Empowering the on-site detection of nucleic acids by integrating CRISPR and digital signal processing.

Addressing the global disparity in cancer care necessitates the development of rapid and affordable nucleic acid (NA) testing technologies. This need is particularly critical for cervical cancer, where molecular detection of human papillomavirus (HPV) has emerged as an accurate screening method. However, implementing this transition in low- and middle-income countries has been challenging due to the high costs and centralized facilities required for current NA tests.

DNA repair-retarded isothermal CRISPR amplification for rapid, sensitive base excision repair enzyme assay.

Background: As a core enzyme in the base excision repair system, uracil DNA glycosylase (UDG) is indispensable in maintaining genomic integrity and normal cell cycles. Its abnormal activity intervenes in cancers and neurodegerative diseases. Previous UDG assays based on isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR/Cas) system were fine in sensitivity, but exposed to complications in assay flow, time, and probe design.

One-pot, one-step, label-free miRNA detection method based on the structural transition of dumbbell probe.

In this study, we present a one-pot, one-step, label-free miRNA detection method through a structural transition of a specially designed dumbbell-shape probe, initiating a rolling circle transition (RCT). In principle, target miRNA binds to right loop of the dumbbell probe (DP), which allows structural change of the DP to circular form, exposing a sequence complementary to the T7 promoter (T7p) previously hidden within the stem. This exposure allows T7 RNA polymerase to initiate RCT, producing a repetitive Mango aptamer sequence.

CRISPR/Cas12a Collateral Cleavage Activity for Sensitive 3'-5' Exonuclease Assay.

This study presents a technique for detecting 3'-5' exonuclease activity through the use of CRISPR/Cas12a. These enzymes, including 3'-5' exonuclease (Exo III), perform crucial roles in various cellular processes and are associated with life expectancy. However, imbalances in their expression can increase susceptibility to diseases such as cancer, particularly under prolonged stress.

Background-filtered telomerase activity assay with cyclic DNA cleavage amplification.

Overexpression of telomerase incites the abnormal proliferation of cancer cells. Thus, it has been regarded as a cancer biomarker and a potential therapeutic target. Existing assays suggest a promising sensing scheme to detect telomerase activity.

Peroxidase-Mimicking Activity of Nanoceria for Label-Free Colorimetric Assay for Exonuclease III Activity.

We present a novel label-free colorimetric method for detecting exonuclease III (Exo III) activity using the peroxidase-mimicking activity of cerium oxide nanoparticles (nanoceria). Exo III, an enzyme that specifically catalyzes the stepwise removal of mononucleotides from the 3'-OH termini of double-stranded DNA, plays a significant role in various cellular and physiological processes, including DNA proofreading and repair. Malfunctions of Exo III have been associated with increased cancer risks.

CRISPR-Enhanced Hydrogel Microparticles for Multiplexed Detection of Nucleic Acids.

CRISPR/Cas systems offer a powerful sensing mechanism to transduce sequence-specific information into amplified analytical signals. However, performing multiplexed CRISPR/Cas assays remains challenging and often requires complex approaches for multiplexed assays. Here, a hydrogel-based CRISPR/Cas12 system termed CLAMP (Cas-Loaded Annotated Micro-Particles) is described.

Characterization and modulation of surface charges to enhance extracellular vesicle isolation in plasma.

Extracellular vesicles (EVs) carry information inherited from parental cells, having significant potential for disease diagnosis. In blood, however, EVs are outnumbered >10-fold by low density lipoproteins (LDLs), yet similar in size and density. These fundamental disadvantages often cause LDL spillover into EV isolates, thus confounding assay results.

The p53-Driven Anticancer Effect of Extract on AGS Gastric Cancer Cells.

Cancer metastasis is directly related to the survival rate of cancer patients. Although cancer metastasis proceeds by the movement of cancer cells, it is fundamentally caused by its resistance to anoikis, a mechanism of apoptosis caused by the loss of adhesion of cancer cells. Therefore, it was found that inhibiting cancer migration and reducing anoikis resistance are important for cancer suppression, and natural compounds can effectively control it.

Ultrasensitive multiplexed miRNA detection based on a self-priming hairpin-triggered isothermal cascade reaction.

We herein describe an ultrasensitive isothermal strategy to detect miRNAs in a multiplexed manner by utilizing a self-priming hairpin-triggered cascade reaction and the adsorption properties of graphene oxide (GO). In principle, a self-priming hairpin probe (SHP) was designed to be opened through binding to the target miRNA and rearranged to serve as a primer. The following extension displaced the target miRNA to be recycled for opening another SHP and produced a double-stranded (ds) SHP with a longer stem region.

Rapid and accurate clinical testing for COVID-19 by nicking and extension chain reaction system-based amplification (NESBA).

We herein describe rapid and accurate clinical testing for COVID-19 by nicking and extension chain reaction system-based amplification (NESBA), an ultrasensitive version of NASBA. The primers to identify SARS-CoV-2 viral RNA were designed to additionally contain the nicking recognition sequence at the 5'-end of conventional NASBA primers, which would enable nicking enzyme-aided exponential amplification of T7 RNA promoter-containing double-stranded DNA (T7DNA). As a consequence of this substantially enhanced amplification power, the NESBA technique was able to ultrasensitively detect SARS-CoV-2 genomic RNA (gRNA) down to 0.

Development of Integrated Systems for On-Site Infection Detection.

The modern healthcare system faces an unrelenting threat from microorganisms, as evidenced by global outbreaks of new viral diseases, emerging antimicrobial resistance, and the rising incidence of healthcare-associated infections (HAIs). An effective response to these threats requires rapid and accurate diagnostic tests that can identify causative pathogens at the point of care (POC). Such tests could eliminate diagnostic uncertainties, facilitating patient triaging, minimizing the empiric use of antimicrobial drugs, and enabling targeted treatments.

Identification of thyroid hormone/thyroid hormone receptor interaction based on aptamer-assisted protein-induced fluorescence enhancement.

We herein describe a novel method to identify thyroid hormone (TH)/thyroid hormone receptor (TR) interaction, termed aptamer-assisted protein-induced fluorescence enhancement (AptPIFE). In this method, a detection probe consisting of an RNA strand incorporating TH-specific aptamer and a Cy3-labeled DNA strand holds TH in close proximity to Cy3. The corresponding TR then binds to the TH near Cy3, consequently stimulating Cy3 to emit a significantly enhanced fluorescence through PIFE phenomenon.

Fluorescence polarization system for rapid COVID-19 diagnosis.

Prompt diagnosis, patient isolation, and contact tracing are key measures to contain the coronavirus disease 2019 (COVID-19). Molecular tests are the current gold standard for COVID-19 detection, but are carried out at central laboratories, delaying treatment and control decisions. Here we describe a portable assay system for rapid, onsite COVID-19 diagnosis.

Fast detection of SARS-CoV-2 RNA via the integration of plasmonic thermocycling and fluorescence detection in a portable device.

The diagnosis of severe acute respiratory syndrome 2 (SARS-CoV-2) infection by quantitative PCR with reverse transcription (RT-qPCR) typically involves bulky instrumentation in centralized laboratories and an assay time of 1-2 h. Here, we show that SARS-CoV-2 RNA can be detected in 17 min via a portable device integrating reverse transcription, fast thermocycling (via plasmonic heating through magneto-plasmonic nanoparticles) and in situ fluorescence detection following magnetic clearance of the nanoparticles. The device correctly classified all nasopharyngeal, oropharyngeal and sputum samples from 75 patients with COVID-19 and 75 healthy controls, with good concordance in fluorescence intensity with standard RT-qPCR (Pearson coefficients > 0.

Portable glucose meter-utilized label-free and washing-free telomerase assay.

We herein describe a portable glucose meter (PGM)-utilized label-free and washing-free method for the facile determination of telomerase activity that relies on the kinase-catalyzed cascade enzymatic reaction (KCER) that transduces the telomerase activity to the glucose level. In the sensor, the telomerase that elongates telomere sequences ((TTAGGG)) from the 3'-terminus of telomerase substrate primer (TSP) consumes deoxynucleoside triphosphate (dNTP), which serves as a phosphate source for KCER promoted by hexokinase and pyruvate kinase. Thus, the presence of telomerase protects KCER from working effectively, resulting in the maintenance of an initial, high glucose level that is readily determined using hand-held PGM.

A hairpin probe-mediated isothermal amplification method to detect target nucleic acid.

We herein describe Hairpin probe-mediated Isothermal Amplification (HIAmp), a novel isothermal method to detect a target nucleic acid. This method employs a hairpin probe (HP) designed to be opened through binding to the target nucleic acid. Upon opening of the HP, the primer binds to the free stem of the opened HP followed by its extension by DNA polymerase, consequently displacing and recycling the target nucleic acid to open another HP and producing an intermediate product (IP) containing a nicking site.

Red Chinese Cabbage Transcriptome Analysis Reveals Structural Genes and Multiple Transcription Factors Regulating Reddish Purple Color.

Reddish purple Chinese cabbage (RPCC) is a popular variety of (AA = 20). It is rich in anthocyanins, which have many health benefits. We detected novel anthocyanins including cyanidin 3-(feruloyl) diglucoside-5-(malonoyl) glucoside and pelargonidin 3-(caffeoyl) diglucoside-5-(malonoyl) glucoside in RPCC.

Colorimetric Assay for Uracil DNA Glycosylase Activity Based on Toehold-Mediated Strand Displacement Circuit.

Herein, a novel enzyme-free and label-free strategy for colorimetric assay of uracil DNA glycosylase (UDG) activity, which relies on a target-activated toehold-mediated strand displacement (TMSD) circuit is described. The strategy employs a detection duplex probe composed of a uracil-containing strand (US) and a catalyst strand (CS). UDG present in a sample will cleave uracil bases within US and destabilize the detection duplex probe, which then leads to the dissociation of the detection duplex, releasing CS.

Label-free and washing-free alkaline phosphatase assay using a personal glucose meter.

We herein describe a personal glucose meter (PGM)-based method for a label-free and washing-free determination of alkaline phosphatase (ALP) activity, which relies on the cascade enzymatic reactions promoted by hexokinase and pyruvate kinase to couple ALP activity with the amount of glucose. In principle, the presence of target ALP scavenges on adenosine 5'-triphosphate (ATP), a phosphate source for hexokinase-catalyzed reactions, and thus suppresses the ensuing cascade enzymatic reactions. As a result, the initial high amount of glucose is maintained and the amount of glucose, which is proportional to ALP activity, is simply measured by a hand-held PGM.

Sensitive detection of DNA from Chlamydia trachomatis by using flap endonuclease-assisted amplification and graphene oxide-based fluorescence signaling.

A simple and sensitive method is described for the determination of DNA. It relies on the use of (a) an invasive reaction that is catalyzed by flap endonuclease 1 (FEN 1), and (b) graphene oxide (GO)-based fluorescence signalling. The presence of target DNA mediates the formation of the invasive structure, and this induces FEN 1 to catalyze multiple cycles of cleavage reaction at the junction, thereby liberating numerous fluorophore-labeled flaps.

Fluorescent S1 nuclease assay utilizing exponential strand displacement amplification.

We herein devise a simple and label-free strategy to determine S1 nuclease activity by exploiting the target-induced inhibition of exponential strand displacement amplification (eSDA). In principle, a DNA probe that is designed to produce a large amount of duplexes through a process of eSDA, is degraded by the catalytic activity of S1 nuclease. This reaction blocks the initiation of eSDA, leading to the quite-reduced fluorescence of a double-stranded DNA specific fluorescent dye, SYBR Green I compared to the one in the absence of S1 nuclease.

Nucleic acid-based fluorescent methods for the determination of DNA repair enzyme activities: A review.

DNA repair pathways are closely associated with the maintenance of genomic integrity, disease outbreak, and the development of therapeutics. Owing to these significances, novel analytical methods for enzymes that are involved in the DNA repair pathways have been actively investigated. This review focuses on discussions on nucleic acid-based methods, especially those based on the fluorescence, for the determination of DNA repair enzymes.