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The shape of eukaryotic cells is determined by the cytoskeleton associated with membrane proteins; however, the detailed mechanism of how the integral morphologies with structural stability is generated and maintained is still not fully understood. Here, based on the Frame‐Guided Assembly (FGA) strategy, we successfully prepared hetero‐liposomes with structural composition similar to that of eukaryotic cells by screening a series of transmembrane peptides as the leading hydrophobic groups (LHGs). It was demonstrated that the conformation and transmembrane mode of the LHGs played dominant roles during the FGA process. The FGA liposomes were formed with excellent stability, which may further provide evidence for the cytoskeleton–membrane protein–lipid bilayer model. Taking advantage of the biocompatibility and stability, the FGA liposomes were also applied to prepare novel drug delivery vehicles, which is promising in diagnostic imaging and cancer therapy applications. [ABSTRACT FROM AUTHOR]

A novel template repairing-PCR technology based on miRNA-primed bypass synthesis at the abasic sites on the PCR template is developed as a sensitive and selective platform for miRNA detection. The assay is expected to show great promise for reverse transcription-free RNA PCR amplification and target-based coding analysis. [ABSTRACT FROM AUTHOR]

Abstract: Enzyme‐based assays have been widely applied in clinical diagnosis for decades. However, the intrinsic limitations of enzymes, such as low operation stability, mediocre sensitivity, and high cost in production and purification, heavily constrain their detection application. Here, an enzyme‐free assay is reported that relies on the strong chelating capability of ethylenediamine tetraacetic acid disodium salt (EDTA•2Na, the chelator) for Au3+ ions, in which the cheap EDTA•2Na labeled by targeting moieties can selectively regulate the growth of plasmonic gold nanoparticles (AuNPs) at the target site subjecting to the concentration of analyte in samples. Independent of ambient temperature and unstable H2O2, EDTA•2Na perform superregulation in AuNPs plasmonic signal generation with distinct tonality and outstanding reliability. Upon integrating with silica nanoparticles as the signal amplifying platform, EDTA•2Na‐regulated bioassay can lead to detection‐sensitivity enhancements exceeding three orders of magnitude in protein detection, compared with the gold‐standard assay. The limit of detection of the HBsAg and alpha fetoprotein (AFP) pushes down to 2.6 × 10−15 and 2.5 × 10−19 g mL−1, respectively. EDTA•2Na‐regulated bioassay is also challenged in the clinical serum sample detection and a good consistency is found with the chemiluminescence immunoassay method in clinics. [ABSTRACT FROM AUTHOR]

Fluorescent signal-based lateral flow immunochromatographic strips (FLFICS) have received great expectations since they effectively improve detection sensitivity with quantitative analysis, and still retain the advantages of simplicity, rapidness, and portability of a common lateral flow immunochromatographic strip (LFICS). Diverse fluorescent reporters have promoted development of FLFICS, such as fluorescent dyes, quantum dots (QDs), an up-converting phosphor (UCP), lanthanide labels, and other fluorescence nanoparticles. In this work, we discuss the different fluorescent reporters applied with LFICS and their unique properties as well as signal amplification strategies helping to enhance detection performance. Benefitting from these sensitive and accurate fluorescent labels, FLFICS commendably satisfies requirements for detecting different disease biomarkers in medical diagnosis, strict supervision of food safety, and water pollution. This work also gives a short introduction to trends in the development of future FLFICS technology. [ABSTRACT FROM AUTHOR]

The detection and analysis of microRNA (miRNA) can provide critical information for biomedical research and clinical diagnosis. In this paper, we constructed a simple method to achieve ultra-sensitive and rapid detection of miRNA based on the double-specific nuclease (DSN) assisting target recovery and horseradish peroxidase (HRP) cascading signal amplification. In the existence of target microRNA-21 (miR-21), the DSN enzyme specifically cut the single-stranded DNA (ssDNA) hybridized with the target miRNA but still kept the target miRNA intact, so there were more free HRP released from the polystyrene microparticles-ssDNA-HRP conjugates (PS-ssDNA-HRP) because of the target recycle and signal amplification. The free HRP in the supernatant were collected to react with the substrate 3,3',5,5'-tetramethylbenzidine (TMB). The color and absorbance changes of HRP-TMB system further resulted in qualitative screening by naked eyes and quantitative detection by the microplate reader with the detection limit as low as 3.1 fM. This assay protocol not only showed high sensitivity and selectivity, but also well distinguished the difference between the miRNA family members, as well as the simple operation, fast detection, naked eye qualitative and microplate reader quantification. It has great application value in the biomedical research and clinical diagnosis. [ABSTRACT FROM AUTHOR]

• A one-step, simple and versatile flow cytometric assay for miRNA detection. • DSN-aided target recycling and cleavage strategy for signal amplification. • Good selectivity and high sensitivity for miRNA detection. • Simultaneous detection of multiple targets of miR-21 and Let-7d in one-step. Considered as the next-generation biomarkers, microRNAs play an important role in the early diagnosis of cancers. Here, we designed a fluorescent signal "removal" sensor for one-step, sensitive and specific detection of multiple microRNAs by flow cytometry (FCM). In this work, single-stranded DNA (ssDNA), working as the interlinkage, immobilized the fluorescent nanosphere (FS) onto the SiO 2 microspheres surface to form the SiO 2 -ssDNA-FS probes. When target miRNAs integrated with SiO 2 -ssDNA-FS probes, the duplex-specific nuclease (DSN) could cleave the ssDNA selectively and release FS with numerous cycles to enhance the fluorescent signal attenuation of SiO 2 -ssDNA-FS, so as to remarkably improve the analysis sensitivity. It achieved a simple, accurate and quantitative microRNA-21 detection for clinical blood samples. Parallel multi-target detection of microRNA-21 and Let-7d was also realized by different color labeled FS. Moreover, our designed sensor was suitable for other targets' detection with the corresponding probes. [ABSTRACT FROM AUTHOR]

DNA nanotechnology has been widely employed in the construction of various functional nanostructures. However, most DNA nanostructures rely on hybridization between multiple single-stranded DNAs. Herein, we report a general strategy for the construction of a double-stranded DNA-ribonucleoprotein (RNP) hybrid nanostructure by folding double-stranded DNA with a covalently bivalent clustered regularly interspaced short palindromic repeats (CRISPR)/nuclease-dead CRISPR-associated protein (dCas) system. In our design, dCas9 and dCas12a can be efficiently fused together through a flexible and stimuli-responsive peptide linker. After activation by guide RNAs, the covalently bivalent dCas9-12a RNPs (staples) can precisely recognize their target sequences in the double-stranded DNA scaffold and pull them together to construct a series of double-stranded DNA-RNP hybrid nanostructures. The genetically encoded hybrid nanostructure can protect genetic information in the folded state, similar to the natural DNA-protein hybrids present in chromosomes, and elicit efficient stimuli-responsive gene transcription in the unfolded form. This rationally developed double-stranded DNA folding and unfolding strategy presents a new avenue for the development of DNA nanotechnology.

The accurate and quantitative detection of microRNAs (miRNAs) as next-generation, reliable biomarkers will provide vital information for cancer research and treatment. However, their unique, intrinsic features pose quite a challenge for miRNA profiling, especially for multiplexed detection. Thus, there is a strong and an ever-growing need to develop an accurate, simple, sensitive and specific miRNA sensing method. Methods: In this study, a simple and novel sensor is presented that uses a flow cytometry (FCM) method based on the double key "unlocked mechanism" and a fluorescence enrichment signal amplification strategy. The "unlocked mechanism" was cleverly designed via using hairpin DNA probes (HDs) labeled by fluorescent particles (FS) as the lock to block part of them, which can specifically hybridize with the probe on polystyrene microparticles (PS). The target miRNA and duplex-specific nuclease (DSN) forming the double key can specifically open the HDs and cleave a single-stranded DNA (ssDNA) into DNA/RNA dimers circularly in order to unlock the special part of the HDs to be specially enriched further on the PS. Results: The designed sensor with a hairpin structure and DSN special performance was found to have a high specificity. The circularly unlocking fluorescent probes and fluorescent signal enrichment can be beneficial for achieving a high sensitivity with a detection limit of 3.39 fM for miRNA-21. Meanwhile, the performance of multiplexing was estimated by simultaneous detection of miR-21 and miR-141, and the method also allowed for miR-21 detection in breast cancer blood samples. Conclusion: The designed sensor based on an "unlocked mechanism" and a signal enrichment strategy resulted in a one-pot, highly specific and sensitive detection of multiplex miRNAs. The whole detection without the need for a complex purification process is based on a FCM and is expected to have a great value in cancer diagnosis and biomedical research., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Nowadays, increasing analytical sensitivity is still a big challenge in constructing membrane-based fluorescence immunochromatography test strips (FICTS). However, the bioactivity of antibody (Ab) immobilized on the test line (T line) of porous nitrocellulose membrane (PNM), which directly influences the analytical sensitivity, is less studied. In this work, a novel amphiphilic hydrophobin (HFBI) protein was introduced to modify the T line to effectively retain the Abs' bioactivity. The results indicated that HFBI could self-assemble on the PNM and immobilize the Abs in the "stand-up" orientation. Compared with the conventional FICTS, the HFBI-modified FICTS with only 0.2 mg/mL of monoclonal Abs on T line enable more accurate quantitative detection and better sensitivity (0.06 ng/mL for prostate specific antigen), which is more than 2 orders of magnitude lower than that of the conventional FICTS with the same concentration of monoclonal Abs on T line. Furthermore, the accuracy of this HFBI-modified FICTS was investigated by testing 150 clinical serum samples and the detection results were coincident with those by electrochemiluminescence immunoassay. Our results provide a novel and promising strategy of Ab immobilization on FICTS for near-patient and point-of-care application.

Identifying the microRNA (miRNA) expression level can provide critical information for early diagnosis of cancers or monitoring the cancer therapeutic efficacy. This paper focused on a kind of gold-nanoparticle-coated polystyrene microbeads (PS@Au microspheres)-based DNA probe as miRNA capture and duplex-specific nuclease (DSN) signal amplification platform based on an RGB value readout for detection of miRNAs. In virtue of the outstanding selectivity and simple experimental operation, 5'-fluorochrome-labeled molecular beacons (MBs) were immobilized on PS@Au microspheres via their 3'-thiol, in the wake of the fluorescence quenching by nanoparticle surface energy transfer (NSET). Target miRNAs were captured by the PS@Au microspheres-based DNA probe through DNA/RNA hybridization. DSN enzyme subsequently selectively cleaved the DNA to recycle the target miRNA and release of fluorophores, thereby triggering the signal amplification with more free fluorophores. The RGB value measurement enabled a detection limit of 50 fM, almost 4 orders of magnitude lower than PS@Au microspheres-based DNA probe detection without DSN. Meanwhile, by different encoding of dyes, miRNA-21 and miRNA-10b were simultaneously detected in the same sample. Considering the ability for quantitation, high sensitivity, and convenient merits, the PS@Au microspheres-based DNA probe and DSN signal amplification platform supplied valuable information for early diagnosis of cancers.

At present, enzyme-linked immunosorbent assay (ELISA) is considered to be the most appropriate approach in clinical biomarker detection, with good specificity, low cost, and straightforward readout. However, unsatisfactory sensitivity severely hampers its wide application in clinical diagnosis. Herein, we designed a new kind of enhanced fluorescence enzyme-linked immunosorbent assay (FELISA) based on the human alpha-thrombin (HAT) triggering fluorescence "turn-on" signals. In this system, detection antibodies (Ab 2 ) and HAT were labeled on the gold nanoparticles (AuNPs) to form the detection probes, and a bisamide derivative of Rhodamine 110 with fluorescence quenched served as the substrate of HAT. After the sandwich immunoreaction, HAT on the sandwich structure could catalyze the cleavage of the fluorescence-quenched substrate, leading to a strong fluorescence signal for sensing ultralow levels of alpha fetoprotein (AFP) and hepatitis B virus surface antigen (HBsAg). Under the optimized reaction conditions, AFP and HBsAg were detected at the ultralow concentrations of 10 -8 ng mL -1 and 5 × 10 -4 IU mL -1 , respectively, which were at least 10 4 times lower than those of the conventional fluorescence assay and 10 6 times lower than those of the conventional ELISA. In addition, we further discussed the efficiency of the sensitive FELISA in clinical serum samples, showing great potential in practical applications.