Integration of the Ligase Chain Reaction with the CRISPR-Cas12a System for Homogeneous, Ultrasensitive, and Visual Detection of microRNA

The ligase chain reaction (LCR), as a classic nucleic acid amplification technique, is popular in the detection of DNA and RNA due to its simplicity, powerfulness, and high specificity. However, homogeneous and ultrasensitive LCR detection is still quite challenging. Herein, we integrate the LCR with a CRISPR-Cas12a system to greatly promote the application of the LCR in a homogeneous fashion. By employing microRNA as the model target, we design LCR probes with specific protospacer adjacent motif sequences and the guide RNA. Then, the LCR is initiated by target microRNA, and the LCR products specifically bind to the guide RNA to activate the Cas12a system, triggering secondary signal amplification to achieve ultrasensitive detection of microRNA without separation steps. Moreover, by virtue of a cationic conjugated polymer, microRNA can not only be visually detected by naked eyes but also be accurately quantified based on RGB ratio analysis of images with no need of sophisticated instruments. The method can quantify microRNA up to 4 orders of magnitude, and the determination limit is 0.4 aM, which is better than those of other reported studies using CRISPR-Cas12a and can be compared with that of the reverse-transcription polymerase chain reaction. This study demonstrates that the CRISPR-Cas12a system can greatly expand the application of the LCR for the homogeneous, ultrasensitive, and visual detection of microRNA, showing great potential in efficient nucleic acid detection and in vitro diagnosis

Conjugated Polymers/DNA Hybrid Materials for Protein Inactivation

Chromophore-assisted light inactivation (CALI) is a powerful tool for analyzing protein functions due to the high degree of spatial and temporal resolution. In this work, we demonstrate a CALI approach based on conjugated polymers (CPs)/DNA hybrid material for protein inactivation. The target protein is conjugated with single-stranded DNA in advance. Single-stranded DNA can form CPs/DNA hybrid material with cationic CPs via electrostatic and hydrophobic interactions. Through the formation of CPs/DNA hybrid material, the target protein that is conjugated with DNA is brought into close proximity to CPs. Under irradiation, CPs harvest light and generate reactive oxygen species (ROS), resulting in the inactivation of the adjacent target protein. This approach can efficiently inactivate any target protein which is conjugated with DNA and has good specificity and universality, providing a new strategy for studies of protein function and adjustment of protein activity

Highly Sensitive and Specific Multiplexed MicroRNA Quantification Using Size-Coded Ligation Chain Reaction

As important regulators of gene expression, microRNAs (miRNAs) are emerging as novel biomarkers with powerful predictive value in diagnosis and prognosis for several diseases, especially for cancers. There is a great demand for flexible multiplexed miRNA quantification methods that can quantify very low levels of miRNA targets with high specificity. For further analysis of miRNA signatures in biological samples, we describe here a highly sensitive and specific method to detect multiple miRNAs simultaneously in total RNA. First, we rationally design one of the DNA probes modified with two ribonucleotides, which can greatly improve the ligation efficiency of DNA probes templated by miRNAs. With the modified DNA probes, the ligation chain reaction (LCR) can be well applied to miRNA detection and as low as 0.2 fM miRNA can be accurately determined. High specificity to clearly discriminate a single nucleotide difference among miRNA sequences can also be achieved. By simply coding the DNA probes with different length of oligo (dA) for different miRNA targets, multiple miRNAs can be simultaneously detected in one LCR reaction. In our proof of principle work, we detect three miRNAs: let-7a, mir-92a, and mir-143, which can also be simultaneously detected in as small as 2 ng of total RNA sample

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

Interpenetration of Donor–Acceptor Hybrid Frameworks for Highly Sensitive Thermal Sensors

Donor–acceptor (D–A) alignment that integrates D–A pairs into the modular and versatile crystalline metal–organic frameworks is a powerful strategy to precisely fabricate multifunctional materials with unique optoelectronic properties and applications at the molecular level. Herein, we reported an unprecedented threefold interpenetrating D–A hybrid framework by incorporating an electron-deficient linear viologen zwitterion into the lead-halide systems. The 1D iodoplumbate nanoribbon and interpenetrating close-packed D–A structure endowed this hitherto unknown semiconductive alignment with the anisotropic conductivity and high stability. When used in a thermal sensor, it presented exceptional electrical properties with a high sensitivity (high thermal index B of 4671 K) and decent temperature coefficient of resistivity (0.72% °C–1) in wide operational temperature ranges (40–220 °C), which are among the best of the related thermistors. This work develops a pathway to bridge the gaps between hybrid materials and electron devices

Interpenetration of Donor–Acceptor Hybrid Frameworks for Highly Sensitive Thermal Sensors

Donor–acceptor (D–A) alignment that integrates D–A pairs into the modular and versatile crystalline metal–organic frameworks is a powerful strategy to precisely fabricate multifunctional materials with unique optoelectronic properties and applications at the molecular level. Herein, we reported an unprecedented threefold interpenetrating D–A hybrid framework by incorporating an electron-deficient linear viologen zwitterion into the lead-halide systems. The 1D iodoplumbate nanoribbon and interpenetrating close-packed D–A structure endowed this hitherto unknown semiconductive alignment with the anisotropic conductivity and high stability. When used in a thermal sensor, it presented exceptional electrical properties with a high sensitivity (high thermal index B of 4671 K) and decent temperature coefficient of resistivity (0.72% °C–1) in wide operational temperature ranges (40–220 °C), which are among the best of the related thermistors. This work develops a pathway to bridge the gaps between hybrid materials and electron devices

Cationic Conjugated Polymers-Induced Quorum Sensing of Bacteria Cells

Bacteria quorum sensing (QS) has attracted significant interest for understanding cell–cell communication and regulating biological functions. In this work, we demonstrate that water-soluble cationic conjugated polymers (PFP-G2) can interact with bacteria to form aggregates through electrostatic interactions. With bacteria coated in the aggregate, PFP-G2 can induce the bacteria QS system and prolong the time duration of QS signal molecules (autoinducer-2 (AI-2)) production. The prolonged AI-2 can bind with specific protein and continuously regulate downstream gene expression. Consequently, the bacteria show a higher survival rate against antibiotics, resulting in decreased antimicrobial susceptibility. Also, AI-2 induced by PFP-G2 can stimulate 55.54 ± 12.03% more biofilm in <i>E. coli</i>. This method can be used to understand cell–cell communication and regulate biological functions, such as the production of signaling molecules, antibiotics, other microbial metabolites, and even virulence

Data_Sheet_1_Comprehensive next-generation sequencing reveals low-grade fibromyxoid sarcoma of the vulva missed by morphological diagnosis: a case report.docx

Low-grade fibromyxoid sarcoma (LGFMS) is a rare soft tissue tumor composed of bland spindled cells in a variably fibrous to myxoid stroma. Its occurrence in the vulva region is rare, and thus, it may not be always taken into account in the differential diagnosis. Here, we describe a 34-year-old woman presented with a right vulvar mass and underwent complete surgical excision. The final pathologic diagnosis revealed LGFMS of the vulva based on the morphological, immunophenotypic, and molecular genetic features. The patient has not experienced a local or metastatic recurrence after 9-month follow-up. Despite being rare, LGFMS of the vulva should be considered when making a diagnosis of vulvar lesions. We also report that the genetic testing by next-generation sequencing (NGS) represents a very useful tool for the differential diagnosis of LGFMS from its mimics. Moreover, we have reviewed the literature on LGFMS of the vulva and summarized the characteristics of the patients, providing assistance for the diagnosis of such patients. Most vulvovaginal LGFMS can be fully removed through surgery. However, ongoing monitoring over the long term is essential as local and/or distant spread can occur decades after the initial diagnosis.</p

Table_1_Comprehensive next-generation sequencing reveals low-grade fibromyxoid sarcoma of the vulva missed by morphological diagnosis: a case report.xlsx

Low-grade fibromyxoid sarcoma (LGFMS) is a rare soft tissue tumor composed of bland spindled cells in a variably fibrous to myxoid stroma. Its occurrence in the vulva region is rare, and thus, it may not be always taken into account in the differential diagnosis. Here, we describe a 34-year-old woman presented with a right vulvar mass and underwent complete surgical excision. The final pathologic diagnosis revealed LGFMS of the vulva based on the morphological, immunophenotypic, and molecular genetic features. The patient has not experienced a local or metastatic recurrence after 9-month follow-up. Despite being rare, LGFMS of the vulva should be considered when making a diagnosis of vulvar lesions. We also report that the genetic testing by next-generation sequencing (NGS) represents a very useful tool for the differential diagnosis of LGFMS from its mimics. Moreover, we have reviewed the literature on LGFMS of the vulva and summarized the characteristics of the patients, providing assistance for the diagnosis of such patients. Most vulvovaginal LGFMS can be fully removed through surgery. However, ongoing monitoring over the long term is essential as local and/or distant spread can occur decades after the initial diagnosis.</p