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Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Illicit drug use has a global concern and effective monitoring and interventions are highly required to combat drug abuse. Wastewater-based epidemiology WBE is an innovative and cost-effective approach to evaluate community-wide drug use trends, compared to traditional population surveys. Here we report for the first time, a novel quantitative community sewage sensor namely DNA-directed immobilization of aptamer sensors, DDIAS for rapid and cost-effective estimation of cocaine use trends via WBE. Thiolated single-stranded DNA ssDNA probe was hybridized with aptamer ssDNA in solution, followed by co-immobilization with 6-mercapto-hexane onto the gold electrodes to control the surface density to effectively bind with cocaine. The concentration pattern of the sampling week is comparable with that from mass spectrometry. Our results demonstrate that the developed DDIAS can be used as community sewage sensors for rapid and cost-effective evaluation of drug use trends, and potentially implemented as a powerful tool for on-site and real-time monitoring of wastewater by un-skilled personnel. Wastewater-based epidemiology WBE has been shown to be an innovative and promising approach for a cost-effective temporal evaluation of drug use trends 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , compared to the conventional population surveys method. In the past decade, a variety of chemicals such as illicit drugs, prescribed pharmaceuticals and new pharmacologically active substances, have been analyzed in wastewater collected from wastewater treatment plants with the aim to evaluate community-wide use trends such as illicit drugs, tobacco and alcohol use 3 , 4 , 7. The spatial difference and temporal drug use changes have been evaluated across Europe via the measurement of specific markers in wastewater from 42 cities in 21 European countries total population The analytical tools for drug use trends utilize mass spectrometry-based techniques due to their robustness, sensitivity and selectivity. The chemical compounds or their metabolites, so-called biomarkers, present in wastewater can be reliably quantified using an internal reference such as a deuterated analogue of target analyte and low-resolution mass spectrometry e. Non-targeted analysis is usually undertaken with the usage of high resolution mass spectrometry However, troublesome sample purification, costly measurements and the requirement for well-trained personnel may burden the assessment process. To this end, there is a great need for novel analytical tools to perform rapid and on-site analysis of wastewater with minimal sample processing by un-skilled personnel. Furthermore, the rapid monitoring with community sensors may minimize the uncertainty of WBE resulting from low stability of certain markers 6 , Biosensing has emerged as a novel and powerful analytical technology in the fields of food safety, drug discovery, healthcare and environmental monitoring A biosensor is a small device with a biological receptor peptide, DNA, protein, aptamer, etc. Electrochemical biosensors, in particular, provide a rapid response time, low cost and ability to be miniaturized with other portable devices for the on-site measurement requiring minimal sample processing even by un-skilled personnel. It is hypothesized that community sensors will play a crucial role in in situ quantitative analysis of chemicals, biomarkers and pathogens in wastewater, for the purpose of evaluation of community-wide drug use trends and monitoring of public health More importantly, community sewage sensors could provide real-time and continuous data for the government agencies in the monitoring of drug use trends, and may serve as an early warning sensing system for the agencies to make effective interventions. In particular, electrochemical sensors have shown a great promise in the detection of wastewater biomarkers such as mitochondrial DNA For instance, we recently developed a new label-free electrochemical DNA E-DNA biosensor utilizing a custom synthesized ferrocenyl intercalator as a transducer, which allows the detection of cancer associated human-specific mitochondrial DNA in wastewater and the identification of potential population biomarkers for monitoring of public health Cocaine abuse is regarded as a global challenge, and the concentration in wastewater has been found to be in the range of 0. It is highly desirable to develop simple, rapid and cost-effective tools for the sensitive detection of cocaine. Sensors could play a vital role in monitoring cocaine abuse due to several advantages over conventional mass spectrometry-based analytical techniques. Aptamer sensors aptasensors have been employed, due to their low cost and simple design, as a new biosensing platform for the detection of cocaine in different matrices in the past decade 15 , 16 , An aptamer is an artificial single-stranded DNA or RNA oligonucleotide that is able to bind a specific molecular target with high affinity. Zhang et al. In this sensing platform, the recognition of cocaine by aptamer-modified electrodes changes the electron transfer resistance associated with the cocaine concentration. Cocaine in undiluted human saliva samples was also detected with a compact, integrated optofluidic system with combination of multiphase liquid-liquid extraction methods Du et al. This electrochemical aptasensor can detect cocaine levels as low as 0. However, the challenges of aptasensors remain on the effective immobilization of ssDNA probe on the electrode surface and prevention from the formation of secondary or tertiary DNA structures and fouling onto the electrode surface. In this paper, we present a novel approach to effectively immobilize aptamer on a gold electrode surface for the electrochemical detection of cocaine in wastewater. A DNA-directed immobilization aptamer sensor DDIAS was further implemented for the detection of small molecules such as cocaine on the basis of previously established immobilization strategy for the detection of proteins The matrix effects on the aptasensor were determined, and DDIAS was used for the detection of cocaine in wastewater prior to a simple pre-concentration step. The evaluation of cocaine occurrence concentration in wastewater demonstrated that the drug use during weekend was larger than that on working days, and the results were comparable with mass spectrometry data. To the best of our knowledge, it is the first time an aptasensor has been reported for the analysis of cocaine in wastewater with an aim of the evaluation of community-wide cocaine use trends via WBE. The DNA-directed immobilization DDI has been shown to be a promising strategy for the elaboration of biosensors to detect protein and antigen 23 , 24 , However, the introduction of probes onto the DNA tail requires complicated synthesis and purification steps, and is likely to influence the affinity of the probe molecules. In our recent report 22 , we implemented the strategy using an aptamer to replace the DNA tailed probe for the specific detection of prostate specific antigen PSA , and in-solution method within DDIAS is able to detect PSA as low as fM range due to a favorable binding medium in a homogeneous solution. Compared to typical direct immobilization of aptamer onto electrodes, DDIAS simplified the elaboration of the biosensor and the linear linkage of DNA allows sufficient access for the active site of the aptamer to recognize the target Furthermore, a filtration device was employed to remove the unbound aptamer in solution pre-assemble onto chips to minimize the spurious effects. In this paper, we aimed to implement on-chip approach within DDIAS for the specific detection of small molecules such as cocaine, in order to demonstrate a generic sensing immobilization strategy that can be applied to monitor other biomarkers within WBE. The binding of cocaine to the aptamers causes a change in overall charge distribution on the electrode surface, which in turn affects the electrostatic barrier that the negatively charged redox couple in solution needs to overcome in order to exchange an electron with the electrode — i. Before binding with cocaine, the DNA aptamer adopts a so-called two-stem loop; however, upon cocaine binding it forms a further loop entrapping the cocaine molecule so-called three-way junction 17 , 26 see illustration in Fig. Therefore, the R ct shifts to a high value upon the formation of three-way junction. This was associated with the fact that the dsDNA aptamer probes within on-chip II has less steric hindrance for cocaine to be pocketed and thus allow more available aptamer to be exposed to bind with cocaine molecules on the electrode surface. Besides, it is likely that the dsDNA may prevent the two-stem loop structure of DNA aptamer fouling onto the electrode surface. Consequently, the on-chip II approach has a great promise for cocaine detection. As shown in Fig. The electrodes modified with ssDNA probe are negatively charged, and the redox maker diffusion to the electrode surface is hindered due to repulsive interactions. Additionally, co-immobilization of MCH serves as limiting non-specific adsorption, which is vital for the detection in complex samples. Keighley et al. The surface densities of ssDNA and dsDNA with the optimized molar fraction are in the low range, which enables efficient attachment of the target molecule as demonstrated by many studies 27 , 28 , However, the optimized molar fraction of dsDNA aptamer probe for targeting cocaine shows an even lower probe density than that of ssDNA for hybridization with complementary DNA. This is associated with the fact that the conformation of aptamer will change to the three-way junction form after binding with cocaine, which may need more available spaces on the surface. Under the optimized conditions, DDIAS was employed to detect various concentrations of cocaine for evaluation of limit of detection LOD and dynamic range. The LOD was estimated to be around 0. Sheng et al. This conformation changes induce significant shift of charge transfer resistance, and LOD at 0. However, the process of assemble and de-assemble 3D DNA nanostructures is difficult to control and the regeneration was not evaluated. Compared to other aptasensors based on the 2D conformation change upon binding with cocaine, our DDIAS is able to achieve a sensitive detection of cocaine without labeling and signal amplification. More importantly, the immobilization strategy was robust and DDIAS allows the re-usability of the sensors. This indicates that developed cocaine aptasensor within DDIAS using on-chip II method is also able to withstand salt solutions and will be beneficial for the detection of cocaine in various samples such as wastewater, which has the same profile of DDIAS using in-solution approach for detection of PSA The heating step unfolds the aptamer and releases the captured cocaine molecules The sensor chips were then re-used to detect cocaine. We also found that the effect of solvent has an effect on the binding efficiency between aptamer and cocaine. Acetonitrile ACN is a widely used stock solvent for commercialized cocaine products for research purposes. Kang et al. Typically, the percentage of ACN at 0. In the lower cocaine concentration range within ACN percentage less than 0. These results are in good agreement with the affinity study described in reference 32 , and this should be taken into account in the development of cocaine aptasensor. Figure 5A shows the matrix effects on the spiked cocaine in buffer, tap water and wastewater. The response from the wastewater was negligible, indicating good anti-fouling properties of the sensor. This suggests that our DDIAS is promising to limit the non-specific adsorption and the high affinity of aptamer could selectively recognize cocaine in wastewater. The optimized DDIAS was used to detect cocaine in wastewater collected daily during one week monitoring, with an aim to quantify cocaine for estimation of drug consumption at the community level. Therefore, wastewater was pre-concentrated times using solid phase extraction SPE in order to allow for the quantification of cocaine using the aptasensor. The calibration curve Fig. The Hill equation assumes a sigmoidal shape that can be approximated well by means of the 4-parameter non-linear logistic equation, which is commonly employed to fit biological dose response curves The cocaine consumption during the weekend is usually higher than those during working days from most of European cities 5 , 7. However, it is essential to take into account other parameters such as rainfall and variable flow of wastewater that can affect concentrations of cocaine in wastewater for the estimation of the real cocaine daily load. Calibration curve of cocaine in wastewater using a non-linear fit Hill 1 for dose growth a and quantification of cocaine concentration in wastewater collected from seven consecutive days within a week using developed DDIAS and mass spectrometry b. In order to validate the results and estimate the cocaine consumption in a defined population, a chromatography assay coupled with tandem triple quadrupole mass spectrometry was performed to quantify the concentration of cocaine in wastewater. Furthermore, we hope to develop a simple portable pre-concentration device for on-site detection of wastewater coupled with an integrated fast and selective sensor assay, which may enable the quantification of the cocaine concentration pattern in real-time. As discussed above, the quantitative mass spectrometry analysis confirmed high selectivity, accuracy and precision of the developed sensor and proved that such sensors can be used for the rapid estimation of the community-wide drug consumption. We believe that this rapidly acquired data could also be vital for estimation of a threshold value for the preliminary indication of cocaine abuse in a defined community, which may be of great help for the government agencies to make effective interventions for illicit drug abuse. As a result, we demonstrated that an aptasensor could be used for cost-effective and fast estimation of community-wide drug use trends using WBE, and with a strong potential to be miniaturized for a portable assay at the site of sample collection. The surface density of aptamer probe was optimized by co-immobilization of MCH with dsDNA for an efficient binding with cocaine. We used the optimized DDIAS to detect cocaine in wastewater collected during 7 consecutive days, and quantification of the cocaine concentration pattern shows that it has higher cocaine consumption during the weekend than that on weekdays. The results are in good agreement with those analyzed by means of mass spectrometry. We believe that community sewage sensors will open a new direction in monitoring of drug use trends and public health such as obesity, cancer and cardiovascular disease. Gold disc working electrodes with a radius of 1. DNA aptamer against cocaine was employed for the detection of cocaine elsewhere 16 , 17 , The reference standard of cocaine Mw Cocaine-D 3 M w All the solvents were of HPLC grade. The buffer for immobilization of ssDNA probe consisted of 0. The influent daily hour composite raw wastewater was collected from a local wastewater treatment plant consecutively in a week from March 10 th to 16 th , The wastewater was filtrated and pre-concentrated with a solid phase extraction SPE , as described elsewhere The samples were analyzed immediately after SPE. Electrodes were electrochemically cleaned in a classical three-electrode cell as described elsewhere 14 , 22 , In brief, electrodes were immersed in H 2 SO 4 0. The aptamers were synthesized with a tail of twelve nucleotides as a complementary sequence to the ssDNA probe. For wastewater measurement, a centrifugal filtration device Amicon Ultra Nitrogen was used as the nebulizing gas, supplied by a high purity nitrogen generator Peak Scientific, UK. Argon MassLynx 4. Cocaine identification was assessed according to the European Guidelines concerning the performance of analytical methods and the interpretation of results. The method detection limit MDL was calculated using the following formula:. The method quantification limit MDL was calculated using the following formula:. The response was linear for cocaine. How to cite this article : Yang, Z. Zuccato, E. Estimating community drug abuse by wastewater analysis. Environ Health Perspect , — Cocaine in surface waters: a new evidence-based tool to monitor community drug abuse. Article Google Scholar. Castiglioni, S. A novel approach for monitoring tobacco use in local communities by wastewater analysis. Tob contr 24 , 38—42 Reid, M. Using biomarkers in wastewater to monitor community drug use: A conceptual approach for dealing with new psychoactive substances. Sci Total Environ , — Ort, C. Spatial differences and temporal changes in illicit drug use in Europe quantified by wastewater analysis. Addiction , — Testing wastewater to detect illicit drugs: State of the art, potential and research needs. Thomas, K. Comparing illicit drug use in 19 European cities through sewage analysis. Daughton, C. Using biomarkers in sewage to monitor community-wide human health: Isoprostanes as conceptual prototype. Sci Total Environ , 16—38 Environ Sci Technol 45 , — Sato, S. Selective immobilization of double stranded DNA on a gold surface through threading intercalation of a naphthalene diimide having dithiolane moieties. Anal Chim Acta , 91—97 Environ Sci Technol 47 , — Kirsch, J. Biosensor technology: recent advances in threat agent detection and medicine. Chem Soc Rev 42 , — Yang, Z. Community sewage sensors for monitoring public health. Environ Sci Technol 49 , — Mokhtarzadeh, A. Nanomaterial-based cocaine aptasensors. Biosens Bioelectron 68 , 95— Stojanovic, M. Aptamer-based colorimetric probe for cocaine. J Am Chem Soc , — Aptamer-based folding fluorescent sensor for cocaine. Zhang, D. Talanta 92 , 65—71 Wagli, P. Microfluidic droplet-based liquid-liquid extraction and on-chip IR spectroscopy detection of cocaine in human saliva. Anal Chem 85 , — Jenkins, A. Comparison of heroin and cocaine concentrations in saliva with concentrations in blood and plasma. J Anal Toxicol 19 , — Du, Y. Anal Chem 82 , — A novel immobilization strategy for electrochemical detection of cancer biomarkers: DNA-directed immobilization of aptamer sensors for sensitive detection of prostate specific antigens. Analyst , — Wacker, R. Performance of antibody microarrays fabricated by either DNA-directed immobilization, direct spotting, or streptavidin-biotin attachment: a comparative study. Analytical Biochemistry , — Chevolot, Y. DNA-based carbohydrate biochips: A platform for surface glyco-engineering. Angew Chem Int Edit 46 , — Chembiochem 5 , — Baker, B. An electronic, aptamer-based small-molecule sensor for the rapid, label-free detection of cocaine in adulterated samples and biological fluids. Keighley, S. Optimization of DNA immobilization on gold electrodes for label-free detection by electrochemical impedance spectroscopy. Biosens Bioelectron 23 , — Zhang, J. Steel, A. Electrochemical quantitation of DNA immobilized on gold. Anal Chem 70 , — Kukol, A. Label-free electrical detection of DNA hybridization for the example of influenza virus gene sequences. Ana Biochem , — Sheng, Q. Ultrasensitive electrochemical cocaine biosensor based on reversible DNA nanostructure. Biosens Bioelectron 51 , — Kang, K. Langmuir 27 , — Gadagkar, S. Computational tools for fitting the Hill equation to dose-response curves. J Pharm Tox Meth 71 , 68—76 Liu, J. Fast colorimetric sensing of adenosine and cocaine based on a general sensor design involving aptamers and nanoparticles. Angew Chem Int Edit 45 , 90—94 Emke, E. Baker, D. Drugs of abuse in wastewater and suspended particulate matter - Further developments in sewage epidemiology. Environ Int 48 , 28—38 Download references. Thet and Dr A. Jenkins for making the potentiostat available in the study. Support from Wessex Water is also acknowledged. You can also search for this author in PubMed Google Scholar. All authors had comments of the manuscript and given approval to the final version of the manuscript. Correspondence to Zhugen Yang or Christopher G. This work is licensed under a Creative Commons Attribution 4. Reprints and permissions. Sci Rep 6 , Download citation. Received : 29 October Accepted : 14 January Published : 15 February Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily. Skip to main content Thank you for visiting nature. Download PDF. Subjects Ecological epidemiology Environmental chemistry. Abstract Illicit drug use has a global concern and effective monitoring and interventions are highly required to combat drug abuse. Urban wastewater analysis as an effective tool for monitoring illegal drugs, including new psychoactive substances, in the Eastern European region Article Open access 17 March Mining chemical information in Swedish wastewaters for simultaneous assessment of population consumption, treatment efficiency and environmental discharge of illicit drugs Article Open access 29 June Longer amplicons provide better sensitivity for electrochemical sensing of viral nucleic acid in water samples using PCB electrodes Article Open access 25 May Introduction Wastewater-based epidemiology WBE has been shown to be an innovative and promising approach for a cost-effective temporal evaluation of drug use trends 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , compared to the conventional population surveys method. Figure 1. Full size image. Figure 2. Figure 3. Figure 4. Effects of solvent on the binding efficiency between cocaine and the aptamer. Figure 5. Figure 6. Full size table. Methods Materials Gold disc working electrodes with a radius of 1. Sample collection and preparation The influent daily hour composite raw wastewater was collected from a local wastewater treatment plant consecutively in a week from March 10 th to 16 th , Additional Information How to cite this article : Yang, Z. References Zuccato, E. Article Google Scholar Castiglioni, S. Article Google Scholar Reid, M. View author publications. Ethics declarations Competing interests The authors declare no competing financial interests. Rights and permissions This work is licensed under a Creative Commons Attribution 4. About this article. Cite this article Yang, Z. Copy to clipboard. Comments By submitting a comment you agree to abide by our Terms and Community Guidelines. Publish with us For authors Language editing services Submit manuscript. Search Search articles by subject, keyword or author. Show results from All journals This journal. Advanced search. Close banner Close. 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