A paper chip biosensor and a preparation method thereof

Abstract

The application belongs to the field of biosensors, and particularly relates to a paper chip biosensor and a preparation method thereof. The paper chip biosensor comprises a capillary microchannel and a biological sensitive unit, the material of the capillary microchannel is selected from paper; the readout signal of the paper chip biosensor is the process of capillary flow; the biological sensitive unit is arranged on one side of the capillary microchannel and used for regulating the behavior of capillary flow. The paper chip biosensor provided by the application uses intelligent molecules as a valve to control the capillary flow process, uses the capillary microchannel as a signal display, and realizes visual quantitative detection of a target. The paper chip sensor not only has the characteristics of simple operation, rapidness, accuracy, directness, strong specificity, but also has low cost and is convenient to carry, and has a wide prospect in the application fields of home diagnosis and treatment, bedside diagnosis and instant detection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biosensors, and particularly relates to a paper chip biosensor and a preparation method thereof. BACKGROUND

[0002] For the detection of lead ions, Li and Lu first discovered in 2000 that 8-17 DNAzyme has high activity to Pb 2+ , but the specificity of 8-17 DNAzyme to metal ions is poor [1] . In 2010, Lu et al. found that the selectivity of GR-5 DNAzyme screened in vitro by Breaker and Joyce to Pb 2+ is 40,000 times higher than that of 8-17 DNAzyme [2] . GR-5 DNAzyme is composed of a Pb 2+ binding enzyme chain and a substrate chain embedded with ribonucleotide "rA". Mao et al. [3] developed a visual method based on GR-5 DNAzyme hydrogel, and encapsulated glucoamylase as a sensor in the hydrogel for signal output for Pb 2+ detection. Jiang et al. [4] designed a portable capillary sensor based on GR-5 DNAzyme for detecting Pb 2+ . However, the price of DNA hydrogel is relatively high, so reducing the amount of hydrogel and thus reducing the cost of manufacturing biosensors is a challenge and problem in this field.

[0003] Compared with capillary glass tube sensors, paper materials have many unique advantages, such as not easy to break, convenient to carry, low cost, suitable for mass production, etc. Therefore, it is urgent to develop a sensitive paper chip sensor based on a small amount of DNA hydrogel, which can solve the problem of easy breakage of capillary glass tube sensors and the problem of DNA hydrogel usage. SUMMARY

[0004] In view of the above technical problems, the present application provides a paper chip biosensor and a preparation method thereof, which uses DNA hydrogel as a valve and a microchannel of the paper chip as a signal display to realize visual and quantitative detection of lead ions.

[0005] The present application is achieved by the following technical solutions:

[0006] A paper chip biosensor, which comprises a capillary microchannel and a biological sensitive unit, and the material of the capillary microchannel is paper;

[0007] The readout signal of the paper chip biosensor is a process of capillary flow; the biosensitive unit is arranged on one side of the capillary microchannel to regulate the behavior of capillary flow.

[0008] Further, the readout signal of the paper chip biosensor is the flow distance of the to-be-tested solution in the capillary microchannel within a certain time, or the time required for the to-be-tested solution to flow a certain distance in the capillary microchannel within a certain time.

[0009] Further, the paper chip biosensor comprises hydrophilic micropores and microchannels arranged on the paper chip, and other regions of the paper chip except the micropores and the microchannels are hydrophobic regions.

[0010] The DNA hydrogel valve of the paper chip is formed by in-situ forming a DNA hydrogel on the micropore or adding a finished DNA hydrogel on the micropore; the DNA hydrogel is cleaved under the stimulation of the corresponding target; the signal readout of the paper chip biosensor is the flow behavior of the solution in the hydrophilic microchannel;

[0011] The hydrogel valve is the biosensitive unit of the paper chip biosensor, and the biosensitive unit comprises aptamers, biological proteins, molecularly imprinted materials and hydrogels.

[0012] Further, the structure of the paper chip is as follows: the micropore is arranged at one end of the paper chip, the micropore and the microchannel are on the same straight line, and a folding line is arranged between the micropore and the microchannel; when the folding line is folded, the micropore falls on one end of the microchannel close to the micropore; the diameter of the micropore ranges from 1.6 mm to 2.5 mm, and the width of the microchannel ranges from 0.8 mm to 1.6 mm.

[0013] Further, when the target is a lead ion, the DNA hydrogel is formed in-situ on the micropore, and the specific method is as follows:

[0014] A DNA-modified polyacrylamide chain mixed solution is prepared: a certain amount of DNA-modified polyacrylamide chain P-SA and DNA-modified polyacrylamide chain P-SB are added to a Tris buffer solution to obtain a DNA-modified polyacrylamide chain mixed solution; in the prepared DNA-modified polyacrylamide chain mixed solution, the concentration of the DNA-modified polyacrylamide chain P-SA is 100-350 μM, and the concentration of the DNA-modified polyacrylamide chain P-SB is 100-350 μM.

[0015] Preparation of a mixed solution of the enzyme strand GR-5 DNAzyme and the substrate strand: the enzyme strand GR-5 DNAzyme and the substrate strand are mixed in a certain ratio to obtain a GR-5 DNAzyme / Substrate mixed solution; in the GR-5 DNAzyme / Substrate mixed solution, the concentration of the enzyme strand GR-5 DNAzyme is 100-350 μM, and the concentration of the substrate strand is 100-350 μM; the base sequence of the GR-5 DNAzyme is shown as SEQ ID NO: 1 in the sequence listing; the base sequence of the substrate strand is shown as SEQ ID NO: 2 in the sequence listing;

[0016] In-situ formation of a hydrogel valve at the microwell: 0.2-1.5 μL of the DNA-modified polyacrylamide chain mixed solution is dropped at the microwell, and after the solution is completely infiltrated into the paper, 0.2-1.5 μL of the GR-5 DNAzyme / Substrate mixed solution is dropped, and after the solution is completely infiltrated into the paper, a DNA hydrogel is formed, i.e., a hydrogel valve in the paper chip is formed;

[0017] Further, when the target is lead ions, the preparation method of the finished DNA hydrogel is as follows:

[0018] In a Tris buffer solution, DNA-modified polyacrylamide chain P-SA, DNA-modified polyacrylamide chain P-SB, enzyme strand GR-5 DNAzyme, and substrate strand Substrate strand are added to obtain a mixed solution; in the mixed solution, the concentration of the DNA-modified polyacrylamide chain P-SA is 100-350 μM, the concentration of the DNA-modified polyacrylamide chain P-SB is 100-350 μM; the concentration of the enzyme strand GR-5 DNAzyme is 100-350 μM, and the concentration of the substrate strand Substrate strand is 100-350 μM; the base sequence of the GR-5 DNAzyme is shown as SEQ ID NO: 1 in the sequence listing; the base sequence of the substrate strand Substrate strand is shown as SEQ ID NO: 2 in the sequence listing;

[0019] In order to ensure that the DNA strands are fully mixed, the mixed solution is heated at 55°C for 1 min, and then cooled to room temperature, and the process is repeated several times, and finally the DNA hydrogel with a three-dimensional network structure is stored at 4°C;

[0020] When detecting, the amount of the DNA hydrogel dropped on the microwell is 1-10 μL.

[0021] Further, the preparation method of the DNA modified polyacrylamide chain P-SA and the DNA modified polyacrylamide chain P-SB is as follows:

[0022] The acrylamide modified DNA chain SA and the DNA chain SB are added into the acrylamide monomer solution with a mass percentage concentration of 4% respectively, and the concentration of the DNA chain SA and the DNA chain SB in the obtained solution is 500 μM. The mixed solution is dried in a vacuum dryer at room temperature for a certain time to remove oxygen, and then fresh prepared initiator ammonium persulfate APS and accelerator TEMED aqueous solution are added. The mass percentage concentration of the ammonium persulfate APS and the accelerator TEMED in the SA polymerization solution and the SB polymerization solution is 0.14% respectively. The base sequence of the DNA chain SA is shown in SEQ ID NO: 3 in the sequence listing, and the base sequence of the DNA chain SB is shown in SEQ ID NO: 4 in the sequence listing.

[0023] In order to make the polymerization reaction complete, the SA polymerization solution and the SB polymerization solution are placed in a vacuum dryer for a certain time to produce the DNA modified polyacrylamide chain P-SA and the DNA modified polyacrylamide chain P-SB.

[0024] The DNA modified polyacrylamide chain P-SA and the DNA modified polyacrylamide chain P-SB are filtered and washed with ultrapure water and an ultrafiltration centrifugal tube to remove monomers and polymers with small molecular weight on the polymer, and are ready for use.

[0025] Further, the paper chip biosensor is used to detect Pb 2+ During detection, the paper chip biosensor is folded along the folding line, and one side of the microwell is placed at the head of the microchannel. The solution to be tested containing lead ions is dropped on the other side of the microwell. After the lead ions react with the hydrogel in the hydrogel valve, the size of the hydrogel mesh is increased, the solution to be tested passes through the valve, penetrates into the microchannel under the microwell, and forms a band on the microchannel.

[0026] The length of the band formed in each experiment is recorded by a video recorder at a certain time after the lead ion solution is dropped, and the concentration of the lead ions is quantitatively analyzed.

[0027] A preparation method of a paper chip biosensor is used to prepare the paper chip biosensor, and the method comprises the following steps:

[0028] Preparation of a paper chip: a hydrophobic region and a hydrophilic region are formed on the paper by wax printing. The hydrophilic region includes a microwell and a microchannel. Except for the microwell and the microchannel, the remaining region of the paper is a hydrophobic region. The microwell is the position of the hydrogel valve.

[0029] Preparation of hydrogel valve in paper chip: forming hydrogel valve of paper chip by forming DNA hydrogel in-situ on the micropore, or dropping finished DNA hydrogel on the micropore;

[0030] Obtaining paper chip sensor.

[0031] Further, when the target is lead ion:

[0032] Forming DNA hydrogel in-situ on the micropore, specifically:

[0033] Preparation of DNA modified polyacrylamide chain mixed solution: adding a certain amount of DNA modified polyacrylamide chain P-SA and DNA modified polyacrylamide chain P-SB in Tris buffer solution to obtain DNA modified polyacrylamide chain mixed solution; the concentration of DNA modified polyacrylamide chain P-SA in the prepared DNA modified polyacrylamide chain mixed solution is 100-350 μM, and the concentration of DNA modified polyacrylamide chain P-SB is 100-350 μM;

[0034] Preparation of mixed solution of enzyme chain GR-5 DNAzyme and substrate chain Substrate: mixing enzyme chain GR-5 DNAzyme and substrate chain Substrate in a certain proportion to obtain GR-5 DNAzyme / Substrate mixed solution; the concentration of enzyme chain GR-5 DNAzyme in the GR-5 DNAzyme / Substrate mixed solution is 100-350 μM, and the concentration of substrate chain Substrate is 100-350 μM;

[0035] Forming hydrogel valve in-situ on the micropore: dropping 0.2-1.5 μL of the DNA modified polyacrylamide chain mixed solution on the micropore, and then dropping 0.2-1.5 μL of the GR-5 DNAzyme / Substrate mixed solution after the solution completely penetrates into the paper, and forming DNA hydrogel after the solution completely penetrates into the paper, that is, forming hydrogel valve in paper chip;

[0036] The preparation method of finished DNA hydrogel is:

[0037] The DNA modified polyacrylamide chain P-SA, the DNA modified polyacrylamide chain P-SB, the enzyme chain GR-5 DNAzyme and the substrate chain Substrate strand are added into a Tris buffer solution to obtain a mixed solution; in the mixed solution, the concentration of the DNA modified polyacrylamide chain P-SA is 100-350 μM, the concentration of the DNA modified polyacrylamide chain P-SB is 100-350 μM; the concentration of the enzyme chain GR-5 DNAzyme is 100-350 μM, and the concentration of the substrate chain Substrate strand is 100-350 μM;

[0038] In order to ensure that the DNA chains are fully mixed, the mixed solution is heated at 55 DEG C for 1 min, and then cooled to room temperature, and the process is repeated several times, and finally the three-dimensional network structure of the DNA hydrogel is formed, and the DNA hydrogel is stored at 4 DEG C;

[0039] In the detection, the amount of the DNA hydrogel dropped on the micropore is 1-10 μL

[0040] The beneficial technical effects of the present application are as follows:

[0041] The method provided by the present application designs a paper-based microfluidic chip, which has a micropore structure and a DNA hydrogel sensitive unit. The paper chip is composed of two parts, one part is a micropore structure, and the DNA hydrogel material is fixed in the micropore, and the other part is a microchannel structure, which records the solution flow process after the target solution reacts with the gel in the micropore, and judges the concentration of the target through the flow rate.

[0042] In addition, the present application uses lead ions as a model target to verify the advancement of the paper chip sensor technology. The existing lead ion detection technology still relies on large equipment devices, which are heavy, high in cost, and require professional technical personnel for operation. The present application realizes lead ion detection by directly naked-eye reading the length of the strip through the fluid transmission of the paper chip capillary effect, which is convenient to carry, low in cost, suitable for mass production, and can be used for environmental monitoring and clinical diagnosis. Compared with the sensor of glass capillary material, the fragility of glass is solved, in addition, the preparation of the gel structure is more controllable, and the portability and operability are effectively improved.

[0043] The Pb 2+ The paper chip sensor has the characteristics of simple operation, rapidness, accuracy, directness, strong specificity, and can realize qualitative detection of lead ions and quantitative analysis of lead ions; the DNA hydrogel is used as a valve, the microchannel of the paper chip is used as a signal display, and the visual quantitative detection of lead ions is realized.

[0044] The Pb 2+The preparation conditions of the polyacrylamide-DNA complex are optimized in the paper chip sensor preparation method, and the yield of the polyacrylamide-DNA complex is improved.

[0045] In view of the relatively high price of the hydrogel, reducing the amount of the hydrogel plays a crucial role in reducing the cost of the paper chip.

[0046] The specificity of the paper chip sensor is evaluated by detecting other different metal ions, and the paper chip sensor is used for detecting real samples, and it is shown that the sensor can be used for detecting Pb 2+ in the real samples, and portable and visual quantitative detection of Pb 2+ in cosmetics can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1a Fig. 1 is a signal generated by detecting 15 μL 100 nM Pb 2+ with different amounts of hydrogel in the embodiment of the present application,

[0048] Figure 1b Fig. 1 is a signal generated by detecting 15 μL 100 nM Pb 2+ with different amounts of hydrogel in the embodiment of the present application,

[0049] Fig. 2 is the optimization of the polyacrylamide-DNA complex in the embodiment of the present application, wherein Fig. 2 (a) is the optimization of the APS concentration, Fig. 2 (b) is the optimization of the acrylamide concentration, Fig. 2 (c) is the optimization of the concentration of TEMED, and Fig. 2 (d) is the yield corresponding to three parallel experiments under the optimal preparation conditions;

[0050] Figure 3 Fig. 3 is a schematic diagram of the paper chip in the embodiment of the present application;

[0051] Figure 4 Fig. 4 (a) is a schematic diagram of detecting Pb 2+ with the DNA hydrogel in the embodiment of the present application; Figure 4 Fig. 4 (b) is a secondary structure of the hydrogel responding to Pb 2+ based on the GR-5 DNAzyme in the embodiment of the present application, which comprises two DNA modified polyacrylamide chains (P-SA, P-SB), an enzyme chain and a substrate chain, and a cleavage site is marked with a black arrow;

[0052] Figure 5 Fig. 5 is a schematic diagram of the experimental scheme for detecting lead ions in the embodiment of the present application;

[0053] Figure 6(a) is a plot of the relationship between the reaction time and the distance of the paper chip biosensor detecting different concentrations of lead ion solution within 2-10 min according to an embodiment of the present application; Figure 6(b) is a histogram of the length of the band after the reaction of the paper chip biosensor detecting Pb2+ according to an embodiment of the present application, wherein the concentrations of Pb2+ in the reaction solution are 1 nM, 10 nM, 50 nM, 100 nM and 500 nM, respectively; the error bar is the average standard error of three repeated experiments (N=3) for each data point. 2+ Figure 7(a) is a plot of the relationship between the reaction time and the distance of the paper chip biosensor detecting different concentrations of lead ion solution within 2-10 min according to an embodiment of the present application; Figure 7(b) is a plot of the linear relationship between the logarithm of the concentration and the length of the band of the paper chip biosensor detecting Pb2+ according to an embodiment of the present application, wherein the concentrations of Pb2+ in the reaction solution are 1 nM, 10 nM, 50 nM, 100 nM and 500 nM, respectively; the error bar is the average standard error of three repeated experiments (N=3) for each data point. 2+ Figure 8(a) is a plot of the relationship between the reaction time and the distance of the paper chip biosensor detecting different concentrations of lead ion solution within 2-10 min according to an embodiment of the present application; Figure 8(b) is a plot of the linear relationship between the logarithm of the concentration and the length of the band of the paper chip biosensor detecting Pb2+ according to an embodiment of the present application, wherein the concentrations of Pb2+ in the reaction solution are 1 nM, 10 nM, 50 nM, 100 nM and 500 nM, respectively; the error bar is the average standard error of three repeated experiments (N=3) for each data point.

[0054] Figure 7 Figure 9 is a histogram of the length of the band after the reaction of the paper chip biosensor detecting Ca2+, Cd2+, Cu2+, Fe2+, Mg2+, Ni2+, blank sample and Pb2+ according to an embodiment of the present application. 2+ 2+ 2+ 3+ 2+ 2+ 2+

[0055] Figure 8 Figure 10 is a photograph of the length of the band after the reaction of the paper chip biosensor detecting samples 2, 3 and 4 according to an embodiment of the present application. DETAILED DESCRIPTION

[0056] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0057] On the contrary, the present application covers any alternatives, modifications, equivalent methods and solutions defined by the claims within the spirit and scope of the present application. Further, in order to make the public have a better understanding of the present application, some specific details are described in the following detailed description of the present application. The present application can also be completely understood without the description of these details by those skilled in the art.

[0058] The paper chip biosensor according to an embodiment of the present application comprises a capillary microchannel and a biological sensitive unit, wherein the material of the capillary microchannel is paper.

[0059] The readout signal of the paper chip biosensor is the process of capillary flow; the biological sensitive unit is arranged on one side of the capillary microchannel and is used to regulate the behavior of capillary flow.

[0060] The readout signal of the paper chip biosensor is the flow distance of the to-be-detected solution in the capillary microchannel within a certain time, or the time required for the to-be-detected solution to flow a certain distance in the capillary microchannel within a certain time.​​​​​​​

[0061] The paper chip biosensor comprises hydrophilic micropores and microchannels arranged on the paper chip, and other regions of the paper chip are hydrophobic regions except the micropores and the microchannels;

[0062] The paper chip biosensor comprises hydrophilic micropores and microchannels arranged on the paper chip, and other regions of the paper chip are hydrophobic regions except the micropores and the microchannels;

[0063] The hydrogel valve is a biological sensitive unit of the paper chip biosensor. The biological sensitive unit comprises aptamers, biological proteins, molecular imprinting materials and hydrogels.

[0064] In the embodiment, the structure of the paper chip is as follows: the micropore is arranged at one end of the paper chip, the micropore and the microchannel are arranged on the same straight line, and a folding line is arranged between the micropore and the microchannel. When the paper chip is folded along the folding line, the micropore falls on one end of the microchannel close to the micropore. The diameter of the micropore ranges from 1.6 mm to 2.5 mm, and the width of the microchannel ranges from 0.8 mm to 1.6 mm.

[0065] In the embodiment, the DNA hydrogel is formed in situ on the micropore, and the specific method is as follows:

[0066] Preparation of a DNA-modified polyacrylamide chain mixed solution: a certain amount of DNA-modified polyacrylamide chain P-SA and DNA-modified polyacrylamide chain P-SB are added into a Tris buffer solution to obtain a DNA-modified polyacrylamide chain mixed solution. In the prepared DNA-modified polyacrylamide chain mixed solution, the concentration of the DNA-modified polyacrylamide chain P-SA is 100-350 μM, and the concentration of the DNA-modified polyacrylamide chain P-SB is 100-350 μM.

[0067] Preparation of a mixed solution of the enzyme strand GR-5 DNAzyme and the substrate strand: the enzyme strand GR-5 DNAzyme and the substrate strand are mixed in a certain ratio to obtain a GR-5 DNAzyme / Substrate mixed solution; in the GR-5 DNAzyme / Substrate mixed solution, the concentration of the enzyme strand GR-5 DNAzyme is 100-350 μM, and the concentration of the substrate strand is 100-350 μM; the base sequence of the GR-5 DNAzyme is shown as SEQ ID NO: 1 in the sequence listing; the base sequence of the substrate strand is shown as SEQ ID NO: 2 in the sequence listing;

[0068] In-situ formation of a hydrogel valve at the micropore: 0.2-1.5 μL (preferably 0.8 μL) of the DNA-modified polyacrylamide chain mixed solution is dropped at the micropore, and after the solution is completely infiltrated into the paper, 0.2-1.5 μL (preferably 0.8 μL) of the GR-5 DNAzyme / Substrate mixed solution is dropped, and after the solution is completely infiltrated into the paper, a DNA hydrogel is formed, i.e., a hydrogel valve in the paper chip is formed;

[0069] Specifically, in the prepared DNA-modified polyacrylamide chain mixed solution, the concentration of the DNA-modified polyacrylamide chain P-SA is 200 μM, and the concentration of the DNA-modified polyacrylamide chain P-SB is 200 μM; in the Tris buffer solution, the concentration of Tris is 10 mM, the concentration of NaCl is 300 mM, and the pH is 7.5; in the GR-5 DNAzyme / Substrate mixed solution, the concentration of the enzyme strand GR-5 DNAzyme is 200 μM, and the concentration of the substrate strand is 200 μM;

[0070] In this embodiment, when the target is lead ions, the preparation method of the finished DNA hydrogel is as follows:

[0071] The DNA modified polyacrylamide chain P-SA, the DNA modified polyacrylamide chain P-SB, the enzyme chain GR-5 DNAzyme and the substrate strand are added into a Tris buffer solution to obtain a mixed solution; in the mixed solution, the concentration of the DNA modified polyacrylamide chain P-SA is 100-350 μM, the concentration of the DNA modified polyacrylamide chain P-SB is 100-350 μM (preferably 200 μM); the concentration of the enzyme chain GR-5 DNAzyme is 100-350 μM, and the concentration of the substrate strand is 100-350 μM (preferably 200 μM); wherein the Tris buffer solution has a Tris concentration of 10 mM and a NaCl concentration of 300 mM, and has a pH of 7.5; the base sequence of the GR-5 DNAzyme is shown as SEQ ID NO: 1 in the sequence listing; and the base sequence of the substrate strand is shown as SEQ ID NO: 2 in the sequence listing.

[0072] In order to ensure that the DNA chains are fully mixed, the mixed solution is heated at 55°C for 1 min, and then cooled to room temperature, and the process is repeated several times (such as 3 times) until a three-dimensional network structure DNA hydrogel is formed, and the DNA hydrogel is stored at 4°C.

[0073] In the detection, the amount of the DNA hydrogel dropped on the micropore is 1-10 μL.

[0074] Gel dosage optimization: 100 nM Pb was detected by using different amounts of hydrogel 2+ According to the signal intensity and background signal after the reaction, the optimal and minimum amount of hydrogel is determined. Figure 1a Signal generated by different amounts of hydrogel to detect 15 μL of 100 nM Pb 2+ , Figure 1b Different amounts of hydrogel were used to detect 15 μL of water, and it was observed whether the hydrogel could completely block the micropore to form a valve. As can be seen from the figure, when the gel dosage used in the present study is 2 μL, 1.6 μL, 1 μL, 0.8 μL, the solution of lead ions with a concentration of 100 nM all have signal generation. Moreover, the less the amount of hydrogel used, the lower the cost. However, when 0.8 μL of hydrogel is used for detection, the micropore cannot be completely "blocked", leading to the fact that pure water dropped into the micropore can also pass through the gel membrane. However, this phenomenon does not occur when 1 μL of hydrogel is used, and 3 experiments have been conducted to verify this. Therefore, the optimal amount of gel is 1 μL.

[0075] In the present embodiment, the preparation method of the DNA modified polyacrylamide chain P-SA and the DNA modified polyacrylamide chain P-SB is as follows:

[0076] The DNA strand SA and the DNA strand SB containing acrylamide modification are added into a 4% acrylamide monomer solution respectively, and the concentration of the DNA strand SA and the DNA strand SB in the obtained solution is 500 μM. The mixed solution is placed in a vacuum dryer at room temperature (≈25°C) for a certain period of time (for example, 10 min) to remove oxygen, and then a freshly prepared initiator ammonium persulfate (APS) and an accelerator TEMED aqueous solution are added. The mass percentage final concentration of ammonium persulfate (APS) and the accelerator TEMED in the SA polymerization solution and the SB polymerization solution is 0.14% respectively. The base sequence of the DNA strand SA is shown in SEQ ID NO: 3 in the sequence listing, and the base sequence of the DNA strand SB is shown in SEQ ID NO: 4 in the sequence listing.

[0077] The SA polymerization solution and the SB polymerization solution are placed in a vacuum dryer for a certain period of time (for example, 15 min) to generate DNA-modified polyacrylamide chains P-SA and P-SB.

[0078] The DNA-modified polyacrylamide chains P-SA and P-SB are filtered and washed with ultrapure water and an ultrafiltration centrifuge tube (100 kDa) to remove monomers and polymers with small molecular weights on the polymer, and are ready for use. Tables 1 and 2 are experimental reagents and materials used in the present application, and DNA sequences:

[0079] Table 1 Experimental reagents and materials

[0080]

[0081]

[0082] Table 2 DNA sequences used in the present application

[0083]

[0084] The DNA strands (SA and SB) are purified by a high-performance liquid chromatography (HPLC) system and are modified with acrylamide at the 5' end. The DNA strands in the table are purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0085] Because the yield is very low when preparing polyacrylamide-DNA complex, the preparation condition is optimized in the present application. In the experiment, three parameters are involved, the concentration of APS, the concentration of TEMED and the concentration of acrylamide, as shown in Fig. 2. After each polymerization experiment, the amount of P-SA and P-SB synthesized is observed, and if the amount is about 30 μL, the preparation condition is the best. As shown in Fig. 2(a), when APS is the initiator, the polymerization degree increases with the decrease of the concentration of APS, and when the concentration of APS is 0.14%, the amount prepared is the most, which is the best preparation condition. As shown in Fig. 2(b), when the amount of acrylamide decreases, the amount prepared gradually decreases, so the concentration of acrylamide is 4%. TEMED is the accelerator, and the effect on the experimental results is the same as that of APS, as shown in Fig. 2(c), the polymerization degree increases with the decrease of the concentration of TEMED. According to the above optimization experiment, the best preparation condition is that the concentration of APS is 0.14%, the concentration of TEMED is 0.14% and the concentration of acrylamide is 4%. Under the best condition, three parallel experiments are carried out to prepare P-SA and P-SB, and the yield can reach more than 85%, as shown in Fig. 2(d).

[0086] In the embodiment, the Pb 2+ The paper chip sensor is used to detect Pb 2+ When detecting, the paper chip is folded along the folding line, one side of the microwell is located at the head of the microchannel, and the solution to be detected containing lead ions is dropped on the other side of the microwell. After the lead ions react with the hydrogel in the hydrogel valve, the size of the hydrogel mesh is increased, the solution to be detected passes through the valve and penetrates into the microchannel under the microwell, and a strip is formed on the microchannel.

[0087] Each detection is recorded by a video recorder at a certain time (10 minutes) after the lead ion solution is dropped, and the length of the strip formed in each experiment is recorded to quantitatively analyze the concentration of lead ions.

[0088] When the solution without lead ions is dropped, the valve will not be opened, and no strip will be generated.

[0089] In the embodiment, the paper used to prepare the paper chip is whatman, 1st grade chromatography filter paper, the structure of the paper chip is designed by using CorelDRAW software, as shown in Fig. 1, and the paper chip is prepared by using the following steps. Figure 3As shown, the left micro-hole is the position of the valve, and the right rectangle is the micro-channel. The length of the micro-channel is 5 cm. The black line between the valve and the micro-channel is the folding position. After printing with a wax jet printer, the paper is then placed in an oven at 130°C for 1 min to melt the wax. Because the filter paper is a porous structure, the wax will penetrate into the paper after melting, forming a hydrophobic region. The hydrophilic region on the paper includes the micro-hole and the micro-channel, and the other region is the hydrophobic region. The width of the micro-channel will be narrowed after the wax melts, and the diameter of the micro-hole will also be reduced. After the hydrogel valve is formed, folding is performed along the black line position, and the valve falls at the head of the micro-channel. When the detection is performed, the other side of the valve is dripped with a lead ion detection solution, and after the reaction with the hydrogel, a strip is formed on the micro-channel.

[0090] Optimization of micro-channel and aperture size: In this experiment, the signal strength generated by the paper chip is used to optimize the size of the micro-channel and aperture. The melting of the wax will cause the size of the micro-channel and aperture to change. The micro-channel will become narrower and shorter, and the diameter of the aperture will decrease. The changes in the size of the micro-channel and aperture of the paper chip of different sizes before and after the melting of the wax are as follows: 1) Before the wax melts, the diameter of the aperture is 3.5 mm, and the width of the micro-channel is 2.5 mm. After being placed in an oven at 130°C for 1 min, the diameter of the aperture becomes 2.93 mm, and the width of the micro-channel becomes 2 mm. 2) The diameter of the aperture of the paper chip is 2.5 mm, and the width of the micro-channel is 1.5 mm. After the wax melts, the diameter of the aperture is 1.95 mm, and the width of the micro-channel is 1.11 mm. 3) Before the wax melts, the diameter of the aperture is 2 mm, and the width of the micro-channel is 1 mm. After the wax melts, the diameter of the aperture is 1.5 mm, and the width of the micro-channel is 0.55 mm.

[0091] A 15 μL pure water droplet is dropped into the aperture to test the signal strength generated by the micro-channel structure of different sizes in the paper chip. A 15 μL pure water droplet is dropped into the micro-hole of the paper chip to observe the length of the strip generated by the paper chip of different sizes. When the width of the micro-channel is 2 mm after the wax melts, the length of the generated strip is 2.22 cm. When the width of the micro-channel is 1.11 mm, the length of the generated strip is 4.24 cm. Considering that the size of the aperture determines the amount of gel, the smaller the amount of gel, the lower the cost. However, when the width of the micro-channel is 0.55 mm and the diameter of the micro-hole is 1.5 mm, the signal is too small, and the length of the generated strip is too short, which is not conducive to the data collection of the subsequent detection experiment. Therefore, the size of the micro-channel is 1.11 mm, and the size of the micro-hole is 1.95 mm.

[0092] The application also provides a preparation method of a paper chip biosensor.

[0093] Preparation of paper chip: hydrophobic and hydrophilic regions are formed on the paper by wax printing, the hydrophilic region includes micropores and microchannels, and the remaining region on the paper is the hydrophobic region except the micropores and the microchannels; the micropores are the positions where the hydrogel valve is formed;

[0094] Preparation of hydrogel valve in paper chip: the hydrogel valve of the paper chip is formed by in-situ forming DNA hydrogel on the micropores or adding finished DNA hydrogel on the micropores;

[0095] Obtaining paper chip sensor.

[0096] In the above method, when the target is lead ions, the DNA hydrogel is formed in-situ on the micropores, and the specific method is as follows:

[0097] Preparation of DNA-modified polyacrylamide chain mixed solution: a certain amount of DNA-modified polyacrylamide chain P-SA and DNA-modified polyacrylamide chain P-SB are added to a Tris buffer solution to obtain a DNA-modified polyacrylamide chain mixed solution; in the prepared DNA-modified polyacrylamide chain mixed solution, the concentration of DNA-modified polyacrylamide chain P-SA is 100-350 μM, and the concentration of DNA-modified polyacrylamide chain P-SB is 100-350 μM;

[0098] Preparation of mixed solution of enzyme chain GR-5 DNAzyme and substrate chain Substrate strand: enzyme chain GR-5 DNAzyme and substrate chain Substrate strand are mixed in a certain proportion to obtain a GR-5 DNAzyme / Substrate mixed solution; in the GR-5 DNAzyme / Substrate mixed solution, the concentration of enzyme chain GR-5 DNAzyme is 100-350 μM, and the concentration of substrate chain Substrate strand is 100-350 μM;

[0099] In-situ formation of hydrogel valve at micropore: 0.2-1.5 μL of the DNA-modified polyacrylamide chain mixed solution is dropped into the micropore, after the solution completely penetrates into the paper, 0.2-1.5 μL of the GR-5 DNAzyme / Substrate mixed solution is dropped, and after the solution completely penetrates into the paper, DNA hydrogel is formed, that is, the hydrogel valve in the paper chip is formed;

[0100] The preparation method of the finished DNA hydrogel is as follows:

[0101] The DNA modified polyacrylamide chain P-SA, the DNA modified polyacrylamide chain P-SB, the enzyme chain GR-5 DNAzyme and the substrate chain Substrate strand are added into a Tris buffer solution to obtain a mixed solution; in the mixed solution, the concentration of the DNA modified polyacrylamide chain P-SA is 100-350 μM, the concentration of the DNA modified polyacrylamide chain P-SB is 100-350 μM; the concentration of the enzyme chain GR-5 DNAzyme is 100-350 μM, and the concentration of the substrate chain Substrate strand is 100-350 μM;

[0102] In order to ensure that the DNA chains are fully mixed, the mixed solution is heated at 55 ℃ for 1 min, and then cooled to room temperature, and the process is repeated several times, and finally the three-dimensional network structure DNA hydrogel is stored at 4 ℃.

[0103] In the detection, the amount of the DNA hydrogel dropped on the micropore is 1-10 μL (preferably 1 μL).

[0104] The Pb 2+ concentration of the sensor prepared by the method can reach 10-6 M. 2+ The detection principle is that the target responsive hydrogel and the capillary action of the paper fiber are utilized to detect Pb 2+ . The GR-5 DNAzyme in the hydrogel serves as a response unit and a crosslinking agent, the hydrogel serves as a valve, and the paper chip serves as a signal reporter, so that a paper chip sensor based on DNA hydrogel is developed for detecting Pb 2+ . The DNA hydrogel valve embedded in the paper chip can convert the Pb 2+ concentration into the visualized flow behavior of the solution on the microchannel. Figure 4 As shown in (a), in the absence of Pb 2+ , the mesh of the hydrogel does not change, the solution does not pass through the valve, and no band is formed on the microchannel. In contrast, in the presence of Pb 2+ , the GR-5 DNAzyme is activated, as shown in (b), the substrate strand is cleaved at the rA site, the mesh size of the hydrogel is increased, part of the hydrogel is broken, the valve is opened, the solution passes through the valve and forms a band on the microchannel. With the increase of the Pb 2+ concentration, the formed band is longer. In 10 min, the length of the band is recorded, and the concentration of Pb 2+ can be visually quantitatively detected. In the experiment, only a small amount (1 μL) of hydrogel is used for each paper chip sensor, and the visual quantitative detection of lead ions can be realized. The paper chip sensor provided in the present study provides a simple, portable, sensitive and low-cost platform for visual detection of lead ions.

[0105] Experimental scheme and feasibility analysis: A micro-hole and a micro-channel are formed by hydrophobic-hydrophilic combination. The micro-hole is the position of the hydrogel valve, and the diameter of the micro-hole is 1.95 mm, and the width of the micro-channel is 1.1 mm. As shown in Figure 5 Figure 1, first, a hydrogel valve is formed at the micro-hole, and then a mixed solution of P-SA, P-SB and Tris-Buffer is dropped, and then a mixed solution of DNAzyme and Substrate is dropped. Similarly, after the solution is completely infiltrated into the paper, the end of the paper chip where the hydrogel valve is located is folded along the crease, and the micro-hole falls on the head of the micro-channel. A solution containing lead ions is dropped on the other side of the hydrogel valve. After the hydrogel reaction, the size of the hydrogel mesh increases, the solution passes through the valve and penetrates into the micro-channel below, forming a band on the micro-channel. Each detection records the length of the band produced after 10 minutes of lead ion solution dropping by using a video recorder. The concentration of lead ions is quantitatively analyzed by recording the length of the band produced in each experiment. When a solution without lead ions is dropped, the valve will not open and no band will be produced.

[0106] For the above experimental scheme, its feasibility is verified by experiments. When water is dropped at the hydrogel valve, no band is produced. When a solution containing lead ions is dropped, the activity of GR-5 DNAzyme is activated, the substrate chain is cleaved, the size of the hydrogel mesh increases, the hydrogel valve opens, the solution penetrates into the micro-channel area below, and a band is formed.

[0107] Linear analysis of paper chip sensor: The concentrations of APS and TEMED are 0.14% and 0.14% respectively, and the concentration of acrylamide is 4%. The polyacrylamide-DNA complex is prepared, and the paper chip with a micro-hole diameter of 1.95 mm and a micro-channel width of 1.11 mm is used. The amount of hydrogel is 1 μL, and the detection of lead ions with different concentrations is carried out. The actual picture of the band length signal produced by the lead ion solution with a detection concentration of 1 nM, 10 nM, 50 nM, 100 nM and 500 nM at a reaction time of 2 min, 4 min, 6 min, 8 min and 10 min is recorded; and then the band length signal produced by the lead ion solution with a detection concentration of 1 nM, 10 nM, 50 nM, 100 nM and 500 nM at a reaction time of 2 min, 4 min, 6 min, 8 min and 10 min is recorded. As seen from the horizontal direction, at the same time, with the increase of the concentration of lead ions, the length of the band gradually increases. As seen from the vertical direction, with the increase of time, the length of the band gradually increases. When the detection concentration is particularly low, the reaction will slow down, and the speed of the signal produced will also slow down. As shown in Figure 6(a), the signals produced at each time are processed, and it can be seen that the slope gradually slows down with the decrease of the concentration of lead ions. In this experiment, the linear analysis is carried out by recording the band length signal produced after 10 minutes of reaction.

[0108] Each concentration of lead ion was tested three times to eliminate the randomness of the experiment, and each test was recorded by a video recorder. As shown in Fig. 6(b), the length of the band signal showed a good linear response to the logarithm of the concentration of lead ion in the range of 1 nM to 500 nM. The limit of detection was 0.3 nM. The linear regression equation was: 2+

[0109]

[0110] The correlation coefficient R 2 = 0.9880.

[0111] By measuring the length of the band after the reaction, the visual quantitative analysis of lead ions was realized. The paper chip sensor used only 1 μL of DNA hydrogel, and the quantitative analysis was performed 10 min after the detection of the lead ion solution, which had the characteristics of simple operation, portability, and rapidness.

[0112] Selective analysis of the paper chip sensor

[0113] To verify the selectivity of the paper chip sensor, different metal ion solutions were dropped at the hydrogel valve, and the length of the band signal was observed. After detecting other different metal ion solutions, no obvious band signal was generated after 10 min of reaction. This was in sharp contrast to the detection of 500 nM lead ion solution. The blank sample in the figure was water. Figure 7 The histogram showing the signals generated by other metal ions is shown. The experimental results show that the paper chip sensor has good specificity for the detection of lead ions.

[0114] Real sample detection: To test the practicability of the paper chip sensor, the spiked recovery method was used to detect the cosmetic water. The cosmetic was detected by ICP (inductively coupled plasma spectrometer) and showed that it contained negligible lead ion concentration. After adding lead ions to the cosmetic, the samples were detected by the paper chip and ICP, respectively. Figure 8 are the actual photos after detecting samples 2, 3, and 4. Table 3 shows the relative standard deviation (RSD) of the detection results and the recovery rate of lead ions in the cosmetic by the paper chip sensor and the ICP method. The recovery rate of the paper chip sensor for detecting Pb 2+ in the cosmetic was 87.5% to 96%. The experimental results show that the paper chip sensor can be used for the detection of Pb 2+ in complex actual samples.

[0115] Table 3 shows the detection of Pb 2+

[0116] ​

[0117] The paper chip biosensor provided by the application takes intelligent molecules as a valve to control the capillary flow process, and takes a capillary microchannel as a signal display to realize visual quantitative detection of a target. The paper chip sensor has the characteristics of simple operation, rapidness, accuracy, intuition, strong specificity, low cost and portability, and has a wide application prospect in home diagnosis and treatment, bedside diagnosis and instant detection.

[0118] The related literatures involved in the background part of the application are as follows:

[0119] [1]LI J,LU Y.A highly sensitive and selective catalytic DNA biosensorfor leadions[J].Journal of the American Chemical Society.2000,122(42):10466-10467.

[0120] [2]LAN T,FURUYA K,LU Y.A highly selective lead sensor based on aclassiclead DNAzyme[J].Chemical Communications.2010,46(22):3896-3898.

[0121] [3]MAO Y.Target-responsive DNA smart hydrogel and gold nanorod constructedinstant detection of new method[D].

[0122] Xiamen University,2017.

[0123] [4]JIANG C,LI Y,WANG H,et al.A portable visual capillary sensor basedonfunctional DNA crosslinked hydrogel for point-of-care detection of lead ion[J].

[0124] Sensors and Actuators B:Chemical.2020,307:127625.

Claims

1. A paper-based biosensor, characterized in that, The paper-chip biosensor includes capillary microchannels and biosensitive units, and the material of the capillary microchannels is paper. The readout signal of the paper chip biosensor is the capillary flow process; the biosensitive unit is placed on one side of the capillary microchannel and is used to regulate the behavior of capillary flow. The readout signal of the paper chip biosensor is the flow distance of the test solution in the capillary microchannel within a certain time, or the time required for the test solution to flow a certain distance in the capillary microchannel within a certain time. The paper-chip biosensor includes hydrophilic micropores and microchannels disposed on the paper chip, and the other areas on the paper chip, except for the micropores and microchannels, are hydrophobic areas. A hydrogel valve for a paper chip is formed by in-situ forming a DNA hydrogel on the micropores or by dropping a finished DNA hydrogel onto the micropores; the DNA hydrogel lyses upon stimulation by a corresponding target; the signal readout of the paper chip biosensor is obtained through the flow behavior of the solution in the hydrophilic microchannels; The hydrogel valve is the biosensitive unit of the paper chip biosensor; The paper chip has the following structure: a microhole is provided at one end of the paper chip, the microhole and the microchannel are on the same straight line, a fold line is provided between the microhole and the microchannel, and when folded along the fold line, the microhole falls on the end of the microchannel that is closer to the microhole; the diameter of the microhole is in the range of 1.6-2.5mm, and the width of the microchannel is in the range of 0.8-1.6mm.

2. The paper-chip biosensor according to claim 1, characterized in that, When the target is lead ions, a DNA hydrogel is formed in situ on the micropores. The specific method is as follows: Preparation of DNA-modified polyacrylamide chain mixed solution: A certain amount of DNA-modified polyacrylamide chain P-SA and DNA-modified polyacrylamide chain P-SB were added to Tris buffer solution to obtain a DNA-modified polyacrylamide chain mixed solution; in the prepared DNA-modified polyacrylamide chain mixed solution, the concentration of DNA-modified polyacrylamide chain P-SA was 100-350 μM, and the concentration of DNA-modified polyacrylamide chain P-SB was 100-350 μM. Preparation of a mixed solution of GR-5 DNAzyme and substrate strand: The GR-5 DNAzyme and substrate strand were mixed in a certain proportion to obtain a GR-5 DNAzyme / Substrate mixed solution; the concentration of the GR-5 DNAzyme in the GR-5 DNAzyme / Substrate mixed solution was 100-350 μM, and the concentration of the substrate strand was 100-350 μM; the base sequence of the GR-5 DNAzyme is shown in SEQ ID NO:1 in the sequence listing; the base sequence of the substrate strand is shown in SEQ ID NO:2 in the sequence listing. In-situ formation of hydrogel valves at micropores: 0.2-1.5 μL of the DNA-modified polyacrylamide chain mixture solution is dropped into the micropores. After the solution has completely penetrated the paper, another 0.2-1.5 μL of the GR-5 DNAzyme / Substrate mixture solution is dropped into the paper. After the solution has completely penetrated the paper, a DNA hydrogel is formed, thus forming the hydrogel valve in the paper chip.

3. The paper-chip biosensor according to claim 1, characterized in that, When the target is lead ions, the preparation method of the finished DNA hydrogel is as follows: DNA-modified polyacrylamide chain P-SA, DNA-modified polyacrylamide chain P-SB, enzyme chain GR-5 DNAzyme, and substrate strand were added to Tris buffer to obtain a mixed solution. In the mixed solution, the concentrations of DNA-modified polyacrylamide chain P-SA, DNA-modified polyacrylamide chain P-SB, GR-5 DNAzyme, and substrate strand were all 100-350 μM. The base sequence of the GR-5 DNAzyme is shown in SEQ ID NO:1 of the sequence listing; the base sequence of the substrate strand is shown in SEQ ID NO:2 of the sequence listing. To ensure that the DNA strands are thoroughly mixed, the mixture was heated at 55°C for 1 minute and then cooled to room temperature. This process was repeated several times to finally form a three-dimensional network structure of DNA hydrogel. The DNA hydrogel was then stored at 4°C. During testing, the amount of DNA hydrogel added to the microwell is 1-10 μL.

4. A paper-based biosensor according to any one of claims 2-3, characterized in that, The preparation methods of the DNA-modified polyacrylamide chain P-SA and the DNA-modified polyacrylamide chain P-SB are as follows: Acrylamide-modified DNA strands SA and SB were added to a 4% (w / w) acrylamide monomer solution, respectively. The resulting solutions contained 500 μM of both DNA strands SA and SB. The mixed solutions were dried in a vacuum desiccator at room temperature for a certain period to remove oxygen. Then, freshly prepared aqueous solutions of initiator ammonium persulfate (APS) and accelerator TEMED were added to obtain SA and SB polymerization solutions, respectively. The mass percentage concentrations of both APS and TEMED in the SA and SB polymerization solutions were 0.14%. The base sequence of the DNA strand SA is shown in SEQ ID NO:3 of the sequence listing; the base sequence of the DNA strand SB is shown in SEQ ID NO:4 of the sequence listing. The SA polymerization solution and the SB polymerization solution are placed in a vacuum dryer and reacted for a certain period of time to produce DNA-modified polyacrylamide chain P-SA and DNA-modified polyacrylamide chain P-SB. The DNA-modified polyacrylamide chain P-SA and the DNA-modified polyacrylamide chain P-SB were filtered and washed with ultrapure water and ultrafiltration centrifuge tubes to remove monomers and polymers with smaller molecular weights from the polymers, and then set aside for later use.

5. A paper-based biosensor according to any one of claims 2-4, characterized in that, Using the paper-chip biosensor to perform Pb 2+ During testing, the device is folded along the fold line, with one side of the micropore placed at the beginning of the microchannel. The test solution containing lead ions is dropped onto the other side of the micropore. After the lead ions react with the hydrogel in the hydrogel valve, the size of the hydrogel mesh increases. The test solution then passes through the valve and permeates into the microchannel below the micropore, forming a strip on the microchannel. Each test was recorded by video recorder after a certain time following the addition of the lead ion solution. By recording the length of the band formed in each experiment, the concentration of lead ions was quantitatively analyzed.

6. A method for preparing a paper-chip biosensor, used to prepare the paper-chip biosensor according to any one of claims 1-3, characterized in that, The method includes: Paper chip fabrication: Hydrophobic and hydrophilic regions are formed on paper using wax printing. The hydrophilic regions include micropores and microchannels. Apart from the micropores and microchannels, the remaining areas on the paper are all hydrophobic regions. The micropores are the locations where hydrogel valves are formed. Preparation of hydrogel valves in paper chips: Hydrogel valves in paper chips are formed by in-situ forming DNA hydrogel on the micropores or by dripping finished DNA hydrogel onto the micropores. Obtain a paper chip sensor.

7. The method for fabricating a paper-based biosensor according to claim 6, characterized in that, When the target is lead ions: The DNA hydrogel is formed in situ on the micropores using the following method: Preparation of a mixed solution of GR-5 DNAzyme and substrate strand: The GR-5 DNAzyme and substrate strand are mixed in a certain proportion to obtain a GR-5 DNAzyme / Substrate mixed solution; the concentration of the GR-5 DNAzyme in the GR-5 DNAzyme / Substrate mixed solution is 100-350 μM, and the concentration of the substrate strand is 100-350 μM. In-situ formation of hydrogel valves at micropores: 0.2-1.5 μL of DNA-modified polyacrylamide chain mixture solution is dropped into the micropores. After the solution has completely penetrated the paper, another 0.2-1.5 μL of the GR-5 DNAzyme / Substrate mixture solution is dropped into the paper. After the solution has completely penetrated the paper, a DNA hydrogel is formed, which forms the hydrogel valve in the paper chip. The preparation method of the finished DNA hydrogel is as follows: DNA-modified polyacrylamide chain P-SA, DNA-modified polyacrylamide chain P-SB, GR-5 DNAzyme, and substrate strand were added to Tris buffer solution to obtain a mixed solution. In the mixed solution, the concentrations of DNA-modified polyacrylamide chain P-SA, DNA-modified polyacrylamide chain P-SB, GR-5 DNAzyme, and substrate strand were 100-350 μM.

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