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.

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.

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.

Advances in optical counting and imaging of micro/nano single-entity reactors for biomolecular analysis.

Ultrasensitive detection of biomarkers is of paramount importance in various fields. Superior to the conventional ensemble measurement-based assays, single-entity assays, especially single-entity detection-based digital assays, not only can reach ultrahigh sensitivity, but also possess the potential to examine the heterogeneities among the individual target molecules within a population. In this review, we summarized the current biomolecular analysis methods that based on optical counting and imaging of the micro/nano-sized single entities that act as the individual reactors (e.g., micro-/nanoparticles, microemulsions, and microwells). We categorize the corresponding techniques as analog and digital single-entity assays and provide detailed information such as the design principles, the analytical performance, and their implementation in biomarker analysis in this work. We have also set critical comments on each technique from these aspects. At last, we reflect on the advantages and limitations of the optical single-entity counting and imaging methods for biomolecular assay and highlight future opportunities in this field.

Programming the trans-cleavage Activity of CRISPR-Cas13a by Single-Strand DNA Blocker and Its Biosensing Application.

The precise and controllable programming of the trans-cleavage activity of the CRISPR-Cas13a systems is significant but challenging for fabricating high-performance biosensing systems toward various kinds of biomolecule targets. In this work, we have demonstrated that under a critical low Mg2+ concentration, a simple and short single-stranded DNA (ssDNA) probe free of any modification can efficiently prevent the assembly of crRNA and LwaCas13a only by partially binding with the crRNA repeat region, thereby blocking the trans-cleavage activity of the LwaCas13a system. Furthermore, we have demonstrated that the blocked trans-cleavage activity of the LwaCas13a system can be recovered by various kinds of biologically important substances as long as they could specifically release the blocker DNA from the crRNA in a target-responsive manner, providing a facile route for the quantification of diverse biomarkers such as enzymes, antigens/proteins, and exosomes. To the best of our knowledge, this is reported for the first time that a simple ssDNA can be employed as the switch element to control the crRNA structure and regulate the trans-cleavage activity of Cas13a, which has enriched the CRISPR-Cas13a sensing toolbox and will greatly expand its application scope.

Nucleic Acid Substrate-Independent DNA Polymerization on the Exosome Membrane: A Mechanism Study and Application in Exosome Analysis.

As generally acknowledged, terminal deoxynucleotidyl transferase (TdT) can only elongate DNA substrates from their 3'-OH ends. Herein, for the first time, we report that TdT-catalyzed DNA polymerization can directly proceed on the exosome membrane without the mediation of any nucleic acids. We prove that both the glycosyl and phenolic hydroxyl groups on the membrane proteins can initiate the DNA polymerization. Accordingly, we have developed powerful strategies for high-sensitive exosome profiling based on a conventional flow cytometer and an emerging CRISPR/Cas system. By using our strategy, the featured membrane protein distributions of different cancer cell-derived exosomes can be figured out, which can clearly distinguish plasma samples of breast cancer patients from those of healthy people. This work paves new ways for exosome profiling and liquid biopsy and expands the understanding of TdT, holding great significance in developing TdT-based sensing systems as well as establishing protein/nucleic acid hybrid biomaterials.

Intraoperative detection of human meningioma using a handheld visible resonance Raman analyzer.

To report for the first time the preliminary results for the evaluation of a VRR-LRR™ analyzer based on visible resonance Raman technique to identify human meningioma grades and margins intraoperatively. Unprocessed primary and recurrent solid human meningeal tissues were collected from 33 patients and underwent Raman analysis during surgeries. A total of 1180 VRR spectra were acquired from fresh solid tissues using a VRR-LRR™ analyzer. A confocal HR Evolution (HORIBA, France SAS) Raman system with 532-nm excitation wavelength was also used to collect data for part of the ex vivo samples after they were thawed from - 80 °C for comparison. The preliminary analysis led to the following observations. (1) The intensity ratio of VRR peaks of protein to fatty acid (I2934/I2888) decreased with the increase of meningioma grade. (2) The ratio of VRR peaks of phosphorylated protein to amid I (I1588/I1639) decreased for the higher grade of meningioma. (3) Three RR vibration modes at 1378, 3174, and 3224 cm-1 which were related to the molecular vibrational bands of oxy-hemeprotein, amide B, and amide A protein significantly changed in peak intensities in the two types of meningioma tissues compared to normal tissue. (4) The changes in the intensities of VRR modes of carotenoids at 1156 and 1524 cm-1 were also found in the meningioma boundary. The VRR-LRR™ analyzer demonstrates a new approach for label-free, rapid, and objective identification of primary human meningioma in quasi-clinical settings. The accuracy for detecting meningioma tissues using support vector machines (SVMs) was over 70% based on Raman peaks of key biomolecules and up to 100% using principal component analysis (PCA).

Amplification-Free and Mix-and-Read Analysis of Multiplexed MicroRNAs on a Single Plasmonic Microbead.

In this work, a single microbead covered with a plasmonic layer is employed as the microreactor for the multiplexed miRNA analysis without nucleic acid amplification. On the plasmonic layer, the S9.6 antibody is adopted as the universal module for binding DNA/miRNA duplexes regardless of the sequence. Meanwhile, there is also a SERS reporter gold nanoparticle (GNP) pool, in which each group of GNPs is labeled with both a Raman coding molecule and a DNA probe for recognizing a given miRNA of interest. The target miRNAs will lead to the specific capture of the corresponding SERS reporter GNPs onto the plasmonic layer, which will enormously enhance the target miRNA-induced SERS signals. Finally, the enhanced SERS signals concentrated on the microbead will be mapped out by a confocal Raman microscope. The proposed method achieves the high-precision sensing of sub-pM target miRNA in a simple mix-and-read format and possesses multiplexed assay capability.

Click Chemistry-Actuated Digital DNA Walker Confined on a Single Particle toward Absolute MicroRNA Quantification.

A versatile single magnetic nanoparticle (MNP)-confined, click chemistry-actuated digital DNA walker (ddWalker) machine is devised for the absolute quantification of microRNA (miRNA). This delicate ddWalker allows one target molecule to fix one DNA walking leg on a single MNP, following the Poisson statistics through a specific click chemical DNA ligation, which will initiate single molecule DNA walking to stepwise cleave the molecular beacon tracks strictly constrained on the leg-hold MNP without cross-particle reaction, fluorescently "lighting up" the exact MNP. Accordingly, the initial miRNA input can be digitally and faithfully reflected by the number of fluorescent-positive MNPs counted by a total internal reflection fluorescent microscope, enabling the absolute and precise miRNA quantification down to the femtomolar level without external calibration. This flexible ddWalker design provides a new digital signaling concept and elegantly expands the toolbox for digital biosensing.

Microchamber-Free Digital Flow Cytometric Analysis of T4 Polynucleotide Kinase Phosphatase Based on Single-Enzyme-to-Single-Bead Space-Confined Reaction.

Digital bioassays have attracted extensive attention in biomedical applications due to their ultrahigh sensitivity. However, traditional digital bioassays require numerous microchambers such as droplets or microwells, which restricts their application scope. Herein, we propose a microchamber-free flow cytometric method for the digital quantification of T4 polynucleotide kinase phosphatase (T4 PNKP) based on an unprecedented phenomenon that each T4 PNKP molecule-catalyzed reaction can be spatially self-confined on a single microbead, which ultimately enables the one-target-to-one-fluorescence-positive microbead digital signal transduction. The digital signal-readout mode can clearly detect T4 PNKP concentrations as low as 1.28 × 10-10 U/µL, making it most sensitive method to date. Significantly, T4 PNKP can be specifically distinguished from other phosphatases and nucleases in complex samples by digitally counting the fluorescence-positive microbeads, which cannot be realized by traditional bulk measurement-based methods. Taking advantage of the novel space-confined enzymatic feature of T4 PNKP, this digital mechanism can use T4 PNKP as the enzyme label to fabricate digital sensing systems toward various biomolecules such as digital enzyme-linked immunosorbent assay (ELISA). Therefore, this work not only enlarges the toolbox for high-sensitivity biomolecule detection but also opens new gates to fabricate next-generation digital assays.

Plasmon-Enhanced Surface-Enhanced Raman Scattering Mapping Concentrated on a Single Bead for Ultrasensitive and Multiplexed Immunoassay.

A single microbead (MB)-concentrated surface-enhanced Raman scattering (SERS) mapping strategy is proposed for ultrasensitive and multiplexed immunoassay with high precision. In this design, the SERS tags are specifically immobilized on the surface of a plasmonic gold nanoparticle (GNP) layer-coated single MB via target protein-mediated immune coupling. By this means, even ultralow target dosage can bring highly concentrated SERS tags on the confined small zone around the single MB, and the target-induced SERS signals are largely enhanced by the plasmonic layer, endowing the proposed strategy with ultrahigh sensitivity to quantify subpicogram per milliliter levels of proteins. Moreover, the per-pixel averaged SERS intensity is adopted for target quantitation through mapping the SERS signals around the MB's surface, achieving greatly improved reproducibility compared with traditional single-point measurement. Benefiting from the intrinsic merits of SERS mapping, this elegant strategy also enables multiplexed immunoassay on a single MB.

New CRISPR-Derived microRNA Sensing Mechanism Based on Cas12a Self-Powered and Rolling Circle Transcription-Unleashed Real-Time crRNA Recruiting.

Current CRISPR-Cas-based nucleic acid sensing methods relying on the preassembled Cas-crRNA complexes are generally limited to the detection of protospacer-adjacent motif (PAM)-containing sequences, and nonspecific backgrounds are inevitable. Herein, we propose a new CRISPR-derived microRNA sensing mechanism based on rolling circle transcription (RCT)-unleashed self-recruiting of crRNA by Cas12a (Cas12a-SCR). In Cas12a-SCR, target microRNA can specifically trigger RCT to produce a long single-strand RNA with numerous pre-crRNA repeats, which can be trimmed and recruited by Cas12a actively. This new target-initiated, real-time producing, trimming, and self-assembling manner of Cas12a-crRNA remarkably suppresses the nonspecific background and relieves the stringent requirement of PAM site in the target sequence. Thus, the universality of the Cas12a-SCR toward different nucleic acid sequences is greatly expanded.

Facile Clamp-Assisted Ligation Strategy for Direct Discrimination and Background-Free Quantification of Site-Specific 5-Formylcytosine.

Quantification of site-specific 5-formylcytosine (5fC) in DNA is highly significant to better understand its biological functions. However, it is still a big challenge to precisely discriminate 5fC from cytosine (C), 5-hydroxymethylcytosine (5hmC), 5-methylcytosine (5mC), and 5-carboxycytosine (5caC) owing to their similar structures that will interfere the quantification of 5fC. To solve this issue, a novel peptide nucleic acid (PNA) clamp-assisted ligation amplification strategy coupled with a 5fC-selective chemical conversion route is employed, through which 5fC can be precisely quantified with other interfering signals completely suppressed. As a result, as low as 200 aM of site-specific 5fC-containing DNA target can be accurately determined at single-base resolution in a background-free manner.

Effect of atmospheric cold plasma treatment on antioxidant activities and reactive oxygen species production in postharvest blueberries during storage.

BACKGROUND: Blueberry is universally acknowledged as a kind of berry rich in antioxidants. Cold plasma, an emerging non-thermal treatment technology, has been proved to be able to maintain or improve the antioxidant level while inactivating the microorganisms on the surface of fruits and vegetables. Postharvest blueberries were treated with atmospheric cold plasma (ACP; 12 kV, 5 kHz) for 0 s (Control), 30 s (ACP-30), 60 s (ACP-60), and 90 s (ACP-90) in this study, and the effects of ACP on the antimicrobial properties, antioxidant activities, and reactive oxygen species (ROS) production were investigated during storage at 4 ± 1 °C for 40 days. RESULTS: Total aerobic bacteria and mold populations on ACP-treated blueberries decreased significantly in a time-dependent manner (P < 0.05), and decreased by 0.34-1.24 and 0.57-0.87 log10 CFU g-1 respectively on ACP-60-treated blueberries during storage. The decay rate of blueberries was decreased by 5.8-11.7% and the decrease of blueberry firmness was slowed down by ACP-60. But the total phenol, anthocyanin, and ascorbic acid contents increased, and superoxide dismutase, catalase, and peroxidase activities were enhanced in ACP-treated blueberries. The free radical scavenging activity and total antioxidant capacity (T-AOC) were enhanced. Hydrogen peroxide (H2 O2 ) and superoxide anion (O2 - ) production rates declined by 27.3% and 41.3% at day 40 of storage, respectively. CONCLUSION: It is suggested that ACP may be a promising non-thermal treatment technology for postharvest sterilization and preservation of blueberry under suitable conditions. © 2020 Society of Chemical Industry.

A Versatile Dynamic Light Scattering Strategy for the Sensitive Detection of Plant MicroRNAs Based on Click-Chemistry-Amplified Aggregation of Gold Nanoparticles.

Plant microRNAs (miRNAs) are naturally 2'-O-methylated at the 3'-terminal; as a consequence, they cannot be efficiently detected by traditional target-triggered polymerization reactions. Here, a simple but robust enzyme-free sensing strategy was developed for plant miRNA analysis by using dynamic light scattering (DLS) to monitor the crosslinking of gold nanoparticles (AuNPs) amplified by click chemical ligation. Combining the enzyme-free cycling chemical-ligation-mediated signal amplification, and the intrinsic outstanding ability of DLS for discriminating the extremely low level of particle aggregation in a large pool of monodisperse AuNPs, high sensitivity was achieved and as low as 78.6 fm plant miRNA could be easily detected.

Variation in the Promoter Region of the MC4R Gene Elucidates the Association of Body Measurement Traits in Hu Sheep.

The melanocortin 4 receptor (MC4R) gene is expressed in the appetite-regulating areas of the brain and is engaged in the leptin signaling pathway. Although previous studies have identified variants in the coding region of the sheep MC4R gene showing significant associations with birth weight, weaning weight, and backfat thickness, no such associations have been reported for the promoter region. Besides, the essential promoter region of the sheep MC4R has not been delineated. In this study, to better understand the transcriptional regulation of MC4R and to elucidate the association between regulatory variants and haplotypes with body measurement traits in sheep, we cloned and characterized the MC4R promoter. We found that the minimal promoter of the gene is located within the region -1207/-880 bp upstream of the first exon. Real-time quantitative PCR (RT-qPCR) data revealed the mRNA expression of the MC4R gene had a significant difference between sex and age. In the association analysis, eight single nucleotide polymorphisms (SNPs) had a significant association with one or more traits (p < 0.05); of these, two SNPs were novel. Notably, individuals with haplotype H1H2 (CT-GA-GT-GA-GT-GA-GA-CG) were heavier in body weight than other haplotypes. Altogether, variations in the MC4R gene promoter, most notably haplotype H1H2, may greatly benefit marker-assisted selection in sheep.

Boosting Luminance Energy Transfer Efficiency in Upconversion Nanoparticles with an Energy-Concentrating Zone.

Despite the successful application of upconversion nanoparticles (UCNPs), their low energy transfer efficiency is still a bottleneck to further applications. Here we design UCNPs with a multilayer structure, including an inert NaYF4 :Gd core and an energy-concentrating zone (ECZ), for efficient energy concentration. The ECZ is composed of an emitting layer of NaYF4 :Yb,Er and an absorption layer of NaYF4 :Nd,Yb with antenna IRDye 800CW to manipulate the energy transfer. The stable and tight packing of 800CW linked originally with a bisphosphonate ligand improves greatly the transfer efficiency. The proximity of the emitting layer to both surface antenna and accepter also decreases energy depletion. Compared to classical UCNPs, the ECZ UCNPs show 3600 times higher luminescence intensity with an energy transfer efficiency near 60 %. In proof-of-concept applications, this type of structure was employed for Hg2+ detection and for photodynamic therapy under hypoxic conditions.

Click Chemical Ligation-Initiated On-Bead DNA Polymerization for the Sensitive Flow Cytometric Detection of 3'-Terminal 2'-O-Methylated Plant MicroRNA.

A versatile flow cytometric strategy is developed for the sensitive detection of plant microRNA (miRNA) by coupling the target-templated click nucleic acid ligation (CNAL) with on-bead terminal enzymatic DNA polymerization (TEP). Unlike ligase-catalyzed ligation reaction, the plant miRNA-templated enzyme-free CNAL between two single-stranded DNA (ssDNA) probes, respectively modified with Aza-dibenzocyclooctyne (Aza-DBCO) and N3, can not only simplify the operation, but also achieve a much higher ligation efficiency. More importantly, the undesirable nonspecific ligation between the Aza-DBCO- and N3-modified ssDNA, can be effectively eliminated by adding Tween-20, which allows the use of cycling CNAL (CCNAL) in a background-free manner. So each plant miRNA can template many rounds of CNAL reaction to produce numerous ligation products, forming efficient signal amplification. The ligated ssDNA can be anchored on the magnetic beads (MBs) with the 3'-OH termini exposed outside. Then terminal deoxynucleotidyl transferase (TdT), a sequence-independent and template-free polymerase, would specifically catalyze the DNA polymerization along these 3'-OH termini on the MBs, forming poly(T) tails up to thousands of nucleotides long. Each poly(T) tail allows specific binding of numerous 6-carboxyfluorescein (FAM)-labeled poly(A)25 oligonucleotides to accumulate a lot of fluorophores on the MBs, leading to the second step of signal amplification. By integrating the advantages of CCNAL-TEP for highly efficient signal amplification and robust MBs signal readout with powerful flow cytometer, high sensitivity is achieved and the detection limit of plant miRNA has been pushed down to a low level of 5 fM with high specificity to well discriminate even single-base difference between miRNA targets.

Edaravone alleviates Alzheimer's disease-type pathologies and cognitive deficits.

Alzheimer's disease (AD) is one of most devastating diseases affecting elderly people. Amyloid-ß (Aß) accumulation and the downstream pathological events such as oxidative stress play critical roles in pathogenesis of AD. Lessons from failures of current clinical trials suggest that targeting multiple key pathways of the AD pathogenesis is necessary to halt the disease progression. Here we show that Edaravone, a free radical scavenger that is marketed for acute ischemic stroke, has a potent capacity of inhibiting Aß aggregation and attenuating Aß-induced oxidation in vitro. When given before or after the onset of Aß deposition via i.p. injection, Edaravone substantially reduces Aß deposition, alleviates oxidative stress, attenuates the downstream pathologies including Tau hyperphosphorylation, glial activation, neuroinflammation, neuronal loss, synaptic dysfunction, and rescues the behavioral deficits of APPswe/PS1 mice. Oral administration of Edaravone also ameliorates the AD-like pathologies and memory deficits of the mice. These findings suggest that Edaravone holds a promise as a therapeutic agent for AD by targeting multiple key pathways of the disease pathogenesis.