Genetically encoded cell-death indicators (GEDI) to detect an early irreversible commitment to neurodegeneration

Cell death is a critical process that occurs normally in health and disease. However, its study is limited due to available technologies that only detect very late stages in the process or specific death mechanisms. Here, we report the development of a family of fluorescent biosensors called genetically encoded death indicators (GEDIs). GEDIs specifically detect an intracellular Ca2+ level that cells achieve early in the cell death process and that marks a stage at which cells are irreversibly committed to die. The time-resolved nature of a GEDI delineates a binary demarcation of cell life and death in real time, reformulating the definition of cell death. We demonstrate that GEDIs acutely and accurately report death of rodent and human neurons in vitro, and show that GEDIs enable an automated imaging platform for single cell detection of neuronal death in vivo in zebrafish larvae. With a quantitative pseudo-ratiometric signal, GEDIs facilitate high-throughput analysis of cell death in time-lapse imaging analysis, providing the necessary resolution and scale to identify early factors leading to cell death in studies of neurodegeneration.

KappaBle fluorescent reporter mice enable dynamic and low-background single-cell detection of NF-kB activity in vivo

Nuclear factor-kB (NF-kB) is a transcription factor with a key role in a great variety of cellular processes from embryonic development to immunity, the outcome of which depends on the fine-tuning of NF-kB activity. The development of sensitive and faithful reporter systems to accurately monitor the activation status of this transcription factor is therefore desirable. To address this need, over the years a number of different approaches have been used to generate NF-kB reporter mice, which can be broadly subdivided into bioluminescence- and fluorescence-based systems. While the former enables whole-body visualization of the activation status of NF-kB, the latter have the potential to allow the analysis of NF-kB activity at single cell level. However, fluorescence-based reporters frequently show poor sensitivity and excessive background or are incompatible with high-throughput flow cytometric analysis. In this work we describe the generation and analysis of ROSA26 knockin NF-kB reporter (KappaBle) mice containing a destabilized EGFP, which showed sensitive, dynamic, and faithful monitoring of NF-kB activity at the single-cell level of various cell types during inflammatory and infectious diseases.

Single-cell detection of Nonsense-Mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that eliminates transcripts containing premature termination codons (PTCs). Half-lives of the mRNAs containing PTCs demonstrates that a small percent escape surveillance and do not degrade. It is not known whether this escape represents variable mRNA degradation within cells or, alternatively cells within the population are resistant. Here we demonstrate a single-cell approach with a bi-directional reporter, which expresses two b-globin genes with or without a PTC in the same cell, to characterize the efficiency of NMD in individual cells. We found a broad range of NMD efficiency in the population; some cells degraded essentially all of the mRNAs, while others escaped NMD almost completely. Characterization of NMD efficiency together with NMD regulators in single cells showed cell-to-cell variability of NMD reflects the differential level of surveillance factors, SMG1 and phosphorylated UPF1. A single-cell fluorescent reporter system that enabled detection of NMD using flow cytometry revealed that this escape occurred either by translational readthrough at the PTC or by failure of mRNA degradation after successful translation termination at the PTC.

Multiplexed and single-cell detection of microRNA with plasmonic nanoparticle assemblies

We present a label-free, low cost and miniatured biosensing platform based on the disassembly of core-satellite plasmonic nanoparticle assemblies. The rapid and selective detection of an exemplary nucleic acid biomarker, has-miRNA-210-3p, was achieved via the strand displacement nucleic acid reaction. Target binding leads to dehybridization of the DNA linkers and changes in the scattering properties of nanostructures as monitored by darkfield microscopy. We demonstrate the ability to detect microRNA expunged from single cells and the potential to multiplex discrete assemblies to enable diverse biological applicability. The work may help translate the applicability of microRNA as diagnostic biomarkers, quantitate their abundance in the microenvironment, and facilitate the study of their correlation or causation to other biomolecules at the single-cell level.

Graphene and its Derivatives-Based Optical Sensors

Being the first successfully prepared two-dimensional material, graphene has attracted extensive attention from researchers due to its excellent properties and extremely wide range of applications. In particular, graphene and its derivatives have displayed several ideal properties, including broadband light absorption, ability to quench fluorescence, excellent biocompatibility, and strong polarization-dependent effects, thus emerging as one of the most popular platforms for optical sensors. Graphene and its derivatives-based optical sensors have numerous advantages, such as high sensitivity, low-cost, fast response time, and small dimensions. In this review, recent developments in graphene and its derivatives-based optical sensors are summarized, covering aspects related to fluorescence, graphene-based substrates for surface-enhanced Raman scattering (SERS), optical fiber biological sensors, and other kinds of graphene-based optical sensors. Various sensing applications, such as single-cell detection, cancer diagnosis, protein, and DNA sensing, are introduced and discussed systematically. Finally, a summary and roadmap of current and future trends are presented in order to provide a prospect for the development of graphene and its derivatives-based optical sensors.

Carbon-based SERS biosensor: from substrate design to sensing and bioapplication

The sensing of bioactive molecules based on photochemical techniques has become one of the fastest-growing scientific fields. Surface-enhanced Raman scattering (SERS) is a highly sensitive technique for the detection of low-concentration molecules, including DNA, microRNA, proteins, blood, and bacteria; single-cell detection and identification; bioimaging; and disease diagnosis, providing abundant structural information for biological analytes. One rapidly developing field of SERS biosensor design is the use of carbon-based nanomaterials as substrate materials, such as zero-dimensional carbon quantum dots, one-dimensional carbon nanotubes, two-dimensional graphene, and graphene oxide (GO) and three-dimensional spatial carbon nanomaterials or carbon-based core-shell nanostructures. In this review, we describe the recent developments in SERS biosensors, in particular carbon-based SERS, for the detection of bioactive molecules. We systematically survey recent developments in carbon nanomaterial-based SERS biosensors, focusing on fundamental principles for carbon-based materials for SERS biosensor design, fabrication, and operation, and provide insights into their rapidly growing future potential in the fields of biomedical and biological engineering, in situ analysis, quantitative analysis, and flexible photoelectric functional materials. As such, this review can play the role of a roadmap to guide researchers toward concepts that can be used in the design of next-generation SERS biosensors while also highlighting current advancements in this field.

Single cell detection using intracellularly-grown-Au-nanoparticles based surface-enhanced Raman scattering spectroscopy for nasopharyngeal cell lines classification

The aim of this study was to evaluate the feasibility of applying intracellularly-grown-Au-nanoparticles (IGAuNPs)-based surface-enhanced Raman scattering (SERS) technology to classify two types of nasopharyngeal cancer (NPC) cell lines (CNE2...

High throughput single-cell detection of multiplex CRISPR-edited gene modifications

CRISPR-Cas9 gene editing has transformed our ability to rapidly interrogate the functional impact of somatic mutations in human cancers. Droplet-based technology enables the analysis of Cas9-introduced gene edits in thousands of single cells. Using this technology, we analyze Ba/F3 cells engineered to express single or multiplexed loss-of-function mutations recurrent in chronic lymphocytic leukemia. Our approach reliably quantifies mutational co-occurrences, zygosity status, and the occurrence of Cas9 edits at single-cell resolution.