**3.2 Quantum dots**

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5-hexachloro- fluorescein (HEX).

*3.1.4 Rhodamine derivatives*

(TAMRA) and Texas red.

*3.1.5 Cyanine derivatives*

including Cy3, Cy5, Cy5.5 and Cy7.

6-carboxyrhodamine (TAMRA) and Texas red.

and can produce strong green fluorescence under the action of laser. Due to its easy synthesis, low cost, low biological and cytotoxicity, high molar absorption coefficient, and high fluorescence quantum yield, fluorescein can be widely used in biological imaging and analysis [48]. However, fluorescein also has some defects, such as high pH sensitivity [49], small stokes [50] and poor light stability [51]. In order to improve the fluorescence performance of fluorescein, many important fluorescein derivatives have been developed by introducing functional group modification to fluorescein [52]. Additionally, fluorescein derivatives contain some active groups, which can bind with bio-receptors to obtain fluorescent dyes labeled probes with high selectivity, good stability and high sensitivity for biosensing [53]. Currently, commercially available fluorescein derivatives dye mainly includes 6-carboxy-fluorescein (FAM), 5-tetrachloro-fluorescein (TET),

It was discovered in the late 1980s that rhodamine and its derivatives are important fluorescent dyes and also belong to xanthene dyes. The molecular structure of rhodamine dyes is very stable, coplanar, and can produce strong red fluorescence under the excitation. They also can bind with bio-receptors to obtain fluorescent dyes labeled probes with high selectivity, good stability and high sensitivity for biosensing. Compared with fluorescein derivatives, rhodamine derivatives have stronger photostability, higher fluorescence quantum yield and lower pH sensitivity. Commercially available rhodamine derivatives dyes for labels in biosensing mainly include 6-carboxyl-x-rhodamine (ROX), tetramethyl-6-carboxyrhodamine

It was discovered in the late 1880s that rhodamine and its derivatives are important fluorescent dyes and also belong to xanthene dyes [54]. The molecular structure of rhodamine dyes is very stable, coplanar, and can produce strong red fluorescence under the excitation [55]. They also can bind with bio-receptors to obtain fluorescent dyes labeled probes with high selectivity, good stability and high sensitivity for biosensing. Compared with fluorescein derivatives, rhodamine derivatives have stronger photostability, higher fluorescence quantum yield and lower pH sensitivity [56]. Commercially available rhodamine derivatives dyes for labels in biosensing mainly includes 6-carboxyl-x-rhodamine (ROX), tetramethyl-

Cyanine dyes was first discovered by Williams in 1856, subsequently, Vogel discovered that these dyes have very good photosensitivity, which promote the development of cyanine dyes [57]. Cyanine dyes and derivatives have excellent spectral characteristics, such as high molar extinction coefficient, high fluorescence quantum yield, and long fluorescence emission wavelength. More importantly, the maximum emission and absorption of these dyes are located in the near-infrared region. In this region, the self-absorption and background fluorescence of biomolecules are relatively small [58]. Thus, cyanine dye derivatives have become the most commonly used fluorescent signal groups in protein, nucleic acid and other biological analysis [59]. Commercially available cyanine derivatives dyes for labels in biosensing mainly refers to N-carboxypentyl-5-sulfonate-3H-indocyanine dyes,

**44**

Quantum dots (QDs) are spherical or quasiballistic semiconductor nanoparticles that bind excitons in the three dimensions, with a diameter no larger than twice the Bohr radius of the excitons of their corresponding semiconductor material, thus confining the motion of electrons, holes, or excitons in three dimensions. Due to the quantum confinement effects, the quantum dots display unique optical and electronic properties compared to the bulk materials.

QDs were firstly synthesized in glass matrix in 1970s and with their fluorescent properties reported [60]. Later some groups studied the novel properties of quantum dots and tried to investigate influences of quantum effects on the optical properties of QDs [61]. In 1980s, CdS nanospheres were synthesized in colloidal solution and the basic theory of QDs were studied [62]. In 1993, the high quality colloidal QDs were prepared first time with uniform size in the solution [63], which provided favorable materials for both theoretical study and practical applications. Since then, various types of QDs with different compositions and properties have been synthesized by the solution growth method.

According to the chemical compositions, QDs can be mainly divided to two groups. Single component QDs, such as metal chalcogenides, [64] attracted much attentions at the early stage of the QDs development. Due to the uniformity, optical and electronic properties of such QDs can be tuned by simply controlling their sizes. Multiple component QDs are either core-shell structural or alloyed. Coreshell structural QDs have a core with one component embedded in another material as a shell, such as CdSe/ZnS [65]. Usually, to reduce the nonradioactive recombination of electron–hole pairs, the material used as the shell has a larger band gap than that in the core, thus improving the fluorescence quantum yield. Coating the same core with different shells adjust the properties of the QDs. Alloyed QDs that have homogeneous or heterogeneous alloyed internal composition, for example, CdSxSe1-x/ZnS [66]. This type of QDs allows tuning the properties by changing the proportion of the component without changing the size. Interestingly, alloyed QDs not only exhibit the original properties of each component, but also display newly additional and adjustable properties because of the merge of the different composites. Now despite classical nanocrystals, there are various new species of QDs that have been prepared, such as perovskite QDs [67] and graphene QDs [68].

Generally, the size of QDs, i.e., in the range of 2–10 nm, endows these nanoparticles high surface-to-volume ratios. The large surface provides rich sites for further functionalization and immobilization of molecules, including nucleic acids and proteins. [69] Importantly, after functionalized with hydrophilic ligands, QDs are soluble and stable in aqueous solution, which is the common environment for biological reactions.

#### *3.2.1 Sensing mechanism*

The most obvious and widely utilized properties of QDs are the optical properties. Compared with organic dyes, QDs display higher fluorescence quantum yield and extinction coefficient, broader excitation spectra, longer lifetimes and tunable fluorescent emissions [70]. As the size of QDs decreases, the band gap between valence band (VB) and conduction band (CB) increases, which means more energy needed for electrons excitations (from ground state in VB to CB) as well as more energy released from the electrons de-excitation (from CB to ground state inVB), leading to the fluorescent emission shift to the high frequency range. In addition, the fluorescence wavelength can also be tuned flexibly by adjusting the structure

and chemical compositions of QDs as mentioned above. These stand out properties make QDs appealing for bio-medical applications including imaging and biosensing [71]. In biosensing systems, QDs, used with or without nano-sized quenchers, are transducers and functionalized with bio-recognition molecules (bio-receptors). Because of the tunable size and broad spectral width, QDs can play as either energy donors or acceptors in the FRET biosensor [72]. Furthermore, due to their high fluorescence intensity, photostability and long lifetimes than conventional organic fluorophores, QDs were also involved in the design of molecular beacons.

**Bioanalytical systems using QDs as labels**. QDs were first applied as bioanalytical labels in 1998, [73] an ultrasensitive bioanalytical system involving QDs was demonstrated for protein imaging. Since then, QDs have been widely applied in various bioanalytical methodologies, e.g., the enzyme-linked immunosorbent assay (ELISA), fluorescence resonance energy transfer (FRET) assay and cell tracking.

QDs can be simply used as fluorescent labels in immunosensors to quantify the biological targets through directly measuring the presence and/or the intensity of the fluorescence. CdTe/SiO2 core-shell structured nanoparticles were labeled with prostate-specific antigen (PSA) detection antibodies for PSA detection. And the fluorescent signals were measured after the specific recognition between PSA and the QD labeled antibodies followed by a magnetic separation to remove unbound QDs. This system represents a typical fluorescent biosensor detecting the presence of QD labels [74]. This strategy design shows great flexibility for employing a variety of fluorescence detectors, e.g., fluorescence spectrometry and handheld UV lamp tests. Furthermore, such design enables not only target quantification but also imaging multiple targets with different QD labels [75].

**Imaging.** QDs have also demonstrated their applicability in biomedical imaging, which is an important tool in diagnosis, visualization, treatment and prognosis of diseases [76]. QDs emit in the visible and near infrared ray wavelengths, with high brightness and excellent photostability, suitable for morphological studies. A representative examples of such applications encapsulated QDs in virus-lick particles as theranostic platforms to image viral behavior in living cells [77]. Furthermore, multifunctional SV40 virus-like particles was constructed encapsulating QDs bearing peptides recognizing early, developmental, and late stages of atherosclerosis, respectively, in live mice [78].

#### *3.2.2 Labeling type*

One of essential challenges to apply QDs in biosensors is immobilization of target recognition biomolecules onto the surface of QDs via stable bonding. In this part, methods for preparing QD-biomolecule conjugates will be presented and modified QDs applied as labels in biosensors are also summarized. Roughly, there are 4 strategies to prepare the QD-based bioconjugates:


**47**

*Dyes as Labels in Biosensing*

fluorescence for detection.

**4.1 Organic dye molecules**

performance in some biosensors.

**4.2 Organometallic complexes**

**4. Dyes in electrochemical biosensors**

of these dyes as labels in the biosensors are also displayed.

*DOI: http://dx.doi.org/10.5772/intechopen.96540*

**3.3 Upconversion fluorescent materials**

as biotin-streptavidin interaction [83].

4.Conjugation via specific biological affinity: some types of biological affinity can be used to bond the QDs with biomolecules strongly and specifically, such

Upconversion fluorescent materials are emerging fluorescent nanoparticles that can convert low frequent exciting light into high frequent emitting light by absorbing two-photons or multi-photons. The luminous mechanism of upconversion nanoparticles (UCNPs) is anti-stokes, which is opposite with the most fluorescent materials, including organic fluorescent dyes, quantum dots, fluorescent proteins, metal complexes, etc [84]. Because of the distinctive luminescence mechanism, UCNPs have some unique advantages, which make up the disadvantage of above other dyes. Firstly, UCNPs have improved biological tissue penetration. Secondly, UCNPs can reduce light damage on biological samples. Thirdly, UCNPs can effectively avoid the disturbance of autofluorescence from biological samples. Therefore,

UCNPs have wide applications in biosensing and imaging [85]. The sensing principle of UCNPs-based probe is widely based on fluorescence resonance energy transfer (FRET) between UCNPs (donor) and other down-conversion fluorophores (acceptor). The reaction between UCNPs-based probe and the target biomolecules will trigger or hinder the FRET process, causing the quenching and enhancement of

Electrochemical biosensor is capable of providing specific quantitative or semiquantitative analytical information using electrochemical transduction elements, e.g., charge-transfer complexes. Low-cost, energy efficient, portable, easy fabrication, and real-time sensing are major advantages of electrochemical biosensing platforms. Among the electrical signal molecules, there are several types of dyes with electrochemical activity, and they will be introduced in this part. In addition, the electrochemical signals generation mechanisms are explained and applications

Methylene blue (MB) is a kind of derivative of phenothiazine and widely used a redox indicator and electron transfer medium in electrochemical analysis. For a typical DNA detection using MB labels, the distance between the MB and the electrode surface is adjusted by the change of conformation of the DNA probes labeled with MB, so that the peak current or the change in impedance can indicate the presence of the target DNA and quantitative concentration. This method is simple and versatile, however easy to be influenced by the solution environment. Despite MB, other organic dyes, such as gentian violet, ethyl green, Hoechst 33258 are also utilized in the electrochemical works. They are not as popular as MB, but show good

Organometallic complexes consist of centrally located metal atoms or ions and completely or partly coordinated organic ligands. The organometallic complexes with transition metals have the advantages of strong redox signal, good chemical

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imaging multiple targets with different QD labels [75].

are 4 strategies to prepare the QD-based bioconjugates:

respectively, in live mice [78].

biomolecules [80].

*3.2.2 Labeling type*

and chemical compositions of QDs as mentioned above. These stand out properties make QDs appealing for bio-medical applications including imaging and biosensing [71]. In biosensing systems, QDs, used with or without nano-sized quenchers, are transducers and functionalized with bio-recognition molecules (bio-receptors). Because of the tunable size and broad spectral width, QDs can play as either energy donors or acceptors in the FRET biosensor [72]. Furthermore, due to their high fluorescence intensity, photostability and long lifetimes than conventional organic

**Bioanalytical systems using QDs as labels**. QDs were first applied as bioanalytical labels in 1998, [73] an ultrasensitive bioanalytical system involving QDs was demonstrated for protein imaging. Since then, QDs have been widely applied in various bioanalytical methodologies, e.g., the enzyme-linked immunosorbent assay (ELISA), fluorescence resonance energy transfer (FRET) assay and cell tracking. QDs can be simply used as fluorescent labels in immunosensors to quantify the biological targets through directly measuring the presence and/or the intensity of the fluorescence. CdTe/SiO2 core-shell structured nanoparticles were labeled with prostate-specific antigen (PSA) detection antibodies for PSA detection. And the fluorescent signals were measured after the specific recognition between PSA and the QD labeled antibodies followed by a magnetic separation to remove unbound QDs. This system represents a typical fluorescent biosensor detecting the presence of QD labels [74]. This strategy design shows great flexibility for employing a variety of fluorescence detectors, e.g., fluorescence spectrometry and handheld UV lamp tests. Furthermore, such design enables not only target quantification but also

**Imaging.** QDs have also demonstrated their applicability in biomedical imaging, which is an important tool in diagnosis, visualization, treatment and prognosis of diseases [76]. QDs emit in the visible and near infrared ray wavelengths, with high brightness and excellent photostability, suitable for morphological studies. A representative examples of such applications encapsulated QDs in virus-lick particles as theranostic platforms to image viral behavior in living cells [77]. Furthermore, multifunctional SV40 virus-like particles was constructed encapsulating QDs bearing peptides recognizing early, developmental, and late stages of atherosclerosis,

One of essential challenges to apply QDs in biosensors is immobilization of target recognition biomolecules onto the surface of QDs via stable bonding. In this part, methods for preparing QD-biomolecule conjugates will be presented and modified QDs applied as labels in biosensors are also summarized. Roughly, there

1.Direct binding: proteins and nucleic acids can be immobilized on the QDs surface directly through interactions between the thiol-groups or imidazolegroups with the metal component of QDs, e.g., alkylthiol terminated DNAs are

2.Conjugation via ligands: QDs can be functionalized firstly by ligands, such as carboxyl groups, hydroxyl groups and amino-groups, then covalently bond to

3.Conjugation via functional shell: QDs are capped with silane shell [81] or copolymers [82], then bond to biomolecules by the functional groups on the outer shell.

linked with QDs surface directly via dative thiol bond [79].

fluorophores, QDs were also involved in the design of molecular beacons.

**46**

4.Conjugation via specific biological affinity: some types of biological affinity can be used to bond the QDs with biomolecules strongly and specifically, such as biotin-streptavidin interaction [83].
