Sensitive and specific CRISPR-Cas12a assisted nanopore with RPA for Monkeypox detection.

Monkeypox virus (MPXV) poses a global health emergency, necessitating rapid, simple, and accurate detection to manage its spread effectively. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique has emerged as a promising next-generation molecular diagnostic approach. Here, we developed a highly sensitive and specific CRISPR-Cas12a assisted nanopore (SCAN) with isothermal recombinase polymerase amplification (RPA) for MPXV detection. The RPA-SCAN method offers a sensitivity unachievable with unamplified SCAN while also addressing the obstacles of PCR-SCAN for point-of-care applications. We demonstrated that size-counting of single molecules enables analysis of reaction-time dependent distribution of the cleaved reporter. Our MPXV-specific RPA assay achieved a limit of detection (LoD) of 19 copies in a 50 µL reaction system. By integrating 2 µL of RPA amplifications into a 20 µL CRISPR reaction, we attained an overall LoD of 16 copies/µL (26.56 aM) of MPXV at a 95% confidence level using the SCAN sensor. We also verified the specificity of RPA-SCAN in distinguishing MPXV from cowpox virus with 100% accuracy. These findings suggest that the isothermal RPA-SCAN device is well-suited for highly sensitive and specific Monkeypox detection. Given its electronic nature and miniaturization potential, the RPA-SCAN system paves the way for diagnosing a wide array of other infectious pathogens at the point of care.

High performance inkjet printed embedded electrochemical sensors for monitoring hypoxia in a gut bilayer microfluidic chip.

Sensing devices have shown tremendous potential for monitoring state-of-the-art organ chip devices. However, challenges like miniaturization while maintaining higher performance, longer operating times for continuous monitoring, and fabrication complexities limit their use. Herein simple, low-cost, and solution-processible inkjet dispenser printing of embedded electrochemical sensors for dissolved oxygen (DO) and reactive oxygen species (ROS) is proposed for monitoring developmental (initially normoxia) and induced hypoxia in a custom-developed gut bilayer microfluidic chip platform for 6 days. The DO sensors showed a high sensitivity of 31.1 nA L mg-1 with a limit of detection (LOD) of 0.67 mg L-1 within the 0-9 mg L-1 range, whereas the ROS sensor had a higher sensitivity of 1.44 nA µm-1 with a limit of detection of 1.7 µm within the 0-300 µm range. The dynamics of the barrier tight junctions are quantified with the help of an in-house developed trans-epithelial-endothelial electrical impedance (TEEI) sensor. Immunofluorescence staining was used to evaluate the expressions of HIF-1α and tight junction protein (TJP) ZO-1. This platform can also be used to enhance bioavailability assays, drug transport studies under an oxygen-controlled environment, and even other barrier organ models, as well as for various applications like toxicity testing, disease modeling and drug screening.

Real-time monitoring of liver fibrosis through embedded sensors in a microphysiological system.

Hepatic fibrosis is a foreshadowing of future adverse events like liver cirrhosis, liver failure, and cancer. Hepatic stellate cell activation is the main event of liver fibrosis, which results in excessive extracellular matrix deposition and hepatic parenchyma's disintegration. Several biochemical and molecular assays have been introduced for in vitro study of the hepatic fibrosis progression. However, they do not forecast real-time events happening to the in vitro models. Trans-epithelial electrical resistance (TEER) is used in cell culture science to measure cell monolayer barrier integrity. Herein, we explored TEER measurement's utility for monitoring fibrosis development in a dynamic cell culture microphysiological system. Immortal HepG2 cells and fibroblasts were co-cultured, and transforming growth factor ß1 (TGF-ß1) was used as a fibrosis stimulus to create a liver fibrosis-on-chip model. A glass chip-based embedded TEER and reactive oxygen species (ROS) sensors were employed to gauge the effect of TGF-ß1 within the microphysiological system, which promotes a positive feedback response in fibrosis development. Furthermore, albumin, Urea, CYP450 measurements, and immunofluorescent microscopy were performed to correlate the following data with embedded sensors responses. We found that chip embedded electrochemical sensors could be used as a potential substitute for conventional end-point assays for studying fibrosis in microphysiological systems.

A fluorescent lateral flow biosensor for the quantitative detection of Vaspin using upconverting nanoparticles.

Vaspin is a protein present in human serum that can cause type-2 diabetes, obesity, and other cardiovascular diseases. We report fluorescent upconverting nanoparticles (UCNPs)-based lateral flow biosensor for ultrasensitive detection of Vaspin. A pair (primary and secondary) of cognate aptamers was used that has duo binding with Vaspin. UCNPs with a diameter of around 100 nm were used as a tag to label a detection probe (secondary aptamer). A primary aptamer (capture probe) was immobilized on the test zone. Sandwich type hybridization reactions among the conjugate probe, target Vaspin, and primary aptamer were performed on the lateral flow biosensor. In the presence of target Vaspin, UCNPs were captured on the test zone of the biosensor and the fluorescent intensity of the captured UCNPs was measured through a colorimetric app under NIR. Fluorescence intensity indicates the quantity of Vaspin present in the sample. A range of Vaspin concentration across 0.1-55 ng ml-1 with a Limit of detection (LOD) 39 pg ml-1 was tested through this UCNPs based LFSA with high sensitivity, reproducibility and repeatability, whereas it's actual range in human blood is from 0.1 to 7 ng ml-1. Therefore, this research provides a well-suited lateral flow strip with an ultrasensitive and low-cost approach for the early diagnosis of type-2 diabetes and this could be applied to any targets with a duo of aptamers generated.