RESUMEN
Homogeneous assays for determining the concentration of small molecules in biological fluids are of importance for monitoring blood levels of critical drugs in patients. We have developed a strand displacement competition assay for the drugs dabigatran, methotrexate, and linezolid, which allows detection and determination of the concentration of the drugs in plasma; however, a surprising kinetic behavior of the assay was observed with an initial rapid change in apparent FRET values. We found that protein-induced fluorescent enhancement or quenching (PIFE/Q) caused the initial change in fluorescence within the first minute after addition of protein, which could be exploited to construct assays for concentration determination within minutes in the low nanomolar range in plasma. A kinetic model for the assay was established, and when taking the new finding into account, the in silico simulations were in good agreement with the experimentally observed results. Utilizing these findings, a simpler assay was constructed for detection of dabigatran, which allowed for detection within minutes without any time dependencies.
Asunto(s)
ADN , Dabigatrán , ADN/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Humanos , ProteínasRESUMEN
Rapid detection and quantification of pharmaceutical drugs directly in human plasma is of major importance for the development of relevant point-of-care testing devices. Here, we report a method for detection and quantification of small molecules in human plasma. An assay employing a small molecule-linked hybridization chain reaction (HCR) has been devised for the detection of the pharmaceutically relevant drugs digoxin (Dig) and methotrexate (MTX). Double modification by small molecule ligands on the initiator strand act as sites to control the rate of the HCR. Upon protein binding to the modified initiator strand, the HCR is greatly inhibited. If the protein is preincubated with a sample containing the small molecule analyte, the protein binding site is occupied by the analyte and the initiator strand will initiate the HCR. This enables efficient detection and quantification of small-molecule analytes in nanomolar concentration even in 50% human plasma within 4 min. Thus, the rapidity and simplicity of this assay has potential for point-of-care testing.
Asunto(s)
Digoxina/sangre , Metotrexato/sangre , Técnicas de Amplificación de Ácido Nucleico/métodos , Anticuerpos/inmunología , Biotina/química , Carbocianinas/química , ADN Complementario/genética , Digoxigenina/inmunología , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Humanos , Límite de Detección , Metotrexato/inmunología , Hibridación de Ácido Nucleico , Pruebas en el Punto de Atención , Estreptavidina/químicaRESUMEN
The most common method for quantifying small-molecule drugs in blood samples is by liquid chromatography in combination with mass spectrometry. Few immuno-based assays are available for the detection of small-molecule drugs in blood. Here we report on a homogeneous assay that enables detection of the concentration of digoxin spiked into in a plasma sample. The assay is based on a shift in the equilibrium of a DNA strand displacement competition reaction, and can be performed in 30 min for concentrations above 10 nM. The equilibrium shift occurs upon binding of anti-digoxigenin antibody. As a model, the assay provides a potential alternative to current small-molecule detection methods used for therapeutic drug monitoring.
Asunto(s)
Antiarrítmicos/sangre , Técnicas Biosensibles/métodos , Cardiotónicos/sangre , ADN/química , Digoxina/sangre , Monitoreo de Drogas/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Humanos , Límite de DetecciónRESUMEN
Direct and rapid intracellular delivery of a functional Cas9/sgRNA complex using ultrasound-powered nanomotors is reported. The Cas9/sgRNA complex is loaded onto the nanomotor surface through a reversible disulfide linkage. A 5â min ultrasound treatment enables the Cas9/sgRNA-loaded nanomotors to directly penetrate through the plasma membrane of GFP-expressing B16F10 cells. The Cas9/sgRNA is released inside the cells to achieve highly effective GFP gene knockout. The acoustic Cas9/sgRNA-loaded nanomotors display more than 80 % GFP knockout within 2â h of cell incubation compared to 30 % knockout using static nanowires. More impressively, the nanomotors enable highly efficient knockout with just 0.6â nm of the Cas9/sgRNA complex. This nanomotor-based intracellular delivery method thus offers an attractive route to overcome physiological barriers for intracellular delivery of functional proteins and RNAs, thus indicating considerable promise for highly efficient therapeutic applications.