Recent Advances in Intrinsically Fluorescent Polydopamine Materials

Abstract

Fluorescence nanoparticles have gained much attention due to their unique properties in the sensing and imaging fields. Among the very successful candidates are fluorescent polydopamine (FPDA) nanoparticles, attributed to their simplicity in tracing and excellent biocompatibility. This article aims to highlight the recent achievements in FPDA materials, especially on the part of luminescence mechanisms. We focus on the intrinsic fluorescence of PDA and will not discuss fluorescent reaction with a fluorometric reagent or coupling reaction with a fluorophore, which may cause more in vivo interferences. We believe that intrinsic FPDA presents great potential in bioapplications.

1. Introduction

Compared with conventional fluorescent materials, fluorescent nanoparticles are receiving increasing interest due to their strong fluorescence, high photostability, water dispersibility, and remarkable biocompatibility, making them widely used in chemical sensing, optical materials, and biological detection [1,2,3]. Fluorescent nanoparticles can be simply defined as fluorescent inorganic nanoparticles (FINs) and fluorescent organic nanoparticles (FONs). Metal nanoparticles, semiconductor nanoparticles, and noble-metal nanoclusters are classic FINs. They generally present a strong fluorescent intensity and high quantum yield but low biocompatibility, which are not suitable for biomedical applications. For example, CdSe quantum dots, which represent semiconductor nanocrystals, have cytotoxicity without surface modification [4], and gold nanoclusters, as one of the typical noble-metal nanoclusters, are also characterized by bad stability and dispersion [5]. In addition, the preparation of FONs also currently has some limitations, including high-temperature conditions and long operation times.

Dopamine (DA), a catecholamine neurotransmitter, plays an important role in regulating many biological processes in the central nervous neurotransmitter [6]. It also can be used as an adhesive material, which can be self-assembled under alkaline and aerobic conditions to generate polydopamine (PDA) [7]. Due to its remarkable biocompatibility and abundant functional groups on the surface, PDA has been widely used in drug delivery, biosensing, and other fields [8,9,10,11]. For instance, Li et al. designed a drug delivery system based on PDA NPs as the photothermal agent and drug delivery carrier for tumor therapy [12]. Furthermore, the endothelial/lysosomal membrane is destroyed by the photothermal effect, thereby overcoming the endothelial/lysosomal barrier and improving the therapeutic effect. However, due to the complex auto-oxidation process and the various oxidation products, the chemical structure and formation mechanism of polydopamine are still controversial, and there are few reports on the development and utilization of polydopamine intrinsic fluorescence. Therefore, researchers are more focused on experimental experience to improve the quantum yield and fluorescence intensity, since the structure–property relationship lacks a solid theoretical foundation. What is more, the PDA structure is similar to a natural Eumelanin polymer, which can be analyzed and studied from a melanin analogy to PDA [13].

As is known to all, melanin is a chemical substance with high photothermal conversion efficiency, where more than 99.9% of the ultraviolet and visible radiation is dissipated by melanin in a non-radiative manner [14]. Therefore, melanin has a low quantum yield, and it is generally considered non-fluorescent even in the early stages of research. With the enrichment and improvement of research methods and measures, melanin is proven to exhibit fluorescence under UV excitation or oxidation conditions [15]. In addition, the quantum yield is a function of the excitation wavelength, and the optimal emission wavelength is at 400–550 nm, which depends on the excitation wavelength [16]. PDA has similar physical and chemical properties to melanin [17]. Most of the absorbed energy is converted into heat in a non-radiative way, which can be attributed to the π-π stacking interaction between the oligomeric units in PDA [18,19]. π-π stacking is a weak interaction (also called a non-covalent interaction) that exists in or between the molecules of some compounds with conjugated structures and can induce fluorescence quenching. In the melanin field, it has been reported to prepare fluorescent polymers by separating the p-conjugated skeleton and inhibiting the interaction between polymers [20]. There are also experiments using hydrogen peroxide to oxidize and cleave melanin polymers to enhance fluorescence [21]. In 2012, Zhang et al. first reported using hydrogen peroxide as an oxidant to prepare fluorescent polydopamine nanoparticles under normal temperature aerobic conditions, and the quantum yield reached 1.2% [22], opening a new chapter in the study of PDA intrinsic fluorescence and making the solution oxidation method of preparing fluorescent polydopamine attract great attention from researchers. Based on this pioneering work, researchers have successfully prepared fluorescent polydopamine using potassium permanganate and manganese dioxide as oxidants [23,24]. However, many mechanism characteristics of the fluorescence enhancement of polydopamine are still unknown. Studies have shown that PDA is composed of three parts: uncycled (catecholamine) and cyclized (indole) units, as well as novel pyrrole carboxylic acid moieties [13]. In these structures, the two cyclized fluorescent units of DHI and its reduced form dimers show remarkable emission characteristics [25]. Another study shows the polydopamine supramolecular aggregate of monomers (consisting primarily of 5,6-dihydroxyindoline and its dione derivative dopaminochrome), which are held together through a combination of charge transfer, π-stacking, and hydrogen-bonding interactions [19]. Therefore, it is now generally believed that reducing the accumulation of π-π stacking will increase the quantum yield and enhance the intrinsic fluorescence of PDA. It was reported that hydroxyl groups can reduce the π-π stacking interaction between oligomeric units in polydopamine nanoparticles. The hydroxyl radicals generated under the action of high concentrations of hydrogen peroxide and sodium hydroxide are added to the double bonds activated by the pyrrole ring and the aromatic ring [26,27,28], which laid a theoretical foundation for the research of solution oxidation. In addition to the method of oxidation by oxidizing agents, UV irradiation photooxidation is also a feasible way to prepare fluorescent polydopamine [29]. It was speculated that the uncyclized catecholamine units in PDA are oxidized by ultraviolet light, resulting in the formation of cyclized fluorescent units, which also confirms that the cyclic side chain can effectively reduce the π-π accumulation effect [20]. The researchers not only enhanced the quantum yield of the fluorescent copolymer through oxidation but also weakened the π-π stacking by doping with N atoms [30,31].

After branched-chain polyethyleneimine (BPEI) achieved good results in enhancing fluorescence [32,33], it has also been used to enhance PDA fluorescence. The abundant amino groups of BPEI can form hydrogen bonds with groups containing O and N, destroying the intermolecular and intramolecular cross-linking of polydopamine. On the other hand, BPEI being alkaline in an aqueous solution is also beneficial to the formation of polydopamine [34]. Moreover, compared with the fluorescent polydopamine prepared by the solution oxidation method, the quantum yield of the PDA-PEI fluorescent polymer is higher (12.5%) [35,36], and the particle size is smaller. The basic idea of solution oxidation and doping with N atoms is to weaken the π-π stacking interaction of PDA. The former has more abundant experiments and applications, but it also exists the problem of a low fluorescence quantum yield (about 0.58–1.2%) [37,38]. The latter’s current experimental method is single, and the research content needs to be further expanded. There are still many gaps in the mechanism and methods of FPDA enhancing fluorescence, and more solid theoretical research on the structure–property relationship is needed.

This review aims to present a comprehensive overview of the relevant advances in the intrinsic fluorescence of PDA from 2012 until now; the research of other coupled fluorophores will not be discussed (Figure 1). At the beginning, we will introduce the fabrication methods of FPDA and discuss the mechanism of synthesis and performance improvements based on mainstream views. In the following section, we will present the recent applications with intrinsic fluorescence of PDA, including in the bioimaging, biomolecule detection, as well as bacteria detection and therapy fields. Benefiting from the excellent biodegradability and biocompatibility, FPDA shows great potential in bioapplications. This review is a powerful supplement to the research on FPDA in intrinsic fluorescence and can provide more ideas for the application of FPDA, thereby inspiring more research interests.

2. Preparation Principle

At present, although there have been many reports on PDA applications, the formation mechanism of PDA has not yet been clarified. However, with an increasing research interest in PDA-based sensing and imaging, better chemical and physical performances are desired. There seems to be a high need to understand the basic structure–property–function relationships and to devise application for drug delivery and other fields [39]. To understand the synthesis method and mechanism of FPDA, it is necessary to clarify the mainstream PDA formation mechanism. In the early stages of this research field, the structural model of melanin plays an important guiding role in studying the self-polymerization of dopamine [40,41,42,43]. DA is oxidized to dopamine quinone by dehydrogenation of catechol under alkaline aerobic conditions. The dopamine quinone is unstable and easy to cyclize to form a leucodopaminechrome yield [44]. Thereafter, leucodopaminechrome is further oxidized to dopaminechrome. The oxidized heterogeneous forms 5,6-dihydroxyindole (DHI), which is easily oxidized to 5,6-indolequinone. DHI attacks dopamine quinone or other quinone oligomers to generate oligomers with mixed structures. Bielawski et al. utilized solid state spectroscopic and crystallographic techniques to suggest the polydopamine supramolecular aggregate of monomers (consisting primarily of 5,6-dihydroxyindoline and its dione derivative dopaminochrome), which are held together through a combination of charge transfer, π-stacking, and hydrogen-bonding interactions [19]. In parallel with these two models, Lee et al. suggested that a combination of non-covalent self-assembly and covalent polymerization is the mechanism of dopamine formation [45]. Therefore, the aggregation of various unit molecules and the intra- and intermolecular coupling lead to the low quantum yield of PDA [46]. To increase the fluorescence intensity and quantum yield of PDA, it is necessary to break the intramolecular coupling, reduce the π-π accumulation of oligomeric units in PDA nanoparticles, and form small molecule oligomers. At present, the solution oxidation method is a common way to synthesize PDA. Zhang et al. used hydrogen peroxide to oxidize PDA to obtain fluorescent polydopamine and open a new chapter in the preparation of FPDA by solution oxidation [22]. The increasing fluorescence intensity and quantum yield (1.2%) attributed to the hydroxyl can effectively reduce the π-π stacking of the conjugated skeleton [28]. Liu et al. employed magnetic iron oxide (Fe₃O₄) nanoparticles as a peroxidase-mimicking enzyme to catalyze hydrogen peroxide to produce hydroxyl radicals which could prepare FPDA in mild conditions [38]. Fe₃O₄ NPs reduce the concentration of H₂O₂ and prevent excessive harmful free radicals from being produced, which benefits the application of FPDA. However, the synthesis of nanoscale enzymes is more complicated and technical, and a convenient way is needed to avoid the use of highly concentrated H₂O₂ (e.g., up to 6%). Xiong et al. used metal ions and H₂O₂ to synthesize water-soluble fluorescent polydopamine nanoparticles in acidic conditions [39]. The prepared PDA displayed excitation-independent emission and excellent photostability. Previous reports suggested that Cu²⁺ ions can both oxidize the catechol groups and induce the aggregation of molecules containing catechol groups due to coordination interaction and may accelerate the polymerization of dopamine [47]. Another general method to prepare FPDA and improve quantum yield is doping with N atoms to destroy the intermolecular and intramolecular cross-linking of polydopamine [48]. In 2015, Liu et al. used polyethyleneimine (PEI) and DA, stirring for 2 h at room temperature to obtain PEI-PDA particles by doping nitrogen atoms for the first time [34]. The product not only exhibits high water dispersibility and strong fluorescence but also has good biocompatibility, which is beneficial to the application of bioimaging. Based on previous research, Zhong et al. used PEI and DA as reactants and improved the preparation process by choosing Tris as the reaction solution and increasing the stirring time to 12 h. The FPDA displayed a higher quantum yield (12.5%) and better photostability (Figure 2) [35]. Compared with the solution oxidation method, the quantum yield of the doping method was greatly improved, but it is important to note that there are few studies on the preparation of FPDA by doping N atoms and few adulterant types.

2.1. Redshift Phenomenon

Oxidation of dopamine by strong oxidants in alkaline aqueous solutions is a common method for FPDA formation. However, uncontrolled autoxidation of dopamine in alkaline aqueous solutions causes the emission wavelength of FPDA to be dependent on the excitation wavelength [29,49,50]. Usually, the optimal emission wavelength increases with the excitation wavelength, which may cause unnecessary interference in multiple biological applications and greatly limit their application as fluorescent probes for biosensing and bioimaging. Especially for imaging, this phenomenon may cause background fluorescence or unclear images. Efforts have been taken to avoid this phenomenon and to inhibit uncontrollable self-polymerization of dopamine (and other catecholamines) in a solution to obtain photostability nanoparticles. One way which is easy for inhibiting the autoxidation of dopamine was to prepare the FPDA in acidic aqueous solutions. Xiong et al. employed hydrogen peroxide and metal ions to prepare fluorescent polydopamine under acidic conditions [39]. Inspired by the Fenton reaction, three metal ions were selected to react with hydrogen peroxide to generate hydroxyl radicals and crack the PDA unit [49]. When the excitation wavelength increases from 350 nm to 452 nm, the best emission light of Cu²⁺-polydopamine does not change and stabilizes at 484 nm. Transition metals can form stable complexes with dopamine through the one-electron oxidation bond in the catechol group [50], which may induce electron transfer to oxygen for the corresponding semiquinones. These corresponding semiquinone substances may continue to be oxidized to undergo disproportionation, coupling, and cyclization [51]. However, this process will be significantly inhibited under acidic conditions and avoid the production of various oxidation products. Zhao et al. chose ammonium persulfate ((NH₄)₂S₂O₈ or APS) as an oxidant to oxidize and polymerize. Dopamine polymerization happened in a citric acid aqueous solution (pH = 1.8) at 180 °C for 24 h in a 20-mL PPL-lined autoclave (Figure 3) [52]. The FPDA aqueous solution showed bright blue-green fluorescence under UV light, and the emission peak was stable when the excitation wavelength was increased from 310 to 360 nm, which indicated that the FPDA had unified chromophores. The small full width at half maximum (FWHM) also indicated unified chromophores in the FPADs. However, it is not always possible to obtain FPDA with good photostability by oxidizing DA under acidic conditions. Chen et al. chose acidic (pH = 5.5) anaerobic conditions as the reaction conditions, and dopamine polymerization happened in plasma-activated water (PAW) [53]. PAW is rich in reactive oxygen species (ROS) (i.e., hydronium and hydroxyl ions), which has been reported to accelerate dopamine polymerization under acidic, basic, and neutral conditions [47,54]. Under different optical properties from other reactions under acidic conditions, the optimal emission changed, with gradually increased excitation wavelengths from 360 to 480 nm. It is speculated that polydopamine formation proceeds via several concurrent pathways, favored by the exceedingly slow oxidation kinetics due to the oxidation ability of PAW being too weak [51]. Another interesting method to control FPDA formation and improve photostability is the use of other groups to occupy amino groups in dopamine that can prevent the generation of branched side chains. Xiong et al. performed dopamine polymerization in glutaraldehyde and glycine based on the well-known Schiff base reaction. The polymerization reaction was initiated by adding an ice-cold NaBH₄ solution and then terminated with excessive mercaptoethanol after 30 min [55]. When the obtained FPDA was excited at different wavelengths in the range of 360 to 440 nm, the purified oligomer had only one emission peak near 485 nm. The amino groups in dopamine were first occupied by glutaraldehyde and glycine, which could prevent the generation of branched side chains. It is a very innovative experimental method which provides a broader idea for improving the photostability and other properties of polydopamine in the future. The method is, however, a multi-step and time-consuming process, and the pH of the reaction process needs to be precisely adjusted to satisfy the Schiff base reaction.

From the above discussion, it can be concluded that acidic conditions are conducive to inhibiting the self-polymerization of dopamine to avoid excitation-independent emission. Secondly, oxidation of dopamine using a suitable strong oxidant is also crucial to impress the photostability, requiring as few other luminescent groups as possible. Preventing the generation of branched side chains may be a promising research direction because of the inhibition of autoxidation of dopamine. On the other hand, it can reduce π-π stacking interaction between the oligomeric units in PDA to improve quantum yield.

2.2. Size Distribution

Thanks to its remarkable physical and chemical properties, abundant surface groups, and good in vivo degradations [56,57], PDA stands out for the development of drug delivery and bioimaging. As a nanomaterial, the particle size of PDA is an important factor affecting its performance. It was found that the particle size of PDA not only affects its loading ability but also its cytotoxicity [58,59,60,61]. The particle size of nanomaterials is related to the cell death pathways. Gold nanoparticles with a smaller particle size (<2 nm) can cross the nuclear membrane and enter the nucleus, resulting in significant cytotoxicity [62]. Furthermore, it was reported that the cancer endothelium has fenestrations between the cells of the vascular system. The size of these gaps ranges from 100 to 780 nm, depending on the tumor type [63,64,65]. Therefore, it is generally considered that the small size of PDA is in favor of impressing its rate of cell endocytosis and its accumulation in cancer cells. For these reasons, it is necessary to reduce the particle size of FPDA. The particle size of nanomaterials is related to many factors (preparation method, pH, temperature, etc.). Fluorescent polydopamine is mostly prepared under alkaline conditions at room temperature and pressure. Thus, it is necessary to consider reducing the particle size of PDA from the type and ratio of raw materials. Lin et al. proposed the use of hydroxyl radical-induced degradation of polydopamine nanoparticles to obtain fluorescent polydopamine with a particle size of 22 ± 3 nm [28]. This is attributed to the addition of hydroxyl groups to the 5,6-dihydroxyindole (DHI) unit of the polydopamine unit, effectively dividing the polydopamine nanoparticles into oligomeric units and able to reduce the π-π stacking interaction between the oligomeric units and inhibit the autonomy of DA and DHI. However, this method is a multi-step and time-consuming process, which limits its preparation and application. Fortunately, there are many ways to generate hydroxyl radicals, including the commonly used tendon method, electrolytic oxidation method, high-power sonication (HPS), and photoelectric catalytic method [66,67,68]. Wang et al. used a micro-plasma anode to treat the dopamine solution and oxidized polydopamine with reactive oxygen species (ROS) generated by the anode to obtain nanoparticles with a uniform particle size of 3.1 nm [37]. At the same time, acidic conditions also inhibited the further growth of FPDA. Hydroxyl radicals are used in these methods to oxidatively cleave the PDA group, which successfully reduces the particle size. It is speculated that the hydroxyl radicals generated by other methods will have a similar effect. In addition to using hydroxyl radicals to crack the PDA, the method of doping N atoms is also a good way to reduce the particle size. Zhao et al. mixed DA, ammonium sulfate, and citric acid using a hydrothermal method to react at 180 °C to obtain an average FPDA particle size of 3.02 nm [52]. However, it is important to note that the material particle size was not as small as possible, as different applications require different particle sizes. When used as a drug carrier, small nanoparticles have a strong tissue-penetrating ability but have a short circulation time in the blood. Although large-diameter nanoparticles have a longer circulation time in the body, the penetration ability in the tissue is slightly weaker. Designing a nanodrug system requires considering the influence of different aspects and organizing the advantages of each component. Therefore, when deciding to construct functional materials, it is necessary to study the optimized size range corresponding to each application. However, mainly because DA oxidation products show a wide size distribution, few studies can accurately control the particle size of fluorescent polydopamine. Polymer capsules have been prepared by the templated assembly, which represents an attractive approach for controlling the particle size [31]. Chen et al. used DA and hydrogen peroxide to prepare FPDA on the PDA capsule, which could achieve precise control of the particle size [69]. Non-fluorescent PDA capsules were prepared by polymerization of DA on templates of SiO₂, and the core was removed by HF. FPDA is synthesized by adding DA and H₂O₂ on templates of non-fluorescent PDA capsules. The size of the FPDA capsules can be controlled by using templates of different diameters, and the cytotoxicity is also extremely low. At the same time, the influence of the application environment of nanomaterials on the particle size of the material cannot be ignored. Using the difference between the tumor micro-environment (low pH, etc.) and the normal physiological environment to construct the responsive transformation of physical and chemical properties is a promising direction. Controlling the particle size of FPDA conveniently has not been fully explored thus far, which represents another promising direction and would further promote the study of many applications of FPDA.

3. Application

Fluorescent PDA nanoparticles have been widely used in the fields of sensing, bioimaging, and even the detection of bacteria due to their outstanding biocompatibility, water solubility, and tunable fluorescence properties. These versatile qualities guarantee adaptable applications under complex environments to achieve different goals. Compared with FINs and fluorescent dyes, FPDA can be easily engineered due to its abundant functional groups to achieve targeted strategies, which makes PDA promising as an extremely useful tool for sensing and imaging.

3.1. Detection of Metal Ions

Fluorescent nanoparticles are receiving great attention nowadays due to their ortho-dihydroxy phenyl structure, which is beneficial for chelating metal ions, making them useful in metal ion detection in the environment and the human body [70,71]. The metal ions chelate with PDA to make the fluorescent polydopamine produce a fluorescent response so that the qualitative and quantitative analysis of metal ions can be performed quickly and sensitively [39,72]. Fluorescence quenching is one of the most common fluorescence responses. There are many reasons for this phenomenon, and the mechanism is also complicated. Studies have shown that the formation of metal ion amine complexes and the quenching activity of the complexes for the FPDA may be the main reason for the fluorescence quenching of FPDA [73]. For instance, Jia et al. synthesized high-photostability PDA-PEI copolymer dots with high selectivity toward Cu²⁺ using a facile one-pot method. The detection of copper ions with a detection limit of 1.6 nM and the FPDA in real samples was evaluated in the detection of copper ions. The fluorescence quenching of the FPDA could be caused by the complexation effect between Cu²⁺ and the amine group, which led to electron transfer from the probe to Cu²⁺ [35]. EDTA (a strong metal ion chelator) was added to the mixture containing PDA-PEI and Cu²⁺. The fluorescence recovered since EDTA has a stronger chelation ability with Cu²⁺ than the amino groups in PDA-PEI. The results confirmed the mechanism for the Cu²⁺ fluorescence quenching the formation of cupric amine complexes and led to electron transfer from the probe to Cu²⁺ [74]. Qi et al. synthesized FPDA with selectivity toward Fe³⁺ using the solution oxidation method. The detection of ferric ions was performed a detection limit of 4.6 nM and confirmed the previous speculation [75]. Due to the oxidative and coordinative properties of Fe³⁺, the intermolecular hydrogen bonds of FPDA are destroyed, and the oxidation produces a planar iron (II) benzo semiquinone structure layered by π-π interactions that can quench fluorescence. In addition to the fluorescence responses of fluorescence quenching, there are also studies showing that metal ions could enhance the fluorescence intensity of FPDA. One study showed that zinc ions can enhance the intensity of fluorescent polydopamine to achieve the sensitive detection of zinc ions with a detection limit of 60 nM, and the measurement was carried out in a complex mixture [38]. It is important to note that the ortho-dihydroxy phenyl structure of PDA is easy to chelate with metal ions, but few metal ions can cause a large fluorescence response mainly due to the large association constants of only a few metal ions with PDA. In practical applications, the samples to be tested often contain multiple metal ions which need to be sensitively detected at the same time. Recently, Zhang et al. prepared three PEI-PDA sensors with three different concentrations of BPEI to form a new chemical sensor array that could distinguish different concentrations of metal ions in biofluids, even in the mixtures with different valence states [76]. The study was inspired not only to start from the fluorescence intensity response but also to develop and utilize the fluorescence lifetime, response mode, etc. and combine some data processing methods for computer analysis to achieve large-scale rapid and sensitive detection.

From the above discussion, it can be concluded that FPDA obtained by doping or solution oxidation can produce a good fluorescence response with metal ions. Furthermore, fluorescent polydopamine shows great potential in detecting metal ions due to mild preparation conditions, meets the requirements of green chemistry, and is easy to carry out compared with other methods. Due to a close correlation between body health and the level of biofluid-derived metal ions [77], the analysis and detection of metal ions in biological fluids is a promising research direction.

3.2. Bioimaging

With the rapid development and utilization of optical instruments and equipment, fluorescence imaging is an important auxiliary modality for biological and medical investigations [78]. Therefore, many kinds of fluorescent probes have been proposed for the visualization of the structure and function in cells [79,80,81,82]. However, most of the commonly used fluorescent probes have disadvantages, such as high cytotoxicity to live cells, poor water dispersibility, and background fluorescence [83,84,85]. PDAs have been proposed as a good material for bioimaging not only because of their excellent water dispersibility and remarkable biocompatibility but also because of their abundant surface groups that allow for connecting other functional groups (e.g., drugs and peptides). Despite many advantages, unfortunately, the low quantum yield and poor photostability of polydopamine limit its use for applications in fluorescence imaging. Wen et al. synthesized starch-based PDA-PEI copolymer dots with strong and stable green-yellow fluorescence using the green and mild methods [86]. Bright green-yellow fluorescence was observed when the cell and copolymer dots culture medium was irradiated by a 405-nm laser, and the fluorescence signals showed that a starch-based PDA-PEI copolymer could be internalized by cells through non-specific cell endocytosis. It is worth noting that with the high incubation concentration of the starch-based PDA-PEI copolymer for 1 day, the cell viability value was still as high as 99.69%, which is an important factor in deciding their suitability for bioimaging. In addition, the researchers also found that the fluorescence intensity was related to the physical and chemical properties of the cell. Han et al. synthesized a label-free ratiometric pH sensor in an alkaline alcohol solution under the ultrasonic radiation method [87]. The as-prepared fluorescent polydopamine showed different intensities of green fluorescence under different pH levels, which could be applied to bioimaging for detecting intracellular pH in live cells in real time (Figure 4). Further research shows the remarkable specificity of FPDA to the pH level. The fluorescence intensity of FPDA is hardly affected by other types of metal ions and biological macromolecules. The change in fluorescence intensity with the pH can be attributed to π-π interactions and decreased electrostatic repulsive interactions between the FPDA in an alkaline environment. Using low-concentration NaOH can easily form 20–30-nm size nanoparticles. As the concentration of NaOH increases, the particle size of the product gradually increases until the concentration of NaOH reaches 2.25 nM, which can be attributed to originating from the congregation of small particles under high-pH conditions.

Similar situations have been reported in other studies of PDA [88]. The pH has a great influence on the polymerization mechanism, leading to changes in particle size. In addition to the changes in the fluorescence intensity of FPDA caused by the pH level, other intracellular substances may also affect the fluorescence intensity. Ma et al. dropped NH₄OH into a mixture of alcohol and water to obtain PDA nanodots which could be further oxidized by hydrogen peroxide to fluorescent polydopamine [89]. Based on the fact that H₂O₂ is the main component of ROS in cells when PDA nanodots get into the cells, there should be a different fluorescence response due to the different concentrations of ROS in tumor cells (high) and normal cells (low), making the PDA effectively distinguish cancer cells from normal cells. When incubating PDA nanodots with normal cells and Hela cells for 4 h, the normal cells displayed weak fluorescence, while the Hela cells had strong fluorescence, which shows it is feasible to distinguish normal cells from cancer cells by the difference in the concentration of ROS in the cell. Compared with the commonly used flow cytometry, this method shows the advantages of simple operation and a low cost, making it a promising application prospect in the identification of tumor cells and tissues. It is worth noting that PDA nanodots that are oxidized by ROS display one-photon and two-photon fluorescence properties, providing a tool for directly observing animal organization structure and activities under free animal activities. In the above studies, PDA nanoparticles or copolymers all enter cells non-specifically through endocytosis, which may limit practical applications. Fortunately, the abundant functional groups on the surface of PDA allow it to be connected to other recognition groups or biological cross-linking agents without the need for additional surface modification. PDA as a drug carrier has been widely reported and applied [90,91,92]. When conducting a drug test evaluation, it is also necessary to connect a fluorescent dye as a probe to ascertain whether the drug enters the cell and releases it. Fluorescent polydopamine can be used as a multifunctional experimental platform for carriers and fluorescent probes at the same time, which is conducive to simplifying the experimental process and combining with other functional groups. Wu et al. prepared highly emissive and biocompatible FPDA with sensitive detection of Fe³⁺ ions and targeted cell imaging of live cells [55]. The cRGD peptides and GPR120 primary antibodies were used for targeted cell imaging and membrane protein labeling, respectively, by the common method of EDC/NHS connection. Because the surface of PDA is rich in carboxyl groups, it can also be easily connected to other types of peptides or proteins through the EDC/NHS connection method. In the above studies, FPDA nanoparticles or copolymers can only enter the cell through endocytosis and imaging in the cytoplasm due to the blocking of nuclear pore complexes [93]. The nucleus plays a key role in the main physiological activities of the cell, including cell metabolism, growth, and differentiation [93,94,95], and real-time in situ observation of the nucleus in the live cell is indispensable for the study of cell behavior and events. Ding et al. have reported polydopamine fluorescent nanoparticles with nucleus imaging in label-free, in situ, real-time, and long-term manners [96]. They incubated N-(3-(Dimethylamino)-propyl)-N-ethyl carbodiimide (EDC), DA, and cells in a cell culture medium for 3 hours and observing them under a confocal microscope by excitation at 405-nm wavelength (Figure 5). The results showed bright blue fluorescence in the nucleus but almost no fluorescence in the cytoplasm. However, the mechanism of the bright fluorescence of FPDA produced by the reaction of EDC and DA was unclear. As a nitrogen-containing biological cross-linking agent, it could be speculated that EDC weakens the π-π stacking by doping with N atoms. More ingeniously, the generated FPDA can only stay in the nucleus due to the existence of nuclear pores and shows a strong nuclear imaging specificity. As mentioned earlier, the method of doping with N atoms can improve the fluorescence intensity and photostability of PDA. Using nitrogen-containing biological cross-linkers or other low-cytotoxic chemicals to generate PDA in situ in cells will be an interesting research direction.

From the above discussion, FPDA shows good application prospects in fluorescence imaging and overcomes the defects of many traditional fluorescent dyes, such as complex preparation operations, a fluorescent background, and high cytotoxicity. However, it was found that the current FPDA bioimaging is at the cell level and has not been applied to in vivo imaging, which is mainly attributed to the lack of relevant imaging equipment or harsh use conditions and environments. On the other hand, the current FPDA quantum yield is not high, and most of the emitted green fluorescence is easily interfered with by the background fluorescence of organisms. Traditional animal experiment methods require slaughtering experimental animals at different time points to obtain data and obtain experimental results at multiple time points. In vivo imaging can continuously monitor animal cell activity in real time, which can acquire more authentic and credible data. It is particularly meaningful to develop a kind of FPDA that can be used as a fluorescent indicator for in vivo imaging. The development of the two-photon fluorescence properties of FPDA may become a future development direction because of the high contrast and resolution of two-photon imaging.

3.3. Detection of Bacteria

In recent years, public health safety issues have received widespread attention from countries around the world. According to estimates by the World Health Organization, 70% of foodborne disease instances worldwide are caused by pathogenic microorganisms each year. Therefore, the development of a sensitive and efficient method for pathogenic bacteria is essential to improve the protection of public health and ensure the safety of people. Fluorescence biosensors are receiving increasing interest recently due to their excellent sensitivity and portability for use on-site compared with conventional polymerase chain reaction (PCR)-based and immunology-based methods, giving them the potential to be used in the rapid and sensitive detection of pathogens [97]. Zhong et al. synthesized PDA-PEI copolymer dots with green emission and great photostability using a facile one-pot method (Figure 6) [36]. Because of the rich carboxyl groups on the surface of PDA, connecting PDA-PEI to the aptamers can specifically recognize Pseudomonas aeruginosa (P. aeruginosa) by the common method of EDC/NHS connection. The amino group on PDA-PEI and the phosphate group on the cell wall form a strong ionic complex, causing fluorescence to be quenched. The experimental results showed high specificity to P. aeruginosa, and the fluorescence intensity was hardly affected by other types of bacteria to use dual-aptamers. The limit of detection (LOD) was calculated to be 1 CFU mL⁻¹ and applied for the detection of P. aeruginosa in real samples (skim milk, orange juice, and a popsicle). In addition to detecting bacteria, fluorescent biosensors also show potential for therapy. As we all know, PDA exhibits strong absorption of ultraviolet and near-infrared (NIR) light and high photothermal conversion efficiency. Therefore, it is also used as a photothermal agent in photothermal therapy for clinical cancer treatment [22,30]. It has been reported that PDA, as a photothermal agent, generates heat under near-infrared light to denature bacterial proteins and kill bacteria [98,99]. Mazrad et al. synthesized PDA-PEI copolymer dots with the sensitive detection of gram bacteria. Since both PEI and PDA are positively charged, PDA-PEI and the negatively charged cell wall are connected by electrostatic action, which leads to strong π-π stacking interactions and quenches fluorescence [100]. It is worth noting that the photothermal effect of the PDA-PEI copolymer dots under near-infrared irradiation leads to a rise in temperature, which inhibits the growth of bacteria and some basic physiological activities [101,102]. Compared with traditional therapies, it displays the advantages of simplicity, convenience, and no drug resistance problems. However, it needs to be pointed out that the specificity of binding to Gram bacteria through electrostatic interaction is not strong enough, and the use of an aptamer connection may result in a distance between the PDA and bacteria and affect heat transfer to the cells. It may be a good idea to choose peptides or proteins that can specifically recognize and enter the bacteria, and the heat in the cell will have a greater impact on the bacteria, leading to a better sterilization effect.

3.4. Detection of Biomolecules

Owing to the excellent optical properties of PDA nanoparticles, they are becoming a kind of popular nanomaterial and have attracted a lot of attention from detecting biomolecules [103,104,105]. PDA nanoparticles as quenchers have been used to construct biosensors for detecting various biomolecules due to their broadband absorption of 300–800 nm [106,107,108,109]. However, the biosensing application based on the intrinsic fluorescence of PDA nanoparticles is still in its infancy [110]. Yildirim et al. reported that DA was oxidized to fluorescence polydopamine under alkaline conditions, and it could be used as a signal molecule to turn on fluorescence. The fluorescence biosensor was used for rapid and sensitive detection of DA with a detection limit of 40 nM [111]. DA sensing experiments showed that sensors still display a high selection to DA under the interference of many common human chemicals, even ascorbic acid and uric acid, which could greatly affect the selectivity of electrochemical methods. Turn-on fluorescence sensing shows huge potential for DA detection due to its simple operation and low cost compared with other detection methods (e.g., electrochemical methods and chromatographic methods). Thereafter, similar turn-on type fluorescent biosensors of DA have also been reported and simplified the experiment process [112]. In addition to the reaction’s raw material as the signal molecule, other molecules also can turn on fluorescence. Jiang et al. designed a sensitive fluorescence sensor composed of fluorescent polydopamine and cobalt oxyhydroxide (CoOOH) with the determination of a-glucosidase activity [113]. The two materials are connected due to the electrostatic effect and meet the requirements of fluorescence resonance energy transfer (FRET), resulting in fluorescence quenching. The ascorbic acid produced by the hydrolysis of 2-O-α-D glucopyranosyl-L-ascorbic acid by glucosidase can reduce CoOOH to Co²⁺. Without the quenching effect of CoOOH, FPDA shows fluorescence intensity that varies with α-glucosidase activity. It is worth noting that the selectivity and sensitivity of fluorescence biosensors were tested in human serum to investigate its practical application. The detection results showed high selectivity and a low detection limit (1.65 U L⁻¹) for the sensor, which has the potential to be applied in biomolecular detection (Figure 7). The above discussions are turn-on fluorescence biosensors, which turn on fluorescence through the presence of signal molecules and perform quantitative detection through fluorescence recovery. Because of the good selectivity and sensitivity of this method, it has been used to detect a variety of biomolecules [114,115,116].

Another regular method for designing fluorescence biosensors is the turn-off mode detection method; that is, by inhibiting the synthesis of the fluorescent probe or quenching the fluorescent probe to realize the fluorescence response, the qualitative and quantitative detection of the substance to be tested is carried out. Zhao et al. employed CoOOH as an oxidant to synthesize fluorescence polydopamine by oxidizing dopamine [117]. Ascorbic acid (AA) can reduce CoOOH to Co²⁺, which renders the DA unable to be oxidized to FPDA due to the absence of the oxidant CoOOH, resulting in a decrease in fluorescence intensity. Therefore, the fluorescence intensity of the PDA could be used to define the AA concentration. The sensing system shows a good linear relationship between fluorescence intensity and AA concentration in the detection range, with a detection limit of 4.8 mM. It is worth noting that CoOOH does not quench the fluorescence as mentioned in the previous article, which can be attributed to the different concentrations of CoOOH (90 μg mL⁻¹ and 580 μg mL⁻¹), and a high concentration of CoOOH can also cause a fluorescence decrease. The effect of MnO₂ is also similar to that of CoOOH [24]. To further improve the sensitivity and selectivity, a fluorescence biosensor of colorimetry dual-mode assay based on manganese dioxide (MnO₂) has recently been developed for the determination of alkaline phosphatase (ALP) activity [118]. Dopamine was oxidized by MnO₂ to form FPDA, which could quench the red fluorescent quantum dots (QDs). Another fluorescent indicator, 3,3′,5,5′-tetramethylbenzidine (TMB), can also be oxidized by MnO₂ to yellow TMBox. When there were ALP and 2-phospho-L-ascorbic acid in the reaction system, MnO₂ would be reduced to Mn²⁺ ions by ascorbic acid, which would prevent the synthesis of FPDA. The sensing system expanded the scope of detection and lowered the detection limit down to 0.015 mU/mL. It was tested in livestock sera and milk to investigate its practical application, consistent with that measured by a commercial ALP assay kit. Similar to turn-on fluorescence sensors, the pros of simple synthesis, good sensitivity, and selectivity, as well as low-cost turn-off fluorescence sensing, showed great potential for biomolecular detection. However, it must also be considered that many factors can quench or weaken fluorescence (pH, reaction time, temperature, etc.), which will limit its wide application in biomolecule detection.

3.5. Other Applications

In addition to the above-mentioned advantages, FPDA and FPDA-based copolymers present other remarkable physicochemical properties, including photoprotective function, strong adhesion, and electrical conduction. Chang et al. synthesized polydopamine-based melanin electronic ink with green fluorescence according to the previous study, with minor modifications [119]. The material was added to the electrophoresis chamber, and electrophoresis drove a single charged nanoparticle to move under the action of an electric field. The movement of the particle was controlled by adjusting the parameters. Surprisingly, the polydopamine-based melanin nanoparticles displayed green fluorescence under 485-nm laser irradiation, and it exhibited an ultra-high resolution (10,000 ppi) and low power consumption (operation voltage of 1 V), which could be attributed to the intrinsic negative charge of −18 mV and high conductivity. The conductivity of synthetic dopamine-melanin has been reported to be 10⁻¹³ S/m in a vacuum [120]. The constructed system provides a fluorescent electronic ink display that has not been reported before. This electronic ink that utilizes the intrinsic fluorescence of polydopamine will have great application prospects in nanoelectronic display systems due to their good water dispersibility, adhesion, and conductivity.

4. Conclusions

This review attempted to give an overview of the recent developments in intrinsic FPDA materials, including luminescence mechanisms, fabrication strategies, and applications for sensing and bioimaging. PDA has been proven to be one of the most powerful coating platforms for surface modification due to its outstanding features, such as good water solubility, a simple fabrication process, and great biocompatibility and biodegradability. Aside from the above advantages, FPDA presented great potential for tracing in bioapplications, owing to the tunable photoluminescence properties. What is more, based on the intrinsic fluorescence of FPDA, there are no requirements for extra reagents like ligands, aptamers, or fluorophores, which is greatly beneficial for bioapplications. Less interference creates better in vivo prospects. However, there are still important challenges in future applications, although impressive achievements have been reached. The chemical structure of FPDA needs to be clarified. Since byproducts seriously affect the fluorescence performance of FPDA, detailed structural information contributes not only to the preparation of novel FPDA materials with excellent properties but also to the control of the morphology and size of FPDA, which mainly affect intracellular diffusion and quantitative imaging. Another potential problem is the long-term in vivo effects of FPDA, including metabolic disturbance and immunogenicity. This is also an important factor in our focus on the intrinsic fluorescence of FPDA, as less reagent interference is much better for systematic in vivo application studies. We believe this article is a powerful supplement to the research on intrinsically fluorescent polydopamine materials which offers a comprehensive review and guides future applications, especially for in vivo tracking.