Search

Fluorescence in situ hybridization (FISH) is the gold standard for visualizing genomic DNA in fixed cells and tissues, but it is incompatible with live-cell imaging, and its combination with RNA imaging is challenging. Consequently, due to its capacity to bind double-stranded DNA (dsDNA) and design flexibility, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) technology has sparked enormous interest over the past decade. In this review, we describe various nucleic acid (NA)- and protein-based (amplified) signal generation methods that achieve imaging of repetitive and single-copy sequences, and even single-nucleotide variants (SNVs), next to highly multiplexed as well as dynamic imaging in live cells. With future progress in the field, the CRISPR-(d)Cas9-based technology promises to break through as a next-generation cell-imaging technique. Adoption of clustered regularly interspaced short palindromic repeats (CRISPR)-based imaging requires consideration of (amplified) signal generation complexity, an appropriate cell delivery mechanism, and the capacity for single-copy sequence imaging. Single-copy sequence imaging is achieved both with and without signal amplification, whereas SNP or single-nucleotide variant (SNV) detection requires signal amplification. CRISPR-based imaging with specificity at the SNV and SNP levels was achieved in both live and fixed cells. The versatility of paired RNA aptamer-based signal generation enables highly multiplexed live-cell imaging of six repetitive sequences. CRISPR-based imaging was used in a clinical setting, allowing the detection of Patau syndrome (in the form of Chr13 trisomy) in patient-derived cells. [ABSTRACT FROM AUTHOR]

Disease treatment with advanced biological therapies such as adalimumab (ADM), although largely beneficial, is still costly and suffers from loss of response. To tackle these aspects, therapeutic drug monitoring (TDM) is proposed to improve treatment dosing and efficacy, but is often associated with long sampling-to-result workflows. Here, we present an in-house constructed ADM-sensor, allowing TDM of ADM at the doctor's office. This biosensor brings fiber optic surface plasmon resonance (FO-SPR), combined with self-powered microfluidics, to a point of care (POC) setting for the first time. After developing a rapid FO-SPR sandwich bioassay for ADM detection on a commercial FO-SPR device, this bioassay was implemented on the fully-integrated ADM-sensor. For the latter, we combined (I) a gold coated fiber optic (FO) probe for bioassay implementation and (II) an FO-SPR readout system with (III) the self-powered iSIMPLE microfluidic technology empowering plasma sample and reagent mixing on the-cartridge as well as connection to the FO-SPR readout system. With a calculated limit of detection (LOD) of 0.35 μg/mL in undiluted plasma, and a total time-to-result (TTR) within 12 min, this innovative biosensor demonstrated a comparable performance to existing POC biosensors for ADM quantification in patient plasma samples, while requiring only 1 μL of plasma. Whereas this study demonstrates great potential for FO-SPR biosensing at the POC using ADM as a model case, it also shows huge potential for bedside TDM of other drugs (e.g. other immunosuppressants, anti-epileptics and antibiotics), as the bioassay is highly amenable to adaptation. • A FO-SPR biosensor was combined with self-powered iSIMPLE technology. • The FO-SPR one-step sandwich bioassay was established for rapid adalimumab detection. • Reagent mixing on the microfluidic cartridge was realized by an optimized mixing design. • Plasma sample dilution was integrated on self-powered iSIMPLE microfluidic cartridge. • The ADM-sensor delivered measurement using only 1 μL of plasma within 12 min. [ABSTRACT FROM AUTHOR]

Therapeutic drug monitoring (TDM) of adalimumab (ADM) at the point-of-care (POC) is key to prevent loss of response but has not been accomplished to date because true POC testing solutions are still lacking. Here, we present a novel "whole blood in – result out" self-powered microfluidic chip for detecting ADM within 30 min to enable TDM at POC. Hereto, we first demonstrated on-chip plasma separation from whole blood, followed by downscaling an ADM ELISA with maintained specificity and sensitivity in plasma. This assay was then performed on a robust and easy-to-use microfluidic chip we designed based on (i)SIMPLE technology, allowing autonomous function upon single finger press activation, which was successfully validated with patient samples. Herein, we prove the potential of our technology to detect targets starting from whole blood introduced directly on-chip and to integrate various immunoassays, both for TDM and other in vitro diagnostics applications, like infectious diseases. [ABSTRACT FROM AUTHOR]