Reusable Fluorescent Nanobiosensor Integrated in a Multiwell Plate for Screening and Quantification of Antidiabetic Drugs

Abstract

A highly stable and reusable fluorescent multisample nanobiosensor for the detection of α-glucosidase inhibitors has been developed by coupling fluorescent liposomal nanoparticles based on conjugated polymers (L-CPNs) to the enzyme α-glucosidase, one of the main target enzymes in the treatment of type 2 diabetes. The mechanism of sensing is based on the fluorescence “turn-on” of L-CPNs by p-nitrophenol (PNP), the end product of the enzymatic hydrolysis of p-nitrophenyl-α-d-glucopyranoside. L-CPNs, composed of lipid vesicles coated with a blue-emitting cationic polyfluorene, were designed and characterized to obtain a good response to PNP. Two nanobiosensor configurations were developed in this study. In the first step, a single-sample nanobiosensor composed of L-CPNs and α-glucosidase entrapped in a sol–gel glass was developed in order to characterize and optimize the device. In the second part, the nanobiosensor was integrated and adapted to a multiwell microplate and the possibility of reusing it and performing multiple measurements simultaneously with samples containing different α-glucosidase inhibitors was investigated. Using super-resolution confocal microscopy, L-CPNs could be visualized within the sol–gel matrix, and the quenching of their fluorescence, induced by the substrate, was directly observed in situ. The device was also shown to be useful not only as a platform for screening of antidiabetic drugs but also for quantifying their presence. The latter application was successfully tested with the currently available drug, acarbose.

Formation of Multicolor Nanogels Based on Cationic Polyfluorenes and Poly(methyl vinyl ether-alt-maleic monoethyl ester): Potential Use as pH-Responsive Fluorescent Drug Carriers

“…Polyanhydrides represent an important class of polymers that has been used for medical purposes including encapsulation of drugs in nanoparticles [11][12][13]. They can be prepared easily from available, low-cost resources and can be manipulated to meet markers for bioimaging and sensing devices and have recently been used to develop multicolor fluorescent liposomal nanoparticles activated by temperature, capable of being excited at the same wavelength [35][36][37][38][39][40][41]. In the present work, we take advantage of the attractive properties of CPEs, together with those of PMVEMA-Es and our previous experience in both types of polymers, to prepare and characterize fluorescent nanoplatforms with emission in different visible bands and potential applications as drug carriers and multiplexed bioimaging agents.…”

“…Our group has synthesized and characterized three fluorene-based cationic CPEs, that can emit fluorescence with high quantum yield and photostability in the blue, green and red regions of the visible spectrum: copoly-((9,9-bis(6 -N,N,N-trimethylammonium)hexyl)-2,7-(fluorene)-alt-1,4-(phenylene)) bromide (HTMA-PFP), copoly-((9,9-bis(6 -N,N,N-trimethylammonium)hexyl)-2,7-(fluorene)-alt-4,7-(2-(phenyl) benzo(d) (1,2,3) triazole)) bromide (HTMA-PFBT), and copoly-((9,9-bis(6 -N,N,N-trimethylammonium)hexyl)-2,7-(fluorene)alt-1,4-(naphtho(2,3c)-1,2,5-thiadiazole)) bromide (HTMA-PFNT) (Scheme 1). These polyfluorenes show interesting properties as fluorescent markers for bioimaging and sensing devices and have recently been used to develop multicolor fluorescent liposomal nanoparticles activated by temperature, capable of being excited at the same wavelength [35][36][37][38][39][40][41]. In the present work, we take advantage of the attractive properties of CPEs, together with those of PMVEMA-Es and our previous experience in both types of polymers, to prepare and characterize fluorescent nanoplatforms with emission in different visible bands and potential applications as drug carriers and multiplexed bioimaging agents.…”

Fluorescent Nanocomposite Hydrogels Based on Conjugated Polymer Nanoparticles as Platforms for Alkaline Phosphatase Detection

“…Recently, conjugated polymer-based nanoparticles (CNPs) have emerged as a new class of fluorescent nanoparticle with interesting properties, such as good biocompatibility, excellent photostability and high quantum efficiency; CNPs also offer the possibility of tunable fluorescence emission by appropriate design of the polymer molecular backbone [ 18 , 19 , 20 , 21 ]. In addition, conjugated polymers, especially those involving fluorene-based systems (known as polyfluorenes), are excellent electron and energy donors; this means that the fluorescence of CNPs can be quenched with suitable acceptors, which gives them great versatility as sensing elements [ 22 , 23 ]. In this regard, micellar nanoparticles based on novel polyfluorene polyelectrolytes have been developed in recent work, as promising materials for the detection of pollutants through photoinduced electron transfer and inner filter effect mechanisms [ 24 ].…”

Disposable Electrochemical Biosensor Based on the Inhibition of Alkaline Phosphatase Encapsulated in Acrylamide Hydrogels

“…The immobilization of ALP in these sensing devices has the advantage of ease of handling and the possibility of miniaturization, allowing in situ measurements. Silica matrices prepared by the sol–gel process are most often used to immobilize enzymes with very satisfactory results, especially in electrochemical and optical biosensor development [ 9 , 10 , 11 ]. This methodology has been used in the past to immobilize ALP to develop fluorescent biosensors capable of detecting enzyme inhibitors such as pesticides and phosphate [ 12 , 13 ].…”