Paper Device Combining CRISPR/Cas12a and Reverse-Transcription Loop-Mediated Isothermal Amplification for SARS-CoV-2 Detection in Wastewater.

Wastewater-based surveillance of the COVID-19 pandemic holds great promise; however, a point-of-use detection method for SARS-CoV-2 in wastewater is lacking. Here, a portable paper device based on CRISPR/Cas12a and reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with excellent sensitivity and specificity was developed for SARS-CoV-2 detection in wastewater. Three primer sets of RT-LAMP and guide RNAs (gRNAs) that could lead Cas12a to recognize target genes via base pairing were used to perform the high-fidelity RT-LAMP to detect the N, E, and S genes of SARS-CoV-2. Due to the trans-cleavage activity of CRISPR/Cas12a after high-fidelity amplicon recognition, carboxyfluorescein-ssDNA-Black Hole Quencher-1 and carboxyfluorescein-ssDNA-biotin probes were adopted to realize different visualization pathways via a fluorescence or lateral flow analysis, respectively. The reactions were integrated into a paper device for simultaneously detecting the N, E, and S genes with limits of detection (LODs) of 25, 310, and 10 copies/mL, respectively. The device achieved a semiquantitative analysis from 0 to 310 copies/mL due to the different LODs of the three genes. Blind experiments demonstrated that the device was suitable for wastewater analysis with 97.7% sensitivity and 82% semiquantitative accuracy. This is the first semiquantitative endpoint detection of SARS-CoV-2 in wastewater via different LODs, demonstrating a promising point-of-use method for wastewater-based surveillance.

Reprogrammed tracrRNAs enable repurposing of RNAs as crRNAs and sequence-specific RNA biosensors.

In type II CRISPR systems, the guide RNA (gRNA) comprises a CRISPR RNA (crRNA) and a hybridized trans-acting CRISPR RNA (tracrRNA), both being essential in guided DNA targeting functions. Although tracrRNAs are diverse in sequence and structure across type II CRISPR systems, the programmability of crRNA-tracrRNA hybridization for Cas9 is not fully understood. Here, we reveal the programmability of crRNA-tracrRNA hybridization for Streptococcus pyogenes Cas9, and in doing so, redefine the capabilities of Cas9 proteins and the sources of crRNAs, providing new biosensing applications for type II CRISPR systems. By reprogramming the crRNA-tracrRNA hybridized sequence, we show that engineered crRNA-tracrRNA interactions can not only enable the design of orthogonal cellular computing devices but also facilitate the hijacking of endogenous small RNAs/mRNAs as crRNAs. We subsequently describe how these re-engineered gRNA pairings can be implemented as RNA sensors, capable of monitoring the transcriptional activity of various environment-responsive genomic genes, or detecting SARS-CoV-2 RNA in vitro, as an Atypical gRNA-activated Transcription Halting Alarm (AGATHA) biosensor.

Micro/nano biomedical devices for point-of-care diagnosis of infectious respiratory diseases.

Corona Virus Disease 2019 (COVID-19) has developed into a global pandemic in the last two years, causing significant impacts on our daily life in many countries. Rapid and accurate detection of COVID-19 is of great importance to both treatments and pandemic management. Till now, a variety of point-of-care testing (POCT) approaches devices, including nucleic acid-based test and immunological detection, have been developed and some of them has been rapidly ruled out for clinical diagnosis of COVID-19 due to the requirement of mass testing. In this review, we provide a summary and commentary on the methods and biomedical devices innovated or renovated for the quick and early diagnosis of COVID-19. In particular, some of micro and nano devices with miniaturized structures, showing outstanding analytical performances such as ultra-sensitivity, rapidness, accuracy and low cost, are discussed in this paper. We also provide our insights on the further implementation of biomedical devices using advanced micro and nano technologies to meet the demand of point-of-care diagnosis and home testing to facilitate pandemic management. In general, our paper provides a comprehensive overview of the latest advances on the POCT device for diagnosis of COVID-19, which may provide insightful knowledge for researcher to further develop novel diagnostic technologies for rapid and on-site detection of pathogens including SARS-CoV-2.

Droplet microfluidics on analysis of pathogenic microbes for wastewater-based epidemiology.

Infectious diseases caused by pathogenic microbes have posed a major health issue for the public, such as the ongoing COVID-19 global pandemic. In recent years, wastewater-based epidemiology (WBE) is emerging as an effective and unbiased method for monitoring public health. Despite its increasing importance, the advancement of WBE requires more competent and streamlined analytical platforms. Herein we discuss the interactions between WBE and droplet microfluidics, focusing on the analysis of pathogens in droplets, which is hard to be tackled by traditional analytical tools. We highlight research works from three aspects, namely, quantitation of pathogen biomarkers in droplets, single-cell analysis in droplets, and living cell biosensors in droplets, as well as providing future perspectives on the synergy between WBE and droplet microfluidics.

Occurrence of various viruses and recent evidence of SARS-CoV-2 in wastewater systems.

Viruses are omnipresent and persistent in wastewater, which poses a risk to human health. In this review, we summarise the different qualitative and quantitative methods for virus analysis in wastewater and systematically discuss the spatial distribution and temporal patterns of various viruses (i.e., enteric viruses, Caliciviridae (Noroviruses (NoVs)), Picornaviridae (Enteroviruses (EVs)), Hepatitis A virus (HAV)), and Adenoviridae (Adenoviruses (AdVs))) in wastewater systems. Then we critically review recent SARS-CoV-2 studies to understand the ongoing COVID-19 pandemic through wastewater surveillance. SARS-CoV-2 genetic material has been detected in wastewater from France, the Netherlands, Australia, Italy, Japan, Spain, Turkey, India, Pakistan, China, and the USA. We then discuss the utility of wastewater-based epidemiology (WBE) to estimate the occurrence, distribution, and genetic diversity of these viruses and generate human health risk assessment. Finally, we not only promote the prevention of viral infectious disease transmission through wastewater but also highlight the potential use of WBE as an early warning system for public health assessment.

Biosensors for wastewater-based epidemiology for monitoring public health.

Public health is attracting increasing attention due to the current global pandemic, and wastewater-based epidemiology (WBE) has emerged as a powerful tool for monitoring of public health by analysis of a variety of biomarkers (e.g., chemicals and pathogens) in wastewater. Rapid development of WBE requires rapid and on-site analytical tools for monitoring of sewage biomarkers to provide immediate decision and intervention. Biosensors have been demonstrated to be highly sensitive and selective tools for the analysis of sewage biomarkers due to their fast response, ease-to-use, low cost and the potential for field-testing. This paper presents biosensors as effective tools for wastewater analysis of potential biomarkers and monitoring of public health via WBE. In particular, we discuss the use of sewage sensors for rapid detection of a range of targets, including rapid monitoring of community-wide illicit drug consumption and pathogens for early warning of infectious diseases outbreaks. Finally, we provide a perspective on the future use of the biosensor technology for WBE to enable rapid on-site monitoring of sewage, which will provide nearly real-time data for public health assessment and effective intervention.

Paper-based devices for rapid diagnostics and testing sewage for early warning of COVID-19 outbreak

Coronavirus disease (COVID-19), caused by SARS-CoV-2, evolved into a global pandemic in 2020, and the outbreak has taken an enormous toll on individuals, families, communities and societies around the world. One practical and effective strategy is to implement rapid case identification based on a rapid testing to respond to this public health crisis. Currently, the available technologies used for rapid diagnostics include RT-PCR, RT-LAMP, ELISA and NGS. Still, due to their different limitations, they are not well suited for rapid diagnosis in a variety of locations. Paper-based devices are alternative approaches to achieve rapid diagnosis, which are cost-effective, highly selective, sensitive, portable, and easy-to-use. In addition to individual virus screening, wastewater-based epidemiology has been emerged to be an effective way for early warning of outbreak within the population, which tests genomic viral sequence to reflect information on the spread and distribution of the virus because SARS-CoV-2 can be shed into wastewater through the feces and urine from infected population. In this paper, we describe paper-based device as a low-cost and rapid sensor for testing of virus for both diagnosis and early warning of outbreak for wastewater-based epidemiology. Most importantly, the device has great potential for real-time detection in the field, without any advanced facilities and well-trained skilled personnel, and provides early warning or timely intervention of an outbreak of pandemic.

An integrated biosensor system with mobile health and wastewater-based epidemiology (iBMW) for COVID-19 pandemic.

The outbreak of coronavirus disease (COVID-19) has caused a significant public health challenge worldwide. A lack of effective methods for screening potential patients, rapidly diagnosing suspected cases, and accurately monitoring of the epidemic in real time to prevent the rapid spread of COVID-19 raises significant difficulties in mitigating the epidemic in many countries. As effective point-of-care diagnosis tools, simple, low-cost and rapid sensors have the potential to greatly accelerate the screening and diagnosis of suspected patients to improve their treatment and care. In particular, there is evidence that multiple pathogens have been detected in sewage, including SARS-CoV-2, providing significant opportunities for the development of advanced sensors for wastewater-based epidemiology that provide an early warning of the pandemic within the population. Sensors could be used to screen potential carriers, provide real-time monitoring and control of the epidemic, and even support targeted drug screening and delivery within the integration of emerging mobile health (mHealth) technology. In this communication, we discuss the feasibility of an integrated point-of-care biosensor system with mobile health for wastewater-based epidemiology (iBMW) for early warning of COVID-19, screening and diagnosis of potential infectors, and improving health care and public health. The iBMW will provide an effective approach to prevent, evaluate and intervene in a fast, affordable and reliable way, thus enabling real-time guidance for the government in providing effective intervention and evaluating the effectiveness of intervention.

Opportunities and Challenges for Biosensors and Nanoscale Analytical Tools for Pandemics: COVID-19.

Biosensors and nanoscale analytical tools have shown huge growth in literature in the past 20 years, with a large number of reports on the topic of 'ultrasensitive', 'cost-effective', and 'early detection' tools with a potential of 'mass-production' cited on the web of science. Yet none of these tools are commercially available in the market or practically viable for mass production and use in pandemic diseases such as coronavirus disease 2019 (COVID-19). In this context, we review the technological challenges and opportunities of current bio/chemical sensors and analytical tools by critically analyzing the bottlenecks which have hindered the implementation of advanced sensing technologies in pandemic diseases. We also describe in brief COVID-19 by comparing it with other pandemic strains such as that of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) for the identification of features that enable biosensing. Moreover, we discuss visualization and characterization tools that can potentially be used not only for sensing applications but also to assist in speeding up the drug discovery and vaccine development process. Furthermore, we discuss the emerging monitoring mechanism, namely wastewater-based epidemiology, for early warning of the outbreak, focusing on sensors for rapid and on-site analysis of SARS-CoV2 in sewage. To conclude, we provide holistic insights into challenges associated with the quick translation of sensing technologies, policies, ethical issues, technology adoption, and an overall outlook of the role of the sensing technologies in pandemics.