| Reagents | Concentration |
|---|---|
| Dry powder | 1 tube |
| A Buffer | 25 μL |
| F-Primer | 2 μL |
| F-Primer | 2 μL |
| ddH2O | 13.5 μL |
| DNA template | 5 μL |
| B Buffer | 2.5 μL |
| Reagents | Concentration |
|---|---|
| Agarose | 0.9 g |
| TAE(1×) | 30 mL |
| Goldview | 2 μL |
（1）Preparation of LB culture medium
| Reagents | Concentration |
|---|---|
| 1% NaCl | 1 g |
| 1% Tryptone | 1 g |
| 0.5% Yeast Extract | 0.5 g |
（1） Turn on the ultra-clean table UV lamp for 30 min in advance, and set the shaker speed and temperature at 220r and 37 ℃.
（2） Open the sterilized culture medium, add 50 μL of kanamycin stock solution (30 mg/mL) to the conical flask, and shake for a thorough mix.
（3）Take 7 mL of culture medium to the test tube.
（4） Add about 30 μL of -80 ℃ lyophilized monoclonal strain into the test tube with 7 mL of culture medium.
（5）Put the test tube inside the shaker and set the parameter at 37 ℃, 220 r for an overnight incubation, which is about 12hr.
（6）Store the culture medium at 4 ℃.
（1） Remove the test tube from the shaker and place it on the ultra-clean table.
（2）Take out the test tube containing the bacterial fluid and transfer 1-2 mL of it into a conical flask containing the sterilized culture medium.
（3） Repeat the procedures with the other test tube and the conical flask.
（4） Place the two conical flasks in the shaker and set the parameter at 37 ℃, 220 rpm incubation, about 3-4 h,OD=0.6.
（5） Preserve the strain.
（1） Take out 1M IPTG (solid powder, filter membrane is used for sterilization after preparation) and melt it at room temperature.
（2） Take out the conical flasks from the shaker and put them into the ultra-clean table together with IPTG.
（3） Add 10 μL IPTG under the surface of the culture medium and repeat the procedure.
（4） Place the conical flasks back into the shaker and incubate at 16 ℃, 220 r for 16 hr.
（1） Centrifuge the bacterial fluid and discard the supernatant.
（2） Add 5mL of 20mM imidazole respectively and mix well with blowing. Then transfer them to the same tube.
（3）Add 200μL PMSF.
（4）Preserve it at -80°C.
（1） Take out the bacteria and melt it at room temperature.
（2） Place the tube that contains the bacterial fluid in a beaker and bathe it with ice water. Then sonicate the tube until the bacterial fluid is clear.
（3） Centrifuge the bacterial fluid (12000×g, 30min).
（4） Equilibrate the Ni column with distilled water and then binding buffer.
（5）Elute the protein with 20mM, 30mM, 60mM, 150mM, 300mM and 500mM imidazole respectively, and collect it at the end.
（1） Using a 10 KDa ultrafiltration tube, add the collection solution (placed on an ice box) (Max=400 μL) and centrifuge at high speed (4 °C, 12000 × g, 4 min).
（2）Discard the effluent, add the collection solution to the ultrafiltration tube, and repeat these steps until the entire collection solution is ultrafiltered.
（3）Add Protein diluent into the ultrafiltration tubes, blow and mix, and ultrafiltrate it. Repeat three times.
（4）Bradford method for concentration measurement.
（5）Sub-pack the solution and store at -80°C.
8% PAGE Glue Formulation：
| Reagents | Concentration |
|---|---|
| 30% Arc-Bis | 5 mL |
| ddH2O | 6.1 mL |
| 5XTBE | 2 mL |
| Aps | 0.15 mL |
| TEMED | 0.07 mL |
| Reagents | Concentration |
|---|---|
| 30% Arc-Bis | 1 ml |
| ddH2O | 3.5 mL |
| 5XTBE | 1.5 mL |
| Aps | 60 μL |
| TEMED | 6 μL |
SDS-PAGE sample system configuration：
| Reagents | Concentration |
|---|---|
| 6X SDS-PAGE Buffer | 8 μL |
| Protein Original Solution | 40 μL |
（1）Place the gel in the electrophoresis chamber. Pour enough electrophoresis buffer (1 X TBE) to cover the gel to prevent overheating of the gel.Carefully remove the comb.
（2）Prepare the protein sample.
（3）Add marker into the first well by using a micropipette. Carefully place the prepared samples into adjacent wells.
（4）Electrophorese the samples at 30V for 20 minutes and 120V for 1hr.
（5）Carefully remove the gel, and take a picture for the gel.
（1）Set up the following 20 μL incubation system:
| Reagents | Concentration |
|---|---|
| Cleavage Buffer | 50 nM |
| FQ probe | 100 nM |
| crRNA | 120 nM |
| Cas protein（Cas12a / Cas14a / CasΦ) | 100 nM |
Dissolved in ddH2O, the mixture was incubated at 37℃ for 30min.
（2）Add following reagents to each protein reaction system after incubation to the certain concentration, and the volume of the overall system is 60μL:
| Reagents | Experimental group | NTC |
|---|---|---|
| Cleavage Buffer | 50 nM | 50 nM |
| Target DNA | 50 nM | - |
（3）Evenly mix the system and add it to 96-hole plates, 20 μL for each. Fluorescence signals were obtained every 2 minutes at 37°C.
Dissolve the dry powders of ssDNA1 and ssDNA2 with PBS and configure them into 240mM solutions.Add ssDNA1 and ssDNA2 in equal proportions and prepared 20μL of each solution with ssDNA concentration of 40uM, 80uM, and 120uM, and the volume difference was complemented with PBS.
Maintain the reaction system at 37 °C for 6h. Examined three sets of samples by 3% agarose gel electrophoresis.
| Ingredient | Dosage |
|---|---|
| Agarose | 0.9g |
| 1 × TAE | 30mL |
Add ssDNA1 and ssDNA2 in equal proportions and prepared 20μL of each solution with ssDNA concentration of 50μM, 100μM, and the volume difference was complemented with PBS. 95℃ to 25℃, One degree per minute, 25℃ for 4hr,Examined two sets of samples by 3% agarose gel electrophoresis.
(1) Add streptavidin to the pre-experimental medium 120mM sample in three tubes, small amount, normal amount and large amount, respectively. Examined three sets of samples by 3% agarose gel electrophoresis.
(2) The group with ssDNA concentration of 50mM in the second experiment was divided into two equal portions. Adding SA according to the proportions in the following table. And examined two sets of samples by 3% agarose gel electrophoresis.
| Group | ssDNA1-biotin:SA |
|---|---|
| 1 | 5:2 |
| 2 | 2:1 |
（1) Configure MgCl2 / NaCl / CaCl2 / NH4Cl / KCl at a concentration of 10mM。Configured 7 reaction systems with the above solutions. As shown in the following table. The temperature conditions for the reaction : 95℃, 10min; 37℃, 110min.
| Groups | Dosage |
|---|---|
| 1 | 1mM Na+ |
| 2 | 1mM K+ |
| 3 | 1mM Na+ and 1mM K+ |
| 4 | 1mM NH4+ |
| 5 | 1mM Mg2+ |
| 6 | 1mM Ca2+ |
| 7 | Negative Control |
(2) On the basis of existing experiments, we reconfigured the reaction solution as showed in the following table. The temperature conditions for the reaction : 95℃, 10min; 37℃, 110min.
| Groups | Dosage |
|---|---|
| 1 | 1mM Mg2+/Ca2+ |
| 2 | 1mM Na+/K+/NH4+ |
| 3 | 1mM Mg2+/Ca2+/Na+/K+/NH4+ |
Examined two sets of samples by 1.5% agarose gel electrophoresis.
| Ingredient | Dosage |
|---|---|
| Agarose | 0.45g |
| 1 × TAE | 30mL |
After adding a variety of cations (as in the above experiments), we continued to adjust the temperature environment of the reaction system, as well as negative control (95℃, 30min; 37℃, 210min), as shown in the following table. Then, examined two sets of samples by 1.5% agarose gel electrophoresis.
| Temperature/℃ | Time |
|---|---|
| 95 | 10min |
| 85 | 10min |
| 80 | 10min |
| 77.5 | 20min |
| 74.5 | 30min |
| 72.5 | 20min |
| 70.5 | 10min |
| 65 | 5min |
| 60 | 5min |
| 50 | 5min |
| 40 | 5min |
| 37 | 230min |
Configure 3 tubes of reaction solution with ingredients in the following table.
The reaction temperature of ssDNA1/2 is consistent with the above table. Then, examined two sets of samples by 1.5% agarose gel electrophoresis.
| Tube 1 | Tube2 | Tube3 |
|---|---|---|
| 1mM Mg2+/Na+/Ca2+/NH4+/K+ | 1mM Mg2+/Na+/Ca2+/NH4+/K+ | 1mM Mg2+/Na+/Ca2+/NH4+/K+ |
| 100mM ssDNA1(NTC) | 100mM ssDNA1/2 | 100mM ssDNA1/2 |
| ssDNA1-biotin:SA= 5:2 | ssDNA1-biotin:SA= 5:2 | ssDNA1-biotin:SA= 5:2 |
| 37℃ / 24h | 37℃ / 24h | 37℃ / 6h |
（1）Configuration of the reaction system:
| Ingredient | Dosage/μL |
|---|---|
| 10 × T4 PNK | 1 |
| 1 × T4 PNK | 1 |
| L2 | 5 |
| ATP(20mM) | 1 |
| ddH2O | 2 |
（2）After the reaction system was configured, place the EP tube in the PCR instrument and carry out the reaction, 37℃, 30min; 70℃, 10min.
（3）Add 5μL L2T to the present reaction system and set the reaction, 90℃, 3min; 65℃, 2min; decrease by one degree per minute after that.
（4）Add the dosage in the following table to the existing reaction system andset the reaction temperature, 37℃, 30min; 70℃, 10min.
| Ingredient | Dosage/μL |
|---|---|
| 10 × T4 ligase | 215 |
| 0.1 × T4 ligase | 1 |
| ATP(20mM) | 1 |
| ddH2O | 615 |
（4）Add the dosage in the following table to 25ul existing reaction system and set the reaction temperature to 4h.
| Ingredient | Dosage/μL |
|---|---|
| 10 × RCA | 5 |
| CasΦ | 1 |
| dNTPs | 5 |
| L2T | 5 |
| ddH2O | 6.5 |
（1）Configure two reaction systems as following 2 tables.
| Ingredient | Dosage/ul |
|---|---|
| RCA Product (4h) | 40 |
| AuNP | 20 |
| MBD-SA | 40 |
（2）React at room temperature and wait for sufficient production of white flocculent. Remove the supernatant, extrace and lyophilize. The freeze-dried samples were photographed in SEM.
MBD-SA was added to the RCA reaction system together with Glu starch, 30°C, 24h, 65°C, 10min.
| Ingredient | Dosage/μL |
|---|---|
| 10 × RCA product | 5 |
| CasΦ | 1 |
| dNTPs | 5 |
| P (0277) | 5 |
| C2 | 2 |
| GCu | 20 |
| ddH2O | 12 |
| Ingredient | Dosage/μL |
|---|---|
| 10 × RCA product | 5 |
| CasΦ | 1 |
| dNTPs | 5 |
| P (0277) | 5 |
| NEB.2 | 5 |
| SAM | 5 |
| M.SSSI | 1 |
| C2 | 2 |
| ddH2O | 21 |
Take appropriate amount of prepared DNA nanosponges and wash with dd, add 10μL of MBD-SA, mix and continue to wash with a small amount of water. Then, add 20μL of the promotion suspension, 20μL of DnaseI and 5μL of Afu Buffer, and react for five minutes at room temperature.
H2O (20ml×2), wash it, add 10μL DnaseI, 10μL Glu starch, 2μL A.B, R.T, 15min.
Take 10μL of DNA nanosponge degradation suspensions, centrifuge at 10000g and remove the supernatant. Add 20μL CasΦ activation fluid (CasΦ+crRNA) and 10μL Glu starch (1mg/ml). Room temperature reaction for one hour and test the blood glucose meter.