*2.2. Properties and Performance*

The critical factors to evaluate the quality of fluorescent probes are absorption cross section, quantum yield, emission rate and photostability. Absorption cross section is used to describe the ability of Pdots to absorb a photon of a particular wavelength and polarization. Studies have shown that the peak absorption cross section of single particles (15 nm in diameter) is about 10–100 times of CdSe Qdots [77]. Moreover, another key property, called quantum yield, is the ratio of the number of photons emitted to the number absorbed; the typical value is below 40% due to aggregation-induced self-quenching [78], and high quantum yield can reach 50–80%. It is generally considered that the brightness of

fluorescent molecules depends on the product of absorption cross section and quantum yield. Photostability is assessed by the photobleaching quantum yield calculated from the ratio of photobleaching photons number to the photons absorbed number. Typical photobleaching quantum yield ranges from 10−<sup>4</sup> to 10−<sup>6</sup> [79]. Additionally, different kinds of Pdots have been proven to have low toxicity, thus Pdots are widely used in biological applications [80]. Several relevant research results about properties of Pdots are given in Figure 3.

**Figure 3.** The properties of Pdots. (**A**) A photograph of various Pdots emitted by ultraviolet light. (**B**) Absorption and emission spectra of various Pdots. Reproduced from Ref. [34] with permission. (**C**) Single-particle images and intensity distributions of Qdot 655 and PBdots. Reproduced from Ref. [81] with permission. (**D**) Fluorescence imaging of MCF-7 cells incubated with anti-EpCAM primary antibody and Pdot-lgG conjugates. The bottom panels show the imaging of cells incubated with Pdot-lgG alone. (**E**) Fluorescence intensity distributions for Pdot-streptavidin-labeled MCF-7 cells and Qdot 565-streptavidin-labeled MCF-7 cells. Reproduced from Ref. [82] with permission. (**F**) Ultrabright FRET-based Pdots with simultaneously high absorption cross section and quantum yield. (**G**) Combined fluorescence microscopy images of MCF-7 cells labeled with PEP/PFPV Pdotstreptavidin and biotinylated primary antibody. Reproduced from Ref. [83] with permission.

The improvement of properties is a constant proposition in biological applications of Pdots. Recently, Zhang et al. reported fluorescence resonance energy transfer (FRET)-based Pdots with both large absorption cross section and high quantum yield [83]. By choosing acceptors that had a greater spectral overlap with donors or mixing different kinds of Pdots to create cascade FRET Pdots, they obtained ultrabright blue-, green- and red-emitting Pdots that were among the brightest Pdots reported in the visible region. In other examples, Kuo et al. found that the photostability of Pdots can be improved by adding 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid (HEPES) or 2-(N-morpholino)ethanesulfonic acid (MES) buffer to quench photoinduced radicals, which aided long-term cell tracking in biological imaging [84]. Chang et al. also designed low-toxic cycloplatinated Pdots, used as a photocatalyst to strengthen the photocatalytic efficiency and stability [85].

#### *2.3. Surface Modification and Biological Functionalization*

## 2.3.1. Encapsulation Method

Silica encapsulation is widely used for surface modification, as other functional groups could be easily attached to silica, which encapsulates particles in a 2–3 nm shell [86,87]. This method greatly promotes biological functionalization of Pdots. However, the silica shell is possible to hydrolyze in biological environments, and the amino groups used to stabilize silica-encapsulated Pdots (10–20 nm in diameter) also cause nonspecific adhesion between Pdots and the cell surface. Another way for surface modification is to encapsulate Pdots using poly (lactic-co-glycolic acid) (PLGA) [88,89] or phospholipids [90,91], which increases nanoparticle stability and reduces nonspecific adhesion. However, the size of nanoparticles modified by PLGA (230–270 nm) and phospholipids (80–100 nm) is too large to apply at the cellular and subcellular levels. Furthermore, the low concentration of fluorescent polymers eventually limits the brightness of nanoparticles, which causes the failure of the encapsulation method to take full advantages of the Pdots.

#### 2.3.2. Amphiphilic Polymer Coprecipitation Method

Chiu's group developed some effective functionalization methods [34,82]. They preadded amphiphilic polymers in organic solvents, and then prepared Pdots by nanoprecipitation. In this process, amphiphilic polymers covered the surface of the hydrophobic nanoparticles, and their hydrophobic ends were randomly bound to hydrophobic Pdots, while the hydrophilic ends were exposed to water. As a result, Pdots with hydrophilic groups were formed to covalently link biomolecules for biological conjugation and functionalization. For example, an amphiphilic polymer, polystyrene-polyethylene glycol-carboxyl (PS-PEG-COOH), was used for surface modification of Pdots [82]. The average diameter of the product was about 15 nm, and more than 80% of the constituents were significantly effective fluorophores. These research results indicated that this strategy can generate efficient nanoparticle probes, since neither the size nor fluorescent properties of Pdots were affected.

Wu et al. used another amphiphilic polymer, poly (styrene-co-maleic anhydride) (PSMA), to realize biological functionalization [73]. The hydrophilic ends were hydrolyzed in an aqueous environment to form Pdots with carboxyl groups, which facilitated further subsequent bioorthogonal labeling by click chemistry (Figure 4A). Dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements showed the typical image and hydrodynamic diameter (~15 nm) of functionalized Pdots. Among different functional groups on the Pdots surface, the carboxyl-functionalized Pdots had a significant increase in mobility in the gel electrophoresis (Figure 4B).

#### 2.3.3. Direct Functionalization

In the above modification methods, functional molecules are non-covalently linked to Pdots, which is the main reason for functional molecules falling from the surface of Pdots, which ultimately affects the performance of functionalized Pdots. To overcome these drawbacks, Zhang et al. developed an alternative direct functionalization method, in which Pdots covalently link functional groups [92]. They synthesized conjugated polymers with different percentages of carboxyl groups and used them directly to prepare functionalized Pdots to avoid extra surface modification procedures. Moreover, they found that the degree of functionalization influences the stability and performance of Pdots. In addition, the low carboxylic acid group density (2.3%) brings the greatest properties, including fluorescence brightness, colloidal stability, non-specific absorption and compact internal structure. Yu et al. reported a cross-linking strategy to form functionalized Pdots with enhanced labeling efficiency and sensitivity for cellular assays (Figure 4C) [93]. In addition, Zhang and Chen et al. developed facile strategies with an optical stimulus to covalently link polyethylene glycol and/or carboxyl functional groups to the Pdots (Figure 4D) [94,95], and demonstrated effective bioconjugation of these functionalized photocross-linkable Pdots for specific cellular labeling.

**Figure 4.** The functionalization methods of Pdots. (**A**) Pdots coprecipitated with amphiphilic polymer PSMA for bioorthogonal labeling via click chemistry. (**B**) Gel electrophoresis of Pdots with various surface functional groups using a 0.7% agarose gel. Reproduced from Ref. [73] with permission. (**C**) Fluorescence microscopy image of microtubules in HeLa cells labeled with cross-linked Pdotstreptavidin. Reproduced from Ref. [93] with permission. (**D**) PEGylated and carboxyl-functionalized Pdots for bioconjugation for specific cellular targeting. Reproduced from Ref. [95] with permission.

#### **3. Application of Pdots Biosensors in Point-of-Care Diagnostics**

Point-of-care diagnostics are analytical assays outside the laboratory in order to ensure the convenience of fast testing for target analytes in patients with the same accuracy as laboratory tests. Recently, Pdots-based biosensors have been used for point-of-care diagnostics due to their superior photophysical properties and efficient energy transfer or electron transfer.

FRET has facilitated tremendous advances in biosensing for point-of-care applications. FRET is a phenomenon in which energy is non-radiatively transferred from a donor fluorophore to an acceptor fluorophore, where the fluorescence of the acceptor is emitted while the fluorescence of the donor is quenched. Nanoscale Pdots enable efficient energy transfer, as the efficiency of FRET strongly depends on the distance between donor and acceptor [96]. Additionally, FRET in Pdots biosensors could enhance the brightness of Pdots and obtain high absorption cross section and great photostability [97]. Specific dyes are added into Pdots to realize corresponding FRET-based biosensing [98], which could be applied to detect various metabolites and physiological information [95], including reactive oxygen species [99–101], pH [102], temperature [103,104] and metal ions [105,106]. In this review, Pdots-based biosensors for biomolecule detection are mainly discussed.
