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A novel single-molecular fluorescent probe was developed for spatiotemporal matrix metalloproteinase-2 (MMP-2) and caspase-3 imaging with distinct fluorescence signals. Due to the multi-Förster resonance energy transfer (FRET) processes, the probe could respond to MMP-2 and caspase-3 independently with high signal-to-noise ratio. Moreover, the overexpression of MMP-2 in cancer cell lines and the cisplatin induced cell apoptosis were spatiotemporal imaged with distinct fluorescence emissions. Because of the independent process of the probe for MMP-2 and caspase-3 imaging, the probe could meet the demands for precise disease diagnosis and cancer theranostic applications, which could extensively simplify the processes for precise cancer diagnosis and imaging. [ABSTRACT FROM AUTHOR]

Point-of-care (POC) testing has become widely used in clinical analysis because of its speed and portability; however, POC tools, such as lateral flow assays, suffer from low specificity, unclear readouts, and susceptibility to environmental and user errors. Herein, we report an ELISA-based competitive volumetric bar-chart chip (CV-chip) that eliminates these limitations. The CV-chip displays the readout in the form of ink bar charts based on direct competition between gases generated by the sample and the internal control. By employing a "competition mode", this platform decreases the potential influence of background resulting from environmental factors and provides visually clear positive or negative results without the requirement of calibration. In addition, the on-chip comparison enables the device to distinguish imperceptible differences (less than 1.3-fold) in human chorionic gonadotropin (hCG) concentrations that are near the cutoff value for pregnancy (∼1.4 ng/mL). We also utilized the ELISA-based CV-chip to successfully detect biomarkers from cancer cells. As a proof-of-concept application in a clinical setting, the CV-chip was employed to evaluate the status of drugs of abuse in 18 patients. For six different drugs, zerofalse-positive and very few false-negative (<2%) results were reported in more than 100 tests. This new ELISA platform offers a clinical diagnostics tool that is portable and easy to use, and provides improved clarity and sensitivity due to the inclusion of a real-time internal control. [ABSTRACT FROM AUTHOR]

The folding of G-quadruplex is hypothesized to undergo a complex process, from the formation of a hairpin structure to a triplex intermediate and to the final G-quadruplex. Currently, no experimental evidence has been found for the hairpin formation, because it folds in the time regime of 10-100 μs, entailing the development of microfluidic mixers with a mixing time of less than 10 fts. In this paper, we reported an ultrarapid micromixer with a mixing time of S.5 μs, which represents the fastest turbulent micromixer to our best knowledge. Evaluations of the micromixer were conducted to confirm its mixing efficiency for small molecules and macromolecules. This new micromixer enabled us to interrogate the hairpin formation in the early folding process of human telomere G-quadruplex The experimental kinetic evidence for the formation of hairpin was obtained for the first time. [ABSTRACT FROM AUTHOR]

Tumor-derived miRNAs in serum are emerging as the new noninvasive biomarkers for the diagnosis of human cancers, especially at their early stage. An ideal method with high sensitivity, excellent selectivity, a simple procedure, and small amounts of starting materials is imperative for the detection of clinic circulating miRNAs. Here, we develop a new method for isothermally sensitive detection of serum miRNAs using hairpin probe-based rolling circle amplification (HP-RCA). This method exhibits ultrahigh sensitivity toward lung cancer-related miR-486-5p with a detection limit of as low as 10 fM and a large dynamic range of 6 orders of magnitude, and it can even discriminate miR-486-5p from both miRNAs with high sequence homology and its precursors (pre-miRNAs). More importantly, this method can directly and accurately distinguish the expression of serum miR-486-5p among six nonsmall-cell lung carcinoma (NSCLC) patients and six healthy persons, holding a great potential for further applications in the clinical diagnosis of lung cancers. [ABSTRACT FROM AUTHOR]

Tracking the folding kinetics of macromolecules under molecular crowding conditions represents a tremendous challenge due to the high viscosity of the solution. In this paper, we report a unique T-type microfluidic mixer with seven consecutive ω-shaped baffles for fast mixing of high-viscosity fluids. Numerical simulations and experimental characterizations proved that the micromixer could achieve a mixing time of 579.4 μs for solutions with viscosities about 33.6 times that of pure water. Over a 1000-fold improvement in mixing dead time was accomplished in comparison to those reported previously. We further used this highly efficient micromixer to track the early folding kinetics of human telomere G-quadruplex under molecular crowding conditions. Results indicated an exponential process in the initial folding phase of G-quadruplex, and the G-quadruplex formed a more compact structure under higher degrees of molecular crowding conditions. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are important regulators of gene expression that control almost every physiological and pathological process. Although the complementarity between the seed region of a miRNA and its target mRNA is usually deemed as the key determinant in the miRNA-target recognition in animals, the mechanism of their recognition still remains enigmatic as more and more exceptions challenge the seed rule. Herein, we employ single-molecule fluorescence resonance energy transfer (smFRET) to investigate human miRNA-induced silencing complex (miRISC)-involved miRNA-target recognition with either perfect base pairing or poor seed match in real time. Our results demonstrate that the recognition between mammalian mIRNA and its target with perfect base pairing proceeds in a two-state model as prokaryotic guide DNA-mediated recognition, suggesting a conserved pattern of guide RNA/DNA strand recognition. In addition to the general rule of mIRNA-target recognition, our results reveal that annealing between miRNA and its target with poor seed match proceeds in a stepwise way, which is in accordance with the increase in the number of conformational states of miRNA-target duplex accommodated by the miRJSC, suggesting the structural plasticity of human miRISC to conciliate the mismatches m seed region. This new dynamic information revealed by smPRET has an important implication for comprehensive understanding of the role of m1RJSC in the target recognition in mammals. [ABSTRACT FROM AUTHOR]

Liver cancer is the third most deadly cancers in the world. Unfortunately, there is no effective treatment. One of the major problems is that most cancers are diagnosed in the later stage, when surgical resection is not feasible. Thus, accurate early diagnosis would significantly improve the clinical outcome of liver cancer. Currently, there are no effective molecular probes to recognize biomarkers that are specffic for liver cancer. The objective of our current study is to identify liver cancer cell-specific molecular probes that could be used for liver cancer recognition and diagnosis. We applied a newly developed cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment) method for the generation of molecular probes for specific recognition of liver cancer cells. The cell-SELEX uses whole live cells as targets to select aptamers (designed DNIVRNA) for cell recognition. In generating aptamers for liver cancer recognition, two liver cell lines were used: a liver cancer cell line BNL 1ME A.7R. 1 (MEAR) and a noncancer cell line, BNL CL2 (BNL). Both cell lines were originally derived from Balb/cJ mice. Through multiple rounds of selection using BNL as a control, we have identified a panel of aptamers that specifically recognize the cancer cell line MEAR with Kd in the nanomolar range. We have also demonstrated that some of the selective aptamers could specifically bind liver cancer cells in a mouse model. There are two major new results (compared with our reported cell-SELEX methodology) in addition to the generation of aptamers specifically for liver cancer. The first one is that our current study demonstrates that cell-based aptamer selection can select specific aptamers for multiple cell lines, even for two cell lines with minor differences (MEAR cell is derived from BNL by chemical inducement); and the second result is that cell-SELEX can be used for adhesive cells and thus open the door for solid tumor selection and investigation. The newly generated cancer-specific aptamers hold great promise as molecular probes for cancer early diagnosis and basic mechanism studies. [ABSTRACT FROM AUTHOR]

In this paper, we describe a new way to generate molecular probes for specific recognition of cancer cells. Molecular medicine will require a large number of probes for molecular recognition and characterization of a variety of diseased cells. Aptamers, single-stranded DNA/RNA probes, are poised to become a chemist's antibody and have the potential to serve as molecular probes for a variety of biomedical applications. By applying newly developed cell-SELEX (cell-based systematic evolution of ligands by exponential enrichment) against whole living cells, panels of aptamers have been evolved from an initial DNA library to characterize target cells at the molecular level. Ramos cells, a B-cell lymphoma cell line, were used as target cells for the generation of effective molecular probes. By taking advantages of the repetitive and broad enrichment strategy, the selected aptamers could bind to target cells and other closely related cell lines in variant patterns with an equilibrium dissociation constant (Kd) in the nanomolar range. Some aptamers could also specifically recognize the target lymphoma cells mixed with normal human bone marrow aspirates. The cell-based SELEX is simple, fast, and robust. The strategies used here will be highly useful for aptamer selection against complex target samples in order to generate a large number of aptamers in a variety of biomedical and biotechnological applications, paving the way for molecular diagnosis, therapy, and biomarker discovery. [ABSTRACT FROM AUTHOR]