LU500332B1 - A Colorimetric Assay and Kit for Detection of Creatine Kinase Using Microfluidic Chips - Google Patents

Abstract

The present invention belongs to the field of protein detection and relates to a colorimetric assay and kit for detection of Creatine Kinase using microfluidic chips. The method includes the following steps: S1. preparation of linear polyacrylamide-DNA polymer; S2. synthesis and modification of AuNPs; S3. design and assembly of microfluidic chip; S4. synthesis of DNA hydrogel; and S5. assay. The present invention utilizes DNA hydrogels for the detection of the target with good stability and high reaction specificity, which can be visualized. The assay using microfluidic chip achieves the purpose of quantitative assay with portability.

Description

| LUSOPBR%0332

DESCRIPTION A Colorimetric Assay and Kit for Detection of Creatine Kinase Using Microfluidic Chips

TECHNICAL FIELD The present invention belongs to the field of protein detection, and specifically, relates to a colorimetric assay and kit for detection of Creatine Kinase using microfluidic chips.

BACKGROUND Acute myocardial infarction (AMI) is a common cause of death worldwide. According to WHO, of the 17.9 million deaths from cardiovascular disease in 2015, about 8.9 million died from ischemic heart disease, and acute myocardial infarction is its leading cause, with a trend toward younger age. Therefore, the rapid and effective diagnosis of acute myocardial infarction is of great clinical importance. Due to the sudden and unpredictable onset of myocardial infarction, there is an urgent need to develop portable tests that can be used for rapid and effective diagnosis in any setting.

Detection of biomarker’s concentrations in body fluids is an effective method for the diagnosis of acute myocardial infarction with a high degree of accuracy and precision. Creatine Kinase MB (CKMB), as one of the cardiac biomarkers, has high specificity and sensitivity, and continuous monitoring of its value can also be used to determine the occurrence of reinfarction. Acute myocardial infarction occurs when the CKMB concentration in the blood rises to more than twice the upper limit of normal (20ng/mL, 44kDa). The level of CKMB in the blood starts to rise in the 4th-6th hours of the infarction and reaches its peak in the 24th hour, at which time its concentration can reach times the normal value and returns to the normal level in 48-72 hours. A variety of immunoassays have been used for biomarker detection, including surface plasmon resonance, fluorescence, and electrochemistry. However, they all require high-cost equipment and specialized technicians, and are often limited by specific operating environments such as laboratories, making their clinical application somewhat restricted. Therefore, there is an urgent need for a portable assay for CKMB. ELISA kits can meet

; LUSOPBR%0332 the requirements of portability and have been widely used for the screening of CKMB, but the method has disadvantages such as long incubation time, many washing steps and low sensitivity. Therefore, there is still an urgent need for an accurate, portable and low- cost assay for CKMB in this field.

SUMMARY In response to the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide a portable, quantitatively scalable DNA hydrogel- based assay using microfluidic chips for the point-of-care testing (POCT) of the marker of myocardial infarction: Creatine Kinase .

To achieve the above, the present invention provides a colorimetric assay and kit for detection of Creatine Kinase using microfluidic chips, comprising the steps of: (1) preparation of linear polyacrylamide-DNA polymer: mixing acrylamide with S1 and S2, respectively, and after vacuum drying, adding ammonium sulfate solution and tetramethylethylenediamine solution, followed by polymerization reaction to obtain PS1 and PS2.

Nucleotide sequences of S1: 5’-Acrydite--3’(SEQ ID NO:1) Nucleotide sequences of S2: 5’-Acrydite--3’(SEQ ID NO:2) (2) synthesis and modification of AuNPs: aqueous HAuCy solution was heated and boiled while stirring, then freshly prepared Trisodium citrate dihydrate solution was quickly added and the mixture continued to boil to obtain AuNPs; BSA was mixed with AuNPs, incubated by mixer, and then resuspended by centrifugation to obtain BSA- AuNPs.

(3) design and assembly of microfluidic chip: using Auto CAD software to draw the chip graphics and prepare the microfluidic chip accordingly, which is made of Polydimethylsiloxane using soft lithography and has a liquid inlet, hydrogel feeding area, detection area and liquid channel; the microfluidic chip of the present invention can be commercially acquired.

(4) synthesis of DNA hydrogel: PS1 and AuNPs were added in a centrifuge tube. then the mixture was incubated at 65°C and 1000 rpm for 10 min. Then add PS2 and mix

; LUSOPRH00332 for another 10 min to ensure uniform mixing; cool the hydrogel to 25 °C and keep it at a constant temperature overnight to obtain DNA hydrogels.

(5) assay The DNA hydrogel was rinsed with PBS solution and placed at the microfluidic chip channel with a pipettor to block the liquid flow; the sample to be measured was added on top of the DNA hydrogel and the chip was tilted after the reaction, using gravity to drive the flow of the reacted liquid into the detection area; after the chip was placed on the exposure plate and photographed, the software was used to analyze the average gray value and calculate the concentration of CKMB in the sample to be measured.

The schematic diagram of the detection process of the present invention is shown in Figure 1. A colorimetric method based on DNA hydrogels for rapid visualization and portable detection of Creatine Kinase is achieved. The two DNA strands (SI and S2) were first copolymerized with acrylamide to form linear DNA-polyacrylamide polymers PS1 and PS2, respectively. S1 contains the CKMB aptamer sequence and is partially complementary to the short S2. When PS1 and PS2 are mixed, S1 and S2 cross-link through base pairing to eventually form a DNA hydrogel. In the absence of CKMB, S1 and S2 can complementarily hybridize smoothly to form a hydrogel. On the contrary, with the presence of CKMB, the aptamer recognizes the target and binds preferentially to it, S1 conformation changes and thus dissociates from S2, leading to DNA hydrogel disintegration and release of encapsulated AuNPs, and the changes of supernatant liquor from colorless to red. Thus, the concentration of CKMB can be indicated by the change in color of the supernatan liquor. The hydrogel was further combined with a microfluidic chip, fixed at the microfluidic channel for portable detection, and the concentration of the target was indicated by analyzing the grayscale value based on the color change generated by the dissociated AuNPs content.

According to a specific embodiment of the present invention, step (1) comprises the following steps: prepare chains PS1 and PS2 with centrifuge tubes containing 2-3% acrylamide, respectively, and add 0.04-0.06% (v/v) ammonium persulfate and 0.04-

0.06% (v/v) tetramethylethylenediamine after vacuum drying at 37°C for 4-8 minutes to carry out the polymerization reaction.

a LUSOPRH00332 According to a specific embodiment of the present invention, step (2) includes the following steps: mixing ultrapure water and 0.8-1.2 mL of 0.8-1.2% HAuCls aqueous solution, heating and boiling at 300-500 rpm, then quickly adding 0.8-1.2 mL of freshly prepared 2-4% trisodium citrate dihydrate solution, the mixture continues to boil for 20- 40 minutes, and the solution cooled to room temperature by avoiding light and then stored at 4°C for backup; BSA was mixed with AuNPs and kept mixing overnight at room temperature, then resuspended in water 2-4 times by centrifugation to obtain BSA- AuNPs.

According to a specific embodiment of the present invention, step (4) comprises the following steps: adding PS1 and AuNPs in a centrifuge tube, incubating the mixture at 60-70°C and 800-1200 rpm for 8-12 min, then adding PS2 and mixing for another 8-12 min to ensure uniform mixture; cooling the hydrogel to 20-30°C and keeping it at a constant temperature overnight to obtain a DNA hydrogel.

According to a specific embodiment of the present invention, step (5) further comprises the step of preparing a standard curve: detecting a series of standard solutions of Creatine Kinase with known concentrations and making a graph with the concentration and the average grayscale value as the horizontal and vertical coordinates, respectively, to produce a standard curve of Creatine Kinase.

According to a specific embodiment of the present invention, the method of the present invention comprises the steps of: (1) preparation of linear polyacrylamide-DNA polymers: chains PS1 and PS2 were prepared using centrifuge tubes containing 2.25% acrylamide, respectively. after vacuum drying at 37°C for 5 min, 0.05% (v/v) ammonium persulfate (0.05 g ammonium persulfate dissolved in 0.5 mL water) and 0.05% (v/v) tetramethylethylenediamine (100 uL TEMED dissolved in 900 pL of water), followed by vacuum drying at 37 °C for 5 min for the polymerization reaction.

(2) synthesis and modification of AuNPs: 99 mL of ultrapure water and 1 mL of 1% aqueous HAuCh solution were added to a 250 mL three-necked round-bottom flask and boiled by heating at 400 rpm. Then 1mL of freshly prepared 3% trisodium citrate dihydrate solution was quickly added and the mixture continued to boil for 30 minutes. The solution was cooled to room temperature away from light and then stored at 4°C for

LUSOPRH00332 backup. And BSA was mixed with AuNPs and incubated overnight at room temperature with mixing, then resuspended in water by centrifugation three times to obtain BSA- AuNPs (3) design and assembly of microfluidic chip: using Auto CAD software to draw the chip graphics and prepare the microfluidic chip accordingly, which is made of Polydimethylsiloxane using soft lithography and has a liquid inlet, hydrogel feeding area, detection area and liquid channel; the microfluidic chip of the present invention can be commercially acquired.

(4) synthesis of DNA hydrogel: PS1 and AuNPs were added in a centrifuge tube. then the mixture was incubated at 65°C and 1000 rpm for 10 min. Then add PS2 and mix for another 10 min to ensure uniform mixing; cool the hydrogel to 25 °C and keep it at a constant temperature overnight to obtain DNA hydrogels.

(5) colorimetry: CKMB solutions of different concentrations were added to DNA hydrogels separately and reacted at 30 °C for 2.5 h at 150 rpm. A color change was observed visually in the hydrogel supernatant liquor, indicating that CKMB caused hydrogel disintegration and release of internal AuNPs (6) readings: In order to obtain more accurate results, the absorbance of the supernatant liquor was also measured by UV-Vis spectrophotometry at 520 nm and the gray value of the analysis system was photographed.

According to the present invention, more specifically, step (1) comprises: first, 26.7 HL and 72 pL of enzyme-free water were added to the centrifuge tubes containing chain S1 and chain S2, respectively, and mixed well. Weigh 12.7 g of MgCl,, add it to the centrifuge tube, and aspirate 1 mL of 50xTAE buffer, dilute it to 10 mL to form 5xTAE buffer, then add it to the centrifuge tube containing MgCl, and dissolve it fully and set aside. Add 10 pL S1, 1.5 pL 30% acrylamide (AA), and 6.5 uL 5xTAF/Mg* to the centrifuge tube and evacuate for 5 min at 37°C. Freshly prepared 0.05% (v/v) ammonium persulfate (0.05 g ammonium persulfate dissolved in 0.5 mL water) and 0.05% (v/v) tetramethylethylenediamine (100 uL TEMED dissolved in 900 uL water) were immediately added, respectively, and then the vacuum was continued for 5 min at 37 °C for polymerization reaction. Linear chains PS1 and PS2 were obtained, respectively.

‘ LUSOPRH00332 According to the present invention, more specifically, step (2) comprises: all glassware used for synthesis is soaked overnight in freshly prepared aqua regia (HNO3:HCI=1:3), then thoroughly washed and dried with ultrapure water. A 250 mL three-necked round-bottom flask was heated with 99 mL of ultrapure water and 1 mL of 1% aqueous HAuCl solution at 400 rpm. During this process, 30mg of trisodium citrate dihydrate was accurately weighed and dissolved in 1ml of ultrapure water and added rapidly after the temperature was raised to 140°C. Maintain this temperature and continue boiling for 30 min. stop heating and store the solution away from light to cool to room temperature. Weigh the BSA according to the concentration of 30mg/mL, mix with the synthesized solution and incubate overnight at room temperature with mixing at 600rpm, then centrifuge at 12000rpm for 20min, remove the supernatant liquor, repeat three times, and finally resuspend in water to obtain the desired AuNPs According to the present invention, preferably, step (3) includes: drawing the chip graphics according to the expected form and producing the microfluidic chip after communicating with Suzhou Tianshuo Automation Technology Company.

Preferably, according to the present invention, step (4) comprises: adding 20 pL PS1 and 20 pL 120 uM BSA-AuNPs in a centrifuge tube. Reacting at 65 °C and 1000 rpm for min. Then add 20 pL PS2 and mixing for another 10 min to ensure uniform mixing. The hydrogels were cooled to 25°C and kept at a constant temperature overnight. Rinse 3 times with PBS buffer to remove unbound AuNPs to obtain DNA hydrogels.

Preferably, according to the present invention, step (5) comprises: adding 80 uL of different concentrations of CKMB solution to the DNA hydrogel separately and reacting at 30°C for 2.5 hours at 150 rpm. The reaction results are recorded by taking pictures on the exposure plate.

According to the present invention, step (5) can also include: taking 50 pL of supernatant liquor, measuring it in a UV-Vis spectrophotometer, obtaining the absorbance value of the supernatant liquor out at 520 nm, processing the data, and obtaining a standard curve. Further, it is applied to the microfluidic chip and the reaction is photographed on the exposure plate for the liquid in the detection area after the reaction to form a file in png format. The grayscale values are detected by image J software to achieve fast and timely readings of the assay results.

, LUSOPRH00332 The present invention also provides a colorimetric assay and kit for detection of Creatine Kinase using microfluidic chips, comprising the following components: (1) the linear polyacrylamide-DNA polymer (2) AuNPs (3) microfluidic chip (4) DNA hydrogel The present invention optimizes the reaction time, the pH of the reaction system, and the concentration of the coating gold during the assay process, allowing for rapid visual detection of Creatine Kinase under optimal experimental conditions. The present invention utilizes DNA hydrogels for the detection of the target with good stability and reaction specificity, which allows visualization of the assay. The use of microfluidic chip for the assay achieves the purpose of portable quantitative assay. The chip has low manufacturing cost, can be reused after detection, has reliable results, can be intelligently read by simple loading operation, and can reach a detection limit of 0.027 nM using cell phone software, which meets the critical value of serum detection at the occurrence of myocardial infarction and is suitable for screening and rapid detection process in the field.

Other features and advantages of the present invention will be described in detail in the subsequent specific implementation section.

BRIEF DESCRIPTION OF THE FIGURES The foregoing and other objects, features and advantages of the present invention will become more apparent by a more detailed description of the exemplary embodiments of the invention in connection with the accompanying drawings.

Figure 1 shows the schematic diagram of the detection method of the present invention.

Figure 2 shows the schematic diagram of the microfluidic chip used in the embodiment of the present invention.

3 LUSOPRH00332 (5) Figure 3 shows the transmission electron microscope, particle size statistical analysis and zeta potential of the AuNPs before and after modification with BSA in the embodiment of the present invention.

Figure 4 shows the detection standard curve of the target in the embodiment of the present invention.

Figure 5 shows the results of the specificity assay in the embodiment of the present invention.

DESCRIPTION OF THE INVENTION Preferred embodiments of the present invention will be described in greater detail below. Although preferred embodiments of the invention are described below, it should be understood, however, that the invention can be implemented in various forms and should not be limited by the embodiments set forth herein.

This embodiment is used to illustrate a DNA hydrogel-based assay using microfluidic chips for the detection of Creatine Kinase in human serum, specifically comprising the steps of: In the embodiment, chloroauric acid (HAuCl4-H20) (>99.5%), trisodium citrate dihydrate, ammonium persulfate (APS), tetramethylethylenediamine (TEMED), and magnesium chloride were purchased from Damao Chemical Reagent Factory (Tianjin, China). Bovine serum albumin (BSA), 10,000x SYBR Green (SG), 30% acrylamide (AA) purchased from Solarbio (Beijing, China). 10x DNA loading buffer, 20 bp DNA ladder purchased from TaKaRa Biotechnology Ltd (Dalian, China). Creatine Kinase (CKMB) was purchased from Prospec (Sionanis, Israel). Troponin I was purchased from Sigma-Aldrich (St. Louis, MO, USA), H-FABP, C-reactive protein (CRP), and calcitonin were purchased from Abcam (Shanghai, China). Other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). The oligonucleotides used in this experiment were synthesized and modified by Shanghai Biotechnology Co., Ltd. and purified by high performance liquid chromatography (HPLC). The sequences of these oligonucleotides are as follows:

9 LUSOPBR%0332 Where specific conditions are not indicated in the embodiment, conventional conditions or those recommended by the manufacturer were followed. The reagents or instruments used, where the manufacturer is not indicated, are conventional products that can be obtained through commercially available purchases.

1) preparation of linear polyacrylamide-DNA polymers: chains PS1 and PS2 were prepared using centrifuge tubes containing 2.25% acrylamide, respectively. after vacuum drying at 37°C for 5 min, 0.05% (v/v) ammonium persulfate (0.05 g ammonium persulfate dissolved in 0.5 mL water) and 0.05% (v/v) tetramethylethylenediamine (100 HL TEMED dissolved in 900 uL of water), followed by vacuum drying at 37 °C for 5 min for the polymerization reaction.

2) synthesis and modification of AuNPs: 99 mL of ultrapure water and 1 mL of 1% aqueous HAuCh solution were added to a 250 mL three-necked round-bottom flask and boiled by heating at 400 rpm. Then 1mL of freshly prepared 3% trisodium citrate dihydrate solution was quickly added and the mixture continued to boil for 30 minutes. The solution was cooled to room temperature away from light and then stored at 4°C for backup. And BSA was mixed with AuNPs and incubated overnight at room temperature with mixing, then resuspended in water by centrifugation three times to obtain BSA- AuNPs. Figure 3 shows the transmission electron microscope, particle size statistical analysis and zeta potential plots of AuNPs before and after modification with BSA.

3) synthesis of DNA hydrogel: 20uL PS1 and 20uL and 120uM AuNPs were added in a centrifuge tube. Then the mixture was incubated at 65°C and 1000 rpm for 10 min. Then add 20uL PS2 and mix for another 10 min to ensure uniform mixing; cool the hydrogel to 25 °C and keep it at a constant temperature overnight to obtain DNA hydrogels before rinsing it 3 times with PBS buffer to remove unbound AuNPs.

0 LUSOPBR%0332 4) design and assembly of microfluidic chip: using Auto CAD software to draw the chip graphics and prepare the microfluidic chip accordingly, which is made of Polydimethylsiloxane (PDMS) using soft lithography and has a liquid inlet, hydrogel feeding area, detection area and liquid channel. As shown in Figure 2, washing the microfluidic chip with PBS buffer and blow drying with nitrogen for backup.

5) assay using microfluidic chip: The DNA hydrogel was rinsed with PBS solution and then placed at the microfluidic chip channel by pipetting with a pipettor to block the liquid flow. Take 50 pL of different concentration gradients of Creatine Kinase solution, add it on top of the DNA hydrogel, and react at 30°C for 1 h. Then the chip was tilted and the reaction liquid flowed into the detection area using gravity to drive the reaction.

6) Production of standard curve: After placing the chip on the exposure plate and taking pictures, the software was used to analyze the average gray value and produce the standard curve, as shown in Figure 4.

7) Specific assay: Other substances elevated in the development of cardiovascular disease (hFABP, cardiac troponin I and C-reactive protein) and common serum enzymes (calcitonin) were investigated using the same experimental method as CKMB. The results of the specific assays are shown in Figure 5.

8) Pre-treatment of human serum samples: Human serum samples were taken and diluted 30-fold using TM buffer as the sample assay matrix. Different concentrations of Creatine Kinase were added to the diluted serum samples as the target solution.

(9) Actual sample detection: The sera containing different concentrations of Creatine Kinase were reacted according to the above steps, and the absorbance values from the analysis were substituted into the standard curve to calculate the content of Creatine Kinase.

The results are shown in Table 1.

Sample Adding of Assay of CKMB RSD Yield Rate (%) CKMB (nM) (%, n= 6) (nM) 1 5 5.28 3.43 105.50

1 LUSOPBR%0332 2 50 53.13 2.54 106.25 3 500 558.66 2.75 96.63 Embodiments of the present invention have been described above, and the foregoing description is exemplary, not exhaustive, and is not limited to the disclosed embodiments.

Without departing from the scope and spirit of the illustrated embodiments, many modifications and changes will be apparent to one of ordinary skill in the art.

20210625 PT1249 Seq.txt

SEQUENCE LISTING LUSOPR 500332 <110> Tianjin Institute of Environmental and Operational Medicine <120> A Colorimetric Assay and Kit for Detection of Creatine Kinase Using Microfluidic Chips <130> PT1249LU <160> 2 <170> BiSSAP 1.3.6 <210> 1 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Seql <400> 1 aaaaaggggg gtgggtgggg gatctcggag gatgctttta gggggttggg 50 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Seq? <400> 2 aaaaacccta aaagcatc 18 Page 1

Claims (6)

1. À colorimetric assay and kit for detection of Creatine Kinase using microfluidic chips comprises the steps of: (1) preparation of linear polyacrylamide-DNA polymer: mixing acrylamide with S1 and S2, respectively, and after vacuum drying, adding ammonium sulfate solution and tetramethylethylenediamine solution, followed by polymerization reaction to obtain PS1 and PS2: nucleotide sequences of S1: 5’-Acrydite--3’(SEQ ID NO:1) nucleotide sequences of S2: 5’-Acrydite--3"(SEQ ID NO:2) (2) synthesis and modification of AuNPs: aqueous HAuCl4 solution was heated and boiled while stirring, then freshly prepared Trisodium citrate dihydrate solution was quickly added and the mixture continued to boil to obtain AuNPs; BSA was mixed with AuNPs, incubated by mixer, and then resuspended by centrifugation to obtain BSA- AuNPs; (3) design and assembly of microfluidic chip: using Auto CAD software to draw the chip graphics and prepare the microfluidic chip accordingly, which is made of Polydimethylsiloxane using soft lithography and has a liquid inlet, hydrogel feeding area, detection area and liquid channel, (4) synthesis of DNA hydrogel: PS1 and AuNPs were added in a centrifuge tube; then the mixture was incubated at 60-70°C and 800-1200 rpm; then add PS2 and incubate and shake again to ensure uniform mixing; cool the hydrogel to 20-30 °C and keep it at a constant temperature overnight to obtain DNA hydrogels; (5) assay the DNA hydrogel was rinsed with PBS solution and placed at the microfluidic chip channel with a pipettor to block the liquid flow; the sample to be measured was added on top of the DNA hydrogel and the chip was tilted after the reaction, using gravity to drive the flow of the reacted liquid into the detection area; after the chip was placed on the exposure plate and photographed, the software was used to analyze the average gray value and calculate the concentration of CKMB in the sample to be measured.

2. The colorimetric assay for detection of Creatine Kinase using microfluidic chips according to claim 1, wherein step (1) comprises the steps of: preparing chains PS1 and

PS2 with centrifuge tubes containing 2-3% acrylamide, respectively and adding 0.04-

0.06% (v/v) ammonium persulfate and 0.04-0.06% (v/v) tetramethylethylenediamine after vacuum drying at 37°C for 4-8 minutes to carry out the polymerization reaction.

3. The colorimetric assay for detection of Creatine Kinase using microfluidic chips according to claim 1, wherein step (2) comprises the steps of: mixing ultrapure water and

0.8-1.2 mL of 0.8-1.2% HAuCl4 aqueous solution, heating and boiling at 300-500 rpm, then quickly adding 0.8-1.2 mL of freshly prepared 2-4% trisodium citrate dihydrate solution, the mixture continues to boil for 20-40 minutes, and the solution is cooled to room temperature by avoiding light and then stored at 4°C for backup; BSA was mixed with AuNPs and kept mixing overnight at room temperature, then resuspended in water 2-4 times by centrifugation to obtain BSA-AuNPs.

4. The colorimetric assay for detection of Creatine Kinase using microfluidic chips according to claim 1, wherein step (4) comprises the steps of: adding PS1 and AuNPs ina centrifuge tube, incubating the mixture at 60-70°C and 800-1200 rpm for 8-12 min, then adding PS2 and mixing for another 8-12 min to ensure uniform mixture; cooling the hydrogel to 20-30°C and keeping it at a constant temperature overnight to obtain a DNA hydrogel.

5. The colorimetric assay for detection of Creatine Kinase using microfluidic chips according to claim 1, wherein step (5) comprises the steps of: preparing a standard curve: detecting a series of standard solutions of Creatine Kinase with known concentrations and making a graph with the concentration and the average grayscale value as the horizontal and vertical coordinates, respectively, to produce a standard curve of Creatine Kinase.

6. The colorimetric assay and kit for detection of Creatine Kinase using microfluidic chips comprises the following parts: (6) the linear polyacrylamide-DNA polymer of claim 1; (7) AuNPs of claim 1; (8) microfluidic chip of claim 1; (9) DNA hydrogel of claim 1.