Luminescent carbon nanostructures for microRNA detection

Trends in Analytical Chemistry 119 (2019) 115613

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Luminescent carbon nanostructures for microRNA detection O.A. Goryacheva a, b, A.M. Vostrikova a, A.A. Kokorina a, E.A. Mordovina a, D.V. Tsyupka a, A.A. Bakal a, A.V. Markin a, R. Shandilya c, P.K. Mishra c, N.V. Beloglazova a, b, d, I.Y. Goryacheva a, * a

Department of General and Inorganic Chemistry, Chemistry Institute, Saratov State University, Astrakhanskaya 83, 410012 Saratov, Russia Ghent University, Faculty of Pharmaceutical Sciences, Centre of Excellence in Mycotoxicology and Public Health, Ottergemsesteenweg 460, 9000 Ghent, Belgium c Department of Molecular Biology, ICMR-National Institute for Research in Environmental Health, Bhopal, 462001, India d Nanotechnology Education and Research Center, South Ural State University, 454080 Chelyabinsk, Russia b

a r t i c l e i n f o

a b s t r a c t

Article history: Available online 27 July 2019

This article reviews the application of luminescent carbon nanostructures (graphene quantum dots, graphene oxide and carbon nanoparticles) for microRNA detection. The main advancement of luminescent carbon nanostructures for application in the microRNA assay (as well as for other short oligonucleotides) in their unique multi-functionality: the high p-electron density of sp2 graphene-based systems allows to use carbon nanostructures as carriers for single-stranded oligonucleotides; surface carboxylic groups provide good solubility; changing of size of structures, amount and location of defects, nature and amount of additives allow to vary luminescent properties. Consequently, the functionality of carbon nanostructures for microRNA detection is higher than of most of nanoparticles, while synthesis is simpler and more cost-effective. Possibility to use carbon nanostructures as emitters, carriers, quenchers and sensitive elements provides wide perspectives for their application as a multifunctional sensing tool. €rster resonance energy transfer, electrochemiluminescence and photoluminescence in combination Fo with different enhancement strategies are discussed. © 2019 Elsevier B.V. All rights reserved.

Keywords: microRNA Luminescent carbon nanostructures Graphene quantum dots Graphene oxide Carbon nanoparticles €rster resonance energy transfer Fo Electrochemiluminescence

1. Introduction MicroRNAs (miRNAs) are single-stranded small noncoding RNAs of 20e25 nucleotides important in the post-transcriptional mechanisms of gene expression [1,2]. miRNAs play a critical role as endogenous gene regulators by mediating the translational repression or promoting the degradation of miRNAs by binding to the complementary sequences located at the 30 -untranslated region (30 -UTRs) of the corresponding target miRNAs [3,4]. MiRNAs are frequently subjected to various genetic alterations as they are located in the fragile genomic regions. The normal expression and function of miRNAs are important for the physiological processes such as differentiation, proliferation, apoptosis and development, while their aberrant expression (both overexpression or downregulation) has been confirmed to be attributed to chromosomal aberrancies, modifications in the transcriptional control, epigenetic alterations and defects in the miRNA biogenesis pathways [5].

* Corresponding author. E-mail address: [email protected] (I.Y. Goryacheva). https://doi.org/10.1016/j.trac.2019.07.024 0165-9936/© 2019 Elsevier B.V. All rights reserved.

miRNAs target about 60% of mammalian genes and present in many human cell types [6]. Due to the ubiquitous nature of miRNAs in the cellular and extracellular compartments, they possess the capability to affect the whole intricate and inter-connected network of the biological processes by regulating multiple genes and pathways [7]. These properties allow miRNAs to play a critical regulatory role in tumor pathogenesis, making them an attractive novel type of biomarkers and potential therapeutic targets for various diseases including cancer [8]. Therefore, new methods for the rapid detection of single and multiple miRNA have been actively pursued both to clarify the regulation of gene expression patterns and as early diagnostics techniques [9e12]. However, due to the low abundance of miRNAs, their small size and similar sequences, their monitoring remains challenging and requires ultra-sensitive and advanced detection techniques [8,13e15]. Effectiveness of miRNA extraction is critical for miRNA detection because of very low target biomarkers concentration and complexity of biological matrixes. The standard procedure is an application of commercial kits (from Macherey-Nagel, ThermoFisher Scientific, Qiagen, Roche, etc.) for quantitative recovery of miRNAs from different samples.

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Existing methods for miRNA detection have been reviewed by Lu et al. [8], Tian et al. [15], Planell-Saguer and Rodicio [16]. The most popular techniques include Northern blotting [17,18], realtime polymerase chain reaction (RT-PCR) [19,20], microarrays [21,22], next generation sequencing [23,24], and NanoString nCounter technology [25,26]. However, each of these methods has its own individual limitations. Northern blotting is complex, has a low detection efficiency, need to be performed in a well-equipped biological laboratory, but is still used as a standard confirmation approach [8,15]. RT-PCR and microarrays are characterized by high sensitivity, but the required equipment and consumables are sophisticated and costly [15]. High-throughput microarray analysis of multiple miRNA in a particular cell or tissue usually combined with a chemical or enzymatic strategy for miRNA labeling [8]. The applicability of next generation sequencing is often limited by requirement of large amount of samples and a complex bioinformatic tool for the analysis of large generated data [27,28]. Precise detection and quantification could be achieved by a NanoString nCounter method, however, the detection is limited up to 800 miRNAs per sample out of the whole transcriptome [29]. Modern methods are based on the development of a sensitive signal output unit, often in a combination with a signal amplification [15]. To improve the sensitivity, different enhancing procedures such as rolling circle amplification [9] enzyme mediated signal amplification [30], isothermal strand-displacement polymerase reaction [31], target catalyzing signal amplification strategy [32] have been incorporated for precise detection of miRNAs. Over the past years, one of the approaches to improve miRNA detection is application of nanoparticles both as multifunctional labels and as carriers for loading of a large amount of low molecular weight probes [33]. One of the most appealing detection strategies is luminescence due to its sensitivity and possibility for multiplexing. Various luminescent nanoparticles were used for miRNA detection, such as different types of semiconductor quantum dots (QDs) [11,12,34,35], also SnO2 nanoparticles [36], rhodaminecoated cobalt ferrite magnetic nanoparticles [37]. In comparison with organic fluorophores, inorganic nanoparticles are photostable, bright, with wide excitation and narrow emission bands. Data related to the use of QDs for miRNA detection was published by us recently [12], whereas the review dealing with the application of nanoparticles for DNA detection was already published by Russ Algar at al [38]. Carbon nanostructures (CNSs), including graphene quantum dots (GQDs), graphene oxide (GO), and carbon nanoparticles (CNPs), are small non-toxic inorganic nanoparticles, cheap to produce, stable in aqueous media while showing the bright and tunable light emission [39,40]. CNPs have been actively examined and used for last ten years, but a significant lack in understanding of the fundamental relationship between the synthesis routes, obtained morphologies and properties limit their application [41,42]. This review is focused on the application of carbon nanostructures both luminescence emitters and quenchers, as well as electrochemiluminescence reagents, for detection of mi-RNA. To the best of our knowledge, this is the first survey related to the application of luminescent carbon nanostructures for miRNA detection. 2. Types of luminescent carbon nanostructures Carbon nanostructures include graphene nanosheets or nanographenes or graphene quantum dots (GQDs), which are either single graphene domains or more often multi-layers (up to 10) graphene formations. GQDs possess the unique structure-related optical, electrical and optoelectrical properties because of their pronounced quantum confinement effect. Due to their stable

photoluminescence, low toxicity and cytotoxicity, high inertness and chemical stability based on the intrinsic inert carbon property, and wide possibilities for surface and structure modification, GQDs are considered as a novel multifunctional material for biological, optoelectronics, energetic and environmental applications. As a contrast to traditional QDs, GQDs are biocompatible, with the enhanced surface grafting and ability to attach components through the pp stacking. GQDs structure, morphology, chemical properties, preparation and modification have been well studied and reviewed [43]. A variety of dopants (nitrogen, sulphur, chlorine, fluorine, etc.), have been introduced to the GQDs structure to diversify their functions and properties. The control of the size and shape of the particles has been realized by means of the preparation parameters, such as energy source, synthesis temperature and time, carbon source concentration and etc. [43,44]. Water-soluble GQDs can be synthesized using different techniques, including hydrothermal (chemical) cutting of oxidized graphene sheets, pyrolysis of organic compounds (such as citric acid [31], or aromatic based plane molecules [45]), hydrothermal treatment of organic compounds, such as pyrene, or 1,5-dinitronaphtalene, etc. QY values of GQDs reported to be varied in a wide range, reaching values of 80%. Due to their nanometer-size, the electronic transport in GQDs is confined in all three spatial dimensions, and GQDs energy levels, and therefore, their optical, electrical, and optoelectrical properties, can be modified using size variation and elemental doping. GQDs have shown promising prospects in bioimaging and biodetection fields [46], however lacking miRNA research. This can be possibly related to instinct, small size, and electronegativity of GQDs preventing the attachment of short nucleic acid sequences to their surface through the pp stacking between the aromatic rings of nucleobases and the hexagonal cells of GQDs [46]. Graphene oxide (GO) or graphene oxide sheets are the single monomolecular nanolayers of graphene with various oxygencontaining functionalities, such as carboxyl, carbonyl, epoxide, and hydroxyl groups [47]. GO are synthesized via the chemical exfoliation of graphite introduced by B.C. Brodie in 1859 [48]. Due to the oxygenated functional groups attached to their basal plane and edges, GO could be dispersed in water [49]. Recently GO have been extensively employed in biological target detection due to their extraordinary distance-dependent fluorescence quenching properties, along with the high photostability and resistance to photobleaching [15]. Very wide possibilities of GO analytical application should be mentioned [50,51]. Carbon nanodots (CNDs), carbon nanoparticles, C-dots [52] or carbon quantum dots [30] is another luminescent carbon-based nanomaterial with the less defined structure compare to GQDs. The most common hypothesis, related to CND' structure, is a presence of carbon atoms in sp2 and sp3 hybridizations and some graphene-like fragments [39,40,53]. The luminescence nature of CNDs could be related to the presence of some organic fragments with high QYs, graphene structures, surface defects etc. The main techniques to synthesize CNDs are hydrothermal [54], microwave [30] or solvotermal [55] treatment of carbon source solution, electrochemical synthesis [56], carbonization in a microreactors [57], etc. Polar groups derived from starting materials allow the particles to be well-dispersed in water and to possess multifarious functionality. Currently, CNDs are experiencing a surge of interest and are used as luminescent labels in various analysis. 3. Carbon nanostructures binding with oligonucleotides The intrinsic properties of a graphene structure, namely the high p-electron density, promote the adsorption of single-stranded oligonucleotides through the pp stacking interactions between the nucleobases and sp2 carbon atoms of the nanomaterials [13,58].

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Therefore, CNSs do not require any additional modification to be used in detection systems [31]. Owing to the rigid structural conformation of the double stranded DNA helix has less binding toward single-stranded ones. As a result, the CNSs possess different affinity towards single- and double-stranded sequences. This is used in the sensors for specific targeting [59]. To improve the DNA probe interaction with the graphene surface, introduction of any functional groups to CNSs' structure promote their covalent binding. For example, amine-terminated nucleic acid sequences could be covalently bound to the carboxyl terminated CNDs obtained from o-phenylenediamine in ethanol solution by the hydrothermal treatment [32], from candle soot by the thermal oxidation [52], or from citric acid e melamine water solution via the microwave synthesis [30] using EDC-NHS chemistry. Glutaraldehyde have been used for the conjugation of an oligoprobe with the CNDs prepared via the hydrothermal reaction between citric acid and ethylenediamine [54]. 4. Detection methods €rster resonance energy transfer 4.1. Fo €rster (or fluorescence) resonance energy transfer (FRET) is one Fo of the most popular and valuable techniques, characterized by a high sensitivity both in homogeneous and heterogeneous formats. FRET is a non-radiative energy transfer from a donor chromophore in the excited state, to an acceptor chromophore in the ground state through the non-radiative dipole-dipole interaction, without any photon or electron involvement [60]. High sensitivity and possibility for a multiplex detection designate the FRET application for miRNA detection. The conditions for FRET realization are: (i) energy of the donor excited state is higher than any possible energy level of an acceptor; (ii) a spectral overlap between the donor emission and the acceptor absorption; (iii) the donor and acceptor should be in a close proximity to each other (ca. 1e20 nm) [61,62]. The sensitivity of FRET to a donor-acceptor distance allows using FRET as a spectroscopic nanoruler [63]. For miRNA detection, carbon nanostructures are used both as energy donors and as energy acceptors (quenchers). 4.1.1. Carbon nanostructures as energy donors Noh et al. [52] used quenching of fluorescence of the CNDs, purified from candle soot by the thermal oxidation, by the black hole quencher 1. In the absence of the target miRNA, the quencher probe contained the black hole quencher 1 and the fluorescent CNDs in the molecular beacon were in a close proximity, which

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resulted in the CNDs' fluorescence quenching. In the presence of the target miRNA (miR124a), its binding to the beacon resulted in the detachment of the quencher oligo from the miR124a sensing probe and the fluorescence recovery (Fig. 1). The system was used as a self-promoted uptake of the miRNA imaging system into cells to sense miR124a expression. Mohamadi and Salimi [54] used the CNDs, obtained via the hydrothermal treatment of citric acid and 1,2-ethylenediamine, as an emissive donor, and MnO2 nanosheets as an acceptor. CNDs covalently labelled with a target miRNA probe were adsorbed onto the MnO2 nanosheets surface via the Van der Waals forces [64]. That resulted in the quenching of the CNDs fluorescence. In the presence of the target miRNA, a specific binding between the probe and the complementary miRNA resulted in detachment of the CNDs-labelled probe from the MnO2 nanosheets, leading to increasing of the CNDs' emission with increasing of the target microRNA concentration. The technique was used for microRNA155 detection in spiked human serum samples and in human breast cancer cell line MCF-7 with the detection limit of 600 cells mL1 [54]. Application of an emissive acceptor (acridone derivative 5,7dinitro-2-sulfo-acridone with the large Stokes shift) allowed Xia et al. [32] to use a ratiometric approach for the detection of the exosomal miRNA-21. The ratiometric approach was able to reduce the environmental fluctuations by calculating the emission intensity ratio at two different wavelengths. The bioprobe was based the CNDs-labeled DNA and the acridone derivative coupled with the target. The acceptor was bound with the double strand DNA via its intercalation. Such construction provided the high FRET efficiency when the bioprobe was assembled. The presence of the target miRNA induced the dissembling of the fluorescent bioprobes leading to the alteration in the fluorescent intensities of the donor and acceptor. Theoretically, a single miRNA-21 could catalyze the disassembly of multiple bioprobes. This strategy was applied for monitoring of the dynamic change in exosomal miRNA-21, which may become a potential tool to distinguish cancer exosomes from non-tumorigenic exosomes. A multifunctional system for the simultaneous detection of two miRNAs was developed by Zhang et al. [46] with GQDs as energy donors. They used the molecular beacon with pyrene moiety and the fluorescent dyes (Cy3 or Cy5). The role of pyrene was the noncovalent attaching of the probe to the GQD surface to guarantee the significant FRET between the GQDs and the fluorescent dyes. The hybridization between the target miRNA and the loop structure of the molecular beacon resulted in the opening of the hairpin structure, increasing the donor-acceptor distance, and thus

Fig. 1. FRET sensing of miRNA124a using the molecular beacon with CNDs as an energy donor and the black hole quencher 1 as an acceptor. Reproduced from Ref. [52] with permission from The Royal Society of Chemistry.

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decreasing the emission intensity of the fluorescent dyes. The authors showed the possibility for the multiplex detection of miRNA155 and miRNA-210 using two different dyes, Cy3 and Cy5. The authors suggested a cascade mechanism of the FRET: the GQDs were excited under 400 nm, and the given emission wavelengths could excite Cy3 to give the emission wavelengths used for exciting Cy5. Thus, Cy3 acted both as an acceptor of the GQDs and as a donor for Cy5.

4.1.2. Carbon nanostructures as energy acceptors Application of carbon nanomaterials as energy acceptors for FRET detection of miRNA is possible due to three main advantages of these graphene-like structures: (i) extraordinary adsorption capacity for single-stranded oligonucleotides, (ii) fast and ultraefficient fluorescence quenching capability, (iii) low affinity to double-stranded oligonucleotide sequences. The last one allows the desorption of fluorophore-labelled nucleic acids from the surface of CNSs in the presence of complementary oligonucleotides [63]. Practically, the intrinsic emission of carbon nanostructures does not play any role in this scheme. The attention-grabbing characteristics of CNSs such as their differential affinity towards single-stranded DNA and DNA/miRNA duplex and their high efficiency of fluorescence quenching have been used for the development of the homogeneous fluorescence sensing platforms [59,66]. The fluorescein-labelled single-stranded DNA adsorbed on the surface of the CNDs, resulting in the fluorescence quenching. Introduction of the miRNA complementary to the probe generated a double-stranded DNA/miRNA hybrid, released from the surface of CNDs and resulted in the fluorescence recovery (Fig. 2). CNDs acted as quenchers of the fluorescein

emission, causing the FRET from the fluorescein molecules (energy donor) to the CNSs (energy acceptors). The similar principle was used in work [65] for the simultaneous detection of miRNA-141 (the donor is cyanine dye Cy5) and miRNA21 (the donor is fluorescein) using GO as an energy acceptor (Fig. 3). The technique was used for the detection of two oncogenic-miRNAs (miRNA-141 and miRNA-21) in blood, urine and saliva. Dong et al. [31] used the similar principle for the detection of three miRNA (miRNA-16, miRNA-21 and miRNA26a) using three different fluorescent dyes (rhodamin, fluorescein and Cy5) as donors and GO monolayer with a size of 70e400 nm as an energy acceptor and single stage probes capturer. For sensitivity enhancement the authors used the isothermal stranddisplacement polymerase reaction amplification. The ability of GO to penetrate through the intracellular membranes and carry absorbed nucleic acid probes has been used to build the FRET sensors for the in-vitro detection of three miRNA (miRNA-21, miRNA-125b and miRNA-96) using the dye-labeled peptide nucleic acid (as the donors fluorescein, rhodamine and Cy5 were used) [67]. GO attached to the probes was able to penetrate through the cell membrane and deliver the cargo probes into cytoplasm, allowing the hybridization of the target miRNA with the dyelabelled probes, detaching them from the GO and thereby recovering the fluorescence intensity (Fig. 4). Therefore, GO could provide multiplexed miRNAs detection, so long as the different probes are labeled with different fluorophores. 4.2. Electrochemiluminescence Electrogenerated chemiluminescence chemiluminescence (ECL) is a result of

or electrothe emission of

Fig. 2. FRET miRNA detection; the energy donor is fluorescein, attached to the single-stranded DNA; CNDs are the energy acceptors. Reproduced from Ref. [59] with permission from The Royal Society of Chemistry.

Fig. 3. The FRET system for detection of two miRNA (miRNA-141 and miRNA-21), the energy donors are fluorescein and Cy5, attached to the single-stranded DNA probes; the energy acceptor is graphene oxide. Adapted with permission from Ref. [65]. Copyright (2019) American Chemical Society.

O.A. Goryacheva et al. / Trends in Analytical Chemistry 119 (2019) 115613

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Fig. 4. The FRETsystem for detecting the intracellular miRNAs. Reprinted with permission from Ref. [67]. Copyright (2019) American Chemical Society.

Fig. 5. Electrochemiluminescence miRNA sensor: (A) nicking enzymes Nb.BbvCI mediated signal amplification (NESA); (B) fabrication of the biosensor: (a) electrode modified with the GO/Au composite, (b) assembly of hairpin probe2, (c) incubation with 6-mercapto-1-hexanol, (d) forming of the signal enhancer; (C) ECL principle [30].

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intermediates, generated during electrochemical reactions in an electronically excited state. GQDs and CNDs were used for the miRNA ECL detection. The possible GQDs' ECL response in the presence of S2O2 8 is shown below [30,68]: CNS þ e / CNS S2O82 þ e / / SO42 þ SO4 CNS þ SO4 / CNS* þ SO42 CNS* / CNS þ hv An ECL biosensor for the miRNA-21 detection was developed based on the hairpin probe1 labelled with the CNDs and the nicking enzymes Nb.BbvCI-mediated signal amplification (NESA) (Fig. 5). First, the hairpin probe1-CNDs formed an Y-junction structure with the assistant probe and the target miRNA. This structure was cleaved by the addition of the nicking enzymes Nb.BbvCI. Subsequently, the released miRNA and assistant probe initiated next recycling process. Numerous intermediate sequences CNDs-DNA were further hybridized with the hairpin probe2 and immobilized on the GO/Au composite on the electrode surface. As a result, the initial ECL intensity enhanced with increasing of the target miRNA [30]. The ability of T7 exonuclease to degrade RNA and DNA from RNA/DNA hybrids together with its inability to degrade both double-stranded or single-stranded RNA22 was used by Zhang et al. [65] to detect miRNA in cell lysates. In this biosensor GQDs were assembled with aminated-3,4,9,10-perylenetetracarboxylic acid

through the p-p stacking. The Fe3O4eAu core-shell nanocomposite was bound to the anchor probe through the AueN bond. Au was used to accelerate the electron transfer, Fe3O4 allowed to realize the magnetic separation. Obtained material on the electrode surface captured of the hairpin probe through AueN bond. The helper DNA was hybridized with the target miRNA to open the hairpin structure. T7 exonuclease digested the hybridized DNA/RNA and released the miRNA to trigger another cycle. ECL signal was further enhanced by the AgNPs-based 3D DNA networks. Immobilizing of GQDs on the surface of an electrode shorten the distance of electronic transmission, improving the efficiency of the ECL response and ensure the stability of the sensor [65]. 4.3. Antenna enhancement of UCNP emission GQDs are able to increase the inherent low luminescence quantum yield of NaYF4:Yb, Er up-converting nanoparticles (UCNPs). It was used by Lourenti et al. [13] to develop a sensor for the specific miRNA sequence, which appears during the Dengue infection. The authors used the pp stacking between the sp2 carbon atoms of the GQDs and the DNA nucleobases anchored on the NaYF4:Yb, Er UCNPs to develop sensors with UCNP luminescence as an analytical signal. Interaction with a single-stranded DNA brought the GQDs close to the surface of UCNPs, resulting in the increase of the upconversion emission. The capacity of the GQDs to absorb near-infrared light allowed transferring the energy to the UCNPs. In the presence of the complementary miRNA sequences, they hybridize with the single-stranded DNA chains, blocking the interaction of GQDs with the UCNPs, and the UCNPs' luminescence enhancement.

Table 1 Examples of luminescent carbon nanostructures application for miRNA detection. Detecting system € rster resonance energy transfer Fo Da e GQDb Ac - Cy3 dye D e CNDd A - 5,7-dinitro-2-sulfo-acridone D e fluorescein A - CND D e fluorescein A e GOe D e CND A - black hole quencher 1 D e CND A e MnO2 nanosheets D e fluorescein and Cy5 dye A - GO D e fluorescein A - GO D - fluorescein, rhodamine and Cy5 A - GO

Analyte

LOD

Detection range

miRNA-155

100 pM

0.1e200 nM

miRNA-21

3.0 fM

let-7 miRNAs

3.5 nM

5e300 nM

miRNA-16

2.1 fM

5 pM to 5 fM

miRNA124a 0.15e20 aM

miRA-21 miRNA-141 miRNA-126

2.0 nM 1.2 nM 3.0 fM

up to 150 nM

miRNA-21, miRNA-125b, miRNA-96

~1 pM

Electrochemiluminescence GQD and S2O2 8

miRNA-155

0.83 fM

2.5 fM to 50 pM

CND and S2O2 8

miR-21

10 aM

10 aM to104 fM

Antenna enhancement of up-converting nanoparticles emission D e GQD DENV-2-vsRNA5 10 fM A - UCNPf a

c d e f

Donor. Graphene quantum dots. Acceptor. Carbon nanodots. Graphene oxide. Upconverting nanoparticles.

Ref.

Ratiometric signal

[32] [59]

P19 cells 0.1 aM

Comments

[46] exosomes

miRNA-155

b

Real sample

isothermal stranddisplacement polymerase reaction amplification visualization microRNA124a expression

[31]

[52]

MCF-7 breast cancer cells, human serum blood, urine, saliva

[54]

lung cancer cell lysate samples (H226, A549, and H358) cytoplasm

[66]

Hela, HK-2, L02 and 22Rv1 cell lysates

[68]

[65]

[67]

signal amplification of the target induced cycling reaction

[30]

[13]

O.A. Goryacheva et al. / Trends in Analytical Chemistry 119 (2019) 115613

5. Conclusions The possibility to use CNSs as carriers, concentrators, quenchers and elements sensitive to target miRNA have been discussed and summarized in Table 1. Summarizing the available data, the main advantage of luminescent carbon nanostructures for miRNA detection is their unique multifunctionality. CNSs possess two main functionality pools. The first functionality is directly related to the high p-electron density of sp2 graphene-based structure. This allows to use CNSs as carriers for single-stranded oligonucleotides as well as for their pre-concentration. The less binding of the doublestranded DNA helix to CNSs toward single-stranded ones leads to the separation of oligonucleotides from the CNSs surface and can be used as an indicator of the formation of double stranded DNA. Surface carboxylic and hydroxyl groups provide their good solubility in water and therefore the possibility to apply these particles in homogeneous assay formats. In the presence of complimentary chains, double strange hybrids form and release from the surface of CNSs, because their affinity to double stranded DNA or well-folded single stranded DNA is much lower. To conclude, CNS is a target miRNA sensitive element. The second functionality is related to the characteristics of the energy levels of carbon nanomaterials. Energy of the electronic levels determines the properties of materials to absorb or emit photons (or electrons), which are related to the emission and quenching properties of CNSs. Changing of the size of nanographene structures, amount and location of defects, nature and amount of additive atoms and groups allow to vary a position of the energy levels, and therefore their PL properties. The common limitation of developed miRNA detection method is sometimes a poor overall correlation between methods as well as relatively poor replication. Also, it is important to mention, only a few studies compared their performances with those of other methods. In addition, because miRNAs vary slightly in guaninecytosine content and length, they have various melting temperatures, which can complicate multiple detections, leading to either false-positive results due to nonspecific hybridization or falsenegative results due to hybridization signals that do not exceed the background. CNSs are perspective for miRNA detection, while their synthesis is simple and cost-effective. The significant limitation of CNSs is their higher level of non-homogeneity. For most of the CNSs synthesis methods, this non-homogenicity is higher than for plasmon gold nanoparticles or semiconductor quantum dots.

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