SEGMENTED FLOW MICROFLUIDICS

Andrew J. DEMELLO 한국생물공학회 2012 한국생물공학회 학술대회 Vol.2012 No.4

High-Throughput, Quantitative Enzyme Kinetic Analysis in Microdroplets Using Stroboscopic Epifluorescence Imaging

Hess, David,Rane, Anandkumar,deMello, Andrew J.,Stavrakis, Stavros American Chemical Society 2015 ANALYTICAL CHEMISTRY - Vol.87 No.9

<P>Droplet-based microfluidic systems offer a range of advantageous features for the investigation of enzyme kinetics, including high time resolution and the ability to probe extremely large numbers of discrete reactions while consuming low sample volumes. Kinetic measurements within droplet-based microfluidic systems are conventionally performed using single point detection schemes. Unfortunately, such an approach prohibits the measurement of an individual droplet over an extended period of time. Accordingly, we present a novel approach for the extensive characterization of enzyme–inhibitor reaction kinetics within a single experiment by tracking individual and rapidly moving droplets as they pass through an extended microfluidic channel. A series of heterogeneous and pL-volume droplets, containing varying concentrations of the fluorogenic substrate resorufin β-<SMALL>d</SMALL>-galactopyranoside and a constant amount of the enzyme β-galactosidase, is produced at frequencies in excess of 150 Hz. By stroboscopic manipulation of the excitation laser light and adoption of a dual view detection system, “blur-free” images containing up to 150 clearly distinguishable droplets per frame are extracted, which allow extraction of kinetic data from all formed droplets. The efficiency of this approach is demonstrated via a Michaelis–Menten analysis which yields a Michaelis constant, <I>K</I><SUB>m</SUB>, of 353 μM. Additionally, the dissociation constant for the competitive inhibitor isopropyl β-<SMALL>d</SMALL>-1-thiogalactopyranoside is extracted using the same method.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancham/2015/ancham.2015.87.issue-9/acs.analchem.5b00766/production/images/medium/ac-2015-00766c_0008.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/ac5b00766'>ACS Electronic Supporting Info</A></P>

Thermoset polyester droplet-based microfluidic devices for high frequency generation

Kim, Jin-young,deMello, Andrew J.,Chang, Soo-Ik,Hong, Jongin,O'Hare, Danny Royal Society of Chemistry 2011 Lab on a chip Vol.11 No.23

<P>The vast majority of droplet-based microfluidic devices are made from polydimethylsiloxane (PDMS). Unfortunately PDMS is not suitable for high frequency droplet generation at high operating pressure due to its low shear modulus. In this paper, we report the fabrication and testing of microfluidic devices using thermoset polyester (TPE). The optical characteristics of the fabricated devices were assessed and substrate resistance to pressure also investigated. TPE devices bonded using an O<SUB>2</SUB> plasma treated PET substrate at 76 °C were shown to function efficiently at pressures up to 18 MPa. TPE material retains many of the attractive features of PDMS such as ease of fabrication but significantly, has superior mechanical properties. The improved resistance of TPE to high pressures enabled investigation of high frequency droplet generation as a function of a wide range of flow-rates with three different oils as continuous phase.</P> <P>Graphic Abstract</P><P>Droplet-based microfluidic devices to withstand high pressure have been successfully fabricated using thermoset polyester (TPE) materials for high frequency generation of droplets. <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c1lc20603f'> </P>

From single-molecule detection to next-generation sequencing: microfluidic droplets for high-throughput nucleic acid analysis

Ding, Yun,Choo, Jaebum,deMello, Andrew J. Springer-Verlag 2017 Microfluidics and Nanofluidics Vol.21 No.3

<P>Droplet-based microfluidic technologies have proved themselves to be of significant utility in the performance of high-throughput chemical and biological experiments. By encapsulating and isolating reagents within femtoliter-nanoliter droplet, millions of (bio) chemical reactions can be processed in a parallel fashion and on ultra-short timescales. Recent applications of such technologies to genetic analysis have suggested significant utility in low-cost, efficient and rapid workflows for DNA amplification, rare mutation detection, antibody screening and next-generation sequencing. To this end, we describe and highlight some of the most interesting recent developments and applications of droplet-based microfluidics in the broad area of nucleic acid analysis. In addition, we also present a cursory description of some of the most essential functional components, which allow the creation of integrated and complex workflows based on flowing streams of droplets.</P>

Integration of monolithic porous polymer with droplet-based microfluidics on a chip for nano/picoliter volume sample analysis

Kim Jin-young,Chang Soo-Ik,deMello Andrew J,O’Hare Danny 나노기술연구협의회 2014 Nano Convergence Vol.1 No.3

In this paper, a porous polymer nanostructure has been integrated with droplet-based microfluidics in a single planar format. Monolithic porous polymer (MPP) was formed selectively within a microfluidic channel. The resulting analyte bands were sequentially comartmentalised into droplets. This device reduces band broadening and the effects of post-column dead volume by the combination of the two techniques. Moreover it offers the precise control of nano/picoliter volume samples. Background

Interfacial Tension-Mediated Droplet Fusion in Rectangular Microchannels

홍종인,최민석,Joshua B. Edel,Andrew J. deMello 한국바이오칩학회 2009 BioChip Journal Vol.3 No.3

We successfully demonstrate the merging of aqueous droplets within a microfluidic channel mediated by a difference in interfacial tension. Interfacial tension is shown to have a significant influence on the hydrodynamic forces associated with a segmented flow in a rectangular microchannel and results in the possibility of merging multiple droplets in a simple fashion. This facility is important in allowing droplet-based microfluidic systems to be used as synthetic tools in complex reaction processing.

Optofluidic platforms based on surface-enhanced Raman scattering

Lim, Chaesung,Hong, Jongin,Chung, Bong Geun,deMello, Andrew J.,Choo, Jaebum Royal Society of Chemistry 2010 The Analyst Vol.135 No.5

<P>We report recent progress in the development of surface-enhanced Raman scattering (SERS)-based optofluidic platforms for the fast and sensitive detection of chemical and biological analytes. In the current context, a SERS-based optofluidic platform is defined as an integrated analytical device composed of a microfluidic element and a sensitive Raman spectrometer. Optofluidic devices for SERS detection normally involve nanocolloid-based microfluidic systems or metal nanostructure-embedded microfluidic systems. In the current review, recent advances in both approaches are surveyed and assessed. Additionally, integrated real-time sensing systems that combine portable Raman spectrometers with microfluidic devices are also reviewed. Such real-time sensing systems have significant utility in environmental monitoring, forensic science and homeland defense applications.</P> <P>Graphic Abstract</P><P>We report recent progress in the development of surface-enhanced Raman scattering-based optofluidic platforms for the fast and sensitive detection of chemical and biological analytes. <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=b919584j'> </P>

Streptavidin-triggered signal amplified fluorescence polarization for analysis of DNA-protein interactions

Choi, Jae-Won,Jo, Byung-Gwan,deMello, Andrew J.,Choo, Jaebum,Kim, Hak Yong The Royal Society of Chemistry 2016 The Analyst Vol.141 No.24

<P>Fluorescence polarization (FP) is a sensitive, robust, and homogeneous assay format, able to probe a diversity of biological molecules and their interactions. Herein, we describe a new FP strategy based on the use of streptavidin as a signal amplifier. Such signal amplified fluorescence polarization (SAFP) was used to monitor the binding affinity of human angiogenin and a single-stranded DNA aptamer. Streptavidin was bound to a biotinylated single-stranded DNA aptamer and the interaction between this complex and Alexa Fluor 488 labelled human angiogenin was measured. A dissociation constant of 135.3 ± 32.9 nM and a limit of detection of 6.3 nM were successfully extracted only when the FP signal was increased (without binding hindrance) <I>via</I> streptavidin. Moreover, the demonstrated approach was specific to target molecules without any non-specific binding. The streptavidin-triggered SAFP method unlike amplification strategies that utilize nanomaterials (such as graphene oxides, carbon nanotubes, and metal nanoparticles) is not compromised by fluorescence quenching, and it is able to operate within nanomolar concentration regimes. Furthermore, unlike the other FP signal amplification strategies that use dual binding DNA probes, the presented method is simple to implement with signal amplification only requiring the binding of streptavidin with biotinylated DNA. This method could be expanded to analyze molecular interactions and it may be a useful tool for FP measurement by reducing the concentration of rare and expensive protein samples.</P>

Analysis of Protein–Protein Interactions by Using Droplet-Based Microfluidics

Srisa-Art, Monpichar,Kang, Dong-Ku,Hong, Jongin,Park, Hyun,Leatherbarrow, Robin J.,Edel, Joshua B.,Chang, Soo-Ik,deMello, Andrew J. WILEY-VCH Verlag 2009 Chembiochem Vol.10 No.10

<P>Every little drop: The K<SUB>D</SUB> values of angiogenin (ANG) interactions as shown by FRET analysis of thousands of pL-sized droplets agree with data from bulk-fluorescence polarization measurements. Importantly, the use of fluorophores does not affect the activity of ANG or the binding of anti-ANG antibodies to ANG. Such an experimental platform could be applied to the high-throughput analysis of protein–protein interactions. <img src='wiley_img/14394227-2009-10-10-CBIC200800841-content.gif' alt='wiley_img/14394227-2009-10-10-CBIC200800841-content'> </P> <B>Graphic Abstract</B> <P>Every little drop: The K<SUB>D</SUB> values of angiogenin (ANG) interactions as shown by FRET analysis of thousands of pL-sized droplets agree with data from bulk-fluorescence polarization measurements. Importantly, the use of fluorophores does not affect the activity of ANG or the binding of anti-ANG antibodies to ANG. Such an experimental platform could be applied to the high-throughput analysis of protein–protein interactions. <img src='wiley_img/14394227-2009-10-10-CBIC200800841-content.gif' alt='wiley_img/14394227-2009-10-10-CBIC200800841-content'> </P>

Design of a solid-state nanopore-based platform for single-molecule spectroscopy

Hong, Jongin,Lee, Yoonjae,Chansin, Guillaume A T,Edel, Joshua B,deMello, Andrew J IOP Pub 2008 Nanotechnology Vol.19 No.16

<P>We numerically assess the light propagation and distribution characteristics of electromagnetic fields on nanopores formed in dielectric and metal/dielectric membranes using a frequency-domain finite element method (3D full-wave electromagnetic field simulation). Results of such studies were used to identify aluminum as an ideal material for use in optically thick metal/dielectric membranes. The comparison between SiN and Al/SiN membranes (with and without a submicron sized aperture) was numerically and experimentally shown to verify the effect of optically thick metal layers on light propagation and fluorescence excitation. The cut-off behavior for Al/SiN membranes with varying pore diameters was investigated in terms of light propagation, distribution of electromagnetic fields, and light attenuation characteristics. </P>