Acknowledgements
This work is supported by Guangdong Scientific and Technological Project (2022A0505030024, 2022B1515120012), National Key Research and Development Program of China (2022YFB3207200), National Natural Science Foundation of China (62105221), Science and Technology Innovation Commission of Shenzhen (JCYJ20220818102618040, JCYJ20200109105608771), and Shenzhen Science and Technology Innovation Commission Sustainable Development Project (KCXFZ20211020163814021).
Yiming Zhang, Zhi Chen and Songrui Wei contributed equally to this work.
Abstract: Real-time polymerase chain reaction (RT-PCR) remains the most prevalent molecular detection technology for sewage analysis but is plagued with numerous disadvantages, such as time consumption, stringent equipment requirements, and susceptibility to false negatives. In this study, we construct an automated robot-driven photoelectrochemical biosensing platform that utilizes the CRISPR/Cas12a system, to achieve fast, ultrasensitive, high specificity detection of biological loads in sewage. The Shennong-1 robot integrates several functional modules, involving sewage sampling and pretreatment to streamline the sewage monitoring. A screen-printed electrode is employed with a vertical graphene-based working electrode and enhanced with surface-deposited Au nanoparticles (NPs). CdTe/ZnS quantum dots (QDs) are further fabricated through the double-stranded DNA anchored on Au nanoparticles. Using cDNA template of Omicron BA.5 spike gene as a model, the photoelectrochemical biosensor demonstrates excellent analytical performance, with a lower detection limit of 2.93×102 zM and an outstanding selectivity at the level of single-base mutation recognition. Furthermore, the rapid, accurate detection of BA.5 in sewage demonstrates the feasibility of the photoelectrochemical platform for sewage monitoring. In conclusion, this platform allows early detection and tracking of infectious disease outbreaks, providing timely data support for public health institutions to take appropriate prevention and control measures.
Keywords: robot automation, CRISPR/Cas12a system, photoelectrochemical biosensor, nucleic acid detection, sewage monitoring
1. Introduction
Sewage, a unique interface between human beings and the natural environment, is generated by human activities and responsible for a considerable biological load, primarily from feces, saliva, and sputum. During the outbreak of the COVID-19 pandemic, numerous studies reported the presence of a large quantity of COVID-19 virus in sewage[1-3]. In this context, biological load, particularly with respect to microbial pathogens, has attracted unprecedented attention[4,5]. On a global scale, microbial pathogens present a substantial obstacle to public health security and place a significant financial burden on the healthcare system[6,7], particularly given that three out of the top ten global health threats identified by the World Health Organization in 2019 were directly linked to pathogens: pandemic influenza, Ebola, and HIV[8]. Sewage is an essential medium for capturing, accumulating, and transmitting pathogens[9-11]; therefore, sewage has become an ideal medium for monitoring the pathogens of the entire community, and a powerful tool called wastewater-based epidemiology has been developed to provide early warning for the emergence of disease and track the outbreak trend[5,12]. Various pathogens have been found in sewage worldwide, including nearly all types of bacteria that can cause human diseases, as well as viruses that have caused significant outbreaks in recent epidemics and pandemics[10,13,14]. Thus, it is extremely essential to establish a sewage monitoring platform for surveillance of various pathogens.
RT-PCR is the most commonly employed molecular detection technique in sewage analysis, alongside other technologies utilized for this purpose[15]. RT-PCR demonstrates advantageous qualities such as early diagnosis, high sensitivity, and high specificity. However, this method has many disadvantages, including its time-consuming nature, substantial technician requirements, the need for laboratory cleanliness, testing equipment requirements, and susceptibility to false negative results that are inconsistent with clinical manifestations[16,17]. In addition, traditional sewage sampling, enrichment, concentration, nucleic acid extraction, and amplification lack the support of automation technology, requiring an investment of manpower and time, significantly reducing the timeliness of sewage monitoring. Therefore, developing a rapid, highly specific, and ultrasensitive surveillance platform for continuous monitoring of biological loads in sewage is necessary to obtain valuable information to effectively support disease treatment, prevention, and control.
Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) technology has led to its wide-spread recognition due to its potential application in molecular biological diagnosis[18,19]. Compared with traditional molecular diagnosis methods, the CRISPR/Cas nucleic acid detection system has higher sensitivity, specificity, and resolution[20]. Moreover, the CRISPR/Cas nucleic acid detection system has a particular universality in biological nucleic acid detection[21]. Different CRISPR/Cas systems, including DETECTR, HOLMES, and SHERLOCK, have been employed in the development of various nucleic acid detection methods[20,22-25]. Recently, researchers have been incorporating CRISPR/Cas system with various detection platforms to enhance the sensitivity of nucleic acid detection methods[26-29]. Photoelectrochemical (PEC) analysis has gained significant recognition as a versatile and promising analytical technology in various fields[30-32]. Because the light emission source and electrical signal readout are completely separate, PEC sensors have notable benefits including high sensitivity, minimal background interference, rapid response time, and affordability[33,34].
Therefore, an automated robot-driven PEC biosensing platform based on a CRISPR/Cas12a system was constructed to meet the need for rapid, accurate detection of biological loads in sewage. The robot has the capacity to automate the complete process, which includes sampling, enrichment, concentration, nucleic acid extraction, and reverse transcription. The electrode used was a screen-printed electrode (SPE), and the working electrode was based on vertical graphene with surface-deposited Au NPs. With the assistance of these Au NPs, double-stranded DNA (dsDNA) was fixed on the electrode surface. Furthermore, CdTe/ZnS QDs were fabricated on the electrode via an amide bond formed between the surface carboxylic acid groups of CdTe/ZnS QDs and amino groups modified at the 5′ end of dsDNA, and the resulting electrode denoted as CdTe/ZnS QDs–dsDNA/Au NPs/rGO (reduced graphene oxide) electrode was obtained. When the Cas12a–crRNA duplex was subjected to the target sequence, it specifically recognized and combined with the target sequence, and trans-cleavage activity of Cas12a was initiated. The dsDNA fixed on the electrode was nonspecifically cut, resulting in CdTe/ZnS QDs detaching from the electrode and reducing the photocurrent. The designed automated robot-driven PEC biosensing platform showed excellent analytical performance in the ultrasensitive detection of Omicron BA.5 and potential application prospects in monitoring sewage.