*5.6. Ion-Selective Field-Effect Transistors (ISFET) Based Biosensors*

The last few years saw a significant change in ion-sensitive field-effect transistor (ISFET)-based devices, a principle initially proposed in the 1970s by Bergveld et al. [132]. This revolutionized the technology at a nanoscale level. These types of prototypes formed using silicon nanowire FETs (SiNWFETs) have been used for a wide range of applications, including pH sensing [133–135], chemical [136–139], and label-free biosensing [133,140–143] applications. The downscaling of these devices has been done by determining the kinetic studies on receptor binding [144] and intracellular recordings of action potentials [145]. The working mechanism is based on the adsorption of charged species on the sensing surface, causing variation in the surface potential and, thus, the current in the FET channel. These devices provide an additional attribute over the conventional ion-selective electrodes by transforming the high-impedance input signal into a low-impedance output signal. The probability of downscaling the dimension and conjugation them with conditioning circuits to detect multifunctional parameters [146] highlights the ability of SiNWFETs to operate as low-cost, efficient, and robust devices.

In one of the examples, the selectivity of the sensing surface is induced to deduce species other than protons. This was carried out to achieve high sensitivity by absorbing the target analyte. The covalent bonding of the linked molecules to perform chemical anchoring has been more than a viable method. The linker binding sites of the selfassembled monolayers (SAMs) are situated in the proximity of the FET surface at a higher density. The topic of SAMs has extensively been studied [49]. In ISFET systems, a technique related to self-assembly of silane monolayers has been used to alter the surfaces of the oxides [133,136,147].

Ion-sensitive field-effect transistors that use silicon nanowires have high dielectric constant gate oxide layers formed with Al2O3 or H2O2. These devices exhibit responses that are sensitive to pH variations and ions present in the electrolyte due to the presence of hydroxyl groups. The complexity of the oxide surface due to its intrinsic non-selective nature makes it challenging to sense ionic species other than protons. The modification of the individual nanowires has been done with thin gold films to increase the specificity via functionalization. It has also been shown that the sodium ion (Na+) detection using SAM of thiol-modified crown ethers has been done, where a response of ≈−44 mV per decade was achieved for the sodium ions in a NaCl solution. The testing process was carried out in the presence of various ions like protons (H+), potassium (K+), and chloride (Cl−) ions, where the voltage difference between the gold-coated nanowire functionalized by the SAM (active) and a gold-coated nanowire was determined. It was seen that the functional SAM was unable to obtain any output from the bare gold-coated nanowire concerning pH and background ionic species. This response increases the credibility of gold in comparison to oxide surfaces when the devices are used for differential measurements.

### **6. Signal Amplification Strategy**

One of the critical capabilities of a biosensor lies in its enhanced performance in terms of the morphological, structural, and electrochemical characteristics of the considered nanostructured material. In normal terms, the sensors are characterized using X-ray diffraction, confocal microscopy, transmission electron microscopy, and voltammetric techniques. The hybrid prototypes used for biosensing applications constitute combined attributes that have generated high sensitivity, selectivity, and rapid and stable response during the detection water pollutants. The synergy observed between the processed materials improved the electrochemical activity, stability of the immobilization of bioreceptor, electron transfer rate and surface area of the electrodes, thus obtaining a high magnitude of the peak current during the detection of different analytes as a typical signal amplification strategy.

The use of metallic nanoparticles with a large electroactive surface area has been employed for electrochemical biosensing applications due to their high electrical conductivity, catalytic properties, fast electron transfer, biocompatible nature, and easy incorporation with different kinds of nanomaterials. Zeinab et al., showcased the use of an ultrasensitive electrochemical aptasensor for quantitative detection of bisphenol A (B.P.A.) via signal amplification strategy

The use of gold-platinum nanoparticles (Au-PtNPs) was carried out by electrodepositing them on acid-oxidized carbon nanotubes (CNTs-COOH)-modified glassy carbon (GC) electrodes. Then, acriflavine was immobilized by covalent bonds at the surface to capture BPA-aptamer by forming phosphoramidite bonds. The aptamer's conformational change occurred once the B.P.A. interacted with the aptamer. Thus, the retardation was carried out for the interfacial electron transfer of acriflavine as a probe. The LOD for this technique was calculated to be as low as 0.035 pM, which resulted from high-density Au-PtNPs and superior electron transfer of acriflavine. The resulting aptasensor also exhibited reasonable specificity, stability, and reproducibility.

Recently, the application of conducting polymer-based materials were made in biosensors for two particular areas, including enhancing the affinity of these sensors as backbone or side groups and using them as immobilization matrices for the bioreceptors with high electrical conductivity and fast electron transfer [44]. Disposable nitrate biosensors were used as nanoarrays to detect nitrate reductase as a target analyze. Bio-recognition element was immobilized within a poly (3, 4-ethylenedioxythiophene) (PEDOT) matrix to produce a quantifiable amperometric response. Superior analytical performance and quick fabrication process, and easy operating principle were obtained with this PEDOT/nitrate reductase nanowire sensor [148].

Dendrimers are synthetic three-dimensional macromolecule polymers with welldefined, highly branched, globular-shaped molecular structures [149]. Poly (propylene imine) dendrimer PPI has been popularized for biosensing applications due to its high biocompatibility and compatibility with host–guest chemistry. Due to the disastrous effects of cholera infection resulting in watery diarrhoea with severe dehydration and death, Tshikalaha et al., developed biosensors to detect cholera toxins in the water. The sensors are operated by co-electrodepositing PPI dendrimer and AuNPs on glassy carbon electrodes. The probe of the anti-cholera toxin was dropped on GCE/PPI/AuNPs and finally blocked with B.S.A. to reduce nonspecific binding[150]. A LOD of 7.2 × <sup>10</sup>−<sup>13</sup> and 4.2 × <sup>10</sup>−<sup>13</sup> g mL−<sup>1</sup> were obtained from SWV and EIS analysis.

Another significant strength in improving electrochemical signals is the core-shell or core-satellite nanostructures. These nanomaterials can be loaded to the core nanoparticle via surface functionalization in chemical ways. For example, heterogeneous magnetic nanoparticles [151] and Mesoporous silica [152] have attracted increasing attention due to their easy magnetic separation with labelled bio-receptors and easy encapsulation of enormous materials in their structural pores, respectively. Ultrasensitive electrochemical biosensors used to detect Ag+ ions were constructed using magnetic Fe3O4@gold core–shell nanoparticles (Fe3O4@Au NPs) for labelling with H.C.R. product and enrichment on the surface of the magnetic gold electrodes [153]. The prototypes showed high selectivity due to their duplex-like DNA. scaffolds structure with specific C–Ag+–C base pairing. They also had attributes like high sensitivity, low LOD of 0.5 fM and a wide dynamic range of 1 fM– 100 pM. Marcos et al. [154] reported a new hybrid nanomaterial platform that included MWCNT and haemoglobin-functionalized mesoporous silica particles with highly sensitive quantification of nitrite and trichloroacetic acid as processed materials. The efficient electron transfer between haemoglobin and the electrode surface can be attributed to certain factors, including the high surface area and protein loading capacity of the mesoporous silica nanoparticles as well as the increased surface area and catalytic properties of MWCNTs.

In regards to the porous materials, metal–organic frameworks (MOFs) are another interesting class of porous crystalline inorganic–organic hybrid materials, as Fe(III)-based MOF (Fe-MOF) was reported [155] to have an excellent stability and redox activity when used as the prime electrode materials [156]. As is shown in Figure 4, a core–shell nanostructured Fe(III)-based metal–organic framework (Fe-MOF) and mesoporous Fe3O4@C nanocapsules (denoted as Fe-MOF@mFe3O4@mC)-based aptasensor was constructed [155]. The EIS was used for detecting the responses, where an advantage of the conformational transition interaction took place. This phenomenon was caused due to two factors: the formation of the G-quadruplex between a single-stranded aptamer and a highly heavy metal ion of Fe-MOF. The proposed aptasensor showcased a decent linear relationship with the logarithm of heavy metals and a low LOD of 2.27 and 6.73 pM toward the detection of metallic ions of Pb2+ and As3+, respectively.

**Figure 4.** Schematic diagram of the preparation process of nanocomposite and its related aptasensor for detecting Pb2+ and As3+ via electrochemical techniques, including (**i**) the preparation of the nanocomposite, (**ii**) the immobilization, and (**iii**) the determination of the heavy metal ions, (reproduced with the permission from [155]).

#### **7. In Situ Monitoring System**

The recent development of various biosensors recommends excellent potential for monitoring water quality in the other treatment water recourses due to their simple, compact design, dispensability, and cheapness. In situ can be considered as online monitoring and offline or portable monitoring. Online monitoring defines as real-time in situ measurements of any sampling for analysis and provides on-field sampling data compared to conventional methods. It is incredibly challenging to monitor water contaminants, primarily chemical pollutants, in online monitoring. It is a more flexible approach and can be conducted from remote locations. An online monitoring system can be constructed utilizing a wireless sensor network (WSN) or an Internet of Things (IoT)-enabled network [25,157,158]. Simultaneous data collections, higher detection, easy monitoring, and sufficient data are the significant advantages of constructing the WSN network for monitoring purposes. Low power consumption and energy harvesting options are essential for developing an online monitoring network.

Pasternak et al., reported [136] a biological oxygen demand (BOD) biosensor, which was self-powered and autonomous for water quality measurement. The energy harvesting system, data logger, and sensing unit were developed continuously to monitor the sample in water (Figure 5). This biosensor can detect urine contamination in water, and the system can run autonomously for five months.

**Figure 5.** Schematic representation of the sensor's proposed biosensor and operating principle. The block diagram of the system shows the energy harvester charging/discharging repeatedly (reproduced with the permission of [159]).

Quek et al. [160] reported an assimilable organic carbon (AOC) based amperometric biosensor for detecting marine microbial fuel cell (MFC) in marine water, where the system was tested for 36 days. The response time, the reproducibility of the signal, and recovery time were good, which are essential for developing an online monitoring system. Bio-recognition elements play a crucial role in developing robust biosensors, which could be helpful for online monitoring. Among the many bio-recognition features, the enzymatic biosensors method is used widely for electrochemical detection, as they have high sensitivity for distinguishing the target elements from interference elements [161]. However, they are a costly method, have an increased duration of immobilization procedure, and have poor durability and stability, which is ascribed to the loss of enzyme activity during the onsite monitoring [82]. Therefore, MFC biosensors are widely used for various target analytes with an extensive range of cells [162]. They have mainly been installed to monitor water quality, but very few commercial prototypes are available for monitoring water toxicity. They can survive under harsh conditions, such as high and low pH, unusual temperature, and salinity [161].

Figure 6 has shown the portable electrochemical EIS based system for monitoring samples from water. Figure 7 shows the schematic block diagram of a standard electrochemical biosensor monitoring system. The sensing parameters would be capacitance, impedance, current, or voltage based on the characteristics of the electrochemical biosensor. The impedance analyzer relates to the sensor to collect the sensor data. It also provides sufficient energy to the sensor. The microcontroller unit manages all the sensing data, sends the data to the cloud server through the base station or the internet and manages the operating condition of the complete sensing system. The energy harvesting unit connects with the power management unit to supply continuous energy to the sensing unit. The microcontroller unit also connects with the wireless communication module, another crucial module for developing an online monitoring system. Different wireless communication modules are available, such as Bluetooth low energy, low power wide area network (LPWAN) wireless modules, SigFox modules, WiFi modules, and ZigBee modules. The modules are characterized based on their bandwidth, data transmission capability, power consumption, and communication range. The communication module solely depends on the installation duration of the network, the number of sensing systems, the content of coverage regions of the networks, and the application.

**Figure 6.** (**a**) Design and fabrication of electrochemical-cell-chips development; (**b**) electrochemical impedance portable platform for EIS measurements; and (**c**) complete portable system for automatic detection (reproduced with the permission of [94]).

**Figure 7.** Schematic diagram of an online monitoring system.
