Fluorescent Indicators for Live-Cell and in Vitro Detection of Inorganic Cadmium Dynamics

Cadmium contamination is a severe threat to the environment and food safety. Thus, there is an urgent need to develop highly sensitive and selective cadmium detection tools. The engineered fluorescent indicator is a powerful tool for the rapid detection of inorganic cadmium in the environment. In this study, the development of yellow fluorescent indicators of cadmium chloride by inserting a fluorescent protein at different positions of the high cadmium-specific repressor and optimizing the flexible linker between the connection points is reported. These indicators provide a fast, sensitive, specific, high dynamic range, and real-time readout of cadmium ion dynamics in solution. Under optimal conditions, the fluorescent indicators N0C0/N1C1 showed a linear response to cadmium concentration within the range from 10/30 to 50/100 nM and with a detection limit of 10/33 nM. Escherichia coli cells containing the indicator were used to further study the response of cadmium ion concentration in living cells. E. coli N1C1 could respond to different concentrations of cadmium ions. This study provides a rapid and straightforward method for cadmium ion detection in vitro and the potential for biological imaging.

Biosensor-based assay of exosome biomarker for early diagnosis of cancer

Cancer imposes a severe threat to people’s health and lives, thus pressing a huge medical and economic burden on individuals and communities. Therefore, early diagnosis of cancer is indispensable in the timely prevention and effective treatment for patients. Exosome has recently become an attractive cancer biomarker in noninvasive early diagnosis because of the unique physiology and pathology functions, which reflects remarkable information regarding the cancer microenvironment, and plays an important role in the occurrence and evolution of cancer. Meanwhile, biosensors have gained great attention for the detection of exosomes due to their superior properties, such as convenient operation, real-time readout, high sensitivity, and remarkable specificity, suggesting promising biomedical applications in the early diagnosis of cancer. In this review, the latest advances of biosensors regarding the assay of exosomes were summarized, and the superiorities of exosomes as markers for the early diagnosis of cancer were evaluated. Moreover, the recent challenges and further opportunities of developing effective biosensors for the early diagnosis of cancer were discussed.

A Robust, Low-Cost Instrument for Real-time Colorimetric Isothermal Nucleic Acid Amplification

The COVID-19 pandemic has highlighted the need for broader access to molecular diagnostics. Colorimetric isothermal nucleic acid amplification assays enable simplified instrumentation over more conventional PCR diagnostic assays and, as such, represent a promising approach for addressing this need. In particular, colorimetric LAMP (loop-mediated isothermal amplification) has received a great deal of interest recently. However, there do not currently exist robust instruments for performing these kinds of assays in high throughput with real-time readout of amplification signals. To address this need, we developed LARI, the LAMP Assay Reader Instrument. We have deployed over 50 LARIs for routine use in R&D and production environments, with over 12,000 assays run to date. In this paper, we present the design and construction of LARI along with thermal, optical, and assay performance characteristics. LARI can be produced for under $1500 and has broad applications in R&D, point-of-care diagnostics, and global health.

Demonstration of a quantitative triplex LAMP assay with an improved probe-based readout for the detection of MRSA

A LAMP assay that simultaneously detects three MRSA genes within a single sample using a quantitative and real-time readout is designed and demonstrated.

Single-molecule assay for proteolytic susceptibility using centrifuge force microscopy:force-induced destabilization of collagen‘s triple helix

Force plays a key role in regulating dynamics of biomolecular structure and interactions, yet techniques are lacking to manipulate and continuously read out this response with high throughput. We present an enzymatic assay for force-dependent accessibility of structure that makes use of a wireless mini-radio centrifuge force microscope (MR.CFM) to provide a real-time readout of kinetics. The microscope is designed for ease of use, fits in a standard centrifuge bucket, and offers high-throughput, video-rate readout of individual proteolytic cleavage events. Proteolysis measurements on thousands of tethered collagen molecules show a load-enhanced trypsin sensitivity, indicating destabilization of the triple helix.

Real-time observation of light-controlled transcription in living cells

Gene expression is a tightly controlled process that is coordinated in space and time. To dissect its dynamic regulation with high temporal resolution, we introduce an optogenetic tool termed BLInCR (Blue Light-Induced Chromatin Recruitment) that combines rapid and reversible light-dependent recruitment of effector proteins with a real-time readout for transcription. We used BLInCR to control the activity of a reporter gene cluster in the human osteosarcoma cell line U2OS by reversibly recruiting the viral transactivator VP16. RNA production was detectable ∼2 minutes after VP16 recruitment and readily decreased when VP16 dissociated from the cluster in the absence of light. Quantitative assessment of the activation process revealed biphasic activation kinetics with a pronounced early phase in cells treated with the histone deacetylase inhibitor SAHA. Comparison with kinetic models for transcription activation suggests that the gene cluster undergoes a maturation process when activated. BLInCR will facilitate the study of transcription dynamics in living cells.