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Carbon Nanodots-Based Nano-Biosensors: Comparison
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Please note this is a comparison between Version 2 by Jessie Wu and Version 3 by Jessie Wu.

Semiconductor quantum dots (QDs) were a modern form of nanostructure that demonstrated excellent qualities for diagnosis and therapy. Controlling QDs size and distribution made it simple to adjust their electrical and optical characteristics. Yet, since certain semiconductor QDs include hazardous substances such as, cadmium, arsenic, selenium, and mercury, they have several disadvantages. One such disadvantage is cytotoxicity. As a result, these QDs are neither environmentally friendly nor biodegradable. On the other hand, since their inception in 2004, carbon nanodots (CNDs) have been recognized as a strong contender to replace the extremely dangerous metallic semiconductor class of quantum dots. This is partly because the characteristics of carbon quantum dots are widely acknowledged to include their nanoscale size, roughly flat or spherical morphologies, great water solubility, broad absorption in the UV-visible light spectrum, and vibrant fluorescence. CNDs have an amorphous or nanocrystalline center, mainly sp2 carbon, graphite grid spacing, and outside oxygenic functional groups, allowing for water solubility and subsequent complexation. 

  • biomarkers
  • carbon nanodots
  • miRNAs

1. Synthesis of Carbon Nanodots

Both “bottom–up” and “top–down” strategies may be used to create semiconductors based on CNDs. Bottom–up strategies encompass thermal decomposition, electrochemical carbonization, microwave irradiation synthesis, and hydrothermal/solvothermal treatment. Top–down methods include laser ablation, ultrasonication, arc discharge and electrochemical oxidation. For both these processes, a strict reaction environment is frequently needed, including high-grade carbon substrates, extreme heat, powerful alkali/acid solutions, and hazardous organic solvents.[1]

1.1. Top–Down Approach

Due to their simplified preparation procedures, top–down approaches are appropriate for the mass manufacturing of CNDs nanomaterials. The top–down approach “cuts” carbon particles including CNTs and graphite into CNDs via an arc discharge, laser ablation, or chemical oxidation. Arc discharge and laser ablation are the most commonly used top–down methods for producing CNDs. Gonçalves et al. used laser ablation in water solution, N-acetyl-l-cysteine, and NH2-polyethylene glycol (PEG200) to create passivated CNDs. Chemical oxidation involves introducing oxygen-containing hydrophilic functional groups into carbon nanostructure complexes by oxidizing them with a potent acid. The carbon nanostructures become water-soluble, which facilitates their discharge into the fluid. Scientists have also produced CNDs by hydrothermally slicing graphene sheets.[2] Wang et al. used graphene oxide as the precursor to creating C-dots using the hydrothermal process with microwave assistance. Carbon dots that are hydrophilic, hydrophobic, or even amphiphilic can be made using microwave-assisted synthesis. A simple one-step microwave-assisted synthesis of hydrophobic C-dots was described by Mitra et al. Using glucose as a starting material, Ma et al. reported the ultrasonic synthesis of N-doped C-dots (Figure 1).[3]
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Figure 1. Top–down and bottom–up approaches for the synthesis of CNDs by using hydrothermal, microwave pyrolysis, thermal decomposition, laser ablation and other different synthesis methods.

1.2. Bottom–Up Approach

In a “bottom–up” approach, CNDs are synthesized from small carbon molecules using microwave, hydrothermal, and pyrolysis methods. Basic principles involve burning and heating carbon precursors. CNDs can be prepared very efficiently through a bottom–up approach using a plethora of starting materials, and the choice of reactants determines their properties, especially in surface coating. Much more important is the fact that the roots of the carbon substrate can have a massive effect on the CNDs’ characteristics, including their sensing capabilities. Another advantage of the bottom–up approach is the easy addition of heteroatoms and other dopants. Sucrose, citric acid, amino acids, and food waste are carbon sources.[4]
Direct pyrolysis, the pyrolytic technique, or the carbonization of precursor materials at high temperatures are standard methods for producing carbon dots. Zhu et al. were the first to employ microwave pyrolysis as a synthesis mode, using a dissolved saccharide and PEG-200. The size of CNDs increased with reaction time as this solution was heated in a 500 W microwave. The yield of CNDs increases, and side reactions are reduced during microwave pyrolysis. Many CNDs variants have been created through direct thermal decomposition, in which precursors are heated in an inert environment until they are carbonized. Solvents are then used to extract them. The carbonization of small molecular precursors is used in the bottom–up synthesis of CND. One of the most common bottom–up synthesis approaches produces CND from a mixture of citric acid and a nitrogen-containing molecule such as urea.[5] When these molecular precursors are pyrolyzed by microwaves or in an autoclave, the synthesis readily produces a black nanopowder of CNDs, which is highly dispersible in water and displays remarkable fluorescent properties. Depending on the conditions, these CNDs can display blue, green, or red emissions, although extensive purification is often needed to isolate CNDs from molecular intermediates produced during the synthesis. Bottom–up methods were efficient routes to produce fluorescent CNDs on a large scale. For example, small molecules and polymers can undergo dehydration and further carbonization to form CNDs.[6]

1.3. Preparation of CNDs Using Green Approach

CNDs synthesized from biological sources play a significant role in biomedical and environmental applications, including bioimaging, biosensing, metal ions detection and electrocatalytic oxidations. Green synthesis has attracted the interest of scientists because it is cost-effective, less hazardous, eco-friendly, less time-consuming, and requires lower temperatures (Table 1). The production of CNDs from mostly reusable substrates includes naturally available raw materials that are relatively cheap and simple to make. CNDs made from natural sources can be used to transform low-value biomass waste into rich and valuable products. The low manufacturing cost and constant availability of raw ingredients for CNDs synthesis have made it a viable procedure for the industry also. Additionally, no dangerous organic solvents are required; instead, an aqueous solution may be used, increasing the CND's water solubility (Figure 2).[7]
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Figure 2. Various natural precursors for the synthesis of CNDs by using hydrothermal, microwave, pyrolysis chemical oxidation and carbonization as green approaches.
Recently, Hashemi et al. manufactured CNDs using a low-cost, simple, and green one-step hydrothermal process, producing luminous CNDs with high quantum yield from red beetroot as an organic source. According to the paper, red beetroot was sliced into small pieces and mixed with deionized water, continuously swirling for 20 min before being sonicated for an hour. The mixture was then placed in a Teflon-lined autoclave and heated in the oven (180° for 10 h). It was then centrifuged (1000 rpm for 30 min) and filtered to obtain the CNDs solution. To obtain a pure CNDs solution, the mixture was dialyzed for three days to remove contaminants.[8] As a result, in the current context, the green synthesis approach of C-dots produces high C-dot yields at a cheap cost because of low-cost raw materials. The simple procedure adopted, as well as the fluorescence qualities found in C-dots derived from environmentally sourced materials, open the way for harmless and biocompatible C-dots to be used in sensing approaches. The study describes a single-step hydrothermal strategy to synthesize colored CNDs from maple leaves to specifically capture cesium ions. The CNDs made emit blue fluorescence and varied in size from 1 to 10 nm. Based on the electron transfer method, these CNDs were successfully employed in glycerol electro-oxidation catalysts and cesium-detecting probes.[9] Arumugham et al. made CNDs using catharanthus roseus (white) leaves as the carbon source without the addition of an oxidizing agent or an encapsulant. These CNDs have excellent antioxidant activity and bioimaging potential against MCF-7 cells as well as strong fluorescence (FL) emission, high water solubility, stability, and non-toxicity, among other properties.[10]
Table 1. List of Various Natural Sources Used in the Preparation of Carbon Nanodots Using Different Green Synthesis Methods.
S.

No.		 Source Method of Synthesis Size Percentage Yield Detection Limit Inference References
1 Banana peel Microwave treatment 5 to 15 nm 16.0% 1.82 × 10–17/mol CNDs are fabricated by the microwave treatment of banana peels in a single pot for the determination of colitoxin DNA in human serum. [11]
2 Sargassum fluitans Hydrothermal 2–8 nm 18.2% - A hydrothermal method is used to produce CNDs from waste seaweed sargassum fluitans (S. fluitans) to detect DNA. [12]
3 Tomato juice Hydrothermal 1.3–3.7 nm 13.9%
Abbreviations: nanometer (nm), millimeter (mm), micrometer (µm), carcinoembryonic antigen (CEA), Escherichia coli (E. coli), Phyllanthus acidus (P. acidus), nanograms (ng).
Kumar et al. simply heated orange juice at 120 °C for 150 min without using any specialized methods or chemicals. These spherical CNDs have a restricted size distribution, as seen by recorded electron microscopy. The hydrothermal technique used for this research is a reactive technique that produces CNDs of good yield and high quality. In a recent article, Saleem et al. present a one-step flexible approach to produce fluorescent CNDs utilizing carrot root species. The synthesized CNDs worked as nano-vehicles for the mitomycin medication delivery. By breaking hydrogen bonds in the moderately acidic extracellular milieu of the tumor, the manufactured CNDs efficiently interacted with the mitomycin drug. This caused the release of mitomycin.[36]
As probes for the detection of heavy metal ions, fluorescent nitrogen-doped CNDs with 5.23% nitrogen content were made utilizing a one-pot microwave processing of lotus roots. The properties of egg yolk oil (EYO) were studied by Zhao et al., who utilized microscopy, spectrophotometry, and chromatography to detect the CNDs that were present in the EYO after it had been extracted and purified using water, dialysis, and ultrafiltration (EYO CNDs). The bleeding periods of mice treated with CNDs were noticeably shorter than those of control animals in tests on liver and tail hemorrhaging. According to coagulation tests, EYO CNDs stimulate and activate the fibrinogen system as well as the intrinsic blood coagulation system. Therefore, EYO CNDs have the capacity to stimulate hemostasis, which may prompt more research into this component of traditional Chinese medicine.[37] Xiao et al. present an inexpensive, easy, and effective microwave pyrolysis method to synthesize highly amino-functionalized fluorescent (CNDs). Through the dehydration of chitosan, the formation and functionalization of CNDs were successfully accomplished. Using a brand-new, quick microwave-assisted method that entails two stages, CNDs with an average size of 9 nm were created from an aqueous solution of raw cashew gum (RCG). A composite of partly depolymerized CG and CNDs was created at the end of the procedure.[38]
In a study, the ecologically friendly one-step electrodeposition method for creating GR-based hybrids was employed which avoids chemically reducing graphene oxide (rGO), which would cause further pollution. The entire process is straightforward and takes only a few minutes. Combining the benefits of GR, CNTs, and CS, the GR/CNTs/CS hybrid was created and might be used to trap organophosphate pesticides. In another study, a simple, cost-effective, and environmentally friendly method for producing ternary nanocomposites of carbon, polydopamine, and gold was demonstrated. The technique did not employ harsh reaction conditions such as those found in hydrothermal or high-temperature techniques. The excellent electrocatalytic activity was demonstrated by the CNTs/PDA/AuNPs modified electrode to oxidize chloramphenicol.[39] One more research study covered a synthesis of multiwall carbon nanotube/Cu2O-CuO ball-like composite (MWCNTs/Cu2O-CuO) adopting a green hydrothermal approach which had been investigated as a novel sorbent for the solid-phase extraction of uranium utilizing inductively coupled plasma mass spectrometry.[40]

2. Carbon Nanodots in Biosensing of mMicroRNAs

Macromolecules and circulating analytes in biological systems must be detected in a way that is efficient, reliable, and inexpensive. Recent developments in the field of biosensors have aided the development of functionalized nanosensors that have the potential to provide a cost-effective, efficient, and quick diagnostic approach for the detection of circulating miRNAs. Along with this, some unique properties—such as biocompatibility, high stability and water dispersibility, and accessible green synthesis, surface functionalization of C-dots that creates a strong interaction between CNDs and biological processes—all make them significant for sensing circulating analytes.[41] Fluorescent, colorimetric, chemiluminescent, and surface plasmon resonance are the most common sensing systems used to detect circulating miRNAs.[42] This is due to the relative ease of making fluorescent CNDs and their photostability, which can be used as low-cost alternatives for sensing significant biomarkers (Table 2). Fluorescence-based analytical approaches allow for the accurate, efficient, and reproducible detection of biomarkers and nucleic acids. Furthermore, changes in fluorescent signals caused by biological events such as nucleic acid probe hybridization are detectable. Thus, fluorescence-based detection technologies have become increasingly popular due to these benefits.[43]
Table 2. List of Various Carbon Nanodots Used in Biosensing of Cell-Free Circulating MiRNAs Using Along with Synthesis Sources, Conjugation Chemistry, Analytical Methods, Target miRNAs, and Detection Limit.
S. No. Carbon Nanomaterial Source and Synthesis Conjugation Chemistry Biomolecule

(Analyte)		 Analytical Method Detection Limit Inference References
1 Carbon nanodots (CNDs) O-phenylene diamine, 2-amino terephthalic acid by solvothermal method EDC-NHS miRNA-21 Fluorescent biosensor 0.03 fM CNDs are synthesized and conjugated via EDC-NHS chemistry to detect miRNA-21. [44]
2 PEI-Carbon dots Polyethyleneimine (PEI) by hydrothermal method - miRNA-21 Fluorescence biosensor - The synthesized CNDs is employed to detect miRNA-21 by fluorescence biosensor. [45]0.3 ng/mL CNDs are synthesized by hydrothermal treatment of tomato juice for the sensing of carcinoembryonic antigen. [13]
3 CNDs/AO Citric acid in formamide π-π conjugation miRNA-92a-3p Fluorometric assay (FRET) 0.14 nM To detect miRNA-92a-3p, CNDs are fabricated and conjugated using π–π conjugation. [46] 4 Limes Pyrolyzing 5-10 nm - - The pyrolyzing process is used to synthesize CNDs for the detection of hepatitis B virus DNA.
4 CNDs–DNA walker Citric acid and urea by microwave-assisted method EDC-NHS miRNA-21

miRNA-155		[ Electrochemiluminescence biosensor14]
33 fM for miRNA-21.

33 aM for miRNA-155 CNDs are created and conjugated via EDC-NHS chemistry to discover miRNA-21 and miRNA-155. [47] 5 Lemon juice Carbonization 6–9 nm - 0.23 mM Carbonization of lemon juice is performed to form CNDs for the detection of l-tyrosine. [15]
5 CNDs Oxidized maple leaf by a pyrolytic method EDC-NHS miRNA-21 Electrochemiluminescence biosensor 21 aM CNDs are synthesized and conjugated via EDC-NHS chemistry to detect miRNA-21 associated with breast cancer. [43]6 Lemon Pyrolyzing 10 nm - 0.0049 µM Synthesis of CNDs from a lemon by the process of pyrolysis for the detection of doxorubicin hydrochloride in human plasma. [16]
6 CNDs Tiger nut milk by carbonization - miRNA-21 Chemiluminescence biosensor 0.721 fM Synthesized CNDs are used to detect miRNA-21 associated with cardiovascular disease. [ 7 Syringa oblata lindl Hydrothermal 1.0–5.0 nm 12.4%, 0.11 μM A hydrothermal method is used to fabricate CNDs from syringa oblata lindl for sensors and cell imaging. [17]
48]
7 CNDs Glutaraldehyde, nitro benzaldehyde by solvothermal method - miRNA-21 Fluorescence sensor 0.03 fM An miRNA-21 associated with breast cancer is identified using a fluorescence sensor that is based on carbon dots. [49] 8 Grapefruit Hydrothermal >30 nm 20% - Grapefruit is used to create CNDs using a hydrothermal process for the detection of E. coli bacteria.
8 CNDs Malic acid centrifugation EDC-NHS[18]
miRNAs Fluorescence 0.03 pM The synthesized CNDs is conjugated via EDC NHS chemistry and used to detect miRNA. [50] 9 Alfalfa and garlic Hydrothermal
9 CNDs1.3–6.9 nm 10% Citric acid by microwave method π-π stacking miRNAs86 nM A hydrothermal method is used to form CNDs from alfalfa and garlic as a fluorescent probe for cysteine, glutathione, and homocysteine. [19]
Fluorescence biosensor 2.78 fM CNDs were synthesized and employed to detect miRNAs by fluorescence biosensor. [51] 10 Catharanthus roseus (white flowering plant)
10 CNDsHydrothermal carbonization - - - Catharanthus roseus (white flowering plant) is hydrothermally carbonized to create CNDs to detect the Al3+ and Fe3+ ions. Tree leaves by hydrothermal method EDC-NHS miRNA-155[10]
Fluorescence biosensor FRET 0.3 aM CNDs were synthesized and conjugated via EDC-NHS chemistry and were used to detect miRNA-155 by fluorescence biosensor. [52] 11 Lemon juice Hydrothermal - -
11- CNDs/BHQ 2 Ethane diamine, p-benzoquinone Maleimide-thiol miRNA-141The one-pot facile hydrothermal approach was used to create highly luminous carbon dots (C-dots) from lemon juice. FRET[20]
16.5 pM miRNA-14 is conjugated with synthesized CNDs via maleimide–thiol conjugation chemistry and detected by a fluorimetry test. [53] 12 Daucus carota Hydrothermal -
127.60% Carbon nanotubes (CNTs) Hydrogen tetrachloroaurate

trihydrate		 EDC-NHS- A hydrothermal method is used to produce CNDs from Daucus carota to detect mitomycin. miRNA-21 Fluorescence biosensor 36 pM[21]
A synthesized CNT is conjugated via EDC-NHS chemistry to detect intracellularly

miRNAs-21. [54] 13 Natural polymer starch Hydrothermal 2.25–3.50 nm - - Hydrothermal treatment of natural polymer starch is performed to produce CNDs. [22]
13 CNDs Pyrolysis synthesis Amine-amine conjugation miRNA-21 Ratiometric fluorescence 1 pM Synthesized CNDs were used to detect miRNA-21 associated with gastrointestinal cancer. [55] 14 P. acidus Hydrothermal 5 nm
14 CNDs12.5% - Citric acid ethylene diamine/carbonization Amine -glutaraldehyde miRNA-155CNDs are produced by a hydrothermal process from P. Acidus for live cell imaging. [23]
FRET 0.1 aM Fabricated CNDs are used to identify miRNA-155 present in cancer cells. [56] 15 Citrus peel powder
15 CNTs (MWCNT/AuNCs)Sand bath heat-assisted method 4.6 ± 0.28nm - - The sand bath heat-assisted method is utilized to form CNDs from citrus peel powder for free radical scavenging and cell imaging. Carboxylic acid-ultrasonic cell

disruption[24]
Thiol conjugation miRNA-155 FRET 33.4 fM CNTs are synthesized and used to detect miRNA-155. [57] 16
16Lentil Hydrothermal 7 ± 4 µm 10% CNDs3.0 µg Citric acid–

hydrothermal		 π-π stacking micro-RNAA hydrothermal method is used to form CNDs from lentils for the colorimetric determination of thioridazine hydrochloride. Fluorescence[25]
  A CNDs is used to detect miRNA by fluorescence method. [58] 17 Rose flowers Hydrothermal 1.0–5.0 nm - 0.02–10 µM CNDs are produced by a hydrothermal process from rose flowers for the determination of diazinon. [26]
18
17 CNTs -   miRNA-21 Electrochemical biosensor 1.95 fM miRNA-21 is detected by a carbon nanotube-based electrochemical biosensor. [59] Saffron Hydrothermal >20 nm 23.6% 1.8 n/mol A hydrothermal method is used to produce CNDs from saffron for the sensing of prilocaine.
18s Carbon nanofibers/SPE -[ Amine-carboxylic acid conjugation miRNA-34a27 Electrochemical biosensor]
54 pM The electrochemical biosensor is utilized to detect miRNA-34a using carbon nanofibers. [60 19 Valerian root Hydrothermal >10 nm 14% 0.6 ng/mL Valerian root has been used to make CNDs using a hydrothermal process for the determination of imipramine. [28]
]
19 DNA-CNDs/CNTs - π-π stacking miRNA-7f Photoelectrochemical biosensor 34 fM A CNTs is used to detect miRNA-7f by a photoelectrochemical method. [61] 20 Rosemary leaves Hydrothermal Approx. 5 nm.
20 Carbon nanoparticles/ssDNA probe Graphite electrode by an electro-oxidation method18% 8 ng/mL Rosemary leaves have been used to make CNDs using a hydrothermal process for the determination of thiabendazole in juices. [ π-π stacking29]
miRNA let-7a Fluorescence 0.35 pM Synthesized carbon nano-particles conjugated via π–π stacking are used to detect miRNA let-7a. [62] 21 Beetroot Microwave 5 & 8 nm 6% & 5% - CNDs made from aqueous beetroot extract by the process of a microwave for in vivo live animal imaging applications. [30]
22 Eutrophic algal blooms Chemical oxidation Approx. 8 nm 13% - Eutrophic algal blooms have been used to make CNDs using chemical oxidation for in vitro imaging. [31]
23 Green tea leaf Pyrolyzation 2 nm 14.8% - Synthesis of CNDs from green tea leaf by the process of pyrolysis for the sensing of gefitinib. [32]
24 Waste tea residue Chemical oxidation 3.2 nm 2.47% Be 0.04 μg /mL Waste tea residue has been used to make CNDs using chemical oxidation for the quantification of tetracycline. [33]
25 Palm shell powder Chemical oxidation 4–10 nm 6.8% 0.079 µM CNDs are synthesized by the chemical oxidation method from palm shell powder for the sensing of nitrophenol. [34]
26 Soybeans Ultrasonic-assisted method 2.4 nm 16.7% 0.9μM An ultrasonic-assisted method is used to produce CNDs from soybeans to detect Fe3+ ions. [35]
Abbreviations: Forster resonance energy transfer (FRET), Single-stranded DNA (ssDNA), Ethyl(dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC-NHS), Screen-printed electrodes (SPEs), Black hole quencher 2 (BHQ-2), Multiwalled carbon nanotube (MWCNT), Gold nanocomposites (AuNCs), Polyethyleneimine (PEI), Acridine orange (AO), Femtomolar (fM), Nanomolar (nM), Attomolar (aM), Picomolar (pM).
When a target interacts with a recognition element, a fluorescent biosensor translates information quantitatively or semi-quantitatively. After hybridizing complementary nucleic acid with its target miRNA, fluorescent-based nucleic acid detection can be generally achieved via signal-on (signal production) and signal-off (signal quenching). For DNA hybridization and tumor marker detection, carbon nanomaterial biosensors based on the FRET mechanism have practical utility in research and clinical practice. A FRET sensing platform for sensitive miRNAs detection using the miRNA-155 probe-labeled C-dots as a fluorophore and MnO2 nanosheets as a quenching agent was also studied. FRET from modified C-dots to MnO2 nanosheets can dramatically reduce the fluorescence of modified C-dots. The quenched fluorescence could be recovered when the target analyte miRNA-155 was introduced.[63] The principal mechanism for sensing miRNA is when the C-dots-miRNA probe is mixed with MnO2 nanosheets, which absorb the C-dots-miRNA probe on its surface. Due to FRET, the fluorescence of the C-dots-miRNA probe decreases as the concentration of MnO2 nanosheets increases.[64] When the complementary target miRNA was added, specific binding occurred between the search and the target miRNA, causing the C-dots labeled miRNA hybrid to separate from the MnO2 nanosheets. As a result, as the concentration of miRNA rises, fluorescence intensity is restored and increased.[65] In a recent report using the colorectal cancer-specific miRNA miR-92a-3p as such a target, the efficacy of a ratiometric fluorescence biosensor made of CNDs and acridine orange is evaluated. The variables that determine the viability of the ratiometric fluorescence bioassay are the charges properties of the DNA probe, target miRNA, CNDs, and AO, as well as the fluorescent properties of CDs and AO. The targeted miRNA has a detection limit for the ratiometric fluorescence biosensor: 0.14 nM.[46] CNDs are also used in paper-based analytical devices (PADs), which were previously reported for detecting circular RNA and miRNA-21 from the hippocampal via probe DNA conjugations and in situ manufacture of blue-emissive CNDs. Using miRNA-21 color analysis, stunning blue-to-green and blue-to-red emission color changes of the PADs are obtained.[66] miRNA-21, a predictor of numerous pathologies including cardiovascular illnesses, is detected sensitively and specifically using an easy CNDs-based electron transfer chemiluminescence biosensor.[48] Shandilya et al. designed and constructed a nanophotonic method employing oligonucleotide-conjugated graphene quantum dot–nanoconjugates, which is a derivative of carbon dots for the quick and precise capture of lncRNAs. The technique provides very selective and precise target lncRNA identification. The data also indicated the method’s great practicality and simplicity in determining lncRNAs selectively.[67] Similarly, Chen et al. created a label-free, enzyme-free fluorescent scheme based on strand displacement amplification (SDA) to detect miRNA with extreme sensitivity utilizing CNDs functionalized with sulfydryl (CDs-SH) as the probe. Based on the catalytic oxidation of -SH into -S-S- by hemin/G-quadruplex, CDs-SH demonstrated an outstanding response to G-quadruplex DNA against other DNAs.[49] In a study using on CNDs, a sensor for miRNA 9-1 recognition was created. On excellent fluorescence QY, water dissolvable, and low-toxicity CNDs, single-strand DNA with the FAM tag was immobilized. As a physical attribute for sensing, the fluorescent quenching of CNDs caused by the transfer of energy of fluorescence resonance among CNDs and FAM was utilized.[58] Jiang et al. proposed a self-assembled tetrahedral DNA nanostructure coupled with gold nanoparticles (AuNPs) and CNDs. The constructed nanostructure enables double fluorescence channels for the parallel estimation of miRNA and telomerase function, which is also easily transported within live cells for in situ scanning by adding an iRGD peptide sequence afterward.[63] Using single-walled carbon nanotubes, a derivative of CNDs that have been sensitized with DNA-CdS emiconductor quantum dots (QDs), a flexible photoelectrochemical biosensors platform has been created. A practical, accurate, and focused biosensor for the direct detection of miRNAs was developed by integrating with cyclic enzymatic multiplication, offering a unique method for miRNA detection.[61] In another study, they presented the selective and sensitive detection of exosomal miRNAs using a ratiometric fluorescent bio-probe based on DNA-labeled carbon dots (DNA-CNDs) and 5,7-dinitro-2-sulfo-acridone coupling with the target-catalyzing signal amplification, in which high FRET between CNDs and DSA boosted the assay’s sensitivity of the bio probe.[68] Similarly, a new strategy was developed for the construction of a dual-emission fluorescent sensor using a new ratiometric nanohybrid fluorescent probe for the detection of miRNA-21 with dual-colored CNDs (blue CNDs and yellow CNDs) as they are provided with the same excitation wavelength (360 nm), two distinct and steady emission signals (409 and 543 nm) were produced as fluorophores and then their applications for ratiometric miRNA-21 sensing and the bioimaging of cancer cells in a microfluidic device were confirmed.[44] The strong and precise binding of DNA probe functionalized B-CNDs to the complementary miRNA-21 target caused probe structural perturbations and changed fluorescence intensity in both wavelengths as miRNA-21 concentration increased. Thus, because of its rapid reaction, high sensitivity, and technical simplicity, the proposed fluorescent nano-biosensor has become a reliable analytical sensing tool.[69] The great sensitivity (because of CNDs’ high brightness), multiplex capability (due to CNDs’ color tunability), and homogeneous assay formats are all key advantages of CND’s performance in fluorescence biosensors for the detection of circulating nucleic acids.[70] Using CNDs as photosensitizers, TiO2 was grown on the edges of gold nanorods (AuNRs) to form dumbbell-shaped structures (AuNRs@end-TiO2), which were then hydrophobically attached to fluorine tin oxide (TiO). FTO was bonded to the electrode surface. As a result, a compact photoelectrochemical miRNA-21 was created. Hairpin probes (HPs) were used to bind to the TiO2-modified FTO electrode surface, while CNDs-modified homologous DNA (CNDs-cDNA) served as the photosensitive label. When targets were present, the miRNA hybridized with the HP, which caused a double-stranded specific nuclease to associate with the miRNA to the homologous segment of the HP. This released the miRNA, potentially starting a new cycle that would result in signal acquisition.[71] Gold nanoparticles conjugated with CNDs have also demonstrated excellent sensing capability. In a study, gold nanoparticles conjugated with CNDs have also shown excellent sensing capabilities. Photo-assisted biofuel cell-based self-powered biosensors (PBFC-SPBs) is also used in biosensing to identify miRNA. The coupling of PBFC-SPBs for miRNA monitoring with a Cu2+/carbon nanotube (Cu2+/CNTs) cathode with laccase-mimicking activity made this possible. When the target was identified, the matched miRNA with the same sequence eluted DNA2/CdS from the electrode, resulting in a weak signal. The method does not require the use of an external power source[72]. CNDs have been also widely used for the fluorescent analysis of various targets, including small molecules such as ions, H2O2, and biomolecules, due to their excellent PL properties. Aptamers were also recognized using CNDs. Aptamers are artificial single-stranded DNA or RNA that have a high affinity for different analytes. Xu et al. created an aptasensor for thrombin detection; it has several aptamer binding sites. Two thrombin aptamers with amino groups were created. They were modified separately on silica nanoparticles and CNDs, and both are capable of recognizing thrombin by forming an intramolecular G-quadruplex.[73]

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