Our enzymatic reaction system (Fig1) is designed as follows:         The reaction system consists of two parts, A and B.
        There are two biological elements in the reaction module A. The first one is the DSN enzyme, a nuclease that degrades the DNA strand in DNA-RNA hybrid double strand but has no effect on single-stranded nucleic acid molecules. The enzyme was fixed on a honeycomb bracket at the bottom of tube A by immobilized enzyme technique (see hardware).. The second biological element is a nucleic acid probe that identifies specific microRNAs in the blood. The probe is a linear DNA-RNA chimeric probe (later called probe-α_LP).         After the target miRNA in the collected blood enters reaction module A, it will bind to the complementary region in the probe and form a locally hybrid double strand of DNA/RNA through base complementary pairing. Subsequently, duplex-specific nuclease (DSN) will cleave DNA in DNA-RNA hybrid duplex, re-releasing the microRNA and a complete sgRNA sequence will be released. The released microRNA can bind to a new probe for a new round of reaction. The released sgRNA sequence then self-folds to form a functional sgRNA, which will enter the reaction module B for the next reaction.
        In reaction module B there are Cas12a protein to be activated, assistant DNA to activate Cas protein and inhibit DNA/sgRNA hybrid double strand, which we call scgRNA-F. There are two small protruded single strands in the DNA that serve as the cleavage site for Cas12a, and fluorescence and fluorescence quenching groups are on both sides of the cleavage site. When the sgRNA in reaction module A enters reaction module B, sgRNA forms a ternary complex with Cas12a and assistant DNA. The complex trans-cleave the inhibit DNA's prominent structure, releasing the sgRNA sequence, while the fluorophore and fluorescence quenching groups separate to emit fluorescence. SgRNA then combines with Cas12a to be activated to carry out a new round of reaction and achieve exponential amplification.

        The parts of our system can be roughly divided into two categories: nucleic acids and proteins. The linear fragment of nucleic acid is short and the sequence is known, so it is synthetic. We groped the dosage of enzymes (see experiments & results) and specified the reaction conditions according to the application scenario and the nature of the enzyme. The most difficult part of constructing this system is the design of probe and reaction system composition. After the system is built, 10E-4nM, 10E-2nM, 10E-1nM and 1nM miRNA were added into the reaction system and fully combined with probe- α _ LP, and then sgRNA was released by DSN enzyme cleavage. Cas12a captured the sgRNA signal and conducted the CONAN system reaction to release the fluorescence signal. (Fig3)

        Unfortunately, the results showed that there is no significant difference in the output signal when the system detects different concentrations of miRNA. The rule is also irregular, and even the curve of low concentration group is above the curve of high concentration group, which means we cannot correctly distinguish different concentrations of input microRNA.
        We analyzed the reason for the poor effect of the first time, and speculated that it’s the functional redundancy of the components of the CONAN system that led to the poor detection effect between groups. In order to change the detection object from DNA to RNA and further optimize the detection threshold, we introduced the DSN enzyme and probe- α. We found that both scgRNA-F and the introduced probe can undertake the task of exponential amplification, and probe- α can also realize the linear amplification, so the existence of scgRNA-F is superfluous. Therefore, we removed the scgRNA-F from the CONAN system and launched our DRJ system.

        To address the inherent flaws of the previous generation system, we redesigned the B module. We removed the scgRNA-F from CONAN system and replaced it with our probe. In this new system, when probe is absolutely excessive, it can realize both linear and exponential signal amplification, and give full play to its function as a component. In this way, the signal amplifies linearly and exponentially through probe identification and Cas12a activation. In addition, we added an additional short single-stranded nucleic acid with a fluorescence quenching group and a fluorophore. When Cas12a is activated, the single-stranded nucleic acid will be cleaved and the fluorescence signal will be released. (Fig 4)

        We replaced scgRNA-F with probe-α_LP and built a new system. We used this system to detect different concentrations of miRNA. MiRNA with the concentration of 10nM, 1nM and 10E-1nM was added into the system respectively and fully combined with probe-α_LP, and then sgRNA was released by DSN enzyme cleavage. Cas12a captured the sgRNA signal and conducted the DRJ system reaction to release the fluorescence signal(Fig5).

        Unfortunately, there was no significant difference between different gradients, and the system still didn’t not correctly reflect the amount of miRNA input. We analyzed the results and doubted the effect of sgRNA with DNA tail, so we decided to use pure RNA sgRNA in the new round of project.

        We used this system to detect different concentrations of miRNA. MiRNA with the concentration of 100nM, 1nM and 10E-2nM was added into the system respectively and fully combined with probe- β _ LP, and then sgRNA was released by DSN enzyme cleavage. Cas12a captured the sgRNA signal and conducted the DRJ system reaction to release the fluorescence signal.(Fig10)
        It can be seen from the diagram that the system can correctly reflect the correct relationship of different input concentrations of miRNA, and has a better improvement than the previous three iterations, which indirectly showed that circular probe improves the work efficiency of DSN. At the same time, we found that there is still a large gap in the curves between 10E-2nM miRNA and 0Nm miRNA, indicating that under our laboratory conditions, the theoretical threshold of this system is lower than that of 10E-2nM, which is an exciting progress compared with the initial threshold of 10E-1nM. At this point, we have successfully constructed a system that can detect specific miRs. The detection system uses fluorescence as the output signal and can also work outside the laboratory with the corresponding hardware. In the next step, we hope to use color changes visible to the naked eye as the output signal and have made efforts to this end. （see experiments & results and Proposed implementation）