Trigging stepwise-strand displacement amplification lights up numerous G-quadruplex for colorimetric signaling of serum microRNAs.

MicroRNAs (miRNAs) play an important biomarker in various biological processes, especially cancer related, yet economic, simple, sensitive and specific methods for miRNA determination are still challenging. In this study, we have developed stepwise-strand displacement amplification (S-SDA)-based colorimetric sensing platform for let-7a miRNA detection in clinical serum samples. Our results demonstrated that the developed S-SDA-based method shows high sensitivity with a detection limit of 63.2 pM and a naked eye detection limit of 0.1 nM. Moreover, the S-SDA amplifier is able to discriminate target miRNAs from their mutants with high accuracy and specificity. With its high sensitivity and selectivity, this method successfully identified healthy individuals from patients with colon cancer by detecting let-7a miRNAs in serum. We believe the colorimetric analysis method will provide a new paradigm for the detection of miRNA with different abundance and show great potential for clinical application in biomedical analysis and early clinical diagnosis.

Branch-Shaped Trapping Device Regulates Accelerated Catalyzed Hairpin Assembly and Its Application for MicroRNA In Situ Imaging.

Enzyme-free DNA strand displacement process is often practical when detecting miRNAs expressed at low levels in living cells. However, the poor kinetics, tedious reaction period, and multicomponent system hamper its in vivo applications to a great extent. Herein, we design a branch-shaped trapping device (BTD)-based spatial confinement reactor and applied it for accelerated miRNA in situ imaging. The reactor consists of a pair of trapped probe-based catalyzed hairpin assembly (T-CHA) reactions attached around the BTD. The trapping device naturally offered CHA reactions a good spatial-confinement effect by integrating the metastable probes (MHPa and MHPb) of the traditional CHA with the four-branched arm of BTD, which greatly improved the localized concentration of probes and shortened their physical distance. The autonomous and progressive walk of miRNA on the four-arm nanoprobes via T-CHA can rapidly tie numerous four-arm nanoprobes into figure-of-eight nanoknots (FENs), yielding strong fluorescence that is proportional to the miRNA expression level. The unique nanoarchitecture of the FEN also benefits the restricted freedom of movement (FOM) in a confined cellular environment, which makes the system ideally suitable for in situ imaging of intracellular miRNAs. In vitro and in situ analyses also demonstrated that the T-CHA overall outperformed the dissociative probe-based CHA (D-CHA) in stability, reaction speed, and amplification sensitivity. The final application of the T-CHA-based four-arm nanoprobe for imagings of both cancer cells and normal cells shows the potential of the platform for accurately and timely revealing miRNA in biological systems.

Intelligent assembly of Y-shaped DNA nanostructures for intracellular microRNA imaging.

Highly sensitive and specific imaging of low-level microRNAs (miRNAs) in cytoplasm is vital for early diagnosis of cancers. In this work, we have developed the amplification strategies for miRNA-155 detection based on the combination the nicked rolling circle amplification (N-RCA) and catalyzed hairpin assembly (CHA). In this system, the target miRNA-155 acts as a polymerase primer to activate N-RCA to produce nicked fragment1 (NF1) and NF2. NF1 acted as new primer could further initiate a new N-RCA reaction over and over. Then, the NF2s could serve as triggers to induce the CHA reaction, and the Y-shaped DNA nanostructure (Y-SDN) was formed. Thus, an amplified fluorescence signal was obtained based on the multiple amplification. Under the optimized experimental conditions, a high sensitivity with a detection limit as low as 1.8 pM at 3σ miRNA-155 and excellent specificity in buffer condition have been achieved by applying this method. Meanwhile, the proposed method enables the application in miRNA-155 detection in human serum. Moreover, we have shown that the method performs well for the intracellular miRNA-155 imaging in cellular environments. Therefore, the present strategy was expected to apply into the clinical disease diagnosis effectively.

miR-454-3p promotes of human glioma cell growth by targeting EGR3.

Gliomas are the most common primary brain tumors in adults and are associated with high mortality rates. In the present study, the aim was to evaluate the role of miR-454-3p in the pathogenesis of human glioma and to explore the underlying mechanism. Reverse transcription-quantitative PCR was performed to compare the expression levels of miR-454-3p in glioma and adjacent normal tissue. The effects of miR-454-3p on cell proliferation was tested by combining MTT and colony formation assays. Dual-luciferase assay was used to identify the target gene of miR-454-3p. The results showed that miR-454-3p was upregulated in glioma tissues where it exerts a positively regulatory role on cell growth. Dual-luciferase assay confirmed Early Growth Response 3 (EGR3) to be a target for miR-454-3p. Overexpression of EGR3 in glioma cells was found to impair miR-454-3p mimic-induced cell proliferation. These results suggested that upregulated miR-454-3p served an important role in glioma tumorigenesis by targeting EGR3, which provided valuable insights into the underlying mechanism of the disease that may lead to possible novel therapeutic strategies.

Capillary electrophoresis inductively coupled plasma mass spectrometry combined with metal tag for ultrasensitively determining trace saxitoxin in seafood.

As one of paralytic shellfish toxins, the saxitoxin (STX) in the aqueous environment can be accumulated by most shellfish, and thus harms human health through the food chain. Therefore, it is crucial to determine trace STX in seafood samples in order to ensure the safety of seafood consumption. In this study, we developed a novel indirect method for ultrasensitively determining trace STX in seafood by using CE-ICP-MS together with Eu3+ chelate labeling. We demonstrated that diethylenetriamine-N,N,N',N″,N″-pentaacetic acid (DTPA) can couple with STX and simultaneously chelate with Eu3+ to realize metallic labeling of STX, and thus realize the ultrasensitive quantification of trace STX with CE-ICP-MS. The proposed method has strong antiinterference ability, good stability, and extremely high sensitivity. It could be used to determine trace STX in seafood samples with an extremely low detection limit of 0.38 fmol (3.8×10-9 M, 100 nL sample injection) and a relative standard deviation (RSD, n = 5) <7%. The success of this study provides an alternative to precise quantification of ultra-trace STX in seafood samples, and further expands the application of ICP-MS.

Rolling circle amplification combined with gold nanoparticles-tag for ultra sensitive and specific quantification of DNA by inductively coupled plasma mass spectrometry.

A novel method for the ultra specific and sensitive detection of DNA in biological samples was described in this paper based on magnetic beads (MBs)-based rolling circle amplification combined with gold nano-particles (AuNPs)-aptamer labeling technique and inductively coupled plasma mass spectrometry (ICP-MS) detection. The proposed assay has an ultra-high sensitivity and stability, excellent specificity and more robust resistibility to the complex matrix due to the utilization of MBs-based rolling circle amplification, AuNPs-aptamer labeling technique and ICP-MS detection. It can be used to determine DNA or single nucleotide polymorphisms (SNP) with an extremely low detection limit of 0.1fM (1.0×10(-16)M) and a discrimination factor for single-base mismatch of 27. The proposed assay has been successfully used to detect DNA in serum sample with a recovery of 91-106% and a relative standard deviation (RSD) <6% (n=6), suggesting that our method is sensitive and reliable. The ultra-high sensitivity and specificity, easiness of fabrication, operational convenience, short analysis time, better stability and robust resistibility to the complex matrix, make the assay a promising alternative for the detection of various DNA sequence in the clinical diagnosis.

A novel phosphatidylserine-functionalized AuNP for the visual detection of free copper ions with high sensitivity and specificity.

In this paper, we have reported on novel phosphatidylserine-functionalized AuNPs (gold nanoparticles) for the visual detection of Cu2+ by employing phosphatidylserine for Cu2+ recognition and AuNPs for signal generation. The phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-l-serine, DOPS) was covalently assembled on the AuNP surface to obtain DOPS-functionalized AuNPs. It was demonstrated that DOPS-functionalized AuNPs could specifically bind Cu2+ and lead to the aggregation of AuNPs, which gave rise to a colour change from wine-red to blue and a new absorption band around 650 nm. This provides a sensing platform for the simple, rapid and field portable colorimetric detection of Cu2+. By using the sensing platform, a selective and sensitive visual biosensor for the detection of Cu2+ was developed. The proposed biosensor has outstanding analytical advantages such as good stability, relatively high sensitivity, low cost and short analysis time. It can be used to detect concentrations of Cu2+ as low as 30 µM in river water by observation with the naked eye and of 1.55 µM Cu2+ in river water by UV-visible spectrophotometry, within 10 min and with a recovery of 98-103% and a relative standard deviation (RSD) < 4% (n = 6). The proposed biosensor is promising for on-site detection of trace Cu2+ in clinical diagnosis or environmental monitoring.