Biological nanoscale fluorescent probes: From structure and performance to bioimaging

2.1 Photoinduced electron transfer (PET)

In PET, there is a linker between the receptor and the fluorophore. The linker connects the fluorophore and the receptor, while also separating the two during fluorophore excitation. Electrons are transferred from the highest occupied molecular orbital (HOMO) of the acceptor (or donor) to the semi-occupied molecular orbital (SOMO) of the fluorophore (or acceptor), resulting in fluorescence quenching. However, if the non-bonded electron pair of the donor is combined with the analyte, the redox potential of the donor increases, and the HOMO energy is lower than the fluorophore, which will cause the fluorescence to be switched on, and that limits the PET process [19,20].

2.2 Fluorescence resonance energy transfer (FRET)

FRET refers to the process in which the excitation energy of a donor transfers to the acceptor when the emission spectrum of the donor overlaps with the excitation spectrum of the acceptor, causing the acceptor to emit fluorescence. This is a non-radiation intermolecular dipole effect. The key factors that determine the fluorescence resonance energy transfer are mainly two points: (1) the emission spectrum of the donor and the excitation spectrum of the acceptor effectively overlap; (2) the distance between the donor and the acceptor needs to be maintained between 1–10 nm. The third one is the relative orientation of donor emission dipole moment and acceptor absorption dipole moment [21].

2.3 Intramolecular charge transfer (ICT)

In ICT system, the fluorophore and the acceptor are directly connected to a single entity. This single entity can provide electrons as an electron donor or accept electrons as an electron acceptor, so it has two functions. The addition of analytes will cause the intensity of the dipole on one side of a single entity to change, thereby affecting the overall intensity change and spectral shift. When the analyte interacts with the acceptor end, the electron-withdrawing property of the acceptor increases, and the emission spectrum shifts red; when the analyte interacts with the donor segment, the electron-donating properties of the donor decrease, and the emission spectrum shifts blue. Compared with the PET mechanism, there is no spectral shift during fluorescence quenching in the PET process, and a significant spectral change occurs in the ICT mechanism [19,20].

3.1 Semiconductor quantum dots (QDs)

Semiconductor QDs are novel fluorescent probes for biological and medical applications, usually composed of nanoscale II-VI and III-V elements. QDs are a kind of nano-particles with quantum domain limiting effect, whose size is usually between 1–10 nm and nearly spherical [22, 23, 24]. Due to the development of quantum confinement, semiconductor QDs have unique optical properties. Compared with organic dyes, QDs have many advantages in fluorescence properties [25]. Firstly, QDs have a broad and continuous excitation spectrum and a narrow and symmetrical fluorescence emission spectrum (Figure 2) [26]. The size of QDs determines the wavelength of fluorescence emission.

Figure 2

Physical properties of QDs. (a) The fluorescence emission spectrum of QDs is adjustable, and multiple QDs can be excited by a single light source. (b) The emission spectrum of QDs can range from near-infrared region to the ultraviolet region. Reprinted image with permission from Ref. [30], Copyright 2015, American Chemical Society.

By changing the size and chemical composition of QDs, the fluorescence emission spectrum of QDs can be adjusted from near-ultraviolet to near-infrared, with the wavelength range of 400–2000 nm [27]. Second, compared with organic dyes, QDs have a more massive stokes shift, which means that the difference between the peak of the excitation wavelength of QDs and the height of the emission wavelength is massive. In this way, the overlap of the excitation spectrum with the emission spectrum and its influence on the fluorescence detection in low signal detection can be avoided [28,29]. Third, QDs have high fluorescence intensity and good stability. Unlike conventional organic fluorescent dyes, which are easily bleached by light, QDs are 100 to 1000 times more resistant to light bleaching than organic dyes and fluorescent proteins [25,26]. In addition, the fluorescence yield of QDs is high, and the fluorescence life generated by QDs is long, and their excited state life lasts 20–50 ns. This enables the QDs to obtain fluorescence signals without background interference during use [22,30]. QDs are used in bioimaging, drug delivery, biosensor, cell tracking, and other fields due to their excellent optical properties [31].

In 1985, Brus first discovered QDs in colloids: ZnS and CdS particles [32]. Then in 1998, Alivisatos and Nie made the first successful use of semiconductor QDs as fluorescent probes in biology [33,34]. Since the synthesized QDs contain heavy metal elements (such as cadmium), they cannot be directly used in biological applications, so they need to be treated with water solubility [33]. Nie et al. used ZnS to encapsulate CdSe and then added a thioglycolic acid solution (Figure 3). On one hand, the sulfhydryl group in thioglycolic acid can be combined with Zn atoms. On the other hand, the carboxylic acid group of thioglycolic acid makes QDs water-soluble. Studies have shown that the optical properties of the obtained QDs have not been changed [34]. At present, there are three main types of methods for synthesizing QDs, namely physical method, chemical method, and biological method. Material synthesis methods include laser physical vapor deposition and laser irradiation of large particles. However, QDs prepared by this method have low yield and poor stability. Therefore, many researchers use chemical processes to synthesize QDs [35]. Chemical synthesis method is mainly divided into an organic synthesis method and an aqueous synthesis method. QDs obtained by this method have good monodispersity and optical properties [30].

Figure 3

(a) Schematic diagram of CdSe QD capped by ZnS covalently coupled with thioglycolic acid and protein. (b) TEM of QD-transferin (an iron transport protein) conjugates. Reprinted image with permission from Ref. [34], Copyright 1998, Science.

3.2 Metal nanoclusters (MNCs)

Fluorescent MNCs are likely to replace organic dyes or QDs [17]. Noble MNCs are typically smaller than 2 nm and consist of several or dozens of atoms [36]. Similar to semiconductor QDs, emission wavelength of noble MNCs can be adjusted from UV-Visible light to near-infrared light, and a single light source can excite the emission wavelength of multiple colors. Emission energy of MNCs can be regulated by determining the number of constituent atoms and ligands. Similarly, noble MNCs have high fluorescence intensity and broad stokes shift [17,37]. However, unlike semiconductor QDs, noble MNCs exhibit low toxicity and ultrafine size [38]. Large nanoparticles can quickly cause kidney barrier effect and cause serious side effects in the body. Noble MNCs can be effectively removed in the body due to their small size [37]. Noble metal nanoparticles have a strong surface plasmon resonance effect due to the electron excitation of the continuous conductive band. In contrast, noble MNCs, due to their small size close to the Fermi wavelength of electrons, will lose their surface plasmon resonance effect, showing discrete electronic structure and molecular properties [17,39,40]. It is important to note that colloid and cluster are often used interactively by researchers. Generally speaking, metal nanoparticles larger than 10 nm are called colloid, while metal nanoparticles smaller than 2 nm are called MNCs [41]. Noble MNCs also have some properties related to size, such as HOMO-LUMO transition, oxidizing reduction, magnetism, and handshape (as is shown in Figure 4) [37,40,41]. Besides, noble MNCs also have an essential property, which can be used as a new type of nano-catalysts in the fine chemical industry, medicine, food additives, and other fields [42]. The ultra-fine size and low toxicity of noble MNCs make their applications in biological imaging and fluorescence labeling very attractive [43].

Figure 4

Properties of Au NCs. Reprinted image with permission from Ref. [40], Copyright 2012, American Chemical Society.

In the 1960s, Malatesta firstly reported the synthesis of Au NCs using phosphine as a ligand. However, the structure of the Au NCs was still unknown [44]. After continuous research, Malatesta deduced the crystal structure of the Au NCs [45]. The precious MNCs were prepared by the template method. They had good biocompatibility and were quickly modified to adapt to specific applications. Therefore, the template method has become the primary method to prepare precious MNCs [46]. At present, proteins, DNA, polymers, dendrimers, and small sulfhydryl molecules are used to prepare noble MNCs.

3.3 Carbon dots (CDs)

In 2004, Xu et al. inadvertently isolated a kind of fluorescent nanoparticles when preparing carbon nanotubes by arc discharge method [47]. Later, in 2006, the Sun research group used the laser ablation method to prepare the fluorescent nanoparticles and named them CDs [48]. CDs are carbon nanoparticles with a particle size of less than 10 nm [49]. CDs are also referred to as carbon quantum dots (CQDS) in some literature. CDs have excellent photoluminescence properties. They show high fluorescence quantum yield, anti-swelling, and anti-bleaching photoluminescence decay, and long concentration range. They could be adjusted from deep ultraviolet to near infrared [50, 51, 52, 53]. Besides, compared with QDs, CDs have good biocompatibility and low toxicity, which can almost be ignored.

Moreover, at a high concentration of CDs, the damage of CDs to organisms is shallow [53]. There are functional groups such as the hydroxyl group, carboxyl group, and carbonyl group on the surface of CDs. These functional groups cause CDs have good water solubility and provide the possibility of functionalization of CDs, which improves the effectiveness of CDs in practical application [51,54]. Due to the high price and large size of QDs, which affect the observation in vivo, QDs are subject to many limitations in practical application [27]. However, CDs with small size, low production costs, and low prices can be produced on a large scale, which is considered as a superior substitute for semiconductor QDs [54].

Synthesis methods of CDs are generally divided into two types: top-down and bottom-up. CDs obtained by the top-down approach tend to have high crystallinity and complete structure [51,55]. However, CDs with amorphous carbon core, large surface functional groups, and doping sites may be obtained by the bottom-up method (Figure 5) [51,55]. Therefore, according to the different carbon cores, CDs are generally divided into graphene quantum dots (GQDs), carbon nanodots (CNDs), and polymer dots (PDs). GQDs are made up of one or more layers of graphene with edges connected by chemical groups and with a degree of crystallinity. CNDs are generally spherical carbon nanoparticles with no lattice. PDs are usually polymerized, assembled, or cross-linked by a linear nonconjugated polymer [56,57]. The photoluminescence mechanism of CDs has long been controversial among researchers. Three theories have been discussed the most, namely quantum confinement theory, molecular state theory, and surface state theory [52,58,59]. The difference of the synthesis method, prepared CDs structure, and performance will be different, so it is important to understand the preparation method of CDs [58].

Figure 5

Synthesis method of CDs. Reprinted image with permission from Ref. [57], Copyright 2015, Springer Nature.

4.1 QDs in biological targeting and biological imaging

Traditional fluorescent materials (such as organic dyes, fluorescent proteins, etc.) are prone to photobleaching, and the signal intensity is low. Multiple light sources are required to excite different fluorophores and other defects, which are subject to many restrictions in actual use [14,60,61]. QDs have vigorous fluorescence intensity and stability and have massive stokes shifts, photo-bleaching resistance, and controllable optical properties. Therefore, in biomedical applications such as bioimaging and targeting labels, QDs are very attractive to researchers [62]. In general, QDs can enter cells four ways: (a) passive absorption through endocytosis; (b) binding or modification with biopolymers, and then internalization through interaction with cell membranes and endocytosis; (c) direct physical manipulation of the cells (such as microin-jection); (d) use of several uptake modes at the same time in combination [63].

In the past ten years, research on the biological imaging of QDs has significantly been developed [30]. Generally speaking, directly prepared QDs have poor biocompatibility, have specific toxicity, and are prone to non-specific adsorption and aggregation during use in the biological environment. Therefore, they need to be modified in biomedicine (usually using natural coupling [64] or encapsulation [65]). Then they can possess good water solubility, biocompatibility, and low cytotoxicity during use [14,66,67]. Gao et al. used amphiphilic triblock copolymers to improve QDs and used them for in vivo imaging in mice and targeted imaging of human prostate cancer cells. Passive targeted imaging is achieved through the penetration and retention of QDs at the tumor site. At the same time, QDs can also be combined with antibodies, and the targeted effect of antibodies and antigens can be used to achieve actively targeted imaging. However, the speed and efficiency of active targeting are significantly higher than the rate and efficiency of passive targeting (Figures 6) [28].

Figure 6

(a) Passive targeted imaging of quantum dots; (b) active targeted imaging of quantum dots . Reprinted image with permission from Ref. [28], Copyright 2004, Springer Nature.

QDs are also used in multi-mode imaging [62,68], multicolor imaging [60,69], and multiphoton imaging [70]. Among them, multicolor imaging can identify individual malignant tumors in a complex biological tissue environment. This application provides detailed molecular information and morphological features in clinical medical diagnosis [60]. Multi-mode imaging combines QDs imaging technology with other imaging technologies (such as nuclear magnetic resonance imaging, computed tomography, etc.) [30]. Shibu et al. used nuclear magnetic resonance imaging technology and near-infrared fluorescence imaging technology to track the fate of modified QDs in mice, and found that kidneys can effectively excrete QDs within 48 h [68]. QDs have more excellent resolution under two-photon excitation and also have higher temperature sensitivity. High-quality thermal gradient images can be recorded in the fluid, and the internal temperature changes of individual cells can also be measured [70].

4.2 Noble MNCs in biotargeting and bioimaging

Although QDs have been successfully applied to cancer targeting and cell imaging in living animals, the mechanism of biodegradation in animals remains unclear due to the toxicity of QDs because of their heavy metal components [71,72]. In addition, the size of QDs is relatively large. Although this allows QDs to have extremely high resolution, it also reduces imaging efficiency and sensitivity, thus limiting their application in clinical imaging [73]. Compared with QDs, noble MNCs not only have the advantages of QDs in biological imaging but also have the characteristics of small size, low toxicity, and good biocompatibility. Therefore, they become more attractive fluorescent probes in biological imaging [72,74,75]. Wu et al. were the first group to use precious MNCs in near-infrared fluorescence imaging. They found that noble MNCs avoided uptake of the reticuloendothelial system due to their small size. Thereby, they passively targeted Hela tumors in tumor-bearing mice through the retention effect (EPR) [73].

Among the precious MNCs, the properties of Au NCs have been studied the most extensively [46,76]. However, due to the small size of Au clusters, the yield of Au NCs, synthesized by traditional methods, is low. Fluorescence intensity is not enough to achieve biological imaging. Therefore, Sun et al. used iron oxidase to assemble two Au NCs. The fluorescence intensity of the cluster is enhanced, and the fluorescence emission is adjustable. This kind of fluorescent probe can be used for whole-body imaging, especially kidneys [77]. Researchers not only use the fluorescence intensity of precious MNCs for biological imaging [78] but also use their fluorescence lifetime for imaging. Shang et al. developed the Fluorescence Lifetime Imaging (FLIM) technology. Through life-gating, cell imaging can be achieved without autofluorescence. FLIM images can not only show the uptake of MNCs by cells but also provide information on the environmental changes of NCs [79].

Due to the small size of precious MNCs, a suitable template preparation (such as glutathione) is chosen to make them quickly cleared by the kidneys and are therefore discharged out of the body after biological imaging [80,81]. While targeting tumor cells, precious MNCs can also provide sufficient information on cancer cells by detecting other substances in the organism (such as a high concentration of reactive oxygen species [82], PH value [83], etc.). Like fluorescent probe imaging with QDs, noble MNCs probe in vivo imaging can also be used with other imaging methods (such as computed tomography, magnetic resonance imaging technology) to achieve multi-mode imaging and provide more tumor information [84], as is shown in Figure 7.

Figure 7

(a) Schematic diagram of Au Gds preparation; (b) Au Gds for trimodal tumor imaging. Reprinted image with permission from Ref. [80], Copyright 2011, WILEY-VCH Verlag GmbH & Co.

4.3 CDs in biotargeting and bioimaging

CDs are used as an excellent fluorescent bio-imaging agent due to their broad absorption spectrum, narrow emission spectrum, adjustable photoluminescence, good chemical stability, and light stability, and photobleaching resistance [85,86]. Compared with QDs and noble MNCs, CDs have the advantages of low toxicity, good biocompatibility, low cost, facile synthesis, easy surface modification, and large-scale preparation [86, 87, 88, 89, 90]. The physical and chemical properties of CDs can be controlled by the reactants, reaction products, and reaction by-products [86]. Passivating CDs by using different organic substances and placing the prepared CDs in an oxidized state can improve the fluorescence quantum yield of the CDs, thereby obtaining higher-quality biological imaging [91].

There are various functional groups on the surface of the CDs. These functional groups not only cause CDs to have good biocompatibility but also provide the possibility of surface modification of the CDs [92]. Hua et al. combined the surface functional groups of CDs with protoporphyrin IX (PpIX) to obtain multifunctional fluorescent CQDs. This kind of CDs can not only perform high-quality nucleolus imaging of fixed cells but it can cause that cells are imaged with high-quality nucleoli (Figure 8) [88]. Due to poor biocompatibility and high toxicity, quantum dots are rarely used for drugs and gene delivery. CDs can realize the dual application of non-viral gene vectors and biological imaging probes at the same time, so that genetic material can be transferred to specific cells of patients to achieve gene therapy [93,94]. Zhou et al. modified the surface of CDs to make it have a positive surface charge, so that the prepared CD can be used as a nanoprobe for biological imaging, and as an efficient non-viral gene carrier to achieve drug and gene transfer. They also found that CDs have high transfection efficiency and cannot enter the nucleus during the transfection process. They speculate that the high transfection efficiency of CDs comes from the strong aggregation of macromolecular pDNA, which prevents its enzymatic hydrolysis during transportation. On the other hand, small size of the prepared CDs and their positive surface charge comtribute to high transfection rate [94].

Figure 8

CDs synthesis schematic diagram and targeting the nucleolus imaging mechanism. Reprinted image with permission from Ref. [88], Copyright 2018, American Chemical Society.

Some researchers are interested in the development of carbon points emitting red fluorescence and their application in near-infrared biological imaging [95, 96, 97]. Li et al. attached electron acceptor groups (rich in sulfoxide/carbonyl groups) to the outer layer and edges of the CDs. These groups lead to increased surface oxidation and discontinuous energy levels, thereby enhancing the infrared absorption band and near-infrared fluorescence emission of CDs. They then successfully applied the prepared CDs to the imaging of the stomach of live mice [95].