**8. Challenges and Future Work**

Although much work has been carried out to detect water quality using electrochemical biosensors, some bottlenecks still need to be addressed. Some challenges in microbial biosensors' detection process are low recognition limits, limited specificity, and high contamination. It also has limitations in mass transmission due to the subsequently limited penetration of substrates and products throughout the cells [163]. These bottlenecks primarily exist and limit the sensitivity during these biosensors' real-time application. These prototypes can only detect certain microorganisms and limited chemicals present in water, thus making them unsuitable for the in situ monitoring of unanticipated shocks in wastewater [164,165]. Additionally, there is currently no verification on the immobilization of enzymes or microorganisms on the surfaces of biosensors during their deployment in harsh environments. They also have low durability, primarily when operated over several hours [165–167]. This makes these prototypes unsuitable for long-term operations during wastewater treatment. Other issues are the requirement of external power sources and additional dissolved oxygen (DO) that deters the exact conductivity and pH probes to measure various parameters [168,169]. These problems lead to the deterioration of their performance over a prolonged period. The real-time monitoring has also been challenging due to the delay in the response of these biosensors, thus hindering the timely action from overcoming the shock effects. For example, when anaerobic granule biosensors were used for the early alarm to detect copper and phenol in the wastewater [170], the delay (6–20 h) in the response time created problems in the practical application and of these prototypes.

Generally, electrochemical sensors have specific attributes like lower detection limit of detection than colourimetric and fluorescent sensors (pvalue 0.05, d-value >0.8) [171]. Some of the primary characteristics of the electrochemical biosensors are their compatibility with modern microfabrication technologies, low input power, roll-to-roll fabrication, and the independence of sample turbidity and colour [172].

Even though the fabricated sensors have been used for multifunctional applications, most focus on the detection criteria lying on the sampled genre like heavy metal ions. Rarely have the sensors been used to detect some multi-analytes like antibiotics, small molecules, and metal ions. Julius et al. [173] displayed the development and implementation of a cell-free in vitro transcription system that deploys RNA output sensors activated by ligand induction (ROSALIND) to detect specific contaminants based on aptamer transcription and fluorescent signal analysis. Importantly, easy storage and distribution can also be carried out with the ROSALIND system, thus making it easier to deploy. This assists in determining their capability to test municipal water supplies and demonstrate their use for monitoring water quality.

#### **9. Conclusions**

There is continuous fear about the risks caused by contaminations or pollutants to human health and marine ecosystems. However, standard analytical techniques are sensitive, accurate, laborious, expensive, and unsuitable for on-site monitoring with complex water sample pretreatment requirements before testing under the guidance of trained personnel. This review assessed the recent progress in developing electrochemical biosensors for water quality sensing applications over the current time. Many of them also have certain advantages over the other methods in detecting the aggregate outcomes of multiple pollutants in water samples. Although electrochemical biosensors have great potential and are very sensitive and cost-effective compared to the standard analytical methods, they still need to reduce their cost and response time performance compared to the other sensors. The research will likely continue by modifying the electrode surfaces and innovative biorecognition elements, using various nanomaterials, conducting polymers, etc., and improving the surface-by-surface modification techniques to enhance electrochemical biosensor sensitivity and selectivity and the quick response. Further integration with intelligent electronics and wireless technologies will significantly benefit the development of biosensors for remote sensing applications or in situ measurements. However, the stability of the electrochemical biosensor remains a challenge that needs additional research to explore and to extend its shelf life. Finally, this review outlines the current methods and technologies in electrochemical biosensors for water quality sensing applications. We think that this review article will be helpful for beginners and a helpful guide that will enhance the awareness of the role that electrochemical biosensors can play in protecting our environment and most valuable water resources.

**Author Contributions:** Conceptualization, Y.H. and M.E.E.A.; resources, Z.H. and A.N.; data curation, Z.H. and A.N.; writing—original draft preparation, Y.H. and M.E.E.A.; writing—review and editing, Y.H., M.E.E.A., S.F. and S.C.M.; supervision, M.E.E.A., S.F. and S.C.M.; project administration, S.F. and M.E.E.A.; funding acquisition, S.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the National Natural Science Foundation of China (No. 62001460), the Natural Science Foundation of Guizhou (Grant No. Qiankehe-ZK[2 022]KEY002), and Shenzhen Key technology projects (JSGG20201103153801005), Shenzhen Municipal Science and Technology Innovation Commission (JCYJ20210324101611031).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors are thankful to their respective institutions/universities for providing valuable support and funding to conduct this research works.

**Conflicts of Interest:** The authors declare no conflict of interest.
