**2.1 Amperometric electrochemical biosensor**

The design of the electrodes and chip is a crucial aspect in the development of an amperometric electrochemical biosensor. In general, designing electrochemical biosensor electrodes necessitate a thorough understanding of fluid flow, particularly its behavior in microscales. Furthermore, it also requires a detailed understanding of mass transport phenomena and microflow mass transport foundations.

Biosensors have been improved using a variety of designs. The design of electrodes in a microfluidic system is a crucial pillar for improved performance. Electrode design necessitates a thorough understanding of electron diffusion phenomena. In most current flows, electron diffusion is the limiting step. Diffusion is generally hampered by crucial parameters such as the electrode surface and the number of active sites available for the target. When a device with a microchannel is utilized as an analytical platform, the analyte is injected into the channel using two alternative methods namely pressure-driven flow and electrokinetic flow [38]. A pressure gradient induces flow in pressure-driven flow, and the nature of the flow is influenced by the channel geometry and flow rates. The Reynolds number is commonly used to express the ratio of inertial and viscous forces:

$$R\_e = \frac{\rho \text{VD}}{\mu}$$

where Re is the Reynolds number, V is the characteristic velocity for the flow, D is the characteristic distance, ρ is the density of the fluid, and µ is the fluid viscosity. Laminar flow occurs at Reynolds numbers below 2300 [38]. Flow in the microchannel is laminar because of the micron-scale size of microchannels, and the low velocity requires fluids to move across the channels. For laminar flow, fluid travels in a steady and time-independent manner at each location. The flow profile is parabolic, in which the velocity of the flow is negligible at the wall surfaces.

Materials used in the design of amperometric electrochemical biosensors are classified as: (1) materials for the electrode and supporting substrate; (2) materials for the immobilization of biological recognition elements; (3) materials for the fabrication of the outer membrane; and (4) biological elements, such as enzymes, antibodies, antigens, mediators, and cofactors.

Solid electrode systems and supporting substrates are frequently constructed with metals and carbon. Due to their superior electrical and mechanical qualities, metals such as platinum, gold, silver, and stainless steel have long been employed as electrochemical electrodes [39]. **Figure 7** depicts the various techniques used for the production of conductive supporting substrates**.**

The basic elements of biosensors are the bioelement and the sensing element. Any organic organism that can detect specific analytes from the medium of interest while remaining unresponsive to any other potentially inquisitive/interfering species is referred to as a bioelement. The signal transducing section of the biosensor is known as the sensing element, and it can take the shape of any magnetic, optical, electrical, or electrochemical transducing mechanism [40].

*Recent Advances in Biosensing in Tissue Engineering and Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.104922*

**Figure 7.**

*A schematic diagram of the techniques used for the production of the conductive supporting substrate.*

#### **2.2 Design and fabrication of a surface plasmon resonance (SPR) biosensor**

A surface plasmon resonance biosensor can be designed and manufactured using a variety of periodic structural patterns. One of the structural patterns employed in the design and fabrication of a surface plasmon resonance biosensor is the nanohole creation procedure using thermal nanoimprint lithography which is discussed in this book chapter.

The stamps for the nanohole array are made by thermal nanoimprint lithography, residual layer etching, (titanium/gold) Ti/Au deposition, and lift-off procedures. For example, if a 10-cm thick glass wafer is utilized for the imprinting process and is coated with a 100-nm thermoplastic polymer layer, it has to be spincoated at 3000 rpm for 30s to obtain this layer. A hot embossing system can be used to perform nanoimprint lithography. The leftover layer is then etched with oxygen (O2) plasma [41].

The polymer is etched uniformly in this procedure until the residual layer is completely removed and the pattern is transferred to the substrate. A metallic titanium (Ti) (adhesion layer, 5 nm)/gold (Au) (50 nm) layer is deposited using electron beam evaporation. Finally, the resist lift-off operation is carried out in an ultrasonic hot acetone bath to obtain the nanohole array structure [42].

#### *2.2.1 Design and fabrication considerations for biosensors*

Studying the target analyte and identifying how it reacts with biological molecules is the first phase in constructing a biosensing device.

Other phases include are as follows:

#### *2.2.1.1 Biological receptor selection*

The sensitivity and selectivity of a biosensor to the analyte of interest are decided by the biological receptor used. As a result, a receptor with a high affinity for the analyte is suggested. It is critical to understand the benefits and drawbacks of various biological receptors in diverse biosensor applications when selecting an appropriate receptor [41, 43, 44].
