Fluorescent signal-based lateral flow immunochromatographic strips (FLFICS) have received great expectations since they combine the quantitative sensitivity of fluorescence analysis and the simplicity, rapidness, and portability of a common lateral flow immunochromatographic strip (LFICS).

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.

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) and HAT were labeled on the gold nanoparticles (AuNPs) to form the detection probes, and a bisamide derivative of Rhodamine 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 ng mL and 5 × 10 IU mL, respectively, which were at least 10 times lower than those of the conventional fluorescence assay and 10 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.

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.

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Recently, immunochromatography test strips (ICTS) have been fully developed for point-of-care testing (POCT). However, the intrinsic limitations including non-quantitative detection of colloidal gold ICTS and low sensitivity of fluorescence ICTS (FICTS) significantly restrict their further application in clinical diagnosis. Taking advantages of rapid colorimetric qualitative detection and fluorescence quantitation, we designed a kind of sensitive and dual-mode magnetic FICTS (mFICTS) based on PLGA@Fe3O4 super-paramagnetic nanosphere (SPMN) probes quenching multiplex fluorescer on the test line through sandwich immunoreactions. Owing to the large number of FeO nanoparticles (about 47) encapsulated in one SPMN, about 2680 Cy5 molecules were quenched by one SPMN on the test line such that to significantly improve the analytical sensitivity as well as the detection of whole blood samples via magnetic separation. Moreover, the aggregation of black SPMN on the test line enabled a quick naked-eye screening in 3 min. For high accuracy breast cancer diagnosis, combined determination of carcinoembryonic antigen (CEA) and carbohydrate antigen (CA153) was performed on one mFICTS with the limits of detection of about 0.06 ng mL and 0.09 U mL, respectively. Then, more than 50 clinical serum samples were investigated for high-resolution screening by mFICTS, and the results were coincident with those obtained by electrochemiluminescence immunoassay (ECLIA). Thus, the designed mFICTS is suitable for point-of-care diagnostics.

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.

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.