified biotin-modified single-stranded DNA strand; the D-TDN consists of four unmodified biotin-modified single-stranded DNA strands. The single-stranded DNA sequence extending from the apex of the B-TDN can bind to the single-stranded DNA sequence extending from the apex of the D-TDN via complementary bases. Figure 2 ).

[0043] The single-stranded DNA in the captured DNA tetrahedron (B-TDN) and the detected DNA tetrahedron (D-TDN) are respectively recorded in the table below, that is, S1-S4 interweave to form B-TDN, and D1-D4 interweave to form D-TDN.

[0044]

[0045]

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

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

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

[0049] Depend on Figure 3 As can be seen, due to the increased number of DNA strands involved in self-assembly, channel 5 exhibits the slowest movement speed, indicating that we have successfully synthesized B-TDN. Figure 3 (5 questions 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 based on the detection of ATP using bifunctional DNA tetrahedrons is as follows:

[0052] 1) The required rectangular chip pattern, including the detection layer and the washing layer, was created using Adobe Illustrator drawing software. This pattern was then 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℃ drying oven for 60 seconds to form a hydrophobic barrier. The paper was cut into rectangles (85mm × 40mm), and then folded in half to form two separate sections, serving as the detection layer with a hydrophilic reaction zone and the washing layer with hydrophilic washing channels, respectively. Each layer measures 40mm × 40mm.

[0053] The reaction zone contains a 9mm diameter circle, and the washing layer contains a corresponding washing channel. One end of the washing channel corresponds to the reaction zone of the detection layer, and the other end is a reservoir for storing waste liquid. The washing channel is used for the washing process. The white areas of the device (reaction zone and washing channel) are hydrophilic areas, and the remaining black or gray areas in each layer serve as hydrophobic barriers to prevent reagent diffusion.

[0054] 2) When the detection layer reaction zone separates from the washing layer washing channel, chitosan (5.0 μL, 2.5 × 10⁻⁶) is introduced into the detection layer reaction zone. -4 After 10 min, add glutaraldehyde (5.0 μL, 2.5%) and react at room temperature for 2 h. During washing, fold the detection layer and washing layer along the axis so that they overlap to form a fluid channel, and wash with PBST washing solution at pH 7.4.

[0055] After washing, the detection layer and washing layer were separated, and after drying, streptavidin (5.0 μL, 5 × 10⁻⁶) was added. -5 Add g / mL) to the reaction zone and react for 15 min. Then, introduce bovine serum albumin (5.0 μL, 0.5%) to block nonspecific sites, and wash with PBST to remove unbound or physically adsorbed molecules, following the washing steps above. Add B-TDN (5.0 μL, 10 g / mL) to the reaction zone. -6 Incubate for 15 min (M). After washing with PBST buffer, add 0.1% biotin (5.0 μL) to block the remaining non-specific sites of streptavidin in the reaction zone. Finally, wash the obtained B-TDN-functionalized microfluidic paper chip analyzer with PBST buffer to remove unbound biotin, and dry it for later use.

[0056] Then, during detection, the tetrahedral D-TDN of the detection DNA is mixed evenly with SG I in the reaction area of ​​the detection layer and reacted for 15 minutes to generate SG-D-TDN (where the final concentration of D-TDN is 10). -6 The final concentration of M, SG I is 0.6 mM, which is 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, i.e., before assembling on 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 (where the final concentration of D-TDN was 10). -6 The final concentration of ATP, SG I was 0.6 mM. Subsequently, different concentrations of ATP (0, 10) were used...-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 (ATP and SG-D-TDN in a volume ratio of 4:1) was added to the reaction zone of the detection layer, and the mixture was reacted at room temperature for 15 min. After washing, the chip device was placed on the stage of a fluorescence spectrophotometer for signal detection. Figure 5 It can be seen that, within a certain concentration range, the fluorescence signal decreases with increasing ATP concentration, and this decreases further within a certain range (around 10 ATP concentration). -12 ~10 -7 Within the M range, the change in fluorescence signal intensity (ΔF = F0 – F) is linearly correlated with ATP concentration.

[0060] Example 4

[0061] The qualitative and / or quantitative detection of ATP in human serum samples was performed using the origami-type microfluidic paper chip analyzer obtained in Examples 1 and 2, as follows:

[0062] Using the origami-type microfluidic paper chip analysis device obtained in Examples 1 and 2, i.e., before assembling on 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 (where the final concentration of D-TDN was 10). -6 The final concentration of SG I was 0.6 mM. Subsequently, different concentrations of ATP (10 mM) diluted with human serum were used. -12 10 -11 10 -10 M) was mixed with SG-D-TDN and incubated for 10 min (the volume ratio of SG-D-TDN to diluted ATP in human serum was 1:4). Finally, 5.0 μL of the mixture was added to the reaction zone of the detection layer and reacted at room temperature for 15 min. After washing, the chip device was placed on the stage of a fluorescence spectrophotometer for signal detection. As shown in Table 1, the spiked recovery rate of ATP in human serum samples ranged from 91.2% to 109.6%, with an RSD of no more than 5%. This indicates that the designed origami device provides reliable analytical results and has broad development potential in detecting ATP content in human serum samples.

[0063]

Claims

1. A paper-folding type microfluidic paper chip analysis device for detecting adenosine triphosphate (ATP), the analysis device comprising a detection layer and a washing layer, characterized in that: The reaction region is functionalized with the captured DNA tetrahedron B-TDN, and the single-stranded DNA sequence extending from any vertex of the tetrahedron formed by the B-TDN is complementary to the single-stranded DNA sequence extending from any vertex of the tetrahedron formed by the detection DNA tetrahedron D-TDN; ATP can bind to the extended single-stranded DNA in the D-TDN, and once ATP binds to the extended single-stranded DNA in the D-TDN, the D-TDN cannot bind to the B-TDN.

2. The origami-type microfluidic paper chip analysis device for detecting adenosine triphosphate as described in claim 1, characterized in that: The capture DNA tetrahedron B-TDN and the detection DNA tetrahedron D-TDN are each assembled from four different single-stranded DNA strands. The B-TDN is formed by the interweaving of three biotin-modified and one unmodified single-stranded DNA strands to form a tetrahedron, and an arm-like chain extends from any vertex of the unmodified single-stranded DNA strand. The D-TDN is formed by the interweaving of four unmodified single-stranded DNA strands to form a tetrahedron, and an arm-like 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 are mixed in equal molar amounts, and then incubated in TM buffer at 95°C for 7 min. After cooling to 4°C, the mixture is incubated for another 25 min to self-assemble into the corresponding DNA tetrahedron.

4. The origami-type microfluidic paper chip analysis device for detecting adenosine triphosphate according to claim 3, characterized in that: When using this product, the mixture of the assembled DNA tetrahedron D-TDN, fluorescent dye, and the sample to be tested is added to the reaction zone; 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 1-4, characterized in that: The single-stranded DNA that assembles to form B-TDN consists of the S1-S4 sequences, and the single-stranded DNA that assembles to form D-TDN consists of the D1-D4 sequences. 。 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 formed by folding cellulose material to create a detection layer with a hydrophilic reaction zone and a washing layer with a hydrophilic washing channel. The detection layer contains a reaction zone, and the washing layer contains a corresponding washing channel. The reaction zone and the washing channel are hydrophilic regions, and the remaining part is a hydrophobic barrier region to prevent reagent diffusion.

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

8. A method for detecting adenosine triphosphate (ATP), characterized in that: Using the origami-type microfluidic paper chip analysis device of claim 1, the detection of DNA tetrahedral D-TDN and fluorescent dye are mixed and reacted, and the sample to be tested is added and incubated before being dropped into the reaction area. Adenosine triphosphate is qualitatively and / or quantitatively detected using the fluorescence signal.

9. The method for detecting adenosine triphosphate according to claim 8, characterized in that: In the reaction zone, when the sample to be tested contains ATP, it can bind to the extended single-stranded DNA in D-TDN, preventing D-TDN from binding to B-TDN and thus reducing the fluorescence signal; when the sample to be tested does not contain ATP, D-TDN bound with SG I specifically binds to B-TDN, thus showing a strong fluorescence signal.

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