Mix-and-Read Digital MicroRNA Analysis Based on Flow Cytometric Counting of Target-Clicked Nanobead Dimer.

A one-step, enzyme-free, and highly sensitive digital microRNA (miRNA) assay is rationally devised based on flow cytometric counting of target miRNA-clicked nanobead dimers via a facile mix-and-read manner. In this strategy, highly efficient miRNA-sandwiched click chemical ligation of two DNA probes may remarkably stabilize and boost the dimer formation between two kinds of fluorescence-coded nanobeads, and the number of as-produced bead dimers will be target dose-responsive, particularly when the trace number of miRNA is much less than that of employed nanobeads. Finally, each fluorescence-coded bead dimer can be easily identified and digitally counted by a powerful flow cytometer (FCM) and accordingly, the amount of target miRNA can be accurately quantified in a digital way. This new digital miRNA assay can be accomplished with a facile mix-and-read operation just by simply mixing the target miRNA with two kinds of preprepared DNA probe-functionalized nanobeads, which do not require any nucleic acid amplification, purification, and complex operation procedures. In spite of the extremely simple one-step operation, benefiting from the low-background but high target-mediated click ligation efficiency, and the powerfully digital statistical capability of FCM, this strategy achieves high sensitivity with a quite low detection limit of 5.2 fM target miRNA as well as high specificity and good generality for miRNA analysis, pioneering a new direction for fabricating digital bioassays.

Selective Ligase-Based Sample Processing-Free Discrimination and Detection of Site-Specific DNA 5-Hydroxymethylcytosine.

Accurate detection of site-specific 5-hydroxymethylcytosine (5hmC) in genomic DNA is of great significance, but it is technically challenging to directly distinguish very low levels of 5hmC from their abundant cytosine/5-methylcytosine (C/5mC) analogues. Herein, we wish to propose a selective ligase-mediated mechanism (SLim) that can directly discriminate 5hmC from C/5mC with a high specificity without the use of any sample processing protocol. In this new design, we discovered that HiFi Taq DNA Ligase can well tolerate the mismatched 5hmC/A base-pairing and then effectively ligate the associated nicking site while the mismatched 5mC/A or C/A pairs cannot be recognized by HiFi Taq DNA Ligase, providing a new way for direct and selective discriminating 5hmC from its similar analogues. Ultrasensitive and selective quantification of site-specific 5hmC is realized by coupling the SLim with polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP).

Click chemical ligation-enabled digital particle counting for multiplexed microRNA analysis.

Digital counting assays, that quantify targets by counting individual signal entities, provide a promising way for the sensitive analysis of biomarkers even at the single-molecule level. Considering the requirements of complex enzyme-catalyzed amplification techniques and specialized instruments in traditional digital counting biosensors, herein, a simple digital counting platform for microRNA (miRNA) analysis is developed by employing the miRNA-templated click chemical ligation to hinge ultrabright quantum dot-doped nanoparticles (QDNPs) on the bottom of microplate well. Compared with the traditional short miRNA-mediated sandwich hybridization mechanism, the click chemistry-mediated ligation featured enhanced stability, achieving higher sensitivity by directly counting the number of QDNPs with a common wide-field fluorescence microscope. Furthermore, enzyme-free cycling click ligation strategy is adopted to push the detection limit of miRNA down to a low level of 8 fM. What is more, taking advantages of the tunable emission wavelength and narrow emission spectra of fluorescent nanoparticles, the platform enables simultaneous detection of multiplex miRNA targets without cross interference. Benefiting from the simple operation, high sensitivity, and good generality, miRNA analysis in complex samples is successfully achieved. This method not only pioneers a new route for digital counting assays but also holds great potential in miRNA-related biological researches.

Aggregation-Augmented Magnetism of Lanthanide-Doped Nanoparticles and Enabling Magnetic Levitation-Based Exosome Sensing.

Due to the presence of unpaired electron orbitals in most lanthanide ions, lanthanide-doped nanoparticles (LnNPs) exhibit paramagnetism. However, as to biosensing applications, the magnetism of LnNPs is so weak that can hardly be employed in target separation. Herein, it is discovered that the magnetism of the LnNPs is highly associated with their concentration in a confined space, enabling aggregation-augmented magnetism to make them susceptive to a conventional magnet. Accordingly, a magnetic levitation (Maglev) sensing system is designed, in which the target exosomes can specifically introduce paramagnetic LnNPs to the microbeads' surface, allowing aggregation-augmented magnetism and further leverage the microbeads' levitation height in the Maglev device to indicate the target exosomes' content. It is demonstrated that this Maglev system can precisely distinguish healthy people's blood samples from those of breast cancer patients. This is the first work to report that LnNPs hold great promise in magnetic separation-based biological sample sorting, and the LnNP-permitted Maglev sensing system is proven to be promising for establishing a new generation of biosensing devices.

Augmented Fluorescence Signaling on a Single BaTiO3 Microbead Optical Booster toward High-Sensitive Biosensing.

In this work, we report a new generation of single microbead bioassay that employs a single BaTiO3 microbead as an optical booster for target biomarker enrichment and optical enhancement toward protein and nucleic acid analysis. The single BaTiO3 microbead can not only concentrate the target molecules by nearly 104-fold but also act as an optical booster to prominently enhance the target-induced fluorescence signal by the whispering gallery mode for improving the excitation efficiency and the microlens effect for promoting the signal collecting efficiency, respectively. Compared with using a conventional single microbead, this optical booster exhibits nearly 2 orders of magnitude higher sensitivity without the assistance of any signal amplification techniques or costly instruments. Moreover, this single microbead optical booster is capable of detecting different kinds of protein and nucleic acid biomarkers in a simple mix-and-read manner, holding great potential for early clinical diagnosis.

Ultrasensitive protein and exosome analysis based on a rolling circle amplification assisted-CRISPR/Cas12a strategy.

CRISPR/Cas12a system has attracted extensive concern in biosensing due to its high specificity and programmability. Nevertheless, existing Cas12a-based assays mainly focus on nucleic acid detection and have limitations in non-nucleic acid biomarker analysis. To broaden the application prospect of the CRISPR/Cas technology, a cascade Cas12a biosensing platform is reported by combining dual-functionalized gold nanoparticles (FGNPs)-assisted rolling circle amplification (RCA) and Cas12a trans-cleavage activity (GAR-Cas) for ultrasensitive protein and exosome analysis. FGNPs serve as a critical component in the transduction of protein or exosome recognition information into nucleic acid amplification events to produce Cas12a activators. In the GAR-Cas assay, by integrating the triple cascade amplification of FGNPs-assisted transduction, RCA, and Cas12a signal amplification, ultralow abundance of target molecules can arouse numerous concatemers to activate Cas12a trans-cleavage activity to release intense fluorescence, allowing the ultrasensitive detection of as low as 1 fg/mL (∼41 aM) cTnI and 5 exosomes per µL. Furthermore, the presented strategy can be applied to detect exosome levels from clinical samples, showing excellent performance in distinguishing cancer patients from healthy individuals. The GAR-Cas sensing platform exhibits great potential in clinical diagnosis and enlarges biosensing toolboxes based on CRISPR/Cas technology for non-nucleic acid target analysis.

Forecasting China carbon price using an error-corrected secondary decomposition hybrid model integrated fuzzy dispersion entropy and deep learning paradigm.

Forecasting China's carbon price accurately can encourage investors and manufacturing industries to take quantitative investments and emission reduction decisions effectively. The inspiration for this paper is developing an error-corrected carbon price forecasting model integrated fuzzy dispersion entropy and deep learning paradigm, named ICEEMDAN-FDE-VMD-PSO-LSTM-EC. Initially, the improved complete ensemble empirical mode decomposition with adaptive noise (ICEEMDAN) is used to primary decompose the original carbon price. Subsequently, the fuzzy dispersion entropy (FDE) is conducted to identify the high-complexity signals. Thirdly, the variational mode decomposition (VMD) and deep learning paradigm of particle swarm optimized long short-term memory (PSO-LSTM) models are employed to secondary decompose the high-complexity signals and perform out-of-sample forecasting. Finally, the error-corrected (EC) method is conducted to re-modify and strengthen the above-predicted accuracy. The results conclude that the forecasting performance of ICEEMDAN-type secondary decomposition models is significantly better than the primary decomposition models, the deep learning PSO-LSTM-type models have superiority in forecasting China carbon price, and the EC method for improving the forecasting accuracy has been proved. Noteworthy, the proposed model presents the best forecasting accuracy, with the forecasting errors RMSE, MAE, MAPE, and Pearson's correlation are 0.0877, 0.0407, 0.0009, and 0.9998, respectively. Especially, the long-term forecasting ability for 750 consecutive trading prices is outstanding. Those conclusions contribute to judging the carbon price characteristics and formulating market regulations.

Tyramine-Invertase Bioconjugate-Amplified Personal Glucose Meter Signaling for Ultrasensitive Immunoassay.

Highly sensitive and facile detection of low levels of protein markers is of great significance for the early diagnosis and efficacy monitoring of diseases. Herein, aided by an efficient tyramine-signal amplification (TSA) mechanism, we wish to report a simple but ultrasensitive immunoassay with signal readout on a portable personal glucose meter (PGM). In this study, the bioconjugates of tyramine and invertase (Tyr-inv), which act as the critical bridge to convert and amplify the protein concentration information into glucose, are prepared following a click chemistry reaction. Then, in the presence of a target protein, the sandwich immunoreaction between the immobilized capture antibody, the target protein, and the horseradish peroxidase (HRP)-conjugated detection antibody is specifically performed in a 96-well microplate. Subsequently, the specifically loaded HRP-conjugated detection antibodies will catalyze the amplified deposition of a large number of Tyr-inv molecules onto adjacent proteins through highly efficient TSA. Then, the deposited invertase, whose dosage can faithfully reflect the original concentration of the target protein, can efficiently convert sucrose to glucose. The amount of finally produced glucose is simply quantified by the PGM, realizing the highly sensitive detection of trace protein markers such as the carcinoembryonic antigen and alpha fetoprotein antigen at the fg/mL level. This method is simple, cost-effective, and ultrasensitive without the requirement of sophisticated instruments or specialized laboratory equipment, which may provide a universal and promising technology for highly sensitive immunoassay for in vitro diagnosis of diseases.

Fuzzy-Inspired Sensing for Time-Domain Brain Stroke Diagnosis: Disease Retrospective Monitoring Strategy.

This paper propose a novel disease retrospective monitoring strategy (DRMS) for optimal brain stroke diagnosis. We describe the disease monitoring process using a fuzzy-based model and demonstrate the use of information at different time points to improve disease diagnosis accuracy under the framework of fuzzy-inspired sensing (FIS). Numerical examples are used to demonstrate how the proposed DRMS can be used to determine the optimal treatment strategy with the least amount of fuzziness.

Composite Nanomaterials of Conjugated Polymers and Upconversion Nanoparticles for NIR-Triggered Photodynamic/Photothermal Synergistic Cancer Therapy.

Phototherapies such as photodynamic therapy (PDT) and photothermal therapy (PTT) have attracted great attention in the field of cancer treatment. However, the individual PDT or PTT makes it difficult to achieve optimal antitumor effects compared to the PDT/PTT combined therapy. Also, the effect of PDT is usually limited by the penetration depth of the UV-vis light source. Herein, we designed and synthesized novel composite nanoparticles UCNPs-CPs, which are constructed from two conjugated polymers and upconversion nanoparticles ß-NaYF4:Yb,Tm (UCNPs) via a coordination reaction. By virtue of the excellent spectral overlap between absorption of conjugated polymers and emission of UCNPs, the UCNPs can absorb NIR light and effectively excite conjugated polymers by energy transfer to produce massive reactive oxygen species under 980 nm excitation and heat energy under 808 nm laser irradiation, achieving photodynamic/photothermal synergistic therapy. The in vitro cellular investigation proves that the dual modal phototherapy exhibits enhanced antitumor ability compared to single PDT or PTT. Furthermore, UCNPs-CPs inhibit tumor growth 100% in a 4T1 breast tumor mice model with both NIR laser irradiation, indicating that UCNPs-CPs is an excellent platform for synergistic PDT/PTT treatment. Thus, this study provides a promising strategy for NIR-triggered dual modal phototherapy.

An immunoassay-like recognition mechanism-based lateral flow strategy for rapid microRNA analysis.

A rapid lateral flow assay (LFA) is developed for the colorimetric and surface-enhanced Raman scattering (SERS) dual-mode detection of microRNA (miRNA) based on the robust immunoassay-like (immuno-like) recognition mechanism of S9.6 antibody to DNA/miRNA duplexes. Different from the traditional target-mediated sandwich-type hybridization-based LFA methods, the formation of S9.6 antibody/miRNA/DNA complexes is more rapid and stable, achieving 40 times higher sensitivity with only 10 min assaying time. Furthermore, taking benefit of the versatility of the immuno-like recognition mode, the multiplexed detection of miRNAs can be realized with the SERS signal readout, providing a versatile LFA design towards sensitive, specific, and multiplexed miRNA analysis.

Digital-like Enzyme Inhibition Mechanism-Based Strategy for the Digital Sensing of Heparin-Specific Biomarkers.

A new microbead (MB)-based digital flow cytometric sensing system is proposed for the sensitive detection of heparin-specific biomarkers, including heparin-binding protein (HBP) and heparinase. This strategy takes advantage of the inherent space-confined enzymatic behavior of T4 polynucleotide kinase phosphatase (T4 PNKP) around a single MB and the heparin's digital-like inhibitory effect on T4 PNKP. By integrating with an on-bead terminal deoxynucleotidyl transferase (TdT)-catalyzed fluorescence signal amplification technology, the concentration of HBP and heparinase can be digitally determined by the number of fluorescence-positive/-negative MBs which can be easily counted by flow cytometry. This is not only the first test to expand the application scenario of T4 PNKP to the digital detection of different biomarkers but also pioneers a new direction for fabricating digital biosensing platforms based on the enzyme inhibition mechanism.

Regulating the trans-Cleavage Activity of CRISPR/Cas12a by Using an Elongation-Caged Single-Stranded DNA Activator and the Biosensing Applications.

The CRISPR/Cas12a system exhibits extraordinary capability in the field of biosensing and molecular diagnosis due to its trans-cleavage ability. However, it is still desirable for precise control and programmable regulation of Cas12a trans-cleavage activity to promote the in-depth studies and application expansion of Cas12a-based sensing platforms. In this work, we have developed a new and robust CRISPR/Cas12a regulation mechanism by endowing the activator with the function of caging crRNA ingeniously. Specifically, we constructed an integrated elongation-caged activator (EL-activator) by extending the ssDNA activator on the 3'-end. We found that appending only about 8 nt that is complementary to the crRNA repeat region is enough to cage the crRNA spacer/repeat region, thus effectively inhibiting Cas12a trans-cleavage activity. The inner inhibition mechanism was further uncovered after a thorough investigation, demonstrating that the EL-activator works by impeding the conformation of crRNA required for Cas12a recognition and destroying its affinity with Cas12a. By further switching on the elongated moiety on the EL-activator using target biomarkers, the blocked trans-cleavage activity of Cas12a can be rapidly recovered. Finally, a versatile sensing platform was established based on the EL-activator regulation mechanism, expanding the conventional Cas12a system that only directly recognizes DNA to the direct detection of enzymes and RNA biomarkers. This work has enriched the CRISPR/Cas12a regulation toolbox and expanded its sensing applications.

A polyoxometalate-based heterojunction nanozyme with peroxidase-mimic catalytic activity for sensitive biomolecule detection.

Nanozymes are a class of nanomaterials that can specifically mimic the structures and catalytic activities as well as overcome limitations of natural enzymes and have hence been considered as a competitive alternative to natural enzymes. At present, plenty of nanozymes, especially those with peroxidase (POD)-like catalytic activity, have been extensively explored for biosensing. In this work, we proposed polyoxometalate-based heterojunction GdP5W30O110@WS2 nanoclusters (NCs) to exert intrinsic POD-like catalytic activity even under harsh catalytic conditions. Detailedly, GdP5W30O110@WS2 NCs possessing conducive POD-like catalytic activity can oxidize chromogenic substrates into colored substances in the presence of H2O2. On the strength of the POD-like catalytic activity of GdP5W30O110@WS2 NCs, a reliable analytical platform is then constructed after the optimization of catalytic conditions for the detection of H2O2, glutathione (GSH) and glucose via a simple TMB colorimetric strategy. This work advances the utilization of versatile polyoxometalate-based nanomaterials for biosensing, dramatically broadening the potential applications of other nanozyme-based biosensors.

A Handheld Visible Resonance Raman Analyzer Used in Intraoperative Detection of Human Glioma.

There is still a lack of reliable intraoperative tools for glioma diagnosis and to guide the maximal safe resection of glioma. We report continuing work on the optical biopsy method to detect glioma grades and assess glioma boundaries intraoperatively using the VRR-LRRTM Raman analyzer, which is based on the visible resonance Raman spectroscopy (VRR) technique. A total of 2220 VRR spectra were collected during surgeries from 63 unprocessed fresh glioma tissues using the VRR-LRRTM Raman analyzer. After the VRR spectral analysis, we found differences in the native molecules in the fingerprint region and in the high-wavenumber region, and differences between normal (control) and different grades of glioma tissues. A principal component analysis-support vector machine (PCA-SVM) machine learning method was used to distinguish glioma tissues from normal tissues and different glioma grades. The accuracy in identifying glioma from normal tissue was over 80%, compared with the gold standard of histopathology reports of glioma. The VRR-LRRTM Raman analyzer may be a new label-free, real-time optical molecular pathology tool aiding in the intraoperative detection of glioma and identification of tumor boundaries, thus helping to guide maximal safe glioma removal and adjacent healthy tissue preservation.

Transcriptomic and metabolomic profiling reveal the mechanism underlying the inhibition of wound healing by ascorbic acid in fresh-cut potato.

Ascorbic acid (AsA) inhibits wound healing in fresh-cut potatoes (FCP); however, the comprehensive regulatory mechanisms of the chemical during wound healing remain unclear. Here, physiobiochemical, transcriptomic, and metabolomic analyses were performed. In total, 685 differentially expressed genes (DEGs) and 1921 differentially accumulated metabolites (DAMs) were identified between control and AsA-treated samples. The level of the majority of DEGs expression and DAMs abundance in AsA-treated samples were similar to data of newly cut samples. The collective data indicated that the AsA treatment inhibited wound healing in FCPs by regulating glutathione metabolism, enhancing starch metabolism, and inhibiting phenylalanine metabolism, sucrose degradation, and fatty acid synthesis. Major genes and metabolites affected by AsA treatment included StGST, StPAL, StPHO1 and StLOX5, and starch, sucrose, and linoleic acid. AsA treatment increased starch content and amylase and lipoxygenase activity and decreased free fatty acid level. Our research provides fundamental insights into wound healing mechanisms in FCP.

A novel full solar light spectrum responsive antimicrobial agent of WS2 quantum dots for photocatalytic wound healing therapy.

Photocatalytic antimicrobial therapy (PCAT) is considered to be a potential therapeutic treatment for bacterial-infection diseases. However, the antibacterial efficiency is unsatisfactory due to the limited application scope of photocatalysis. In this work, full-spectrum responsive tungsten disulfide quantum dots (WS2 QDs) are prepared for killing bacteria and enabling wound healing through photocatalytic reactive oxygen species (ROS) generation and glutathione (GSH) depletion. On the one hand, these ultrasmall WS2 QDs exhibit an excellent full spectrum (UV-Vis-NIR)-responsive photocatalytic effect by hindering the recombination of electron-hole pairs, thereby achieving the full use of the energy spectrum. Furthermore, the full-spectrum photocatalytic property of the as-prepared WS2 QDs can be effectively strengthened by redox reaction to deplete GSH for accelerated wound healing. In a word, the as-prepared nanoplatform exhibits the ability to act as an admirable antibacterial reagent with full-spectrum catalytic performance for photocatalytic wound healing therapy. Therefore, this work will not only provide an effective full-spectrum photocatalytic reagent for anti-bacteria therapy and wound healing, but also provide a rational idea for the development of other novel antibacterial agents for applications in the biomedical field.

Integration of transcriptome, miRNA and degradome sequencing reveals the early browning response in fresh-cut apple.

Surface browning negatively impacts the shelf-life of fresh-cut apple. Herein, we found that the browning of fresh-cut apple aggravated rapidly after 24 h post-cutting, then the transcriptomic and miRNA expression profiles of fresh-cut apple immediately after cutting (T0) and 24 h post-cutting (T24) were analyzed to explore the molecular mechanism of early browning response. A total of 3156 differentially expressed mRNAs (DEGs) and 23 differentially expressed miRNAs (DEmiRNAs) were identified in T24 vs T0. Most DEGs related to respiratory, energy, antioxidant, lipid and secondary metabolism were activated in the early stage of browning. There were 63 target genes of 10 DEmiRNAs validated by degradome sequencing and among them, mdm-miR156aa_L + 1_1 targets 12-oxophytodienoate reductase, ptc-miR6478_R-1 targets patatin-like protein, mdm-miR156aa_L + 1_1 and mdm-miR156aa_L + 1_2 co-target SPLs might participate in the early browning response through regulating antioxidant, lipid and secondary metabolism. Our results will be beneficial for the technological innovation of browning amelioration for fresh-cut apple.

A versatile CRISPR/Cas12a-based biosensing platform coupled with a target-protected transcription strategy.

Besides the critical role in gene editing, CRISPR/Cas system also brings a new signal amplification mechanism to the development of next generation biosensing technologies. Herein, we have developed a versatile CRISPR/Cas12a sensing platform by combining a target protection-based transcription amplification strategy with the Cas12a-based signal amplification mechanism, which allows for the sensitive detection of both nucleic acid and non-nucleic acid targets. In this design, a rationally designed transcription template sequence is able to avoid Exonuclease I (Exo I) degradation only in the existence of the target-mediated binding events including either nucleic acid hybridization or protein-based affinity interactions. This target binding-induced protection effect can facilitate the subsequent transcription amplification to generate crRNA and activate the subsequent Cas12a trans-cleavage signal amplification mechanism to yield target dosage-responsive fluorescence signal. In contrast, if the target is absent, the protection-free transcription template will be completely digested by Exo I, thus no fluorescence response is produced. This new strategy well eliminates the T7 polymerase-associated non-specific transcription background and realizes the sensitive detection of various kinds of biomolecules including microRNA, protein, as well as exosome, broadening the application scenarios of CRISPR/Cas system in the field of bioanalysis and biosensing.