Discussion: Embracing microfluidics to advance environmental science and technology.

Microfluidics, also called lab-on-a-chip, represents an emerging research platform that permits more precise and manipulation of samples at the microscale or even down to the nanoscale (nanofluidic) including picoliter droplets, microparticles, and microbes within miniaturized and highly integrated devices. This groundbreaking technology has made significant strides across multiple disciplines by providing an unprecedented view of physical, chemical, and biological events, fostering a holistic and an in-depth understanding of complex systems. The application of microfluidics to address the challenges in environmental science is likely to contribute to our better understanding, however, it's not yet fully developed. To raise researchers' interest, this discussion first delineates the valuable and underutilized environmental applications of microfluidic technology, ranging from environmental surveillance to acting as microreactors for investigating interfacial dynamic processes, and facilitating high-throughput bioassays. We highlight, with examples, how rationally designed microfluidic devices lead to new insights into the advancement of environmental science and technology. We then critically review the key challenges that hinder the practical adoption of microfluidic technologies. Specifically, we discuss the extent to which microfluidics accurately reflect realistic environmental scenarios, outline the areas to be improved, and propose strategies to overcome bottlenecks that impede the broad application of microfluidics. We also envision new opportunities and future research directions, aiming to provide guidelines for the broader utilization of microfluidics in environmental studies.

Efficient DNA walker guided by ordered cruciform-shaped DNA track for ultrasensitive and rapid electrochemical detection of lead ion.

The rational design of DNA tracks is an effective pathway to guide the autonomous movement and high-efficiency recognition in DNA walkers, showing outstanding advantages for the cascade signal amplification of electrochemical biosensors. However, the uncontrolled distance between two adjacent tracks on the electrode could increase the risk of derailment and interruption of the reaction. Hence, a novel four-way balanced cruciform-shaped DNA track (C-DNT) was designed as a structured pathway to improve the effectiveness and stability of the reaction in DNA walkers. In this work, two kinds of cruciform-shaped DNA were interconnected as a robust structure that could avoid the invalid movement of the designed DNA walker on the electrode. When hairpin H2 was introduced onto the electrode, the strand displacement reaction (SDR) effectively triggered movements of the DNA walker along the cruciform-shaped track while leaving ferrocene (Fc) on the electrode, leading to a significant enhancement of the electrochemical signal. This design enabled the walker to move in an excellent organized and controllable manner, thus enhancing the reaction speed and walking efficiency. Compared to other walkers moving on random tracks, the reaction time of the C-DNT-based DNA walker could be reduced to 20 min. Lead ion (Pb2+) was used as a model target to evaluate the analytical performance of this biosensor, which exhibited a low detection limit of 0.033 pM along with a wide detection ranging from 0.1 pM to 500 nM. This strategy presented a novel concept for designing a high-performance DNA walker-based sensing platform for the detection of contaminants.

Dual-Emission Single Sensing Element-Assembled Fluorescent Sensor Arrays for the Rapid Discrimination of Multiple Surfactants in Environments.

Surfactants are considered as typical emerging pollutants, their extensive use of in disinfectants has hugely threatened the ecosystem and human health, particularly during the pandemic of coronavirus disease-19 (COVID-19), whereas the rapid discrimination of multiple surfactants in environments is still a great challenge. Herein, we designed a fluorescent sensor array based on luminescent metal-organic frameworks (UiO-66-NH2@Au NCs) for the specific discrimination of six surfactants (AOS, SDS, SDSO, MES, SDBS, and Tween-20). Wherein, UiO-66-NH2@Au NCs were fabricated by integrating UiO-66-NH2 (2-aminoterephthalic acid-anchored-MOFs based on zirconium ions) with gold nanoclusters (Au NCs), which exhibited a dual-emission features, showing good luminescence. Interestingly, due to the interactions of surfactants and UiO-66-NH2@Au NCs, the surfactants can differentially regulate the fluorescence property of UiO-66-NH2@Au NCs, producing diverse fluorescent "fingerprints", which were further identified by pattern recognition methods. The proposed fluorescence sensor array achieved 100% accuracy in identifying various surfactants and multicomponent mixtures, with the detection limit in the range of 0.0032 to 0.0315 mM for six pollutants, which was successfully employed in the discrimination of surfactants in real environmental waters. More importantly, our findings provided a new avenue in rapid detection of surfactants, rendering a promising technique for environmental monitoring against trace multicontaminants.

Poly-adenine-mediated tetrahedral DNA nanostructure with multiple target-recognition sites for ultrasensitive and rapid electrochemical detection of Aflatoxin B1.

Tetrahedral DNA nanostructures (TDNs) are widely used in the development of electrochemical biosensors due to their structural stability, programmability, and strong interfacial orderliness. However, the complex modifications on the electrode and the single vertex target recognition of the TDNs limit their applications in electrochemical biosensing. Herein, we developed a universal detection system based on a novel polyadenine-based tetrahedral DNA nanostructure (ATDN) using Aflatoxin B1 (AFB1) as the model target for analysis. In the absence of target AFB1, the signal probes (SP) modified with ferrocene would be anchored by five aptamers on ATDN. The target capture by aptamers led to a release of SP from the electrode surface, resulting in a significant reduction of the electrochemical signal. This new nanostructure was not only dispensed with multi-step electrode modifications and strong mechanical rigidity but also had five modification sites which enhanced the detection sensitivity for the target. As a result, this biosensor shows good analytical performance in the linear range of 1 fg mL-1 to 1 ng mL-1, exhibiting a low detection limit of 0.33 fg mL-1. Satisfactory accuracy has also been demonstrated through good recoveries (95.2%-98.9%). The proposed new tetrahedral DNA nanostructure can provide a more rapid and sensitive alternative to previous electrochemical sensors based on the conventional TDN. Since DNA sequences can be designed flexibly, the sensing platform in this strategy can be extended to detect various targets in different fields.

Review of paper-based microfluidic analytical devices for in-field testing of pathogens.

Pathogens cause various infectious diseases and high morbidity and mortality which is a global public health threat. The highly sensitive and specific detection is of significant importance for the effective treatment and intervention to minimise the impact. However, conventional detection methods including culture and molecular method gravely depend on expensive equipment and well-trained skilled personnel, limiting in the laboratory. It remains challenging to adapt in resource-limiting areas, e.g., low and middle-income countries (LMICs). To this end, low-cost, rapid, and sensitive detection tools with the capability of field testing e.g., a portable device for identification and quantification of pathogens, has attracted increasing attentions. Recently, paper-based microfluidic analytical devices (µPADs) have shown a promising tool for rapid and on-site diagnosis, providing a cost-effective and sensitive analytical approach for pathogens detection. The fast turn-round data collection may also contribute to better understanding of the risks and insights on mitigation method. In this paper, critical developments of µPADs for in-field detection of pathogens both for clinical diagnostics and environmental surveillance are reviewed. The future development, and challenges of µPADs for rapid and onsite detection of pathogens are discussed, including using the cross-disciplinary development with, emerging techniques such as deep learning and Internet of Things (IoT).

Printable biosensors towards next-generation point-of-care testing: paper substrate as an example.

Printable biosensors have gained numerous exciting advancements towards downstream applications in fundamental biomedical research, healthcare, food safety, environmental monitoring and governance, and to name a few. Particularly, paper-based printable biosensors have gained rising popularity in providing affordable platforms due to their merits, such as cost-effective, accurate, simple, and efficient detection of diseases for clinical diagnosis. In addition to advantages and opportunities in point-of-care detection, printable biosensors are also facing challenges. Herein, this review aims to provide a systematic summary of the development of printable biosensors, with a special focus on paper-based printable biosensors. Different types of substrates for printable biosensors are highlighted with a focus on paper substrates which have superior properties like low-cost, simple, flexible, lightweight, recyclable, etc. In addition, current printing technologies to fabricate paper-based sensors, including wax printing, photolithography, screen printing, inkjet printing, and laser printing summarize, are discussed, together with strategies for biomolecular fabrication on substrates and transducers. Finally, we also discuss the challenges and possible future perspectives, hoping to provide researchers and clinicians with informative insights into paper-based printable biosensors for smart and effective point-of-care detection.

Tailoring the whole-cell sensing spectrum with cyborgian redox machinery.

Whole-cell biosensors are an important class of analytical tools that offer the advantages of low cost, facile operation, and unique reproduction/regeneration ability. However, it has always been quite challenging to expand the sensing spectrum of the host. Here, a new approach to extend the cell sensing spectrum with biomineralized nanoparticles is developed. The nano-biohybrid design comprise biomineralized FeS nanoparticles firmly anchored onto the bacterium, Shewanella oneidensis MR-1, wherein the nanoparticles are wired to the cellular electron transfer machinery (MtrCAB/OmcA) of the bacterium, forming an artificial cyborgian redox machinery consisting of FeS-MtrCAB/OmcA-FccA. Strikingly, with this cyborgian redox machinery, the sensing spectrum of FeS hybridized S. oneidensis MR-1 cell is successfully expanded to enable whole-cell electrochemical detection of Vitamin B12, while an unhybridized native cell is incapable of sensing. This proof-of-concept nano-biohybrid design offers a new perspective on manipulating the microbial toolkit for an expanded sensing spectrum in whole-cell biosensors.

Entropy-Driven Three-Dimensional DNA Nanofireworks for Simultaneous Real-Time Imaging of Telomerase and MicroRNA in Living Cells.

Real-time monitoring of different types of intracellular tumor-related biomarkers is of key importance for the identification of tumor cells. However, it is hampered by the low abundance of biomarkers, inefficient free diffusion of reactants, and complex cytoplasmic milieu. Herein, we present a stable and general method for in situ imaging of microRNA-21 and telomerase utilizing simple highly integrated dual tetrahedral DNA nanostructures (TDNs) that can naturally enter cells, which could initiate to form the three-dimensional (3D) higher-order DNA superstructures (DNA nanofireworks, DNFs) through a reliable target-triggered entropy-driven strand displacement reaction in living cells for remarkable signal amplification. Importantly, the excellent biostability, biocompatibility, and sensitivity of this approach benefited from (i) the precise multidirectional arrangement of probes with a pure DNA structure and (ii) the local target concentration enhanced by the spatially confined microdomain inside the DNFs. This strategy provides a pivotal molecular toolbox for broad applications such as biomedical imaging and early precise cancer diagnosis.

Soil Microbial Fuel Cell Based Self-Powered Cathodic Biosensor for Sensitive Detection of Heavy Metals.

Soil microbial fuel cells (SMFCs) are an innovative device for soil-powered biosensors. However, the traditional SMFC sensors relied on anodic biosensing which might be unstable for long-term and continuous monitoring of toxic pollutants. Here, a carbon-felt-based cathodic SMFC biosensor was developed and applied for soil-powered long-term sensing of heavy metal ions. The SMFC-based biosensor generated output voltage about 400 mV with the external load of 1000 Ω. Upon the injection of metal ions, the voltage of the SMFC was increased sharply and quickly reached a stable output within 2~5 min. The metal ions of Cd2+, Zn2+, Pb2+, or Hg2+ ranging from 0.5 to 30 mg/L could be quantified by using this SMFC biosensor. As the anode was immersed in the deep soil, this SMFC-based biosensor was able to monitor efficiently for four months under repeated metal ions detection without significant decrease on the output voltage. This finding demonstrated the clear potential of the cathodic SMFC biosensor, which can be further implemented as a low-cost self-powered biosensor.

Self-Assembled Microfiber-Like Biohydrogel for Ultrasensitive Whole-Cell Electrochemical Biosensing in Microdroplets.

A novel microfiber-like biohydrogel was fabricated by a facile approach relying on electroactive bacteria-induced graphene oxide reduction and confined self-assembly in a capillary tube. The microfiber-like biohydrogel (d = ∼1 mm) embedded high-density living cells and activated efficient electron exchange between cells and the conductive graphene network. Further, a miniature whole-cell electrochemical biosensing system was developed and applied for fumarate detection under -0.6 V (vs Ag/AgCl) applied potential. Taking advantage of its small size, high local cell density, and excellent electron exchange, this microfiber-like biohydrogel-based sensing system reached a linear calibration curve (R2 = 0.999) ranging from 1 nM to 10 mM. The limit of detection obtained was 0.60 nM, which was over 1300 times lower than a traditional biosensor for fumarate detection in 0.2 µL microdroplets. This work opened a new dimension for miniature whole-cell electrochemical sensing system design, which provided the possibility for bioelectrochemical detection in small volumes or three-dimensional local detection at high spatial resolutions.

Ultrathin Graphdiyne/Graphene Heterostructure as a Robust Electrochemical Sensing Platform.

Graphdiyne (GDY) has been considered as an appealing electrode material for electrochemical sensing because of its alkyne-rich structure and high degrees of π-conjugation, which shows great affinity to heavy metal ions and pollutant molecules via d-π and π-π interactions. However, the low surface area and poor conductivity of bulk GDY limit its electrochemical performance. Herein, a two-dimensional ultrathin GDY/graphene (GDY/G) nanostructure was synthesized and used as an electrode material for electrochemical sensing. Graphene plays the role of an epitaxy template for few-layered GDY growth and conductive layers. The formed few-layered GDY with a high surface area possesses abundant affinity sites toward heavy metal ions (Cd2+, Pb2+) and toxic molecules, for example, nitrobenzene and 4-nitrophenol, via d-π and π-π interactions, respectively. Moreover, hemin as a key part of the enzyme catalytic motif was immobilized on GDY/G via π-π interactions. The artificial enzyme mimic hemin/GDY/G-modified electrode exhibited promising ascorbic acid and uric acid detection performance with excellent sensitivity and selectivity, a good linear range, and reproducibility. More importantly, real sample detection and the feasibility of this electrochemical sensor as a wearable biosensor were demonstrated.

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.

Biosensors for rapid detection of bacterial pathogens in water, food and environment.

Conventional techniques (e.g., culture-based method) for bacterial detection typically require a central laboratory and well-trained technicians, which may take several hours or days. However, recent developments within various disciplines of science and engineering have led to a major paradigm shift in how microorganisms can be detected. The analytical sensors which are widely used for medical applications in the literature are being extended for rapid and on-site monitoring of the bacterial pathogens in food, water and the environment. Especially, within the low-resource settings such as low and middle-income countries, due to the advantages of low cost, rapidness and potential for field-testing, their use is indispensable for sustainable development of the regions. Within this context, this paper discusses analytical methods and biosensors which can be used to ensure food safety, water quality and environmental monitoring. In brief, most of our discussion is focused on various rapid sensors including biosensors and microfluidic chips. The analytical performances such as the sensitivity, specificity and usability of these sensors, as well as a brief comparison with the conventional techniques for bacteria detection, form the core part of the discussion. Furthermore, we provide a holistic viewpoint on how future research should focus on exploring the synergy of different sensing technologies by developing an integrated multiplexed, sensitive and accurate sensors that will enable rapid detection for food safety, water and environmental monitoring.

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.

AuAg nanocages/graphdiyne for rapid elimination and detection of trace pathogenic bacteria.

We prepared a biocompatible AuAg nanocages/graphdiyne @ polyethylene glycol (AuAg/GDY@PEG) composite. The combination of AuAg and GDY to obtain a synergistically enhanced photothermal effect, and the antibacterial effect of GDY and AuAg are used in combined anti-infective therapy. The in vitro antibacterial activity of AuAg/GDY@PEG was investigated, showing an impressive broad-spectrum antibacterial activity with the killing rate > 99.999%. Based on the photothermal conversion ability of AuAg/GDY@PEG, a simple photothermal immunoassay for pathogenic bacteria was successfully established. Sandwich immune response was performed on a microporous plate, the microplate containing the antibody binds specifically to the bacterium being tested, which then binds to the material with the antibody on its surface, and the signal was a change in temperature under 808 nm near-infrared light. The limit of detection (LOD) for S. typhimurium detection is 103 CFU mL-1, with a range of 103-107 CFU mL-1. This method is accurate, rapid and low-cost, which can be used for on-site detection of pathogenic bacteria in food.

Paper microfluidic implementation of loop mediated isothermal amplification for early diagnosis of hepatitis C virus.

The early diagnosis of active hepatitis C virus (HCV) infection remains a significant barrier to the treatment of the disease and to preventing the associated significant morbidity and mortality seen, worldwide. Current testing is delayed due to the high cost, long turnaround times and high expertise needed in centralised diagnostic laboratories. Here we demonstrate a user-friendly, low-cost pan-genotypic assay, based upon reverse transcriptase loop mediated isothermal amplification (RT-LAMP). We developed a prototype device for point-of-care use, comprising a LAMP amplification chamber and lateral flow nucleic acid detection strips, giving a visually-read, user-friendly result in <40 min. The developed assay fulfils the current guidelines recommended by World Health Organisation and is manufactured at minimal cost using simple, portable equipment. Further development of the diagnostic test will facilitate linkage between disease diagnosis and treatment, greatly improving patient care pathways and reducing loss to follow-up, so assisting in the global elimination strategy.

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.