Brij
Mohan
*a,
Sandeep
Kumar
b,
Suresh
Kumar
*c,
Krunal
Modi
d,
Deependra
Tyagi
e,
Dimitri
Papukashvili
e,
Nino
Rcheulishvili
e and
Armando J. L.
Pombeiro
a
aCentro de Química Estrutural, Institute of Molecular Sciences, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. E-mail: brizharry17@gmail.com
bSchool of Science, Harbin Institute of Technology (Shenzhen), Shenzhen 518055, China
cDepartment of Chemistry, SUS Government PG College, Matak Majri, Karnal, 132041, Haryana, India. E-mail: sureshprocha@gmail.com
dDepartment of Humanity and Science, School of Engineering, Indrashil University, Mehsana-382740, Gujarat, India
eSouthern University of Science and Technology, Shenzhen 518000, China
First published on 8th December 2022
MicroRNAs (miRNAs) with nucleotides are a class of endogenous small RNAs and can play crucial functions in diagnosing diseases. In particular, the group of miRNAs is responsible for information related to the cell and disease. Among various techniques for miRNA detection, the hybridization chain reaction (HCR) strategy shows potential and efficiency. This review summarizes and studies the most efficient HCR strategy for detecting miRNA using nanomaterials due to their ultrasensitive detection and excellent performances. In addition, signal amplification for the sensitive detection of miRNA due to the chain reaction has been studied. The key factors, such as limit of detection (LOD), linear range, the importance of the strategy, limitations or challenges, and future perspective are described. Finally, the study will provide new findings for developing a miRNA detection strategy applicable to disease diagnosis.
Recently, the ligation chain reaction with HCR for enzyme-free signal amplification strategies has been used to quantify microRNAs.27,28 Also, incorporating a 3D DNA layer on the electrode interface has been used to achieve ladder HCR and improved electrochemical signals for miRNA detection.29 However, it remains a challenge for researchers to ascertain the quantity of miRNA, mainly due to the short sequence and low abundance of miRNA.30 However, various techniques, such as microarrays, Northern blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), are commonly employed for quantitative RNA measurement,31 but these typically all have limitations, such as lengthy procedures, the requirement for a large sample, and complicated and sophisticated instrumentation, that must be addressed.32
The most critical issues, such as the sensitivity and performances of biosensors for miRNA detection, have been resolved using Au-loaded nanoporous superparamagnetic Fe2O3, which can provide an active surface for miRNA adsorption.33 In addition, it has been used to develop rapid and inexpensive miRNA biosensor strategies.34 With the rapid growth in this research area, the hybridization chain reaction (HCR) provides an advanced, efficient, isothermal signal amplification that has been widely used in miRNA detection. It involves a linear elongation of double-stranded nicked DNA, single-stranded initiator DNA, and two hairpin fuel DNAs. Efforts have been made to explore HCR in recent times, given the utility of HCR in miRNA detection. Therefore, there is an urgent need to review all such articles for further improvement and for the utilization of the HCR in detecting miRNA.35,36
In recent years, various techniques, namely cyclic voltammetry (CV), Raman scattering, infrared, ultraviolet, visible, and fluorescence spectroscopy, mass spectrometry, surface plasmon resonance, and electrochemical-based approaches, have been used to detect biospecies.37–39 In addition, various inorganic and hybrid materials have been utilized for miRNA detection. In particular, nanomaterials have attracted considerable attention for detecting miRNAs among these materials due to their active surface area and robust sensing properties. In addition, the electropositive nature of metal-based nanomaterials (nanoparticles, nanorods, nanosheets, nanowires, nanoflares, nanotags, and nanoclusters) provides a suitable platform for miRNA recognition.40,41 Moreover, the two hairpin probes in miRNAs show interactions, resulting in the signal changes due to the HCR. In addition, biomolecules accumulation of nanomaterials will lead to signal amplification in miRNA detection. For example, AuNPs and magnetic 3D DNA walkers provided a solution for the colorimetric detection of miRNA. In addition, NPs with multiple metal ions have shown multianalyte detection with enhanced specificity and sensitivity.42 Hence, nanomaterials for HCR-based detection have allowed insights into the roles of various materials in signal amplification. In addition, it has created interest in researchers to collect information about miRNA binding to the unfolded H1 probe through SDR and then hybridization (Fig. 1).43
Fig. 1 Materials for HCR-based miRNA detection and to gain working insights.43 |
Several review articles have been published in the past years summarizing materials for detecting miRNA.44 Despite these valuable studies, insights into the HCR-based signal amplification techniques have gained limited attention.45,46 Hence, studies into the insights provided by HCR-assisted signal amplification strategies are needed, mainly because of the extensive research in this field. Therefore, we have reviewed and analyzed HCR-assisted signal amplification strategies in this article to overview the new developments in this field for miRNA detection. Furthermore, we have explored several combined nanomaterials based on miRNA biosensors using HCR as representative examples, and finally, the future trends in miRNA detection are briefly discussed.
Moreover, miRNA detection is based on the interactions for miRNA hybridization via signal changes and could lead to biorecognition and hairpin-shaped probes. The active surface in fabricated nanomaterials is helpful for a rapid HCR in miRNA recognition.52,53 The presence of functional sites in DNA-fabricated nanomaterials allows the hybridization of miRNA and follows a Watson–Crick base-pairing.54 This could result in rigid DNA–RNA or RNA–RNA hybrid structures. Therefore, it offers rapid and sensitive electrochemical, optical, or mechanical-based detection. Hence, biomolecule-fabricated NPs have advanced features for detecting miRNA with high sensitivity through hairpin-shaped nucleic acid probes. In addition, a loop is present that includes the target miRNA and an active surface acting as a stem in NPs.55 Therefore, the interactions between the active surfaces of fabricated nanomaterials and miRNA could result in loop dissociation, thus supporting the HCR. The presence of new functional sites in fabricated nanomaterials provides benefits in engineered nanomaterials biosensing.56 This strategy has thus emerged as a promising tool for HCR-based miRNA detection due to its active surface, nano-size surface:volume ratio, and enhanced optical-electronic properties.
Immobilizing DNA molecules and amino acids could be useful for the fabrication or modulation of nanomaterials. Such fabricated nanomaterials (<10 nm) were found to exhibit better properties, such as water solubility, biocompatibility, photobleaching, and chemical resistance compared to traditional fluorophores.57 Also, nanomaterials with sizes of 1–100 nm showed applications in cancer detection due to their Tyndall effect or light-scattering properties. Furthermore, AgNPs, AuNPs, and magnetic nanocomposites have shown advanced applications in the colorimetric detection of miRNA.58
HCR-based miRNA detection produces double-strand molecules with overlapping hairpin pairs with partial complementarities. In addition, the detection process may include one DNA initiator and a pair of DNA hairpins (H1 and H2). The existing DNA hairpin pair gets opened in the solution with the initiator binding. It can be observed that the designed length plays a crucial role in the working and sensing mechanisms for HCR detection assembly. Therefore, using HCR to detect miRNA is a potential useful tool, and precise signals could be achieved by designing DNA hairpins.61
The electrochemical detection of miRNA offers a sensitive surface for electrochemical signals, which could be regarded as offering miRNA detection. However, photobleaching in the case of fluorescence and preparation of the electrochemical surface in detection can be complicated. Therefore, an appropriate hairpin design is crucial for miRNA's enhanced specific and sensitive detection.62 In addition, a fluorescence HCR assay to detect miRNA was designed by heating DNA H1 and DNA H2 at a particular temperature and then cooling them at room temperature, which led to activating the miRNA detection process.63 Upon miRNA addition with the designed hairpins, changes in the fluorescence intensity signals could be observed. A pair of hairpin DNA probes enriched with fluorophores was treated with miRNA in solution. Each miRNA molecule was triggered due to HCR between two hairpins, H1* and H2*, with accumulated changes in the signals.64
Various nanomaterials show excellent physicochemical properties and can be functionalized to develop fascinating biosensing platforms. Moreover, graphene-based nanomaterials have exhibited excellent detection for miRNA by forming π-conjugated supramolecular assemblies.65 For example, adding graphene oxide (GO) to the solution after the HCR resulted in the absorption of the two hairpins on the GO surface due to π–π stacking interactions.66 The sensitivity of this method was based mainly on the surface prepared for electrodes. The electrochemical HCR miRNA detection results could be read through the interaction with the target. In one study into the working mechanism of an HCR electrochemical sensor for miRNA detection, methylene blue (MB) and mesoporous silica containers (MSNs) were utilized for the signal release and helped in the sensitive electrochemical detection of miRNA. The DNA molecules acted as gate molecules to hold the hairpins for MB when miRNA was not present. In the presence of miRNA, hairpin 1 (H1) was released from the MSNs due to the miRNA interaction. Then MB was released due to the gate opening by the DNA. Moreover, the electrode surface was hybridized and resulted in an HCR amplification process (Fig. 2).67
Fig. 2 Silica containers and MB-based HCR electrochemical assay for miRNA detection; reproduced with permission; Copyright©2019, American Chemical Society.67 |
Fig. 3 Representation of a biosensor based on DHCR for miRNA detection. Reproduced with permission Copyright©2020, American Chemical Society.74 |
Immobilized surface plasmon resonance and HCR can be utilized for an economical, robust, and straightforward biosensor for miRNA detection. The use of gold nanoparticles to detect miRNA (miR-17) is a method with good specificity for detection via hairpin probes, in combination with HCR to amplify the signal to extend the dynamic range of quantization. This process is rapid and required just 1 h for completion and displayed a very low LOD, nearly equal to 1 pM or 50 amol per measurement.75 A rapid electrochemical assay was developed for the HCR-based detection of microRNA-122. It was observed that hairpin DNA (hpDNA) gets opened on the gold electrode surface when miR-122 is present. The hpDNA helped to trigger HCR via the cross-opening of helper DNA hairpins and their hybridization (Fig. 4). The high density of hpDNA on the electrode resulted in HCR signal amplification. The LOD of the electrochemical assay for miR-122 detection was found to be 53 aM. The assay was also noted to selective and could detect exosomal miR-122 with high-valuable efficiency in cancer diagnostics.76
Fig. 4 Electrochemical sensing of exosomal MicroRNA based on HCR signal amplification. Reproduced with permission Copyright©2020, American Chemical Society.76 |
In another example, Guao et al. recently described using time-gated Förster resonance energy transfer (TG-FRET) for microRNA analysis. The technique was used for terbium donors and dye acceptors in HCR for miR-20a and miR-21. The author's method provided excellent results for quantifying microRNA with a low LOD with a minimum of 240 amol of microRNA. The technique proved efficient at distinguishing between homologous microRNAs with high target specificity. The multiplexing of measurements in a FRET pair at excitation wavelength allowed the simultaneous quantification of miR-20a and miR-21, even at low concentrations of 30 and 300 pM. The developed HCR was applied equally to serum-free and serum-containing samples without RNase inhibitors. It can work in various living systems and can be applied for advanced nucleic acid biosensing.78
For example, MoS2 nanosheets enriched with MBs were used by Zhang et al. to develop hybridization chain reaction (HCR)-based miRNA detection. The authors used MoS2 nanosheets to capture MBs as an adsorption probe. In addition, the MoS2 nanosheets also worked as selective fluorescence quenching probes required for reducing background signals. The presence of target miRNAs triggered the HCR process, leading to many products. However, the products obtained from HCR as nanowires chains had a G-quadruplex abundance, which could not be adsorbed on the MoS2 surface and detached. Hence, Thioflavin T could be attached to the G-quadruplex and produced an electrochemical signal analyzed by fluorescence spectroscopy. This method could achieve a low detection for miRNA to a minimum of 4.2 pM in a wide linear range from 0.1 to 100 nM.79 In addition, Jia et al. used MoS2 quantum dots (QDs) in an HCR of G-quadruplex enzymatic catalysis for microRNA analysis via the inner filter effect. It was observed that the target microRNA triggered the HCR of two DNA probes and generated double-stranded DNA (dsDNA). The long dsDNA thus produced had many hemin/G-quadruplex enzymes with hemin. In the presence of hydrogen peroxide, these DNAzymes directly oxidized o-phenylenediamine into 2,3-diaminophenazine; this oxidation through the inner filter effect resulted in the fluorescence quenching of the MoS2 QDs. The electrochemical response of these QDs was directly proportional to the amount of miRNA. This approach was found to have a low LOD of 42 fM and can be used for practical applications.80 Ding et al. designed silicon nanoparticles (SiNPs) for detecting miRNA by fluorescence quenching due to the inner filter effects (IFEs). The HCR between DNA hairpin probes with G-quadruplex sequences led to horseradish peroxidase (HRP)-mimicking DNAzyme formation for targeting miRNA. This then catalyzed the H2O2-mediated oxidation of o-phenylenediamine to 2,3-diaminophenazine. The absorption and emission band overlapping resulted in fluorescence quenching.81 Also, Ying et al. demonstrated an HRP-based HCR strategy for detecting miRNA-155 with color change responses with an LOD of 31.8 fM. CP immobilized at the microplate played an active role in capturing miR-155 and the 3′ end of the reporter probe, while the 5′ end initiated the HCR. This resulted in signal amplification via dsDNA polymers having multiple fluorescein isothiocyanates. The observed colorimetric changes from colorless to blue color for miRNA detection could have been due to the antibody interactions on the microplate through the tetramethylbenzidine/H2O2 system.82 In another example, Feng et al. developed an HCR-based electrochemical miRNA-21 detection method by combining the DNA-generated current and target-triggered HCR. The immobilization of thiol-modified hairpin CP on a Au electrode resulted in miRNA-21 conformational change. This conformational change was responsible for HCR initiation for long DNA strands generated on the electrode surface. Also, the reaction between the DNA phosphate backbone and molybdate led to the redox probe molybdophosphate and generation of an electrochemical.83
miRNA detection in the presence of DNA has been widely explored and gained considerable attention.84 Zhou et al. developed a nonlinear HCR-based electrochemical method to detect microRNAs using Y-shaped DNA integration. Y-Shaped DNA consists of three sequences Y1, Y2, and Y3. These can act as stable and specific units for the detection of miRNA. It was reported that a competitive hybridization reaction occurs between miRNA and Y-shaped DNA when target miRNA is present. This was demonstrated by the freeing of the Y3 probe followed by the dissociation of the Y-shaped DNA structure. Subsequently, triggers blocked by Y3 become exposed, resulting in the onset of nonlinear HCR. This resulted in electrochemical changes in the signal through an amplification reaction, which could be recorded. The biosensor developed in this technique could detect microRNAs (miRNAs) up to an LOD of 0.3334 fM. The linear range was from 1 fM to 10 pM. Furthermore, the unique Y-shaped DNA turned out to be helpful to the biosensor for identifying single-base mutations.85
It is important to improve the reliability of miRNA for detecting progression-motivated disease diagnosis applications. Hence, developing an accelerated DNA nanoprobe for miRNA for the in situ monitoring of biofluids with a spatial strategy could provide a highly efficient and significant approach. In addition, a fast response would allow the nanoprobes to monitor the process of exosome endocytosis.87 For example, Zhuang et al. reported improved in situ hybridization methods for detecting miRNA through HCR. The in situ HCR method could detect miRNA even from mouse retinas. Furthermore, this process could be used to detect two microRNAs simultaneously, and even miRNA and mRNA could be detected simultaneously.88
Moreover, the combination of Fe magnetic nanoprobes with DNAs could be used for the HCR detection of microRNA-141 and microRNA-21 at a low level. For instance, SH-modified hairpin capture probes (CPs) were hybridized with miRNAs on an Au electrode, leading to conformation changes of the CPs. Furthermore, this triggered HCR to generate plentiful bonding sequences of the magnetic nanoprobes.89 Zhu et al. designed a Ru(bpy)32+-based ratiometric ECL–EC hybrid biosensor for the sensitive and low detection of miRNA-133a with an LOD of 12.17 aM. The Au–S bond in the DNA tetrahedron nanostructure and two Ru(bpy)32+-labeled H1 and H2 hairpins were utilized as ECL probes and fuel strands for an HCR. The MB with the 5′ end of the miRNA provided the internal reference signals.90 Extracellular vehicle (EV) cancer biomarkers can provide parent molecular information early on. For example, Wu et al. developed an EV-derived HCR-based strategy for detecting inherent in situ miRNAs. A modularized DNAzyme-amplified two-stage cascaded HCR circuit created the detector amplifier. This was followed by HCR1 as an analyte-generated output and HCR2 as a trigger input. Moreover, the designed modular CHCR–DNAzyme circuit acted as a “plug-and-play” sensing mode for detecting miRNA. Interestingly the developed model worked in vitro in different cells by the amplified detection of the miRNA biomarkers in the EVs.91
Au-based nanomaterials have demonstrated improved biosensing performance that would be helpful for disease identification.92 Recently, Yuan et al. reported a ratiometric electrochemical assay for detecting miRNA from thionine (Thi) and ferrocene (Fc). First, a Au electrode was developed through a Au–S reaction through a thiol-modified and ferrocene-labeled hairpin CP. Then, the HCR-based target detection of miRNA was accompanied by a hybridization of the chain probes and unfolding of the miRNA-DNA duplexes hairpin and Kamchatka crab duplex-specific, resulting in the miRNA release. The far electrode distance of Fc led to the Fc signal-off state, and the residual fragment process on the electrode surface was responsible for the HCR for generating dsDNA. The in situ HCR had a primer, HDNA, and HDNA′ probes for capturing numerous DNA/Au NPs/Thi, resulting in the signal-on state of Thi. The observed dual-amplification mechanism signal-off for Fc and signal-on for Thi provided a sensitive HCR technique for detecting miRNA-141 with an LOD of 11 aM.93
Yang et al. designed a strategy to introduce a branched HCR circuit. The use of a terbium(II) organic gel (TOG) electrode elevated the biosensor sensitivity by a bHCR circuit for miRNA-141 detection with a low LOD of 0.18 fM. The advancement in this work was a one-step approach to modify nucleic acids to electrodes to introduce the DNA structure. This method was reported to be more precise and can avoid errors that may arise in the stepwise modification methods due to the electrode's drawback of low-molecular-weight nucleic sequences.94 In another example, Zhang et al. designed polydopamine (PDA)-encapsulated photonic crystal (PhC) barcodes for target-triggering cycle amplification and HCR for detecting miRNA with an LOD of 8.0 fM. Moreover, the barcodes showed structural colors for different encoding miRNAs that could immobilize biomolecules for helping the reaction with amino-modified hairpin probes (H1) and could initiate HCR for cycle amplification.95
miRNAs as riveting RNAs have significance in gene regulation and specific roles in certain pathological and physiological or pathological processes. The use of Au nanomaterials-based sensors provides a tool for the rapid and sensitive detection of the miRNA assay.96 For example, Lu et al. designed an ECL biosensor by immobilizing a CP on Fe3O4@SiO2@AuNPs to detect femtomolar miRNA-141 with a low LOD of 0.03 fM. The HCR-assisted cascade amplification and Faraday cage-type strategy through GO and signal unit from the material (Ru(phen)32+-HCR/GO) were allowed via nucleic acid hybridization. The large surface area and electronic transport properties in this system were responsible for the enhanced signal amplification. In addition, GO concentration on the electrode surface resulted in the sensor's outer Helmholtz plane (OHP) extension.97 Also, Zheng et al. reported that Fe3O4@SiO2@AuNPs-cDNA nanomaterials coated by hairpin cDNA could be used to detect miRNA-126 with an LOD of 2 fM. The ECL signal unit was fixed through DNA and HCR-Ru(phen)32+ for target miRNA-126, which opened the stem-loop structure of cDNA.98 In another example, Fan et al. reported target HCR-based detection for microRNA with an LOD of 4.2 fM. The GO has far-reaching significance in signal change for HCR miRNA detection, which was achieved by a helicase-assisted GO-based reaction platform for microRNA (miRNA) detection.99
Nanomaterials-based electrochemical sensors hold excellent promise for fast miRNAs detection in real samples. Combining G-quadruplex DNA probes and single-stranded anchor DNA could help seal the pores.100 Furthermore, the combination of materials can provide new materials for biosensing applications. For example, a magnetic beads and duplex-specific nuclease enzyme combination showed an enhanced detection potential for miRNAs-21 with an LOD of 170 aM.101 Tang et al. designed DNA/Fe3O4 nanosheets as a triple-amplification assay for detecting miRNA let-7a with an LOD of 13 aM. The magnetic nanosheet networks were initiated by the target miRNA-associated HCR. Also, DNA-combined networks were reported to catalyze peroxidase for a colorimetric reaction.102 Nanomaterials with CdSe QDs showed enhanced signals for detecting miRNA. In addition, the hybrid materials were helpful for surface programmatic chain reactions and multiple amplification.103 In another example, a nucleic acid framework designed by Qu et al. acted as a multiple miRNAs sensor. The surface was modulated with DNA probes via lateral interactions that enabled a programmable tailoring of the enhanced kinetics and hybridization efficiency for sensing. The microassay framework combined with the HCR amplification strategy was used to detect miRNA (e.g., FNA-miR-652, FNA-miR-627, and FNA-miR-629) biomarkers in gastric cancer.104
In another example, Zhu et al. designed flower-like gold nanostructures (HFGNs) as an electrochemical sensor for detecting miRNA-21 with an LOD of 0.12 fM in a linear range from 1 fM to 1 nM. The HFGNs-deposited ITO first captured DNA (DNA-1) on its surface, and then HCR was attached to the electrode through a target miRNA-mediated sandwich hybridization for signal amplification.105 Xue et al. recently designed a label-free DNA dendrimers HCR-mediated multiple G-quadruplex to detect miRNAs. The hairpin switch probe was employed for enhancing the weak signals of the split G-quadruplex of double-stranded DNAs and nonlinear HCR assembly.106 Nanomaterials have been used for various biomedical faces due to their active biological immobilization properties. Hence, HCR-based miRNA detection provide an effective strategy for detecting miRNA, and the observed results can be evaluated with easy handling (Fig. 5). The current early detection of diseases is often ineffective due to the significant population needing testing. An HCR-based sensor could be helpful in this regard for the effective and early detection of miRNA to provide disease information. Moreover, HCR-based outcomes are effective with nanomaterials (Table 1).107
Sr. no. | Materials/electrode | Target | LOD | Linear range | Ref. |
---|---|---|---|---|---|
1 | Gold electrode surface | miR-122 | 53 aM | 76 | |
2 | MoS2 nanosheets | miRNAs | 4.2 pM | 0.1 to 100 nM | 79 |
3 | MoS2 quantum dots | miRNA | 42 fM | — | 80 |
4 | CP immobilized | miRNA-155 | 31.8 fM | — | 82 |
5 | Y-Shaped DNA integration | miRNAs | 0.3334 fM | 1 fM to 10 pM | 85 |
6 | Ru(bpy)32+-based ratiometric ECL–EC hybrid biosensor | miRNA-133a | 12.17 aM | — | 90 |
7 | DNA/Au electrode | miRNA-141 | 11 aM | — | 93 |
8 | Terbium(II) organic gel | miRNA-141 | 0.18 fM | — | 94 |
9 | Polydopamine (PDA) encapsulated photonic crystal | miRNAs | 8.0 fM | — | 95 |
10 | Fe3O4@SiO2@AuNPs | miRNA-141 | 0.03 fM | — | 97 |
11 | Fe3O4@SiO2@AuNPs-cDNA | miRNA-126 | 2 fM | — | 98 |
Furthermore, HCR-based miRNA detection and the use of this technique for disease diagnosis will be highly beneficial for society. This can be achieved by using various newly designed material matrices. This approach will have significant scientific importance to aid developing a technology that can overcome several issues in diagnosis. The design of HCR-based detection for miRNA will help avoid the need for the use of expensive and complex techniques for clinical applications.109 In particular, while different amplification techniques based on enzymes and nuclease have been potentially utilized for miRNA detection, these generally still suffer some challenges and limitations, such as selection and the selectivity of the analyte. The feasible guidelines for real-world applications are still lacking. By solving these limitations, the materials science for detecting multiple miRNAs will be advanced and solutions could be utilized for real-world applications. The significant challenges are discussed as following.
1. Other issues, such as the concentrations of the hairpins and leakage, are still challenging and need to be resolved to address the background leakage. Moreover, DNA present during the HCR of the hairpin without miRNA could be subject to an unnecessary chain reaction. These unwanted factors could be responsible for false-positive signals. Various factors, such as temperature, concentration, solvent choice, and pressure, must be controlled for the HCR assay.
2. It was seen that the HCR-based biosensors follow the random diffusion of DNA and displacement. Therefore, it could take a longer time to overcome the slow kinetics. Thus, designing a model for rapid miRNA detection is still challenging.
3. The lack of sensitivity with current amplification technologies needs to be improved. In addition, the detection of multiple miRNAs is still a challenge. Therefore, these imitations need a combined detection technology with enhanced signals and probes.
4. The development of portable detection devices is an urgent need and a challenge for portable detection tools to meet real-world applications.
5. Also, the low concentration of miRNAs in vivo could limit the detection process. On the other hand, the in vitro detection of miRNA could lead to degradation and affect the efficiency of the detection techniques. Hence, there is an urgent need to develop potential and efficient detection methods for in vivo detection. This could be achieved by developing fabricated or hybrid materials.
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