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.

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.

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.

Paper-based microfluidics for DNA diagnostics of malaria in low resource underserved rural communities.

Rapid, low-cost, species-specific diagnosis, based upon DNA testing, is becoming important in the treatment of patients with infectious diseases. Here, we demonstrate an innovation that uses origami to enable multiplexed, sensitive assays that rival polymerase chain reactions (PCR) laboratory assays and provide high-quality, fast precision diagnostics for malaria. The paper-based microfluidic technology proposed here combines vertical flow sample-processing steps, including paper folding for whole-blood sample preparation, with an isothermal amplification and a lateral flow detection, incorporating a simple visualization system. Studies were performed in village schools in Uganda with individual diagnoses being completed in <50 min (faster than the standard laboratory-based PCR). The tests, which enabled the diagnosis of malaria species in patients from a finger prick of whole blood, were both highly sensitive and specific, detecting malaria in 98% of infected individuals in a double-blind first-in-human study. Our method was more sensitive than other field-based, benchmark techniques, including optical microscopy and industry standard rapid immunodiagnostic tests, both performed by experienced local healthcare teams (which detected malaria in 86% and 83% of cases, respectively). All assays were independently validated using a real-time double-blinded reference PCR assay. We not only demonstrate that advanced, low-cost DNA-based sensors can be implemented in underserved communities at the point of need but also highlight the challenges associated with developing and implementing new diagnostic technologies in the field, without access to laboratories or infrastructure.

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.

Nanomaterial-based aptamer sensors for analysis of illicit drugs and evaluation of drugs consumption for wastewater-based epidemiology.

The abuse of illicit drugs usually associated with dramatic crimes may cause significant problems for the whole society. Wastewater-based epidemiology (WBE) has been demonstrated to be a novel and cost-effective way to evaluate the abuse of illicit drugs at the community level, and has been used as a routine method for monitoring and played a significant role for combating the crimes in some countries, e.g. China. The method can also provide temporal and spatial variation of drugs of abuse. The detection methods mainly remain on the conventional liquid chromatography coupled with mass spectrometry, which is extremely sensitive and selective, however needs advanced facility and well-trained personals, thus limit it in the lab. As an alternative, sensors have emerged to be a powerful analytical tool for a wide spectrum of analytes, in particular aptamer sensors (aptasensors) have attracted increasing attention and could act as an efficient tool in this field due to the excellent characteristics of selectivity, sensitivity, low cost, miniaturization, easy-to-use, and automation. In this review, we will briefly introduce the context, specific assessment process and applications of WBE and the recent progress of illicit drug aptasensors, in particular focusing on optical and electrochemical sensors. We then highlight several recent aptasensors for illicit drugs in new technology integration and discuss the feasibility of these aptasensor for WBE. We will summarize the challenges and propose our insights and opportunity on aptasensor for WBE to evaluate community-wide drug use trends and public health.

Bioaccumulation of Hg in Rice Leaf Facilitates Selenium Bioaccumulation in Rice (Oryza sativa L.) Leaf in the Wanshan Mercury Mine.

Mercury (Hg) bioaccumulation in rice poses a health issue for rice consumers. In rice paddies, selenium (Se) can decrease the bioavailability of Hg through forming the less bioavailable Hg selenides (HgSe) in soil. Rice leaves can directly uptake a substantial amount of elemental Hg from the atmosphere, however, whether the bioaccumulation of Hg in rice leaves can affect the bioaccumulation of Se in rice plants is not known. Here, we conducted field and controlled studies to investigate the bioaccumulation of Hg and Se in the rice-soil system. In the field study, we observed a significantly positive correlation between Hg concentrations and BAFs of Se in rice leaves (r2 = 0.60, p < 0.01) collected from the Wanshan Mercury Mine, SW China, suggesting that the bioaccumulation of atmospheric Hg in rice leaves can facilitate the uptake of soil Se, perhaps through the formation of Hg-Se complex in rice leaves. This conclusion was supported by the controlled study, which observed significantly higher concentrations and BAFs of Se in rice leaf at a high atmospheric Hg site at WMM, compared to a low atmospheric Hg site in Guiyang, SW China.

Subsequent monitoring of ferric ion and ascorbic acid using graphdiyne quantum dots-based optical sensors.

Graphdiyne (GDY) as an emerging carbon nanomaterial has attracted increasing attention because of its uniformly distributed pores, highly π-conjugated, and tunable electronic properties. These excellent characteristics have been widely explored in the fields of energy storage and catalysts, yet there is no report on the development of sensors based on the outstanding optical property of GDY. In this paper, a new sensing mechanism is reported built upon the synergistic effect between inner filter effect and photoinduced electron transfer. We constructed a novel nanosensor based upon the newly-synthesized nanomaterial and demonstrated a sensitive and selective detection for both Fe3+ ion and ascorbic acid, enabling the measurements in real clinical samples. For the first time fluorescent graphdiyne oxide quantum dots (GDYO-QDs) were prepared using a facile ultrasonic protocol and they were characterized with a range of techniques, showing a strong blue-green emission with 14.6% quantum yield. The emission is quenched efficiently by Fe3+ and recovered by ascorbic acid (AA). We have fabricated an off/on fluorescent nanosensors based on this unique property. The nanosensors are able to detect Fe3+ as low as 95 nmol L-1 with a promising dynamic range from 0.25 to 200 µmol L-1. The LOD of AA was 2.5 µmol L-1, with range of 10-500 µmol L-1. It showed a promising capability to detect Fe3+ and AA in serum samples. Graphical abstract.

Monitoring Genetic Population Biomarkers for Wastewater-Based Epidemiology.

We report a rapid "sample-to-answer" platform that can be used for the quantitative monitoring of genetic biomarkers within communities through the analysis of wastewater. The assay is based on the loop-mediated isothermal amplification (LAMP) of nucleic acid biomarkers and shows for the first time the ability to rapidly quantify human-specific mitochondrial DNA (mtDNA) from raw untreated wastewater samples. mtDNA provides a model population biomarker associated with carcinogenesis including breast, renal and gastric cancers. To enable a sample-to-answer, field-based technology, we integrated a filter to remove solid impurities and perform DNA extraction and enrichment into a low cost lateral flow-based test. We demonstrated mtDNA detection over seven consecutive days, achieving a limit of detection of 40 copies of human genomic DNA per reaction volume. The assay can be performed at the site of sample collection, with minimal user intervention, yielding results within 45 min and providing a method to monitor public health from wastewater.

A novel immobilization strategy for electrochemical detection of cancer biomarkers: DNA-directed immobilization of aptamer sensors for sensitive detection of prostate specific antigens.

We report on a novel strategy for DNA aptamer immobilization to develop sensitive electrochemical detection of a protein biomarker, with prostate specific antigen (PSA) as a case biomarker. Thiolated single-stranded DNA (ssDNA) was co-immobilized with 3-mercapto-1-propanol on gold electrodes, and used as a scaffold for DNA aptamer attachment through hybridization of the aptamer overhang (so-called "DNA-directed immobilization aptamer sensors", DDIAS). In the approach, the complementary DNA aptamer against PSA was assembled by the probe ssDNA onto the electrode to detect PSA; or the probe ssDNA directly hybridized with a complementary DNA aptamer/PSA complex following their pre-incubation in solution, so-called 'on-chip' and 'in-solution' methods, respectively. A double stranded DNA intercalator with a ferrocenyl (Fc) redox marker was synthesized to evaluate the feasibility of the strategy. The results demonstrate that the 'in-solution' method offers a favourable medium (in a homogeneous solution) for the binding between the aptamer and PSA, which shows to be more efficient than the 'on-chip' approach. DDIAS shows promising analytical performance under optimized conditions, with a limit of detection in the range of fM and low non-specific adsorption.

A novel DNA biosensor using a ferrocenyl intercalator applied to the potential detection of human population biomarkers in wastewater.

A new label-free electrochemical DNA (E-DNA) biosensor using a custom synthesized ferrocenyl (Fc) double-stranded DNA intercalator as a redox marker is presented. Single-stranded DNA (ssDNA) was co-immobilized on gold electrodes with 6-mecarpto-hexanol to control the surface density of the ssDNA probe, and hybridized with complementary DNA. The binding of the Fc intercalator to dsDNA was measured by differential pulse voltammetry. This new biosensor was optimized to allow the detection of single base pair mismatched sequences, able to detect as low as 10 pM target ssDNA with a dynamic range from 10 pM to 100 nM. DNA extracted from wastewater was analyzed by quantitative polymerase chain reaction targeting human-specific mitochondrial DNA (mtDNA). The aim of this approach is to enable the analysis of population biomarkers in wastewater for the evaluation of public health using wastewater-based epidemiology (WBE). The E-DNA biosensor was employed to detect human-specific mtDNA from wastewater before and after PCR amplification. The results demonstrate the feasibility of detecting human DNA biomarkers in wastewater using the developed biosensor, which may allow the further development of DNA population biomarkers for public health using WBE.

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.

Characterization of three amino-functionalized surfaces and evaluation of antibody immobilization for the multiplex detection of tumor markers involved in colorectal cancer.

Antibody microarrays are powerful and high-throughput tools for screening and identifying tumor markers from small sample volumes of only a few microliters. Optimization of surface chemistry and spotting conditions are crucial parameters to enhance antibodies' immobilization efficiency and to maintain their biological activity. Here, we report the implementation of an antibody microarray for the detection of tumor markers involved in colorectal cancer. Three-dimensional microstructured glass slides were functionalized with three different aminated molecules ((3-aminopropyl)dimethylethoxysilane (APDMES), Jeffamine, and chitosan) varying in their chain length, their amine density, and their hydrophilic/hydrophobic balance. The physicochemical properties of the resulting surfaces were characterized. Antibody immobilization efficiency through physical interaction was studied as a function of surface properties as well as a function of the immobilization conditions. The results show that surface energy, steric hindrance, and pH of spotting buffer have great effects on protein immobilization. Under optimal conditions, biological activities of four immobilized antitumor marker antibodies were evaluated in multiplex immunoassay for the detection of the corresponding tumor markers. Results indicated that the chitosan functionalized surface displayed the highest binding capacity and allowed to retain maximal biological activity of the four tested antibody/antigen systems. Thus, we successfully demonstrated the application of amino-based surface modification for antibody microarrays to efficiently detect tumor markers.