Metal-free quantum dots such as carbon and BN quantum dots (CQDs, BNQDs) have multiple advantages over conventional semiconductor quantum dots. It is believed that CQDs and BNQDs exhibit higher photophysical, chemical, and photochemical stability. Besides, CQDs and BNQDs are non-toxic and more biocompatible [
39,
85]. It was reported that BNQDs are more biocompatible than carbon QDs as the latter would undesirably interact with biomolecules (DNA, proteins, or enzymes) that would compromise the intrinsic properties of these biomolecules [
70]. Besides, BNQDs are more tolerant of different pH environments in the range of 2–12, with no significant effect on the fluorescence of BNQDs [
40]. The cytotoxicity and biocompatibility of BNQDs were studied; excellent biocompatibility was shown [
23,
36,
39,
40,
44,
45,
47,
57,
81].
1.1. Bioimaging
Organic dyes are popular for bioimaging applications for their relatively low cost and good biocompatibility. However, organic dyes are limited by photobleaching where their fluorescence brightness is degrading under the prolonged irradiation of excitation laser light. The use of nanoparticles such as BNQDs with strong fluorescence, photostable, nonblinking, and nonbleaching would be a good alternative for cellular imaging probes. Lin et al. have reported on the use of BNQDs for the imaging of MDCK-II (Madin-Darby canine kidney) cells by confocal microscopy in the FITC (fluorescein isothiocyanate) mode as shown in . The BNQDs were internalized into cells but did not penetrate the cell nuclei [
23]. A similar study was performed on different types of cells by others [
39,
40,
44,
46,
57,
81]. Jung et al. [
47] studied the internalization of edge-hydroxylated BNQDs (EH-BNQDs) by MCF-7 breast cancer cells and PC-3 prostate cancer cells. Blue fluorescence in the perinuclear region was demonstrated. The locations of EH-BNQDs inside the cells were compared with the locations of intracellular endosomes that were labeled with Lysotracker (red dyes), which resulted in a purple color due to the overlapping of blue and red fluorescence under confocal microscope proving the successful endocytosis of QDs. A similar study was performed on HeLa cells [
40].
Figure 8. Confocal microscopy images of mammalian cells. (
a) Agglomerated BNQDs surrounding each nucleus (cells are stained by BNQDs only). (
b) Individual nucleus stained blue with DAPI (4’, 6-diamidino-2-phenylindole). (
c) BNQDs with green luminescence surrounding the nuclei. (
d) The overlay image of cells stained with DAPI and BNQDs. Reproduced from [
23] with permission from 2013 Wiley-VCH Verlag GmbH & Co. KGaA (Weinheim, Germany).
Thangasamy et al. [
50] reported the imaging of bacterial cells (Gram-negative and Gram-positive) using BNQDs. Both types of cells were stained with 100 µg/mL of BNQDs. According to images from epi-fluorescence microscopy and confocal microscopy, the internalization of BNQDs occurred for Gram-negative cells but not for Gram-positive cells. It is to be noted that the Gram-negative cells possess an extra lipid layer outside the peptidoglycan layer, which Gram-positive cells lack. This interesting interaction of BNQDs with the outer lipid layer of Gram-negative cells can be used for Gram-negative cell identification and selective staining from environmentally mixed bacterial samples.
BNQDs exhibit a long fluorescent lifetime (FLT) and a unique excitation-dependent emission property. Therefore, BNQDs can be a good candidate for multiplex fluorescence imaging when used in conjunction with organic dyes. The combined fluorescent signals can be resolved spectrally and temporally based on the emission wavelength and the FLT, respectively. For multiple probes having overlapping emission wavelengths but different fluorescent lifetimes, both spectral and temporal imaging can be combined. Dehghani et al. [
45] reported the use of two-photon excitation features of BNQDs for multiplex cell imaging. The fluorescence lifetime imaging microscopy (FLIM) was used to accurately distinguish the emission signals of BNQDs with longer FLT in comparison to relatively shorter cell autofluorescence and those from organic dyes. They reported the use of two nuclear stains: DAPI (4’, 6-diamidino-2-phenylindole) with blue and Sytox with green fluorescence, both with shorter fluorescent decay lifetimes, to achieve the temporally and spectrally resolved fluorescent signals as shown in . They co-stained the RL-14 cells with b-BNQDs/DAPI (b-BNQDs: blue-emitting BNQDs) and g-BNQD/Sytox green (g-BNQDs: green-emitting BNQDs) separately. The emission of BNQDs and dyes was not separated by spectral analysis of the image, but this issue was resolved by using the difference in fluorescence lifetimes of BNQDs and dyes using the FLIM technique.
Figure 9. Spectral imaging (
A–
C,
G–
I) and fluorescence lifetime imaging (
D–
F,
J–
L) of RL-14 cells separately treated with b-BNQDs/DAPI and g-BNQDs/Sytox green. No emission was detected in panels B and G co-stained with QDs and stains. Fluorescence signals from b-BNQDs and g-BNQDs were resolved using spectrally matched detection ranges as seen in panels A and H. The fluorescence lifetimes were obtained by bi-exponential fitting and mapped to a false-color scale from 0 ns (blue) to >4 ns (red). The resulting false-color shows the difference between signals originating from BNQDs (panels D and J) and organic dyes (
E,
K). Reproduced from [
45] with permission from Copyright © 2018 American Chemical Society.
1.2. Biosensing
Angizi et al. reported the use of BNQDs for the sensitive and selective detection of vitamin C (VC) [
55]. This was performed by a modified screen-printed gold electrode (GSPE) with functionalized BNQDs. BNQDs are superior to carbon nanostructures in terms of the linear range of sensing and the detection limit. For example, a detection limit of 0.45 µM was recorded in Angizi et al.’s work while using the BNQDs/Au system, significantly lower than 3 µM for the cellulose acetate/graphite system. The high electrocatalytic activity for electro-oxidation of VC was reported and the oxidation occurred at the surface of the BN QD/Au electrode at a lower positive potential than the GSPE. Later, Jerome et al. [
77] reported a rapid response sensor (1.8 sec) to detect vitamin C (ascorbic acid, AA) using BNQDs and polyluminol (Plu) coated glassy carbon electrode (GCE). As-prepared hybrid Plu/BNQDs coated GCE was reported to show improved electrocatalytic activity for AA oxidation at 0.2 V using the amperometry detection method. Further, they carried out the interference effects in the presence of uric acid, dopamine, and glucose which did not respond to the Plu/BNQDs/GCE sensors, indicating good selectivity of the sensor for AA. In another study, Kong et al. [
80] reported a fluorescent “on-off-on” sensor to detect AA with Fe
3+ as a medium. The fluorescence of BNQDs was quenched due to the inner filter effect between Fe
3+ and BNQDs. Then, the fluorescence of BNQDs was restored with increasing AA due to the oxidation-reduction between Fe
3+ and AA. This technique enabled the detection of AA in the concentration range 1–100 µM with a detection limit of 0.0833 µM under the optimized experimental conditions at pH 6. The sensing selectivity of BNQDs to Fe
3+ ions was confirmed by comparing data obtained by using other metal ions. Other metal ions had a negligible effect on quenching BNQD fluorescence. The selectivity of BNQDs on AA sensing was also verified by replacing AA with other interfering substances, such as uric acid, dopamine, and glucose. These substances have shown little recovering effect on the fluorescence of BNQDs. All these data support the fact that BNQDs exhibit high selectivity for Fe
3+ and AA.
Dehghani et al. [
45] reported the use of g-BNQDs passivated with polyethylene glycol, for the biosensing of intracellular pH variations and their distribution inside cells. This was performed by confocal microscopy and FLIM techniques. It is to be noted that the increase in incubation time will cause the micro-environment of the cells to become more acidic due to the formation of more endosomes. The acidic environment would result in a shorter FLT. The authors reported the translocation and accumulation of g-BNQDs at endosomes over 4 h, which resulted in the change of cell morphology. The quenching of photoluminescence of BNQDs in an acidic medium can be utilized for detecting changes in metabolic activity inside human cells. Owing to the Warburg effect [
86], lactate production in cancer cells during glycolysis is high. Therefore, the extracellular pH of tumor cells is often acidic, leading to a high extracellular acidification rate (ECAR) compared to benign cells. Radhakrishnan et al. [
58] reported the use of the glycolytic inhibitor, 3-bromopyruvate(3-BP), to suppress ECAR. This was demonstrated by monitoring the increase in fluorescence from FBNQDs, as a method to detect the glycolytic activity in cancer cells. In this case, the green fluorescence channel of flow cytometry was used to detect the enhanced fluorescent signal after introducing 3-BP to cancer cells. There was no considerable change in fluorescence in benign cells due to a less active glycolytic pathway. In another study, Yola et al. [
41] reported a stable, repeatable, reusable, and selective imprinted biosensor based on BNQDs for cardiac Troponin-I (cTnI) detection in plasma samples. The cTnI is widely used for the diagnosis of acute myocardial infarction (AMI) diseases. Later, the same group [
87] reported the preparation of a novel voltammetry sensor for the detection of various organophosphate pesticides in water samples based on BNQDs on graphene oxide.
Owing to the strong inner filter effect between 2,4,6-trinitrophenol (TNP) and BNQDs, the fluorescence intensity of BNQDs can be reduced upon quenching by TNP. By this mean, TNP can be selectively and sensitively detected in the concentration range of 0.25−200 μM, with a detection limit of 0.14 μM when BNQDs are used as fluorescence probes [
49]. Therefore, BNQDs can be used as the turn-off sensors for the rapid detection of TNP from natural water sources without tedious pretreatment processes. Similarly, Peng et al. [
67] reported a “switch on” nanosensor for sensitive assay of glutathione (GSH). GSH can regulate the inner filter effect of MnO
2 nanosheets (NS) on BNQDs. Owing to the superior light absorption capability of redox-able MnO
2NS centered at 380 nm, the fluorescence of BNQDs (with a maximum emission wavelength 380 nm) can be selectively quenched. For an optimal inner filter effect to take place between the two substances, the maximum absorption of one should overlap the maximum emission of the other. However, the introduction of GSH will help to recover the fluorescence of BNQDs, dependent on the concentration of GSH, by weakening the inner filter effect as initiated by the decomposition of MnO
2 to Mn
2+. This MnO
2NS/BNQDs nanoprobe was reported to exhibit good selectivity on GSH detection in the range of 0.5–250 µM with a detection limit of 160 nm in human plasma samples. They reported the use of other interference substances such as histidine, glutamic acid, cysteine, vitamin C, sodium chloride, potassium chloride, etc., instead of GSH in the same concentration which did not show much change in fluorescence recovery, thus suggesting the need for the separation of interfering reductants from real samples. The sensing principle is shown in .
Figure 10. Schematic principle of glutathione (GSH) regulated IFE (inner filter effect) of MnO
2NS on BNQDs for sensing. The observed fluorescence of BNQDs is quenched with the MnO
2NS presence and later recovered with GSH application. Reproduced from [
67] with permission from copyright © 2019 American Chemical Society.
In addition to the inner filter effect, fluorescence resonance energy transfer (FRET) on BNQDs also offers interesting application. For example, FRET between BNQDs and gold nanoparticles (AuNPs) has enabled the fabrication of rapid, label-free, and highly sensitive fluorescence-based biosensors [
88]. Such sensors were used to detect acetylcholinesterase (AChE), a key enzyme in the biological nerve conduction system, whose activities have been connected to several diseases such as muscular paralysis, convulsions, and bronchial constriction. As reported, AChE can hydrolyze acetylthiocholine (ATCh) to generate thiocholine (TCh) whose thiol (-SH) group can reduce chloroauric acid (HAuCl
4) to AuNPs. The formation of TCh-BNQDs/AuNPs aggregates can quench the fluorescence of BNQDs via FRET. An inhibitor, paraoxon, was used to lower the activity of AChE and decreased the fluorescence quenching of BNQDs. In this way, a simple ‘one-pot’ FRET-based biosensor was reported to be constructed to assess AChE activity and its inhibitor by analyzing the fluorescence of BNQDs.