A sample-to-answer electrochemical biosensor system for biomarker detection

We interfaced with a painless blood collection device and integrated on-chip blood-to-plasma separation with an electronic bead-based biomarker detection assay to enable true sample-to-answer detection of biomarkers.

Isoprocurcumenol Supports Keratinocyte Growth and Survival through Epidermal Growth Factor Receptor Activation

Although proliferation of keratinocytes, a major type of skin cells, is a key factor in maintaining the function of skin, their ability to proliferate tends to diminish with age. To solve such a problem, researchers in medical and skin cosmetic fields have tried to utilize epidermal growth factor (EGF), but achieved limited success. Therefore, a small natural compound that can mimic the activity of EGF is highly desired in both medical and cosmetic fields. Here, using the modified biosensor system, we observed that natural small-compound isoprocurcumenol, which is a terpenoid molecule derived from turmeric, can activate EGFR signaling. It increased the phosphorylation of ERK and AKT, and upregulated the expression of genes related to cell growth and proliferation, such as c-myc, c-jun, c-fos, and egr-1. In addition, isoprocurcumenol induced the proliferation of keratinocytes in both physical and UVB-induced cellular damage, indicative of its function in skin regeneration. These findings reveal that EGF-like isoprocurcumenol promotes the proliferation of keratinocytes and further suggest its potential as an ingredient for medical and cosmetics use.

A dual-reporter system for investigating and optimizing protein translation and folding in E. coli

Strategies for investigating and optimizing the expression and folding of proteins for biotechnological and pharmaceutical purposes are in high demand. Here, we describe a dual-reporter biosensor system that simultaneously assesses in vivo protein translation and protein folding, thereby enabling rapid screening of mutant libraries. We have validated the dual-reporter system on five different proteins and find an excellent correlation between reporter signals and the levels of protein expression and solubility of the proteins. We further demonstrate the applicability of the dual-reporter system as a screening assay for deep mutational scanning experiments. The system enables high throughput selection of protein variants with high expression levels and altered protein stability. Next generation sequencing analysis of the resulting libraries of protein variants show a good correlation between computationally predicted and experimentally determined protein stabilities. We furthermore show that the mutational experimental data obtained using this system may be useful for protein structure calculations.

Physical Surface Modification of Carbon-Nanotube/Polydimethylsiloxane Composite Electrodes for High-Sensitivity DNA Detection

The chemical modification of electrode surfaces has attracted significant attention for lowering the limit of detection or for improving the recognition of biomolecules; however, the chemical processes are complex, dangerous, and difficult to control. Therefore, instead of the chemical process, we physically modified the surface of carbon-nanotube/polydimethylsiloxane composite electrodes by dip coating them with functionalized multi-walled carbon nanotubes (F-MWCNTs). These electrodes are used as working electrodes in electrochemistry, where they act as a recognition layer for sequence-specific DNA sensing through π–π interactions. The F-MWCNT-modified electrodes showed a limit of detection of 19.9 fM, which was 1250 times lower than that of pristine carbon/polydimethylsiloxane electrodes in a previous study, with a broad linear range of 1–1000 pM. The physically modified electrode was very stable during the electrode regeneration process after DNA detection. Our method paves the way for utilizing physical modification to significantly lower the limit of detection of a biosensor system as an alternative to chemical processes.

Versatile Cell and Animal Models for Advanced Investigation of Lead Poisoning

The heavy metal, lead (Pb) can irreversibly damage the human nervous system. To help understand Pb-induced damage, we applied a genetically encoded Förster resonance energy transfer (FRET)-based Pb biosensor Met-lead 1.44 M1 to two living systems to monitor the concentration of Pb: induced pluripotent stem cell (iPSC)-derived cardiomyocytes as a semi-tissue platform and Drosophila melanogaster fruit flies as an in vivo animal model. Different FRET imaging modalities were used to obtain FRET signals, which represented the presence of Pb in the tested samples in different spatial dimensions. Using iPSC-derived cardiomyocytes, the relationship between beating activity (20–24 beats per minute, bpm) determined from the fluctuation of fluorescent signals and the concentrations of Pb represented by the FRET emission ratio values of Met-lead 1.44 M1 was revealed from simultaneous measurements. Pb (50 μM) affected the beating activity of cardiomyocytes, whereas two drugs that stop the entry of Pb differentially affected this beating activity: verapamil (2 μM) did not reverse the cessation of beating, whereas 2-APB (50 μM) partially restored this activity (16 bpm). The results clearly demonstrate the potential of this biosensor system as an anti-Pb drug screening application. In the Drosophila model, Pb was detected within the adult brain or larval central nervous system (Cha-gal4 > UAS-Met-lead 1.44 M1) using fast epifluorescence and high-resolution two-photon 3D FRET ratio image systems. The tissue-specific expression of Pb biosensors provides an excellent opportunity to explore the possible Pb-specific populations within living organisms. We believe that this integrated Pb biosensor system can be applied to the prevention of Pb poisoning and advanced research on Pb neurotoxicology.

Abstract 123: Study Of The Role Of Myosin-binding Protein C In The Structure-function Of Intact Cardiac Muscles Through The Use Of A Sarcomere Activation Biosensor

Cardiomyopathies affect as many as 1 in 500 adults, with hypertrophic cardiomyopathy (HCM) being the most commonly inherited heart disease. Although there are various genetic mutations which cause HCM, 40-50% of mutations identified in patients with this disease are found in the cardiac myosin-binding protein C (MyBP-C) gene. Thus, understanding this protein's role in sarcomere activation is critical for the development of effective therapeutic strategies. Previous work has identified MyBP-C as a key modulator of the sarcomere through inter-myofilament signaling. Although these studies have provided valuable information on the function of MyBP-C protein, the bulk of this work has been done with either in vitro protein recombination or permeabilized muscle. Unfortunately, these systems lack significant physiological features of muscle, such as load and intact excitation-contraction coupling mechanisms. Therefore, a large gap exists in the ability to monitor muscle sarcomere activation in an intact system in real time. In order to further advance the study of sarcomere activation in HCM disease models, specifically those targeting MyBP-C, a cardiac specific myofilament-targeted FRET based biosensor has been designed and validated. This new sarcomere activation biosensor reports real-time myofilament dynamics during physiological twitch contractions in live cardiac muscle. This allows for the in vivo biophysical tracking of FRET-detected conformation changes in TnC, which reflect the ensemble of regulatory events which lead to sarcomere activation. Currently, the biosensor is being utilized to investigate changes in sarcomere activation in MyBP-C knockout mice. We find that intact cardiac muscle under load shows slowed isometric twitch relaxation kinetics in MyBP-C knockout mice versus controls. In concert with thin and thick filament modifying small molecules, this biosensor system will be used to investigate sarcomere-based mechanisms of intact cardiac muscle performance in health and disease.

Versatile Cell and Animal Models for Advanced Investigation of Lead Poisoning

The heavy metal lead (Pb) can irreversibly damage the human nervous system. To help understand Pb-induced damage, we have developed practical applications for genetically encoded Pb biosensors in cardiac cells and insect central nervous tissue. We applied the optimized fluorescence resonance energy transfer (FRET)-based Pb biosensor Met-lead 1.44 M1 to two living systems to monitor the concentration of Pb: induced pluripotent stem cell (iPSC)-derived cardiomyocytes as a semi-tissue platform, and Drosophila melanogaster fruit flies as an in vivo animal model. Different FRET imaging modalities were used to obtain FRET signals, which repre-sented the presence of Pb in the tested samples in different spatial dimensions. Pb was effectively sensed in two living models producing Met-led 1.44 M1. In iPSC-derived cardiomyocytes, the relationship between beating rate determined from the fluctuation of fluorescent signals and the concentrations of Pb represented by the FRET emission ratio values of Met-lead 1.44 M1 demonstrated the potential of this fluorescence biosensor system for anti-Pb drug screening. In the Drosophila model, Pb was detected within the adult brain or larval central nervous system using fast epifluorescence and high-resolution two-photon 3D FRET ratio image sys-tems. The optimized Pb biosensor together with FRET microscopy can be used for specific applications to de-tect Pb with a limit of detection of 10 nM (2 ppb). We believe that this integrated Pb biosensor system can be applied to the prevention of Pb poisoning.