*2.1.1 Sensing mechanism*

*Dyes and Pigments - Novel Applications and Waste Treatment*

*Scheme of typical (a) label-free and (b) label-based biosensors.*

period of the biosensor history, typical biosensing processes were usually realized by measuring the transduced mechanical, electrical, or optical signals without any labels, as illustrated in **Figure 1a**. Such label-free biosensors can provide direct information without complicated sample preparation steps. In contrast, label-based biosensors utilizing additional operation processes for higher signal-to-noise ratios and for a broader range of sensing/transducer systems. Conventional biosensing labels are optical molecules or radioactive elements borrowed from bio-analytical systems such as gel electrophoresis and enzyme linked immunosorbent assays. A typical strategy of sandwich assay for antibody-based detection is shown in

Due to the fast development of nanotechnology and material science, nanomaterials are widely adopted as biosensing labels, many of which are dyes in senses of optics or electrochemistry. The special size of these nanomaterial dyes provides unique properties that greatly improve the performances of relevant biosensors. In this chapter, we focus on dyes used as biosensing labels and discuss their properties,

Colorimetry technique is a practical and direct analytical method to determine the concentration of colored analyte depending on the color change in solution. The colorimetric biosensing strategy based on this principle has become one of the most popular and important strategies due to its simplicity, visualization, low cost and non-destruction. Based on the strategies of signal generation, colorimetric assays can mainly be divided into two groups, i.e., assays based on enzymes for chromogenic reactions, and assays based on colored labels. Herein, we focus on the later

As a successful example to employ nanomaterial and nanotechnology to solve

the biological problems, colloidal gold has been introduced in the biosensing fields more than 20 years with lots of amazing works being reported. Colorimetric biosensors based on gold nanoparticles (GNPs) have greatly developed in both

applications and how they improve the biosensing properties.

**2. Dyes in colorimetric biosensors**

group in which dyes play a crucial role.

scientific study and commercial applications.

**2.1 Gold nanoparticles**

**36**

**Figure 1b**.

**Figure 1.**

**Surface plasmon resonance.** Various GNP-based colorimetric biosensors were built depending on the same principle, i.e., surface plasmon resonance (SPR). SPR is a prominent spectroscopic feature that results in an intense and sharp absorption band in the visible range of noble metal nanoparticles that have an adequate density of free electrons. Localized SPR is an effect that the electron cloud of the nanoparticle sense and start to oscillate at the same frequency as the electromagnetic field of the incident light (**Figure 2a**). During this process the incident light is scattered and converted into heat, both leading to the intensity attenuation of the incident light. Meanwhile, localized SPR produce an electric field on the nanomaterial surface, which can be utilized for labeling several kinds of biosensors. The SPR-induced color is determined by several factors including the size, shape, modified ligands and aggregation state. In colorimetric biosensors, the aggregation state of GNPs is interested. As shown in **Figure 2b**, in bioanalytical assays, the combination of bio-receptors (or targets) labeled with GNPs can induce the isolated GNP assembly as well as the interparticle coupling of the surface plasmon, resulting in the color of solution transferring from red to blue.

**Surface-enhanced Raman spectroscopy.** Based on the SPR effect, GNPs have also been used in other types of biosensors, e.g., surface-enhanced Raman spectroscopy (SERS) based sensing. SERS as a versatile finger-print vibrational technology has been widely utilized in analytical chemistry, [8] electrochemistry, [9] and media diagnostics. [10] However, the mechanism of SERS is still

#### **Figure 2.**

*(a) Localized SPR band formation. Red and gray circles represent negative and positive electron clouds, respectively. (b) GNP agglomeration leads to the shift of SPR absorption band.*

under debating [11]. Regarding to the mechanism of SERS, there are two broadly accepted opinions: electromagnetic (EM) theory and chemical enhancement (CE) mechanism [12]. The EM enhancement considers the molecules as point dipoles, which react with the local enhanced electric field on or near the surface [13]. The CE mechanism attributes the SERS intensity to molecular resonance through the interaction with the metal surface, thereby changing the molecular polarizability, resulting in enhancement such as resonance Raman scattering [14]. SERS does not occur on any metal surfaces, among which GNPs are currently the most widely used SERS substrate. SERS-based biosensors involving GNPs have been widely used in the sensitive and selective detection of antigens, [15] aptamers, [16] tumor biomarkers, [17] as well as *Staphylococcus aureus* [18].

**Lateral flow system.** The most well known biosensing application of GNPs must be the pregnancy test sticks, which belong to the most commercialized type of biosensor, i.e., the lateral flow (LF) system. LF biosensors are paper-based devices permit low-cost and rapid diagnostics with moderate robustness, specificity and sensitivity. The ease-of-synthesize, stability, biocompatibility and tunable size make GNPs suitable label for LF systems. Moreover, the SPR effect endows GNPs an intense red color that can be either detected qualitatively by naked eye or measured quantitatively using spectrometers for lower detection limits. Because of these properties, GNPs are the most widely used optical label in LF systems [19]. A standard and conventional LF strip consists of four main sections made of membranes, papers or glass fibers, including a sample pad for sample loading, a conjugate pad impregnated with bioreceptor-modified labels (usually GNPs), a detection pad/membrane where test line (to show whether the target is exist in the sample) and control line (to show whether the LF assay works well) are printed, and an absorption pad at the end of the strip, as illustrated in **Figure 3**. Except for GNPs, other colored materials are also used in LF biosensors, including carbon dots and latex particles.

#### *2.1.2 Labeling type*

Three principle methods for modifying GNPs are briefly illustrated in **Figure 4**: (1) A simple and common way to immobilize the biomolecules on the GNPs is labeling through the sulfur-gold interactions; [20] (2) Ligands can be capped on the GNPs in the process of particle growth or though ligands replace after the synthesis; (3) Ligands can be absorbed on the GNPs surface directly via non-covalent interactions.

In 1997, the first GNP-based colorimetric biosensor was reported [21]. In this work, the hybridization of DNA probes capped on GNP surface and target DNA

**39**

strategies [25].

**Figure 4.**

**2.3 Latex particles**

**2.2 Carbon nanoparticles**

*Dyes as Labels in Biosensing*

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

*Illustration of different GNP conjugation methods.*

resulted in the formation of GNP agglomeration, accompanying a visually redto-purple color change. This is the most typical biosensor that uses biochemical reactions to induce the cross-linked aggregation of GNPs. Since then, a new generation of medical diagnostic technology based on the nanomaterials has begun. Oligonucleotides, peptides, antibodies and aptamers have been labeled with GNPs for the colorimetric detection of different targets based on the similar principle. Such strategy designs usually achieve naomolar detection limits, which is limited by the signal-to-noise ratio related to the intrinsic properties of labels and sensors [22]. To amplify the signals and optimize the performance of biosensor, the biochemical and molecular amplification methods are introduced in the biosensing process. Via duplex-specific nuclease-assisted amplification method, a colorimetric method was developed for microRNA dection based on GNPs aggregation [23]. DNAzymeassisted target recycling was utilized, combined with surface plasmons of GNPs coupling in the colorimetric biosensor, obtaining a fast and simple detection of genetic targets with 50 pM sensitivity [24]. Using imaging-based analysis instead of spectrographic analysis has higher signal-to-noise ratio and thus potentially lower detection limit. A dark-field microscope based methodology was applied for the sensing of GNP aggregation, obtaining a detection limit of 43 aM of DNA, which was 5–9 orders of magnitude lower than conventional colorimetric sensor based

Carbon nanoparticles, also named colloidal carbon, can be visually detected in a qualitative or semi-quantitative manner and thus being used as colored labels. Compared to GNPs, carbon nanoparticles have several excellent properties, e.g., high stability, nontoxicity, ease-of-preparation, and ease-of-modification [26]. The dark black color of carbon dots endows a high signal-to-noise ratio, allowing sensitivities below the low picomolar range even by visual inspection [27].

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.

**Figure 3.** *Schematic of a typical lateral-flow immunological detection system.*

*Dyes and Pigments - Novel Applications and Waste Treatment*

biomarkers, [17] as well as *Staphylococcus aureus* [18].

under debating [11]. Regarding to the mechanism of SERS, there are two broadly accepted opinions: electromagnetic (EM) theory and chemical enhancement (CE) mechanism [12]. The EM enhancement considers the molecules as point dipoles, which react with the local enhanced electric field on or near the surface [13]. The CE mechanism attributes the SERS intensity to molecular resonance through the interaction with the metal surface, thereby changing the molecular polarizability, resulting in enhancement such as resonance Raman scattering [14]. SERS does not occur on any metal surfaces, among which GNPs are currently the most widely used SERS substrate. SERS-based biosensors involving GNPs have been widely used in the sensitive and selective detection of antigens, [15] aptamers, [16] tumor

**Lateral flow system.** The most well known biosensing application of GNPs must be the pregnancy test sticks, which belong to the most commercialized type of biosensor, i.e., the lateral flow (LF) system. LF biosensors are paper-based devices permit low-cost and rapid diagnostics with moderate robustness, specificity and sensitivity. The ease-of-synthesize, stability, biocompatibility and tunable size make GNPs suitable label for LF systems. Moreover, the SPR effect endows GNPs an intense red color that can be either detected qualitatively by naked eye or measured quantitatively using spectrometers for lower detection limits. Because of these properties, GNPs are the most widely used optical label in LF systems [19]. A standard and conventional LF strip consists of four main sections made of membranes, papers or glass fibers, including a sample pad for sample loading, a conjugate pad impregnated with bioreceptor-modified labels (usually GNPs), a detection pad/membrane where test line (to show whether the target is exist in the sample) and control line (to show whether the LF assay works well) are printed, and an absorption pad at the end of the strip, as illustrated in **Figure 3**. Except for GNPs, other colored materials are also used in LF biosensors, including carbon dots

Three principle methods for modifying GNPs are briefly illustrated in **Figure 4**:

In 1997, the first GNP-based colorimetric biosensor was reported [21]. In this work, the hybridization of DNA probes capped on GNP surface and target DNA

(1) A simple and common way to immobilize the biomolecules on the GNPs is labeling through the sulfur-gold interactions; [20] (2) Ligands can be capped on the GNPs in the process of particle growth or though ligands replace after the synthesis; (3) Ligands can be absorbed on the GNPs surface directly via non-covalent

**38**

**Figure 3.**

*Schematic of a typical lateral-flow immunological detection system.*

and latex particles.

*2.1.2 Labeling type*

interactions.

**Figure 4.** *Illustration of different GNP conjugation methods.*

resulted in the formation of GNP agglomeration, accompanying a visually redto-purple color change. This is the most typical biosensor that uses biochemical reactions to induce the cross-linked aggregation of GNPs. Since then, a new generation of medical diagnostic technology based on the nanomaterials has begun. Oligonucleotides, peptides, antibodies and aptamers have been labeled with GNPs for the colorimetric detection of different targets based on the similar principle. Such strategy designs usually achieve naomolar detection limits, which is limited by the signal-to-noise ratio related to the intrinsic properties of labels and sensors [22]. To amplify the signals and optimize the performance of biosensor, the biochemical and molecular amplification methods are introduced in the biosensing process. Via duplex-specific nuclease-assisted amplification method, a colorimetric method was developed for microRNA dection based on GNPs aggregation [23]. DNAzymeassisted target recycling was utilized, combined with surface plasmons of GNPs coupling in the colorimetric biosensor, obtaining a fast and simple detection of genetic targets with 50 pM sensitivity [24]. Using imaging-based analysis instead of spectrographic analysis has higher signal-to-noise ratio and thus potentially lower detection limit. A dark-field microscope based methodology was applied for the sensing of GNP aggregation, obtaining a detection limit of 43 aM of DNA, which was 5–9 orders of magnitude lower than conventional colorimetric sensor based strategies [25].
