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]
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 MnO
2 nanosheets as a quenching agent was also studied. FRET from modified C-dots to MnO
2 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 MnO
2 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 MnO
2 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 MnO
2 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, TiO
2 was grown on the edges of gold nanorods (AuNRs) to form dumbbell-shaped structures (AuNRs@end-TiO
2), 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 TiO
2-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 Cu
2+/carbon nanotube (Cu
2+/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, H
2O
2, 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]