Combining nanoflares biosensor and mathematical resolution technique for multi-class mycotoxin analysis in complex food matrices.

A multi-functional nanoflares biosensor of spherical gold nanoparticle (Au NP) modified by fluorophore-labeled oligonucleotides (ONS) was designed for ultra-sensitive multi-target mycotoxin analysis in food. Au NP was densely modified with multiplex highly oriented hairpins of oligonucleotides (ONS), each ONS was hybridized to a reporter with a distinct fluorophore label and specifically affiliative to its corresponding mycotoxin target. The fluorescent signals of reporters were pre-quenched by Au NP based on ONS hairpin structures and recovered when exposed to ONS's targets. Excitation-emission matrix (EEM) fluorescence detection was performed in EX and EM wavelength of 200-800 nm. Heavily overlapping spectra of fluorophores, mycotoxins and backgrounds were resolved by alternative trilinear decomposition (ATLD) algorithm, pure spectra of specific fluorophore responding to mycotoxin target can be extracted out for quantitative analysis. Four mycotoxins (Aflatoxin B1, zearalenone, Fumonisins B1, ochratoxin A) were simultaneously quantified at extremely low level with limit of detection <0.02 µg kg-1, the average recovery accuracies were higher than 91.7 % in various matrices of cereals, nuts, edible oils. This study realized an important breakthrough of the application of nanoflares biosensor and maybe promising to be as an alternative strategy for onsite mycotoxins monitoring of food.

Amplified FRET Nanoflares: An Endogenous mRNA-Powered Nanomachine for Intracellular MicroRNA Imaging.

It is of great value to detect biological molecules in live cells. However, probes for imaging low-abundance targets in live cells are limited by the one-to-one signal-triggered model. Here, we introduce the concept of the amplified FRET nanoflare, which employs high-abundance endogenous mRNA as fuel strands to amplify the detection of low abundance intracellular miRNA. As far as we know, this is the first report of an endogenous mRNA-powered nanomachine for intracellular molecular detection. We experimentally prove the mechanism of the nanomachine and demonstrate its specificity and sensitivity. The proposed amplified FRET nanoflare can act as an excellent intracellular molecular detection strategy that is promising for biological and medical applications.

Biomedical applications of nanoflares: Targeted intracellular fluorescence probes.

Nanoflares are intracellular probes consisting of oligonucleotides immobilized on various nanoparticles that can recognize intracellular nucleic acids or other analytes, thus releasing a fluorescent reporter dye. Single-stranded DNA (ssDNA) complementary to mRNA for a target gene is constructed containing a 3'-thiol for binding to gold nanoparticles. The ssDNA "recognition sequence" is prehybridized to a shorter DNA complement containing a fluorescent dye that is quenched. The functionalized gold nanoparticles are easily taken up into cells. When the ssDNA recognizes its complementary target, the fluorescent dye is released inside the cells. Different intracellular targets can be detected by nanoflares, such as mRNAs coding for genes over-expressed in cancer (epithelial-mesenchymal transition, oncogenes, thymidine kinase, telomerase, etc.), intracellular levels of ATP, pH values and inorganic ions can also be measured. Advantages include high transfection efficiency, enzymatic stability, good optical properties, biocompatibility, high selectivity and specificity. Multiplexed assays and FRET-based systems have been designed.

DNA-Coated Gold Nanoparticles for the Detection of mRNA in Live Hydra Vulgaris Animals.

Advances in nanoparticle design have led to the development of nanoparticulate systems that can sense intracellular molecules, alter cellular processes, and release drugs to specific targets in vitro. In this work, we demonstrate that oligonucleotide-coated gold nanoparticles are suitable for the detection of mRNA in live Hydra vulgaris, a model organism, without affecting the animal's integrity. We specifically focus on the detection of Hymyc1 mRNA, which is responsible for the regulation of the balance between stem cell self-renewal and differentiation. Myc deregulation is found in more than half of human cancers, thus the ability to detect in vivo related mRNAs through innovative fluorescent systems is of outmost interest.

Development of functionalized gold nanoparticles as nanoflare probes for rapid detection of classical swine fever virus.

Classical swine fever (CSF) is a devastating viral disease affecting pigs that causes major economic losses worldwide. Conventional assays to identify classical swine fever virus (CSFV) face challenges, such as the required molecular amplification of the target molecules via polymerase chain reaction (PCR). We designed a gold nanoflare probe to directly detect CSFV. Gold nanoparticles (AuNPs) were conjugated with a pair of complementary DNA sequences that specifically recognized and captured CSFV RNA, resulting in a fluorescence signal to indicate the existence of CSFV. The constructed nanocomposite was then utilized in a quantitative analysis to recognize the virus sequence present at amounts as low as 50 pg/µL. The CSFV-AuNP probe enabled real-time, quantitative detection of native CSFV in response to doses of the specific RNA sequence (CSFV NS2) that indicated active viral replication of CSFV Shimen in macrophages after 12, 24, and 48 h. The potential diagnostic applications of the probe were demonstrated by measuring CSFV without nucleic acid amplification in samples from seven types of tissue samples, specifically heart, spleen, kidney, liver, lymph, intestine, and muscle samples obtained from one pig confirmed to suffer CSF. The speed, sensitivity, and versatility of this CSFV-AuNP biosensor make it an ideal candidate for further application in the prevention and control of animal epidemic diseases.

Sensing of Vimentin mRNA in 2D and 3D Models of Wounded Skin Using DNA-Coated Gold Nanoparticles.

Wound healing is a highly complex biological process, which is accompanied by changes in cell phenotype, variations in protein expression, and the production of active biomolecules. Currently, the detection of proteins in cells is done by immunostaining where the proteins in fixed cells are detected by labeled antibodies. However, immunostaining cannot provide information about dynamic processes in living cells, within the whole tissue. Here, an easy method is presented to detect the transition of epithelial to mesenchymal cells during wound healing. The method employs DNA-coated gold nanoparticle fluorescent nanoprobes to sense the production of Vimentin mRNA expressed in mesenchymal cells. Fluorescence microscopy is used to achieve temporal detection of Vimentin mRNA in wounds. 3D light-sheet microscopy is utilized to observe the dynamic expression of Vimentin mRNA spatially around the wounded site in skin tissue. The use of DNA-gold nanoprobes to detect mRNA expression during wound healing opens up new possibilities for the study of real-time mechanisms in complex biological processes.

Two-Color-Based Nanoflares for Multiplexed MicroRNAs Imaging in Live Cells.

MicroRNAs (miRNAs) have become an ideal biomarker candidate for early diagnosis of diseases. But various diseases involve changes in the expression of different miRNAs. Therefore, multiplexed assay of miRNAs in live cells can provide critical information for our better understanding of their roles in cells and further validating of their function in clinical diagnoses. Simultaneous detection of multiple biomarkers could effectively improve the accuracy of early cancer diagnosis. Here, we develop the two-color-based nanoflares for simultaneously detecting two distinct miRNA targets inside live cells. The nanoflares consist of gold nanoparticles (AuNPs) functionalized with a dense shell of recognition sequences hybridized to two short fluorophore-labeled DNA molecules, termed "flares". In this conformation, the close proximity of the fluorophore to the AuNPs surface leads to quenching of the fluorescence. However, when target miRNAs bind to the recognition sequence, the concomitant displacement of the flare can be detected as a corresponding increase in fluorescence. The results demonstrate that the two-color-based nanoflares can simultaneously detect miR-21 and miR-141 expression levels in various live cancer cells successfully. Compared to the traditional single-color-based nanoflares, the two-color-based nanoflares could offer more reliable and practical information for cancer detection, improving the accuracy of early disease diagnosis.

Stress granules assembly affects detection of mRNA in living cells by the NanoFlares; an important aspect of the technology.

The recently announced new methodologies to detect mRNA molecules in single cells offer opportunities for research, medicine and molecular diagnostics. The NanoFlare RNA Detection Probes are tools for characterizing RNA content (not localization) using fluorescence-based approaches in living cells. Combined with flow cytometry, NanoFlares have expanded the available possibilities of quantitative analysis of mRNA level in a single cell. Herein we present that in some cases, the specific NanoFlare probes (SmartFlares) detect different amounts of mRNA compared to qPCR. Using the previously published model, in which we studied influence of BCR-ABL oncogene on BRCA1 mRNA translation, we found that the NanoFlare-mediated measurement of mRNA was affected by the assembly of stress granules, structures which store mRNA in complexes with RNA binding proteins. With the usage of chemical compounds we confirmed that under conditions supporting assembly of stress granules, the detection of mRNAs by these probes was decreased, whereas disassembly resulted in the increased mRNAs detection. Altogether, we showed that assembly of stress granules could interfere with mRNA accessibility to the NanoFlare RNA Detection Probes, indicating that the SmartFlares could recognize only the translationally active pool of mRNA, contrary to qPCR. This can significantly influence the quality of obtained data and should be taken into consideration while planning the analysis of mRNA markers using NanoFlares.

NanoFlares for the detection, isolation, and culture of live tumor cells from human blood.

Metastasis portends a poor prognosis for cancer patients. Primary tumor cells disseminate through the bloodstream before the appearance of detectable metastatic lesions. The analysis of cancer cells in blood­so-called circulating tumor cells (CTCs)­may provide unprecedented opportunities for metastatic risk assessment and investigation. NanoFlares are nanoconstructs that enable live-cell detection of intracellular mRNA. NanoFlares, when coupled with flow cytometry, can be used to fluorescently detect genetic markers of CTCs in the context of whole blood. They allow one to detect as few as 100 live cancer cells per mL of blood and subsequently culture those cells. This technique can also be used to detect CTCs in a murine model of metastatic breast cancer. As such, NanoFlares provide, to our knowledge, the first genetic-based approach for detecting, isolating, and characterizing live cancer cells from blood and may provide new opportunities for cancer diagnosis, prognosis, and personalized therapy.