**3.4 Hybrid chain reaction (HCR)**

The hybridized chain reaction proposed by Dirks and Pierce in 2004 is an isothermal signal amplification technology based on DNA strand displacement reaction [33]. Single strand promoter DNA (target miRNA) binds to the stemloop nucleic acid probe and causes conformation changing of hairpin DNA. The unfolded hairpin structure can unfold a new DNA hairpin. Two kinds of stem-loop probes were alternately hybridized to form double-stranded DNA containing a large number of repeat units [34]. This method has the advantages of constant temperature, efficient signal amplification, and without the requirement of enzyme. It has been applied to the detection of DNA or RNA.

Wang *et al.* [35] combined CHA with HCR to design a sensing system with six DNA hairpins (**Figure 4**). Target miRNA first catalyzed CHA and formed numerous double-stranded products (H1∙H2) containing initiator sequences to initiate downstream HCR circuit. The resultant dsDNA products then triggered subsequently autonomous cross-hybridization reactions to form HCR copolymer (H3∙H4∙H5∙H6). The resultant HCR copolymer carries many donor-acceptor pairs that can generate FRET signal. The synergistic amplification effect of CHA-HCR system significantly increased the selectivity and sensitivity of miRNA detection.

Exploiting the signal amplification function of protein with multiple binding sites, Huang *et al.* [36] used streptavidin (SA) as a protein scaffold and four biotinylated hairpin DNA probes to construct a DNA tetrads probe. When miRNA was

#### **Figure 4.**

*Schematic of the isothermal CHA–HCR cascaded circuit for miRNAs assay. Adapted with permission from ref 35. Copyright 2018 Royal Society of Chemistry.*

present, miRNA unfolded the Cy3-labeled hairpin H1, triggered HCR, and hybridized with Cy5-labeled H2 to form a cross-linked hydrogel network, generating the signal. It is proved that the DNA tetrad was a highly robust delivery agent and could realize the sensitive imaging of miRNA.

To improved the stability of miRNA sensing system, Wu and coworkers [37] designed a DNA probe composed of tripartite Y-shaped DNA structures, folate probe FAP and hairpin probe H1, H2. MiRNA triggered H1 and H2 hairpin probes to assemble HCR, separating Cy5 and BHQ-2 labeled on H1 to recover fluorescence signal. This method was proved to have high sensitivity with a sub-picomolar limit *in vitro*, and the probe had high stability *in vivo*.

### **3.5 Entropy-driven DNA catalysis (EDC)**

Entropy-driven DNA catalysis (EDC) exponentially amplifies DNA signal by target-induced entropy change of pre-design sensing system [38]. EDC is a simple, rapid, and enzyme-free isothermal signal amplification technology based on toehold exchange mechanism and adaptable to different low-abundance targets due to its modular design and tunability.

A EDC system usually is composed of a three-strand substrate complex (output strand and signal strand are complementary with link strand) and a fuel strand [39]. In the absence of targets, the sensing system does not work because the toehold domain in substrate that binds to the fuel strand has been protected. As a catalyst, miRNA can combine with the substrate link strand to replace the signal strand, and then the fuel strand replaced miRNA to be recycled. The production of liberated molecules leads to the increase of entropy, repeating the abovementioned strand displacement reaction to generate amplified signals.

Liang *et al.* [40] constructed an entropy-driven DNA nanomachine. A threestranded substrate complex (A/B/C) and an affinity ligand (L) were modified on the AuNP surface, respectively. Target miRNA hybridized with L to replace B from C. A fuel strand (F) bound to C and the C-F complex departed from the AuNP surface, restoring the fluorescence of the FAM-labeled C strand. Thus, such entropydriven catalytic DNA nanomachine operated automatically and progressively to realize signal amplification. The assay had superior sensitivity (LOD = 8 pM) due to the accelerated intramolecular reaction.

To avoiding the addition of external enzyme or fuel transfection, Lu *et al.* [41] developed a NIR-controlled DNA sensing system based on entropy-driven catalysis to detect intracellular miRNA. Hollow copper sulfide nanoparticle (HCuSNPs) served as the photothermal conversion agent and a carrier. An entropy-driven DNA probe and DNA fuel were conjugated to HCuSNPs. Under the irradiation of the near-infrared laser, target miRNA-155 recognized toe1 and combined with the probe, replaced Cy3-DNA and exposed toe2 that initiates toehold-mediated strand displacement reactions. Cy3-DNA was released and its fluorescence was recovered. This method possessed facile design and its sensitivity is two orders of magnitude higher than that of molecular beacons (MBs).

#### **3.6 DNAzyme-mediated assays**

DNAzyme is a kind of DNA with catalytic function and structure recognition ability. It was screened by Breaker and Joyce through the systematic evolution of ligands by exponential enrichment (SELEX) technology in 1994. The single strand, simulating the function of enzymes *in vivo*, can catalyze different chemical reactions, including nucleic acid cleavage, nucleic acid ligation, phosphorylation, porphyrin metallization enzyme activity, and peroxidase activity. It has high

#### *Novel Biosensing Strategies for the* in Vivo *Detection of microRNA DOI: http://dx.doi.org/10.5772/intechopen.93937*

catalytic efficiency, simple modification of fluorescent dyes, and strong chemical stability. Also, compared with the traditional protease, DNAzyme can be denaturated repeatedly and renatured without loss of enzyme activity. DNAzyme catalytic amplification technology is a constant temperature amplification technology, which is especially suitable for high sensitivity detection of intracellular targets.

Wu *et al.* [42] constructed a signal-enhanced split DNAzyme probe loaded on gold nanoparticles for miRNA detection in living cells. They split Mg2+-dependent DNAzyme into two nucleic acid strands, which were hybridized with the substrate to form a complex. The fluorescence of the complex was quenched without target miRNA. In the presence of miRNA, two split strands hybridized with target miRNA to form a secondary structure with catalytic activity, cleaving the substrate to separate fluorescence reporter and quenching groups and restoring fluorescence. The released miRNA targeted the next DNAzyme probe and switched on recognitioncleavage-release cycles to produce signal amplification. In this experiment, split DNAzyme serves as both recognition element and signal reporter. As a carrier, gold nanoparticles increase the biological affinity of nanoprobe and avoid the degradation of the nucleic acid probe in the process of transport into cells. This method improved the detection sensitivity and specificity. Additionally, it had low cytotoxicity, high enzymatic degradation resistance which is effective for detection in living cells.

Yang *et al.* [43] integrated DNAzyme, its substrate, and recognition strand into a FAM-labeled hairpin-locked-DNAzyme probe. The probe was immobilized on surface of gold nanoparticles. The catalytic activity of DNAzyme was inhibited and the fluorescence of FAM was quenched by gold nanoparticles. When target miRNA hybridized with the hairpin probe, the change of the probe structure activated the DNAzyme to cleave the substrate strand and made the FAM-labeled substrate strand emit fluorescence. After the miRNA was released, it entered the next cycle and generated amplification signals. This design significantly reduced fluorescent background signal. The detection limit of the target miRNA was 25 pM. It can be applied to the *in vivo* detection of different types of miRNA.

Although AuNP-DNA probes are highly sensitive and selective, they suffer from the aggregation of AuNPs in the complex intracellular environment. To overcome this limitation, there is highly desirable for homogeneous DNA (composed entirely of DNAs) sensing system. Xue *et al.* [44] utilized a Y-shaped backbone-rigidified triangular DNA scaffold (YTDS) to develop a self-powered DNAzyme walker (**Figure 5**). This sensing platform consists of YTDS (carrier), nuclide aptamer (transportation), and a locked M-DNAzyme-substrate complex (recognition and signal reporter). The binding of miRNA trigger DNAzyme walker to perform selfpowered stepwise walking and amplify the signal at the same time. The detection platform has the advantages of efficient delivery without any transfection agent and amplification of output signal without any protein enzyme.

To effectively protect the probe from degradation by nuclease and greatly improve its cell permeability, Li *et al.* [45] constructed a DNAzyme probe based on the tetrahedral nanostructure. FAM and Dabcyl were labeled the linker strand and partial complementary strand, respectively. Catalytic activity of DNAzyme was effectively silenced by the locking strand in the absence of target miRNA. The target miRNA hybridized with the locking strand to release DNAzyme. With the assist of Na+ cofactor, the substrate strand was cleaved and the fluorescence was recovered. Activated DNAzyme could compete with inactive DNAzyme for the next locking strand, starting the next hybridization, and generating amplified signals circularly. The LOD of the DNAzyme probe is 16 pM. It possessed high specificity and distinguished target miRNA from its family members.

The catalytic activity of DNAzyme depends on the concentration of its cofactor Mg2+. However, the content of Mg2+ in the cell is too low to support the long-time

**Figure 5.**

*(A) The preparation routes of Ap-YTDS-DzW. (B) Ap-YTDS-DzW imaging of miRNAs in vivo. Reprinted with permission from ref 44. Copyright 2019 American Chemical Society.*

catalytic reactions used for the signal amplification. To circumvent this limitation, Wei *et al.* [46] constructed a smart autocatalytic DNAzyme imaging machinery to execute magnetic resonance imaging (MRI) of miRNA *in vivo*. The imaging system composed of honeycomb-like MnO2 nanosponge (hMNS), HCR, and autocatalytic DNAzyme. In this system, hMNS act as three roles, that is, nanocarrier, DNAzyme cofactors, and MRI agents. The multifunctional hMNS effectively delivered the system into the cells and was degraded into Mn2+ by intracellular glutathione (GSH) as a DNAzyme cofactor. MiR-21 activates HCR amplification to produce DNAzyme nanowires, mediating the automatic catalytic accumulation of the new trigger and the retroaction to the original HCR sensor. This robust assay can accurately locate miRNAs *in vivo* and enhance the amplification signal.
