**1. Introduction**

Among the most interesting and promising nanomaterials are colloidal semiconducting quantum dots (QDs). These nanostructures have found already several commercial applications in displays [1], light concentrators [2], photovoltaics [3] and as optical probes in various bio-applications [4,5]. The main reasons for their still growing success are: broad absorption band (several hundreds of nm), narrow emission band (below 40 nm), high quantum yield (up to 95%), possibility of emission band tuning over a wide range of wavelengths (350–2000 nm) and high resistivity of optical properties on external physico-chemical conditions, e.g., pH, temperature or power of the excitation beam [6]. QDs have a high surface to volume ratio, which can be controlled by QDs size but also by the nanostructures shape. This high surface area equips them with much more functional groups compared to organic compounds [7]. This makes QDs much more reactive and thus more effective in biological sensing [8].

The high quality QDs are typically grown as hydrophobic structures. In consequence, to make them useful for biological or medical application, additional post growth treatment is usually needed. This treatment includes QDs functionalization and in many cases bioconjugation (see Figure 1). One serious reason why QDs still do not dominate over organic markers (i.e., Green Fluorescent Protein, Rhodamine) lies in the absence of widely tested and already accepted protocols for QDs functionalization and bioconjugation [9]. There is also another reason to not use the QDs in biological sensing, especially in case of in vivo imaging, namely their toxicity. The QDs toxicity is a subject widely discussed recently in the literature [10]. The main reason affecting QDs toxicity is an aggregation of nanostructures in cells, organs, tissue etc. [11]. This is an even more serious problem than the chemical toxicity, due to dissociation of Cd atoms from CdSe/CdS QDs, and this is true for any types of nanostructures: semiconducting, dielectric or metallic nanostructures. Nevertheless, fortunately the QDs toxicity becomes a much less serious problem when the QDs are used for external sensing or for some in vitro applications when we can use their potential without barriers.

Concluding the above discussion, it can be seen that real benefits coming from extraordinary properties of QDs must be always compared to drawbacks of using inorganic probes in biological systems. In other words, for some specific applications QDs are an excellent choice, or very bad idea [12–14]. Among the applications where incontestably the advantages of QDs are utilized are sensing systems.

In this review, we present the main physical and chemical mechanisms used for detection of various species (bacteria, cells, nucleic acids, molecules, ions, etc.) with utilizing of QDs. We present the most successful examples of QDs applications in biology and medicine as optical and electrochemical sensors. Finally, we focus on the perspectives for further development in this field.

**Figure 1.** (**a**) TEM image of hydrophilic CdSe quantum dots. Schematic structure of selected quantum dot after synthesis (**a1**), after surface functionalization (with examples of most typical functional groups) (**a2**) and after bioconjugation (with examples of most common biomolecules used for detection/targeting) (**a3**). (**b**,**<sup>c</sup>**) Digital images of CdSe quantum dots dispersed in water with and without laser excitation.

#### **2. Comparison between Optical Properties of Organic Dyes and QDs**

In comparison to organic dyes, QDs have the spectral position of absorption and emission dependent on their size (so-called Quantum Size Effect) [15]. During synthesis, this effect enables continuous tuning of the emission peak position in a wide range of wavelengths (Figure 2a). Moreover, the broad absorption of QDs allows free selection of the excitation wavelength and thus straightforward separation of the excitation and emission signal (Figure 2b,c) [16]. The fluorescence lifetimes of organic dyes are commonly too short for efficient temporal discrimination of short-lived autofluorescence of biological objects. In the case of QDs, the emission decay time can be tuned or selected with a proper choice of QDs composition (giving times up to several microseconds). This enables straightforward temporal discrimination of the signal from cellular autofluorescence and scattered excitation light by time-gated measurements, thereby enhancing detection sensitivity [17]. In contrast to conventional dyes, QDs emitting different colors (and functionalized with different groups) can be simultaneously excited by a single excitation wavelength. This makes QDs suitable for multiplex testing by simultaneously detecting multiple signals [18]. Moreover, QDs characterize with extremely high chemical stability and photostability (stability against chemical reactions induced by the incoming radiation). In addition, QDs are free from photobleaching [19] (Figure 2d) what is one of their most important advantages.

**Figure 2.** (**a**) Emission spectra from CdS QDs (left side) and PbS QDs (right side) with different size and chemical composition; (**b**) absorbance and emission spectra of CdSe/CdS quantum dots; (**c**) emission and absorption spectra of Rhodamine; (**d**) emission intensity vs illumination time for CdSe/CdS QDs and Rhodamine.

#### **3. Fundamentals of QDs Sensing Phenomena**

The unique optical properties of QDs make them attractive fluorophores that can be used both in vitro and in vivo in various biological studies, where traditional fluorescent labels based on organic molecules do not provide long-term stability, high enough intensity or where simultaneous detection of many signals is needed [7]. In sensors, the signal detection bases on a registration of the change in one of the physical properties (optical, thermal, mechanical, magnetic, electrical) of sensing material induced by the interaction with the analyte. Changing in optical properties of QDs like emission color, intensity, polarization or emission kinetics can be used as the principle in optical sensors system (Figure 3). In addition, obtained changes can be recorded directly by human senses or indirectly *via* the signal transformation, amplification, and visualization. All these factors determine sensors construction and their mechanism of action in the detection of various substances [20,21].

**Figure 3.** Signal processing characteristics for living organisms and sensor machines.

#### **4. Basic Strategies for Analyte Detection**

The use of QDs for sensors construction requires adjusting their optical properties adequately to the needs that arise their shape, size, the color of emission, position of the absorption band. Moreover, to ge<sup>t</sup> specificity of QDs in their sensing action, the surface modification—called functionalization—must be applied first [22,23]. Functionalization is the process of attaching, exchanging already attached chemical molecules present on the surface of quantum dots. Chemical and physical methods used for this purpose, include processes such as exchange of ligands, silanisation, the creation of additional coatings or dendrimeric structures [24]. The presence of ligands at the QDs surfaces affect their size, shape and physico-chemical properties, e.g., surface charge and chemical reactivity. Surface modifications allow the control of colloidal stability of QDs and their dispersion in non-polar environments (organic solvents in which they are most commonly synthesized) and polar (e.g., water, in which solubility is necessary for biological and medical applications). Moreover, the surface attached ligands determine the possibility of QDs conjugation to biological molecules (bioconjugation) or to determine their potential in applications where QDs must be embedded within the matrix [25,26].

In order to achieve high selectivity of QDs sensor, QDs are coupled to various vectors specific for an analyte. Wales et al. constructed a sensor for the selective detection of dicofol, a substance used to kill mites. For this purpose, they used CdS QDs with glutathione on their surface, whose both aminoand carboxyl- functional groups interact with chloride groups in the dicofol structure, thus leading to an increase in fluorescence intensity, which was directly proportional to the dicofol concentration in the studied sample [27,28].

The QDs-based sensors can be designed in several ways, depending on demands regarding their sensitivity, types of detected analytes, costs or complexity of their preparation. Figure 4 shows the examples of preparation protocols used in QDs-based optical sensors.

In all cases, the protocol starts with the appropriate modification of QDs surface selectivity. As a result, QDs are targeted to determine a particular analyte. An important aspect is also the preparation of substrates which can take an active part in the detection protocol. Strategies (a) and (b) differ in Stages III and IV, which occur in reverse order. While in Strategy (a) Step III is the deposition of QDs, in Strategy (b) it is Step IV. This stage can be made using methods such as layer-by-layer [29], sol-gel [30] or electrochemical method [31]. Stage IV in Strategy (a) and III in Strategy (b) are a conjugation of the analyte, which may be possible thanks to the previously prepared and targeted substrate. Jie et al. proposed the coupling of the analyte with the previously prepared substrate, based on CdSe nanocomposites, using antibodies selective for an antigen called human IgG [32].

The final step in all strategies is QDs stimulation, which is used to detect the analyte. As a result, both the qualitative and quantitative assessment of the presence of the designated substance is possible. It is also possible to combine the first two strategies, resulting in Strategy (c), which uses the Förster resonance energy transfer between optical centers (QDs + QDs or dye).

**Figure 4.** Three examples of the strategy of QDs-based optical sensors (strategy **a**—modification of substrate with QDs directed to detection of analyte, strategy **b**—modification of substrate for detection of analyte-QDs complex, strategy **c**—using the analyte labeled with appropriate fluorophore).

The presented strategies differ in the number of steps that complicate detection and require a lot of user experience.

#### **5. Physico-Chemical Mechanisms Used for Analyte Detection**

One of the most popular mechanisms used for detection of analyte relies on emission quenching from QDs. In this mechanism, due to the interaction of the QDs surface with the analyte, the QDs emission intensity decreases (Figure 5a) [33]. Another mechanism relies on an increase of QDs emission due to passivation of QDs surface by analyte (Figure 5b), e.g., addition of bovine serum albumin or nucleic acids resulted in increasing emission from CdS dots coated with mercaptoacetic acid [34].

The third mechanism, which can be used for analyte detection, is stimulated aggregation (Figure 5c). In this mechanism, due to an interaction of the analyte with the QDs surface, the surface ligands are detached and QDs aggregate. The aggregation can be also induced by analyte stimulated bonds formation between functionalized QDs [35].

There is also a very rarely used mechanism of analyte detection based on modification of the nanostructures' growth process by introduction of the analyte during the nanostructures' growth. Due to this perturbation, the nanostructures can have different emission or other properties which can be detected (Figure 5d). There is also the fifth mechanism commonly used for analyte detection based on changes in QDs optical properties. The changes come from excitation energy transfer from QDs to other optical center (QDs or dye). In consequences, the color of emission changes or emission decay time of donor is reduced (Figure 5e) [36,37].

**Figure 5.** Examples of physico-chemical mechanisms used for analyte optical detection—emission bleaching (**a**), increase of emission (**b**), emission localization (**c**), nanostructures growth's modification (**d**), emission change (**e**).
