key: cord-0736022-6plkpfjr authors: Ramírez-Chavarría, Roberto G.; Castillo-Villanueva, Elizabeth; Alvarez-Serna, Bryan E.; Carrillo-Reyes, Julián; Ramírez-Zamora, Rosa María; Buitrón, Germán; Alvarez-Icaza, Luis title: Loop-mediated isothermal amplification-based electrochemical sensor for detecting SARS-CoV-2 in wastewater samples date: 2022-02-28 journal: J Environ Chem Eng DOI: 10.1016/j.jece.2022.107488 sha: bba123582176b8612936c085d567c2e9b13e67b1 doc_id: 736022 cord_uid: 6plkpfjr The current pandemic COVID-19 caused by the coronavirus SARS-CoV-2, has generated different economic, social and public health problems. Moreover, wastewater-based epidemiology could be a predictor of the virus rate of spread to alert on new outbreaks. To assist in epidemiological surveillance, this work introduces a simple, low-cost and affordable electrochemical sensor to specifically detect N and ORF1ab genes of the SARS-CoV-2 genome. The proposed sensor works based on screen-printed electrodes acting as a disposable test strip, where the reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction takes place. Electrochemical detection relies upon methylene blue as a redox intercalator probe, to provide a diffusion-controlled current encoding the presence and concentration of RT-LAMP products, namely amplicons or double-stranded DNA. We test the performance of the sensor by testing real wastewater samples using end-point and time course measurements. Results show the ability of the electrochemical test strip to specifically detect and quantify RT-LAMP amplicons below to ~ 2.5 × 10(−6) ng/μL exhibiting high reproducibility. In this sense, our RT-LAMP electrochemical sensor is an attractive, efficient and powerful tool for rapid and reliable wastewater-based epidemiology studies. The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses, has rapidly spread worldwide bringing serious consequences for human life and the global economy [1] . Since the outbreak, SARS-CoV-2 pandemic has infected more than 239 million people worldwide resulting in death of about 4.8 million people (https://covid19.who.int/). Hence, curbing the spread of infection is paramount at present. Testing is a key starting point to contain COVID-19 transmission and accurate SARS-CoV-2 detections. Nevertheless, clinical testing is more expensive and time consuming to detect new outbreaks than testing of wastewater for the presence of SARS-CoV-2 [2, 3, 4, 5] . Currently, for detecting and quantifying SARS-CoV-2 one can nd, rapid antigen detection (RAD) [6] , rapid detection of antibodies (RDA) [7] and molecular detection [8] . This latter is the most reliable method to detect the presence SARS-CoV-2 due to its specicity [9, 10] . On the other hand, testing of wastewater for the presence of SARS-CoV-2 nucleic acid as a surveillance and management tool is essential to slow the spread of the virus [11] . SARS-CoV-2 may enter wastewater systems from pathogen shedding in human waste, resulting in a potentially fecal-oral transmission with a serious health consequence [12, 13, 14] . Recently, several groups in dierent countries isolated and detected the genetic material of SARS-CoV-2 in wastewater using reverse transcription quantitative polymerase chain reaction (RT-qPCR), as a gold standard technique [15, 16, 17] . However, one important problem is that RT-qPCR still requires expensive laboratory infrastructure and skilled technicians or scientists to complete the assay. Furthermore, 2 J o u r n a l P r e -p r o o f more eorts are needed to develop rapid and accurate detection tools for wastewater surveillance and management of the SARS-CoV-2 spread using molecular diagnostics in limited-resources settings [18] . In this context, it results imperative the application of versatile and aordable tools to detect viral or microbiological pathogens in environmental samples in a fast and sensitive manner [19] . The nucleic acid based isothermal amplication methods have been extensively deployed as a sensitive and straightforward techniques [20] . Specically, loopmediated isothermal amplication with simultaneous reverse-transcription (RT-LAMP) allows for rapid and analytically sensitive detection of nucleic acids within one hour that requires only a heat source [21] . Several groups are currently developing LAMP-based protocols for the detection of SARS-CoV-2 in clinical samples [22, 23, 24, 25] . However, scarce information is reported about RT-LAMP technique as a cheaper and faster option for monitoring the genetic material of SARS-CoV-2 in wastewater-based epidemiology [26] , and its integration with attractive sensing schemes. Among the wide variety of transduction mechanisms for detecting nucleic acid amplication, one can nd electrochemical-based sensors [27, 28] , optical devices [29] , colorimetric assays [30] , luminescence-based sensors [31] , and surface plasmon resonance apparatus [32] , to mention only a few. Particularly, electrochemical transduction has demonstrated its ability to provide a cost-eective alternative to circumvent manufacturing and integration processes to robust devices. Moreover, electrochemical sensors exhibit several advantages such as low-cost, portability, miniaturization and high reliability, ideal for in-situ measurements [33, 34] . Nevertheless, common electrochemical biosensors need a labeled receptor to be immobilized on the sensitive element or electrode [35, 36] . Due to the COVID-19 pandemic, several investigations have been devoted to simplify the experimental processes and methods to provide aordable and versatile platforms suitable for a easy-to-develop sensors in resource-limited or eld settings [37] . For instance, the work 3 J o u r n a l P r e -p r o o f in [38] shows the trends in electrochemical sensors for rapid detection of SARS-CoV-2 from human samples focusing on viral nucleic acid, immunoglobulin, antigen, and the entire viral particles. Monitoring and detecting SARS-CoV-2, however, remain a challenging task, even more when testing environmental samples due to its complex structure. To overcome the diculties encountered in classical benchtop equipment and methods, herein we report the development and potentiality of an electrochemical sensor for detecting SARS-CoV-2 in wastewater samples. To the best of our knowledge, the sensor innovates in the following aspects: By working around a RT-LAMP reaction, it is a cost-eective and less-time consuming alternative to the classical RT-PCR amplication, without losing specicity. It works around screen-printed electrodes (SPEs) and minimal instrumentation, which is a current trend for eld-deployable and low-cost detection systems. The sensor is primarily devoted to retrieve end-point results for detecting RT-LAMP amplicons, and additionally, shows promising results for real-time quantication. Its potentiality is demonstrated by measuring real wastewater samples for a current sanitary problem, which is an underestimated application for electrochemical detection devices. Summarizing, the proposed device acts as a test strip to detect, previously concentrated and extracted, nucleic acid fragments of SARS-CoV-2 by monitoring the diusion- The rest of the paper is organized as follows. In Section 2 we introduce the methods for sample collection, RNA concentration and extraction, as well as the monitoring of RT-LAMP reaction with electrochemical transduction. Section 3 shows the experimental results to assess the performance of the proposal alongside a thorough discussion. Finally, Section 4 is devoted to the conclusions. Figure 1 shows the device workow comprising four main stages: i) wastewater sampling, ii) RNA concentration, iii) RT-LAMP mixture, and iv) the electrochemical monitoring of RT-LAMP reaction. First, the samples are collected and then the nucleic acids are extracted and concentrated using a custom-developed method [18] . Together these methodologies take a time of 1h 30 min in the laboratory with minimum infrastructure. Afterwards, the RNA is mixed with the RT-LAMP primers and methylene blue (MB) as a redox intercalator for the electrochemical transduction. Hence, a micro-volume sample is drop cast over the surface of custom fabricated screen-printed electrodes (SPEs), wherein the RT-LAMP reaction takes place by controlling the local temperature at 63°C . Thereby, the resultant diusion-controlled current, promoted by the redox process, is monitored by a portable potentiostat to provide a measure of the RT-LAMP reaction. The amplication and monitoring take approximately 30 minutes. Finally, the peak current change %∆I p encodes the concentration of the nucleic acids according to the sensor model. Jointly, the turnaround time for a complete experiment is 2 hours. To validate the electrochemical monitoring performance, a colorimetric assay was simultaneously performed on the evaluated wastewater samples. The sampling was carried out at two wastewater treatment plants (WTTP) in the metropolitan area of the City of Queretaro, Mexico (see Table 1 ). In South and Santa Rosa plants, the inuent was sampled. Samples were collected from the period between May 31 and June 7, 2021. The inuent samples (500 mL) were collected during the morning (9-11 am) and kept at 4°C until their use. J o u r n a l P r e -p r o o f Samples were concentrated the same day of sampling, using the electronegative membrane method owing its detection limit for SARS-CoV-2 genes [18] . Briey, the pH of samples was adjusted to 3.5 with 2N HCl and then were ltered through a negatively charged nitrocellulose membrane (0.45 µm pore diameter, Millipore, Netherlands). According to the manufacturer's instructions, the membranes were cut and used directly in the RNeasy Power Microbiome extraction kit (Qiagen, Germany) for RNA extraction. RNA was stored at -20°C until its use. Table 2 . RT-LAMP reactions were monitored by the electrochemical sensor using MB at 6 µM at 63°C in uniform temperature for 30 min. Also, the reactions of RT-LAMP assays were checked on 1% agarose gel stained with SYBR Safe DNA Gel Stain for the presence of ladder pattern, and the products were also veried by sequencing. Afterwards, the concentration of the RT-LAMP amplied products was estimated using a Nanodrop spectrophotometer. Finally, to test the specicity of the amplication, the products of the RT-LAMP reactions were validated by sequencing (Genbank OM522662). For electrochemical monitoring, MB was added to the RT-LAMP mixture as an electroactive intercalator [40] , exhibiting strong and specic binding ability to dsDNA amplicon without inhibiting the RT-LAMP process. A negative control template (NTC) was composed by RT-LAMP master mix and MB without genome owing the well-know performance of the RNA concentration and extraction [18] . The detection strategy relies upon the measurement of a change in the faradaic current, promoted by the free-to-diuse state of the MB as the RT-LAMP progresses. Thus, the current amplitude is attenuated because the MB becomes less active to an electron exchange following the complex formation with DNA amplicon compared with its free counterpart. As the sensing element, a three electrode electrochemical cell based on screen-printed electrodes (SPEs) was used. This conguration allowed to be treated as a disposable test strip. Therein, the working and counter electrodes were fabricated using carbon paste; whereas, the reference elec- where I m and I 0 are the measured peak currents in the presence of amplicons and for the NTC, respectively. Following this rationale, Figure 2 Table 1 ) and veried the results using gel electrophoresis. Firstly, electrochemical measurements were performed following an end-point procedure. That is, the voltammograms were measured after 30 minutes of the RT-LAMP reaction. Subsequently, we computed the peak current change % ∆I p as in (1) . Figure 3 (a) depicts the results retrieved by our sensor for these three samples. One can see, the peak current change decreased almost 55%, which reects the success of the RT-LAMP reaction. It makes sense, the intercalation of MB to double-stranded amplicons signicantly reduced the concentration of free MB at the electrode surface, and hence, diminished the peak current with respect to the negative control sample. To verify that the end-point measurements were reliable we performed an electrophoresis test on a 1% agarose gel for RT-LAMP N and ORF1ab reaction products, respectively. Fig. 3(b) , one can observe that only the positive reactions resulted in a ladder pattern, while the NTCs did not show any detectable amplicons. Ultimately, by using the calibration curve shown in Fig. 2(a) , we computed the estimated concentrations by our sensor for two SARS-CoV-2 genes, N and ORF1ab, in three samples. To validate the electrochemical sensor, we concurrently performed a colorimetric assay as shown in Fig. 2(c) . Therein, one can see the negative reactions indicated in pink, and how the positive reactions change the color to yellow. As expected, this eect is due to the presence of phenol red within the RT-LAMP reaction mix, which allows a straightforward dierentiation among positive and negative samples. Finally, the concentration of both, electrechmical and colorimetric assays, was veried with the Nanodrop spectrophotometer. Table 3 summarizes the concentration results given by its mean value and uncertainty. These results thereby allow us to conrm that the proposed sensor reproduces well the Nanodrop measurements and agree 12 J o u r n a l P r e -p r o o f with the colorimetric readings. Thus, the sensor accuracy is above 90%, with the largest error for the sample IPS, which is the one more concentrated. End-point measurements allowed to measure the concentration of the amplicons for the N and ORF1ab genes after the amplication process promoted by the RT-LAMP reaction. One should keep in mind that, those concentrations are not the number of copies in the total RNA isolated from the wastewater samples. Hence, this experiment was useful to validate the sensor to only detect the presence of SARS-CoV-2 genome, and to corroborate the concentration of the dsDNA products given by the redox current change due to the RT-LAMP amplication. Though this behavior was to be expected, it is convenient to focus on the last three samples. Therefore, the inset of Fig. 4 The use of wastewater surveillance as an epidemiological tool has raised more interest in detecting the increment of the viral load, which could be related to disease spread in the population. Another approach though, is the near-source tracking, applying the surveillance on a small spatial scale, in vulnerable or higher risk groups, like people in prisons, schools, hospitals and factories. Therein, the only detection of the virus in their wastewater or sewage system is a valuable tool to prevent a local outbreak, followed by targeted clinical tests [45] . For instance, using a conventional RT-qPCR approach monitoring the sewage system for a prison, Carrillo-Reyes et al. [18] were able to detect the presence of the SARS-CoV-2 previous the report of clinical cases by the local health authorities. Following the current trends, the proposed electrochemical sensor showed promising results for detecting SARS-CoV-2 in real wastewater samples. The main advantages of the device are that, it does not require sophisticated infrastructure to succeed; and the electrochemical detection preserve acceptable sensitivity compared with optical methods, but its instrumentation is cost-eective for eld-deployable devices. Following this rationale, the described approach is at least 5000 USD cheaper than a classical setup by replacing the thermocylcer and the optical detection apparatus. In further studies, the proposed sensor can be modied to integrate all the methods, such as concentration and to detect SARS-CoV-2 in low-resource settings for surveillance the COVID-19 spread in environmental scenarios. Finally, the device could be further improved to be an integrated system for stand-alone measurements, and can be extended for detecting other pathogens. Impact of COVID-19 on the social, economic, 17 energy domains: Lessons learnt from a global pandemic COVID-19: Transmission, prevention, and potential therapeutic opportunities Detection of SARS-CoV-2 in Dierent Types of Clinical Specimens First conrmed detection of SARS-CoV-2 in untreated wastewater in Australia: a proof of concept for the wastewater surveillance of COVID-19 in the community Presence of SARS-Coronavirus-2 RNA in Sewage and Correlation with Reported COVID-19 Prevalence in the Early Stage of the Epidemic in The Netherlands Low performance of rapid antigen detection test as frontline testing for COVID-19 diagnosis Antibody responses to SARS-CoV-2 in patients with COVID-19 Strategies and perspectives to develop SARS-CoV-2 detection methods and diagnostics Summary of the available molecular methods for detection of SARS-CoV-2 during the ongoing pandemic Comparison of 12 molecular detection assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Concerns and strategies for wastewater treatment during COVID-19 pandemic to stop plausible transmission Review on the contamination of wastewater by COVID-19 virus: Impact and treatment A year into the COVID-19 pandemic: Rethinking of wastewater monitoring as a preemptive approach SARS-CoV-2 in wastewater: potential health risk, but also data source SARS-CoV-2 in water services: Presence and impacts Presence and infectivity of SARS-CoV-2 virus in wastewaters and rivers Detection of SARS-CoV-2 in wastewater in japan during a COVID-19 outbreak Surveillance of SARS-CoV-2 in sewage and wastewater treatment plants in Mexico Recent advances in biosensors for detecting viruses in water and wastewater Loop-Mediated Isothermal Amplication (LAMP): A Rapid, Sensitive, Specic, and Cost-Eective Point-of-Care Test for Coronaviruses in the Context of COVID-19 Pandemic Loop-mediated isothermal amplication of DNA Clinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection RT-LAMP for rapid diagnosis of coronavirus SARS-CoV-2 Rapid detection of novel coronavirus/severe acute respiratory syndrome coronavirus 2 (sars-cov-2) by reverse transcription-loop-mediated isothermal amplication Rapid Detection of COVID-19 Coronavirus Using a Reverse Transcriptional Loop-Mediated Isothermal Amplication (RT-LAMP) Diagnostic Platform RT-LAMP: A Cheaper, Simpler and Faster Alternative for the Detection of SARS-CoV-2 in Wastewater A nanoscale genosensor for early detection of COVID-19 by voltammetric determination of RNA-dependent RNA polymerase (RdRP) sequence of SARS-CoV-2 virus An electrochemical biosensor for 21 of the SARS-CoV-2 RNA HRPZyme assisted recognition of SARS-CoV-2 infection by optical measurement (HARIOM) Colorimetric detection of SARS-CoV-2 and drug-resistant ph1n1 using crispr/dcas9 Entropy-driven amplied electrochemiluminescence biosensor for RdRp gene of SARS-CoV-2 detection with self-assembled DNA tetrahedron scaolds Biosensing amplication by hybridization chain reaction on phase-sensitive surface plasmon resonance Microbial electrochemical sensors for volatile fatty acid measurement in high strength wastewaters: A review Recent advances in biosensors for detecting viruses in water and wastewater Prospects and challenges of using electrochemical immunosensors as an alternative detection method for SARS-CoV-2 wastewater-based epidemiology An integrated biosensor system with mobile health and wastewater-based epidemiology (iBMW) for COVID-19 pandemic Point of care detection of covid-19: Advancement in biosensing and diagnostic methods Electrochemical sensors for the detection of sars-cov-2 virus A Single and Two-Stage, Closed-Tube, Molecular Test for the 2019 Novel Coronavirus (COVID-19) at Home, Clinic, and Points of Entry Real-time electrochemical lamp: a rational comparative study of dierent dna intercalating and non-intercalating redox probes Time-constant-domain spectroscopy: An impedance-based method for sensing bio-23 in suspension Real-time electrochemical detection of pathogen DNA using electrostatic interaction of a redox probe Wastewater-Based Epidemiology for Community Monitoring of SARS-CoV-2: Progress and Challenges Revisiting the sigmoidal curve tting applied to quantitative real-time pcr data Innovation in wastewater near-source tracking for rapid identication of COVID-19 in schools This work was supported by the grant DGAPA-UNAM-PAPIIT TA100221.