High-Fidelity Sensitive Tracing Circulating Tumor Cell Telomerase Activity.

Dynamic tracing of intracellular telomerase activity plays a crucial role in cancer cell recognition and correspondingly in earlier cancer diagnosis and personalized precision therapy. However, due to the complexity of the required reaction system and insufficient loading of reaction components into cells, achieving a high-fidelity determination of telomerase activity is still a challenge. Herein, an Aptamer-Liposome mediated Telomerase activated poly-Molecular beacon Arborescent Nanoassembly(ALTMAN) approach was described for direct high-fidelity visualization of telomerase activity. Briefly, intracellular telomerase activates molecular beacons, causing their hairpin structures to unfold and produce fluorescent signals. Furthermore, multiple molecular beacons can self-assemble, forming arborescent nanostructures and leading to exponential amplification of fluorescent signals. Integrating the enzyme-free isothermal signal amplification successfully increased the sensitivity and reduced interference by leveraging the skillful design of the molecular beacon and the extension of the telomerase-activated TTAGGG repeat sequence. The proposed approach enabled ultrasensitive visualization of activated telomerase exclusively with a prominent detection limit of 2 cells·µL-1 and realized real-time imaging of telomerase activity in living cancer cells including blood samples from breast cancer patients and urine samples from bladder cancer patients. This approach opens an avenue for establishing a telomerase activity determination and in situ monitoring technique that can facilitate both telomerase fundamental biological studies and cancer diagnostics.

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.

Rolling Circular Amplification (RCA)-Assisted CRISPR/Cas9 Cleavage (RACE) for Highly Specific Detection of Multiple Extracellular Vesicle MicroRNAs.

Multiplexed detection of extracellular vesicle (EV)-derived microRNAs (miRNAs) plays a critical role in facilitating disease diagnosis and prognosis evaluation. Herein, we developed a highly specific nucleic acid detection platform for simultaneous quantification of several EV-derived miRNAs in constant temperature by integrating the advantages of a clustered regularly interspaced short palindromic repeats/CRISPR associated nucleases (CRISPR/Cas) system and rolling circular amplification (RCA) techniques. Particularly, the proposed approach demonstrated single-base resolution attributed to the dual-specific recognition from both padlock probe-mediated ligation and protospacer adjacent motif (PAM)-triggered cleavage. The high consistency between the proposed approach RCA-assisted CRISPR/Cas9 cleavage (RACE) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) in detecting EV-derived miRNAs' abundance from both cultured cancer cells and clinical lung cancer patients validated its robustness, revealing its potentials in the screening, diagnosis, and prognosis of various diseases. In summary, RACE is a powerful tool for multiplexed, specific detection of nucleic acids in point-of-care diagnostics and field-deployable analysis.

Disposable 3D GNAs/AuNPs DNA-Circuit Strip for miRNAs Dynamic Quantification.

Real-time quantitative monitoring of miRNAs plays an essential role in diagnosis and therapeutics. Herein, a DSN-coupled graphene nanoarray/gold nanoparticles (GNAs/AuNPs) carbon paper (CP) electrode for the dynamic, sensitive, and real-time analysis of miRNAs is reported. GNAs are vertically grown on the conductive CP by radio frequency plasma enhanced chemical vapor deposition, and AuNPs are electrodeposited on CP/GNAs to build a 3D ultrasensitive sensing interface with large specific surface area, good conductivity and biocompatibility. The dynamic quantitative monitoring of microRNA-21 (miR-21) is realized by cyclic voltammetry with a series of different concentrations within 16 min, and this 3D GNAs/AuNPs DNA-circuit strip shows good performance for the simultaneous detection of miR-21 and miR-155, and the detection limits are as low as 21.4 and 30.3 am, respectively. Moreover, comparable detection results are achieved for clinical samples between the proposed sensor and qRT-PCR, suggesting the reliability of the constructed sensor. This ultrasensitive sensing and disposable DNA-circuit strip with 3D structure can efficiently shorten the diffusion distance between reactive biomolecules and the sensing interface, enhance the hybridization of probes and improve the sensitivity of the biosensor, holding great promise for the rapid, quantitative and dynamic monitoring of multiple low concentrations of biomolecules in point-of-care clinical analysis.

High-Fidelity Determination and Tracing of Small Extracellular Vesicle Cargoes.

Direct tracing of small extracellular vesicle (sEV) cargoes holds unprecedented importance for elucidating the mechanisms involved in intercellular communication. However, high-fidelity determination of sEVs' molecular cargoes in situ has yet to be achieved due to the difficulty in transporting molecular probes into intact sEVs. Herein, a fLuorescent Intracellular-Guided Hairpin-Tetrahedron (fLIGHT) nanoprobe is described for direct visualization of sEV microRNAs in situ. Integrating the advantages of nondestructive sEV penetration via DNA origami and single-nucleotide discrimination as well as wash-free fluorescence readout using a hairpin probe, the proposed approach enables high-fidelity fluorescence visualization of sEVs' microRNA without RNA extraction or leakage, demonstrating the potential of on-site tracing of sEV cargoes. This strategy opens an avenue to establishing universal molecular detection and labeling platforms that can facilitate both sEV-derived fundamental biological studies and molecular diagnostics.

Rapid and sensitive exosome detection with CRISPR/Cas12a.

Numerous studies have shown that exosomes are closely related to the pathogenesis of various diseases, especially cancers. Therefore, a rapid and sensitive method for exosome detection will be of great importance for the diagnosis and prognosis of diseases. We report here a method for exosome detection based on the CD63 aptamer and clustered regular interspaced short palindromic repeats (CRISPR)/Cas12a system. This method consists mainly of exosomal membrane protein recognition based on the CD63 aptamer and signal amplification based on CRISPR/Cas12a. The CD63 aptamer, as an easily adaptable nucleic acid strand, is responsible for the conversion of the amounts of exosomes into nucleic acid detection, whereas CRISPR/Cas12a is responsible for highly specific nucleic acid signal amplification. The detection range of the method was determined as 3 × 103-6 × 107 particles per microliter. Additionally, we successfully applied this method to detect exosomes in clinical samples from both healthy individuals and patients with lung cancer, and the results were highly consistent with those obtained by nanoparticle tracking analysis. In general, this method provides a highly sensitive and specific method for the detection of exosomes and offers an avenue toward future exosome-based diagnosis of diseases.

Dynamic Monitoring of MicroRNA-DNA Hybridization Using DNAase-Triggered Signal Amplification.

Dynamically monitoring microRNA (miRNA)-DNA reactions is critical for elucidating various biological processes. However, traditional strategies fail to capture this dynamic event because the original targets are preamplified. In the present study, we developed an amplification-free strategy for real-time monitoring of miRNA-DNA hybridization that integrates the advantages of both duplex-specific nuclease (DSN)-triggered signal amplification and single-stranded DNA probe coating facilitated by reduced graphene oxide. DSN-mediated miRNA recognition was found to consist of two phases: hybridization and hybridization cleavage. In the presence of miRNA and DSN, hybridization of a 22-mer miRNA-DNA could be completed within 7 min by observing the angle increase in a surface plasmon resonance (SPR) biosensor. The subsequent hybridization-cleavage process could be visualized as a gradual SPR angle decrease that occurred until all coated probes were hydrolyzed. In addition, for miRNA-21 detection, the proposed linear signal amplification assay demonstrated a sensitivity of 3 fM over a dynamic range of 5 orders of magnitude.

Commercial Strip-Inspired One-Pot CRISPR-Based Chip for Multiplexed Detection of Respiratory Viruses.

The absence of sensitive, multiplexed, and point-of-care assays poses a critical obstacle in promptly responding to emerging human respiratory virus (HRV) pandemics. Herein, RECOGNIZER (re-building commercial pregnancy strips via large-size nanoflowers), an innovative one-pot CRISPR assay, is presented that employs commercially available strips to identify several types of HRVs. The superiority of the RECOGNIZER assay mainly relies on two aspects: (i) DNA nanoflowers possessing a high surface-to-volume ratio and well-defined surface allow for a considerable probe loading density and minimized non-specific interaction, achieving an impressive signal-to-noise proportion exceeding tenfold at 1 nM target. (ii) The design of the one-pot reaction, multi-channel chip, and custom-made app enables the rapid, sample-to-answer, and multiplexed analysis of four HRVs in 25 min. This assay demonstrates a sensitivity of 5.42 pM for synthetic SARS-CoV-2 RNA and 10 copies µL-1 for SARS-CoV-2 plasmids after pre-amplification. Finally, the proposed approach indicated 100% accuracy in 50 clinical swab samples, demonstrating the robust performance in distinguishing SARS-CoV-2 from other HRVs. The versatility and scalability of RECOGNIZER renders it a user-friendly platform for virus infection monitoring, offering significant potential for improving pandemic response efforts.

Enzyme-free and copper-free strategy based on cyclic click chemical-triggered hairpin stacking circuit for accurate detection of circulating microRNAs.

Accurate detection of circulating microRNAs (miRNAs) plays a vital role in the diagnosis of various diseases. However, enzyme-free amplification detection remains challenging. Here, we report an enzyme-free fluorescence resonance energy transfer assay termed "3C-TASK" (cyclic click chemical-triggered hairpin stacking kit) for the detection of circulating miRNA. In this strategy, the miRNA could initiate copper-free click chemical ligation reactions and the ligated products then trigger another hairpin stacking circuit. The first signal amplification was achieved through the recycling of the target miRNA in the click chemical ligation circuit, and the second signal amplification was realized through the recycling of ligated probes in a hairpin stacking circuit driven by thermodynamics. The two-step chain reaction event triggered by miRNAs was quantified by the fluorescence signal value so that accurate detection of target miRNA could be achieved. The 3C-TASK was easily controlled because no enzyme was involved in the entire procedure. Although simple, this strategy showed sensitivity with a detection limit of 8.63 pM and specificity for distinguishing miRNA sequences with single-base variations. In addition, the applicability of this method in complex biological samples was verified by detecting target miRNA in diluted plasma samples. Hence, our method achieved sensitive and specific detection of miRNA and may offer a new perspective for the broader application of enzyme-free chemical reaction and DNA circuits in biosensing.

Programming a DNA tetrahedral nanomachine as an integrative tool for intracellular microRNA biosensing and stimulus-unlocked target regulation.

The synchronous detection and regulation of microRNAs (miRNAs) are essential for the early tumor diagnosis and treatment but remains a challenge. An integrative DNA tetrahedral nanomachine was self-assembled for sensitive detection and negative feedback regulation of intracellular miRNAs. This nanomachine comprised a DNA tetrahedron nanostructure as the framework, and a miRNA inhibitor-controlled allosteric DNAzyme as the core. The DNA tetrahedron brought the DNAzyme and the substrate in spatial proximity and facilitated the cellular uptake of DNAzyme. In allosteric regulation of DNAzyme, the locked tetrahedral DNAzyme (L-tetra-D) and active tetrahedral DNAzyme (A-Tetra-D) were controlled by miRNA inhibitor. The combination of miRNA inhibitor and target could trigger the conformational change from L-tetra-D to A-Tetra-D. A-Tetra-D cleaved the substrate and released fluorescence for intracellular miRNA biosensing. The DNA tetrahedral nanomachine showed excellent sensitivity (with detection limit down to 0.77 pM), specificity (with one-base mismatch discrimination), biocompatibility and stability. Simultaneously, miRNA stimulus-unlocked inhibitor introduced by our nanomachine exhibited the synchronous regulation of target cells, of which regulatory performance has been verified by the upregulated levels of downstream genes/proteins and the increased cellular apoptosis. Our study demonstrated that the DNA tetrahedral nanomachine is a promising biosense-and-treat tool for the synchronous detection and regulation of intracellular miRNA, and is expected to be applied in the early diagnosis and tailored management of cancers.

A dye-assisted paper-based point-of-care assay for fast and reliable blood grouping.

Fast and simultaneous forward and reverse blood grouping has long remained elusive. Forward blood grouping detects antigens on red blood cells, whereas reverse grouping identifies specific antibodies present in plasma. We developed a paper-based assay using immobilized antibodies and bromocresol green dye for rapid and reliable blood grouping, where dye-assisted color changes corresponding to distinct blood components provide a visual readout. ABO antigens and five major Rhesus antigens could be detected within 30 s, and simultaneous forward and reverse ABO blood grouping using small volumes (100 µl) of whole blood was achieved within 2 min through on-chip plasma separation without centrifugation. A machine-learning method was developed to classify the spectral plots corresponding to dye-based color changes, which enabled reproducible automatic grouping. Using optimized operating parameters, the dye-assisted paper assay exhibited comparable accuracy and reproducibility to the classical gel-card assays in grouping 3550 human blood samples. When translated to the assembly line and low-cost manufacturing, the proposed approach may be developed into a cost-effective and robust universal blood-grouping platform.

Duplex-specific nuclease-mediated bioanalysis.

Duplex-specific nucleases (DSNs) are promising tools for bioanalysis because of their unique ability to cleave DNA within duplexes while keeping a single strand intact. There is prevalent use of DSNs in both biomedical and biological science applications, such as cDNA library construction, circulating miRNA detection, telomeric overhang detection, and SNP recognition. We present an overview of the current knowledge of DSNs, with special emphasis on DSN-mediated isothermal signal amplification strategies for trace miRNA detection. Continued innovation to address key challenges, such as amplification-free approaches, will open up new avenues in the field of miRNA profiling, offering opportunities for improved personalized medicine, preventive medicine, and translational medicine.