**2.3 Latex particles**

Colored latex particles are also often used as labels in the colorimetric biosensor. Latex particles are natural or synthetic polymer nano- and micro-particles that suspend stably in water, and the polystyrene particles are used mostly.

There are three ways to prepare colored latex particles by dying latex particles with different types of dyes molecules: (1) co-polymerization of polymer monomer with dyes; (2) cross-linking the dyes on particles surface by covalent bonds; (3) physical embedding or absorption dyes in particles. After the dying, usually dyes on the surface are removed in order to functionalize the active groups (sulfhydryl groups, amino groups and carboxyl groups) on the latex particles for further labeling the biomolecules [28].

Benefit its wide variety of sources, low cost and easy to be functionalized, the latex particles are applied as probes in immunochromatographic analysis quite early [29]. The good properties enable them are still used now. A lateral flow immunoassay was developed by covalent functionalizing the antigens on colored latex particles for the visual diagnosis of canine visceral leishmaniasis [30]. A latex particles-GNPs composites labeled with antibodies were synthesized as probed for the immunochromatographic test. The nanocomposites amplified the binding capacity of GNPs with target antigens and improved the sensitivity 2 orders of magnitude compared with GNPs-antibodies probes [31].

### **3. Dyes in fluorescent biosensors**

In the field of biotechnology, diagnosis and drug discovery, fluorescent assay is by far the most popular methodology because of not only its sensitivity and versatility but also the high commercialization of fluorescent labels [32]. In addition to the new fluorescent nanomaterials (e.g., upconversion fluorescent materials and aggregation-induced emission (AIE) materials that are described further below), new spectroscopic sensors have also been developed based on rising technologies such as fiber optics, LEDs and fast imaging devices, all of which contributed to the fast development as well as high interdisciplinarity of fluorescent biosensors.

#### **3.1 Organic dyes**

Organic fluorescent dyes are a class of organic molecules that contain a fluorescent core skeleton with a large conjugate system and some auxochrome or active group (such as carboxyl, amino, amide, etc). The fluorescent core skeleton enables them absorb a certain excitation light and emitting it as fluorescence. The auxochrome or active group is capable of altering wavelengths and enhance fluorescence or labeling them to bio-receptors for recognizing various biomolecules in biosensing [33]. Briefly, the fluorescent dyes labeled bio-receptors, also called fluorescent probes, can recognize various biomolecules and then convert the recognition events into fluorescent signal output to achieve biosensing or imaging.

Currently, there are many kinds of organic fluorescent dyes, most of which can be used to label bio-receptors for biosensing and imaging. Here, some major organic fluorescent dyes labels are introduced, including fluorescein derivatives, rhodamine derivatives, cyanine derivatives and other commonly used organic fluorescent dyes.

#### *3.1.1 Sensing mechanism*

The signal conversion mechanism in sensing process are various, such as electron transfer quenching or fluorescence recovery, fluorescence resonance energy transfer (FRET), or monomer-excimer emission conversion with pyrene fluorophores. The following will introduce the major signal conversion mechanism involved in the fluorescence biosensing process.

**41**

**Figure 5.**

*EB staining for nucleic acids detection.*

*Dyes as Labels in Biosensing*

EB for detection.

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

homogeneous reaction processes with high sensitivity.

**Nucleic acid intercalating dyes.** Nucleic acid intercalating dyes [34–36] is a special kind of organic fluorescent dyes that have no fluoresce or the fluorescence is weak in solution, which may be caused by the quenching of solvent. However, when they are embedded in specific DNA structures, the fluorescence intensity will increase significantly, due to the protection of the hydrophobic groups of the oligonucleotide (**Figure 5**). Commercially available nucleic acid intercalating dyes for labels in biosensing mainly include ethidium bromide (EB), thioflavin T (ThT), N-methylporphyrin dipropionic acid IX (NMM) and triphenylmethane dyes. The most classical biosensing application is EB staining-mediated gel electrophoresis for nucleic acids detection [37]. Nucleic acid fragments can be separated in gel under the action of electric field, then EB contains a tricyclic planar group can insert between nucleic acid stacking bases, resulting in increased fluorescence intensity of

An emerging biosensing strategy is designed based on the G-quadruplex (G4) DNA structure and corresponding intercalating dyes such as ThT, NMM, etc. (**Figure 6**) [38]. G4 DNA structure is formed from DNA guanine-rich sequences, which has been confirmed to be stably present in human live cell [39]. Therefore, endogenous G4 DNA can be easily detected by using intercalating dyes targeting G4 DNA. Additionally, G4 DNA structures can be formed by the amplicons of any kinds of DNA amplification methods that produce single-stranded DNA, making G4 structure a convenient cascade amplification tool (a molecular amplification followed by a signal amplification) that can be applied in homogeneous and isothermal bioanalytical assays. Moreover, the formation or consume of the G4 structure after binding to the target molecules will change the interaction between G4 and intercalating dyes, resulting in increased or decreased fluorescence intensity for detection. **Fluorescence resonance energy transfer.** Measuring the presence of labels always means employing tedious operation steps for separation and washing. Today, homogeneous reaction processes are highly preferred due to its potential for pointof-care applications. FRET assays are frequently used in biosensors due to achieve

#### *Dyes as Labels in Biosensing DOI: http://dx.doi.org/10.5772/intechopen.96540*

*Dyes and Pigments - Novel Applications and Waste Treatment*

magnitude compared with GNPs-antibodies probes [31].

**3. Dyes in fluorescent biosensors**

**3.1 Organic dyes**

fluorescent dyes.

*3.1.1 Sensing mechanism*

ing the biomolecules [28].

There are three ways to prepare colored latex particles by dying latex particles with different types of dyes molecules: (1) co-polymerization of polymer monomer with dyes; (2) cross-linking the dyes on particles surface by covalent bonds; (3) physical embedding or absorption dyes in particles. After the dying, usually dyes on the surface are removed in order to functionalize the active groups (sulfhydryl groups, amino groups and carboxyl groups) on the latex particles for further label-

Benefit its wide variety of sources, low cost and easy to be functionalized, the latex particles are applied as probes in immunochromatographic analysis quite early [29]. The good properties enable them are still used now. A lateral flow immunoassay was developed by covalent functionalizing the antigens on colored latex particles for the visual diagnosis of canine visceral leishmaniasis [30]. A latex particles-GNPs composites labeled with antibodies were synthesized as probed for the immunochromatographic test. The nanocomposites amplified the binding capacity of GNPs with target antigens and improved the sensitivity 2 orders of

In the field of biotechnology, diagnosis and drug discovery, fluorescent assay is by far the most popular methodology because of not only its sensitivity and versatility but also the high commercialization of fluorescent labels [32]. In addition to the new fluorescent nanomaterials (e.g., upconversion fluorescent materials and aggregation-induced emission (AIE) materials that are described further below), new spectroscopic sensors have also been developed based on rising technologies such as fiber optics, LEDs and fast imaging devices, all of which contributed to the fast development as well as high interdisciplinarity of fluorescent biosensors.

Organic fluorescent dyes are a class of organic molecules that contain a fluorescent core skeleton with a large conjugate system and some auxochrome or active group (such as carboxyl, amino, amide, etc). The fluorescent core skeleton enables them absorb a certain excitation light and emitting it as fluorescence. The auxochrome or active group is capable of altering wavelengths and enhance fluorescence or labeling them to bio-receptors for recognizing various biomolecules in biosensing [33]. Briefly, the fluorescent dyes labeled bio-receptors, also called fluorescent probes, can recognize various biomolecules and then convert the recognition events

Currently, there are many kinds of organic fluorescent dyes, most of which can be used to label bio-receptors for biosensing and imaging. Here, some major organic fluorescent dyes labels are introduced, including fluorescein derivatives, rhodamine derivatives, cyanine derivatives and other commonly used organic

The signal conversion mechanism in sensing process are various, such as electron transfer quenching or fluorescence recovery, fluorescence resonance energy transfer (FRET), or monomer-excimer emission conversion with pyrene fluorophores. The following will introduce the major signal conversion mechanism

into fluorescent signal output to achieve biosensing or imaging.

involved in the fluorescence biosensing process.

**40**

**Nucleic acid intercalating dyes.** Nucleic acid intercalating dyes [34–36] is a special kind of organic fluorescent dyes that have no fluoresce or the fluorescence is weak in solution, which may be caused by the quenching of solvent. However, when they are embedded in specific DNA structures, the fluorescence intensity will increase significantly, due to the protection of the hydrophobic groups of the oligonucleotide (**Figure 5**). Commercially available nucleic acid intercalating dyes for labels in biosensing mainly include ethidium bromide (EB), thioflavin T (ThT), N-methylporphyrin dipropionic acid IX (NMM) and triphenylmethane dyes. The most classical biosensing application is EB staining-mediated gel electrophoresis for nucleic acids detection [37]. Nucleic acid fragments can be separated in gel under the action of electric field, then EB contains a tricyclic planar group can insert between nucleic acid stacking bases, resulting in increased fluorescence intensity of EB for detection.

An emerging biosensing strategy is designed based on the G-quadruplex (G4) DNA structure and corresponding intercalating dyes such as ThT, NMM, etc. (**Figure 6**) [38]. G4 DNA structure is formed from DNA guanine-rich sequences, which has been confirmed to be stably present in human live cell [39]. Therefore, endogenous G4 DNA can be easily detected by using intercalating dyes targeting G4 DNA. Additionally, G4 DNA structures can be formed by the amplicons of any kinds of DNA amplification methods that produce single-stranded DNA, making G4 structure a convenient cascade amplification tool (a molecular amplification followed by a signal amplification) that can be applied in homogeneous and isothermal bioanalytical assays. Moreover, the formation or consume of the G4 structure after binding to the target molecules will change the interaction between G4 and intercalating dyes, resulting in increased or decreased fluorescence intensity for detection.

**Fluorescence resonance energy transfer.** Measuring the presence of labels always means employing tedious operation steps for separation and washing. Today, homogeneous reaction processes are highly preferred due to its potential for pointof-care applications. FRET assays are frequently used in biosensors due to achieve homogeneous reaction processes with high sensitivity.

**Figure 5.** *EB staining for nucleic acids detection.*

*Dyes and Pigments - Novel Applications and Waste Treatment*

**Figure 6.**

*Fluorescence enhancement based on the intercalating dyes and G4 structure.*

**Figure 7.** *Schematic illustration of fluorescence resonance energy transfer.*

A FRET technique includes an energy transfer between two fluorophores, i.e., from high energy donor to a lower energy acceptor (**Figure 7**). The FRET occurs when the donor and acceptor are close to each other, approximately between 1–10 nm, and this distance meets the dimensions of biological molecules. Since the FRET is sensitive to the relative distance between donors and acceptors, when biological acceptor labeled with donors (or acceptors) comes close to the biological target labeled with acceptors (or donors), FRET signals can be detected.

Rather than labels the donor and the acceptor on different biomolecules, a molecular beacon (MB) utilized a donor linked with an acceptor through a bioresponsive probe, further simply the design of FRET biosensors [40]. Typically, a MB is a single-stranded oligonucleotide probe labeled with a fluorophore at its one end and a quencher at the other. Due to the length and/or the secondary structure of the oligonucleotide, the probe holds the fluorophore and quencher close to each other, thus inducing a quenching. Upon the hybridization between the probe and the target, the distance between the fluorophore and the quencher changes, restoring the fluorescence [41] (**Figure 8**).

**43**

*Dyes as Labels in Biosensing*

*3.1.2 Labeling type*

**Figure 8.**

fluorescent sensing mechanisms.

*3.1.3 Fluorescein derivatives*

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

*Illustration of a molecular beacon and examples of its applications.*

**Monomer-Excimer.** Generally, when the distance and position between fluorescent dyes with the same or different structures are appropriate, the excited state fluorescent dye and the other ground state fluorescent dye would form an excimer. Therefore, the fluorescence emission intensity of the original monomer weakens or disappears, and the fluorescence emission of the formed excimer appears [42, 43]. For monomer-excimer based biosensing process, the reaction between the fluorescent dyes-labeled probe and the target biomolecules will trigger or hinder the monomer-excimer process, causing the variation of fluorescence emission spectra.

The labeling type between organic fluorescent dyes and bio-receptors can generally be divided into two types: covalent and non-covalent (such as intercalation, groove binding or electrostatic interaction). Different labeling method has different

Covalently binding labeled fluorescent probes including single-labeled fluorescent

Fluorescein and its derivatives are one class of xanthene dyes. Fluorescein was first produced by Von Bayer in 1871, which has a good rigid coplanar structure

probe and dual-labeled fluorescent probe. Single-labeled fluorescent probes are obtained by covalently binding single fluorescent dye molecules to bio-receptors. The single-labeled probe sensing mechanism may be summarized as follows: when the bio-receptor of the fluorescent probe recognizes the target molecules, the fluorescence properties of dyes would be changed, such as changes in fluorescence intensity and fluorescence anisotropy, thus converting the recognition process into a measurable fluorescence signal [44–46]. Dual-labeled fluorescent probes are obtained by covalently binding dual fluorescent dye molecules (or a dye and a quencher) to bio-receptors. The dual-labeled probes are all distance dependent, the rearrangement of the probe structure after binding to the target molecules will change the distance between the two labels, resulting in changes in the fluorescence properties of the system. Non-covalently binding fluorescent probes mainly refer to nucleic acid fluorescent probes that obtained by the binding of intercalating dyes and DNA. As mentioned above, when the nucleic acid intercalating dye binds to a specific DNA structure, the fluorescent signal changes. Based on this principle, a series of biosensors have been developed. Compared with covalently binding fluorescent probes, the non-covalently binding fluorescent probes will not affect the binding affinity of the probe to the target, and also have the advantages of easy operation and low cost [47].

*Dyes and Pigments - Novel Applications and Waste Treatment*

*Fluorescence enhancement based on the intercalating dyes and G4 structure.*

**Figure 6.**

A FRET technique includes an energy transfer between two fluorophores, i.e., from high energy donor to a lower energy acceptor (**Figure 7**). The FRET occurs when the donor and acceptor are close to each other, approximately between 1–10 nm, and this distance meets the dimensions of biological molecules. Since the FRET is sensitive to the relative distance between donors and acceptors, when biological acceptor labeled with donors (or acceptors) comes close to the biological

target labeled with acceptors (or donors), FRET signals can be detected.

*Schematic illustration of fluorescence resonance energy transfer.*

Rather than labels the donor and the acceptor on different biomolecules, a molecular beacon (MB) utilized a donor linked with an acceptor through a bioresponsive probe, further simply the design of FRET biosensors [40]. Typically, a MB is a single-stranded oligonucleotide probe labeled with a fluorophore at its one end and a quencher at the other. Due to the length and/or the secondary structure of the oligonucleotide, the probe holds the fluorophore and quencher close to each other, thus inducing a quenching. Upon the hybridization between the probe and the target, the distance between the fluorophore and the quencher changes, restoring

**42**

**Figure 7.**

the fluorescence [41] (**Figure 8**).

**Figure 8.** *Illustration of a molecular beacon and examples of its applications.*

**Monomer-Excimer.** Generally, when the distance and position between fluorescent dyes with the same or different structures are appropriate, the excited state fluorescent dye and the other ground state fluorescent dye would form an excimer. Therefore, the fluorescence emission intensity of the original monomer weakens or disappears, and the fluorescence emission of the formed excimer appears [42, 43]. For monomer-excimer based biosensing process, the reaction between the fluorescent dyes-labeled probe and the target biomolecules will trigger or hinder the monomer-excimer process, causing the variation of fluorescence emission spectra.

## *3.1.2 Labeling type*

The labeling type between organic fluorescent dyes and bio-receptors can generally be divided into two types: covalent and non-covalent (such as intercalation, groove binding or electrostatic interaction). Different labeling method has different fluorescent sensing mechanisms.

Covalently binding labeled fluorescent probes including single-labeled fluorescent probe and dual-labeled fluorescent probe. Single-labeled fluorescent probes are obtained by covalently binding single fluorescent dye molecules to bio-receptors. The single-labeled probe sensing mechanism may be summarized as follows: when the bio-receptor of the fluorescent probe recognizes the target molecules, the fluorescence properties of dyes would be changed, such as changes in fluorescence intensity and fluorescence anisotropy, thus converting the recognition process into a measurable fluorescence signal [44–46]. Dual-labeled fluorescent probes are obtained by covalently binding dual fluorescent dye molecules (or a dye and a quencher) to bio-receptors. The dual-labeled probes are all distance dependent, the rearrangement of the probe structure after binding to the target molecules will change the distance between the two labels, resulting in changes in the fluorescence properties of the system.

Non-covalently binding fluorescent probes mainly refer to nucleic acid fluorescent probes that obtained by the binding of intercalating dyes and DNA. As mentioned above, when the nucleic acid intercalating dye binds to a specific DNA structure, the fluorescent signal changes. Based on this principle, a series of biosensors have been developed. Compared with covalently binding fluorescent probes, the non-covalently binding fluorescent probes will not affect the binding affinity of the probe to the target, and also have the advantages of easy operation and low cost [47].

#### *3.1.3 Fluorescein derivatives*

Fluorescein and its derivatives are one class of xanthene dyes. Fluorescein was first produced by Von Bayer in 1871, which has a good rigid coplanar structure

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), 5-hexachloro- fluorescein (HEX).

#### *3.1.4 Rhodamine derivatives*

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 (TAMRA) and Texas red.

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-6-carboxyrhodamine (TAMRA) and Texas red.

#### *3.1.5 Cyanine derivatives*

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, including Cy3, Cy5, Cy5.5 and Cy7.

**45**

*3.2.1 Sensing mechanism*

*Dyes as Labels in Biosensing*

**3.2 Quantum dots**

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

electronic properties compared to the bulk materials.

been synthesized by the solution growth method.

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

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

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.

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
