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Multisomes are multicompartmental structures formed by a lipid-stabilized network of aqueous droplets, which are contained by an outer oil phase. These biomimetic structures are emerging as a versatile platform for soft matter and synthetic biology applications. While several methods for producing multisomes have been described, including microfluidic techniques, approaches for generating biocompatible, monodisperse multisomes in a reproducible manner remain challenging to implement due to low throughput and complex device fabrication. Here, we report on a robust method for the dynamically controlled generation of multisomes with controllable sizes and high monodispersity from lipid-based double emulsions. The described microfluidic approach entails the use of three different phases forming a water/oil/water (W/O/W) double emulsion stabilized by lipid layers. We employ a gradient of glycerol concentration between the inner core and outer phase to drive the directed osmosis, allowing the swelling of lamellar lipid layers resulting in the formation of small aqueous daughter droplets at the interface of the inner aqueous core. By adding increasing concentrations of glycerol to the outer aqueous phase and subsequently varying the osmotic gradient, we show that key structural parameters, including the size of the internal droplets, can be specifically controlled. Finally, we show that this approach can be used to generate multisomes encapsulating small-molecule cargo, with potential applications in synthetic biology, drug delivery, and as carriers for active materials in the food and cosmetics industries.

Cell-free protein synthesis has been widely used as a “breadboard” for design of synthetic genetic networks. However, due to a severe lack of modularity, forward engineering of genetic networks remains challenging. Here, we demonstrate how a combination of optimal experimental design and microfluidics allows us to devise dynamic cell-free gene expression experiments providing maximum information content for subsequent non-linear model identification. Importantly, we reveal that applying this methodology to a library of genetic circuits, that share common elements, further increases the information content of the data resulting in higher accuracy of model parameters. To show modularity of model parameters, we design a pulse decoder and bistable switch, and predict their behaviour both qualitatively and quantitatively. Finally, we update the parameter database and indicate that network topology affects parameter estimation accuracy. Utilizing our methodology provides us with more accurate model parameters, a necessity for forward engineering of complex genetic networks.

Spatiotemporal organization of distinct biological processes in cytomimetic compartments is a crucial step towards engineering functional artificial cells. Mimicking controlled bi-directional molecular communication inside artificial cells remains a considerable challenge. Here we present photoswitchable molecular transport between programmable membraneless organelle-like DNA coacervates in a synthetic microcompartment. We use droplet microfluidics to fabricate membraneless non-fusing DNA coacervates by liquid-liquid phase separation in a water-in-oil droplet, and employ the interior DNA coacervates as artificial organelles to imitate intracellular communication via photo-regulated uni- and bi-directional transfer of biomolecules. Our results highlight a promising new route to assembly of multicompartment artificial cells with functional networks.

Ultrasensitivity is a ubiquitous emergent property of biochemical reaction networks. The design and construction of synthetic reaction networks exhibiting ultrasensitivity has been challenging, but would greatly expand the potential properties of life-like materials. Herein, we exploit a general and modular strategy to reversibly regulate the activity of enzymes using light and show how ultrasensitivity arises in simple out-of-equilibrium enzymatic systems upon incorporation of reversible photoswitchable inhibitors (PIs). Utilizing a chromophore/warhead strategy, PIs of the protease α-chymotrypsin were synthesized, which led to the discovery of inhibitors with large differences in inhibition constants (Ki) for the different photoisomers. A microfluidic flow setup was used to study enzymatic reactions under out-of-equilibrium conditions by continuous addition and removal of reagents. Upon irradiation of the continuously stirred tank reactor with different light pulse sequences, i.e., varying the pulse duration or frequency of UV and blue light irradiation, reversible switching between photoisomers resulted in ultrasensitive responses in enzymatic activity as well as frequency filtering of input signals. This general and modular strategy enables reversible and tunable control over the kinetic rates of individual enzyme-catalyzed reactions and makes a programmable linkage of enzymes to a wide range of network topologies feasible.

A droplet-based microfluidics technique is used to produce monodisperse, 80 μm collagen–gelatin beads with tunable mechanical properties in the range of 1–10 kPa after photo-crosslinking. The gel beads are porous, mechanically robust and stable in buffer, but can be degraded enzymatically. Encapsulated fibroblast cells maintain 70% viability after one-week encapsulation and preliminary results show that the degree of spreading of cells in gels is correlated with the stiffness of the material.

Coacervates have been widely studied as model compartments in protocell research. Complex coacervates composed of disordered proteins and RNA have also been shown to play an important role in cellular processes. Herein, we report on a microfluidic strategy for constructing monodisperse coacervate droplets encapsulated within uniform unilamellar liposomes. These structures represent a bottom‐up approach to hierarchically structured protocells, as demonstrated by storage and release of DNA from the encapsulated coacervates as well as localized transcription.

The formation of cytomimetic protocells that capture the physicochemical aspects of living cells is an important goal in bottom-up synthetic biology. Here, we recreated the crowded cytoplasm in liposome-based protocells and studied the kinetics of cell-free gene expression in these crowded containers. We found that diffusion of key components is affected not only by macromolecular crowding but also by enzymatic activity in the protocell. Surprisingly, size-dependent diffusion in crowded conditions yielded two distinct maxima for protein synthesis, reflecting the differential impact of crowding on transcription and translation. Our experimental data show, for the first time, that macromolecular crowding induces a switch from reaction to diffusion control and that this switch depends on the sizes of the macromolecules involved. These results highlight the need to control the physical environment in the design of synthetic cells.

Summary Microfluidics permit the automated manipulation of fluids at the microscale with high throughput and spatiotemporal precision, enabling the generation of large, multidimensional datasets. Machine intelligence provides powerful predictive tools with the ability to learn from data. The analysis of microfluidics-generated data via machine learning has been applied in a variety of contexts, achieving impressive results. Here, we elaborate on the potential of operating microfluidic platforms via closed-loop data-driven models by leveraging multimodal monitoring and data-acquisition instrumentation. We believe this approach will provide a robust framework for fundamental explorations in materials science and biomedicine, with implications in fields such as drug discovery, nanomaterials, in vitro organ modeling, and developmental biology. We identify challenges and propose research strategies in the context of the prediction and optimization of chemical reactions and materials syntheses and the development of the next generation of more robust and functional organs-on-chips and emerging organoids-on-chips.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The compartmentalization of cell-free gene expression systems in liposomes provides an attractive route to the formation of protocells, but these models do not capture the physical (crowded) environment found in living systems. Here, we present a microfluidics-based route to produce monodisperse liposomes that can shrink almost 3 orders of magnitude without compromising their stability. We demonstrate that our strategy is compatible with cell-free gene expression and show increased protein production rates in crowded liposome protocells.

The number of circulating tumor cells (CTCs) in blood is strongly correlated with the progress of metastatic cancer. Current methods to detect CTCs are based on immunostaining or discrimination of physical properties. Herein, a label-free method is presented exploiting the abnormal metabolic behavior of cancer cells. A single-cell analysis technique is used to measure the secretion of acid from individual living tumor cells compartmentalized in microfluidically prepared, monodisperse, picoliter (pL) droplets. As few as 10 tumor cells can be detected in a background of 200 000 white blood cells and proof-of-concept data is shown on the detection of CTCs in the blood of metastatic patients.

Cell-free protein synthesis (CFPS) is a robust platform for the simple, rapid, and cost-effective in vitro production of proteins, as well as an important tool for cell-free synthetic biology research. Here, a microfluidic clay microgel system is reported, which creates compartmentalized microenvironments for CFPS capable of high-yield and repeated protein synthesis, as well as an artificial cell-like structure. As an advantageous platform for CFPS, a modular manner to prepare clay microgels with rationally designed functions is demonstrated: (i) gene/clay microgels enhance protein expression, (ii) gene/clay/magnetic nanoparticle microgels enable a repeated protein production system, and (iii) gene/clay microgels in microfluidic droplets serve as a cell-like structure. Beyond CFPS, considering the compatibility of clay microgels with hydrophilic functional materials, our clay microgels will provide a more general platform for preparing a variety of functional materials such as encapsulating drugs and cells, enabling more biomedical applications.

Contains fulltext : 140715.pdf (Publisher’s version ) (Open Access) Droplet-based microfluidics is increasingly used for biological applications, where the recovery of cells or particles after an experiment or assay is desirable. Here, we present an electro-demulsification chip which circumvents the use of harsh chemicals and multiple washing/centrifugation steps and offers a mild way for extracting cells and polymer particles into an aqueous phase from microfluidic water-in-oil emulsions.

We report a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic device is used to generate and manipulate

Fully integrated: Mass spectrometry has been integrated into a detection scheme for microdroplets that are created within microfluidic channels (see picture, scale bar 200 microm). This technique allows droplets to be identified based on the compounds they contain, and combines fluorescence screening with MS analysis. These experiments indicate how similar approaches can be applied to the ambitious goals of on-chip protein evolution and chemical synthesis.

Contains fulltext : 103459.pdf (Publisher’s version ) (Open Access)

Here, the formation of eutectic Gallium-Indium (EGaIn) liquid-metal microdroplets, both spherical and non-spherical, in microfluidic devices at room temperature is reported. Monodisperse microdroplets were created in an aqueous polyethylene glycol (PEG) solution, in oxygenated and in deoxygenated silicone oil. The volume of the droplets depends on the channel dimensions and flow rates applied, varying between 0.5 and 4 nL. Non-spherical droplets were formed in oxygenated silicone oil due to the instantaneous formation of an oxide layer. These metal "micro-rice" droplets retained their shape and did not spontaneously reflow to form shapes of the lowest interfacial energy on egress from the channel, unlike in aqueous PEG solution and in deoxygenated silicone oil. Liquid-metal droplets with such tunable morphology can potentially be used in MEMS devices for optical and electrical switches, valves and micropumps. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A microfluidic device capable of exploiting the permeability of small molecules through polydimethylsiloxane (PDMS) has been fabricated in order to control the contents of microdroplets stored in storage wells. We demonstrate that protein precipitation and crystallization can be triggered by delivery of ethanol from a reservoir channel, thus controlling the protein solubility in microdroplets. Likewise quorum sensing in bacteria was triggered by delivery of the auto-inducer N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) through the PDMS membrane of the device.

Water-in-oil microdroplets in microfluidics are well-defined individual picoliter reaction compartments and, as such, have great potential for quantitative high-throughput biological screening. This, however, depends upon contents of the droplets not leaking out into the oil phase. To assess the mechanism of possible leaking, the retention of various fluorescein derivatives from droplets formed in mineral oil and stored for hours in a reservoir on chip was studied. Leaking into the oil phase was observed and was shown to be dependent on the nature of the compounds and on the concentration of the silicone-based polymeric surfactant Abil EM 90 used. In experiments in which droplets filled with fluorescein were mixed with droplets filled with only buffer, the rate of efflux from filled droplets to empty droplets was dependent on the number of neighboring droplets of different composition. Buffer droplets with five fluorescein-containing neighbors took up the fluorophore 4.5 times faster than buffer droplets without fluorescein neighbors. The addition of bovine serum albumin (BSA) substantially reduced leaking. A formulation with 5% BSA reduces leaking of the fluorophore from 45% to 3%. Inclusion of BSA enabled experiments to be carried out over periods up to 18 h, without substantial leaking (

In droplet-based microfluidics, non-ionic, high-molecular weight surfactants are required to stabilize droplet interfaces. One of the most common structures that imparts stability as well as biocompatibility to water-in-oil droplets is a triblock copolymer surfactant composed of perfluoropolyether (PFPE) and polyethylene glycol (PEG) blocks. However, the fast growing applications of microdroplets in biology would benefit from a larger choice of specialized surfactants. PEG as a hydrophilic moiety, however, is a very limited tool in surfactant modification as one can only vary the molecular weight and chain-end functionalization. In contrast, linear polyglycerol offers further side-chain functionalization to create custom-tailored, biocompatible droplet interfaces. Herein, we describe the synthesis and characterization of polyglycerol-based triblock surfactants with tailored side-chain composition, and exemplify their application in cell encapsulation and in vitro gene expression studies in droplet-based microfluidics.

Water-in-oil-in-water (w/o/w) microfluidics double emulsions offer a new route to compartmentalise reagents into isolated aqueous microenvironments while maintaining an aqueous carrier fluid phase; this enables compatibility with commercial flow cytometry systems such as fluorescence-activated cell sorting (FACS). Double emulsion (inner core) deformation under hydrodynamic focusing conditions that mimic the environment double emulsions experience in flow cytometry applications is of particular importance for droplet stability and cell viability. This paper reports on an experimental study of the dynamic deformation of aqueous cores of w/o/w double emulsions under hydrodynamic focusing, with the sheath flow directed at 45° to the sample flow. A number of factors affecting the inner core deformation and recovery were examined. Deformation was found to depend significantly on the core or shell viscosity, the droplet-to-sheath flow velocity ratio, and core and shell sizes. Core deformation was found to depend more on the type of surfactant rather concentration with high molecular weight surfactant exhibiting a negligible effect on deformation whereas low molecular weight surfactant enhancing deformation at low concentrations due to their lateral mobility at the interface.

The flow environment in inner cores of water-in-oil-in-water (w/o/w) microfluidic double emulsions has a significant impact on industrial applications of such systems. For example, in the case of shear sensitive cells compartmentalised in the cores, high shear conditions may be deleterious. This study reports on the flow characteristics of w/o/w inner cores in comparison to those in single water-in-oil (w/o) microdroplets of equal size moving in the same microchannel, resolved by means of micro-particle image velocimetry (μPIV). The multiphase flow system employed in the study had a viscosity ratio, λ, between aqueous and oil phase of the order of unity (λ = 0.78) and both single droplets and inner cores of double emulsions filled the channel. This configuration resulted in a weak recirculating flow inside the w/o single droplet: the measured flow field exhibited a uniform low velocity flow field in the central region surrounded by small regions of reversed flow near the channel walls. This flow topology was maintained in the inner cores of w/o/w double emulsions for intermediate capillary numbers (Ca) ranging from 10(-3) to 10(-2), and core morphologies varying from large plugs to pancake cores. The core morphology affected the magnitude and distribution of the velocity in the droplets. The similarity in the flow topology resulted from the fact that inner cores were located at the back of the outer droplet in such a way that inner and outer interfaces were in contact for over half of core surface area and separated only by a thin lubricating film. Both single droplets and inner cores exhibited a narrow shear rate distribution characterised by small regions of maximum shear confined near the channel walls. Shear rate magnitude values were found to be an order of magnitude lower than those in the channel and hence capable of reducing stresses in flow cytometry to far below reported values for cell damage. Hence, it can be concluded that double emulsions are suitable candidates to substitute single droplets in flow cytometry to protect the screened items and are compatible with the commercial flow cytometry systems.

Surface adhesion is regulated by sparsely grafting responsive hydrophilic polymer chains on superhydrophobic surfaces but without obviously changing the wettability. We study experimentally how adhesion of superhydrophobic surfaces affects liquid slip. The slip length of water on such surfaces decays quickly as the adhesive force increases. This intrinsic dependence is theoretically explained based on scaling descriptions for specific geometries. A slip length range of 87 μm can be achieved reversibly by changing the temperature below and above the low critical solution temperature (LCST) of the grafted temperature-sensitive polymer. The results shed light on the intrinsic mechanism of liquid slip on textured surfaces and have important implications in the design of smart microfluidic and biofluidic devices, in which the regulation of fluid flow is highly desirable.

A series of water-soluble macro-initiators is synthesized to avoid radical loss in microfluidic on-chip photo cross-linking of hyaluronic acid methacrylate-containing water-in-oil emulsions. Their superior performance over known photo-initiators through the generation of water-soluble radicals and excellent biocompatibility are demonstrated.

Item does not contain fulltext Here, we present a platform to detect cytokine (IL-2, IFN-gamma, TNF-alpha) secretion of single, activated T-cells in droplets over time. We use a novel droplet-based microfluidic approach to encapsulate cells in monodisperse agarose droplets together with functionalized cytokine-capture beads for subsequent binding and detection of secreted cytokines from single cells. This method allows high-throughput detection of cellular heterogeneity and maps subsets within cell populations with specific functions.

This paper presents a droplet-based microfluidic platform for miniaturized combinatorial synthesis. As a proof of concept, a library of small molecules for early stage drug screening was produced. We present an efficient strategy for producing a 7 × 3 library of potential thrombin inhibitors that can be utilized for other combinatorial synthesis applications. Picolitre droplets containing the first type of reagent (reagents A(1), A(2), …, A(m)) were formed individually in identical microfluidic chips and then stored off chip with the aid of stabilizing surfactants. These droplets were then mixed to form a library of droplets containing reagents A(1-m), each individually compartmentalized, which was reinjected into a second microfluidic chip and combinatorially fused with picolitre droplets containing the second reagent (reagents B(1), B(2), …, B(n)) that were formed on chip. The concept was demonstrated with a three-component Ugi-type reaction involving an amine (reagents A(1-3)), an aldehyde (reagents B(1-7)), and an isocyanide (held constant), to synthesize a library of small molecules with potential thrombin inhibitory activity. Our technique produced 10(6) droplets of each reaction at a rate of 2.3 kHz. Each droplet had a reaction volume of 3.1 pL, at least six orders of magnitude lower than conventional techniques. The droplets can then be divided into aliquots for different downstream screening applications. In addition to medicinal chemistry applications, this combinatorial droplet-based approach holds great potential for other applications that involve sampling large areas of chemical parameter space with minimal reagent consumption; such an approach could be beneficial when optimizing reaction conditions or performing combinatorial reactions aimed at producing novel materials.

We have experimentally determined the optimal flow velocities to characterize or count single molecules by using a simple microfluidic device to perform two-color coincidence detection (TCCD) and single pair Förster resonance energy transfer (spFRET) using confocal fluorescence spectroscopy on molecules traveling at speeds of up to 10 cm s(-1). We show that flowing single fluorophores at ≥0.5 cm s(-1) reduces the photophysical processes competing with fluorescence, enabling the use of high excitation irradiances to partially compensate for the short residence time within the confocal volume (10-200 μs). Under these conditions, the data acquisition rate can be increased by a maximum of 38-fold using TCCD at 5 cm s(-1) or 18-fold using spFRET at 2 cm s(-1), when compared with diffusion. While structural characterization requires more photons to be collected per event and so necessitates the use of slower speeds (2 cm s(-1) for TCCD and 1 cm s(-1) for spFRET), a considerable enhancement in the event rate could still be obtained (33-fold for TCCD and 16-fold for spFRET). Using flow under optimized conditions, analytes could be rapidly quantified over a dynamic range of up to 4 orders of magnitude by direct molecule counting; a 50 fM dual-labeled model sample can be detected with 99.5% statistical confidence in around 8 s using TCCD and a flow velocity of 5 cm s(-1).

Microgel particles are formed from aqueous-two-phase-system (ATPS) droplets in poly(dimethylsiloxane) (PDMS) microfluidic devices. The droplets consist of a dextran core and a photopolymerizable poly(ethylene glycol) diacrylate (PEGDA) shell. Upon UV exposure, the ATPS droplets undergo a shape-transformation yielding PEGDA microgel particles containing a socket.

In this paper we introduce a simple droplet-based microfluidic system consisting of two separate devices to encapsulate and culture microalgae, in contrast to cultivation in bulk liquid medium. This microdroplet technology has been used to monitor the growth of individual microalgal cells in a constant environment for extended periods of time. Single cells from three species of green microalgae, (two freshwater species Chlamydomonas reinhardtii and Chlorella vulgaris, and one saline species Dunaliella tertiolecta), were encapsulated and incubated in microdroplet compartments of diameter of ∼80 μm, and their growth analysed over 10 days. In all cases, the doubling time of microalgae grown in microdroplets was similar to growth in bulk. The growth of C. reinhardtii in microdroplets of varying diameters and with different initial cell numbers per droplet was investigated, as well as the effect of varying medium conditions such as pH and nitrogen concentration. This methodology offers the opportunity to study characteristics over time of individual cells and colonies, as well as to screen large numbers of them.

We report the use of microfluidics for the production of monodisperse oil-in-water droplets functionalized by a pH responsive branched co-polymer surfactant. The droplet functionality facilitates the reversible aggregation of the micron-sized droplets into macroscopic engineered emulsions in response to solution pH changes. Co-injection of dye-loaded and non-dyed droplets into acidic water yields bi-colored dumbbell-shaped aggregates that disassemble into their constituent droplet building blocks upon an increase in pH. Optical tweezers are used to study and quantify the pH dependent interactions of individual droplets.

Microdroplets in microfluidics offer a great number of opportunities in chemical and biological research. They provide a compartment in which species or reactions can be isolated, they are monodisperse and therefore suitable for quantitative studies, they offer the possibility to work with extremely small volumes, single cells, or single molecules, and are suitable for high-throughput experiments. The aim of this Review is to show the importance of these features in enabling new experiments in biology and chemistry. The recent advances in device fabrication are highlighted as are the remaining technological challenges. Examples are presented to show how compartmentalization, monodispersity, single-molecule sensitivity, and high throughput have been exploited in experiments that would have been extremely difficult outside the microfluidics platform.

Here we present a novel surface modification method based on the sequential layer-by-layer deposition of polyelectrolytes yielding hydrophilic microchannels in PDMS-based microfluidic devices. The coatings are long-term stable and allow for the generation of monodisperse oil-in-water microdroplets even several months after the channel surface treatment. Due to the robustness of the polyelectrolyte multilayers ultra-high flow rates can be applied, making high-throughput droplet formation in the jetting mode possible. Furthermore, we successfully used our method to selectively modify the surface properties in certain areas of assembled microchannels. The resulting partially hydrophilic, partially hydrophobic microfluidic devices allow for the production of monodisperse water-in-oil-in-water double emulsions.

Using microfluidic techniques and a novel fluorous-tagged palladium catalyst, we generated droplet reactors with catalytically active walls and used these compartments for small molecule synthesis.

Microdroplets in microfluidics offer a great number of opportunities in chemical and biological research. They provide a compartment in which species or reactions can be isolated, they are monodisperse and therefore suitable for quantitative studies, they offer the possibility to work with extremely small volumes, single cells, or single molecules, and are suitable for high-throughput experiments. The aim of this Review is to show the importance of these features in enabling new experiments in biology and chemistry. The recent advances in device fabrication are highlighted as are the remaining technological challenges. Examples are presented to show how compartmentalization, monodispersity, single-molecule sensitivity, and high throughput have been exploited in experiments that would have been extremely difficult outside the microfluidics platform.

Here we present the design, fabrication and operation of a microfluidic device to trap droplets in a large array of droplet pairs in a controlled manner with the aim of studying the transport of small molecules across the resultant surfactant bilayers formed between the droplet pairs.

There has been an increasing drive toward miniaturizing and accelerating experiments with droplet-based microfluidics across the chemical disciplines. Current applications take advantage of the numerous techniques for manipulating nano- to femtoliter droplets within microfluidic devices. To expand the range of possible applications, we have developed a method for compartmentalizing pure compounds within droplets, at a gradient of concentrations, starting from chemical mixtures. In this technique, a mixture is injected into an ultra performance liquid chromatography (UPLC) system, and droplets are generated from the LC output at a frequency high enough to fraction each compound into approximately 10(5) droplets, compartmentalizing pure compounds into a sequence of droplets with a range of concentrations spanning 2-3 orders of magnitude. Here we used fluorescent dyes to quantify the concentration profile of the droplet collections, and to demonstrate the correspondence between the concentration profile of the droplets and the compound elution profile monitored with a UV absorbance detector, allowing the use of compounds that are not fluorescently labeled but show UV absorbance. Hence this technique is applicable to a wide variety of applications that require both compound purity and the ability to probe a variety of concentrations, such as drug screening and titrations.