**2. Plasmonic nanostructures in biosensing**

Biosensors are significant in a variety of scientific domains, including clinical diagnostics, medical diagnostics, illicit drug detection, food quality and safety, and *Application of Plasmonic Nanostructures in Molecular Diagnostics and Biosensor Technology… DOI: http://dx.doi.org/10.5772/intechopen.108319*

**Figure 1.** *General schematic for biosensor as adopted from literature [19].*

environmental evaluation [17]. Biosensor is an analytical device, which consists of two basic components: the recognition unit employed to capture the specific target and the transducer that converts the biomolecule interaction into an electrical, chemical, or optical signal. The types of biosensor depend on the types of output signal measured and quantified in real-time [18]. As depicted in **Figure 1**, the identification of stimulus is released after the interaction of the sensing surface with the analyte and converts it into a detectable signal.

### **2.1 Types of biosensors**

Biosensors can be classified according to the output as shown in **Figure 2**. In addition, all these types share a common working principle. Furthermore, electrochemical biosensors are sub-classified into impedimetric, voltammetric, potentiometric, and amperometric. Among these, bio-sensing types, electrochemical sensor is considered as conventional sensor and has been under an extensive search for years. It has attracted interest in several fields such as health, food, and agriculture [3]. However, plasmonic biosensing possesses many advantages over traditional (electrochemical) sensors due to their (i) label-free detection, (ii) real-time, (iii) short response time, and (iv) simple sample preparation.

### **2.2 Plasmonic bio-sensing mechanism and sensing principle**

### *2.2.1 Surface plasmon resonance (SPR)*

Surface plasmon resonance (SPR) biosensor (**Figure 3**) is one of the important tools for examining the kinetics of biomolecular interaction with the surface and they offer a unique real-time and label-free measurements, non-invasive nature with high detection sensitivity [21]. SPR is a frequently used optical technique for tracking changes in a sensor layer's refractive index (RI) after interaction with a target molecule [3]. SPR has been widely used in various detection of biological and chemical analytes, for environmental and agricultural monitoring [22]. It is a metal-based film

#### **Figure 2.**

*Types of biosensor based on their transducer identity [18].*

#### **Figure 3.**

*Schematic of SPR biosensor experimental set up (a) [20], adopted from literature [10] copyrights, SPR (a) and LSPR (b) sensing mechanism (c).*

sensor, made of gold (Au), which is used to characterize biomolecular interactions [23]. SPR systems are based on an optical phenomena pioneered by Wood in the 1980s, which is a gold standard method. The most successful plasmonic up to date, based on optical label-free sensing technology. Wang et al. used hepatitis B surface (HBs) antigen as the target molecule and gold nanorods (AuNRs) to realize LSPR for an HBV sensor [3]. Ever since, there has been ongoing research on developments of label-free, real-time, and ultra-sensitive SPR for the sensing of small and large biomolecules which is based on monitoring of refractive index (RI) changes in the surrounding environment (surface chip sensor) caused by biomolecular interaction around sensing area [24]. These technologies have been widely used in drug screening *Application of Plasmonic Nanostructures in Molecular Diagnostics and Biosensor Technology… DOI: http://dx.doi.org/10.5772/intechopen.108319*

and in other biomedical disciplines. Bai and co-workers presented an SPR-based biosensor for the detection of the avian influenza virus (AIV) H5N1. Employing a chosen aptamer as the recognition element [3].

**Figure 3(a)** and **(b)** depicts the SPR sensing mechanism, it can be observed as electromagnetic surface waves that are solution of Max equation. SPR occurs at the interface of the bulk materials with positive dielectric constant and of a negative dielectric constant of the metals (precious metals: Au, Ag) [25]. The electron clouds propagate horizontal (x-y) plane to hundreds of micrometers along the metal-dielectric interface and their lateral extensions and eventually decay exponentially away into both sides of the interface in z-axis [3].

In contrast to SPR, LSPR sensing mechanism (**Figure 3(c)**), the electromagnetic waves are confined and no-propagating surface plasmon at the surface of an isolated metallic nanostructure. As the curve metallic surface of nanoparticle, the interaction between electromagnetic waves applies the restoring force to oscillating electron cloud and amplifies the electromagnetic resonance (EM) field of the metal interface due to resonance. In addition, the light-matter interaction is where LSPR originates [26]. When it comes to plasmonic nanostructures, surface plasmons are restricted to a narrow area near the nanostructures and sensitive to RI changes.

The EM field enhancement can scrutinized and quantified on the metallic surface may be explained using Mie Theory (Eq. (1)), which is frequently used for electromagnetic simulations.

Mie theory;

$$\hat{\mathcal{O}} = 4\,\pi r^3 \frac{\in - \in m}{\in + 2 \in m} \tag{1}$$

where R and ∂ is a permeability and the radius of the metal nanoparticles. The EM field is sensitive to the changes of RI of the dielectric layer, which has a potential to be used as sensing layer for SPR based sensor realization. Kretschmann and Reather pioneered the creation of SPR-based sensors in 1968 by introducing the traditional prismbased structure, while Liedberg et al. reported the first experimental demonstration of exploiting the phenomenon for sensing. Plenty of companies to manufacture point-ofcars (POCs) facilities have utilized these technologies (SPR) sensor mechanism.

### *2.2.2 Localized surface plasmon resonance biosensors (LSPR)*

The bioanalytical community has been interested in localized surface plasmon resonance (LSPR) phenomenon. LSPR has many of the same benefits as traditional propagating SPR, but with a few key differences. In comparison to SPR, LSPR has the following benefits: a high aspect ratio that allows for a larger interaction surface area for immobilizing the sensing elements; a miniature probe to produce compact devices; and broad applicability and compatibility with several phenomena, including fluorescence, Raman, and IR spectroscopy. In LSPR, the interaction of the electromagnetic waves and sub-wave metal NPs gives a rise to non-propagating oscillations of the collected electron conducted cloud against metal positive core, this phenomenon is localized surface Plasmon [27]. LSPR operates with the same merits as traditional propagating SPR, however with additional crucial advantages. One of the examples, they are more suitable to microchip integration, faster response time and have much better spatial resolution [28]. Moreover, LSPR has high aspect ratio, thus allowing more interaction surface area for immobilizing the sensing elements to

obtain compact devices and wide stability, compatibility, and applicability towards phenomenon such fluorescence, and Raman and IR spectroscopy to name the few. Researchers have devoted efforts in contribution to the development of noble metalbased LSPR on a planer surface [28]. However, so far, the development of LSPR is restricted at the laboratory scale due to the fundamental limits of noble metals such as their high price and their high production cost [26].

### *2.2.3 Plasmonic nanostructures in biosensing*

Nanostructures offer a wide range of potential uses in biosensors because of their distinctive size-tuneable and shape-dependent physicochemical features. The field of optical biosensors enters a new age with the incorporation of nanostructures and useful biological molecules (such as antibodies, nucleic acids, and peptides) [22]. Loideau et al. have reported the Ag and Ag@Au NPs based LSPR for application in naked-eye biosensing, utilizing color change from cyan to green [28]. In the last two decades, Plasmon resonance in gold nanoparticles (Au NPs) has been the subject of intense research efforts. However, the inflated cost for precious metal such as Ag and Au has been a great concern.

Recently, due to the inflated cost, production cost of noble metals, researchers have paid a considerable attention to the development of non-noble metal and semiconductor materials. As potential replacements for plasmonic noble metals, low-cost and resource-rich non-noble metal plasmonic materials have drawn significant interest [29]. The commonly used non-noble plasmonic material reports are copper-based, Aluminum, semiconductor, and graphene-based LSPR. Non-noble metal like bismuth (Bi) have similar plasmonic properties as precious metal. Chen et al. have reported the synthesis of non-noble (Bi/BiVO4) as photoanode sensing materials for application in detection of H2O2 [30]. Zhu et al. reported sponge-like surface-enhanced scattering (SERS) substrate in which reduced graphene oxide used to wrap the Ag nanotube for detection of dithiocarbomates pesticide [31]. Among the support surface for LSPR, glass substrates have been the most popular and been attracted considerable attention for LSPR sensor platform. The LSRP sensing mechanism has been recently been utilized in sensing the eloba virus by Tsang et al. and the nanoparticle have been able to sense the virus at lowest LOD level. While and Li et al. reported the detection of SARS-COV-2 (COVID-19) [29].

### *2.2.4 Bottom-up fabrication methods developments of plasmonic biosensors*

Among the fabrication approaches for plasmon biosensors, bottom-up approach has attracted prodigious interest due to control over the structure, shape, and size as compared to top-down approach. Top-down approach uses lithographic etching, which is associated with undesirable structures. The unique and extremely sensitive nanostructures of SPR and LSPR properties, together with generality of fabrication method used obviate the undesirable optical and structural effect associated lithographical prepared nanostructures for sensing applications. The size and shape of the nanostructures are of great interest, due to their huge influences in fine-tuning the sensitivity due to an enhanced interaction surface area and electric properties compared to the bulk material counterparts. A novel way to get around the constraints of a traditional SPR biosensor's detection limit, sensitivity, selectivity, and throughput provided by recent developments in nanofabrication techniques and nanoparticle syntheses.

## *Application of Plasmonic Nanostructures in Molecular Diagnostics and Biosensor Technology… DOI: http://dx.doi.org/10.5772/intechopen.108319*

Before every step in plasmonic nanostructures developments is the fabrication approach. The fabrication method influences the physical and chemical identity/ nature of plasmonic materials, which is the size, and structural morphology to name few. These properties turn to determine the plasmonic biosensor activity. Nanotechnology and nanostructures have shown a great interest in contributing towards the nanoplasmonic materials, which entails biosensing and biological application, through manipulating the sensitivity [32]. Nanostructures as the driving force of nanotechnology, hold the futuristic technological developments of portable devices, and drive the next technology generation such as the fourth industrial revolution (4IR) and machine learning [33, 34].

The recent progress in the fabrication of advanced smart nanostructures find a widely application in the environmental and biological disciplines. In addition, the effort to fabricate nanoplasmonic biosensors based on LSPR mostly noble metal (Au or Ag), to reduce cost, expensive equipment, and these materials exhibited unmatched characteristic features that can be utilized [35]. In this regard, the optical characteristics of metallic nanostructures such as Au and Ag nanoparticles (NPs) have been developed and utilized to create simple, fast-responding, and low-cost nanodiagnostic and nanotherapeutic smart systems due to their chemical stability and biocompatibility [36]. In fact, due to their intense interactions with light, AuNPs and AgNPs are specifically studied for their optical features [37].

Masterson et al. reported the bottom-up synthesis of AuNPs, AuNRs [38]. There are many methods used to fabricate plasmon nanostructures with various morphologies, that is, nanoplates, nanorods, nanosphere, nanoarrays, etc. [39] as shown in **Table 1**. Kim and co-workers reported the nanoarrays using electron beam lithography, for detection of avian DNA-Influenza utilizing the SPR [45]. In 2018, Liu et al. reported the Au nanoplates using the hydrothermal synthesis for the sensing of, [46].

## *2.2.5 The limitation of plasmonic biosensors*

The inability to consistently detect minor changes in refractive index brought on by substances in low concentration at the sensor surface is one of the primary issues preventing the further development of SPR applications. The expense of noble metals used in plasmonic biosensors. In addition, there are still many obstacles to overcome, both now and in the future, despite the recent boom in the development of nanomaterial-based plastic sensors for POC facilities applications. Among the technology bottleneck, researcher mostly discuss the plasmonic biosensors that are urgent which limits the development of biosensors. The first one is the Covid-19 global pandemic,


#### **Table 1.**

*Summarized list of methods for the synthesis of various nanostructures morphology and their applications.*

which requires rapid, POC diagnostic facility to urgent identify contraction of the (SARS-COv-2) virus. Another challenge I to quickly develop the accurate, reliable plasmonic biosensor that is flexible in realization of 4IR and machine-learning tool that can easily use to predict the sensing properties of the nanoparticles [44].
