Developing Biosensors for SARS-CoV-2 Wastewater-Based Epidemiology: A Systematic Review of Trends, Limitations and Future Perspectives

Abstract

Wastewater-based epidemiology (WBE) permits the sustainable surveillance of pathogens in large populations and does not discriminate between symptomatic and asymptomatic groups. WBE allows health authorities and policymakers to make swift decisions to limit the impact of local and regional disease outbreaks, minimise the spread of infection and mitigate the effects of pathogen importation. Biosensors are an exciting addition to conventional WBE analytical approaches. Combined with sentinel surveillance programs, biosensors can be reactive to novel variants of a virus in the community. However, progress developing biosensors for wastewater surveillance is severely limited compared to advances in clinical diagnostics, with a lack of well-developed biosensors currently being available. Whilst the field of biosensors is vast, this review focuses on trends in monitoring SARS-CoV-2 in wastewater over a key period (2020–2021). We explore the complexities involved in sampling within wastewater networks, the options for target selection, and reflect on the ethical considerations and limitations of this approach by highlighting the complex transdisciplinary connections needed. The outlook for WBE biosensors is assessed to be on a positive trajectory as current technical challenges are overcome. Finally, we outline the current status and where further development is needed to have a systematic feedback mechanism which would allow wastewater biosensors to be kept current and relevant to emergent pathogens.

1. Introduction

Wastewater-based epidemiology (WBE) is a rapidly developing surveillance tool. It permits the sustainable monitoring of wastewater systems for the early detection of health hazards circulating within a defined population, and alerts the appropriate health authorities to the potential danger. This becomes especially important in the case of epidemics and the eradication of specific disease agents. For example, the ease of the restrictions following the COVID-19 pandemic allowed for the re-emergence of enteroviruses, such as the enterovirus-D68 [1] and the vaccine-derived type 2 poliovirus [2,3,4]. The United Kingdom (UK) was declared polio-free in 2003. However, the decrease in the number of vaccinated people and rise in global movement following the COVID-19 pandemic, allowed the importation and spread of vaccine-derived type 2 poliovirus. Consequently, cases of polio were detected once again in London and other cosmopolitan cities around the UK [5,6]. WBE was originally designed to detect environmental concentrations of pharmaceuticals and to track the use of illicit substances [7]; further, its success is contingent on identifying good targets (i.e., metabolites, specific indicator chemicals, biomarkers, biological agents) that will be selectively and specifically detected as analyte(s) of interest in the complex matrix of wastewater. WBE relies on appropriate sampling, purification and/or extraction from the wastewater and subsequent detection of the target (analyte of interest). Following these steps, data must be processed and information relayed to the appropriate health bodies, to inform interventions as required (Figure 1A).

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), brought the practical applications and importance of WBE to the fore. Although the global COVID-19 pandemic is in decline, understanding and learning from these episodes remains vital in developing a rapid response to existing or new pathogens that might arise in the future. Viruses are ideal candidates for WBE, owing to their inability to proliferate outside a living host. SARS-CoV-2 is a positive-sense single-stranded RNA ((+)ssRNA) coronavirus, with around 79.6% identity to the SARS-CoV virus responsible for the 2003 SARS epidemic [8]. The SARS-CoV-2 genome is around 30 kb in length, and encodes for four structural proteins common to coronaviruses (spike, S; envelope, E; membrane, M; nucleocapsid, N), two overlapping ORFs (ORF1a and ORF1b) (Figure 1B), as well as accessory proteins including 3a, 6, 7a, 7b, 8 and 10 [9,10]. ORF1a and ORF1b are translated to form polypeptides, which become cleaved to generate 16 non-structural proteins (NSPs). These include components of the RNA-dependent RNA polymerase (RdRp) complex, used for viral genome replication and transcription. WBE works on the principle that SARS-CoV-2 is excreted by the host, particularly within faeces [11]. Waste converging at wastewater catchment points represents a pooled sample for community or population-level analysis. Importantly, SARS-CoV-2 reliably persists in wastewater samples enabling detection for surveillance and epidemiological studies [12]. Further, because the virus can be shed in faeces even after four weeks of a negative nasopharyngeal test [13], SARS-CoV-2 WBE can be used as both an early and long-term surveillance tool. Notwithstanding the current difficulty of correlating SARS-CoV-2 detected viral particles from wastewater to infectivity, WBE provides a complete picture of ongoing viral load within a population [14,15,16]. In comparison, since only a subset of infected individuals present with symptoms [17], and with an incubation period of anywhere between a few days and over a week [18], the screening of symptomatic individuals through clinical testing misses those individuals that are asymptomatic or pre-symptomatic. WBE can be used as an estimation of the total number of positive cases within a population [15]. Despite the benefit of offering a holistic surveillance approach, viral WBE techniques are limited in their capacity and flexibility. Specifically, SARS-CoV-2 conventional analytical detection techniques, such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR), when deployed on a large scale are time consuming, slow and can quickly become expensive.

The use of biosensors may provide simple, cost-effective, on-site and real-time detection, permitting frequent sampling and greater geographic granularity than is currently possible. Importantly, they could provide a sustainable surveillance alternative for less developed countries. Biosensors are analytical devices that use a recognition element(s) (usually of biological origin) to detect a target(s) of biological origin and provide a readable output signal. Biosensors, therefore, have the potential to represent a robust and rapid approach to SARS-CoV-2 wastewater surveillance, particularly when combined with a sentinel whole genome sequencing network to ensure that biosensors remain agile to changes in the viral sequence and detection of novel variants of concern.

Excellent reviews have been carried out on WBE [19,20], biosensors for SARS-CoV-2 point-of-care diagnostics [21,22], biosensors for WBE with a focus on a wide range of analytes of interest [23,24], and other analytical methods for SARS-CoV-2 detection in wastewater [25,26]. However, due to the tremendous progress on SARS-CoV-2 biosensor for diagnostic applications, and the lack of similar progress on SARS-CoV-2 biosensors for wastewater surveillance, this review focuses on the key parameters to consider, and methods and trends towards developing bespoke biosensors for SARS-CoV-2 WBE. It emphasises the progress made on existing diagnostic biosensors while recognising that wastewater is a complex matrix. Specifically, the review focuses on viral nucleic acids as the target of interest. It discusses target amplification requirements, the use of biorecognition elements for target detection, as well as highlighting the trends in SARS-CoV-2 detection and devices, coupled with deployment and ethical considerations. Finally, it acknowledges the limitations of biosensors for SARS-CoV-2 WBE as well as their potential for revolutionising WBE, and summarises possible ways forward.

2. WBE—Approaches to Monitoring Infectious Diseases

WBE was developed over 20 years ago and its application has been gaining interest in recent years, (Figure 2). For example, WBE was adopted for the Environmental Surveillance (ES) of poliovirus, emerging from the “Global Polio Eradication Initiative” launched in 1988. Active ES was implemented in Egypt in 2000 [27] and in Israel in 2013, where a linear relationship was observed between the number of people actively shedding poliovirus, and the occurrence of poliovirus in the sampled sewage [28,29,30]. WBE also succeeded in monitoring the prevalence of HepA in Scandinavia [31]. Analysis of human sewage from Florianopolis, Brazil, identified evidence of SARS-CoV-2 RNA circulating 56 days in advance of the first clinically confirmed case in South America and 91 days in advance of the first clinically confirmed case in Brazil [32], highlighting how wastewater analysis can play a central role in monitoring diseases from a very early stage. This has value in alerting public health authorities to the need to take swift action towards early intervention, control and mitigation of disease outbreak.

Many of the techniques associated with WBE for monitoring infectious disease, particularly in relation to SARS-CoV-2 WBE, need optimisation [20,33,34]; additionally, there is currently a lack of consensus and standardization in the SARS-CoV-2 WBE methodologies [11,35,36]. To understand the range of parameters, consistencies and inconsistencies between different studies, we looked at a wide range of randomly selected SARS-CoV-2 WBE publications between 2020/2021 and compared their methods (Supplementary Materials). For example, although samples are predominantly taken from Wastewater Treatment Works (WwTWs) (Figure 3A and Supplementary Materials), sample collection and handling approaches differ (Figure 3B,C). Likewise, approaches to extraction and concentration vary (Figure 3D,E), meaning viral/RNA recovery efficiency is inconsistent [37,38,39] and the choice of reference viruses (surrogates) is erratic (Supplementary Materials). The conventional method for the detection of SARS-CoV-2 from clinical samples uses variations of polymerase chain reaction (PCR). The commonly used reverse transcription-quantitative polymerase chain reaction (RT-qPCR) approach has been applied to over 80% of published SARS-CoV-2 studies considered in this review (Figure 3F), showing it to be remarkably robust (Supplementary Materials). Further, over half of the PCR tested samples were successfully coupled with sequencing for target validation, either through Sanger sequencing or next generation sequencing (NGS) of PCR amplicons, or whole genome sequencing of the virus (Figure 3F). Although RT-qPCR is proving effective at SARS-CoV-2 WBE, there is currently no uniformity in the choice of targets analysed, thus making data comparison even more difficult [33,39,40] (Figure 3G).

In addition to RNA sequencing supporting target validation for WBE, whole genome sequencing of clinical samples, and genomic epidemiology, have represented a significant tool in the response to the COVID-19 pandemic, with the UK’s COVID-19 Genomics UK Consortium (COG-UK) providing significant coverage across the UK for clinical and community cases [40,41]. It is through such work, and through collaborations of a National and International nature, that novel variants such as the B.1.351 lineage first detected in South Africa [42], and the B.1.1.7 lineage first described in the UK [43], have been identified and monitored. This approach has been successfully demonstrated for WBE, utilising similar rapid assays and protocols that have been developed for whole genome sequencing of clinical SARS-CoV-2 samples, most notably by the ARTIC network [44]. Indeed, despite the large-scale RNA degradation expected of viral RNA over time in wastewater, a number of studies have shown high levels of genome coverage from samples isolated from wastewater samples [45,46,47,48]. Such analyses are essential for fully understanding and profiling novel variants, but can be coupled with more rapid typing approaches for swift identification of circulating virus and known variants. However, whilst detailed information is obtained from such RNA sequencing analysis, and it ensures RT-qPCR remains responsive to the detection of any variants present, a simple to use, inexpensive, rapid detection approach for SARS-CoV-2 and its variants in wastewater would be of significant value to improve sampling frequency and geographic granularity.

Figure 3. SARS-CoV-2 analysis from wastewater (2020/2021 period). (A) Wastewater sampling location. Wastewater treatment work (WwTW) and Wastewater resource recovery facility (WRRF) (B) Wastewater sampling method. (C) Post sampling steps prior to specialist laboratory analysis. (D) Specialist laboratory sample extraction methods. The vast majority of the sample extractions were made via a commercially available kit, while others used more conventional methods and some used a combination of both. Qiagen kits are the most commonly used kits within these studies, followed by combinations of Trizol LS/Commercial kit (T/Com), Trizol LS/Chloroform (T/Chl) and Macherey Nagel kit (MG). Less commonly used were kits from ZymoResearch (ZR) and Thermo Fisher Scientific (TFS). (E) Specialist laboratory sample concentration technique. The predominant approach was concentration via filtration, followed by the use of electronegative membranes (EN memb) and polyethylene glycol (PEG). Less commonly used techniques include concentration via flocculation (Floc), glycine (G), direct sludge extraction (SE), silica columns or silica milk (Si) and automated concentrating pipette (Pi). (F) SARS-CoV-2 detection methods. (G) SARS-CoV-2 target gene detected. Data based on information provided within 18 peer reviewed publications from the 2020/2021 period [15,32,33,37,39,45,46,49,50,51,52,53,54,55,56,57,58,59]. No information (NI). Graphics were generated using Excel.

Figure 3. SARS-CoV-2 analysis from wastewater (2020/2021 period). (A) Wastewater sampling location. Wastewater treatment work (WwTW) and Wastewater resource recovery facility (WRRF) (B) Wastewater sampling method. (C) Post sampling steps prior to specialist laboratory analysis. (D) Specialist laboratory sample extraction methods. The vast majority of the sample extractions were made via a commercially available kit, while others used more conventional methods and some used a combination of both. Qiagen kits are the most commonly used kits within these studies, followed by combinations of Trizol LS/Commercial kit (T/Com), Trizol LS/Chloroform (T/Chl) and Macherey Nagel kit (MG). Less commonly used were kits from ZymoResearch (ZR) and Thermo Fisher Scientific (TFS). (E) Specialist laboratory sample concentration technique. The predominant approach was concentration via filtration, followed by the use of electronegative membranes (EN memb) and polyethylene glycol (PEG). Less commonly used techniques include concentration via flocculation (Floc), glycine (G), direct sludge extraction (SE), silica columns or silica milk (Si) and automated concentrating pipette (Pi). (F) SARS-CoV-2 detection methods. (G) SARS-CoV-2 target gene detected. Data based on information provided within 18 peer reviewed publications from the 2020/2021 period [15,32,33,37,39,45,46,49,50,51,52,53,54,55,56,57,58,59]. No information (NI). Graphics were generated using Excel.

3. Biosensors for SARS-CoV-2 WBE

Biosensors are suitable bioanalytical devices for SARS-CoV-2 WBE. They have the potential to overcome key limitations of conventional WBE by combining the high selectivity and specificity of PCR-based approaches with simple to use, low-cost, rapid, on-site target detection [24,25,60,61,62,63,64]. Biosensors have found applications in clinical, pharmaceutical, food, environmental and security industries [61,65]. Unlike other methods of viral detection in environmental samples, which require specific storage conditions enabling sample stability to be retained during transportation to specialist laboratories for processing and analysis (resulting in outputs being obtained hours to days after sampling), biosensors can be portable, simple to use, deployed on-site, and give a readout (signal) in minutes. Biosensors therefore represent one of the swiftest bioanalytical devices for detecting pathogens in environmental samples [60]. Point-of-care (POC) biosensors, with a high degree of sensitivity, specificity and short detection time, have been developed for SARS-CoV-2 targets and related biomarkers, and their performance validated against SARS-CoV-2 clinical samples [21,66,67,68,69]. These biosensors are portable, simple to operate and require less sophisticated equipment for operation than conventional molecular analytics. Nevertheless, SARS-CoV-2 biosensors have predominantly been developed for use on clinical specimens (Figure 4A), and unlike clinical specimens that are easily extracted and detected, wastewater samples require pre-processing steps to remove impurities before isolating targets for detection. Further, the likelihood of a more heterogeneous population of viruses in the WBE samples compared to those from a clinical source, places a greater emphasis on the need for selectivity. Indeed, if prevalence is high, a biosensor would have to deal with a complex matrix of infections, whilst if prevalence is low, it would have to deal with a low viral load. In addition, adsorption of the biorecognition element to the surface of the biosensor [70,71], and the stability of the biosensor interface and potential decrease in signal over time [72], are important considerations that are reviewed in depth elsewhere. Such additional complexities make a large difference in the design and fabrication of biosensors for wastewater, making it challenging to repurpose biosensors for clinical applications towards WBE.

3.1. Target Considerations for a SARS-CoV-2 Wastewater Biosensor

The choice of target for SARS-CoV-2 WBE is crucial to the sensitivity and specificity of biosensor fabrication. Within the publications reviewed here, the most commonly used bioanalytical method for detecting SARS-CoV-2 in wastewater is RT-qPCR, and despite recommendations from the World Health Organization (WHO) to use a combination of the S and RdRp genes [101,102], the predominant target of interest is the N gene (Figure 3G, left), which has a low mutation rate [103] and is highly expressed during infection [104]; however, there is no consensus on the region of the N gene targeted [37,49] (Figure 3G, right). Interestingly, amongst the published WBE reports highlighted in the Supplementary Materials, the S gene appears to be less frequently targeted in wastewater samples than is the case for clinical samples (Figure 3G and Figure 4A). The S gene encodes a homotrimeric glycoprotein complex that facilitates viral cell entry through affinity of the receptor binding domain (RBD) with mammalian Angiotensin Converting Enzyme-2 (ACE2) receptors [105]. The spike proteins sit on the virus surface, giving the coronavirus its characteristic “crown”-like appearance, and plays a significant role in immune responses. For this reason, the S gene is a common site for vaccine treatment [106]. The S gene, however, is highly susceptible to mutation, with a number of variants on the spike glycoprotein (e.g., K417N, E484K, and N501Y in the RBD) identified with the potential to reduce the efficiency of antibody neutralization and support vaccine escape [107]. For the detection of SARS-CoV-2 in WBE, using a method such as RT-qPCR, a target with low mutation rate is preferable; mutations can result in failure to amplify the target, as seen with S gene target amplification failure in the B.1.1.7 lineage identified in the UK in November 2020 [108,109]. The importance of monitoring potential variants of concern (VOC) and variants under investigation (VUI) that carry mutations with the potential to escape treatments and vaccines should therefore not be underestimated. Sentinel surveillance operations such as COG-UK are able to identify novel VOCs, which should be targeted in addition to a more general estimation of prevalence of SARS-CoV-2, with the COG-UK RNA sequencing data informing changes to the RT-qPCR assay to support specific VOC/VUI detection [110]. This establishes a mechanism for early identification of variants spread in the community that have the potential to bypass vaccination programs. The ability to identify the community spread of VOCs and VUIs as early as possible is essential for a robust public health response to be levied. Given these considerations, a wastewater biosensor with output capabilities akin to RT-qPCR, detecting both stable SARS-CoV-2 gene(s) as well as any VOC/VUI, would represent the ideal scenario.

In addition to directly considering the gene target(s) for SARS-CoV-2 detection, the viral concentration method applied to the wastewater sample is vital to the success, reproducibility, reliability, sensitivity and specificity of target detection. For SARS-CoV-2 WBE, a range of different concentration approaches have been used (Figure 3E; Supplementary Materials), and just under 50% have involved filtration methods. For example, using a centrifugal ultrafiltration concentration method, and RT-qPCR detection, the RdRp complex genes exhibited the highest specificity compared to the E, M, and N genes, whilst the M-gene exhibited the highest sensitivity [33]. Another study showed that RdRp was more sensitive to detection compared to ORF1ab and N genes following an ultrafiltration concentration technique [49]. An alternative study which, like 17% of the SARS-CoV-2 WBE studies that used a polyethylene glycol (PEG) sample concentration technique (Figure 3E; Supplementary Materials), noted that the N gene was more sensitive and specific to detection [37]. However, RT-qPCR assay sensitivity may decrease over time as the SARS-CoV-2 sequence changes [111], further illustrating the need for SARS-CoV-2 whole genome sequencing data to ensure that detection assays (be they RT-qPCR-based or any associated application as a biosensor) remain relevant.

Collectively, it is evident that the genetic detection of SARS-CoV-2 in wastewater is possible, and a biosensor capable of doing this would be of significant advantage. In considering biosensor target(s), it would be crucial to screen SARS-CoV-2 targets together with other parameters that can potentially impact the output of a biosensor, particularly sensitivity and specificity, before the final fabrication. This facilitates the identification of suitable gene targets, including a standardised positive control. Importantly, a biosensor that incorporates the simultaneous detection of multiple SARS-CoV-2 targets, together with a positive control, in a single reaction, would not only ensure the reliability of the biosensor output, but would also support the parallel assessment of prevalence and identification of VOCs/VUIs. Such a biosensor, capable of providing near real-time outputs on multiple targets in one step, would have recognised benefits over current specialist laboratory-based analytical approaches. In addition, it would provide the potential for the rapid detection of VOC and VUI sequences in a population much earlier than would be possible through clinical testing. Indeed, high sensitivity is required to detect potentially low prevalence VOCs and VUIs within the community and to allow early interventions, such as surge testing to prevent onward spread. Such biosensor detection would be reliant on the feedback of data through a sentinel program of whole genome sequencing of cases across the globe to allow for designs against novel variants to be incorporated. Therefore, a biosensor system that can be rapidly repurposed, allowing for the swift inclusion of new targets, would be of clear benefit.

3.2. Target Amplification for a SARS-CoV-2 Wastewater Biosensor

The RT-qPCR detection limits of SARS-CoV-2 in wastewater are estimated to be 2 copies/mL wastewater [112]. Target amplification is therefore a central requirement of efficient and sensitive detection. The ability to increase the copy of target nucleic acids for detection at constant temperature (isothermal reaction) offers the possibility of rapid readout, flexibility and simplicity; these aspects are key features of an ideal biosensor. Before the COVID-19 pandemic, a specific high-sensitivity enzymatic reporter unlocking assay (SHERLOCK), which allows multiplex and ultrasensitive detection of nucleic acids in a one-pot assay in less than 30 min, was developed for clinical samples [113]. The method employs recombinase-mediated polymerase pre-amplification of the target, followed by Cas13 or Cas12 detection. The specificity of SHERLOCK was applied to distinguish between two Zika viruses based on a single nucleotide difference in the target [114]. More recently, SHERLOCK was applied to detect the SARS-CoV-2 N gene in clinical samples [115]. Further, a reverse transcription-loop mediated isothermal amplification assay (RT-LAMP) combined with CRISPR Cas12 assay (DETECTR: DNA Endonuclease-Targeted CRISPR Trans Reporter and iSCAN: in vitro Specific CRISPR-based Assay for Nucleic acids detection) facilitated the rapid detection of SARS-CoV-2 N and E genes within 30–40 min, compared to 120 min for RT-PCR [73,74]. Importantly, whilst these assays employed relatively simple equipment and protocols, coupled to a simple readout that could be easily interpreted, the limit of detection for these assays on clinical samples was 10 RNA copies/µL, which is an order of magnitude greater than may be anticipated in wastewater. This adds to the complexity of converting such clinical biosensors to wastewater applications.

The organic matrix that is present in clinical samples is very different in terms of structural, chemical and biological complexity to a wastewater sample. A typical SARS-CoV-2 clinical sample will generally comprise of material extracted from a single individual, resulting in limited non-viral organic material and no structural complexity. However, dependent on where wastewater is taken in the treatment process, wastewater comprises samples from many individuals, mixed and adhered with a range of inorganic and organic materials, with many structural components and surface types present. Given that wastewater is a complex matrix, and that other coronaviruses may interfere with SARS-CoV-2 detection, high specificity is also required. Of relevance, RT-LAMP assays can discriminate between the SARS-CoV-2 N gene from that of other human coronaviruses, thus offering the high specificity required [116,117]. Despite the many advantages of RT-LAMP, the assay is prone to false-positive readout and needs a further step to validate the result [116,117]. Nonetheless, RT-LAMP has been successfully applied to raw wastewater, directly amplifying SARS-CoV-2 E and N genes, without the need for sample concentration and RNA extraction [118], illustrating the potential for nucleic acid-based tests to detect SARS-CoV-2 from raw wastewater. Finally, the incubation temperature for RT-LAMP reactions is >60 °C. This limits point-of-care and wastewater surveillance applications, especially in low resource settings. Fabricating a biosensor system that can amplify target nucleic acids isothermally under ambient temperature, without the need for heating equipment, would therefore reduce the economic consideration of deploying a biosensor for wastewater surveillance.

3.3. Biorecognition Elements and Detection Approaches for a SARS-CoV-2 Wastewater Biosensor

For biosensor functioning, the role of the biorecognition element is to detect the target, either present within the sample or following amplification. The majority of biosensors for SARS-CoV-2 are fabricated with antibodies or nucleic acids as the biorecognition element (Figure 4C), whilst the principal detection approaches being used are optical (colorimetric, fluorescent and luminescent) and electrochemical signals (Figure 4D), paper-based devices and nanotechnology-based biosensors are also gaining traction [66,75,119]. Amongst the nucleic acid biorecognition elements are ssDNA, aptamers, probes, and Cas-sgRNA [66,75,76,77]. For example, using thio-modified antisense ssDNA-capped gold nanoparticles (AuNPs) as the biorecognition element, it was shown to be possible to circumvent the gene amplification step and achieve a low detection limit of 6 copies/µL on clinical samples. Similarly, a colorimetric biosensor that utilized the optical properties of plasmonic AuNPs simultaneously targeted two regions of the N gene, and the resulting signal was based on a naked eye visualization of precipitation of the gold nanoparticles [75]. Further, a modular dual-signal (colorimetric and luminescent) Toehold RNA biosensor developed to target the SARS-CoV-2 ORF1ab (Nsp13) gene detected 100 copies of the viral RNA in a relatively short time (30 min); the naked eye colorimetric signal was based on the LacZ system [36]. However, given the possible low levels of SARS-CoV-2 in wastewater [112], amplification likely remains a necessary step prior to detection by the biorecognition element. In addition, since stability of the biorecognition element can impact on signal strength over time [120], additional design considerations (e.g., the use of thiol-based monolayers to prolong stability) may also be required [121].

Whilst a range of detection approaches (electrical, magnetic, and plasmonic) have been described for SARS-CoV-2 lab-on-chip platforms [122], nucleic acid biorecognition elements offer the advantage of distinguishing between SARS-CoV-2 lineages at the nucleotide level; this is of importance given the rate of SARS-CoV-2 mutations. For post-pandemic surveillance, this advantage can be leveraged to design a wastewater biosensor that can potentially detect multiple known mutated regions of SARS-CoV-2, thus programming a biosensor output to inform on the lineage of a detected or predominant variant of the virus in wastewater. A similar approach was employed to distinguish between the American Zika virus and African Zika virus, by leveraging the presence of a protospacer adjacent motif (PAM) site in the American-lineage of the virus [123]. The clustered regularly interspaced short palindromic repeat (CRISPR; Cas12 and Cas13 for ssDNA and RNA target, respectively) approach is gaining significant attention in wastewater monitoring owing to its sensitivity and specificity towards SARS-CoV-2 detection [74].

3.4. Trends in Device Considerations for a SARS-CoV-2 Wastewater Biosensor

Learning from point-of-care biosensors, it is worth considering the criteria developed for their use: REASSURED (Real-time connectivity, Ease of specimen collection, Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free or simple Environmentally friendly, Deliverable to end-user) characteristics [21,124]. Paper-based microfluidics biosensors for SARS-CoV-2 meet the REASSURED criteria but are designed for immunoassays [68,78,125], rather than using a nucleic acid detection approach. For paper-based microfluidic biosensors, the signal intensity of the output is greatly influenced by the paper pore size and charge, as demonstrated for the Zika virus [126]. Specifically, this study showed that paper pore size, and perhaps charge, affected the migration of amplicons and RT-LAMP reaction components through a microfluidic chip. Therefore, using an alternative physical support, which would allow alteration in the pore size and charge, would improve biosensor sensitivity especially towards wastewater monitoring.

In addition to the REASSURED design criteria, there are four further “M4” aspects to consider that greatly expand the usefulness of any biosensor device: Modularity, Multiplexing, Multifunctionality, and Miniaturisation (or Minimisation). A Modular biosensor will ensure flexibility in detecting the same or different viral targets using a different biorecognition element or detection approach. With this feature, a biosensor developed for detecting SARS-CoV-2 in wastewater could easily be adapted for use as a surveillance tool for detecting other viruses in environmental or clinical settings. Multiplexing ensures that more than one viral target can be detected in a single assay to increase the reliability of the assay output and reduce false-positive readouts. This also allows for the detection of multiple specific variants of interest, and can be modified as new VOC/VUIs are identified. Due to the pre-processing steps involved in wastewater analysis, it is equally important to consider designing SARS-CoV-2 biosensors for WBE to be Multifunctional—highly sensitive and resistant to inhibitors or interference in wastewater—to allow the direct detection of targets from raw wastewater in a one-pot integrated process of sample input through to detection. Lastly, a biosensor must be Miniaturised (or Minimised) to reduce costs, be portable, and allow easy transportation and deployment into a sewage system.

To decrease the time taken to obtain an output, some studies have circumvented conventional RNA extraction and purification processes to reduce the completion time of SARS-CoV-2 target detection, [118,127,128], while others have performed direct detection without target amplification [35]. The majority of such biosensors have been developed for clinical and point-of-care applications, which are designed to function within the context of a very different sample matrix to wastewater. For a SARS-CoV-2 wastewater biosensor, more rigour is required due to the complexity of wastewater, which in turn affects the sensitivity and specificity requirements of a biosensor developed specially for WBE. Nevertheless, with the challenges of developing biosensors for SARS-CoV-2 WBE, it will be appealing to reduce the steps involved, from sampling through to signal detection, without compromising on accuracy and reliability.

4. Considerations for SARS-CoV-2 Wastewater Biosensor Deployment

As part of developing a biosensor for WBE, deployment requirements, such as wastewater sampling and positioning within the sewer network, as well as ethics, need to be thoroughly considered [129]. Such deployment considerations affect the associated epidemiological modelling possible based on biosensor output. This, in turn, impacts the value of the biosensor for WBE, and consequently its capability as a tool for epidemic/pandemic management.

4.1. Wastewater Network Considerations

Sewer networks use the flow of water to transport human waste to a Wastewater Treatment Works (WwTWs) (Figure 5). They involve a network of progressively larger pipes taking flows from homes with pipe gradients to ideally maintain self-cleansing velocities. This becomes a problem when sewers receive material they were not designed to transport (such as the modern issues of clogging with wet wipes and fats, oils and grease (FOG) deposits, also known as fatbergs). Where topography does not allow gravity flow, pumping stations are used to move the wastewater uphill and to the next part of the network [130]. In countries such as the United Kingdom, there are many older combined networks that receive surface runoff. When storage capacity is full, this gives rise to discharges of dilute sewage in combined sewer overflows [131].

Most applications of SARS-CoV-2 WBE to date have considered samples collected at centralised WwTWs at the end of sewerage networks serving whole communities (Supplementary Materials, Figure 1, Figure 3A and Figure 5). While this provides a holistic overview of infection, this has a disadvantage of not resolving “hot spots” of detection. Additionally, the long travel times of samples within the sewage network can cause degradation of viral particles and other chemical transformations that may affect testing efficacy, as well as delaying detection. Due to the manner in which sewers are laid, often with different areas of a city served by separate trunk mains (large sewers), there is the potential to sample up-catchment of the main treatment works to improve the granularity of the data collected [132]. It has therefore been suggested that WwTWs sampling is supplemented with two more units of analysis at the neighbourhood or congregate living facilities level, and protocols for how this could be applied to cities have been proposed [132] (Figure 5).

For wastewater biosensors to be deployed within the sewer network, practical considerations need to be taken into account. Firstly, sewers are dangerous environments; in addition to pathogens in sewage, there are many risks such as vermin, sharps and toxic gases (e.g., hydrogen sulphide) [133]. Sewage can also become corrosive under reduced conditions, especially in sewer networks with long residence times [134]. Combined sewers are at particular risk from flash flooding. Human access is, therefore, restricted and requires specialised training and equipment as storm flows can give high velocity and surcharged pipes [135]. Biosensors must therefore be designed in such a way as to limit the need for human intervention. Careful consideration must therefore be given to ease of access for the installation and removal of biosensors, as well as for the development of robust communication links access to well-maintained power supplies for any pre-treatment steps. Vandalism and theft are also a concern in any deployed equipment. These challenges mean that deployment would be easiest at water company infrastructure, such as pumping stations, where secure, safe access to wastewater and power supplies are available. However, with the location of these facilities dictated by topography, they may not always be located close to smaller units of analysis, impacting the granularity of the data obtained.

As part of developing a biosensor for WBE of SARS-CoV-2, the challenge of relating the data from the biosensor to population size, to enable modelling, needs to be considered. WwTWs serve different sized populations, ranging from a few hundreds to several million people [136]. Populations are also not static, but fluctuate daily (commuters) and weekly (tourism). In addition, flow rates in sewers fluctuate diurnally in relation to domestic water use and industrial activities [137]. In combined systems, storm flows can be many times higher than dry weather flow, and groundwater infiltration (and exfiltration) can be significant in some catchments [138]. Water companies are aware of, through domestic and commercial charging, the approximate number of customers in a catchment. In addition, volumes of flow from different sources are routinely standardised to a population by dividing by a hydraulic population equivalent (typically 150 L/d per person in the UK), and the organic load can also be related to a population through the organic population equivalent (60 g/d O₂ per person) using the biochemical oxygen demand test (BOD) [139]. Other routinely measured parameters in sewage, such as ammoniacal nitrogen, can also be used to estimate population, but concentrations can be easily affected by sewer processes [139]. However, BOD and other tests are usually only routinely conducted at WwTWs in the context of process control and compliance with environmental standards for effluents, not in sewer sub-catchments. On a more sophisticated level, endogenous biomarkers can be used to normalise sewage flows to an estimated population, but while this works well on a city level it can contribute to high uncertainties (40%) in smaller populations [136]. What is clear is that data on other parameters in the wastewater will be required alongside SARS-CoV-2 detection to enable useful monitoring and predictive modelling to be accurate. This could take the form of including biomarker detectors as part of a multifunctional biosensor, or a separate sensor for physio-chemical parameters.

4.2. Wastewater Matrix Factors

The sewage matrix can contain thousands of chemical and biological targets to analyse and mine for data on public health [140], but that very rich source of information can be problematic when it comes to the analysis of specific compounds or biological factors, due to interference [141]. Traditionally, after wastewater samples are taken for WBE, prolonged and expensive clean-up procedures are required before target analysis can be performed [139]. Such clean-up procedures may involve the removal of interferents from the matrix to allow for a “clean” signal, as well as usually requiring target amplification to allow for a strong signal [142]. Biosensors developed for SARS-CoV-2 WBE will therefore likely be required to perform the clean-up procedures before target amplification and detection to ensure suitability for field deployment. While nucleic acid-based detection protocols bring a high degree of specificity to the analysis, environmental DNA and RNA molecules are directly affected by the chemical makeup of the environmental matrix. For example, SARS-CoV-1 RNA was found to survive in urine for 17 days at 20 °C, but in domestic sewage it persisted for only three days at 20 °C [143]. The SARS-CoV-2 lifespan in sewage is still not well understood, and further studies are required to determine the survival of the virus in water and wastewater under different operational conditions [144]. This illustrates how temperature and matrix chemistry greatly affect the persistence of the viral nucleic acid, and demonstrates the value of a real-time biosensor which would negate the need for sample storage.

In addition to biofouling and clogging, sewers are chemically aggressive environments, with industrial wastes and reduced conditions producing corrosive concentrations of sulphide and other compounds. There is a wide variation in flow rates; many old networks receive urban runoff creating sudden storm flows, surcharging pipes and creating high flow velocities, whilst low flows (or no flow) also occur, especially at night and in sewers serving small catchments. An in situ biosensor would require the ability to handle wastewater samples in this hostile, constantly changing and challenging environment, and to function over a large calibration range to deal with the changes in wastewater concentration between wet and dry weather flows. Whilst deployment within a sewage network can enhance data granularity for epidemiological modelling, the wastewater matrix can be considered more standardised within specific settings of a WwTW. A balance is therefore likely between the suitability of deployment location, complexity of the wastewater matrix factors at the given site, and the functioning of the biosensor for WBE.

4.3. Wastewater Sampling: Options and Challenges

Current wastewater sampling methods for SARS-CoV-2 WBE involve manual sampling, usually at a WwTWs, and can take up to 72 h from sample collection to reporting the qPCR result [145]. A manually operated and transportable biosensor would allow for immediate field analysis and reporting, rather than requiring transportation to a specialist laboratory for analysis. While manual deployment of a SARS-CoV-2 biosensor would be responsive rather than prognostic, it is ideally suited to surveillance purposes. Manually operated biosensors would have the benefit of being entirely mobile, allowing for sampling at any location of interest, such as when new information arises (e.g., sampling at a school or assisted living facility when outbreak concern is raised), and returning data within minutes. Although it would not need specialist laboratory equipment and technicians, it would require a trained operator, albeit in the field rather than a laboratory.

Whilst a manual wastewater biosensor could significantly support the SARS-CoV-2 WBE effort, samples collected can be either composite or grab. Both composite and grab samples have been used for sampling at WwTWs for SARS-CoV-2 WBE studies (Figure 3B; Supplementary Materials), with composite samples alone representing just under 50% of the samples analysed. A composite sample is a mixed sample created from smaller subsamples taken over a defined period of time (usually over 24 h). By comparison, a grab sample is a single sample collected at a set time. It is suggested that 24 h composite samples provide the most reliable average concentration of SARS-CoV-2 RNA in wastewater, while timed grab samples allow the accurate portrayal of the peak daily faecal load in wastewater, which can be more useful for modelling [146]. However, of the grab samples taken for SARS-CoV-2 WBE studies (Supplementary Materials), just over 50% provided details of the collection time (Figure 3B). Such inconsistencies and discrepancies in reporting sampling conditions complicate the understanding of the most suitable sample approach for SARS-CoV-2 WBE.

Once samples have been collected, they are transported and stored before analysis at a specialist laboratory. For the samples taken for SARS-CoV-2 WBE studies (Supplementary Materials), only 50% provided details regarding sample transport conditions (i.e., samples were refrigerated/on ice (Figure 3C)), whilst ~95% provided details on sample storage conditions (although the conditions themselves varied). Routinely, no pre-treatment is undertaken at the collection point, with all sample preparation occurring at the specialist laboratory. Whether composite or grab samples are taken, the fact that routine automated sampling technology is already widely utilised by the wastewater industry provides a major benefit to biosensor usage in WBE. If a biosensor could be integrated into a “traditional” automated sampler, it could greatly reduce barriers in the adoption of such technology by water authorities. Deployment of a biosensor is then only restricted by the financial and logistical constraints of requiring an electrical source and the security of the sampler/biosensor system. For example, if the preferred sample locations are midway up a catchment area, there may be the requirement for new infrastructure to accommodate the biosensor (e.g., a secure cabinet with electrical supply over a man hole). However, automated samplers are common place at WwTWs, so deployment in such locations could be immediate once a system-compatible wastewater biosensor is available.

An in-line real-time SARS-CoV-2 biosensor would provide a step change in WBE monitoring [147] and the predictive modelling of hotspots, since results can be provided in real-time. Despite these advantages, sewerage networks are challenging environments for deploying biosensors, where fouling from biofilms and physical insult from materials in the wastewater occur [148]. Deployment, maintenance, battery replacement, data download, sensor recovery and vandalism also pose challenges. Transmitted signals can be difficult to receive from underground sources and health and safety considerations generally minimise entry into confined spaces. Additionally, anything deployed in the sewer system would require ATEX rating for explosive atmospheres (EU directive 94/9/EC). Water companies are risk adverse when it comes to allowing unattended foreign objects into their sewers, where malfunction may later lead to blockages and issues in the network. So, while an in-line biosensor may be feasible at fixed water utility assets, such as WwTWs and pumping stations, which are secure and have energy sources and pose fewer problems than in pipelines, it is currently considered more feasible to use the manual or automated collection of a sample from a sewer and then perform the analysis with the biosensor for areas upstream in the catchment.

In summary, the deployment of biosensors for these units of analysis could range from real-time fully automated in-line sampling, to manual collection with in situ manual analysis. In situ manual analysis approaches are currently being trialled, with an aim to further develop this approach to begin to fit the fully automated model. The ultimate aim for SARS-CoV-2 WBE would be a continuous automated biosensor analysis for viral genome targets and variant types at the three units of analysis, for each catchment. The development of novel biosensor technologies for application and evaluation in these complex systems is an essential aspect of realising this ambition.

4.4. Ethical Considerations

In addition to scientific and technical sampling considerations, WBE can pose ethical dilemmas, particularly when high-resolution sampling for source detection is undertaken, which can result in stigmatisation of institutions or communities [19]. To address such ethical concerns, there are public health surveillance guidelines from the World Health Organization (WHO) [149] and also WBE-specific guidelines developed by the European Cooperation in Science and Technology Network SCORE (Sewage Analysis CORe group Europe) [150]. More recently, specific SARS-CoV-2 ethics guidance has been published by Hrudey et al. [151] and the requirement of a social license to operate wastewater surveillance [152]. While the legal ownership status of sewage is unclear, and individual human participants in WBE are not identifiable, the SCORE WBE guidelines suggest that the standard procedures for protecting participants, such as anonymising data and gaining informed consent from stakeholders, should be applied. In the case of institutions, this might be the School Head, Prison Governor, Hospital Director or relevant government body. Such ethical considerations are to be balanced with the cost/benefit ratios for the disease, which will be important to calculate in more detail in the future. For example, the intensive environmental surveillance of wild poliovirus in Israel and Egypt provided critical information that better informed the use of vaccine type, leading to better public health outcomes [27,29]. With the ability to monitor disease prevalence and ‘real time’ variant tracking, environmental surveillance techniques, which may lead to the successful eradication of a disease [28], need to be discussed openly alongside ethical concerns. The ethical implications of each study should be carefully evaluated in light of the disease risk, and all samples should follow best practice guidelines for anonymising and protecting an individual’s data.

5. Summary and Outlook

WBE has gained considerable profile as a sentinel surveillance tool for SARS-CoV-2. Initial concerns around genome stability within wastewater proved unfounded, and the specialist analytical detection techniques of RT-qPCR and RNA sequencing, usually applied to clinical samples, have also been successfully applied to samples from wastewater. However, at their heart, RT-qPCR and sequencing proved unsustainable as they are laborious, time-consuming, and expensive. In addition, the delay in obtaining sample results as a consequence of the required transport time and need for testing at a specialist laboratory distant from a wastewater sampling site, impacts on the utility of the surveillance network if it does not allow for public health authorities to react early to potential threats. In contrast, biosensors can be designed to give rapid results in an automated fashion, allowing for the possibility of a near real-time in situ sustainable monitoring network when combined with WBE, and bypassing the requirement for routine use of RT-qPCR (Figure 6).

Difficulties regarding the development of biosensors for SARS-Co-2 WBE include the complexity of the sample matrix and the likely low levels of target present. Fabrication of a biosensor would therefore need to consider the incorporation of any pre-processing requirement, prior to target amplification and detection. Further, the “M4” features of Modularity, Multiplexing, Multifunctionality, and Miniaturisation provide a framework when developing a biosensor for WBE. For example, biosensors detecting target genes of SARS-CoV-2, as well as VOCs, VUIs and a positive control, demonstrate the value of multiplexing. Nevertheless, while a biosensor could replace the need for RT-qPCR at times, whole genome sequencing would still need to remain an essential aspect to any future pathogen surveillance network in order to ensure that biosensor design could be reactive to VOCs and VUIs as they are identified. With this in mind, a multiplex biosensor system that allows for the rapid inclusion of additional targets would be of significant value.

In addition to the technological development of the wastewater biosensor, deployment for WBE needs to consider a range of factors relating to the environmental conditions in sewers, the sewerage network configuration, population and ethics. For example, the rapid evolution seen in SARS-CoV2 throughout the pandemic necessitates a sustainable, regenerative approach to biosensor development to account for emerging variants of concern. This would also alleviate issues with environmental waste from obsolete single-use biosensors. The biosensor casing would need to be able to withstand the rapidly changing and often aggressive environmental conditions in sewers, and the design should also minimise the requirement for human entry into sewers during deployment and retrieval. Relating the biosensor sample to a population size should be generally possible from water utility records combined with existing flow gauging, but this also needs to consider the dynamic movements of human population, water use and other contributions to sewer flow from industry and storm events. An ideal scenario could consider a multifunctional biosensor, incorporating sensors to monitor organic loads or other biomarkers which could provide surrogate estimates of population.

The granularity of the unit of analysis and timescale potentially offered by biosensors is epidemiologically attractive, but may also be constrained by the practical issues of services required and the sewer environment alongside ethical considerations. There are ongoing moves by water companies to install more flow monitoring devices in sewers and this may provide more opportunities for greater deployment of other monitoring equipment, such as biosensors, in the future. The optimal strategy for WBE may be low resolution routine sampling at fixed assets for monitoring pathogens in the general population, augmented with high resolution sampling with greater human resource input for significant public health crises, like pandemics.

Finally, to realise the goal of developing biosensors for SARS-CoV-2 WBE, a broad range of transdisciplinary skills are needed, from molecular biologists to civil engineers, bioinformaticians to wastewater infrastructure operators [153]. Whilst WBE was no doubt a growing field prior to the pandemic, COVID-19 has provided the impetus needed to connect these diverse skills sets together, focused around addressing a major global challenge (Figure 6). The energy of individuals coming together to share their collective skills, and unite in tackling a common goal for the greater public good, is not to be underestimated. With such transdisciplinary connections made and embedded at a time of crisis, significant progress is now being made towards realising the ambition. Yet, with such connections now in place, the future for biosensors for WBE looks set to extend far beyond SARS-CoV-2.