Experiment Period：2024.5.1-2024.5.15

Experimenters: Ruiying Wang, Lanxin Huang

Experiment Purpose:

To verify the feasibility of target participation in DDSD self-enhancement system.

Experimental Procedures:

(1) Sample preparation (20 uL)

(2) Electrophoresis

Experiment Period：2024.4.7

Experimenters: Chu Zibin, She Ao, Wang Ruiying, Huang Lanxin, Cai Leyan

Experiment Purpose:

To prepare materials and equipment for subsequent characterization of the single-molecule platform.

Experimental Procedures:

1. Slide Preparation

2. Single-Molecule Microscope (Use in Complete Dark Room)

Experimental period: 2024.4.9-2024.5.28

Experimenters: She Ao, Liu Yiqun, He Tianru, Wang Junlan, Wang Ruiying, He Zitong, Wang He, Zhang Jinling, and Chu Zibin.

Experimental purpose:

1. Mimicking the amplification capacity of a fluorescence self-enhanced primary amplification system based on polymerase chain substitution reaction by synthesizing targets.
2. Verification of the ability of qPCR fluorescence to character.
3. The whole process of detection was simulated at the synthetic target level, and the detection capability, sensitivity and applicability of the detection system were verified by starting different concentrations of target molecules.

Experimental procedure:

(1) Construction of a fluorescence self-enhanced primary amplification system based on polymerase chain substitution reaction

Specifically, the reaction process is as follows: the target chain I and the receiving probe Rep complete the chain substitution reaction under the catalytic action of DNA polymerase Bst to generate Rep-I and H, and the H chain further completes the chain substitution reaction with the digital probe DDSD under the catalytic action of DNA polymerase Bst to generate DDSD-H, the weighted chain wt and the target chain I, and the regenerated target chain I continues to participate in the above reaction to realize the first-stage self-enhancement; furthermore, the generated weighted chain Wt continues to have chain substitution reaction with RP to separate the fluorescent group from the quenching group to realize the self-enhancement. Further, the generated weight chain Wt and RP continue to undergo the chain substitution reaction to separate the fluorescent group from the quenching group to realize self-enhancement of fluorescence, and at the same time, there exists a quantitative relationship between the starting target concentration and the final number of fluorescent molecules.

(2) Determination and dilution of nucleic acid content of samples required for the system

1. Centrifuge tubes containing the DNA molecule powder synthesized by the company are briefly centrifuged before opening, appropriate amount of enzyme-free water is added according to the markings on the tube, mixed, and diluted 20-fold in a new tube.
2. Nano Ultra Trace Nucleic Acid Protein Detector before using the upper and lower lenses each using non-enzymatic water rinse wash 3 times, wipe the mirror paper dry, repeat 3 times.
3. A blank of $1.5\,\mathrm{μL}$ enzyme-free water was taken and wiped clean with microscope paper after calibration.
4. Take the DNA dilution, make 3 parallel measurements at A260 to obtain the mean value and calculate the concentration (formula: concentration $=\cdot$mean value*dilution*nm number/OD value)
5. After the measurement, wash the upper and lower lenses 3 times, and turn off the instrument.
6. A batch of 10 μM nucleic acid samples to be used is prepared by diluting each tube with enzyme-free water according to the concentration of DNA molecules in each tube.A batch of $10\,\mathrm{μM}$ nucleic acid samples to be used was prepared..

(3) The system probes were prepared (all in microliters, the initial concentration of each sample was $10\,\mathrm{μM}$)

1. Probe spiking

A	SA1	A1	buffer	H2O
	6	10	5	29
B	3BD*	ND	NA4*	buffer	H2O
	10	24	8	5	3
	2BD*	ND	NA4*	buffer	H2O
	10	16	8	5	11
	1BD*	ND	NA4*	buffer	H2O
	10	8	8	5	19
F-a	fluorescent probe1	fluorescent probe2	H2O	Buffer
	10	10	25	5

After adding the sample, put it into the PCR instrument for annealing operation, and then put it into the $4^{\circ}\mathrm{C}$ for preservation.

2. Dilution of $\mathrm{NA4^{*}.}$ into different concentration gradients

Concentration gradient1:

1	2	3	4	5	6	7
25 nM	12.5 nM	6.25 nM	3.125 nM	1.56 nM	0.78 nM	Blank

Concentration gradient2:

1	2	3	4	5	6	7
500 nM	250 nM	125 nM	62.5 nM	31.25 nM	15.625 nM	Blank

3. Polymerase self-reinforcing binding chain substitution system

A blank of $1.5\,\mathrm{μL}$ enzyme-free water was taken and wiped clean with microscope paper after calibration.

A	B	F-α	NA4*	dNTP	buffer	Bst3.0	H20
1	1	1	1	1	2	1	12

System spiking: sequentially add A, B, different concentration gradients of $\mathrm{NA4^{*}.}$ dNTP, buffer, Bst3.0, H2O, put into the PCR instrument after adding, select $37^{\circ}\mathrm{C}$ program, keep warm for 30 min, then add F-a and carry out Q-PCR.(3 parallels are made for each $\mathrm{NA4^{*}.}$ dilution)

(4) Q-PCR Detection

1. Setup Program: Open Rotor-GeneQ software and set q-PCR reaction conditions: annealing temperature ($25^{\circ}\mathrm{C}$), cycle period (5s), fluorescence type, Gein value.
2. Sample preparation: Add F-α sequentially to the pcr tubes after the above reaction, centrifuge, and mix the liquid well
3. Quickly load the centrifuge tube into the q-PCR instrument and start running the instrument.
4. Record the data and analyze the results.

Experiment result:

Experimental period: 2024.4.7-2024.5.10

Experimenters: She Ao, Chu Zibin, Wang Ruiying, He Zitong

Experimental purpose:

1. To simulate the amplification ability of fluorescence self-enhanced secondary amplification system based on crispr system and polymerase chain substitution reaction by synthesizing targets, and to realize highly sensitive detection of target nucleic acid sequences.
2. Using QPCR assay to verify the amplification effect under the participation of different concentrations of targets.
3. Simulate the whole process of detection at the level of synthetic targets, and verify the detection capability and applicability of the detection system by starting different concentrations of target molecules.

Experimental procedure:

(1) Construction of fluorescence self-enhanced secondary amplification system based on crispr system and polymerase chain substitution reaction

The specific reaction process is as follows: the target chain I initially completes the chain substitution reaction with the receiver probe Rep under the catalytic action of DNA polymerase Bst to form Rep-I and H; the H chain further completes the chain substitution reaction with the digital probe DDSD under the catalytic action of DNA polymerase Bst to form DDSD-H, the weighted strand wt, and the target strand I, and the regenerated target strand I continues to take part in the above reaction, realizing the first-stage self-enhancement; furthermore, the generated weighted strand wt and crRNA jointly activate the cas12a enzyme paracrine activity. Further, the generated weight chain wt and crRNA together activate the side activity of cas12a enzyme, and efficiently shear the fluorescent chain RP, so that the fluorescent group and the quenching group are separated to show fluorescence. Secondary self-enhancement was realized, and there was a quantitative relationship between the starting target concentration and the number of final fluorescent molecules.

(2) Determination and calculation of nucleic acid concentration

1. Nano Instrument Procedure

1. Mix the DNA tubes synthesized by the company and centrifuge them (12k, 2 min), remove them smoothly, add appropriate amount of non-enzymatic water according to the markings on the tubes, mix them well, and dilute them 20 times in the new tubes.
2. Wash the upper and lower lenses of the Nano instrument three times with enzyme-free water and dry them with microscope paper, and repeat the process 2-3 times.
3. Take 1.7 μL of water for blank calibration, and wipe with mirror paper after calibration.
4. Take the DNA dilution, measure it 3 times in parallel at A260 wavelength to get its average value and calculate the concentration, the formula is: concentration $=\cdot$ average value*dilution $^*\mathrm{nm}$ number/OD value.
5. After the measurement, wash the upper and lower lenses 3 times, and exit the homepage.

2. The absorbance measured using the Nano instrument for partial detection of concentration is shown in the table below:

PS: Dilutions of 20$\times$, $\mathbf{nm}$ and OD values can be checked on the DNA tube.

1. Preparation of Rep and DDSD chains

After spiking, put the sample into the PCR instrument for annealing, and then put it into the $4^{\circ}\mathrm{C}$ for storage.

2. Gradient dilution of target chain $\mathrm{NA4^{*}}$ (I)

3. Preparation of CrRNA dilution solution

Dilute CrRNA to the appropriate concentration with ultrapure water in an ultraclean bench.

4. Mixing of Cas-12a enzyme with crRNA

In an ice box, we premixed the cas-12a enzyme solution with the crRNA solution at a volume ratio of 1:2 and left it in the ice box for a while.

5. System spiking (3 parallels per NA4* dilution)

1. First, polymerization, add H2O, different dilutions of $\mathrm{NA4^{*}}$, dNTP, A, B, R2.1buffer, Bst3.0, centrifugation and mixing. (without Cas12a, CrRNA and RP first), PCR $37^{\circ}\mathrm{C}$ reaction for 30 min.

2. Mix Cas12a and CrRNA 1:2 in advance to form $3\mathrm{μL}$ (note the use of an ice box).

3. Add RP and Cas12a and CrRNA mixture to the wall.

4. QPCR reaction is performed immediately after uniform centrifugation.

   (4) qPCR procedure

1. Remove the sample rack from the PCR instrument, place it in the freezer to cool down, and place the PCR tubes (be careful to use the appropriate size of PCR tubes $20\mathrm{μL}$.);

2. Open the Rotor-Gene Q software and click New to create a new file. 3;

3. Select Locking Ring Attached and fill in the volume $20\mathrm{μL}$; 4. Click Edit Profile.

4. Click Edit Profile to define the reaction conditions. 5:

5. Include: Annealing Temperature ($25~^{\circ}\mathrm{C}$), Cycles per second (usually 5 s), Number of cycles.

6. Edit the fluorescence type (ROX), click “OK” to confirm, and click Edit Gain to edit the Gain value to 9. 7. Click run start, the run start screen appears;

7. Click run start, the file storage location will appear, name the file;

8. After loading the tubes into the instrument, click “Save” and the machine will run.

Caveat:

1. The QPCR instrument should be loaded as quickly as possible.

2. Since the QPCR Instrument does not tolerate high temperatures, the sample racks should be frozen in the freezer before use.

3. Unused wells in the sample rack should be filled with an equal volume of nuclease-free water or other suitable buffer.

• qPCR instrument use process.

• Take out the sample rack of the PCR instrument, place the PCR tubes, and put them in the freezer to cool down (pay attention to choose the right size of PCR tubes $20\mathrm{μL}$/$50\mathrm{μL}$).

• Open Rotor-Gene Q, click New to create a new file.

• Select Locking Ring Attached and fill in the volume ($20\mathrm{μL}$/$50\mathrm{μL}$).

• Click Edit Profile boundary reaction conditions. Include: annealing temperature ($25^{\circ}\mathrm{C}$), cycles per second (usually 5s), number of cycles.

• Edit the fluorescence type (ROX, etc.), click OK to confirm, click Edit Gain to edit the Gain value.

• Click rum start, the file storage location will appear, name the file.

• After loading the tube into the instrument, click “Save”, the machine will run.

Results:

Experimental period: 2024.5.1-2024.6.30

Experimenters: She Ao, Wang Ruiying, Chu Zibin, Wang Junlan, He Zitong

Experimental purpose:

1. To evaluate the amplification capability of a fluorescence self-amplifying secondary amplification system based on the CRISPR system and polymerase chain displacement reaction by synthesizing target simulations.
2. To validate the detection capability of the single-molecule platform and the analytical capacity of downstream data processing programs.
3. Simulate the entire detection process at the synthesized target level, validating the detection system's capability and applicability by using starting target molecules at different concentrations.

Experimental procedure:

(1) Construction of a fluorescence self-amplifying secondary amplification system based on the CRISPR system and polymerase chain displacement reaction.

The specific reaction process involves target strand I initiating a strand displacement reaction with the receptor probe Rep under the catalysis of DNA polymerase Bst, producing Rep-I and H. The H strand then undergoes another strand displacement reaction with the digital probe DDSD, also catalyzed by DNA polymerase Bst, generating DDSD-H, the weight strand wt, and target strand I. The regenerated target strand I continues to participate in the above reactions, achieving primary self-amplification. Furthermore, the generated weight strand wt, together with crRNA, activates the auxiliary activity of the Cas12a enzyme, efficiently cleaving the fluorescent strand RP to separate the fluorophore from the quencher, resulting in fluorescence. This achieves secondary self-amplification, with a quantitative relationship between the initial target concentration and the final number of fluorescent molecules.

(2) Determination and dilution of nucleic acid content required for the system.

1. Before opening the centrifuge tube containing the DNA oligonucleotide powder synthesized by the company, briefly centrifuge it. Add an appropriate amount of nuclease-free water as indicated on the tube, mix well, and dilute it 20 times into a new tube.
2. Before using the NanoDrop micro-volume nucleic acid and protein detector, rinse both the upper and lower lenses with nuclease-free water three times, then wipe them dry with lens paper, repeating this process three times.
3. Take 1.5 μL of nuclease-free water for blank calibration, and after calibration, wipe the lenses clean with lens paper.
4. Take the DNA dilution solution and measure it in parallel three times at a wavelength of A260 to obtain the average value, then calculate the concentration using the formula: concentration = average value * dilution factor * nm number / OD value.
5. After measurement, rinse the top and bottom lenses three times with water, then turn off the instrument.
6. Dilute each tube of DNA solution using nuclease-free water to prepare a batch of nucleic acid samples with a concentration of 10 μM.

The absorbance values measured using the Nano instrument for concentration detection are shown in the table below:

PS: Dilution factor20×,The nm value and OD values can be checked on the DNA tube.

(3) Preparation of the system probes (all units are in microliters, and the initial concentration of each sample is 10 μM)

1. Preparation of Rep and DDSD chains: Before opening the centrifuge tube containing the DNA oligonucleotide powder synthesized by the company, briefly centrifuge it. Add an appropriate amount of nuclease-free water as indicated on the tube, mix well, and dilute it 20 times into a new tube.
2. Gradient dilution of the target strand NA4*(I):

1	2	3	4	5	6	7	8	9	10
100 nM	50 nM	25 nM	12.5 nM	6.25 nM	3.125 nM	1.5625 nM	0.7813 nM	0.3906nM	Blank

3. The preparation of CrRNA dilution solution: Use ultrapure water in a clean bench to dilute CrRNA to the appropriate concentration.
4. Mixing Cas12a enzyme with crRNA: Take the DNA dilution solution and measure it in parallel three times at a wavelength of A260 to obtain the average value, then calculate the concentration using the formula: concentration = average value * dilution factor * nm number / OD value.
5. Sample addition to the system (perform three replicates for each NA4* dilution): Components of the fluorescence self-amplifying secondary amplification system based on the CRISPR system and polymerase chain displacement reaction are as follows (all units in μL):

NA4*	A	B	dNTP	R2.1buffer	Bst3.0	Cas12a	CrRNA	RP	H2O
1	1	1	1	2	1	1	2	9	9

Step 1: Sample addition (add to the wall of PCR tube): H2O 9 + different dilution NA4* 1 + A 1 + B 1 + dNTP 1 + R2.1 buffer 2 + Bst 3.0 1; briefly centrifuge to mix evenly, then incubate at 37°C for about 30 minutes.

Step 2: Take 3 μL of the pre-mixed Cas12a enzyme and crRNA solution, add it to the PCR tube (gently pipette a few times), and start timing for 100 seconds. After the time is up, immediately add 5 μL of EDTA solution (gently pipette a few times) to terminate the reaction.

6. Single-molecule platform characterization: Dilute each tube of DNA solution using nuclease-free water to prepare a batch of nucleic acid samples with a concentration of 10 μM.

a. Sample preparation for detection: Prepare the detection slide, attach the detection chamber, inject 25 μL of the solution to be tested, and let it sit for about 30 minutes.

b. Single-molecule platform preparation: Sequentially power on the single-molecule platform equipment and keep it in standby mode.

c. Sample characterization: After loading the sample, adjust the single-molecule platform for optimal clarity. Randomly select three fields of view for each sample chamber and capture 100 continuous images per selected view. Use the self-developed fluorescence spot analysis software to analyze the images and calculate the average number of fluorescence spots.

d. Generate the detection report.

Experimental results:

Experimental period: 2024.4.22-2024.6.18

Experimenters: She Ao, He Zitong, Wang Ruiying, Wang He and Zhang Jinling.

Experimental purpose:

The model cancer cells were recovered, expanded, cultured and passaged, microRNA extracted and NANO nucleic acid content determined for subsequent testing.

Experimental procedure:

(1) Cell recovery:

1. Preheat the culture medium 30 min in advance (37℃ water bath).
2. Find the desired cells in the liquid nitrogen tank.
3. Add 1 mL of culture medium to the centrifuge tube.
4. Take the frozen tube into the water bath and thaw it quickly.
5. Transfer the frozen cells to the centrifuge tube and add 1 mL of medium to rinse the inside.
6. Blow the 2 mL centrifuge tube and centrifuge (3 min, manually close).
7. Take the aeration bottle, open it up next to the flame, and add 4 mL of medium into it.
8. Take the centrifuged tube, pour off the supernatant and wash the residual liquid with a gun tip.
9. Add 1 mL of culture medium and blow the suspension.

(2) Expansion and Passage:

Cultivate the above cells in a suitable temperature and gas environment until the cells grow against the wall and reach a certain density. When the cells covered most of the culture surface, the cells were treated with a digestive enzyme such as trypsin to detach them from the surface of the vessel and form a cell suspension. Afterwards, the cell suspension was transferred to a new culture vessel, replenished with fresh medium, and the culture continued. The cell growth status was regularly observed and the medium was changed as needed to maintain healthy cell growth. When the cell density reaches saturation again, repeat the above passaging process. The whole expansion process needs to strictly follow the aseptic operation procedures to prevent contamination.

(3) microRNA extraction:

Anhydrous ethanol should be added to the rinsing solution RW and deproteinizing solution MRD before the first use, please refer to the label on the bottle for the amount to be added.

(4) Extraction of miRNA-enriched fraction in tissues or cells.

1. Sample processing

a.Tissue:Grind the tissue in liquid nitrogen. Add 1 ml of lysate MZ for every 30-50 mg of animal tissue or 100 mg of plant tissue and homogenize with a homogenizer. The sample volume should not exceed one-tenth of the volume of lysate MZ.

b. Monolayer culture cells: Lysate cells directly in the culture plate by adding Lysate MZ, add 1 ml MZ per 10 cm2 area, and pump several times with a sampler.

Note:The amount of lysate MZ added is determined by the area of the culture flask, not by the number of cells. Insufficient addition may result in DNA contamination in the extracted RNA.

c. Cell suspension: Centrifuge the cells at 2100 rpm (400×g) for 5 min and discard the supernatant. Add 1 ml of lysate MZ and mix by shaking with an oscillator or pipetting several times. Do not wash the cells before adding lysate MZ to avoid degradation of mRNA.

2. Transfer the liquid in the tube to the culture flask, cover it, label it (cell name - your name - date), and put it into the incubator.
3. Leave the homogenized sample at room temperature for 5 min to allow complete separation of the nucleic acid-protein complex.
4. Optional: Centrifuge the sample at 12000 rpm (~13400×g) at 4°C for 5 min, remove the supernatant and transfer to a new RNase-free tube.

Note: If the sample contains more proteins, fats, polysaccharides or muscle, plant nodules, etc., they can be removed by centrifugation in this step. The precipitate obtained by centrifugation includes extracellular membranes, polysaccharides, high molecular weight DNA, and RNA is present in the supernatant solution.


5. Add 200 μl of chloroform, cap the tube, shake vigorously for 15 sec, and leave for 5 min at room temperature.
6. Centrifuge the sample at 12,000 rpm (~13,400×g) at 4°C for 15 min. The sample will be divided into three layers: a yellow organic phase, an intermediate layer, and a colorless aqueous phase, with the RNA predominantly in the aqueous phase, which is about 50% of the volume of the lysate used, MZ Reagent. Transfer the aqueous phase to a new tube and proceed to the next step.
7. Measure the volume of the transfer solution, slowly add 0.43 times the volume of the transfer solution of anhydrous ethanol (e.g.: 500 μl of the transfer solution plus 215 μl of anhydrous ethanol), mixing (at this time, precipitation may occur). Transfer the obtained solution and precipitate together to the adsorption column miRspin, centrifuge at 12,000 rpm (~13,400 × g) for 30 sec at room temperature, if all the solution and mixture cannot be added to the adsorption column miRspin at one time, please transfer it in two times, after centrifugation, discard the adsorption column miRspin, and keep the effluent.
8. Measure the volume of effluent, slowly add 0.75 times the effluent volume of anhydrous ethanol (e.g., 700 μl of effluent plus 525 μl of anhydrous ethanol), and mix well (at this time, a precipitate may appear). Transfer the obtained solution and precipitate together into the adsorption column miRelute, centrifuge at 12000 rpm (~13400×g) for 30 sec at room temperature, if you can not add all the solution and mixture into the adsorption column miRelute at one time, please transfer it in two times, and discard the effluent after centrifugation, keep the adsorption column miRelute.
9. Add 500 μl of deproteinizing solution MRD into the adsorption column miRelute (please check whether ethanol has been added first), let it stand for 2 min at room temperature, and then centrifuge it at 12000 rpm (~13400×g) for 30 sec at room temperature, and discard the waste liquid.
10. Add 500 μl of rinsing solution RW (please check whether ethanol has been added first) to the adsorption column miRelute, let it stand for 2 min at room temperature, centrifuge at 12000 rpm (~134,00×g) for 30 sec at room temperature, and discard the waste solution.
11. Repeat step 9.
12. Place the adsorbent column miRelute into a 2 ml collection tube and centrifuge at 12,000 rpm (~13,400 × g) for 1 min at room temperature to remove residual liquid.

Note: The purpose of this step is to remove the residual rinse solution from the adsorbent column. After centrifugation, leave the adsorbent column miRelute at room temperature for a few moments or place it on an ultra-clean benchtop and ventilate for a few moments to allow sufficient drying. If there is any rinse solution left, it may affect the subsequent RT and other experimental operations.

13. Transfer the adsorption column miRelute into a new RNase-Free 1.5 ml centrifuge tube, add 15-30 μl of RNase-Free ddH20, leave it at room temperature for 2 min, and centrifuge it at 12,000 rpm (~13,400×g) for 2 min at room temperature.

Note: The volume of elution buffer should not be less than 15 μl, and too small a volume will affect the recovery efficiency. The RNA should be stored at -70℃ to prevent degradation. Note: If you want to increase the RNA yield, repeat the above step once.

(5) Determination and calculation of nucleic acid concentration (NANO):

1. Mix the RNA tube and centrifuge it, add appropriate amount of water according to the marking on the tube, mix it well and dilute it 20 times in a new tube.
2. Wash the upper and lower lenses of Nano with water three times each, wipe dry with microscope paper, and repeat three times.
3. Take 1.5 μl of water to do blank correction, wipe the mirror paper clean after correction.
4. Take the RNA dilution solution, measure it 3 times in parallel at A260 wavelength to get its average value and calculate the concentration (the formula is: concentration=average valuedilution timesnm number/OD value).
5. After the measurement, wash the upper and lower lenses 3 times, and exit the homepage.

Experimental period: 2024.6.1-2024.6.15

Experimenters: Ao She, Ruiying Wang.

Experimental purpose:

1. To validate the fluorescence amplification ability of the secondary amplification system at the cellular level using model cancer cells.
2. To verify the feasibility and accuracy of single-molecule platform characterization and data analysis at the cellular level.

Experimental procedure:

Preparation of system probes (all units are microliters, and the initial concentration of each sample is 10μM).

Preparation of Rep and DDSD chains:

After the addition of samples into the PCR instrument for annealing operation, and then placed in 4 ℃ storage.

(1) Sample preparation for model cancer cell assay (NA4*)

Prepare microRNA extracts of model cancer cells with known microRNA concentration prepared in the previous part and dilute them accordingly.

(2) Mixing of Cas-12a enzyme with crRNA

In the ice box, we pre-mixed the cas-12a enzyme solution with the crRNA solution at a volume ratio of 1:2 and left it in the ice box temporarily

(3) System spiking

The components of the fluorescence self-enhanced secondary amplification system based on the Crispr system and polymerase chain substitution reaction were as follows (all units are μL):

1. The first step of adding samples: (PCR tube wall adding samples) H2O 9+NA4* 1+A 1+B 1+dNTP 1+R2.1buffer 2+Bst3.0 1; briefly centrifuged to mix uniformly and then polymerized at 37 ℃ for about 30 min.
2. The second step of adding samples: Aspirate 3 μL of Cas12a enzyme and crRNA solution mixed proportionally, hit into the PCR tube (gently blow a few times) at the same time to start the clock for 100 s, and immediately after the time is over, add 5 μL of EDTA solution (gently blow a few times) to terminate the reaction.

(4) Single-molecule platform characterization

1. Preparation of test samples: Prepare test slides - paste the test chamber - inject 25 μL of the solution to be detected - leave it for about 30 min.
2. Single-molecule platform preparation: Start the single-molecule platform instrument in sequence and keep it in the ready-to-use state.
3. Sample characterization: After loading the samples, adjust the single-molecule platform to the best clarity, randomly select three clear fields of view for each sample chamber for dynamic photography (each selected field of view for 100 consecutive shots); use self-developed numerical analysis software to analyze the image and derive the average number of fluorescent dots.
4. Derive the test report

Experimental results:

hsa-mir-21 target test cell sample:

hsa-mir-141 target test blood sample: