A multi-cycle signal amplification-mediated single quantum dot nanosensor for PIWI-interacting RNA detection.

We construct a single quantum dot-based nanosensor for piRNA detection based on ligation-mediated multi-cycle signal amplification. This nanosensor is homogenous, selective, and sensitive with a detection limit of 0.104 fM. Moreover, it can detect the endogenous piRNA level in different cell lines, and discriminate cancer tissues from normal tissues.

Methylation-Powered Assembly of a Single Quantum Dot-Based FRET Nanosensor for Antibody-Free and Enzyme-Free Monitoring of Locus-Specific N6-Methyladenosine in Clinical Tissues.

N6-Methyladenosine (m6A) is the most pervasive and evolutionarily conserved epitranscriptomic modification in long noncoding RNA (lncRNA), and its dysregulation may induce aberrant transcription and translation programs. Herein, we demonstrate the methylation-powered assembly of a single quantum dot (QD)-based fluorescence resonance energy transfer (FRET) nanosensor for antibody- and enzyme-free monitoring of locus-specific m6A in clinical tissues. The m6A-sensitive DNAzyme VMC10 is employed to identify a specific m6A site in lncRNA, and it catalyzes the hydrolytic cleavage of unmethylated lncRNA. The cleaved lncRNA fails to trigger the subsequent catalytic hairpin assembly (CHA) reaction due to the energy barrier. In contrast, when m6A-lncRNA is present, the methyl group in m6A protects lncRNA from VMC10-mediated cleavage. With the aid of an assistant probe, the retained intact m6A-lncRNA is released from the VMC10/lncRNA complex and subsequently triggers the CHA reaction, generating abundant AF647/biotin dual-labeled duplexes. The assembly of AF647/biotin dual-labeled duplexes onto 605QD results in efficient FRET between 605QD and AF647. The FRET signal can be simply quantified by single-molecule detection. Notably, this assay can be implemented in an antibody-free and enzyme-free manner. This nanosensor can sensitively quantify target m6A with a detection limit of 0.47 fM, and it can discriminate as low as a 0.001% m6A level from excess coexisting counterparts. Importantly, this nanosensor can monitor the cellular m6A level with single-cell sensitivity and profile target m6A expression in breast cancer and healthy para-cancerous tissues, providing a powerful tool for studying the physiological and pathological functions of m6A.

A Bioinspired Flexible Sensor for Electrochemical Probing of Dynamic Redox Disequilibrium in Cancer Cells.

Malignant tumors pose a serious risk to human health. Ascorbic acid (AA) has potential for tumor therapy; however, the mechanism underlying the ability of AA to selectively kill tumor cells remains unclear. AA can cause redox disequilibrium in tumor cells, resulting in the release of abundant reactive oxygen species, represented by hydrogen peroxide (H2 O2 ). Therefore, the detection of H2 O2 changes can provide insight into the selective killing mechanism of AA against tumor cells. In this work, inspired by the ion-exchange mechanism in coral formation, a flexible H2 O2 sensor (PtNFs/CoPi@CC) is constructed to monitor the dynamics of H2 O2 in the cell microenvironment, which exhibits excellent sensitivity and spatiotemporal resolution. Moreover, the findings suggest that dehydroascorbic acid (DHA), the oxidation product of AA, is highly possible the substance that actually acts on tumor cells in AA therapy. Additionally, the intracellular redox disequilibrium and H2 O2 release caused by DHA are positively correlated with the abundance and activity of glucose transporter 1 (GLUT1). In conclusion, this work has revealed the potential mechanism underlying the ability of AA to selectively kill tumor cells through the construction and use of PtNFs/CoPi@CC. The findings provide new insights into the clinical application of AA.

Patterning amyloid-ß aggregation under the effect of acetylcholinesterase using a biological nanopore - an in vitro study.

Aggregation of amyloid-ß peptide (Aß) is hypothesized to be the primary cause of Alzheimer's disease (AD) progression. Aß aggregation has been widely studied using conventional sensing tools like emission fluorescence, electron microscopy, mass spectroscopy, and circular dichroism. However, none of these techniques can provide cost-efficient, highly sensitive quantification of Aß aggregation kinetics at the molecular level. Among the influences on Aß aggregation of interest to disease progression is the acceleration of Aß aggregation by acetylcholinesterase (AChE), which is present in the brain and inflicts the fast progression of disease due to its direct interaction with Aß. In this work, we demonstrate the ability of a biological nanopore to map and quantify AChE accelerated aggregation of Aß monomers to mixed oligomers and small soluble aggregates with single-molecule precision. This method will allow future work on testing direct and indirect effects of therapeutic drugs on AChE accelerated Aß aggregation as well as disease prognosis.

Automatically showing microbial growth kinetics with a high-performance microbial growth analyzer.

It is difficult to show microbial growth kinetics online when they grow in complex matrices. We presented a novel strategy to address this challenge by developing a high-performance microbial growth analyzer (HPMGA), which employed a unique 32-channel capacitively coupled contactless conductivity detector as a sensing element and fixed with a CellStatz software. It was capable of online showing accurate and repeatable growth curves of well-dispersed and bad-dispersed microbes, whether they grew in homogeneous simple culture broth or heterogeneous complex matrices. Moreover, it could automatically report key growth kinetics parameters. In comparison to optical density (OD), plate counting and broth microdilution (BMD) methods, we demonstrated its practicability in five scenarios: 1) the illustration of the growth, growth rate, and acceleration curves of Escherichia coli (E. coli); 2) the antimicrobial susceptibility testing (AST) of Oxacillin against Staphylococcus aureus (S. aureus); 3) the determination of Ag nanoparticle toxicity on Providencia rettgeri (P. rettgeri); 4) the characterization of milk fermentation; and 5) the enumeration of viable pathogenic Vibrio in shrimp body. Results highlighted that the HPMGA method had the advantages of universality and effectivity. This technology would significantly facilitate the routine analysis of microbial growth in many fields (biology, medicine, clinic, life, food, environment, and ecology), paving an avenue for microbiologists to achieve research goals that have been inhibited for years due to a lack of practical analytical methods.

EvIPqPCR, Target Circulating Tumorous Extracellular Vesicles for Detection of Pancreatic Cancer.

Pancreatic cancer patients predominantly present with advanced disease at diagnosis, contributing to its high mortality. A noninvasive, fast screening method to detect this disease is an unmet need. Tumor-derived extracellular vesicles (tdEVs) bearing information from parental cells have emerged as a promising cancer diagnostic biomarker. However, most tdEV-based assays have impractical sample volumes and time-consuming, complex, and costly techniques. To overcome these limitations, we developed a novel diagnostic method for pancreatic cancer screening. Our approach utilizes the mitochondrial DNA to nuclear DNA ratio of EVs as a collective cell-specific characteristic. We introduce EvIPqPCR, a fast method that combines immunoprecipitation (IP) and qPCR quantification to detect tumor-derived EVs directly from serum. Importantly, our method employs DNA isolation-free and duplexing probes for qPCR, saving at least 3 h. This technique has the potential to serve as a translational assay for cancer screening with a weak correlation to prognosis biomarkers and sufficient discriminatory power among healthy controls, pancreatitis, and pancreatic cancer cases.

Reining in Cas13a activity with N-terminal removable tags expands Cas13a based molecular sensing and enables precise gene interference.

Activation of Cas13 is exclusively dependent on crRNA-target RNA hybridization according to the canonical mode of Cas13 action. Upon activation Cas13 can cleave both target RNA and any surrounding RNA. The latter has been well adopted by therapeutic gene interference and biosensor development. This work for the first time, rationale designs and validates a multi-component controlled activation system of Cas13 by N-terminus tagging. A composite SUMO tag comprised of His, Twinstrep, and Smt3 tags fully suppresses target dependent activation of Cas13a by interfering with crRNA docking. The suppression releases upon proteases mediated proteolytic cleavage. The modular composition of the composite tag can be altered to fulfill customized response to alternative proteases. The biosensor SUMO-Cas13a is able to resolve a broad concentration range of protease Ulp1 with a calculated LOD of 48.8pg/µL in aqueous buffer. Further, in accordance with this finding Cas13a was successfully programmed to exert target gene knock down preferentially in SUMO protease high cell types. In summary the discovered regulatory component not only fulfills Cas13a based protease detection for the first time, but also delivers a novel strategy for multi-component controlled activation of Cas13a toward temporal and spacial precision.

Towards hospital-on-chip supported by 2D MXenes-based 5th generation intelligent biosensors.

Existing public health emergencies due to fatal/infectious diseases such as coronavirus disease (COVID-19) and monkeypox have raised the paradigm of 5th generation portable intelligent and multifunctional biosensors embedded on a single chip. The state-of-the-art 5th generation biosensors are concerned with integrating advanced functional materials with controllable physicochemical attributes and optimal machine processability. In this direction, 2D metal carbides and nitrides (MXenes), owing to their enhanced effective surface area, tunable physicochemical properties, and rich surface functionalities, have shown promising performances in biosensing flatlands. Moreover, their hybridization with diversified nanomaterials caters to their associated challenges for the commercialization of stability due to restacking and oxidation. MXenes and its hybrid biosensors have demonstrated intelligent and lab-on-chip prospects for determining diverse biomarkers/pathogens related to fatal and infectious diseases. Recently, on-site detection has been clubbed with solution-on-chip MXenes by interfacing biosensors with modern-age technologies, including 5G communication, internet-of-medical-things (IoMT), artificial intelligence (AI), and data clouding to progress toward hospital-on-chip (HOC) modules. This review comprehensively summarizes the state-of-the-art MXene fabrication, advancements in physicochemical properties to architect biosensors, and the progress of MXene-based lab-on-chip biosensors toward HOC solutions. Besides, it discusses sustainable aspects, practical challenges and alternative solutions associated with these modules to develop personalized and remote healthcare solutions for every individual in the world.

Monocrystalline Labeling Enables Stable Plasmonic Enhancement for Isolation-Free Extracellular Vesicle Analysis.

Sensitive detection of extracellular vesicles (EVs) as emerging biomarkers has shown great promises for disease diagnosis. Plasmonic metal nanostructures conjugated with molecules that bind specific biomarker targets are widely used for EVs sensing but involve tradeoffs between particle-size-dependent signal intensity and conjugation efficiency. One solution to this problem would be to induce nucleation on nanoparticles that have successfully bound a target biomarker to permit in situ nanoparticle growth for signal amplification, but approaches that are evaluated to date require harsh conditions or lack nucleation specificity, prohibiting their effective use with most biological specimens. This study describes a one-step in situ strategy to induce monocrystalline copper shell growth on gold nanorod probes without decreasing signal by disrupting probe-target interactions or lipid bilayer integrity to enable EV biomarker detections. This approach increases the detected nanoparticle signal about two orders of magnitude after a 10 min copper nanoshell growth reaction. This has significant implications for improved disease detection, as indicated by the ability of a novel immunoassay using this approach to detect low abundance EVs carrying a pathogen-derived biomarker, after their direct capture from serum, to facilitate the diagnosis of tuberculosis cases in a diagnostically challenging pediatric cohort.

Sensitive and rapid determination of heat shock protein 70 using lateral flow immunostrips and upconversion nanoparticle fluorescence probes.

Heat shock protein 70 (Hsp70), belonging to the heat shock protein (HSP) family, is reported to be a potential diagnostic biomarker. In this work, a lateral flow immunostrip was fabricated for the sensitive and rapid determination of Hsp70 by the incorporation of fluorescence and upconversion nanoparticle probes. The upconversion nanoparticles (UCNPs, size ∼39 nm, λex = 980 nm; λem = 540 nm) consisting of a NaYF4:Yb/Er core and polyacrylic acid-modified shell were covalently coupled with Hsp70 antibodies to form the signal probe, which was characterized by dynamic light scattering and zeta potential analyses. The lateral flow assay (LFA) was constructed based on the sandwich-type immunoassay using a sample pad, a test pad, and an adsorption pad on a PVP backing. Hsp70 antibody, IgG antibody and the signal probe were separately dropped on the test zone, the control zone of the test pad, and the sample pad, respectively. In the sandwich LFA, since two antibodies bind to Hsp70 antigenic epitopes, i.e. specific binding, it provided superior specificity and high sensitivity, making it an ideal sensing platform for complex samples like serum Hsp70 samples. The important parameters for the preparation of the lateral flow immunostrips were optimized. Under the optimized conditions, Hsp70 can be detected using the increased fluorescence intensity of UCNPs with a wide linear range from 0.11 to 12 ng mL-1, low detection limit of 0.06 ng mL-1, small sample volume (120 µL), short assay time (15 min) and good reproducibility. The fluorescence method was successfully applied in the determination of Hsp70 in serum samples with good recovery. By combining the accessibility of the lateral flow immunostrips and upconversion nanoparticles, the fluorescence method can serve as a point-of-care testing method for protein assays with high sensitivity and fast detection.

Identification of PDXDC1 as a novel pleiotropic susceptibility locus shared between lumbar spine bone mineral density and birth weight.

An increasing number of epidemiological studies have suggested that birth weight (BW) may be a determinant of bone health later in life, although the underlying genetic mechanism remains unclear. Here, we applied a pleiotropic conditional false discovery rate (cFDR) approach to the genome-wide association study (GWAS) summary statistics for lumbar spine bone mineral density (LS BMD) and BW, aiming to identify novel susceptibility variants shared between these two traits. We detected 5 novel potential pleiotropic loci which are located at or near 7 different genes (NTAN1, PDXDC1, CACNA1G, JAG1, FAT1P1, CCDC170, ESR1), among which PDXDC1 and FAT1P1 have not previously been linked to these phenotypes. To partially validate the findings, we demonstrated that the expression of PDXDC1 was dramatically reduced in ovariectomized (OVX) mice in comparison with sham-operated (SHAM) mice in both the growth plate and trabecula bone. Furthermore, immunohistochemistry assay with serial sections showed that both osteoclasts and osteoblasts express PDXDC1, supporting its potential role in bone metabolism. In conclusion, our study provides insights into some shared genetic mechanisms for BMD and BW as well as a novel potential therapeutic target for the prevention of OP in the early stages of the disease development. KEY MESSAGES : We investigated pleiotropy-informed enrichment between LS BMD and BW. We identified genetic variants related to both LS BMD and BW by utilizing a cFDR approach. PDXDC1 is a novel pleiotropic gene which may be related to both LS BMD and BW. Elevated expression of PDXDC1 is related to higher BMD and lower ratio n-6/n-3 PUFA indicating a bone protective effect of PDXDC1.

CRISPR-based assay reveals SARS-CoV-2 RNA dynamic changes and redistribution patterns in non-human primate model.

Mounting evidence indicates that SARS-CoV-2 can infect multiple systemic tissues, but few studies have evaluated SARS-CoV-2 RNA dynamics in multiple specimen types due to their reduced accessibility and diminished performance of RT-qPCR with non-respiratory specimens. Here, we employed an ultrasensitive CRISPR-RT-PCR assay to analyze longitudinal mucosal (nasal, buccal, pharyngeal, and rectal), plasma, and breath samples from SARS-CoV-2-infected non-human primates (NHPs) to detect dynamic changes in SARS-CoV-2 RNA level and distribution among these specimens. We observed that CRISPR-RT-PCR results consistently detected SARS-CoV-2 RNA in all sample types at most time points post-infection, and that SARS-CoV-2 infection dose and administration route did not markedly affect the CRISPR-RT-PCR signal detected in most specimen types. However, consistent RT-qPCR positive results were restricted to nasal, pharyngeal, and rectal swab samples, and tended to decrease earlier than CRISPR-RT-PCR results, reflecting lower assay sensitivity. SARS-CoV-2 RNA was detectable in both pulmonary and extrapulmonary specimens from early to late infection by CRISPR-RT-PCR, albeit with different abundance and kinetics, with SARS-CoV-2 RNA increases detected in plasma and rectal samples trailing those detected in upper respiratory tract samples. CRISPR-RT-PCR assays for SARS-CoV-2 RNA in non-respiratory specimens may thus permit direct diagnosis of suspected COVID-19 cases missed by RT-PCR, while tracking SARS-CoV-2 RNA in minimally invasive alternate specimens may better evaluate the progression and resolution of SARS-CoV-2 infections.

Exploring nano-enabled CRISPR-Cas-powered strategies for efficient diagnostics and treatment of infectious diseases.

Biomedical researchers have subsequently been inspired the development of new approaches for precisely changing an organism's genomic DNA in order to investigate customized diagnostics and therapeutics utilizing genetic engineering techniques. Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) is one such technique that has emerged as a safe, targeted, and effective pharmaceutical treatment against a wide range of disease-causing organisms, including bacteria, fungi, parasites, and viruses, as well as genetic abnormalities. The recent discovery of very flexible engineered nucleic acid binding proteins has changed the scientific area of genome editing in a revolutionary way. Since current genetic engineering technique relies on viral vectors, issues about immunogenicity, insertional oncogenesis, retention, and targeted delivery remain unanswered. The use of nanotechnology has the potential to improve the safety and efficacy of CRISPR/Cas9 component distribution by employing tailored polymeric nanoparticles. The combination of two (CRISPR/Cas9 and nanotechnology) offers the potential to open new therapeutic paths. Considering the benefits, demand, and constraints, the goal of this research is to acquire more about the biology of CRISPR technology, as well as aspects of selective and effective diagnostics and therapies for infectious illnesses and other metabolic disorders. This review advocated combining nanomedicine (nanomedicine) with a CRISPR/Cas enabled sensing system to perform early-stage diagnostics and selective therapy of specific infectious disorders. Such a Nano-CRISPR-powered nanomedicine and sensing system would allow for successful infectious illness control, even on a personal level. This comprehensive study also discusses the current obstacles and potential of the predicted technology. Supplementary Information: The online version contains supplementary material available at 10.1007/s40097-022-00472-7.

2D metal carbides and nitrides (MXenes) for sensors and biosensors.

MXenes are layered two-dimensional (2D) materials discovered in 2011 (Ti3C2X) and are otherwise called 2D transition metal carbides, carbonitrides, and nitrides. These 2D layered materials have been in the limelight for a decade due to their interesting properties such as large surface area, high ion transport, biocompatibility, and low diffusion barrier. Therefore, MXenes are widely preferred by researchers for applications in electronics, sensing, biosensing, electrocatalysis, super-capacitors and fuel cells. There are a number of methods available for the bulk synthesis of MXene-based nanomaterials. In addition, the possibility of structural modification as required and its outstanding surface chemistry offer a fascinating interface for the development of novel biosensors. In this review, we specifically discuss important MXene synthesis routes. Moreover, critical parameters such as surface functionalization that can dictate the mechanical, electronic, magnetic, and optical properties of MXenes are also discussed. Following this, methods available for the surface functionalization and modification strategies of MXenes are also discussed. Furthermore, the emergence of gas, electrochemical, and optical biosensors based on MXenes since its first report is discussed in detail. Finally, future directions of MXenes biosensors for critical applications are discussed.

Environmental noise reduction for tunable resistive pulse sensing of extracellular vesicles.

Extracellular vesicles (EVs) bearing biomolecules from parental cells can represent a novel source of disease biomarkers and are under intensive study for their clinical potential. Tunable resistive pulse sensing (TRPS) quantifies the magnitude of a small ionic resistive pulse current to determine the size, concentration, and zeta potential of EVs. Environmental noise is a common limiting factor that affects the precision of sensing devices. TRPS is particularly vulnerable to environmental noise, including both mechanical and electrical. The upper detection limit of the TRPS relies on the physical size of the elastomeric tunable nanopore. The lower limit relies on the electrical signal-to-noise ratio. Guided by simulation, we designed an external device to suppress environmental noise for TRPS measurement. Both mechanical and electrical environmental noise reductions were observed after using the shield. The study also validated the noise reduction function of the shield by quantifying EVs from different cell origins. Detection of EVs smaller than 200 nm was improved by using the shield; which was reported challenging for conventional quantification methods. The study highlighted a feasible approach to solve environmental noise challenges for TRPS based EV quantification.

Nanopore-based disease diagnosis using pathogen-derived tryptic peptides from serum.

Nanopore sensors have shown great utility in nucleic acid detection and sequencing approaches. Recent studies also indicate that current signatures produced by peptide-nanopore interactions can distinguish high purity peptide mixtures, but the utility of nanopore sensors in clinical applications still needs to be explored due to the inherent complexity of clinical specimens. To fill this gap between research and clinical nanopore applications, we describe a methodology to select peptide biomarkers suitable for use in an immunoprecipitation-coupled nanopore (IP-NP) assay, based on their pathogen specificity, antigenicity, charge, water solubility and ability to produce a characteristic nanopore interaction signature. Using tuberculosis as a proof-of-principle example in a disease that can be challenging to diagnose, we demonstrate that a peptide identified by this approach produced high-affinity antibodies and yielded a characteristic peptide signature that was detectable over a broad linear range, to detect and quantify a pathogen-derived peptide from digested human serum samples with high sensitivity and specificity. This nanopore signal distinguished serum from a TB case, non-disease controls, and from a TB-case after extended anti-TB treatment. We believe this assay approach should be readily adaptable to other infectious and chronic diseases that can be diagnosed by peptide biomarkers.

Lighting up ATP in cells and tissues using a simple aptamer-based fluorescent probe.

Extracellular ATP as a purinergic signaling molecule, together with ATP receptor, are playing an important role in tumor growth, therapy resistance, and host immunity suppression. Meanwhile ATP is a crucial indicator for cellular energy status and viability, thus a vital variable for tissue regeneration and in vitro tissue engineering. Most recent studies on COVID-19 virus suggest infection caused ATP deficit and release as a major characterization at the early stage of the disease and major causes for disease complications. Thus, imaging ATP molecule in both cellular and extracellular contexts has many applications in biology, engineering, and clinics. A sensitive and selective fluorescence "signal-on" probe for ATP detection was constructed, based on the base recognition between a black hole quencher (BHQ)-labeled aptamer oligonucleotide and a fluorophore (Cy5)-labeled reporter flare. The probe was able to detect ATP in solution with single digit µM detection limit. With the assistance of lipofectamine, this probe efficiently entered and shined in the model cells U2OS within 3 h. Further application of the probe in specific scenery, cardio-tissue engineering, was also tested where the ATP aptamer complex was able to sense cellular ATP status in a semi-quantitative manner, representing a novel approach for selection of functional cardiomyocytes for tissue engineering. At last a slight change in probe configuration in which a flexible intermolecular A14 linker was introduced granted regeneration capability. These data support the application of this probe in multiple circumstances where ATP measurement or imaging is on demand.