**2. Overview of traditional miRNA detection methods**

#### **2.1 Northern blotting**

Northern blotting, invented by Alwine in 1979, is the first established method to identify and detect miRNA. It is widely used to detect the expression of miRNAs of various sizes from long primitive miRNAs to mature miRNAs. In the process, the miRNA was separated by polyacrylamide gel electrophoresis in total RNA, then transferred to the imprinted membrane, hybridized with the radionuclide labeled probe. The RNA molecule of interest is detected by the signal of labeled probe. This method can detect both the quantity and the length of miRNA, but it has some defects such as cumbersome operation, low sensitivity, time-consuming, and large sample consumption, which limits its application in clinical diagnosis.

To improve the detection sensitivity, Válóczi *et al.* [2] employed locked nucleic acid (LNA) probe instead of traditional oligonucleotide probe to enhance the affinity and stability of the nucleotide double strand. Pall *et al.* [3] utilized 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride crosslinking method instead of the traditional UV crosslinking method to improve the binding efficiency of miRNA and nylon membrane. The method of biotin labeling instead of isotope labeling was used to conduct chemiluminescence detection and reduce radioactive pollution [4].

#### **2.2 Microarray**

The microarray, developed in the early 1990s, enables the high-throughput miRNA detection in a parallel fashion. In this method, the target miRNA is incubated and hybridized with multiple probes (complementary to the target miRNA sequence) on a chip. After removing the non-hybridized part, the signal can be detected and analyzed by fluorescence scanning or northern blotting [5]. DNA probe-based microarray usually consumes a large amount of samples, and has disadvantages such as low sensitivity and specificity and false positives caused by cross-reactions. However, LNA probe can reduce the consumption of starting materials and improve the sensitivity and accuracy of microarray [6]. Furthermore, liquid suspension microsphere hybridization can effectively avoid cross-reaction in the solid chip to decrease the occurrence of false positives.

#### **2.3 qRT-PCR**

Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the main methods to detect low abundance miRNA with high sensitivity and accuracy [7]. The principle of this method is to reversely transcribe miRNA into the corresponding cDNA that is used as a template to initiate real-time PCR, and then indirectly analyze miRNA by detecting the signal of amplified products [8].

Because the miRNA sequence is short (18 ~ 25 nt) and similar to the length of PCR primers, researchers overcome this shortcoming by introducing stem-ring

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

primers for reverse transcription [9] or adding poly (A) into RNA to initiate reverse transcription and dyeing with SYBR Green. Besides, pri-miRNA and pre-miRNA can be introduced into qRT-PCR, causing inaccurate quantification [10]. qRT-PCR usually requires complex primer design and precise reaction temperature control, thereby greatly increasing the cost and complexity of the experiment.

Although the traditional miRNA detection methods are widely used in miRNA detection, there are still some shortcomings such as complex operation, low sensitivity, poor specificity, and large sample consumption. These shortcomings greatly limited the application of these methods in clinical diagnosis and treatment. Importantly, these approaches are only applied to the *in vitro* measurement of miRNA.

## **3. Emerging sensing techniques in microRNA detection** *in Vivo*

#### **3.1 Hybridization probe assay**

Hybridization probe assay is a simple and direct detection method without amplification of target miRNA. The principle of this method is as follows: firstly, miRNA is fixed in the tissue or cell. Secondly, signal-labeled nucleic acid probes are added and hybridized with the miRNA based on the principle of complementary pairing. Finally, the position of target miRNA to be detected in the tissue or cell is displayed by certain detection means.

With the principle of hybridization, Wang *et al.* [11] combined the excellent fluorescence-quenching ability of Ti3C2 nanosheets with a double-labeled DNA fluorescent probes to design a chimeric DNA-functionalized Ti3C2 nanoprobe. When this probe crosses the cell membrane, TAMRA-labeled plasma membrane glycoprotein MUC1 aptamer interacted with MUC1 and peeled off from Ti3C2, lighting up red fluorescence. In the cytoplasm, FAM-tagged hairpin DNA hybridized with miR-21, leading to green fluorescence recovery. This strategy can perform *in vitro* measurement of miR-21 and MUC1 with nanomolar sensitivity and *in situ* simultaneous imaging of dual biomarkers in living cells. Although Ti3C2 greatly reduces the fluorescence background, the sensitivity of the probe is still limited because one target miRNA only restore the fluorescence of one fluorescent molecule.

To further improve the detection and imaging sensitivity, fluorescence doublelabeling and double-quenching strategy is undoubtedly a good design. Molecular beacon (MB) is hairpin-structure DNA probe. Its two ends are labeled with two identical fluorescent molecules. When MB is in a close state, self-quenching effect between two fluorescent molecules occurs. Graphene oxide (GO) is a good fluorescence quencher. The electrostatic interaction between DNA probe and GO also quenches effectively the fluorescence of DNA probe. Based on the aforementioned properties of MB and GO, Yang *et al.* [12] designed a MB-GO fluorescent sensor for sensitive imaging of intracellular miRNA (**Figure 1**). Two Cy5 fluorophores were tagged at the both ends of MB. MB was electrostatically adsorbed on the surface of GO through the π-π stacking effect. Two Cy5 fluorophores was quenched through their self-quenching effect and cy5-GO resonance energy transfer, which greatly reduced the fluorescence background of sensing system. Once target miRNA hybridize with MB probes, hairpin-structure MBs were unfolded to separate two Cy5 molecules and the MB-miRNA complex with rigid structure was released from the surface of GO, recovering fluorescence signal and significantly enhancing the detection sensitivity (Limit of detection, LOD = 30 pM). This method also visualized miRNA-21 in cancer cells and three xenograft tumor models.

#### **Figure 1.**

*Illustration of 2Cy5-MB-GO complex sensing miRNA in living cells. Reprinted with permission from ref 12. Copyright 2018 American Chemical Society.*

Another approach to enhance the sensitivity of hybridization probe assay is to use the plasmon coupling effect of assembled nanostructures, especially, dissymmetric nanostructures. Xu *et al.* [13] developed a plasmonic nanorod probe by miR-21-directed *in situ* self-assembly of DNA-modified Gold nanorods (AuNR) in living cells. The formation of NR@PS-PAA dimers gives rise to intense chiroplasmonic signal and surface-enhanced Raman scattering (SERS). The LOD were 0.081 fmol/10μgˑRNA (CD signal) and 1.12 fmol/10μgˑRNA (SERS signal), respectively. Highly sensitive quantitative analysis and *in situ* imaging of microRNA in cells are achieved.

Although the introduction of nanomaterials (gold nanoparticles, graphene oxide, upconversion nanoparticles, MnO2 nanosheets, quantum dots, silver nanoparticles, noble metal nanoclusters, and silica nanoparticles) remarkably enhanced the sensitivity of hybridization probe assay, the lack of signal amplification limits its practical application in the sensitive detection of intracellular miRNA.

#### **3.2 Strand displacement reaction (SDR)**

Strand displacement reaction (SDR) is a dynamic process of hybridizationdriven DNA strand exchange accompanied by branch migration [14]. In this process, a single-stranded reactant (input, target miRNA) reacts with multi-stranded DNA complex and releases another single-stranded product (output signal) and a new DNA complex. This process operates autonomously through a series of reversible DNA hybridization and dissociation steps to produce numerous output strands, thereby generating cascaded signal amplification [15, 16].

Using SDR, Wang *et al.* [17] constructed a double-stranded fuel catalyzed DNA molecular machine. The machine was divided into two parts: the first part was composed of the Cy5-labeled signal strand and BHQ-2 labeled complementary quenching strand. The second part was double-strand fuel (DSF), consisting of fuel strand and protected fuel. The target miRNA bound to the quenched strand and triggered the first SDR to obtain the miRNA-quenching double-strand, releasing the signal strand to recover the fluorescence. At this time, DSF initiated the second SDR, the dominated fuel hybridized with quencher strand, replacing miRNA to produce double-stranded waste and intermediated miRNA protected fuel. The

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

new signal strand can combine with the protected fuel to release miRNA again and repeated the above-mentioned cycle reaction. Thus, the target miRNA was continuously released from intermediate 2 and circularly participated in the subsequent reaction to achieve signal amplification. This method has high sensitivity, strong signal amplification ability, and relatively low background signal.

Ma *et al.* [18] similarly developed an artificial intelligence signal amplification system (AISA). The system also consists of a fluorescence-quenching double-strand and fuel-protection double strand. It underwent three-steps reaction: miRNA replaced signal strand and combined with quenching strand, fuel strand replaced miRNA and combined with quenching strand, and new signal strand replaced miRNA to achieve the reuse of it (the last two steps of reaction could be recycled to produce amplified signal). Based on this principle, they built two AISA systems to detect HsamiR-100 and Hsa-miR-484, respectively. Their versatility and feasibility were proved to be actively used in disease diagnosis and treatment, evaluation, stem cell tracking, and other fields, but the design of the probe was cumbersome.

In recent years, the combination of SDR and nanomaterials for miRNA detection has become a hot spot. Li *et al.* [19] constructed dsDNA-AuNPs nanoprobes by means of using toehold strand displacement reaction (TSDR) to attain the amplification detection of trace let-7a in living cells (**Figure 2**). The molecular catalytic machine contained dsDNA-AuNPs nanoprobes and DNA fuel strands, while the dsDNA contained an unfolded hairpin sequence labeled with FAM and TAMRA at both ends. Binding to the linker strand of the dsDNA, the target miRNA replaced the hairpin to restore its folded structure. Forster resonance energy transfer (FRET) signal occurred between the fluorescent groups. At the same time, the target miRNA was recycled by cascaded strand displacement reactions realizing the amplification and detection signal of the target miRNA. Zhang *et al.* [20] developed a kind of dendritic mesoporous silica nanoparticles encapsulated by tumor cell

#### **Figure 2.**

*Mechanism of in vivo imaging microRNA of the DNA-fueled and catalytic molecular machine. Reprinted with permission from ref 19. Copyright 2017 American Chemical Society.*

(MCF-7) membrane (DMSN-DP@CM) and simultaneously modified with DNA photoacoustic probe and fuel strand. The target miRNA triggered the release of the near-infrared fluorescence strand from the quenched strand and the fuel strand further replaced the target miRNA. The alternate replacement of target miRNA and DNA fuel strand generated an amplified photoacoustic signal ratio to realize the miRNA detection.

Surface-enhanced Raman spectroscopy (SERS) refers to that when molecules approach the metal surface of nanostructures, the Raman signal of molecules is enhanced. Gold nanoparticles with the core-satellite structure are important SERS substrate structures, which are composed of a single Au (or Ag) inside and multiple Au (or Ag) linked outside. The core-satellite structure is combined with SDR to detect miRNA and produce the SERS signal.

Li *et al.* [21] designed the core-satellite plasma rulers (PRs) for the quantitative determination of miRNA-21 in living cells. Several small gold nanoparticles were assembled on large gold nanoparticles with dsDNA to form a core-satellite. MiRNA-21 initiated a single hybridization event, leading to the destruction of core-satellite structural components. The plasma displacement between the core and the satellite initiated the wavelength changing of scattering intensity. The expression and the amount of miRNA-21 could be detected via statistical analysis of the wavelength signal. This method enabled highly sensitive detection of the intracellular miRNA without side effect.

Gold nanorods have strong and controllable plasma resonance properties, which can be widely used in photothermal therapy and amplification detection of miRNA. Qu *et al.* [22] used the assembly of Ag2S on the surface of platinum (Pt)-modified gold nanorods (AuNR-Pt) to form a nuclear-satellite structure (AuNR-Pt@Ag2S). The probe can complete the near-infrared imaging of miRNA *in vivo*. Target miRNA bound to the complementary DNA sequence and released the strand modified with Ag2S, thereby destroying the core-satellite structure and recovering the fluorescence. The LOD was 0.0082 amol ngRNA −1, providing a multifunctional nanoplatform for tumor diagnosis and treatment *in vivo*.

Inspired by Qu's work, Yan *et al.* [23] utilized the electrostatic interaction between polyethylenimine (PEI)-modified gold nanorod and nucleic acid probe to construct a Fuel Improved miRNA Explorer (FIRE) sensing platform. The detection system included a double-strand DNA labeled with BHQ-2, Cy5 fluorescencequenching group and a fuel strand that realizing the cycling of miRNA for signal amplification. The target miRNA triggered the release of dsDNA to recover fluorescence. In this design, the preparation of FIRE is simple via electrostatic interactions. Moreover, AuNRs enhanced the photoacoustic imaging and real-time monitoring capabilities of FIRE, resulting in sensitive detection of miRNA and effective tumor treatment effect.

In addition to the strand displacement reaction catalyzed by fuel strand, toehold-mediated strand displacement is another commonly used amplification strategy. A novel catalytic self-assembly nanosensor based on quantum dots was constructed [24] to detect miRNA *in vivo*. As an efficient catalyst, miRNA triggered the toehold-mediated strand displacement cascade reaction to produce multiple Cy5/biotin double-labeled dsDNA, which were assembled on a 605QD surface, resulting in significant FRET signal. The nanosensor was simple to operate and has high specificity and sensitivity. Also, the synergistic effect between telomerase and the catalytic strand was helpful to distinguish cancer cells from normal cells, which provided a valuable method for clinical diagnosis.

Enzyme-mediated strand displacement reaction enables the exponential accumulation of DNA products through the continuous polymerization-nickingdisplacement cycle process catalyzed by polymerases. Based on the amplification *Novel Biosensing Strategies for the* in Vivo *Detection of microRNA DOI: http://dx.doi.org/10.5772/intechopen.93937*

methods, Yang *et al.* [25] proposed a novel strategy by utilizing branched DNA ligation enhanced isothermal strand displacement polymerization (B-ICSDP) for *in vitro* quantitation and intracellular miRNA imaging. This internal Y-shaped DNA structure consisted of a circular DNA scaffold and three repeatable molecular beacons (MB). MiRNA triggered the conformation conversion of MB and recovered the fluorescence. Additionally, polymerase-based cyclic SDR produced a large number of extended Y-type DNA structures (one DNA scaffold could hybridize with three MBs), thus generating amplified signals. This method had the advantages of high sensitivity, high specificity, and simple design. Besides, it could be used to detect single or multiple target miRNAs in living cells.

Peng's team [26] developed a telomerase-catalyzed FRET ratio probe for accurate miRNA detection. AuNPs were modified with capture probe containing recognition sequence and telomerase primer located at the 5′ of capture probe strand. The detection probe (a molecular beacon labeled with donor FAM and acceptor TARMA) hybridized with the capture probe, separating the fluorescent donor and acceptor and causing low FRET signal. Once miRNA specifically recognized and hybridized the capture probe. The detection probe was then replaced by miRNA to form a stem-ring structure. Thus, the FAM and TAMRA were brought in close proximity to produce high FRET signal. In addition, the capture probe was extended with telomerase primers and hybridized with the catalytic strand to displace target miRNA. The released miRNA also triggered the above-mentioned detection system. This method had low background signal and can detect low abundance miRNA molecules in living cells.

### **3.3 Catalytic hairpin assembly (CHA)**

Catalytic hairpin assembly (CHA) is an enzyme-free, hairpin fuel-driven, and automomous nucleic acid amplification technology. A CHA system needs to design two hairpin structures according to the sequence of target miRNA [27]. One segment of the first hairpin is complementary to the target miRNA sequence [28]. Its hairpin structure can be unfolded by miRNA, and then form a complementary structure with another hairpin probe. The target miRNA will be replaced and dissociated, which can further catalyze CHA between other hairpin probes, forming a cycle to generate amplification signal. The catalytic hairpin assembly has been widely used in nucleic acid detection due to its enzyme-free and target-recyclable advantages.

Like SDR, CHA usually employs nanomaterials as a scaffold and carrier to deliver DNA probes into living cells. As shown in **Figure 3**, Liu *et al.* [29] developed a core-satellite nanoprobe (AuNPs-AuNDs CS). Hairpin1 (H1) was immobilized on the Au nanoparticle surface (AuNPs-H1, satellites) and hairpin2 (H2) was assembled on the surface of the plasmonic gold nanodumbbells (AuNDs-H2, core) via Au − S bonds. MiRNA triggered CHA by targeting AuNPs-H1 (AuNPs-H1-miRNA). AuNDs-H2 then hybridized with AuNPs-H1-miRNA to form a core-satellite nanostructure, whereas target miRNA was displaced from AuNPs-H1-miRNA. The released miRNA originated the next assembly of AuNPs-AuNDs CS, generating an amplified signal. This design engineered metallic nanoparticle aggregates to increase electromagnetic hot spots, thereby realizing the highly-sensitive SERS detection of low abundant miRNA.

Wang *et al.* [30] modified the fluorescence-labeled hairpin H1 to the surface of AuNPs. The fluorescence of H1 was quenched by AuNPs. When the probes delivered into cells, the hybridization between H1 and miRNA unfolded H1. The exposed single-strand domain of H1 induced H2 (hairpin fuel) hybridization and displaced miR-21. The released miR-21 participated in the subsequent reaction cycle. The

#### **Figure 3.**

*Schematic representation of (A) the Assembly process of AuNPs-AuNDs-CS and (B) AuNPs-AuNDs-CS sensing miRNA and imaging in living cells. Reprinted with permission from ref 29. Copyright 2018 American Chemical Society.*

sensitivity of hairpin-fuelled catalytic nanobeacons is two orders of magnitude lower than that of noncatalytic nanobeacons.

Aruni *et al.* [31] exploited a genetically encoded means to design a RNA-based CHA circuits for imaging miRNA in living cells. In this design, Broccoli, a specific RNA aptamer for DFHBI-1 T, was used as fluorescence reporter. It was split into two non-fluorescent parts (Broc and Coli). Broc was attached to the 5′ end of H1, while Coli was conjugated to the 3′ end of H2. The target miRNA initiated CHA and hybridized with H1. The unfolded H1 then hybridized with H2 to displace target miRNA and form dsDNA. The recombinant Broccoli activated the dye DFHBI-1 T to emit fluorescence, and the target miRNA can be recycled to catalyze the fluorescence of ten to hundreds of broccolis, achieving signal amplification. This strategy provided a new opportunity for the application of gene-encoded RNA circuits in cells.

To improve the kinetics and efficiency of CHA in the complex intracellular environment, inspired by spatial-confinement effects of cells, Liu *et al.* [32] constructed a three-dimensional DNA nanocube to implement the localized hairpin-DNA cascade signal amplification. The hairpin H1 and H2 labeled with Cy3 and Cy5 were modified in the cube, respectively. MiRNA could specifically unfold H1 and then unfold H2. The rapid and efficient imaging of miRNA could be monitored

by FRET. This cascade amplifier significantly improved the speed (7 times faster) and efficiency (2.6 times higher) of signal amplification. Furthermore, this probe possesses good cell permeability, good nuclease resistance, and the ability to avoid false-positive signals.
