Photothermal Detection of Individual Gold Nanoparticles: Perspectives for High‐Throughput Screening

We use photothermal microscopy to detect and image individual gold nanoparticles that are either embedded in a polymer film or immobilized in an aqueous environment. Reducing the numerical aperture of the detection optics allows us to achieve a 200‐fold‐enlarged detection volume while still retaining sufficient detectivity. We characterize the capabilities of this approach for the detection of gold colloids with a diameter of 20 nm, with emphasis on practical aspects that are important for high‐throughput‐screening applications. The extended detection volume in combination with the stability of the photothermal signal are major advantages compared to fluorescence‐based approaches, which are limited by photoblinking and photobleaching. Careful consideration is given to the trade‐off between the maximum increase in local temperature that can be tolerated by a biological specimen and the minimum integration time needed to reliably determine whether a given volume contains a target species. We find that our approach has the potential to increase the detection‐limited flow rate (i.e. the limit given by the detection volume divided by the minimum detection time) by two to three orders of magnitude.     

A High‐Throughput Assay for Arylamine Halogenation Based on a Peroxidase‐Mediated Quinone–Amine Coupling with Applications in the Screening of Enzymatic Halogenations

Arylhalides are important building blocks in many fine chemicals, pharmaceuticals and agrochemicals, and there has been increasing interest in the development of more “green” halogenation methods based on enzyme catalysis. However, the screening and development of new enzymes for biohalogenation has been hampered by a lack of high‐throughput screening methods. Described herein is the development of a colorimetric assay for detecting both chemical and enzymatic arylamine halogenation reactions in an aqueous environment. The assay is based on the unique UV/Vis spectrum created by the formation of an ortho‐benzoquinone‐amine adduct, which is produced by the peroxidase‐catalysed benzoquinone generation, followed by Michael addition of either a halogenated or non‐halogenated arylamine. This assay is sensitive, rapid and amenable to high‐throughput screening platforms. We have also shown this assay to be easily coupled to a flavin‐dependent halogenase, which currently lacks any convenient colorimetric assay, in a “one‐pot” workflow.     

Design of a Full‐Profile‐Matching Solution for High‐Throughput Analysis of Multiphase Samples Through Powder X‐ray Diffraction

Finding a clear route to new structures : The design of an adaptable time warping (ATW) methodology (see figure) for automatically, quickly, and reliably deciphering X‐ray diffraction patterns is described.

     

Fluorogenic stereochemical probes for transaldolases

Transaldolase catalyzes the transfer of dihydroxyacetone from, for example, fructose 6-phosphate to erythrose 4-phosphate. As a potential probe for assaying fluorescent transaldolase, 6-O-coumarinyl-fructose (1) was prepared in six steps from D-fructose. The corresponding 6-O-coumarinyl-5-deoxy derivative 2 was prepared stereoselectively from acrolein and tert-butyl acetate by a chemoenzymatic route involving Amano PS lipase for the kinetic resolution of tert-butyl 3-hydroxypent-4-enoate (7) and E. coli transketolase for assembly of the final product. The corresponding stereoisomer related to D-tagatose was obtained by a chemical synthesis starting from D-ribose. Indeed, transaldolases catalyze the retro-aldolization of substrate 1 to give dihydroxyacetone and 3-O-coumarinyl-glyceraldehyde. The latter primary product undergoes a beta-elimination in the presence of bovine serum albumin (BSA) to give the strongly fluorescent product umbelliferone. A similar reaction is obtained with the 5-deoxy analogue 2, but there is almost no reaction with its stereoisomer 3. The stereoselectivity of transaldolases can be readily measured by the relative rates of fluorescence development in the presence of the latter pair of diastereomeric substrates.     

On‐Bead Screens Sample Narrower Affinity Ranges of Protein–Ligand Interactions Compared to Equivalent Solution Assays

Conceptually, on‐bead screening is one of the most efficient high‐throughput screening (HTS) methods. One of its inherent advantages is that the solid support has a dual function: it serves as a synthesis platform and as a screening compartment. Compound purification, cleavage and storage and extensive liquid handling are not necessary in bead‐based HTS. Since the establishment of one‐bead one‐compound library synthesis, the properties of polymer beads in chemical reactions have been thoroughly investigated. However, the characterization of the kinetics and thermodynamics of protein–ligand interactions on the beads used for screening has received much less attention. Consequently, the majority of reported on‐bead screens are based on empirically derived procedures, independent of measured equilibrium constants and rate constants of protein binding to ligands on beads. More often than not, on‐bead screens reveal apparent high affinity binders through strong protein complexation on the matrix of the solid support. After decoding, resynthesis, and solution testing the primary hits turn out to be unexpectedly weak binders, or may even fall out of the detection limit of the solution assay. Only a quantitative comparison of on‐bead binding and solution binding events will allow systematically investigating affinity differences as function of protein and small molecule properties. This will open up routes for optimized bead materials, blocking conditions and other improved assay procedures. By making use of the unique features of our previously introduced confocal nanoscanning (CONA) method, we investigated the kinetic and thermodynamic properties of protein–ligand interactions on TentaGel beads, a popular solid support for on‐bead screening. The data obtained from these experiments allowed us to determine dissociation constants for the interaction of bead‐immobilized ligands with soluble proteins. Our results therefore provide, for the first time, a comparison of on‐bead versus solution binding thermodynamics. Our data indicate that affinity ranges found in on‐bead screening are indeed narrower compared to equivalent interactions in homogeneous solution. A thorough physico‐chemical understanding of the molecular recognition between proteins and surface bound ligands will further strengthen the role of on‐bead screening as an ultimately cost‐effective method in hit and lead finding.     

Particle Display: A Quantitative Screening Method for Generating High‐Affinity Aptamers

We report an aptamer discovery technology that reproducibly yields higher affinity aptamers in fewer rounds compared to conventional selection. Our method (termed particle display) transforms libraries of solution‐phase aptamers into “aptamer particles”, each displaying many copies of a single sequence on its surface. We then use fluorescence‐activated cell sorting (FACS) to individually measure the relative affinities of >10⁸ aptamer particles and sort them in a high‐throughput manner. Through mathematical analysis, we identified experimental parameters that enable optimal screening, and demonstrate enrichment performance that exceeds the theoretical maximum achievable with conventional selection by many orders of magnitude. We used particle display to obtain high‐affinity DNA aptamers for four different protein targets in three rounds, including proteins for which previous DNA aptamer selection efforts have been unsuccessful. We believe particle display offers an extraordinarily efficient mechanism for generating high‐quality aptamers in a rapid and economic manner, towards accelerated exploration of the human proteome.     

A High‐Throughput Method for Determining the Stability of Pigment Dispersions

Summary: An approach for the high‐throughput preparation and characterisation of aqueous pigment dispersions is described and evaluated. The use of ultrasonication as a rapid technique for dispersing pigments using polymeric dispersants was developed. The results are comparable to those obtained using time‐intensive conventional high‐energy ball mill processing. The quality of the pigment dispersion was evaluated in a high‐throughput fashion using digital image analysis and the results correlated with particle sizes, determined by photo‐correlation spectroscopy.

Cuvettes containing dispersions of varying particle size.     

Acetylcholinesterase‐capped Mesoporous Silica Nanoparticles Controlled by the Presence of Inhibitors

Two different acetylcholinesterase (AChE)‐capped mesoporous silica nanoparticles (MSNs), S1‐AChE and S2‐AChE , were prepared and characterized. MSNs were loaded with rhodamine B and the external surface was functionalized with either pyridostigmine derivative P1 (to yield solid S1 ) or neostigmine derivative P2 (to obtain S2 ). The final capped materials were obtained by coordinating grafted P1 or P2 with AChE′s active sites (to give S1‐AChE and S2‐AChE , respectively). Both materials were able to release rhodamine B in the presence of diisopropylfluorophosphate (DFP) or neostigmine in a concentration‐dependent manner via the competitive displacement of AChE through DFP and neostigmine coordination with the AChE‘s active sites. The responses of S1‐AChE and S2‐AChE were also tested with other enzyme inhibitors and substrates. These studies suggest that S1‐AChE nanoparticles can be used for the selective detection of nerve agent simulant DFP and paraoxon.     

High‐Throughput Reaction Optimisation and Activity Screening of Ferrocene‐Based Lewis Acid–Catalyst Complexes by Using Continuous‐Flow Reaction Detection Mass Spectrometry

Optimising synthetic conversions and assessing catalyst performance is a tedious and laborious endeavour. Herein, we present an automated alternative to the commonly applied sequential approaches that are used to increase catalyst discovery process efficiencies by increasing the number of entities that can be tested. This new approach combines conversion of the reactants and determination of product formation into a single comprehensive reaction detection system that can be operated with minimal catalyst and reactant consumption. With this approach, rudimentary reaction conditions can be quickly optimised and the same system can then be used to screen for the optimal homogenous catalyst in a selected solution‐phase synthetic conversion. The system, which is composed of standard HPLC components, can be used to screen catalyst libraries at a repetition rate of five minutes and can be run unsupervised. The sensitive mass spectrometric detection that is implemented in the reaction detection methodology can be used for the simultaneous monitoring of reactants, catalysts and product ions. In the experiments, the three‐component reaction that gives a substituted 2‐imidazoline was optimised. Afterwards, the same method was used to assess a library of ferrocene‐based Lewis acid catalysts for performance in the aforementioned conversion in six different solvents. We demonstrate the feasibility of using this methodology to directly compare the performance results obtained in different solvents by calibrating the solvent‐specific MS responses.     

High‐Throughput Development of a Hybrid‐Type Fluorescent Glutamate Sensor for Analysis of Synaptic Transmission

Fluorescent sensors are powerful tools for visualizing cellular molecular dynamics. We present a high‐throughput screening system, designated hybrid‐type fluorescence indicator development (HyFInD), to identify optimal position‐specific fluorophore labeling in hybrid‐type sensors consisting of combinations of ligand‐binding protein mutants with small molecular fluorophores. We screened sensors for glutamate among hybrid molecules obtained by the reaction of four cysteine‐reactive fluorescence probes with a set of cysteine‐scanning mutants of the 274 amino acid S1S2 domain of AMPA‐type glutamate receptor GluA2 subunit. HyFInD identified a glutamate‐responsive probe (enhanced glutamate optical sensor: eEOS) with a dynamic range >2400 %, good photostability, and high selectivity. When eEOS was specifically tethered to neuronal surfaces, it reliably visualized the spatiotemporal dynamics of glutamate release at single synapses, revealing synapse‐to‐synapse heterogeneity of short‐term plasticity.     

Detection and Analysis of Low‐Abundance Cell‐Surface Biomarkers Using Enzymatic Amplification in Microfluidic Droplets

Finding the few : Cell‐surface proteins are useful disease biomarkers, but current high‐throughput methods are limited to detecting cells expressing more than several hundred proteins. Enzymatic amplification in microfluidic droplets (see picture) is a high‐throughput method for detection and analysis of cell‐surface biomarkers expressed at very low levels on individual human cells. Droplet optical labels allow concurrent analysis of several samples.