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The CRISPR/Cas sensors represent a new approach in molecular diagnostics. Based on the programmable trans-cleavage of Type V & VI Cas proteins to degrade single-strand DNA/RNA reporters they detect nucleic acids with single-nucleotide specificity. However, their clinical usefulness is limited by their pM level sensitivity, which is insufficient unless integrated with preamplification. To overcome this limitation, we designed a bifunctional circular DNA amplifier to report nucleic acid detection events and simultaneously facilitate an autocatalytic reaction network. This minimalistic design converts a classic CRISPR/Cas sensor into a DNA amplifier-enhanced CRISPR/Cas autocatalytic sensor (DANCER), without the need for additional amplification reaction or device. DANCER achieved 1 aM level (6 times improvement), within 15 mins, at room temperature, and with 11 orders of magnitude detection range. When combined with a colorimetric lateral flow assay, DANCER realized point-of-care testing of circulating tumor DNA in plasma of a colorectal cancer-mouse.

Mammalian oocytes develop and mature in a mutually dependent relationship with surrounding cumulus cells. The oocyte actively regulates cumulus cell differentiation and function by secreting soluble paracrine oocyte-secreted factors (OSFs). We characterized the molecular mechanisms by which two model OSFs, cumulin and BMP15, regulate oocyte maturation and cumulus-oocyte cooperativity. Exposure to these OSFs during maturation altered the proteomic and multispectral autofluorescence profiles of both the oocyte and cumulus cells. In oocytes, cumulin significantly upregulated proteins involved in nuclear function. In cumulus cells, both OSFs elicited marked upregulation of a variety of metabolic processes (mostly anabolic), including lipid, nucleotide, and carbohydrate metabolism, while mitochondrial metabolic processes were downregulated. The mitochondrial changes were validated by functional assays confirming altered mitochondrial morphology, respiration, and content, whilst maintaining ATP homeostasis. Collectively, these data demonstrate that OSFs remodel cumulus cell metabolism during oocyte maturation in preparation for ensuing fertilization and embryonic development.HIGHLIGHTSDuring oocyte maturation, oocyte-secreted factors promote cell cooperativity between the oocyte and cumulus cells by altering the molecular composition of both cell types.Oocyte-secreted factors downregulate protein catabolic processes, and upregulate DNA binding, translation, and ribosome assembly in oocytes.Oocyte-secreted factors alter mitochondrial number, morphology, and function in cumulus cells.Oocyte-secreted factors further enhance metabolic plasticity in cumulus cells by upregulating anabolic pathways for macromolecules and small molecule organics.The oocyte, via oocyte-secreted factors, instructs cumulus cells to increase metabolic workload on its behalf, thereby subduing oocyte metabolism.

AimTo evaluate if suitably designed computed tomography (CT) radiomic signatures are sensitive to tumour transformation, and able to predict disease free survival (DFS) and overall survival (OS) time in patients with pancreatic cancer.MethodEthical approval by UNSW review board was obtained for this retrospective analysis. This study consisted of 27 patients with pancreatic cancer. Unsupervised principal component analysis was employed to evaluate the sensitivity of radiomic signatures to cancer presence and treatment. Further, optimised radiomic signatures were discovered using swarm intelligence and assessed for their capability to predict DFS and OS based on Kaplan-Meier analysis and receiver-operator characteristics (ROC) curves.ResultsWe found that appropriate two radiomic signature are sensitive to cancer presence (area under the curve, AUC=0.95) and cancer treatment, respectively. Two other optimized radiomics signatures showed significant correlations with DFS and OS, respectively (pConclusionThe CT radiomics signatures are an independent biomarkers which are modified when cancer is present and can help to estimate DFS and OS in patients. These signatures have the potential to be used to support clinical decision-making in pancreatic cancer treatment.

Tracking trace amounts of analytes directly from low volumes of complex biological samples remains an ongoing challenge in precision diagnostics, as the commonly used immunosorbent assays have limited sensitivity. Herein, a CRISPR/Cas12a assisted on-fibre immunosensor (CAFI) was developed based on an antibody-analyte-aptamer sandwich structure, in which a single strand DNA aptamer was applied to detect the analyte while triggering the CRISPR/Cas12a fluorescent detection system to amplify the analyte signal. This novel CAFI biosensing system was fabricated on a glass fibre surface with an antifouling PEG polymer brush modified for the detection of a spectrum of small molecules from complex media. In comparison with a conventional ELISA system, CAFI has a 1,000-fold higher sensitivity with the limit of detection for IFN-γ down to 1 fg mL

Molecular beam epitaxy-grown GaN with different Mn concentrations (5-23×1019 cm-3) and codoped with Si were investigated by cathodoluminescence (CL) spectroscopy and optical transmission measurements. In the GaN:Mn, an intense absorption peak at 1.414 +/- 0.002 eV was observed. This peak was attributed to an internal 5T 2→ 5E transition of the deep neutral Mn3+ state since its intensity scaled with the Mn3+ concentration. The CL measurements showed that Mn-doping concentrations around 1020 cm -3 had three effects on the emission spectrum: (i) the donor bound exciton at 3.460 eV was reduced by more than one order of magnitude, (ii) the donor-acceptor-pair band at 3.27 eV was completely quenched and (iii) the yellow luminescence centered at 2.2 eV was the strongly decreased. The latter two effects were attributed to a reduced concentration of VGa. In the infrared spectral range, three broad, Mn-doping related CL emission bands centered at 1.01 ± 0.02 eV, 1.09 ± 0.02 eV and 1.25 ± 0.03 eV were observed. These bands might be related to deep donor complexes, which are generated as a result of the heavy Mn-doping, rather than internal transitions at the Mn atom.

Moderately and heavily Mg-doped GaN were studied by a combination of post-growth annealing processes and electron beam irradiation techniques during cathodoluminescence (CL) to elucidate the chemical origin of the recombination centers responsible for the main optical emission lines. The shallow donor at 20-30 meV below the conduction band, which is involved in the donor-acceptor-pair (DAP) emission at 3.27 eV, was attributed to a hydrogen-related center, presumably a (VN-H) complex. Due to the small dissociation energy (

Over the last two decades, fluorescence-based detection has become one of the leading sensing technologies in biomedical, biological, and related sciences. Its sensitivity makes it possible to detect a single biomolecule through labelling with a suitable fluorophore. Two principal fluorophore properties, brightness and photostability, are fundamentally important to achieve a high level of sensitivity, and in many conventional fluorophores these often fall short of the requirements.. Among the methods used to improve the sensitivity of fluorescence detection, the metal-enhanced fluorescence (MEF) technique has been recently actively developed. The MEF phenomenon occurs when an excited fluorophore is located in close proximity to metals, and it is particularly pronounced near noble metal nanostructures.. Electrons in such metal nanostructures exhibit strong resonances often located in the visible part of the spectrum (also known as surface plasmon resonance). They can interact with proximal fluorophores, modifying their optical properties and producing increased quantum yield (fluorescence efficiency) and improved photostability. It has been experimentally demonstrated that the MEF technique can increase fluorescence intensity up to several hundreds times. This chapter provides an overview of MEF, covering its basic theory, synthesis of metal nanostructures, and biological applications based on MEF. We focus on silver nanostructures because their strong surface plasmon resonance in the visible matches the absorption and emission bands of most fluorophores. We discuss traditional planar MEF substrates as well as silver nanostructures deposited on micrometre-sized silica beads, generating a fluorescence enhancement of more than one order of magnitude. This achievement allowed us to demonstrate for the first time MEF immunoassays on silica beads by using high-throughput flow cytometry. Furthermore, we discovered that these silver nanostructure-coated silica beads are able to modify the luminescence decay lifetime of lanthanide fluorophores for time-gated luminescence bioimaging applications. These developments open up a broad range of opportunities for ultrasensitive and low-background fluorescence detection using lanthanide fluorophores. © 2011 by Pan Stanford Publishing Pte. Ltd. All rights reserved.