Mitochondria-Targeted Fluorescent Nanoparticles with Large Stokes Shift for Long-Term BioImaging.

Mitochondria (MITO) play a significant role in various physiological processes and are a key organelle associated with different human diseases including cancer, diabetes mellitus, atherosclerosis, Alzheimer's disease, etc. Thus, detecting the activity of MITO in real time is becoming more and more important. Herein, a novel class of amphiphilic aggregation-induced emission (AIE) active probe fluorescence (AC-QC nanoparticles) based on a quinoxalinone scaffold was developed for imaging MITO. AC-QC nanoparticles possess an excellent ability to monitor MITO in real-time. This probe demonstrated the following advantages: (1) lower cytotoxicity; (2) superior photostability; and (3) good performance in long-term imaging in vitro. Each result of these indicates that self-assembled AC-QC nanoparticles can be used as effective and promising MITO-targeted fluorescent probes.

Rapid One-Pot Microwave Assisted Green Synthesis Nitrogen Doped Carbon Quantum Dots as Fluorescent Precursor for Estimation of Modafinil as Post-Covid Neurological Drug in Human Plasma with Greenness Assessments.

The neuro-stimulant anti-narcoleptic drug as modafinil (MOD) is used to treatment neurological conditions caused by COVID-19. MOD was used to treatment narcolepsy, shift-work sleep disorder, and obstructive sleep apnea-related sleepiness. So, an innovative, quick, economical, selective, and ecologically friendly procedure was carried out. A highly sensitive N@CQDs technique was created from green Eruca sativa leaves in about 4 min using microwave synthesis at 700 w. The quantum yield of the synthesized N@CQDs was found to be 41.39%. By increasing the concentration of MOD, the quantum dots' fluorescence intensity was gradually quenched. After being excited at 445 nm, the fluorescence reading was recorded at 515 nm. The linear range was found to be in the range 50 - 700 ng mL-1 with lower limit of quantitation (LOQ) equal to 45.00 ng mL-1. The current method was fully validated and bio analytically according to (US-FDA and ICH) guidelines. Full characterization of the N@CQDs has been conducted by high resolution transmission electron microscope (HRTEM), Zeta potential measurement, fluorescence, UV-VIS, and FTIR spectroscopy. Various experimental variables including pH, QDs concentration and the reaction time were optimized. The proposed study is simply implemented for the therapeutic drug monitoring system (TDMS) and various clinical laboratories for further pharmacokinetic research.

Spatially Addressable Multiplex Biodetection by Calibrated Micro/Nanostructured Surfaces.

A challenge of any biosensing technology is the detection of very low concentrations of analytes. The fluorescence interference contrast (FLIC) technique improves the fluorescence-based sensitivity by selectively amplifying, or suppressing, the emission of a fluorophore-labeled biomolecule immobilized on a transparent layer placed on top of a mirror basal surface. The standing wave of the reflected emission light means that the height of the transparent layer operates as a surface-embedded optical filter for the fluorescence signal. FLIC extreme sensitivity to wavelength is also its main problem: small, e.g., 10 nm range, variations of the vertical position of the fluorophore can translate in unwanted suppression of the detection signal. Herein, we introduce the concept of quasi-circular lenticular microstructured domes operating as continuous-mode optical filters, generating fluorescent concentric rings, with diameters determined by the wavelengths of the fluorescence light, in turn modulated by FLIC. The critical component of the lenticular structures was the shallow sloping side wall, which allowed the simultaneous separation of fluorescent patterns for virtually any fluorophore wavelength. Purposefully designed microstructures with either stepwise or continuous-slope dome geometries were fabricated to modulate the intensity and the lateral position of a fluorescence signal. The simulation of FLIC effects induced by the lenticular microstructures was confirmed by the measurement of the fluorescence profile for three fluorescent dyes, as well as high-resolution fluorescence scanning using stimulated emission depletion (STED) microscopy. The high sensitivity of the spatially addressable FLIC technology was further validated on a diagnostically important target, i.e., the receptor-binding domain (RBD) of the SARS-Cov2 via the detection of RBD:anti-S1-antibody.

Detection of SARS-CoV-2 receptor binding domain using fluorescence probe and DNA flowers enabled by rolling circle amplification.

Using rolling circle amplification (RCA) and two different ways of signal readout, we developed analytical methods to detect the receptor-binding domain (RBD) of SARS-CoV-2 spike protein (S protein). We modified streptavidin-coated magnetic beads with an aptamer of RBD through a biotin-tagged complementary DNA strand (biotin-cDNA). Binding of RBD caused the aptamer to dissociate from the biotin-cDNA, making the cDNA available to initiate RCA on the magnetic beads. Detection of RBD was achieved using a dual signal output. For fluorescence signaling, the RCA products were mixed with a dsDNA probe labeled with fluorophore and quencher. Hybridization of the RCA products caused the dsDNA to separate and to emit fluorescence (λex = 488 nm, λem = 520 nm). To generate easily detectable UV-vis absorbance signal, the RCA amplification was extended to produce DNA flower to encapsulate horseradish peroxidase (HRP). The HRP-encapsulated DNA flower catalyzed a colorimetric reaction between H2O2 and 3,3',5,5'-tetramethylbenzidine (TMB) to generate an optical signal (λabs = 450 nm). The fluorescence and colorimetric assays for RBD have low detection limits (0.11 pg mL-1 and 0.904 pg mL-1) and a wide linear range (0.001-100 ng mL-1). For detection of RBD in human saliva, the recovery was 93.0-100% for the fluorescence assay and 87.2-107% for the colorimetric assay. By combining fluorescence and colorimetric detection with RCA, detection of the target RBD in human saliva was achieved with high sensitivity and selectivity.

An ESIPT-based ratiometric fluorescent probe for detecting H2O2 in water environment and biosystems.

The outbreak of the COVID-19 has resulted in a great increase in the use of H2O2 disinfectant, which is listed as one of the commonly used disinfectants for COVID-19 by the U.S. Environmental Protection Agency. However, excessive use of H2O2 disinfectant can threaten human health and damage the water environment. Therefore, it's of great importance to detect H2O2 in aquatic environments and biological systems. Herein, we proposed a novel ESIPT ratio fluorescent probe (named probe 1) for detecting H2O2 in water environment and biosystems. Probe 1 emits blue fluorescence as the introduction of the phenylboronic acid disrupts the ESIPT process. After reacting with H2O2, the phenylboronic acid is oxidatively removed, and the ESIPT process is restored, which makes the fluorescence emission wavelength red-shifted. Probe 1 exhibited a short response time, high sensitivity, and a large Stokes shift to H2O2. Importantly, it has been successfully used to detect H2O2 not only in actual water samples, but also endogenous and exogenous H2O2 in living cells. The characteristics of probe 1 have a wide range of applications in environmental and biological systems.

Role of the Probe Sequence/Structure in Developing an Ultra-Efficient Label-Free COVID-19 Detection Method Based on Competitive Dual-Emission Ratiometric DNA-Templated Silver Nanoclusters as Single Fluorescent Probes.

We report the development of a label-, antibody-, enzyme-, and amplification-free ratiometric fluorescent biosensor for low-cost and rapid (less than 12 min) diagnosis of COVID-19 from isolated RNA samples. The biosensor is designed on the basis of cytosine-modified antisense oligonucleotides specific for either N gene or RdRP gene that can form silver nanoclusters (AgNCs) with both green and red emission on an oligonucleotide via a one-step synthesis process. The presence of the target RNA sequence of SARS-CoV-2 causes a dual-emission ratiometric signal transduction, resulting in a limit of detection of 0.30 to 10.0 nM and appropriate linear ranges with no need for any further amplification, fluorophore, or design with a special DNA fragment. With this strategy, five different ratiometric fluorescent probes are designed, and how the T/C ratio, the length of the stem region, and the number of cytosines in the loop structure and at the 3' end of the cluster-stabilizing template can affect the biosensor sensitivity is investigated. Furthermore, the effect of graphene oxide (GO) on the ratiometric behavior of nanoclusters is demonstrated and the concentration-/time-dependent new competitive mechanism between aggregation-caused quenching (ACQ) and aggregation-induced emission enhancement (AIE) for the developed ssDNA-AgNCs/GO nanohybrids is proposed. Finally, the performance of the designed ratiometric biosensor has been validated using the RNA extract obtained from more than 150 clinical samples, and the results have been confirmed by the FDA-approved reverse transcription-polymerase chain reaction (RT-PCR) diagnostic method. The diagnostic sensitivity and specificity of the best probe is more than >90%, with an area under the receiver operating characteristic (ROC) curve of 0.978.

Measurement of Protein Kinetics Using a Liquid Phase-Based Biosensing Platform.

Macromolecular association is crucial to many fields in biomedical sciences, including drug development, gene editing, and diagnostics. In particular, protein-protein association and dissociation rate constants are typically determined using surface plasmon resonance systems, which require costly instrumentation and cumbersome procedures (e.g., blocking, washing, and separation). Herein, we demonstrate that protein-binding constants can be readily determined using a real-time biosensing platform facilitated by graphene oxide-modified microwell plates and fluorophore-labeled proteins, where the fluorescent probes remain highly fluorescent during protein association, whereas fluorescent bioprobes that are not associated with their counterparts are quenched by graphene oxide. Binding data of three pairs of proteins were systematically determined employing this single-step platform and compared with those data reported by the suppliers or the literature, suggesting that this approach is comparable and consistent with the existing ones. Such pairs include (i) human immunoglobulin G (H-IgG)-fluorophore-labeled anti-H-IgG, (ii) prostate-specific antigen (PSA)-quantum dot-labeled anti-PSA, and (iii) anti-RBD-fluorophore-labeled SARS-CoV-2 spike receptor-binding domain recombinant protein. We also offer an open-source software that automatically determines the binding kinetics constants of proteins. This Technical Note introduces a simple, yet effective, platform to determine relevant information on protein kinetics, which can be performed using a microwell plate reader and economical materials like graphene oxide. We foresee a new generation of diagnostics based on our affordable protein kinetics analysis.

Blood identified and quantified in formalin fixed paraffin embedded lung sections using eosin fluorescence.

Eosin Y is a common stain in histology. Although usually used for colourimetric imaging where the dye is used to stain pink/red a range of structures in the tissue, Eosin Y is also a fluorochrome, and has been used in this manner for decades. In this study our aim was to investigate the fluorescence properties of the dye to enable quantification of structures within formalin-fixed paraffin-embedded (FFPE) tissue sections. To do this, FFPE sections of hamster tissue were prepared with haematoxylin and eosin Y dyes. Spectral detection on a confocal laser scanning microscope was used to obtain the fluorescence emission spectra of the eosin Y under blue light. This showed clear spectral differences between the red blood cells and congealed blood, compared to the rest of the section. The spectra were so distinct that it was possible to discern these in fluorescence and multi-photon microscopy. An image analysis algorithm was used to quantify the red blood cells. These analyses could have broad applications in histopathology where differentiation is required, such as the analysis of clotting disorders to haemorrhage or damage from infectious disease.

Highly Efficient Red/NIR-Emissive Fluorescent Probe with Polarity-Sensitive Character for Visualizing Cellular Lipid Droplets and Determining Their Polarity.

Lipid droplets (LDs), which are ubiquitous organelles existing in almost all eukaryotic cells, have attracted a lot of attention in the field of cell biology over the last decade. For the biological study of LDs via fluorescence imaging, the superior LD fluorescent probes with environmental polarity-sensitive character are highly desired and powerful but are very scarce. Herein, we have newly developed such a kind of fluorescent probe named LDs-Red which enables us to visualize LDs and to further reveal their polarity information. This fluorescent probe displays the advantages of intense red/near-infrared emission, high LD staining specificity, and good photostability; thus, it would be very useful for LD fluorescence imaging application. As a result, the three-dimensional confocal imaging to visualize spatial distribution of LDs and the multicolor confocal imaging to simultaneously observe LDs and other cellular organelles have been realized using this new LD fluorescent probe. Furthermore, the polarity-sensitive emission character of this probe enables us to quantitatively determine the LD polarity via spectral scan imaging. Consequently, the cancer cells (HepG2, HeLa, and Panc02) displaying lower polarity of LDs than the normal cells (L929, U251, and HT22) have been systematically demonstrated. In addition, this polarity-sensitive probe displaying shorter fluorescence wavelengths in cancer cells than in normal cells has an important and potential ability to distinguish them.

Basic Fluorescent Protonation-Type pH Probe Sensitive to Small ΔpKa of Methanol and Ethanol.

An optical pH probe is a simple and effective tool for determining an accurate pH value in its localized area. However, basic pH probes with pKBH+ values above 8 have rarely been reported, although many components with high pKa such as arginine play important roles in vivo. Herein, we introduce novel colorimetric and fluorescent basic probes 1-5, which are designed using push-pull-type aminoquinoline and aminobenzoquinoline fluorophores, with pKBH+ values ranging from 8.4 to 9.9. After the basicity of the remarkably sensitive basic probe 4 was tuned, it was able to successfully distinguish between the pKa values of MeOH (15.5) and EtOH (15.9), thus displaying selective protonation and fluorescence enhancement in MeOH over EtOH. Our pH probes can be used to detect MeOH poisoning in commercial EtOH products such as hand sanitizers, providing an effective solution to this problem observed during the COVID-19 pandemic.

Bead-Based Multiplexed Droplet Digital Polymerase Chain Reaction in a Single Tube Using Universal Sequences: An Ultrasensitive, Cross-Reaction-Free, and High-Throughput Strategy.

The multiplexed digital polymerase chain reaction (PCR) is widely used in molecular diagnosis owing to its high sensitivity and throughput for multiple target detection compared with the single-plexed digital PCR; however, current multiplexed digital PCR technologies lack efficient coding strategies that do not compromise the sensitivity and signal-to-noise (S/N) ratio. Hence, we propose a fluorescent-encoded bead-based multiplexed droplet digital PCR method for ultra-high coding capacity, along with the creative design of universal sequences (primer and fluorescent TaqMan probe) for ultra-sensitivity and high S/N ratios. First, pre-amplification is used to introduce universal primers and universal fluorescent TaqMan probes to reduce primer interference and background noise, as well as to enrich regions of interest in targeted analytes. Second, fluorescent-encoded beads (FEBs), coupled with the corresponding target sequence-specific capture probes through streptavidin-biotin conjugation, are used to partition amplicons via hybridization according to the Poisson distribution. Finally, FEBs mixed with digital PCR mixes are isolated into droplets generated via Sapphire chips (Naica Crystal Digital PCR system) to complete the digital PCR and result analysis. For proof of concept, we demonstrate that this method achieves high S/N ratios in a 5-plexed assay for influenza viruses and SARS-CoV-2 at concentrations below 10 copies and even close to a single molecule per reaction without cross-reaction, further verifying the possibility of clinical actual sample detection with 100% accuracy, which paves the way for the realization of digital PCR with ultrahigh coding capacity and ultra-sensitivity.

Application of meso-CF3-Fluorophore BODIPY with Phenyl and Pyrazolyl Substituents for Lifetime Visualization of Lysosomes.

A bright far-red emitting unsymmetrical meso-CF3-BODIPY fluorescent dye with phenyl and pyrazolyl substituents was synthesized by condensation of trifluoropyrrolylethanol with pyrazolyl-pyrrole, with subsequent oxidation and complexation of the formed dipyrromethane. This BODIPY dye exhibits optical absorption at λab ≈ 610-620 nm and emission at λem ≈ 640-650 nm. The BODIPY was studied on Ehrlich carcinoma cells as a lysosome-specific fluorescent dye that allows intravital staining of cell structures with subsequent real-time monitoring of changes occurring in the cells. It was also shown that the rate of uptake by cells, the rate of intracellular transport into lysosomes, and the rate of saturation of cells with the dye depend on its concentration in the culture medium. A concentration of 5 µM was chosen as the most suitable BODIPY concentration for fluorescent staining of living cell lysosomes, while a concentration of 100 µM was found to be toxic to Ehrlich carcinoma cells.

A Self-Immolative Fluorescent Probe for Selective Detection of SARS-CoV-2 Main Protease.

Existing tools to detect and visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suffer from low selectivity, poor cell permeability, and high cytotoxicity. Here we report a novel self-immolative fluorescent probe (MP590) for the highly selective and sensitive detection of the SARS-CoV-2 main protease (Mpro). This fluorescent probe was prepared by connecting a Mpro-cleavable peptide (N-acetyl-Abu-Tle-Leu-Gln) with a fluorophore (i.e., resorufin) via a self-immolative aromatic linker. Fluorescent titration results show that MP590 can detect Mpro with a limit of detection (LoD) of 35 nM and is selective over interferents such as hemoglobin, bovine serum albumin (BSA), thrombin, amylase, SARS-CoV-2 papain-like protease (PLpro), and trypsin. The cell imaging data indicate that this probe can report Mpro in HEK 293T cells transfected with a Mpro expression plasmid as well as in TMPRSS2-VeroE6 cells infected with SARS-CoV-2. Our results suggest that MP590 can both measure and monitor Mpro activity and quantitatively evaluate Mpro inhibition in infected cells, making it an important tool for diagnostic and therapeutic research on SARS-CoV-2.

Fluorescent Platforms for RNA Chemical Biology Research.

Efficient detection and observation of dynamic RNA changes remain a tremendous challenge. However, the continuous development of fluorescence applications in recent years enhances the efficacy of RNA imaging. Here we summarize some of these developments from different aspects. For example, single-molecule fluorescence in situ hybridization (smFISH) can detect low abundance RNA at the subcellular level. A relatively new aptamer, Mango, is widely applied to label and track RNA activities in living cells. Molecular beacons (MBs) are valid for quantifying both endogenous and exogenous mRNA and microRNA (miRNA). Covalent binding enzyme labeling fluorescent group with RNA of interest (ROI) partially overcomes the RNA length limitation associated with oligonucleotide synthesis. Forced intercalation (FIT) probes are resistant to nuclease degradation upon binding to target RNA and are used to visualize mRNA and messenger ribonucleoprotein (mRNP) activities. We also summarize the importance of some fluorescence spectroscopic techniques in exploring the function and movement of RNA. Single-molecule fluorescence resonance energy transfer (smFRET) has been employed to investigate the dynamic changes of biomolecules by covalently linking biotin to RNA, and a focus on dye selection increases FRET efficiency. Furthermore, the applications of fluorescence assays in drug discovery and drug delivery have been discussed. Fluorescence imaging can also combine with RNA nanotechnology to target tumors. The invention of novel antibacterial drugs targeting non-coding RNAs (ncRNAs) is also possible with steady-state fluorescence-monitored ligand-binding assay and the T-box riboswitch fluorescence anisotropy assay. More recently, COVID-19 tests using fluorescent clustered regularly interspaced short palindromic repeat (CRISPR) technology have been demonstrated to be efficient and clinically useful. In summary, fluorescence assays have significant applications in both fundamental and clinical research and will facilitate the process of RNA-targeted new drug discovery, therefore deserving further development and updating.

Multiplexed reverse-transcriptase quantitative polymerase chain reaction using plasmonic nanoparticles for point-of-care COVID-19 diagnosis.

Quantitative polymerase chain reaction (qPCR) offers the capabilities of real-time monitoring of amplified products, fast detection, and quantitation of infectious units, but poses technical hurdles for point-of-care miniaturization compared with end-point polymerase chain reaction. Here we demonstrate plasmonic thermocycling, in which rapid heating of the solution is achieved via infrared excitation of nanoparticles, successfully performing reverse-transcriptase qPCR (RT-qPCR) in a reaction vessel containing polymerase chain reaction chemistry, fluorescent probes and plasmonic nanoparticles. The method could rapidly detect SARS-CoV-2 RNA from human saliva and nasal specimens with 100% sensitivity and 100% specificity, as well as two distinct SARS-CoV-2 variants. The use of small optical components for both thermocycling and multiplexed fluorescence monitoring renders the instrument amenable to point-of-care use. Overall, this study demonstrates that plasmonic nanoparticles with compact optics can be used to achieve real-time and multiplexed RT-qPCR on clinical specimens, towards the goal of rapid and accurate molecular clinical diagnostics in decentralized settings.

Aggregation-Induced Emission Fluorophore-Incorporated Curcumin-Based Ratiometric Nanoprobe for Hypochlorite Detection in Food Matrices.

The development of efficient, economic, reliable, and accurate monitoring of hypochlorite (ClO-) in food matrices is in great demand for food safety assessment, particularly during its massive use against the COVID-19 epidemic. Here, we prepared an aggregation-induced emission (AIE) fluorophore tetraphenylethylene (TPE)-incorporated curcumin-based hybrid ratiometric fluorescence nanoprobe (Curcumin/TPE@HyNPs) through amphiphilic phospholipid polymer-powered nanoprecipitation, which exhibited a fast, highly sensitive, and selective response to the residual ClO- in real food matrices. Because of the inner filter effect (IFE) from curcumin toward TPE inside the nanoprobe, the bright fluorescence of TPE aggregation at ∼437 nm was effectively quenched, along with an enhanced fluorescence of curcumin at ∼478 nm. Once there was a ClO- residue in food matrices, ClO- triggered the oxidation of o-methoxyphenol inside curcumin and led to the almost complete absorption collapse, thereby terminating curcumin fluorescence at ∼478 nm and the IFE process. Accordingly, the fluorescence of TPE at ∼437 nm was recovered. In this case, a ratiometric fluorescent response of Curcumin/TPE@HyNPs toward the residual ClO- in food matrices (e.g., milk) was proposed with a low detection limit of 0.353 µM and a rapid response time of 140.0 s. Notably, the phospholipid polymer as the protection layer effectively reduced/evaded the nonspecific binding of signal reporters inside the nanoprobe, facilitating it to directly monitor the residual ClO- in real food matrices. This work provided a novel approach to utilize the unconventional AIE luminophors for constructing the efficient and reliable early warning mechanisms toward various food contaminants.

Rapid and Highly Sensitive Detection of Target DNA Related to COVID-19 Virus With a Fluorescent Bio-conjugated Probe via a FRET Mechanism.

A novel cyanine 3 (Cy3)-based bio-conjugated sensor has been developed to detect target DNA or extracted RNA from COVID -19 samples using the fluorescence resonance energy transfer (FRET) experiment. A special sequence of the COVID -19 genome was selected as a complementary DNA (target DNA) part. The opposite chain of this target sequence was designed in 2 parts; one part was attached to the Cy3 organic dye (capture DNA or Cy3- DNA), and the other part was attached to the BHQ2 molecule (quencher DNA or BHQ2- DNA). The Cy3 molecule acts as a donor pair, and BHQ2 acts as an acceptor pair in the FRET experiment. The capture DNA and quencher DNA can form a sandwiched complex in the presence of target DNA. The formation of the entitled sandwiched hybrid causes the decrement of emission intensity of the Cy3 donor in bio-conjugated Cy3-DNA via energy transfer from Cy3 (as a donor) to BHQ2 (as an acceptor). Indeed, in the presence of non-complementary DNA, the pairing of DNA strands does not occur, the FRET phenomenon does not exist, and therefore fluorescence intensity of Cy3 does not decrease. Moreover, this biosensor was successfully applied to analyze real samples containing extracted RNA of COVID -19 prepared for the reverse transcriptase-polymerase chain reaction (RT-PCR) test, and the results were promising.

Multiplex Target-Redundant RT-LAMP for Robust Detection of SARS-CoV-2 Using Fluorescent Universal Displacement Probes.

The increasing prevalence of variant lineages during the COVID-19 pandemic has the potential to disrupt molecular diagnostics due to mismatches between primers and variant templates. Point-of-care molecular diagnostics, which often lack the complete functionality of their high-throughput laboratory counterparts, are particularly susceptible to this type of disruption, which can result in false-negative results. To address this challenge, we have developed a robust Loop Mediated Isothermal Amplification assay with single tube multiplexed multitarget redundancy and an internal amplification control. A convenient and cost-effective target-specific fluorescence detection system allows amplifications to be grouped by signal using adaptable probes for pooled reporting of SARS-CoV-2 target amplifications or differentiation of the Internal Amplification Control. Over the course of the pandemic, primer coverage of viral lineages by the three redundant sub-assays has varied from assay to assay as they have diverged from the Wuhan-Hu-1 isolate sequence, but aggregate coverage has remained high for all variant sequences analyzed, with a minimum of 97.4% (Variant of Interest: Eta). In three instances (Delta, Gamma, Eta), a high-frequency mismatch with one of the three sub-assays was observed, but overall coverage remained high due to multitarget redundancy. When challenged with extracted human samples the multiplex assay showed 87% or better sensitivity (of 30 positive samples), with 100% sensitivity for samples containing greater than 30 copies of viral RNA per reaction (of 21 positive samples), and 100% specificity (of 60 negative samples). These results are further evidence that conventional laboratory methodologies can be leveraged at the point of care for robust performance and diagnostic stability over time. IMPORTANCE The COVID-19 pandemic has had tremendous impact, and the ability to perform molecular diagnostics in resource limited settings has emerged as a key resource for mitigating spread of the disease. One challenge in COVID-19 diagnosis, as well as other viruses, is ongoing mutation that can allow viruses to evade detection by diagnostic tests. We developed a test that detects multiple parts of the virus genome in a single test to reduce the chance of missing a virus due to mutation, and it is designed to be simpler and faster than typical laboratory tests while maintaining high sensitivity. This capability is enabled by a novel fluorescent probe technology that works with a simple constant temperature reaction condition.

Telomere G-triplex lights up Thioflavin T for RNA detection: new wine in an old bottle.

Few reports are found working on the features and functions of the human telomere G-triplex (ht-G3) though the telomere G-quadruplex has been intensely studied and widely implemented to develop various biosensors. We herein report that ht-G3 lights up Thioflavin T (ThT) and establish a sensitive biosensing platform for RNA detection by introducing a target recycling strategy. An optimal condition was selected out for ht-G3 to promote ThT to generate a strong fluorescence. Accordingly, an ht-G3-based molecular beacon was successfully designed against the corresponding RNA sequence of the SARS-CoV-2 N-gene. The sensitivity for the non-amplified RNA target achieves 0.01 nM, improved 100 times over the conventional ThT-based method. We believe this ht-G3/ThT-based label-free strategy could be widely applied for RNA detection.

RT-LAMP assay combining multi-fluorescent probes for SARS-CoV-2 RNA detection and variant differentiation.

Simple and accurate testing tools for SARS-CoV-2 viral RNA detection are essential for the prevention of the spread of the virus and timely governmental actions. Herein, we present a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the simultaneous detection of ORF1ab and N gene fragments of SARS-CoV-2 in one pot. Using two primer sets and two molecular beacon (MB) probes respectively labelled with different fluorophore, positive results were obtained with a limit of detection of 20 and 2 copies/µL for ORF1ab and N gene fragments, respectively. Moreover, the RT-LAMP based assay was applied to detect single-site differences in S genes using two one-step displacement (OSD) probes targeting wild-type and mutant (P681R mutation was chosen as model) genes. Through that, the wild type strain and P681R mutant variant were well distinguished from each other, and a preliminary observation was also made on other mutations at this site such as P681H. The proposed method has high sensitivity for quantification and high specificity for mutation differentiation. In addition, it does not require accurate sophisticated thermal cycler instrumentation and can be used in clinical settings in resource-limited regions.