An ultra-red fluorescent biosensor for highly sensitive and rapid detection of biliverdin.

The important role of BV in clinical diagnostics of liver-related diseases has been established in veterinary medicine. However, the sensitivity and selectivity of the current BV assays remain relatively low compromising its wider application in clinical diagnosis. Herein, we developed a rapid and sensitive BV-detecting biosensor based on a novel far-red fluorescent protein smURFP, which produced fluorescence only through specific interaction with its cofactor BV. In our study, the binding of BV to smURFP was then systematically optimized based on the structures of the smURFP + BV complex to increase the sensitivity of our biosensor. A wide linear range from 0 µM to 25 µM was obtained in both chicken and human serum. The limit of detection (LOD) and limit of quantification (LOQ) for BV was as low as 0.4 nM and 1.5 nM in human serum, and 0.4 nM and 1.2 nM in chicken serum. To our knowledge, this is the lowest LOD that has ever been reported for a BV biosensor. Our study sheds light on the biological and clinical analysis of BV.

A Rapid and Ultrasensitive Thrombin Biosensor Based on a Rationally Designed Trifunctional Protein.

Rapid and sensitive detection of thrombin is imperative for the early diagnosis, prevention, and treatment of thrombin-related diseases. Here, an ultrasensitive and rapid thrombin biosensor is developed based on rationally designed trifunctional protein HTs, comprising three functional units, including a far-red fluorescent protein smURFP, hydrophobin HGFI, and a thrombin cleavage site (TCS). smURFP is used as a detection signal to eliminate any interference from the autofluorescence of sample matrix to increase detection sensitivity. HGFI serve as an adhesive unit to allow rapid immobilization of HTs on a multiwall plate. The TCS linking HGFI and smURFP function as a sensing element to recognize and detect thrombin. HTs immobilization is symmetrically optimized and characterized. Thrombin assay reveals the specific recognition of active thrombin in samples and the hydrolysis of the immobilized HTs, resulting in a decrease in the fluorescence intensity of the sample in a thrombin concentration-dependent manner. The limit of detection (LOD) is as low as 0.2 am in the serum. To the authors' knowledge, this is the lowest LOD ever reported for any thrombin biosensor. This study sheds light on the engineering of multifunctional proteins for biosensing.

Hepatic Premalignant Alterations Triggered by Human Nephrotoxin Aristolochic Acid I in Canines.

Aristolochic acid I (AAI) existing in plant drugs from Aristolochia species is an environmental human carcinogen associated with urothelial cancer. Although gene association network analysis demonstrated gene expression profile changes in the liver of human TP53 knock-in mice after acute AAI exposure, to date, whether AAI causes hepatic tumorigenesis is still not confirmed. Here, we show that hepatic premalignant alterations appeared in canines after a 10-day AAI oral administration (3 mg/kg/day). We observed c-Myc oncoprotein and oncofetal RNA-binding protein Lin28B overexpressions accompanied by cancer progenitor-like cell formation in the liver by AAI exposure. Meanwhile, we found that forkhead box O1 (FOXO1) was robustly phosphorylated, thereby shuttling into the cytoplasm of hepatocytes. Furthermore, utilizing microarray and qRT-PCR analysis, we confirmed that microRNA expression significantly dysregulated in the liver treated with AAI. Among them, we particularly focused on the members in let-7 miRNAs and miR-23a clusters, the downstream of c-Myc and IL6 receptor (IL6R) signaling pathway linking the premalignant alteration. Strikingly, when IL6 was added in vitro, IL6R/NF-κB signaling activation contributed to the increase of FOXO1 phosphorylation by the let-7b inhibitor. Therefore, it highlights the new insight into the interplay of the network in hepatic tumorigenesis by AAI exposure, and also suggests that anti-premalignant therapy may be crucial for preventing AAI-induced hepatocarcinogenesis.