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Biofilms are ubiquitous in nature and in the man-made environment. Given their harmful effects on human health, an in-depth understanding of biofilms and the monitoring of their formation and growth are important. Particularly relevant for many metabolic processes and survival strategies of biofilms is their extracellular pH. However, most conventional techniques are not suited for minimally invasive pH measurements of living biofilms. Here, a fluorescent nanosensor is presented for ratiometric measurements of pH in biofilms in the range of pH 4.5–9.5 using confocal laser scanning microscopy. The nanosensor consists of biocompatible polystyrene nanoparticles loaded with pH-inert dye Nile Red and is surface functionalized with a pH-responsive fluorescein dye. Its performance was validated by fluorometrically monitoring the time-dependent changes in pH in E. coli biofilms after glucose inoculation at 37 °C and 4 °C. This revealed a temperature-dependent decrease in pH over a 4-h period caused by the acidifying glucose metabolism of E. coli. These studies demonstrate the applicability of this nanosensor to characterize the chemical microenvironment in biofilms with fluorescence methods.
Nano- and microparticles are of increasing importance for a wide range of applications in the material and life sciences. Examples are their use as carriers for dye molecules and drugs, multichromophoric reporters for signal enhancement strategies in optical assays, targeted probes for bioimaging, and biosensors. All these applications require surface functionalization of the particles with e.g., ligands (to tune the dispersibility and prevent unspecific interactions), crosslinkers, sensor dyes, or analyte recognition moieties like biomolecules, and subsequently, the knowledge of the chemical nature and total number of surface groups as well as the number of groups accessible for coupling reactions. Particularly attractive for the latter are sensitive and fast photometric or fluorometric assays, which can be read out with simple, inexpensive instrumentation. Here, we present a novel family of cleavable photometric and multimodal reporters for the quantification of conjugatable amino and carboxyl surface groups on nano- and microparticles. These probes allow for the determination of particle-bound labels, unbound reporters in the supernatant, and reporters cleaved off from the particle surface as well as the remaining thiol groups on the particles by photometry and inductively coupled optical emission spectrometry (32S ICP-OES).3 Comparison of the performance of these cleavable reporters with conventional labels, utilizing changes in intensity and/or colour of absorption and/or emission, underlines the analytical potential of this versatile concept which elegantly circumvents signal distortions by light scattering and encoding dyes, and enables straightforward validation by method comparison.
Aggregation-induced emission (AIE) has been meanwhile observed for many dye classes and particularly for fluorophores containing propeller-like groups. Herein, we report on the AIE characteristics of a series of four hydrophobic pyrrolidinylvinylquinoxaline (PVQ) derivatives with phenyl, pyrrolyl, indolyl, and methoxythienyl substituents used to systematically vary the torsion angle between this substituent at the quinoxaline C2 position and the planar PVQ moiety. These molecules, which are accessible via four- or five-component one-pot syntheses, were spectroscopically studied in organic solvents and solvent−water mixtures, as dye aggregates, solids, and entrapped in polystyrene particles (PSP). Steady-state and time-resolved fluorescence measurements revealed a strong fluorescence enhancement for all dyes in ethanol−water mixtures of high water content, accompanying the formation of dye aggregates with sizes of a few hundred nm, overcoming polarity and H-bonding-induced fluorescence quenching of the chargetransfer-type emission of these PVQ dyes. The size and shape of these dye aggregates and the size of the AIE effect are controlled by the water content and the substituent-dependent torsion angle that influences the nucleation process and the packing of the molecules during aggregation. Staining of 1 μm-sized carboxy-functionalized PSP with the PVQ dyes resulted also in a considerable increase in the fluorescence quantum yield and lifetime, reflecting the combined influence of the restricted molecular motion and the reduced polarity of the dye microenvironment.
Merocyanine–triarylamine bichromophores are readily synthesized by sequentially Pd-catalyzed insertion alkynylation–Michael–Suzuki four-component reactions. White-light emissive systems form upon aggregation in 1 : 99 and 0.1 : 99.9 vol% CH2Cl2–cyclohexane mixtures, ascribed to aggregation-induced dual emission (AIDE) in combination with partial energy transfer between both chromophore units as supported by spectroscopic studies.
We present a comparative study of the spectroscopic properties of the donor–acceptor–donor substituted dyes triphenylamine-allylidenemalononitrile-julolidine (TMJ) and triphenylamine-allylidenemalononitriletriphenylamine (TMT), bearing one and two propeller-like triphenylamine donor moieties, in solvents of varying polarity and viscosity and in the aggregated and solid state. Our results reveal control of the aggregation-induced spectroscopic changes and the packing motifs of the dye molecules in the solid state by the chemical nature and structure of the second nitrogen-containing donor, i.e., a planar and a rigid julolidine or a twisted triphenyl group. Assuming that the TMT and TMJ aggregates show a comparable arrangement of the molecules to the respective crystals, these different molecular interactions in the solid state are responsible for aggregation induced emission (AIE) in the case of TMT and its absence for TMJ. Moreover, a versatile strategy for the fluorescence enhancement of only weakly emissive AIE dyes is shown, turning these dyes into bright nanoscale fluorescent reporters by using them as stains for preformed polymer particles.
N-Benzyl aroyl-S,N-ketene acetals can be readily synthesized by condensation of aroyl chlorides and N-Benzyl 2-methyl benzothiazolium salts in good to excellent yields, yielding a library of 35 chromophores with bright solid-state emission and aggregation-induced emission characteristics.
Varying the substituent from electron-donating to electronwithdrawing enables the tuning of the solid-state emission Color from deep blue to red.
Two sets of polystyrene nanoparticles (PSNPs) with comparable core sizes but different carboxyl group densities were made and separated using asymmetric flow field flow fractionation (AF4), capillary electrophoresis (CE), and the off-line hyphenation of both methods. Our results revealed the significant potential of two-dimensional off-line AF4-CE hyphenation to improve the separation and demonstrated for the first time, the applicability of CE to determine the functional group density of nanoparticles (NPs). Compared to the result acquired with conductometric titration, the result obtained with synthesized 100 nm sized PSNPs revealed only a slight deviation of 1.7%. Commercial 100 nm sized PSNPs yielded a deviation of 4.6 %. For 60 nm sized PSNPs, a larger deviation of 10.6 % between both methods was observed, which is attributed to the lower separation resolution.