Multiarmed DNA jumper and metal-organic frameworks-functionalized paper-based bioplatform for small extracellular vesicle-derived miRNAs assay.

Small extracellular vesicle-derived microRNAs (sEV-miRNAs) have emerged as promising noninvasive biomarkers for early cancer diagnosis. Herein, we developed a molecular probe based on three-dimensional (3D) multiarmed DNA tetrahedral jumpers (mDNA-Js)-assisted DNAzyme activated by Na+, combined with a disposable paper-based electrode modified with a Zr-MOF-rGO-Au NP nanocomplex (ZrGA) to fabricate a novel biosensor for sEV-miRNAs Assay. Zr-MOF tightly wrapped by rGO was prepared via a one-step method, and it effectively aids electron transfer and maximizes the effective reaction area. In addition, the mechanically rigid, and nanoscale-addressable mDNA-Js assembled from the bottom up ensure the distance and orientation between fixed biological probes as well as avoid probe entanglement, considerably improving the efficiency of molecular hybridization. The fabricated bioplatform achieved the sensitive detection of sEV-miR-21 with a detection limit of 34.6 aM and a dynamic range from100 aM to 0.2 µM. In clinical blood sample tests, the proposed bioplatform showed results highly consistent with those of qRT-PCRs and the signal increased proportionally with the NSCLC staging. The proposed biosensor with a portable wireless USB-type analyzer is promising for the fast, easy, low-cost, and highly sensitive detection of various nucleic acids and their mutation derivatives, making it ideal for POC biosensing.

A Universal Strategy for Enhancing the Circulating miRNAs' Detection Performance of Rolling Circle Amplification by Using a Dual-Terminal Stem-Loop Padlock.

Rolling circle amplification (RCA) is one of the most promising nucleic acid detection technologies and has been widely used in the molecular diagnosis of disease. Padlock probes are often used to form circular templates, which are the core of RCA. However, RCA often suffers from insufficient specificity and sensitivity. Here we report a reconstruction strategy for conventional padlock probes to promote their overall performance in nucleic acid detection while maintaining probe functions uncompromised. When two rationally designed stem-loops were strategically placed at the two terminals of linear padlock probes, the specificity of target recognition was enhanced and the negative signal was significantly delayed. Our design achieved the best single-base discrimination compared with other structures and over a 1000-fold higher sensitivity than that of the conventional padlock probe, validating the effectiveness of this reconstruction. In addition, the underlying mechanisms of our design were elucidated through molecular dynamics simulations, and the versatility was validated with longer and shorter padlocks targeting the same target, as well as five additional targets (four miRNAs and dengue virus - 2 RNA mimic (DENV-2)). Finally, clinical applicability in multiplex detection was demonstrated by testing real plasma samples. Our exploration of the structures of nucleic acids provided another perspective for developing high-performance detection systems, improving the efficacy of practical detection strategies, and advancing clinical diagnostic research.

Modularized Enzymatic Tandem Reaction for tsRNA Detection.

tRNA-derived small RNA (tsRNA) has emerged as a new biomarker for early diagnosis and prognosis prediction of breast cancer. Like the detection of other small non-coding RNAs, the traditional DNA circuit could be used for the tsRNA detection. However, the highly coupling DNA strands in the circuit increase the difficulty of design and could raise a false-positive signal. Here, we demonstrated a versatile modularized enzymatic tandem reaction, namely, reverse-transcribed nicking exponential truncation (RT-NExT). This enzymatic reaction was constructed by cohesive modules, which can work independently or in assembly. Each module could amplify and initiate the downstream module. The RT-NExT reaction could detect 10-18 M ts-66 or ts-86 within 10 min and exhibited excellent consistency to the qRT-PCR when measuring the tsRNA expression level of breast cancer or healthy patients. RT-NExT provides an appealing detection strategy for further research on the clinical diagnosis with tsRNAs.

Phage Endolysin LysP108 Showed Promising Antibacterial Potential Against Methicillin-resistant Staphylococcus aureus.

As a potential antibacterial agent, endolysin can directly lyse Gram-positive bacteria from the outside and does not lead to drug resistance. Considering that XN108 is the first reported methicillin-resistant Staphylococcus aureus (MRSA) strain in mainland China with a vancomycin MIC that exceeds 8 µg mL-1, we conducted a systematic study on its phage-encoded endolysin LysP108. Standard plate counting method revealed that LysP108 could lyse S. aureus and Pseudomonas aeruginosa with damaged outer membrane, resulting in a significant reduction in the number of live bacteria. Scanning electron microscopy results showed that S. aureus cells could be lysed directly from the outside by LysP108. Live/dead bacteria staining results indicated that LysP108 possessed strong bactericidal ability, with an anti-bacterial rate of approximately 90%. Crystal violet staining results implied that LysP108 could also inhibit and destroy bacterial biofilms. In vivo animal experiments suggested that the area of subcutaneous abscess of mice infected with MRSA was significantly reduced after the combined injection of LysP108 and vancomycin in comparison with monotherapy. The synergistic antibacterial effects of LysP108 and vancomycin were confirmed. Therefore, the present data strongly support the idea that endolysin LysP108 exhibits promising antibacterial potential to be used as a candidate for the treatment of infections caused by MRSA.

Lateral flow assay of methicillin-resistant Staphylococcus aureus using bacteriophage cellular wall-binding domain as recognition agent.

As one of the most common and noticeable superbugs, methicillin-resistant Staphylococcus aureus (MRSA) has long been a major threat to public health. To meet the demand for effective diagnosis of MRSA-induced infection, it is urgent to establish rapid assay method for this type of pathogen. In this study, an aqueous soluble cellular wall-binding domain (CWBD) protein from bacteriophage P108 was obtained with a recombinant expression technique. It can act as a wide-spectrum binding agent for all MRSA strains and exclude the interference from methicillin-susceptible strains of Staphylococcus aureus and other species of bacteria. To establish a lateral flow assay (LFA) method for MRSA, CWBD-coupled time-resolved fluorescent microspheres (FMs) were used as signal probes for tracing MRSA, and a nitrocellulose membrane immobilized with porcine IgG was used to capture MRSA. With the LFA based on sandwich format, MRSA can be assayed within 10 min with a broad linear range of 6.6 × 102-6.6 × 107 CFU/mL. Its application potential has been demonstrated by assaying different types of bacteria-contaminated real samples. The results suggest that the LFA strip using recombinant CWBD as the recognition agent provides a rapid, portable, cost-effective approach for point-of-care testing of MRSA.

Recombinant Bacteriophage Cell-Binding Domain Proteins for Broad-Spectrum Recognition of Methicillin-Resistant Staphylococcus aureus Strains.

Methicillin-resistant Staphylococcus aureus (MRSA) has been well-recognized as one of the most common multiresistant bacteria threatening human health. Broad-spectrum recognition of multiple MRSA strains can meet the urgent demands for efficient diagnosis and subsequent decision of relevant treatment of MRSA-induced infections. Here, recombinant cell-binding domain (CBD) and green fluorescent protein-fused CBD of MRSA bacteriophage were expressed in soluble form. Distinct from the strain-specific MRSA bacteriophage, both recombinant CBD proteins displayed broad-spectrum recognition capability toward all five staphylococcal cassette chromosome mec types of MRSA. Furthermore, they did not display any lytic activity toward the host bacteria, which facilitated the capture of whole MRSA cells with ideal flexibility for downstream manipulation and tracing. For demonstration of their application potential, a flow cytometry method employing the recombinant CBD proteins as the recognition agents was established to detect MRSA within a dynamic range of 1.5 × 102 to 1.5 × 106 cfu mL-1. The method can exclude potential interference from methicillin-sensitive Staphylococcus aureus strains and other bacterial species. The recombinant CBD proteins were also successfully employed in antibiotic susceptibility testing of MRSA with a microplate-based method. The obtained results were consistent with those by the standard broth microdilution method. The satisfying results demonstrated their great application potential in clinical diagnosis and treatment of MRSA-induced infections.

Nonlytic Recombinant Phage Tail Fiber Protein for Specific Recognition of Pseudomonas aeruginosa.

Rapid and accurate bacterial detection is crucial to an early diagnosis for treating various infectious diseases. A recombinant tail fiber protein (P069) of the Pseudomonas aeruginosa ( P. aeruginosa) phage was expressed in Escherichia coli. After renaturation at a low temperature, the inclusion body of P069 was successfully transformed to an aqueous soluble protein that retained the capacity for recognizing P. aeruginosa. The recombinant P069 did not show lytic activity to P. aeruginosa, which facilitated the capture and manipulation of bacterial whole cells with a high flexibility for downstream identification and detection. Bioluminescent and fluorescent methods using this biorecognition element allowed P. aeruginosa detection with the detection limits of 6.7 × 102 CFU mL-1 and 1.7 × 102 CFU mL-1, respectively. Moreover, the specificity investigations showed that P069 was a species-specific protein. Therefore, it avoided the potential false negative results originating from the excessive high specificity of phage toward a given strain. It has been successfully applied to detect P. aeruginosa in spiked samples with acceptable recovery values ranging from 88% to 98%. The above results demonstrate that P069 is an ideal biorecognition element for the detection of P. aeruginosa in complicated sample matrixes.