A personal glucose meter-utilized strategy for portable and label-free detection of hydrogen peroxide.

Rapid and precise detection of hydrogen peroxide (H2O2) holds great significance since it is linked to numerous physiological and inorganic catalytic processes. We herein developed a label-free and washing-free strategy to detect H2O2 by employing a hand-held personal glucose meter (PGM) as a signal readout device. By focusing on the fact that the reduced redox mediator ([Fe(CN)6]4-) itself is responsible for the final PGM signal, we developed a new PGM-based strategy to detect H2O2 by utilizing the target H2O2-mediated oxidation of [Fe(CN)6]4- to [Fe(CN)6]3- in the presence of horseradish peroxidase (HRP) and monitoring the reduced PGM signal in response to the target amount. Based on this straightforward and facile design principle, H2O2 was successfully determined down to 3.63 µM with high specificity against various non-target molecules. We further demonstrated that this strategy could be expanded to identify another model target choline by detecting H2O2 produced through its oxidation promoted by choline oxidase. Moreover, we verified its practical applicability by reliably determining extracellular H2O2 released from the breast cancer cell line, MDA-MB-231. This work could evolve into versatile PGM-based platform technology to identify various non-glucose target molecules by employing their corresponding oxidase enzymes, greatly advancing the portable biosensing technologies.

A novel ultrasensitive RNase H assay based on phosphorothioated-terminal hairpin formation and self-priming extension reaction.

We herein present a novel ultrasensitive RNase H assay based on phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP) reaction. The detection probe employed as a key component in this technique serves as a substrate for RNase H and triggers the PS-THSP reaction upon the RNase H-mediated degradation of the probe. As a consequence, a large number of long concatemeric amplification products could be produced and used to identify the RNase H activity through the fluorescence signals produced by the nucleic acid-specific fluorescent dye, SYTO 9. Importantly, the use of the gp32 protein allowed the PS-THSP reaction to be performed at 37 °C, ultimately enabling an isothermal one-step RNase H assay. Based on this sophisticated design principle, the RNase H activity was very sensitively detected, down to 0.000237 U mL-1 with high specificity. We further verified its practical applicability through its successful application to the screening of RNase H inhibitors. With its operational convenience and excellent analytical performance, this technique could serve as a new platform for RNase H assay in a wide range of biological applications.

Multifunctional self-priming hairpin probe-based isothermal nucleic acid amplification and its applications for COVID-19 diagnosis.

We herein present a multifunctional self-priming hairpin probe-based isothermal amplification, termed MSH, enabling one-pot detection of target nucleic acids. The sophisticatedly designed multifunctional self-priming hairpin (MSH) probe recognizes the target and rearranges to prime itself, triggering the amplification reaction powered by the continuously repeated extension, nicking, and target recycling. As a consequence, a large number of double-stranded DNA (dsDNA) amplicons are produced that could be monitored in real-time using a dsDNA-intercalating dye. Based on this unique design approach, the nucleocapsid (N) and the open reading frame 1 ab (ORF1ab) genes of SARS-CoV-2 were successfully detected down to 1.664 fM and 0.770 fM, respectively. The practical applicability of our method was validated by accurately diagnosing 60 clinical samples with 93.33% sensitivity and 96.67% specificity. This isothermal one-pot MSH technique holds great promise as a point-of-care testing protocol for the reliable detection of a wide spectrum of pathogens, particularly in resource-limited settings.

A novel electrochemical strategy to detect hydrogen peroxide by utilizing peroxidase-mimicking activity of cerium oxide/graphene oxide nanocomposites.

We herein describe a novel electrochemical strategy to detect hydrogen peroxide (H2O2) by utilizing the peroxidase-mimicking activity of cerium oxide nanoparticles (CeO2 NP) and reduced graphene oxide (rGO). Particularly, CeO2 NP/rGO nanocomposites were deposited on the commercial electrode by a very convenient and direct electrochemical reduction of graphene oxide. Due to the peroxidase-mimicking activity of CeO2 NP and the outstanding electrochemical properties of reduced graphene oxide, the reduction current of H2O2 was greatly enhanced. Based on this strategy, we reliably determined H2O2 down to 1.67 µM with excellent specificity and further validated its practical capabilities by robustly detecting H2O2 present in heterogeneous human serum samples. We believe that this work could serve as a new facile platform for H2O2 detection.

An ultrasensitive label-free RNase H assay based on in vitro transcription of fluorogenic light-up aptamer.

Herein, we proposed a label-free method to identify RNase H activity by utilizing in vitro transcription of fluorogenic light-up aptamers. In this work, we employed the specially designed two pivotal components of the hairpin substrate probe (HP) containing an RNA/DNA chimeric stem region and the template probe (TP) as a transcription template, and the RNase H activity was made to lead to the formation of a complete ds T7 promoter. T7 RNA polymerase could then promote in vitro transcription to generate numerous light-up RNA aptamers that result in significant fluorescence enhancements upon binding to the cognate fluorogenic dye. By leveraging this deliberate design principle, we identified RNase H activity ultrasensitively as low as 0.000156 U mL-1 with excellent specificity against non-target enzymes. We further demonstrated that the strategy can also reliably identify RNase H activity in heterogeneous biological samples such as cell lysates, ensuring its robust practical applicability. This work would provide invaluable insight for the development of innovative biosensing systems utilizing in vitro transcription of light-up aptamers, and it could be broadened to construct other assays by appropriately redesigning the HPs.

A CRISPR/Cas12 trans-cleavage reporter enabling label-free colorimetric detection of SARS-CoV-2 and its variants.

We present a label-free colorimetric CRISPR/Cas-based method enabling affordable molecular diagnostics for SARS-CoV-2. This technique utilizes 3,3'-diethylthiadicarbocyanine iodide (DISC2(5)) which exhibits a distinct color transition from purple to blue when it forms dimers by inserting into the duplex of the thymidine adenine (TA) repeat sequence. Loop-mediated isothermal amplification (LAMP) or recombinase polymerase amplification (RPA) was used to amplify target samples, which were subsequently subjected to the CRISPR/Cas12a system. The target amplicons would activate Cas12a to degrade nearby TA repeat sequences, preserving DISC2(5) in its free form to display purple as opposed to blue in the absence of the target. Based on this design approach, SARS-CoV-2 RNA was colorimetrically detected very sensitively down to 2 copies/µL, and delta and omicron variants of SARS-CoV-2 were also successfully identified. The practical diagnostic utility of this method was further validated by reliably identifying 179 clinical samples including 20 variant samples with 100% clinical sensitivity and specificity. This technique has the potential to become a promising CRISPR-based colorimetric platform for molecular diagnostics of a wide range of target pathogens.

A label-free and washing-free method to detect biological thiols on a personal glucose meter utilizing glucose oxidase-mimicking activity of gold nanoparticles.

We herein developed a label-free and washing-free method to detect biological thiols (biothiols) on a personal glucose meter (PGM) utilizing the intrinsic glucose oxidase (GOx)-mimicking activity of gold nanoparticles (AuNPs). By focusing on the fact that this activity could be diminished by target biothiols through their binding onto the AuNP surface, we correlated the concentration of biothiols with that of glucose readily measurable on a PGM and successfully determined cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) down to 0.116, 0.059, and 0.133 µM, respectively, with high specificity against non-target biomolecules. We further demonstrated its practical applicability by reliably detecting target biothiol in heterogeneous human serum. Due to the meritorious features of PGM such as simplicity, portability, and cost-effectiveness, we believe that this work could serve as a powerful platform for biothiol detection in point-of-care settings.

Plasmonic digital PCR for discriminative detection of SARS-CoV-2 variants.

We developed a novel strategy for discriminative detection of SARS-CoV-2 variants based on the plasmonic photothermal effect of gold nanofilms and digital polymerase chain reaction (dPCR) technology. This method consists of the gold nanofilm-based dPCR chip fabrication for ultrafast heating and cooling cycles by the plasmonic photothermal effect, the LED quencher immobilization through the interfacing compound on the surface of the gold nanofilm to prevent photoquenching of PCR signaling dye, and the discriminative detection of the variant viruses from the COVID-19 clinical samples by photothermal cycles with fabricated dPCR chips and a portable plasmonic PCR device. Compared to conventional sequencing or RT-qPCR-based variant detection methods, this technology can be effectively applied to point-of-care testing by enabling ultrafast quantitative analysis with a small device. With this method, we successfully detected the delta variant and the omicron variant with a high sensitivity of 10 copies from COVID-19 patients' clinical samples within 25 min, including reverse transcription. This method can be applied universally to rapid and accurate point-of-care testing for various pandemic viruses as well as the coronavirus.

Fully Automated Multiple Standard Addition on a Centrifugal Microfluidic System.

We herein describe a novel centrifugal microfluidic system to achieve multiple standard additions, which could minimize the effects of matrix interference and consequently lead to more accurate and reliable measurements of analyte concentrations in complex samples. The system leverages laser-irradiated ferrowax microvalves to automatically control fluid transfer on the disc without the need for external pumps or pressure systems, simplifying the procedures and eliminating the need for manual intervention. The disc incorporates metering chambers with rationally designed varying sizes, which could lead to the formation of six standard addition samples very rapidly in just 2.5 min. The final solutions are designed to contain a target component at gradually increasing concentrations but have an equal final volume containing the same amount of an analyte solution, thereby equalizing the matrix effect that is supposedly caused by the unknown components in the analyte solution. By utilizing this design principle, we were able to successfully quantify a model target component, salivary thiocyanate ions, that could be used as a biomarker for exposure to tobacco smoke. Our centrifugal microfluidic system holds great promise as a powerful analytical tool to achieve fully automated diagnostic microsystems involving a standard addition process.

One-pot, ultrasensitive, and multiplex detection of SARS-CoV-2 genes utilizing self-priming hairpin-mediated isothermal amplification.

The global pandemic resulting from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its emerging variants highlights the need for convenient and accurate detection protocols to facilitate timely prevention and management of the disease. Herein, we propose a new self-priming hairpin-mediated isothermal amplification (SIAM) protocol enabling one-pot and ultrasensitive identification of SARS-CoV-2 in a multiplexed way. This approach works by targeting a specific RNA sequence with a self-priming hairpin (SP) probe and promoting continuously repeated extension and nicking reactions to produce numerous trigger molecules, which could specifically bind to molecular beacons (MBs) and produce fluorescent signals. Under an isothermal condition of 37 °C, this technique allowed for the simultaneous identification of the spike (S) and nucleocapsid (N) genes of SARS-CoV-2 down to single copy/µL levels. We further validated the practical diagnostic capabilities of the SIAM method by accurately testing 20 clinical samples with 100% sensitivity and specificity. The SIAM method has a lot of potential to be a reliable nucleic acid testing protocol to identify infections caused by a wide range of pathogens.

Synergistic Effect of Calcination and Sintering on the Reduction of Grain Boundary Resistance of LATP Solid Electrolyte.

The NASICON-type Li1.4Al0.4Ti1.6(PO4)3 (LATP) solid electrolyte is a promising candidate for next-generation lithium-ion batteries due to its high stability in air and moisture, as well as high bulk ion conductivity. However, the grain boundary resistance of LATP limits its overall ionic conductivity, which remains a major obstacle to the commercialization of all-solid-state batteries. In this study, we made efforts to solve the problem by promoting the minimization of voids and the formation of well-defined grain boundaries by controlling the temperature of two heat treatments during the synthesis process. The crystallization temperature was confirmed through thermogravimetric analysis/DTA analysis, and the degree of crystallization was confirmed using XRD analysis. The formation of grain boundaries and the presence of voids were evaluated by cross-sectional SEM images after sintering. After sintering, the LA_900 °C sample, characterized by a high degree of crystallization and well-formed grain boundaries without voids, demonstrated a low bulk and grain boundary resistance, which was confirmed by electrochemical impedance spectroscopy analysis. The result was an ionic conductivity of 1.72 × 10-4 S/cm. These results provide valuable insights into the facile synthesis of LATP.

Elution-free DNA detection using CRISPR/Cas9-mediated light-up aptamer transcription: Toward all-in-one DNA purification and detection tube.

Accurate and efficient detection of DNA is crucial for disease diagnosis and health monitoring. The traditional methods for DNA analysis involve multiple steps, including sample preparation, lysis, extraction, amplification, and detection. In this study, we present a one-step elution-free DNA analysis method based on the combination of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated light-up aptamer transcription (CLAT) assay and a DNA-capturing poly(2-dimethylaminomethyl styrene) (pDMAMS)-coated tube. The sample solution and lysis buffer are added to the pDMAMS-coated tube, and the DNA is efficiently captured on the surface via electrostatic interaction and directly detected by CLAT assay. The ability of the CRISPR/Cas9 system to specifically recognize DNA enables direct detection of DNA captured on the pDMAMS-coated tube. The combination of CLAT assay and pDMAMS-coated tube simplifies DNA detection in a single tube without the need for complicated extraction steps, improving sensitivity. Our platform demonstrated attomolar sensitivity in the detection of target DNA in cell lysate (0.92 aM), urine (7.7 aM), and plasma (94.6 aM) samples within 1 h. The practical applicability of this method was further demonstrated in experiments with tumor-bearing mice. We believe that this approach brings us closer to an all-in-one DNA purification and detection tube system and has potential applications in tissue and liquid biopsies, as well as various other DNA sensing applications.

Ultrasensitive Isothermal Detection of SARS-CoV-2 Based on Self-Priming Hairpin-Utilized Amplification of the G-Rich Sequence.

The outbreak of the novel coronavirus disease 2019 (COVID-19) pandemic induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of fatalities all over the world. Unquestionably, the effective and timely testing for infected individuals is the most imperative for the prevention of the ongoing pandemic. Herein, a new method was established for detecting SARS-CoV-2 based on the self-priming hairpin-utilized isothermal amplification of the G-rich sequence (SHIAG). In this strategy, the target RNA binding to the hairpin probe (HP) was uniquely devised to lead to the self-priming-mediated extension followed by the continuously repeated nicking and extension reactions, consequently generating abundant G-rich sequences from the intended reaction capable of producing fluorescence signals upon specifically interacting with thioflavin T (ThT). Based on the unique isothermal design concept, we successfully identified SARS-CoV-2 genomic RNA (gRNA) as low as 0.19 fM with excellent selectivity by applying only a single HP and further verified its practical diagnostic capability by reliably testing a total of 100 clinical specimens for COVID-19 with 100% clinical sensitivity and specificity. This study would provide notable insights into the design and evolution of new isothermal strategies for the sensitive and facile detection of SARS-CoV-2 under resource constraints.

A strategy of enhancing the ionic conductivity of Li7La3Zr2O12 under accurate sintering conditions.

Garnet-type Li7La3Zr2O12 (LLZO) oxide solid electrolytes are spotlighted as solid electrolytes for lithium-ion secondary batteries due to their high thermal and electrochemical stability. However, LLZO has a low ionic conductivity compared to liquid electrolytes, which is one of the biggest problems hindering the commercialization of all-solid-state batteries (ASSBs). Essential conditions for improving the ionic conductivity can be classified into two factors: (1) formation of a cubic LLZO phase related to bulk ionic conductivity and (2) formation of grain boundaries for low interfacial resistance. In this work, cubic LLZO phase formation conditions were first confirmed by TGA-DTA analysis. The LLZO phase was pre-formed via a holding range of furnace temperature profile (HRFTP) found by TGA-DTA analysis. The pre-formed LLZO phase could stabilize the cubic LLZO phase after a sintering process. This was confirmed by XRD analysis. Stabilized cubic LLZO under HRFTP conditions could enhance the bulk ionic conductivity, the main factor affecting the total ionic conductivity. In addition, to confirm the characteristics of sintering temperature changes, the grain boundaries of LLZO surfaces and the color of LZO pellets were investigated by SEM in detail. By setting the holding time process at 600 °C, the pre-formed LLZO phase stabilized the cubic LLZO phase formation after the sintering process. By optimizing the sintering temperature, both bulk and grain boundary ionic conductivities were improved. As a result, an ionic conductivity of 1.87 × 10-4 S cm-1 of the cubic LLZO phase was confirmed by EIS analysis. These results provide an insight into the reproducibility of the facile synthesis of LLZO. This strategy can be successfully applied to next-generation ASSBs.

Smartphone-Based SARS-CoV-2 and Variants Detection System using Colorimetric DNAzyme Reaction Triggered by Loop-Mediated Isothermal Amplification (LAMP) with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).

Coronavirus disease (COVID-19) has affected people for over two years. Moreover, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has raised concerns regarding its accurate diagnosis. Here, we report a colorimetric DNAzyme reaction triggered by loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR), referred to as DAMPR assay for detecting SARS-CoV-2 and variants genes with attomolar sensitivity within an hour. The CRISPR-associated protein 9 (Cas9) system eliminated false-positive signals of LAMP products, improving the accuracy of DAMPR assay. Further, we fabricated a portable DAMPR assay system using a three-dimensional printing technique and developed a machine learning (ML)-based smartphone application to routinely check diagnostic results of SARS-CoV-2 and variants. Among blind tests of 136 clinical samples, the proposed system successfully diagnosed COVID-19 patients with a clinical sensitivity and specificity of 100% each. More importantly, the D614G (variant-common), T478K (delta-specific), and A67V (omicron-specific) mutations of the SARS-CoV-2 S gene were detected selectively, enabling the diagnosis of 70 SARS-CoV-2 delta or omicron variant patients. The DAMPR assay system is expected to be employed for on-site, rapid, accurate detection of SARS-CoV-2 and its variants gene and employed in the diagnosis of various infectious diseases.

Polychromatic Quantum Dot Array to Compose a Community Signal Ensemble for Multiplexed miRNA Detection.

We herein describe a polychromatic quantum dot array (PQDA) to compose a community signal ensemble enabling accurate and precise quantification of miRNAs in a multiplexed manner. Advanced multicomponent ultrahigh-resolution patterning technique achieved by capsulation-assisted transfer printing following self-assembly-based poly(methyl methacrylate) (PMMA) patterning is utilized to manufacture the PQDA, which is designed to discharge a target miRNAs-specific set of fluorescent quantum dots (QDs) through the activity of duplex-specific nuclease (DSN). On the basis of the community signal ensemble produced by the discharged QD profiles, target miRNAs are very specifically identified down to a femtomolar level (1.27 fM) in a multiplexed manner over a wide dynamic range of up to 6 orders of magnitude. The practical diagnostic capability of this strategy is also demonstrated by reliably identifying breast cancer-specific miRNAs from heterogeneous cancer cell lysates.

Palindromic hyperbranched rolling circle amplification enabling ultrasensitive microRNA detection.

We herein describe a palindromic hyperbranched rolling circle amplification (PH-RCA) reaction and its application for ultrasensitive detection of microRNAs (miRNAs). In this strategy, target miRNAs bind to a dumb-bell probe (DP) and initiate the RCA reactions, concomitantly converting the dumb-bell structure to the circular form, which then allows the annealing of the palindromic primers to promote an additional two RCA reactions. As a consequence of the RCA reactions promoted by both target miRNAs and palindromic primers, multiple long concatenated DNA strands would be produced. Importantly, the palindromic primers can also bind to numerous palindromic domains of the long linear single DNA strands, consequently promoting highly branched simultaneous extension reactions at multiple sites. By detecting the fluorescence signals resulting from the amplified DNA products, we successfully identified target miRNA under isothermal conditions with excellent specificity. The PH-RCA technique developed in this work would greatly advance the conventional RCA reaction and HRCA reaction by significantly enhancing the sensitivity and reducing the reaction time within 30 min.

Ligation-free isothermal nucleic acid amplification.

In this study, we uncover a ligation-free DNA extension method in two adjacent fragmented probes, which are hybridized to target RNA, for developing a ligation-free nucleic acid amplification reaction. In this reaction, DNA elongation occurs from a forward probe to a phosphorothioated-hairpin probe in the presence of target RNA regardless of ligation. The second DNA elongation then occurs simultaneously at the nick site of the phosphorothioated probe and the self-priming region. Therefore, the binding site of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a is repeatedly amplified, inducing a fluorescence signal in the presence of CRISPR-Cas12a. This ligation-free isothermal gene amplification method enables the detection of target RNA with 49.2 fM sensitivity. Moreover, two types of mRNA detection are feasible, thus, demonstrating the potential of this method for cancer companion diagnostics. Notably, the proposed method also demonstrates efficacy when applied for the detection of mRNA extracted from human cells and tumor-bearing mouse tissue and urine samples. Hence, this newly developed ligation-free isothermal nucleic acid amplification system is expected to be widely used in a variety of gene detection platforms.

A novel method for miRNA detection based on target-triggered transcription of a light-up RNA aptamer.

We herein describe a novel method for miRNA detection based on target-triggered transcription of a light-up RNA aptamer (TTRApt), consequently producing a highly enhanced fluorescence signal through specific binding of a light-up RNA aptamer to the corresponding dye. Using this strategy, we successfully identified target miRNA down to 59.4 aM with excellent specificity.

Multiplexed miRNA detection based on target-triggered transcription of multicolor fluorogenic RNA aptamers.

We herein describe a new multicolor fluorogenic RNA aptasensor to accomplish multiplexed detection of miRNAs. The stem-loop primer (SL primer) entailing a fluorogenic RNA aptamer (FRA) antisense sequence is designed to anneal to target miRNA at its 3' overhang, which would be reverse transcribed by reverse transcriptase (RT) to produce the cDNA sequence followed by the degradation of target miRNA. The T7 promoter-containing primer (T7 primer) is then annealed to the 3' end of the extended cDNA sequence and the following RT-promoted extension in both directions produces the T7 promoter-containing double-stranded DNA (T7 dsDNA). T7 RNA polymerase finally transcribes the T7 dsDNA to produce a large number of RNA transcripts containing FRA sequence, which would produce intense fluorescence signals by forming fluorescent complexes with cognate fluorogens, reflecting the amount of target miRNAs. Based on this unique design principle employing the SL primers to encode several different FRAs with distinct fluorescence profiles, target miRNAs were very specifically determined in a multiplexed manner down to a subpicomolar level. The practical applicability of this technique was also verified by reliably quantifying target miRNAs in serum and human cancer cell lysates.