False Positive Signals Research Articles

An entropy-driven signal-off DNA circuit for label-free, visual detection of small molecules with enhanced accuracy.

An entropy-driven DNA circuit offers an efficient means of sensitive analyte detection with signal amplification. In this article, we rationally engineered an aptamer-based entropy-driven signal-off DNA circuit for colorimetric detection of small molecules. The proposed signal-off DNA circuit is activated by target small molecule binding to drive the collapse of G-quadruplex DNAzyme, accompanied by the colour change of the detection solution from dark blue to light blue. Entropy-driven recycling hybridization significantly magnified the input signal of the target small molecule. Such an assay enables naked-eye detection of adenosine triphosphate and oxytetracycline at concentrations as low as 0.5 μM and 1 μM respectively. Moreover, when compared with the signal-on DNA circuit, the entropy-driven signal-off DNA circuit for colorimetric detection has two advantages. Firstly, unlike in the signal-on DNA circuit, the unavoidable formation of waste complexes in the absence of a target in the signal-off DNA circuit has no influence on target detection performance as its background signal is only determined by the substrate complex. Secondly, the signal-on DNA circuit cannot distinguish false-positive signals generated by invasive catalysts (e.g., HRP, serum, Fe3O4), while the signal-off DNA circuit can distinguish those signals as undesired signals. Overall, the signal-off DNA circuit affords a novel strategy for sensitive and accurate detection of small molecules.

An Endogenous miRNA-Initiated Hybridization Chain Reaction and Subsequent DNAzyme Activation for Cellular Theranostics

An Endogenous miRNA-Initiated Hybridization Chain Reaction and Subsequent DNAzyme Activation for Cellular Theranostics

Diurnal variation in DLCO and non-standardized study procedures may cause a false positive safety signal in clinical trials

Diffusing capacity for carbon monoxide (DLCO) was measured in a phase I single ascending dose study after inhalation of AZD8154 or placebo in healthy participants at baseline (DLCOBaseline) and follow-up (DLCOFollow-up) 6 days after dosing. Initially, DLCOFollow-up timepoint was 2 h earlier than the DLCOBaseline timepoint and clinically significant decreases in DLCOFollow-up (absolute change up to 19% from baseline and DLCO%predicted values less than 70) were observed then. The observed reduction in DLCOFollow-up was confirmed as a false positive finding after alignment of DLCO timings. As a consequence, when DLCO is used in clinical studies, measurements should be strictly standardized in relation to time of the day.

Rapid Detection of blaKPC, blaNDM, blaOXA-48-like and blaIMP Carbapenemases in Enterobacterales Using Recombinase Polymerase Amplification Combined With Lateral Flow Strip.

The emergence of carbapenemase-producing Enterobacterales (CPE) infections is a major global public health threat. Rapid and accurate detection of pathogenic bacteria is essential to optimize treatment and timely avoid further transmission of these bacteria. Here, we aimed to develop a rapid on site visualization detection method for CPE using improved recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) method, based on four most popular carbapenemase genes: bla KPC, bla NDM, bla OXA-48-like, and bla IMP. All available allelic variants of the above carbapenemases were downloaded from the β-lactamase database, and the conserved regions were used as targets for RPA assay. Five primer sets were designed targeting to each carbapenemase gene and the RPA amplification products were analyzed by agarose gel electrophoresis. FITC-labeled specific probes were selected, combined with the best performance primer set (Biotin-labeled on the reverse primer), and detected by RPA-LFS. Mismatches were made to exclude the false positive signals interference. This assay was evaluated in 207 clinically validated carbapenem-resistant Enterobacterales (CRE) isolates and made a comparison with conventional PCR. Results showed that the established RPA-LFS assay for CPE could be realized within 30 min at a constant temperature of 37°C and visually detected amplification products without the need for special equipment. This assay could specifically differentiate the four classes of carbapenemases without cross-reactivity and shared a minimum detection limit of 100 fg/reaction (for bla KPC, bla NDM, and bla OXA-48-like) or 1000 fg/reaction (for bla IMP), which is ten times more sensitive than PCR. Furthermore, the detection of 207 pre-validated clinically CRE strains using the RPA-LFS method resulted in 134 bla KPC, 69 bla NDM, 3 bla OXA-48-like, and 1 bla IMP. The results of the RPA-LFS assay were in consistent with PCR, indicating that this method shared high sensitivity and specificity. Therefore, the RPA-LFS method for CPE may be a simple, specific, and sensitive method for the rapid diagnosis of carbapenemase Enterobacterales.

Establishing diagnostic algorithms for SARS-CoV-2 nucleic acid testing in clinical practice.

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic. Our laboratory initially used a two-step molecular assay, first reported by Corman et al, for SARS-CoV-2 identification (the Taiwan Center for Disease Control [T-CDC] method). As rapid and accurate diagnosis of COVID-19 is required to control the spread of this infectious disease, the current study evaluated three commercially available assays, including the TaqPath COVID-19 Combo kit, the cobas SARS-CoV-2 test, and the Rendu 2019-nCoV Assay kit, to establish diagnostic algorithms for clinical laboratories. A total of 790 clinical specimens, including nasopharyngeal swabs, throat swabs, sputum, saliva, stool, endotracheal aspirate, and serum were obtained from patients who were suspected or already confirmed to have COVID-19 at the Taipei Veterans General Hospital from February to May 2020. These specimens were tested for SARS-CoV-2 using the different assays and the performance variance between the assays was analyzed. Of the assays we evaluated, the T-CDC method and the TaqPath COVID-19 Combo kit require lots of hands-on practical laboratory work, while the cobas SARS-CoV-2 test and the Rendu 2019-nCoV Assay kit are fully automated detection systems. The T-CDC method and the TaqPath COVID-19 Combo kit showed similar detection sensitivity; however, the T-CDC method frequently delivered false-positive signals for envelope (E) and/or RNA-dependent RNA polymerase (RdRP) gene detection, thus increasing the risk of reporting false-positive results. A manual test-based testing strategy combining the T-CDC method and the TaqPath COVID-19 Combo kit was developed, which demonstrated excellent concordance rates (>99%) with the cobas and Rendu automatic systems. There were a few cases showing discrepant results, which may be due to the varied detection sensitivities as well as targets among the different platforms. Moreover, the concordance rate between the cobas and Rendu assays was 100%. Based on our evaluation, two SARS-CoV-2 diagnostic algorithms, one focusing on the manual assays and the other on the automatic platforms, were proposed. Our results provide valuable information that allows clinical laboratories to implement optimal diagnostic strategies for SARS-CoV-2 testing based on their clinical needs, such as test volume, turn-around time, and staff/resource limitations.

Selection shapes the landscape of functional variation in wild house mice

BackgroundThrough human-aided dispersal over the last ~ 10,000 years, house mice (Mus musculus) have recently colonized diverse habitats across the globe, promoting the emergence of new traits that confer adaptive advantages in distinct environments. Despite their status as the premier mammalian model system, the impact of this demographic and selective history on the global patterning of disease-relevant trait variation in wild mouse populations is poorly understood.ResultsHere, we leveraged 154 whole-genome sequences from diverse wild house mouse populations to survey the geographic organization of functional variation and systematically identify signals of positive selection. We show that a significant proportion of wild mouse variation is private to single populations, including numerous predicted functional alleles. In addition, we report strong signals of positive selection at many genes associated with both complex and Mendelian diseases in humans. Notably, we detect a significant excess of selection signals at disease-associated genes relative to null expectations, pointing to the important role of adaptation in shaping the landscape of functional variation in wild mouse populations. We also uncover strong signals of selection at multiple genes involved in starch digestion, including Mgam and Amy1. We speculate that the successful emergence of the human-mouse commensalism may have been facilitated, in part, by dietary adaptations at these loci. Finally, our work uncovers multiple cryptic structural variants that manifest as putative signals of positive selection, highlighting an important and under-appreciated source of false-positive signals in genome-wide selection scans.ConclusionsOverall, our findings highlight the role of adaptation in shaping wild mouse genetic variation at human disease-associated genes. Our work also highlights the biomedical relevance of wild mouse genetic diversity and underscores the potential for targeted sampling of mice from specific populations as a strategy for developing effective new mouse models of both rare and common human diseases.

RTP: One Effective Platform to Probe Reactive Compound Transformation Products and Its Applications for a Reactive Plasticizer BADGE.

Reactive compounds, such as covalent toxicants/drugs, have their ubiquitous occurrences and are known to react with protein or DNA in human beings, but their reactions with endogenous metabolites are rarely understood. Currently, a viable platform is demanded for discovering their reaction products since their efficacy/toxicity may be altered after the reaction. We aim to develop a platform for identifying unknown abiotic or biotransformation products for these reactive compounds. Based on stable isotope-labeling (SIL) metabolomics, we have developed a novel and robust analytical platform, reactive compound transformation profiler (RTP), which can automatically analyze preannotated high-resolution mass spectrometry (LC-HRMS) data sets and uncover probable transformation products. Generally, RTP consists of four complementary steps: (1) selecting peak pairs of light and heavy-labeled products, (2) defining the "core structure mass" for possible reaction search, (3) constructing an endogenous metabolite reaction database, and (4) developing algorithms to propose the potential transformation products by searching against the database with a single-/multiple-site reaction. Its performance was validated using the reactive plasticizer bisphenol A diglycidyl ether (BADGE) in several sample matrices. This platform enabled the identification of novel transformation products while also demonstrating its capacity to filter out the false-positive signals and provide product annotation. The RTP is freely accessible at https://github.com/FangLabNTU/Reactive-Compound-Transformation-Profiler-RTP-.

SuperSelective primer pairs for sensitive detection of rare somatic mutations

SuperSelective primers, by virtue of their unique design, enable the selective exponential amplification of rare DNA fragments containing somatic mutations in the presence of abundant closely related wild-type DNA fragments. However, when a SuperSelective primer is used in conjunction with a conventional reverse primer, linear amplification of the abundant wild-type fragments occurs, and this may lead to a late arising signal that can be confused with the late arising signal from the rare mutant fragments. We have discovered that the use of a pair of SuperSelective primers, one specific for the target mutation in a plus strand, and the other specific for the same mutation in the complementary minus strand, but both possessing 3′-terminal nucleotides that are complementary to the mutation, significantly suppresses the linear amplification of the related wild-type sequence, and prevents the generation of false mutant sequences due to mis-incorporation by the DNA polymerase. As a consequence, the absence of mutant fragments in a sample does not give rise to a false-positive signal, and the presence of mutant fragments in a sample is clearly distinguishable as a true-positive signal. The use of SuperSelective primer pairs should enhance the sensitivity of multiplex PCR assays that identify and quantitate somatic mutations in liquid biopsies obtained from patients with cancer, thereby enabling the choice of a targeted therapy, the determination of its effectiveness over time, and the substitution of a more appropriate therapy as new mutations arise.

Membrane-Activated Fluorescent Probe for High-Fidelity Imaging of Mitochondrial Membrane Potential.

Mitochondrial membrane potential (ΔΨm) is a key indicator of cell health or injury due to its vital roles in adenosine 5'-triphosphate synthesis. Thus, monitoring ΔΨm is of great significance for the assessment of cell status, diagnosis of diseases, and medicament screening. Cationic fluorescent probes suffer from severe photobleaching or false positive signals due to the luminescence of the probe on non-mitochondria. Herein, we report a lipophilic cationic fluorescent probe [1-methyl-2-(4-(1,2,2-triphenylvinyl)styryl)-β-naphthothiazol-1-ium trifluoromethanesulfonate (TPE-NT)] with the features of aggregation-induced emission and intramolecular charge transfer for imaging ΔΨm in live cells. TPE-NT is enriched on the surface of the mitochondrial inner membrane due to the negative ΔΨm, and its fluorescence is activated in the high-viscosity microenvironment. The false positive signals of emission from TPE-NT on non-mitochondria are therefore effectively eliminated. Moreover, TPE-NT exhibits a Stokes shift of >200 nm, near-infrared (∼675 nm) emission, excellent photostability, and low cytotoxicity, which facilitate real-time imaging in live cells. Cell imaging confirmed that the probe can rapidly and reliably report mitochondrial depolarization (decrement of ΔΨm) during cell damage caused by CCCP and H2O2 as well as mitochondrial polarization (increment of ΔΨm) by oligomycin. Furthermore, the probe successfully detected the reduction of ΔΨm in these cell models of hypoxia, heat damage, acidification, aging, inflammation, mitophagy, and apoptosis caused by hypoxia, heatstroke, lactate/pyruvate, doxorubicin, lipopolysaccharide, rapamycin, monensin, and nystatin, respectively.

Peptide nucleic acid–dependent artifact can lead to false-positive triplex gene editing signals

Triplex gene editing relies on binding a stable peptide nucleic acid (PNA) sequence to a chromosomal target, which alters the helical structure of DNA to stimulate site-specific recombination with a single-strand DNA (ssDNA) donor template and elicits gene correction. Here, we assessed whether the codelivery of PNA and donor template encapsulated in Poly Lactic-co-Glycolic Acid (PLGA)-based nanoparticles can correct sickle cell disease and x-linked severe combined immunodeficiency. However, through this process we have identified a false-positive PCR artifact due to the intrinsic capability of PNAs to aggregate with ssDNA donor templates. Here, we show that the combination of PNA and donor templates but not either agent alone results in different degrees of aggregation that result in varying but highly reproducible levels of false-positive signal. We have identified this phenomenon in vitro and confirmed that the PNA sequences producing the highest supposed correction in vitro are not active in vivo in both disease models, which highlights the importance of interrogating and eliminating carryover of ssDNA donor templates in assessing various gene editing technologies such as PNA-mediated gene editing.

Stimuli-Induced Upgrade of Nuclease-Resistant DNA Nanostructure Composed of a Single Molecular Beacon for Detecting Mutant Genes.

As a kind of cell-free DNA in the bloodstream liberated from tumor cells, circulating tumor DNAs (ctDNAs) have been recognized as promising biomarkers in the field of early cancer diagnosis. However, robust, sensitive, and accurate detection of ctDNA in serum remains extremely challenging, especially toward the mutant KRAS gene, one of the most frequently mutated genes. Although DNA oligonucleotides as emerging practical signaling materials have been developed as sensitive and accurate tools, some intrinsic defects need to be overcome, such as fragility in complex biological environments. In this work, on the basis of the hydrophilicity-promoted assembly, a core/shell DNA nanostructure (DNS-MB) probe is constructed from only one hairpin-shaped probe (cholesterol-modified palindromic molecular beacon, Chol-PMB) for the amplification detection of KRAS mutation in serum without the need for any auxiliary probe. Chol-PMB is designed to recognize target DNA and serve as a polymerization primer and template, and thus target species can initiate polymerization-based strand displacement amplification (SDA). Moreover, target DNA is able to induce further aggregation of DNS-MB particles due to the enzymatic cross-linking effect, leading to a structural upgrade. The DNS-MB probe exhibits a detection limit of 50 fM and a wide quantitative range (from 50 fM to 160 nM). In addition, single nucleotide polymorphisms can be discriminated, such as mutant KRAS G12D (KRAS-M), providing a desirable platform for screening ctDNAs. More excitingly, because the termini of DNA components are hidden inward from nuclease attack, DNS-MB circumvents a false-positive signal even in freshly sampled serum and is suitable for application in the complex biological milieu. As a proof of concept, the DNS-MB probe is expected to provide useful insight into the development of simple and degradation-resistant DNA probes for substantially amplified detection of ctDNAs in complex serum, showing potential applications in the field of early tumor diagnosis.

A pH-Reversible Fluorescent Probe for in Situ Imaging of Extracellular Vesicles and Their Secretion from Living Cells.

Our knowledge in how extracellular vesicles (EVs) are secreted from cells remains inadequate due to the limited technologies available for visualizing them in situ. We report a pH-reversible boron dipyrromethene (BODIPY) fluorescent probe for confocal imaging of EVs secreted from living cells without inducing severe cytotoxicity. This probe predominantly assumes a non-fluorescent leuco-BODIPY form under basic conditions, but it gradually switches to its fluorescent parent BODIPY form upon acidification; such pH transition empowers the imaging of acidic EVs (such as CD81-enriched exosomes and extracellular multivesicular bodies) in weakly basic culture medium and intracellular acidic precursor EVs in weakly basic cytoplasm, with minimal false positive signals frequently encountered for "always-on" dyes. Joint application of this probe with plasmid transfection reveals the secretion of some EVs from cellular pseudopodia via microtubule trackways. This probe may provide mechanistic insights into the extracellular transport of EVs and support the development of EV-based nanomedicines.

A Facile Strategy for the Ion Current and Fluorescence Dual-Lock in Detection: Naphthalic Anhydride Azide (NAA)-Modified Biomimetic Nanochannel Sensor towards H2S

Inspired by biological channels, the electric signal-based artificial nanochannel system exhibits high sensitivity in various analyses. However, ion current may be affected by many other factors, leading to false-positive signals. For reliable detection, in this work, we apply a facile strategy to combine both current signal and fluorescence. Fluorescent probes were conjugated to the nanochannel surface by covalent bonds. By utilizing the specific reduction of azide groups in the probe to amino groups by H2S, a synchronizing change in fluorescence and nanochannel surface charge was established. As a result, both transmembrane ion current and fluorescence intensity showed significant changes. The photoelectric double-checked locking from temporal and spatial variation validly confirmed the response process and protected detection accuracy. The work may provide new ideas for the development of more sophisticated current and fluorescence dual-index nanochannel systems.

Generation of False-Positive SARS-CoV-2 Antigen Results with Testing Conditions outside Manufacturer Recommendations: A Scientific Approach to Pandemic Misinformation.

ABSTRACTAntigen-based rapid diagnostics tests (Ag-RDTs) are useful tools for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. However, misleading demonstrations of the Abbott Panbio coronavirus disease 2019 (COVID-19) Ag-RDT on social media claimed that SARS-CoV-2 antigen could be detected in municipal water and food products. To offer a scientific rebuttal to pandemic misinformation and disinformation, this study explored the impact of using the Panbio SARS-CoV-2 assay with conditions falling outside manufacturer recommendations. Using Panbio, various water and food products, laboratory buffers, and SARS-CoV-2-negative clinical specimens were tested with and without manufacturer buffer. Additional experiments were conducted to assess the role of each Panbio buffer component (tricine, NaCl, pH, and Tween 20) as well as the impact of temperature (4°C, 20°C, and 45°C) and humidity (90%) on assay performance. Direct sample testing (without the kit buffer) resulted in false-positive signals resembling those obtained with SARS-CoV-2 positive controls tested under proper conditions. The likely explanation of these artifacts is nonspecific interactions between the SARS-CoV-2-specific conjugated and capture antibodies, as proteinase K treatment abrogated this phenomenon, and thermal shift assays showed pH-induced conformational changes under conditions promoting artifact formation. Omitting, altering, and reverse engineering the kit buffer all supported the importance of maintaining buffering capacity, ionic strength, and pH for accurate kit function. Interestingly, the Panbio assay could tolerate some extremes of temperature and humidity outside manufacturer claims. Our data support strict adherence to manufacturer instructions to avoid false-positive SARS-CoV-2 Ag-RDT reactions, otherwise resulting in anxiety, overuse of public health resources, and dissemination of misinformation.IMPORTANCE With the Panbio severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen test being deployed in over 120 countries worldwide, understanding conditions required for its ideal performance is critical. Recently on social media, this kit was shown to generate false positives when manufacturer recommendations were not followed. While erroneous results from improper use of a test may not be surprising to some health care professionals, understanding why false positives occur can help reduce the propagation of misinformation and provide a scientific rebuttal for these aberrant findings. This study demonstrated that the kit buffer’s pH, ionic strength, and buffering capacity were critical components to ensure proper kit function and avoid generation of false-positive results. Typically, false positives arise from cross-reacting or interfering substances; however, this study demonstrated a mechanism where false positives were generated under conditions favoring nonspecific interactions between the two antibodies designed for SARS-CoV-2 antigen detection. Following the manufacturer instructions is critical for accurate test results.

Rapid and sensitive recombinase polymerase amplification combined with lateral flow strips for detecting Candida albicans

Owing to modern lifestyles and increasing amounts of medical intervention, clinical infections caused by conditionally pathogenic fungi are becoming increasingly serious. Among these, Candida albicans is the most common. Therefore, the rapid and accurate detection of this pathogenic fungus is important to guiding the selection of clinical therapeutic agents. Recombinase polymerase amplification (RPA) combined with lateral flow strips (LFS) is a promising molecular detection method with the advantages of rapidity, simplicity of operation and high sensitivity. However, this simplicity brings with it the inherent and non-negligible risk of false-positive signals from primer-dimers. In this study, primer-dependent artifacts were eliminated by using probes in the RPA reaction, introducing specific base substitutions to the primer and probe sequences and analyzing and screening the formation of primer-probe complexes. These measures were rigorously tested for efficacy, leading to the creation of an improved RPA-LFS system. The standardized method enabled the specific detection of C. albicans within 25 min at 37 °C without interference. The system had a detection limit of 1 CFU per reaction without DNA purification or 102 fg genomic DNA/50 μL. The detection sensitivity was not affected by the presence of other fungal DNA. The RPA-LFS method can therefore be used to detect clinical samples, and the results are accurate and consistent in comparison with those obtained using quantitative PCR. This study provides a paradigm for eliminating the risk of false-positive primer dimers in isothermal amplification assays and establishes a simple and easy method for the detection of C. albicans.

Essence - A New Electrochemical Platform

There is a great demand for a fast, on-site screening test, especially during the COVID-19 pandemic. However, most point-of-care (POC) sensors usually suffer from insufficient sensitivity and selectivity. Often these devices have a very high rate of false positives and false negatives. The incorrect data could result in low customer usage (both the patient and the doctor) due to decreased confidence in the results. Portable ELISA-based kits can have low rates of false-positive and false negatives. However, ELISA is inherently complicated and often requires a specialist on-site to ensure the success of each sample's high costs and time.Here we show a new electrochemical platform called ESSENCE. ESSENCE is an Electrochemical Sensor that uses a Shear-Enhanced, flow-through Nanoporous Capacitive Electrode. ESSENCE can overcome current electrochemical sensors' selectivity and sensitivity limitations. The ESSENCE sensor architecture is a microfluidic channel sandwiched between two gold micro-electrode glass slides, formed a non-planar interdigitated porous electrode that improved electric field penetration compared with the traditional planar interdigitated electrode. The porous electrode structure used in this research has a high Zeta potential compared to the bare glass surface. This results in a significantly smaller Debye double-layer (EDL) length than in a clear channel. Due to a smaller EDL length, the relaxation frequency increases, which shifts the EIS signal to a higher frequency range (1kHz to 100MHz) and results in a fast response with a higher signal-to-noise ratio in Electrochemical Impedance Spectroscopy(EIS) measurements.Further, the modular fabrication of the ESSENCE chip allows ESSENCE to target different biomolecules from DNAs to protein cancer biomarkers by simply changing the packed transducer material. However, the packing material’s conductivity represents a significant challenge to the electrochemical response of ESSENCE. The high conductivity of the transducer material shorts the system leading to a pure resistive response. Impedance spectroscopy (EIS) response of ESSENCE cannot be distinguished enough to determine if the sensor is working. ESSENCE has a switchable electrode system for usage in multiple configurations like a material electrode, clear electrode, and working electrode to overcome these limitations. For carbon nanotube (CNT) transducers with functionalized oligos on the CNT, a 20-fold jump in the signal is noticed from the working electrode configuration compared to the other configurations. It is worthwhile to note that depending on the biomolecule (DNA or Protein), the surface charge transfer resistance (Rct) in the EIS signal of ESSENCE can either show a decrease (DNA) or increase (protein) on the binding of the target bio-molecule (target DNA to DNA or target protein to protein). The change in the Rct depends on the charge of the molecule. It has been seen that on the binding of a target protein, the active area of the electrode surface decreased, leading to a reduction in current and a concomitant increase in Rct. On binding of the target DNA, the capture-target DNA pair essentially becomes a part of the electrode due to its charge. This increase in charge is more significant than any reduction in the surface area due to the DNA's binding. This leads to an increase in current to the packed electrode, leading to a concomitant decrease in Rct.The fluid flow through the porous electrode increases mixing, minimizes diffusion, and generates a significantly enhanced fluidic shear force. This force e can unbind most of the false-positive signals generated by non-specific binding, such as physical adhesive or biofouling. The porous electrode also provides a high surface area to volume ratio to capture the target analyte leading to increased sensitivity. We have an automatic fluidic control system in ESSENCE that minimized any errors in the operation of ESSENCE. Our system has high selectivity and sensitivity for DNA (fM sensitivity, selective against non-target DNA), breast cancer biomarker proteins (p53, pg/L sensitivity, selective against non-target HER2). Figure 1

Long-Wave Infrared Imaging for Intraoperative Cancer Detection-What is the True Temperature of a Cancer?

During cancer operations, the cancer itself is often hard to delineate-buried beneath healthy tissue and lacking discernable differences from the surrounding healthy organ. Long-wave infrared, or thermal, imaging poses a unique solution to this problem, allowing for the real-time label-free visualization of temperature deviations within the depth of tissues. The current study evaluated this technology for intraoperative cancer detection. In this diagnostic study, patients with gastrointestinal, hepatobiliary, and renal cancers underwent long-wave infrared imaging of the malignancy during routine operations. It was found that 74% were clearly identifiable as hypothermic anomalies. The average temperature difference was 2.4°C (range 0.7 to 5.0) relative to the surrounding tissue. Cancers as deep as 3.3cm from the surgical surface were visualized. Yet, 79% of the images had clinically relevant false positive signals [median 3 per image (range 0 to 10)] establishing an accuracy of 47%. Analysis suggests that the degree of temperature difference was primarily determined by features within the cancer and not peritumoral changes in the surrounding tissue. These findings provide important information on the unexpected hypothermal properties of intra-abdominal cancers, directions for future use of intraoperative long-wave infrared imaging, and new knowledge about the in vivo thermal energy expenditure of cancers and peritumoral tissue.

Rapid identification of Atlantic salmon (Salmo salar) based on loop-mediated isothermal amplification (LAMP) using self-quenching fluorogenic approach

Loop-mediated isothermal amplification (LAMP) has emerged as an important diagnostic tool for fish authenticity purpose. While the sequence-independent detection methods greatly increased the likelihood of detecting false-positive signals. Instead, target-specific detection of LAMP amplicons can be achieved through the use of target-specific probes or modified primers as biorecognition elements. The present work selected Salmo salar as a case study, and developed a novel LAMP assay coupled with the self-quenching approach for accurate species identification.Specifically, the loop primer (LB-6) was identified as the candidate primer to design the self-quenching element. The fluorophore attached to LB-6 is self-quenched in unbound state. After binding to the dumb bell shaped DNA specifically, the FAM fluorophore is de-quenched, resulting in the fluorescence release. With the novel assay, as little as 5 pg of S. salar DNA could be detected. Moreover, the self-quenching assay embraces a high tolerance to impurities and the positive LAMP results can always be obtained, regardless of the DNA extraction methods and sample treatments. While significant difference with the Ct value according to both the extraction methods and sample treatments, highlighted also an improved LAMP amplification using DNA with higher purity.

Endogenous miRNA-Activated DNA Nanomachine for Intracellular miRNA Imaging and Gene Silencing.

The development of multifunctional nanoplatforms that integrate both diagnostic and therapeutic functions has always been extremely desirable and challenging in the cancer combat. Here, we report an endogenous miRNA-activated DNA nanomachine (EMDN) in living cells for concurrent sensitive miRNA imaging and activatable gene silencing. EMDN is constructed by interval hybridization of two functional DNA monomers (R/HP and F) to a DNA nanowire generated by hybridization chain reaction. After the target cell-specific transportation of EMDN, intracellular let-7a miRNA initiates the DNA nanomachine by DNA strand displacement cascades, resulting in an amplified fluorescence resonance energy-transfer signal and the release of many free HP sequences. The restoration of HP hairpin structures further activates the split-DNAzyme to identify and cleave the EGR-1 mRNA to realize gene silencing therapy. The proposed EMDN shows efficient cell internalization, good biological stability, rapid reaction kinetics, and the ability to avoid false-positive signals, thus ensuring reliable miRNA imaging in living cells. Meanwhile, the controlled activation of the split-DNAzyme activity regulated by the intracellular specific miRNA may be promising in the precise treatment of cancer. Collectively, this strategy provides a valuable nanoplatform for early clinical diagnosis and activatable gene therapy of tumors.

Two-dimensional Ti2C MXene-induced photocurrent polarity switching photoelectrochemical biosensing platform for ultrasensitive and selective detection of soluble CD146

Two-dimensional (2D) materials with unique structures and fascinating photoelectric properties are typically employed as supporting materials but they have not been mentioned in the context of polarity-reversal-mode photoelectrochemical (PEC) biosensors. Herein, a novel 2D Ti2C MXene-induced photocurrent polarity switching PEC biosensing platform was constructed for the selective and ultrasensitive detection of soluble CD146 (sCD146). Ag2S quantum dots (QDs)-fixed indium-tin oxide slices were used as the photocathode electrodes, and the capture antibody (Ab1) was assembled on the electrode. When the target sCD146 existed, 2D Ti2C MXene labeled with the detection antibody (Ab2) was introduced onto the modified electrode through a typical antigen-antibody sandwich immune reaction, resulting in a polarity switch from a cathodic to an anodic photocurrent. The outstanding optical and electron-transfer capability of the 2D Ti2C MXene made it beneficial to establish a high-performance photocurrent polarity switching PEC biosensor for sCD146 assay, and a linear response range of 0.1–1000 pg/mL with a detection limit of 18 fg/mL was obtained. Furthermore, the developed PEC biosensing strategy possessed excellent anti-interference ability for precluding the false positive or negative signals. The designed 2D Ti2C MXene opens up a new two-dimensional photocurrent-polarity switcher for constructing high-performance PEC biosensors, and the environmentally friendly 2D Ti2C MXene//Ag2S QDs photocurrent polarity switching system could overcome the harmfulness of conventional toxic semiconductors for operators and the environment, offering great potential applications in biological analysis and disease diagnosis.