*5.1. Emission Bleaching*

One of the basic mechanisms of an analyte detection is emission bleaching (Figure 5a). This mechanism can be induced by different physical phenomena schematically shown in Figure 6. In the first example, the absorption band of the analyte overlaps with the emission band of QDs. In such a case, the emission from QDs is absorbed by an analyte, which results in bleaching QDs emission. (Figure 6a). Another mechanism (Figure 6b) bases on a charge transfer from an analyte to QDs. When the additional electron or hole appears in an excited QDs the Auger processes lead to QDs ionization or charging. In both cases, the dot became non-emissive (dark). The third mechanism, which is responsible for QDs emission quenching under the interaction with the analyte is analyte-induced degradation of the QDs surface providing to emission decrease. As an example, thiol coated ZnS QDs in the presence of peptides showed a significant emission quenching [38].

**Figure 6.** Examples of physico-chemical mechanisms responsible for quantum dots emission quenching—energy transfer from QD to analyte (**a**), charge transfer from analyte to QD (**b**), degradation of QD surface (**c**).

#### *5.2. Emission Change—Nonradiative Förster Resonance Energy Transfer (FRET)*

When two optically active centers (donor and acceptor) are in close proximity to each other, (typically 1–10 nm) and an absorption spectrum of the acceptor overlaps an emission spectrum of the donor, the non-radiative transfer of the excitation energy from the donor to acceptor appears. This phenomenon is called FRET (Förster Resonance Energy Transfer). For the experiments using the FRET approach, photostable emitters must be used, characterized by a quantum efficiency greater than 0.1 and a high brightness (one in which the absorbance coefficient *ε* is greater than 50.000 <sup>M</sup>−1·cm<sup>−</sup>1) [39]. All these conditions are perfectly fulfilled by the quantum dots. The effectiveness of FRET is inversely proportional to the distance between the donor and acceptor and defined as:

$$K\_{FRET} = \frac{1}{1 + \left(\frac{R}{K\_0}\right)^{6}} \tag{1}$$

where: *KFRET*—FRET efficiency; *R*—distance between donor and acceptor; *R0*—distance at which the FRET efficiency is equal to 0.5 [40].

The highest sensitivity of FRET signal is for a distance between the donor and acceptor in the range from 0.5R0 to 1.5R0 [40]. For years researchers have been using the FRET mechanism to monitor intracellular interactions, due to its sensitivity to molecular rearrangements in the 1–10 nm range (this is the scale correlating with the size of biological macromolecules and the possibility of creating bonds between them) [41].

The universality of FRET method allows its use in nanosystems as well [42]. FRET yield is typically measured by observing one of the three parameters of the fluorescent donor: fluorescence intensity, spectral response or average fluorescence lifetime. Moreover, FRET has found application in many sensing systems giving the possibility of applying it to three analyte detection strategies. Figure 7 shows different processes which can be detected with use of FRET. The first mechanism uses analyte as optically active acceptor. In this case, the analyte attachment as well analyte removal can be observed as a change in the optical signal. The other strategy uses the analyte as the emission quencher and was also discussed in the previous paragraph [43]. The third strategy is more complex and uses a multistep

energy-transfer phenomenon. Detection using FRET between the QDs, as donors, directed to a linker with an acceptor, associated, e.g., with a receptor protein, is widely used to study the receptor-ligand interactions and changes in protein conformation after binding to the target analyte [44]. Thanks to this, in analytics consisting of several acceptors, QDs can interact with only one of them, which significantly improves the efficiency and sensitivity of the FRET method [45].

**Figure 7.** Different detection possibilities with use of nonradiative energy-transfer phenomena.

#### *5.3. Analyte Stimulated QDs Aggregation*

The colloidal QDs solution is sensitive to the presence of additional charges either on QDs surface or in the solvent, which may result in QDs aggregation. The charges may be introduced or induced by an analyte, which ultimately is manifested by QDs aggregation [46–48]. In the absence of analyte, a fluorescence comes from the whole volume of the QDs solution, while after aggregation caused by the analyte, the emission is localized [19]. This type of stimulation belongs to qualitative tests.

#### **6. Photoelectrochemical and Electrochemical Methods of Analyte Detection**

The chemical detection method is usually signaled by the following ways: competition binding assay of labelled and unlabeled analytes, using labelled molecules specific for immobilized analytes, sandwich formation or enzyme immunoassay, where enzymatically active substrate is added that changes color or fluorescence after interaction with enzyme-related analyzes [49].

Mo et al. [50] used a redox mechanism in the detection of hydroquinone in water samples. They have observed that ZnS QDs cannot react with hydroquinone. When hydroquinone and K2S2O8 were added into ZnSe QDs solution, no new photoluminescence (PL) peak was observed. Comparing with the pure ZnSe QDs solution, the PL intensity of the mixture decreased. This result reveals that hydroquinone oxidation product can efficiently quench the fluorescence emission of ZnSe QDs by energy transfer in electrochemiluminescence mechanism.

The development of new, reliable, fast and efficient methods for detecting anthropogenic and natural substances, both organic and inorganic, is a huge challenge for modern analytical chemistry and diagnostics. An alternative to such methods are electrochemical strategies using semiconducting quantum dots. The growing interest in the construction of electrochemical devices using quantum dots results from their aforementioned properties. Due to these features, small changes in the external environment lead to grea<sup>t</sup> changes in particle properties and electron transfer. Based on these significant changes, quantum dots are prone to engaging in heterogeneous redox chemistry with the surrounding environment. QDs are also used as carriers of biomacromolecules in bioanalytics. For this purpose, the chemical functionalization of QDs is carried out by means of a functional cap layer that allows the molecules to be trapped. The immobilization of biomolecules (e.g., the enzyme catalyzing the redox reaction) on the surface of semiconductor QDs causes QDs to promote direct electron transfer between biomolecules and the surface of the electrode, which significantly affects the operation of the system by enhancing the sensitivity due to signal amplification. A tremendous increase of development of electrochemical sensors based on QDs has been observed over the past decades due to the simplicity of implementation, high selectivity, and specificity of the system, low cost and the possibility of miniaturization [51]. Moreover, research carried out by Bard et al. revealed that CdS QDs could also act as multi-electron donors or acceptors at a given potential due to trapping of holes and electrons within the particle [52]. On the other hand, the surface structures of QDs also play a key role in determining the properties of the particles [53].

Liu et al. [54] described an electrochemical assay strategy for specific recognition of tumor cells. For this purpose, gold nanoparticles (AuNPs) have been assembled onto the indium tin oxide (ITO) substrate to create a specific, biocompatible interface to effective capture of tumor cells. CdSe/ZnS QDs labelled on the cell surface have been used as an amplified signal during the square wave stripping voltammetry (SWSV). The developed biosensing platform shown good analytical performance with a broad linear range, good selectivity and low limit of detection (LOD).

Electrochemiluminescence (ECL) is a method which aims to convert electric energy into radiation energy, in which electrochemically generated intermediate products undergo a high energy electron transfer reaction to generate excited states, resulting in the emission of a measurable luminescence signal [55]. As a form of luminescence (light emission without heat), ECL is characterized by the fact that light emission occurs when an appropriate potential is applied to the electrode, as a result of which the oxidation or reduction reaction takes place. There are several features that distinguish ECL from other techniques, e.g., chemiluminescence (CL). It is clear that the electrochemical reaction that takes place allows for precise time control. This means that the emission of light can be delayed to the desired moment, e.g., an immune reaction or an enzymatic reaction. Another advantage of ECL is the ability to control the location of the reaction, which means that there is the possibility of limiting the emission of light to a specific area relative to the detector. Electrochemiluminescence may occur as a result of two independent processes: annihilation of ions and co-reactant ECL. The annihilation of ions consists in creating states of excited molecules due to the transfer of electrons between radical ions on the surface of the electrode. The ECL co-reactant is due to the use of anode or cathodic potential in a solution containing phosphor and co-agen<sup>t</sup> molecules. Depending on the potential application, the phosphor or co-reactant molecules can be reduced or oxidized to form radical ions and medium compounds, followed by decomposition and formation of excited states that cause light emission [56].

#### **7. Applications of QDs-Based Sensors**

#### *7.1. Detection of Ions*

Fast and reliable detection and recognition of ions in the environment is extremely important in modern medicine and environmental protection. Among the ions, heavy metal ions such as mercury, cadmium and lead due to their high toxicity and negative health effects (cardiovascular diseases, cancer, liver, kidney and central nervous system disorders, reproductive and neurological disorders) require constant control concentration and rapid response in view of its possible reduction [57–61].

Hydrophilic QDs have been demonstrated to be a promising sensor probe for fluorescence-based sensing of heavy metal ions [57] such as Pb2+ [62], Cd2+ [63], Cu2+ [64], Hg2+ [65], Fe3+ [62], etc. Table 1 shows an exemplary strategies for heavy metal ions determination with using of QDs.


**Table 1.** QDs-based sensors for heavy metal ions determination.

\* LOD—limit of detection

Detection of ions with the use of photoluminescent-induced changes in the QDs involves the use of a number of ligands—derivatives of thioalkyl, mercaptoacetic or dihydrolipoic acids. The affinity of the thiol group to QDs results in self-assembly of the ligands on the surface of the dots, as a result of which the hydrophilic carboxylic groups are exposed on the surface towards the surrounding aqueous solution [6]. Chen and Rosenzweig proposed a method for detection of Zn2+ and Cu2+ ions. They exploit the fact that surface-modified QDs with mercaptoacetic acid show high sensitivity and selectivity to Cu2+ copper ions present in the mixture. The result of adding Cu2+ to the ligand-QDs complex is a reduction of the intensity of PL QDs. Such constructed sensor exhibited high LOD 0.8 μM [13]. Selective quenching PL was also used by Li et al. [58] They constructed sensor sensitive to the presence of mercury ions Hg2+ In this measuring system, CdSe/ZnS QDs have been modified with sulfur calixarene (S-Calix). The linear range of this system was found as 0–3 × 10−<sup>5</sup> M with a LOD 15 nM.

Zhou et al. [57] presented a ratiometric fluorescence sensor for real-time and on-site detection of Fe3+ ions based on CdTe QDs-doped hydrogel optical fiber with a broad linear range from 0 to 3.5 μM and high LOD 14 nM. The ratiometric configuration of the proposed sensor provides a built-in calibration to eliminate the analyte-independent interferences. Two types of CdTe QDs, which possessed different emission bands, have been synthesized for ratiometric measurements. One of the QDs, coated with thioglycolic acid, exhibits green emission and is insensitive to metal ions, thus serving as a reference. The other QDs as the specific recognition element, coated with N-acetyl-l-cysteine, are red emissive and show high selectivity of fluorescence quenching towards Fe3+ ions. To avoid mutual interference, the green emissive QDs and red emissive QDs are doped in discrete sections of the hydrogel optical fiber. As a result, it has been observed a decrease in PL intensity.
