Highly sensitive non-stripping gold nanoparticles-based chemiluminescent detection of DNA hybridization coupled to magnetic beads

We have developed a new nanoparticle (NP)-based chemiluminescent (CL) method for the sensitive detection of DNA hybridization. The ''sandwich-type'' DNA technique comprises NH 2 -functionalized capture DNA immobilized on magnetic beads that can hybridize with one end of the target DNA. The other end of the target is recognized by a reporter DNA probe labeled with Au NPs on its 3-terminus. The hybridization events were monitored by a simple and direct luminol-AgNO 3 CL reaction with a detection limit of 0.5 pM through a non-stripping Au NPs catalysis. Moreover, the CL reaction of non-stripping Au NPs-luminol-AgNO 3 offers the advantages of being simpler and faster as compared to previous stripping Ag NPs and CuS methods. This simple and easy-to-use technique holds great promise for clinical applications of DNA hybridization

Effect of the Concentration Difference between Magnesium Ions and Total Ribonucleotide Triphosphates in Governing the Specificity of T7 RNA Polymerase-Based Rolling Circle Transcription for Quantitative Detection

T7 RNA polymerase-based rolling circle transcription (RCT) is a more powerful tool than universal runoff transcription and traditional DNA polymerase-based rolling circle amplification (RCA). However, RCT is rarely employed in quantitative detection due to its poor specificity for small single-stranded DNA (ssDNA), which can be transcribed efficiently by T7 RNA polymerase even without a promoter. Herein we show that the concentration difference between Mg²⁺ and total ribonucleotide triphosphates (rNTPs) radically governs the specificity of T7 RNA polymerase. Only when the total rNTP concentration is 9 mM greater than the Mg²⁺ concentration can T7 RNA polymerase transcribe ssDNA specifically and efficiently. This knowledge improves our traditional understanding of T7 RNA polymerase and makes convenient application of RCT in quantitative detection possible. Subsequently, an RCT-based label-free chemiluminescence method for microRNA detection was designed to test the capability of this sensing platform. Using this simple method, microRNA as low as 20 amol could be quantitatively detected. The results reveal that the developed sensing platform holds great potential for further applications in the quantitative detection of a variety of targets

xMAP Array Microspheres Based Stem–Loop Structured Probes as Conformational Switches for Multiplexing Detection of miRNAs

We have designed and evaluated novel stem-loop-structured probes for fluorescence detection of multiple microRNA (miRNA) targets. In the initial stage, the probes are in a closed stem conformation, shielding sterically a biotin label from being accessible to a fluorescence reporter. After hybridizing with target miRNAs, the probes undergo a conformational switch, restoring accessibility of the biotin to streptavidin–phycoerythin (SA–PE) for signal readout. Apparently, the bulky nature of the reporter SA–PE facilitates shielding of the biotin label in the absence of the target, thereby the stem–loop-structured probes allow sensitive detection of unlabeled miRNA targets, and xMAP array microspheres further realize simultaneous detection of multiple analytes using one fluorescence dye, SA–PE, for final readout. Here we demonstrated a successful multiplex assay for quantitative measurement of miRNA21, miRNA222, miRNA20a, and miRNA223, which are associated with nonsmall cell lung cancer. The approach can be extended to detecting an increasing number of targets for various indications. We believe such advancements represent a significant improvement for early disease diagnosis and prognosis

Bioluminescence Imaging of Carbon Monoxide in Living Cells and Nude Mice Based on Pd⁰‑Mediated Tsuji–Trost Reaction

Carbon monoxide (CO) is highly toxic and lethal to humans and animals because of its strong affinity for hemoglobin, while this “silent killer” is constantly generated in the body as a cell-signaling molecule of the gasotransmitter family in various pathological and physiological conditions. Up to now, designing fluorescent probes for real-time imaging of CO in living species is a continuous challenge due to background interference, light scattering, and photoactivation/photobleaching. Herein, a novel type of bioluminescence probe (allyl-luciferin) was synthesized and exploited to realize CO imaging with high signal-to-noise ratios. Based on Pd⁰-mediated Tsuji–Trost reaction, allyl-luciferin specifically reacted with CO to yield D-luciferin and thus generate a turn-on bioluminescence response, exhibiting high selectivity against bioactive small molecules such as reactive nitrogen, oxygen, and sulfur species. Furthermore, the new probe can be easily employed to detect exogenous CO in Huh7 cells and MDA-MB-231 cells, and CO production was enhanced greatly in these living cells after pretreatment with [Ru­(CO)₃Cl-(glycinate)] (CORM-3). Through the use of PdCl₂-containing liposomes to improve poor membrane permeability of PdCl₂, endogenous CO stimulated by heme was also seen clearly. In addition, the probe was successfully used to monitor exogenous and endogenous CO in nude mice. Overall, our data proved that the allyl-luciferin is a promising tool for exogenous and endogenous CO detection and imaging within living species. This is the first demonstration of bioluminescence imaging obtained by a probe for CO. We anticipate that the good imaging properties of allyl-luciferin presented in this study will provide a potentially powerful approach for illuminating CO functions in the future