ABSTRACT
The gene encoding PTEN is one of the most frequently mutated tumor suppressor-encoding genes in human cancer. While PTEN's function in tumor suppression is well established, its relationship to anti-microbial immunity remains unknown. Here we found a pivotal role for PTEN in the induction of type I interferon, the hallmark of antiviral innate immunity, that was independent of the pathway of the kinases PI(3)K and Akt. PTEN controlled the import of IRF3, a master transcription factor responsible for IFN-ß production, into the nucleus. We further identified a PTEN-controlled negative phosphorylation site at Ser97 of IRF3 and found that release from this negative regulation via the phosphatase activity of PTEN was essential for the activation of IRF3 and its import into the nucleus. Our study identifies crosstalk between PTEN and IRF3 in tumor suppression and innate immunity.
Subject(s)
Immunity, Innate/immunology , Interferon Regulatory Factor-3/immunology , Interferon Type I/immunology , PTEN Phosphohydrolase/immunology , Respirovirus Infections/immunology , Rhabdoviridae Infections/immunology , Animals , Cell Line , Cell Line, Tumor , Cell Nucleus , Cell Proliferation , Cytokines/immunology , Dendritic Cells/immunology , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique , Gene Transfer Techniques , HEK293 Cells , Humans , Immunoblotting , Immunoprecipitation , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-7/genetics , MCF-7 Cells , Macrophages/immunology , Mass Spectrometry , Mice , Microscopy, Confocal , Mutagenesis, Site-Directed , PTEN Phosphohydrolase/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sendai virus , VesiculovirusABSTRACT
The maintenance of lysosome homeostasis is crucial for cell growth. Lysosome-dependent degradation and metabolism sustain tumor cell survival. Here, we demonstrate that CCDC50 serves as a lysophagy receptor, promoting tumor progression and invasion by controlling lysosomal integrity and renewal. CCDC50 monitors lysosomal damage, recognizes galectin-3 and K63-linked polyubiquitination on damaged lysosomes, and specifically targets them for autophagy-dependent degradation. CCDC50 deficiency causes the accumulation of ruptured lysosomes, impaired autophagic flux, and superfluous reactive oxygen species, consequently leading to cell death and tumor suppression. CCDC50 expression is associated with malignancy, progression to metastasis, and poor overall survival in human melanoma. Targeting CCDC50 suppresses tumor growth and lung metastasis, and enhances the effect of BRAFV600E inhibition. Thus, we demonstrate critical roles of CCDC50-mediated clearance of damaged lysosomes in supporting tumor growth, hereby identifying a potential therapeutic target of melanoma.
ABSTRACT
DNA assemblies are commonly used in biosensing, particularly for the detection and imaging of microRNAs (miRNAs), which are biomarkers associated with tumor progression. However, the difficulty lies in the exploration of high-sensitivity analytical techniques for miRNA due to its limited presence in living cells. In this study, we introduced a DNA nanosphere (DS) enhanced catalytic hairpin assembly (CHA) system for the detection and imaging of intracellular miR-21. The single-stranded DNA with four palindromic portions and extending sequences at the terminal was annealed for assembling DS, which avoided the complex sequence design and high cost of long DNA strands. Benefiting from the multiple modification sites of DS, functional hairpins H1 (modified with Cy3 and BHQ2) and H2 were grafted onto the surface of DS for assembling DS-H1-H2 using a hybridization reaction. The DS-H1-H2 system utilized spatial confinement and the CHA reaction to amplify fluorescence signals of Cy3. This enabled highly sensitive and rapid detection of miR-21 in the range from 0.05 to 3.5 nM. The system achieved a limit of determination (LOD) of 2.0 pM, which was 56 times lower than that of the control CHA circuit with freedom hairpins. Additionally, the sensitivity was improved by 8 times. Moreover, DS-H1-H2 also showed an excellent imaging capability for endogenous miR-21 in tumor cells. This was due to enhanced cell internalization efficiency, accelerated reaction kinetics, and improved biostability. The imaging strategy was shown to effectively monitor the dynamic content of miR-21 in live cancer cells and differentiate various cells. In general, the simple nanostructure DS not only enhanced the detection and imaging capability of the conventional probe but also could be easily integrated with the reported DNA-free probe, indicating a wide range of potential applications.
Subject(s)
Biosensing Techniques , DNA, Catalytic , MicroRNAs , Nanospheres , Neoplasms , MicroRNAs/genetics , MicroRNAs/chemistry , DNA/genetics , DNA/chemistry , Nucleic Acid Hybridization , DNA Probes/chemistry , Biosensing Techniques/methods , Limit of DetectionABSTRACT
Chirality is a widespread phenomenon in nature and in living organisms and plays an important role in living systems. The sensitive discrimination of chiral molecular enantiomers remains a challenge in the fields of chemistry and biology. Establishing a simple, fast, and efficient strategy to discriminate the spatial configuration of chiral molecular enantiomers is of great significance. Chiral perovskite nanocrystals (PNCs) have attracted much attention because of their excellent optical activity. However, it is a challenge to prepare perovskites with both chiral and fluorescence properties for chiral sensing. In this work, we synthesized two chiral fluorescent perovskite nanocrystal assembly (PNA) enantiomers by using l- or d-phenylalanine (Phe) as chiral ligands. PNA exhibited good fluorescence recognition for l- and d-proline (Pro). Homochiral interaction led to fluorescence enhancement, while heterochiral interaction led to fluorescence quenching, and there is a good linear relationship between the fluorescence changing rate and l- or d-Pro concentration. Mechanism studies show that homochiral interaction-induced fluorescence enhancement is attributed to the disassembly of chiral PNA, while no disassembly of chiral PNA was found in heterochiral interaction-induced fluorescence quenching, which is attributed to the substitution of Phe on the surface of chiral PNA by heterochiral Pro. This work suggests that chiral perovskite can be used for chiral fluorescence sensing; it will inspire the development of chiral nanomaterials and chiral optical sensors.
ABSTRACT
Tile-based DNA self-assembly provides a versatile approach for the construction of a wide range of nanostructures for various applications such as nanomedicine and advanced materials. The inter-tile interactions are primarily programmed by base pairing, particularly Watson-Crick base pairing. To further expand the tool box for DNA nanotechnology, herein, we have designed DNA tiles that contain both ligands and aptamers. Upon ligand-aptamer binding, tiles associate into geometrically well-defined nanostructures. This strategy has been demonstrated by the assembly of a series of DNA nanostructures, which have been thoroughly characterized by gel electrophoresis and atomic force microscopy. This new inter-tile cohesion could bring new potentials to DNA self-assembly in the future. For example, the addition of free ligand could modulate the nanostructure formation. In the case of biological ligands, DNA self-assembly could be related to the presence of certain ligands.
Subject(s)
DNA , Oligonucleotides , Ligands , Base Pairing , Microscopy, Atomic ForceABSTRACT
Owing to the excellent structural rigidity and programmable reaction sites, DNA nanostructures are more and more widely used, but they are limited by high cost, strict sequence requirements, and time-consuming preparation. Herein, a general signal amplifier based on a micelle-supported entropy-driven circuit (MEDC) was designed and prepared for sensitive quantification of biomarkers. By modifying a hydrophobic cholesterol molecule onto a hydrophilic DNA strand, the amphiphilic DNA strand was first prepared and then self-assembled into DNA micelles (DMs) driven by hydrophobic effects. The as-developed DM showed unique advantages of sequence-independence, easy preparation, and low cost. Subsequently, amplifier units DMF and DMTD were successfully fabricated by connecting fuel strands and three-strand duplexes (TDs) to DMs, respectively. Finally, the MEDC was triggered by microRNA-155 (miR-155), which herein acted as a model analyte, resulting in dynamic self-assembly of poly-DNA micelles (PDMs) and causing the recovery of cyanine 3 (Cy3) fluorescence as the DMTD dissociated. Benefiting from the "diffusion effect", the MEDC herein had a nearly 2.9-fold increase in sensitivity and a nearly 97-fold reduction in detection limit compared to conventional EDC. This amplifier exhibited excellent sensitivity of microRNAs, such as miR-155 detection in a dynamic range from 0.05 to 4 nM with a detection limit of 3.1 pM, and demonstrated outstanding selectivity with the distinguishing ability of a single-base mismatched sequence of microRNAs. Overall, the proposed strategy demonstrated that this sequence-independent DNA nanostructure improved the performance of traditional DNA probes and provided a versatile method for the development of DNA nanotechnology in biosensing.
ABSTRACT
System leakage critically confines the development of cascade DNA systems that need to be implemented in a strict order-by-order manner. In principle, ternary DNA reactants, composed of three single-strand DNA (ssDNA) with a strict equimolar ratio (1:1:1), have been indispensable for successfully cascading upstream entropy-driven DNA circuit (EDC) with downstream circuits, and system leakage will occur with any unbalance of the molar ratio. In this work, we proposed "splitting-reconstruction" and "protection-release" strategies on the potential downstream circuit initiator derived from upstream EDC to guide the construction of EDC-involved cascade systems independent of system leakage derived from unpurified reactants. Both the reconstructed and released downstream circuit initiators were in compliance with the principle of the cascade AND logic gate. Using these two strategies, two cascade systemsâEDC2-4WJ-TMSDR and EDC3-HCRâwere developed to carry out the designed order, which did not require that the ratio of 1:1:1 be maintained. Furthermore, the inherent property of the upstream EDC could transfer into the downstream circuit, endowing the developed cascade systems with a more powerful signal amplification ability for the sensitive detection of the corresponding initiator strand. These two strategies may provide new insights into the process of constructing EDC-like circuit-involved high-order DNA networks.
Subject(s)
DNA, Single-Stranded , DNA , DNA/genetics , DNA, Single-Stranded/genetics , Entropy , LogicABSTRACT
Challenges remained in precisely real-time monitoring of apoptotic molecular events at the subcellular level. Herein, we developed a new type of intelligent DNA biocomputing nanodevices (iDBNs) that responded to mitochondrial microRNA-21 (miR-21) and microRNA-10b (miR-10b) simultaneously which were produced during cell apoptosis. By hybridizing two hairpins (H1 and H2) onto DNA nanospheres (DNSs) that had been previously modified with mitochondria-targeted triphenylphosphine (TPP) motifs, iDBNs were assembled in which two localized catalytic hairpins self-assembly (CHA) reactions occurred upon the co-stimulation of mitochondrial miR-21 and miR-10b to perform AND logic operations, outputting fluorescence resonance energy transfer (FRET) signals for sensitive intracellular imaging during cell apoptosis. Owing to the spatial confinement effects of DNSs, it was discovered that iDBNs had a high efficiency and speed of logic operations by high local concentrations of H1 and H2, making the simultaneous real-time responses of mitochondrial miR-21 and miR-10b reliable and sensitive during cell apoptosis. These results demonstrated that iDBNs were simultaneously responsive to multiple biomarkers, which greatly improved the detection accuracy to identify the cell apoptosis, demonstrating that iDBNs are highly effective and reliable for the diagnosis of major disease and screening of anticancer drugs.
Subject(s)
MicroRNAs , MicroRNAs/genetics , DNA , Apoptosis , BiomarkersABSTRACT
BACKGROUND: We aimed to evaluate whether extracellular vesicles (EV)-derived microRNAs (miRNAs) can be used as biomarkers for advanced adenoma (AA) and colorectal cancer (CRC). METHODS: We detected the changes in the plasma EV-delivered miRNA profiles in healthy donor (HD), AA patient, and I-II stage CRC patient groups using miRNA deep sequencing assay. We performed the TaqMan miRNA assay using 173 plasma samples (two independent cohorts) from HDs, AA patients, and CRC patients to identify the candidate miRNA(s). The accuracy of candidate miRNA(s) in diagnosing AA and CRC was determined using the area under the receiver-operating characteristic curve (AUC) values. Logistic regression analysis was performed to evaluate the association of candidate miRNA(s) as an independent factor for the diagnosis of AA and CRC. The role of candidate miRNA(s) in the malignant progression of CRC was explored using functional assays. RESULTS: We screened and identified four prospective EV-delivered miRNAs, including miR-185-5p, which were significantly upregulated or downregulated in AA vs. HD and CRC vs. AA groups. In two independent cohorts, miR-185-5p was the best potential biomarker with the AUCs of 0.737 (Cohort I) and 0.720 (Cohort II) for AA vs. HD diagnosis, 0.887 (Cohort I) and 0.803 (Cohort II) for CRC vs. HD diagnosis, and 0.700 (Cohort I) and 0.631 (Cohort II) for CRC vs. AA diagnosis. Finally, we demonstrated that the upregulated expression of miR-185-5p promoted the malignant progression of CRC. CONCLUSION: EV-delivered miR-185-5p in the plasma of patients is a promising diagnostic biomarker for colorectal AA and CRC. Trial registration The study protocol was approved by the Ethics Committee of Changzheng Hospital, Naval Medical University, China (Ethics No. 2022SL005, Registration No. of China Clinical Trial Registration Center: ChiCTR220061592).
Subject(s)
Adenoma , Colorectal Neoplasms , Extracellular Vesicles , MicroRNAs , Humans , Prospective Studies , MicroRNAs/genetics , Adenoma/diagnosis , Adenoma/genetics , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/geneticsABSTRACT
Monitoring tumor biomarkers is crucial for cancer diagnosis, progression monitoring, and treatment. However, identifying single or multiple biomarkers with the same spatial locations can cause false-positive feedback. Herein, we integrated the DNA tetrahedron (DT) structures with logic-responsive and signal amplifying capability to construct transmembrane DNA logic nanodevices (TDLNs) for the in situ sequential imaging of transmembrane glycoprotein mucin 1 (MUC1) and cytoplasmic microRNA-21 (miR-21) to cell identifications. The TDLNs were developed by encoding two metastable hairpin DNAs (namely, H1 and H2) in a DT scaffold, in which the triggering toeholds of H1 for miR-21 were sealed by the MUC1-specific aptamer (MUC1-apt). The TDLNs not only had the function of signal amplification owing to the localized catalytic hairpin assembly (CHA) reaction through spatial constraints effect of DT structures but also performed an AND logic operation to output a green Cy3 signal in MCF-7 cells, where MUC1 protein and miR-21 were simultaneously expressed. These results showed that the newly developed TDLNs have better molecular targeting and recognition ability so as to be easily identify cell types and diagnose cancer early.
Subject(s)
Biosensing Techniques , DNA, Catalytic , MicroRNAs , Neoplasms , Biosensing Techniques/methods , Catalysis , DNA/chemistry , DNA, Catalytic/metabolism , Humans , MCF-7 Cells , MicroRNAs/genetics , Nanotechnology , Neoplasms/diagnostic imagingABSTRACT
Carbon dots have promising prospects for analytical and monitoring purposes, but are greatly hindered by the aggregation-induced luminescence quenching owing to the π-π interaction or the non-radiation-excited radical complex formation. Herein hydrothermally prepared orange-yellow fluorescent carbon dots (O-CDs) show an aggregation-induced fluorescence enhancement (AIFE) with Cu2+ owing to the complexation of Cu(II) and the O-CDs. Cu2+ was then sensitively and selectively detected in the linear range from 0.02 to 30 µM with the detection limit of 14 nM, making the detection of Cu2+ in fresh water and E. coli lysate successful, showing that the as-prepared O-CDs could be well applied to the environmental monitoring of heavy metals.
Subject(s)
Carbon , Quantum Dots , Copper , Escherichia coli , Fluorescent Dyes , Spectrometry, FluorescenceABSTRACT
DNA logic nanodevices have prospects in molecular recognitions but still face challenges in achieving DNA computation-controlled regulation in specific compartments of living cells. By incorporating the i-motif sequence and ATP aptamers into a Y-shaped DNA (Y-DNA) structure, and applying gold nanoparticles (AuNPs) as the transporting carrier, herein we present a new type of DNA logic nanodevices to monitor the ATP levels in lysosomes of living cells. Triple energy transfers including dual fluorescent resonance energy transfers (FRETs) and a nanometal surface energy transfer (NSET) occurred in the DNA logic nanodevices. It was identified that the proposed nanodevices perform an AND logic operation to output FRET signals only when an endogenous proton and ATP simultaneously exist in the cellular microenvironment. Owing to the use of the i-motif sequence, the nanodevices have lysosome-recognizing capacity without causing alkalization of the acidic organelle, making DNA computation-controlled regulation at the level of cellular organelles achievable. These DNA logic nanodevices show high application prospects in lysosome-related cellular function and disease treatment.
Subject(s)
Gold , Metal Nanoparticles , Adenosine Triphosphate , DNA , Logic , LysosomesABSTRACT
MicroRNAs (miRNAs) are found in extremely low concentrations in cells, so highly sensitive quantitation is a great challenge. Herein, a simple dual-amplification strategy involving target-activated catalytic hairpin assembly (CHA) coupled with multiple fluorophores concentrated on one X-shaped DNA is reported. In this strategy, four hairpin probes (H1, H2, H3, and H4) are modified with FAM and BHQ1 at both sticky ends, while a circulating hairpin probe (H0) is used to activate CHA circuits once it binds to complementary sequences in the target miR-21 (T). The powerful dual-amplification cascades in Förster resonance energy transfer (FRET)-based nonenzymatic nucleic acid circuits are triggered by T-H0-activated formation of the X-shaped DNA nanostructure, freeing T-H0 for the next CHA reaction cycle. CHA circuits increase the fluorescence due to the wide distance between FAM and BHQ1 in the formed X-shaped DNA nanostructure, resulting in signal amplification and highly sensitive detection of miR-21, with a limit of detection (LOD, 3σ) of 0.025 nM, which is 25.6 or 57.6 times lower than that obtained through a single-amplification strategy without multiple fluorophores on one X-shaped DNA or CHA circuit. Furthermore, this cascade reaction was completed in 45 min, effectively avoiding target degradation. This new enzyme-free signal amplification strategy holds promising potential for sensitively detecting different DNA or RNA sequences by simply adapting the fragment of the H0 sequence complementary to the target.
Subject(s)
Biosensing Techniques , DNA, Catalytic , MicroRNAs , Nanostructures , DNA , Limit of DetectionABSTRACT
Interferon-inducible p200 family protein IFI204 was reported to be involved in DNA sensing, and subsequently induces the production of type I interferons and proinflammatory mediators. However, its function in the regulation of antiviral innate immune signaling pathway remains unclear. Here we reported a novel role of IFI204 that specifically inhibits the IRF7-mediated type I interferons response during viral infection. IFI204 and other p200 family proteins are highly expressed in mouse hepatitis coronavirus-infected bone marrow-derived dendritic cells. The abundant IFI204 could significantly interact with IRF7 in nucleus by its HIN domain and prevent the binding of IRF7 with its corresponding promoter. Moreover, other p200 family proteins that possess HIN domain could also inhibit the IRF7-mediated type I interferons. These results reveal that, besides the positive regulation function in type I interferon response at the early stage of DNA virus infection, the interferon-inducible p200 family proteins such as IFI204 could also negatively regulate the IRF7-mediated type I interferon response after RNA virus infection to avoid unnecessary host damage from hyper-inflammatory responses.
Subject(s)
Coronavirus Infections/immunology , Coronavirus/immunology , Interferon Regulatory Factor-7/metabolism , Interferon Type I/immunology , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , 3T3 Cells , A549 Cells , Animals , Cell Line , Coronavirus Infections/pathology , HEK293 Cells , Humans , Immunity, Innate/immunology , Inflammation/immunology , Inflammation/pathology , Interferon Regulatory Factor-7/genetics , Mice , RAW 264.7 CellsABSTRACT
The expression level of nucleic acids is closely related to a variety of diseases. Herein, a highly sensitive detection of a nucleic acid based on a CoOOH-luminol chemiluminescence (CL) system without the addition of oxidants was proposed by the toehold-mediated strand displacement reaction (TSDR) and the liposome dual signal amplification strategy with the hybrid probe formed by linking soft nanoballs (SNBs) to magnetic beads (MBs) through DNA hybridization. Inspired by the free radical scavenging effect of the as-prepared carbon dots (CDs), CDs were successfully employed to quench the CL intensity of the CoOOH-luminol system. And the CDs were further encapsulated into liposomes to construct SNBs, which avoided the complex modification of CDs to maintain their original properties, as well as loaded a large number of CDs to scavenge free radicals to achieve signal amplification. Based on this, target DNA (tDNA) could be sensitively detected based on the reduced CL intensity, which achieved a dynamic detection range from 0.1 nM to 20 nM with a limit of detection as low as 59 pM (3σ/k), showing amazing promise in the biosensing of nucleic acid biomarkers.
Subject(s)
Biosensing Techniques , Nucleic Acids , Biomarkers , Carbon , Limit of Detection , Luminescent Measurements , Nucleic Acid Hybridization , Reactive Oxygen SpeciesABSTRACT
DNA-based nanomachines have aroused tremendous interest because of their potential applications in bioimaging, biocomputing, and diagnostic treatment. Herein, we constructed a novel exonuclease III-propelled and signal-amplified stochastic DNA walker that autonomously walked on a spherical particle-based 3D track through a burnt-bridge mechanism, during which nanosurface energy transfer (NSET) occurred between the fluorescent dye modified on hairpin DNA and the surface of gold nanoparticles (AuNPs). As a proof of concept, this stochastic DNA walker achieves prominent detection performance of HIV DNA in the range of 0.05-1.2 nM with a detection limit of 12.7 pM and satisfactory recovery in blood serum, showing high promise in biosensing applications with complicated media.
Subject(s)
Biosensing Techniques , HIV Infections , Metal Nanoparticles , DNA/genetics , Energy Transfer , Exodeoxyribonucleases/metabolism , Gold , Humans , Limit of Detection , WalkersABSTRACT
OBJECTIVE: To determine the impact of smoking status in the prediction of stroke using CHADS2 and CHA2DS2-VASc schemes. METHODS: Five hundred twenty-eight consecutive patients with arrhythmic symptoms and without any documented arrhythmia from Queen Mary Hospital, Hong Kong, were followed up to determine the incidence of ischemic stroke, new-onset atrial fibrillation (AF), or all-cause mortality. Smoking status was classified into nonsmokers and smokers. The pairwise comparisons of C-statistics for outcomes were performed. RESULTS: During a median follow-up period of 6.2 years, 65 (12.3%) individuals developed ischemic stroke. Smokers experienced higher annual incidence of stroke, a new-onset AF, and all-cause death compare to nonsmokers, with corresponding hazard ratio (HR) of stroke, AF, and all-cause death being 2.51 (95% confidence intervals, CI 1.36als, CIse death bein 1.15a3.24), and 1.95 (95% CI 1.161.95 (95% CIath being 2.51 (95% confidence corr2 and CHA2DS2-VASc for stroke were 0.60 (95% CI 0.51 for stp = 0.09) and 0.59 (95% CI 0.50 (95%, p = 0.15) respectively, whereas the C-statistics of CHADS2 and CHA2DS2-VASc were 0.66 (95% CI 0.61 were 0p = 0.005), 0.75 (95% CI 0.7 CI 0.7p < 0.0001), respectively among nonsmokers. After incorporating smoking, both the CHADS2-smoking and CHA2DS2-VASc-smoking achieved better C-statistics for new-onset ischemic stroke prediction superior to baseline score systems in male groups. CONCLUSION: Cigarette smoking status has impact on stroke stratification using CHADS2 and CHA2DS2-VASc scheme. The discrimination of the CHADS2 and CHA2DS2-VASc scheme for stroke can be significantly improved if smoking status is additionally considered.
Subject(s)
Atrial Fibrillation , Cigarette Smoking , Stroke , Humans , Male , Risk Assessment , Risk Factors , Stroke/epidemiologyABSTRACT
Isothermal nucleic acid amplification technology has been widely adopted for analytical chemistry with the purpose of sensitivity improvement. Herein we present an ultrasensitive concatenated hybridization chain reaction (C-HCR) based surface-enhanced Raman scattering (SERS) immunoassay by forming antibody-antigen-aptamer heterosandwich structures with the model analyte of total prostate specific antigens (tPSA). In the C-HCR, two HCRs, one proceeds with two hairpins and the other with four biotin-modified hairpins, are coupled, making the formation of DNA nanofirecrackers with the lengths longer than 200 nm and more than four hundred million binding sites of streptavidin modified enzymes. These types of DNA nanofirecrackers through the aptamer encoded linker strand to form heterosandwich structures could provide a general signal application platform such as enzyme catalysis with high amplification efficiency. As a proof of concept, the Au@Ag core-shell nanostructure based SERS immunoassay with excellent signal amplification has been developed by employing the streptavidin modified alkaline phosphatase (SA-ALP) through its catalysis of 2-phospho-l-ascorbic acid trisodium salt (AAP) to form Au@Ag core-shell nanostructures via the formation of ascorbic acid (AA) to reduce AgNO3 and deposition of silver element on gold nanorods (AuNRs). The newly developed method has a detection limit as low as 0.94 fg/mL and has successfully achieved the detection of serum samples from clinical patients, which was consistent with the clinical test results, showing that this C-HCR strategy to form DNA nanofirecrackers has great potential in clinical applications.
Subject(s)
DNA/chemistry , Immunoassay/methods , Prostate-Specific Antigen/blood , Antigen-Antibody Complex/chemistry , Aptamers, Nucleotide/chemistry , DNA/metabolism , Gold/chemistry , Humans , Limit of Detection , Nanostructures/chemistry , Nanotubes/chemistry , Nucleic Acid Amplification Techniques , Nucleic Acid Hybridization , Prostate-Specific Antigen/analysis , Prostate-Specific Antigen/chemistry , Silver/chemistry , Spectrum Analysis, RamanABSTRACT
As an important biomarker for early diagnosis of cancers, sensitive detection and high-resolution imaging of microRNA-21 in cancer cells have become important and challengeable. In this work, highly sensitive detection and spatial imaging of intracellular microRNA-21 were realized by the reduced signal background through vertical polarization excitation with a polarizer. The lateral local surface plasmon resonance property of gold nanorods (AuNRs) displayed a pronounced green color with low scattering intensity, which was adjusted to red color with strong scattering intensity when the core-satellite gold nanoparticle (AuNP) assembly was constructed on the side of AuNRs through a catalyzed hairpin assembly (CHA) circuit in the presence of microRNA-21. This unique approach allows for effectively reducing the strong background signal to improve the sensitivity of detection. Additionally, the proposed strategy can not only realize the sensitive detection of microRNA-21 with the limit of detection as low as 2 pM (3σ) but also achieve the high spatial imaging of cancer cells, which provided a specific strategy for the construction and imaging of intracellular imaging probes. It is believed that the simple and sensitive approach on the basis of lateral local surface plasmon resonance property of anisotropic AuNRs with excellent sensitivity combined with high spatial imaging holds promising potentials to visualize intracellular microRNAs with low abundance.
Subject(s)
Biosensing Techniques , Gold/chemistry , Metal Nanoparticles/chemistry , MicroRNAs/analysis , Optical Imaging , Surface Plasmon Resonance , Anisotropy , Cells, Cultured , Hep G2 Cells , HumansABSTRACT
Fluorescent labeled single-stranded DNA (ssDNA) molecules physisorbed on graphene oxide (GO) have been extensively explored as a useful sensing platform. However, this approach faces challenges when applied to complex biological samples due to heavy nonspecific desorption of nontarget molecules from GO. To overcome this problem, we introduced a capture DNA (cDNA) fragment with a poly adenine (poly-A) extension into the physisorption system that greatly reduces nonspecific desorption and false positive signal due to strong binding between poly-A and GO. Fluorescence from the dye can be effectively quenched by BHQ, which thus provides a second guarantee of anti-interference to avoid possible nonspecific poly-A DNA displacement. As a proof of concept, we have successfully developed a novel DNA-adsorbing GO nanocomplex probe (DNA-GO nanocomplex probe). This probe has a high anti-interference capability and low background due to the presence of both GO and black hole quencher (BHQ) as a dual-quencher that reduces the background in live cell imaging due to resonance energy transfer (RET). We then employed the DNA-GO nanocomplex probe for simultaneous detection of miR-630 and miR-21 and also for simultaneous in situ dynamic monitoring of intracellular miR-630 and miR-21 in apoptotic cells. We discovered that miR-630 expression was up-regulated during the first 120 min. This simple but powerful protocol has great potential in precise detection and imaging of various substances in complex biological samples with improved accuracy.