Electrochemical biosensors are advantageous analytical devices or tools that convert a bio-signal by biorecognition elements into a significant electrical response. Biosensors have many advantages over the conventional lab-based method, including low costs, portability, fast response time, less usage of reagents, and the capability to continuous monitor the complex wastewater. Such sensors significantly benefit from sensing the minimum level in polluted water, such as wastewater.
Water is an essential part of all the living beings on earth, but in recent times, anthropogenic activities have increased immensely, which are the major causes of water pollution, disturbing the marine biodiversity and leading to a tremendous water shortage 
. Even though the chemicals and water nutrients are crucial to our day-to-day lives, the excessive amount threatens humans, aquatic life, and animals. The pollution of water and habitat degradation are the causes of the escalating water shortage and the reasons for the deterioration in marine biodiversity. Although freshwater accessibility has deteriorated over the past decades, water demand has risen, particularly in warm areas with minimal rainfall. Recently, 71% of the world’s inhabitants, equal to 4.3 billion, were dealing with water shortages for several months 
. Although water demand sharply increased, massive water pollution increased water scarcity and declining water quality in the past decades.
The characteristics of water pollution are comprised of their physical presence, chemical parameters, and richness of microorganisms. The concentration and composition of ingredients in water differ extensively. They can be categorized into four distinct classifications, such as (i) inorganic chemicals, (ii) nutrients, (iii) microorganisms’ pollution, and (iv) organic pollutants. They can bring about harmful ecological consequences, for example, the interference of internal secretion and hormone systems, stimulation of genotoxicity and cytotoxicity, and hazardous effects 
. The strength of ingredients in water is essential for selecting, designing, and operational treatment processes and recycling waste. The variable quantity of contaminants in effluent over time also increases the attention to emerging technologies for monitoring the water and applying reasonably priced and real-time approaches 
. This review is mainly focused on monitoring heavy metals, nutrients, organic pollutants, biochemical oxygen demand, and microorganisms. Heavy metals in soil and water are considered environmental contaminants with elevated toxicity, easy accretion, and complicated degradation 
. Nutrients bring about water eutrophication. Organic pollutants, particularly persistent organic pollutants (POPs), have harshly harmful impacts on human health and the environment with their complex degradation and potential bioaccumulation 
. The biochemical oxygen demand (BOD) is the essential supervisory index to measure organic water contamination and demonstrate water quality 
. Water quality monitoring is critical and closely related to our life and production.
Conventional analytical techniques or laboratory-based procedures, such as gas chromatography (GC), high-performance liquid chromatography (HPLC), atomic absorption spectroscopy (AAS), atomic fluorescence spectrometry (AFS), and inductively coupled plasma mass spectrometry (ICP-MS), are sensitive, precise, and consistent. They are regularly used to measure water parameters with the help of trained operators. However, they are involved with bulky and costly instrumentation, take much time for sample preparation, and are unsuitable for in situ measurements, especially requiring trained operators’ help and transporting the water samples to laboratories for assessment 
. Additionally, they cannot asses the accumulative toxicity or nutrient value of multiple chemicals or pollutants in a sample, which is a crucial objective of water quality monitoring applications 
. Many property indicators are regularly used to determine the different qualities of water for settling or recycling. Many of them are laboratory-based techniques, which require complex pretreatment, and consequently, the methods are sluggish and expensive 
. These characteristics encourage developing new technologies that are more low-cost, portable, sensitive, and efficient in the on-site real-time detection of multi-contaminants containing a wide variety of materials 
. The significant challenges of developing a portable biosensing device are inadequate sensitivity and poor selectivity during the on-site detection. The significant level of noises can come on chemical components level from the sampling field and ambient environments can be variable due to the harsh environments or diurnal variations. These are the major obstacles where the researchers are putting lot of attentions on how to avoid these for generating a reliable and portable biosensing output signal. The portable biosensing method is successfully utilized for other applications, such as pesticide residues in fruits and vegetables 
, POC Detection for biomedical application 
, chemical and biological pollutants in water 
In recent years, the advancement of electrochemical biosensors for detecting environmental pollutants has received considerable attention 
. Biosensors have many advantages over the conventional lab-based method, including low costs, portability, fast response time, less usage of reagents, and the capability to continuous monitor the complex wastewater 
. Such sensors significantly benefit from sensing the minimum level in polluted water, such as wastewater. Biosensors are also compact and miniaturized devices that facilitate the advancement of portable sensing systems to monitor on-site effluents 
. Bearing in mind the wide range of bio-recognition elements (including enzymatic, immunochemical, non-enzymatic receptor, whole-cell and DNA elements, and molecularly imprinted polymer (MIP)), the various types of biosensors can be classified as (i) electrochemical 
, (iii) piezoelectric 
, (ii) optical 
, and (iv) thermal biosensors 
based on their working principles and transducing mechanisms 
, but the current review paper will cover the topics which are related to electrochemical biosensing. An electrochemical biosensor is based on the interactions between the immobilized bio-recognition element on its surface with binding molecules (the analyte of interest) and generating the changes in electrochemical properties, further translating into a meaningful electrical signal. The electrochemical methods offer rapid detection, fabrication, excellent sensitivity, and low cost.
Moreover, by operating at a wide range of potential, it is possible to simultaneously determine multiple analytes with different electrochemical potentials. Electrochemical biosensors’ efficiency in monitoring water pollutants’ presence relied on bio-recognition elements, transducers, and immobilization techniques, which offer us the classification criterion. In comparison with optical methods, electrochemical transduction has advantages for analyzing turbid samples because it is non-sensitive to light. For optical sensing, they are likely to be interference from environmental effects, costly, and susceptible to physical damage.
2. Electrochemical Biosensors
Electrochemistry is essential for achieving the biosensing process in various biomarker analyses. Thus, electrochemical biosensing has attracted widespread attention in various applications due to its considerable advantages. Electrochemical biosensors react with the analyte of interest or molecules to produce an electrical signal proportionate to the analyte concentration. A conventional electrochemical biosensor comprises a reference electrode and a sensing electrode (working electrode) separated by an electrolyte. In most applications, the electrochemical biosensors consist of a three-electrode system with the reference electrode connected to a potentiostat, and the circuit can be completed by adding a counter electrode for flowing the current. These sensing devices are inexpensive, low-cost electrochemical cells that can be produced, portable, and easy to use, and can be operated with reduced power consumption. It requires electronic components for detecting the target analytes, unlike optical sensors. The following sections describe a range of elements and techniques of the electrochemical biosensor for biosensing applications.
3. Surface Modification Technique
Surface chemistry plays a considerable role in electrochemical biosensors to link the biological recognition element (BRE) on top of the sensing surface and prevent the substrate electrode from nonspecific interactions. In addition, the functionalization of the surface is conducive to noise control and sensitivity enhancement. BRE used in electrochemical sensors mainly consists of enzymes, antibodies, DNA/RNA, aptamers, and whole cells 
, which define a biosensor’s sensitivity and selectivity. Immobilization techniques, such as adsorption, encapsulation in polymers or gel, chemical crosslinking, self-assembled monolayer, covalent linking, affinity, and electrodeposition, have been widely investigated for detecting various analysts in complex water samples. The surface modification of BRE on the electrodes usually involves one or more strategies.
Adsorption is a straightforward method of modifying the surface of the electrode to a specific recognition element in an entirely arbitrary way. Every biological recognition element needs to achieve the best conditions. Most proteins usually achieve the best surface coverage on uncharged surfaces under the neutral pH and functional ionic strength, using a specific 5−20 μg/mL concentration 
. Yang et al. 
have developed an impedimetric-based immunosensor by the adsorption method of anti-E. coli
antibodies against the integrated microelectrode arrays for detecting the E. coli
O157:H7. Surface modification of the surface receptor proteins G and A can be produced by many bacterial strains that can promote receptor binding. Each protein, such as A and G, can certainly be capable of binding 4 and 2 molecules of IgG. An analogous method utilizes or takes advantage of the strong binding of the glycoprotein avidin for biotin-functionalizing the receptors due to the intense alienation of the avidin/biotin complex. The detection level of E. coli
was 1.3 × 10−15
M, which was as low as 10−100 CFU mL−1
in concentration. It may be identified on the avidin-modified developed electrodes using biotinylated anti-E. coli
as the targeted recognition ligand.
3.2. Self-Assembled Monolayers (SAMs)
Self-Assembled Monolayers (SAMs) are chemisorbed and ordered with various layers formed by the natural arrangement of thiolated molecules on the location of metallic interfaces. The most extensively used methods consist of SAMs with n-alkanethiols on noble metals 
, SAMs with carboxylate on the oxide surfaces 
, and SAMs with silane on the glass/silicon surfaces 
. Xia et al., immersed the sidewall of the silica core into AuNSM colloid, forming a self-assembled AuNSM monolayer for sensitive wavelength-modulated localized surface plasmon resonance (LSPR) for detecting the mercury (II) 
. The label-free sensor obtained a very low LOD of 0.7 nM owing to the near field coupling improvement by the proximity distance of two types of gold nanoparticles-DNA conjugates.
3.3. Covalent Attachment
Covalent attachment is another approach for the covalent coupling to the ligand recognitions to electrochemical biosensor’s interfaces, and improvements from the arrays of protein help form the most favorable conditions. A commonly used crosslinking molecule is carboxylic acid (C(=O) OH) groups on the electrode’s surface as the biorecognition element with amine functional groups for exploiting the amide bond formation using the techniques of EDC/NHS chemistry. Likewise, this coupling approach has been effectively applied in various three-dimensional supports, such as agarose, aldehyde−agarose, and carboxymethylated dextran-based modified electrodes 
. Carbon-based materials that reduce graphene oxide and carbon nanotubes can be adjusted with carboxylic acid through π−π stacking interaction. Furthermore, some researchers have lately proposed integrating covalent functional groups using diazonium chemistry 
The electrochemical deposition was crucial in preparing nanomaterials reliably and cost-effectively with mild physicochemical conditions. Furthermore, noble metals, mixed metal oxides, carbon materials, or conducting polymers can be deposited on the electrode with high deposition speed, straightforward scale-up techniques and commercial feasibility with standard maintenance. This method helps form the hybrid films with the controlled thickness and morphology, modifying the process parameters, controlling the bath conditions (solvent, pH, temperature), and effectively regulating the electrolyte formula 
. For example, new properties immediately stand out when poly(3,4-ethylenedioxythiophene) associates with one or more components deposited as films 
. Table 1
shows the various characteristics of the surface modification techniques for the BRE in electrochemical biosensors.
Table 1. Surface modification techniques of BRE in electrochemical biosensors.
|Surface Modification Technique
||simple and direct
||low immobilization efficiency
|Encapsulation in polymers or gel
||necessary surface treatment and low immobilization efficiency
||simple and high stability
||the strict control of conditions and nonspecific interaction
||simple and controllable BRE density
||possible nonspecific interaction
||necessary surface treatment and low immobilization efficiency
||simple and high stability
||necessary surface treatment and possible nonspecific interaction
||Reliable, cost-effective, and easy fabrication and maintenance
||possible nonspecific interaction