Todd Duncombe | Joint Bioenergy Institute (original) (raw)

Papers by Todd Duncombe

Insert a brief two or three sentence description of the hardware and the design files.

Insert a brief two or three sentence description of the hardware and the design files.

2017 IEEE 30th International Conference on Micro Electro Mechanical Systems (MEMS), 2017

Droplet microfluidics affords researchers the ability to rapidly generate discretized conditions ... more Droplet microfluidics affords researchers the ability to rapidly generate discretized conditions at kHz rates cheaply and controllably. We leveraged a simple microwell array to enable a parallel process of droplet microarraying, droplet sample deposition through surfactant removal, and thermally actuated device disassembly for facile integration with MALDI mass spectrometry analysis.

<strong>HardwareX manuscript templates</strong> Word document (.docx) OpenDocument Te... more <strong>HardwareX manuscript templates</strong> Word document (.docx) OpenDocument Text (.odt) LaTeX (.tex, overleaf.com) HardwareX manuscripts are submitted at Editorial Manager. Additional instructions can be found in the Guide for Authors. If there is an error in the instructions or something is not clear, contact Co-Editor-In-Chief Todd Duncombe.

SSRN Electronic Journal, 2022

Nature Protocols, 2016

No two cells are the same. Cell-to-cell variation affects biological systems, from development 1 ... more No two cells are the same. Cell-to-cell variation affects biological systems, from development 1 to stem cell biology 2 to cancer 3,4. Unfortunately, conventional bulk measurements mask the biology occurring in each individual cell 5. Single-cell measurement techniques capture cell-to-cell variation, and microfluidic tools have a central role in bringing biochemical assays to single-cell resolution. The small device length scales and precision fluid control can maintain high local concentrations of single-cell lysates 6. In particular, RNA sequencing and genotyping on ten to thousands of single cells is possible, owing to microwell arrays and pneumatic microfluidic valves that control RT-PCR 1,7-11. These tools are making contributions such as elucidating genome diversity and identifying mutations that occur during gametogenesis 1. The study of events that occur at low frequency has also benefited 5. Isolation of rare circulating tumor cells with micropillar arrays 12,13 and RNA analysis in those cells offer the prospect of an enhanced cancer taxonomy for clinical medicine. Nevertheless, even with large strides in single-cell genomics and transcriptomics, nucleic acid expression levels do not always correlate with protein expression levels 14,15 , which drive cell fate. Consequently, advances in single-cell proteomic assays will complement advances in nucleic acid and imaging assays. For a more in-depth treatment of microfluidic single-cell analyses-including signaling dynamics 16 and lineage tracing using microscopy 17-we direct the reader to a recent review of these topics 6. Here we describe a single-cell resolution western blot assay with enhanced selectivity as compared with immunoassays 18,19 , which rely on only antibody binding for detection 20-23. Performing a protein sizing (electrophoretic mobility) stage with a subsequent immunoassay stage can identify protein isoforms and off-target signals that are not discernible with immunoassays alone. scWB performs thousands of concurrent single-cell western blots on one microdevice within a 4-6 h workflow. scWB has been applied to studies ranging from stem cell differentiation 18 to variations in cancer cell responses 19. Development of the protocol The scWB protocol presented here builds upon the efforts of our group to miniaturize western blotting assays for rapid, sensitive and selective quantitative analysis of protein expression 24. The scWB assay leverages microfabrication techniques to pattern microwells that accommodate single cells in a thin PAG 18. A key innovation is UV immobilization (blotting) of resolved proteins in the sieving gel 25 , thus maintaining high local protein concentrations for in-gel immunoprobing. Furthermore, photopatterning of hundreds to thousands of 1-mm-long pore-gradient microgels 26 extends scWB to analyses of wide molecular mass ranges 27. We have characterized cell lysis, PAGE and immunoprobing performance of scWB 19,28 , and critical insight from our findings is included throughout this protocol. scWB is designed to be an economical assay compatible with bench-top equipment commonly found in well-equipped life science laboratories and is adaptable to the needs of users seeking to directly assay proteins in single mammalian cells. Overview of the procedure The procedure begins with a step-by-step guide to scWB microdevice fabrication, including surface silanization of standard glass microscope slides and epoxy-based negative photoresist (SU-8) mold casting of microwells in a thin PAG layered on the glass microscope slide. Options are provided to create either a PAGE gel of uniform pore size, using chemical polymerization with an SU-8 silicon (Si) mold, or a PAGE gel of gradient pore size, using a grayscale photopatterning technique with an SU-8 glass mold. To facilitate in-gel immunoprobing of immobilized proteins with large antibodies, we also describe an alternative gel formulation that incorporates an acid-labile cross-linker chemistry 27. Once the microdevice has been fabricated, the scWB assay workflow comprises five stages (Fig. 1): sedimentation of cells, in-well chemical cell lysis and protein solubilization, PAGE of each cell lysate, photoactivated protein immobilization and in-gel immunoprobing.

Analytical Chemistry, 2016

High throughput, efficient, and readily adoptable analytical tools for the validation and selecti... more High throughput, efficient, and readily adoptable analytical tools for the validation and selection of reliable antibody reagents would impact the life sciences, clinical chemistry, and clinical medicine. To directly quantify antibody−antigen association and dissociation rate constants, k on and k off , in a single experiment, we introduce a microfluidic free-standing kinetic polyacrylamide gel electrophoresis (fsKPAGE) assay. Here, an antibody is immobilized in zones along the length of a single freestanding polyacrylamide gel lane of varying cross-sectional width. Fluorescently labeled antigen is electrophoresed through each immobilized antibody zone, with local cross-sectional area determining the local electric field strength and, thus, the local interaction time between immobilized antibody and electromigrating antigen. Upon crossing, the interaction yields immobilized immunocomplex. The k on is quantified by assessing the amount of immunocomplex formed at each interaction time. To quantify k off , immobilized zones of fluorescently labeled immunocomplex are subjected to a buffer dilution and monitored over time. We determine k on and k off for prostate-specific antigen (PSA) and make a comparison to gold-standard values. The fsKPAGE assay determines k on and k off in a single experiment of less than 20 min, using 45 ng of often limited antibody material and standard laboratory equipment. We see the fsKPAGE assay as forming the basis for rapid, quantitative antibody-screening tools.

This repository describes how to use HardwareX's approved file repositories. A file repositor... more This repository describes how to use HardwareX's approved file repositories. A file repository accompanies the submission of a manuscript using the Editorial Manager online interface and HardwareX's manuscript template. > Zenodo Instructions<br> > Open Science Framework Instructions<br> > Mendeley Data Instructions When submitting to HardwareX, the complete set of design files must be either uploaded to an approved online repository, uploaded at the time of submission on the online Editorial Manager submission interface as supplementary materials (CAD files, videos…), or included in the body of the manuscript (e.g. figures). The three external approved online repositories are Mendeley Data, the Open Science Framework, and Zenodo. The Open Science Framework and Zenodo repositories must be fully public at the time of submission. The Mendeley Data repository can be public or embargoed for up to one year during the review process. Once accepted the Mendeley ...

We describe a platform for high-throughput electrophoretic mobility shift assays (EMSAs) for iden... more We describe a platform for high-throughput electrophoretic mobility shift assays (EMSAs) for identification and characterization of molecular binding reactions. A photopatterned free-standing polyacrylamide gel array comprised of 8 mmscale polyacrylamide gel strips acts as a chassis for 96 concurrent EMSAs. The high-throughput EMSAs was employed to assess binding of the Vc2 cyclic-di-GMP riboswitch to its ligand. In optimizing the riboswitch EMSAs on the free-standing polyacrylamide gel array, three design considerations were made: minimizing sample injection dispersion, mitigating evaporation from the open free-standing polyacrylamide gel structures during electrophoresis, and controlling unit-to-unit variation across the large-format free-standing polyacrylamide gel array. Optimized electrophoretic mobility shift conditions allowed for 10% difference in mobility shift baseline resolution within 3 min. The powerful 96-plex EMSAs increased the throughput to ∼10 data/ min, notably mo...

2017 IEEE 30th International Conference on Micro Electro Mechanical Systems (MEMS), 2017

We present the self-assembly of fluorinated-Au nanoparticle films as a mass-producible fabricatio... more We present the self-assembly of fluorinated-Au nanoparticle films as a mass-producible fabrication methodology for generating nanostructure-initiator mass spectrometry substrates. The nanostructured surfaces enable the direct high sensitivity detection of peptides (20 fmol) and other small molecules using laser desorption ionization. Further, through a photolithographic liftoff technique we can realize micropatterned fluorinated-Au nanoparticle films. These micropatterns create a discrete wettability patterns, allowing us to passively fractionate hydrophobic molecules of interest from high-salt background environments for robust and predictable MS.

We report here for the first time a new open-microchannel architecture for rapid protein electrop... more We report here for the first time a new open-microchannel architecture for rapid protein electrophoresis. Owing to its open architecture, this technology is optimized for interfacing with automated robotic controllers and downstream processing (e.g., sample spotters, immunological probing, mass spectroscopy). We show the design, fabrication and initial feasibility testing of an open-microchannel array with favorable attributes for up-scaling into high-throughput electrophoretic separations. This multiplexed open-channel platform is uniquely well-suited for massively parallelized proteomics, a major unrealized goal from bioanalytical technology.

Author(s): Duncombe, Todd Andrew | Advisor(s): Herr, Amy E | Abstract: Seminal bioanalytical tech... more Author(s): Duncombe, Todd Andrew | Advisor(s): Herr, Amy E | Abstract: Seminal bioanalytical technologies for high-throughput analysis, such as flow cytometry and capillary electrophoresis, were leveraging microfluidic physical phenomena long before the advent of the term “microfluidics”. Transitioning from the initial solid-state micro-electronic fabrication approaches, microfluidic fabrication has moved towards polymer based technologies that are amenable to a rapid design, prototype, and test development cycle. In my dissertation, I took advantage of these features to create new tools for performing electrophoresis-based protein assays over a range of applications, including, rapid low-power electrophoretic immunoassays, open-microfluidic ‘soft-MEMS’ platform for high-throughput protein analysis, and spatially a temporally controlled separation media for enhanced single-cell western blotting assays.Rapid low-power electrophoretic immunoassay: To reduce the power requirements for ...

2012 Solid-State, Actuators, and Microsystems Workshop Technical Digest, May 23, 2012

We report a new open-channel architecture that enables the utility of microfluidics without sacri... more We report a new open-channel architecture that enables the utility of microfluidics without sacrificing accessibilityfor high performance protein electrophoresis. We utilize photo-patterned free-standing polyacrylamide gels as both an open-microchannel and a sieving matrix for protein sizing. The open format allows for easy downstream sample access for immunoblotting after rapid microfluidic protein separations are performed. For the purposes of multiplexing, the assay footprint is minimized to a single channel through the implementation of moving boundary electrophoresis protein separations.

We introduce the UV−vis spectra-activated droplet sorter (UVADS) for high-throughput label-free c... more We introduce the UV−vis spectra-activated droplet sorter (UVADS) for high-throughput label-free chemical identification and enzyme screening. In contrast to previous absorbancebased droplet sorters that relied on single-wavelength absorbance in the visible range, our platform collects full UV−vis spectra from 200 to 1050 nm at up to 2100 spectra per second. Our custombuilt open-source software application, "SpectraSorter," enables real-time data processing, analysis, visualization, and selection of droplets for sorting with any set of UV−vis spectral features. An optimized UV−vis detection region extended the absorbance path length for droplets and allowed for the direct protein quantification down to 10 μM of bovine serum albumin at 280 nm. UV−vis spectral data can distinguish a variety of different chemicals or spurious events (such as air bubbles) that are inaccessible at a single wavelength. The platform is used to measure ergothionase enzyme activity from monoclonal microcolonies isolated in droplets. In a label-free manner, we directly measure the ergothioneine substrate to thiourocanic acid product conversion while tracking the microcolony formation. UVADS represents an important new tool for high-throughput label-free in-droplet chemical analysis.

Communications Biology

In the field of bottom-up synthetic biology, lipid membranes are the scaffold to create minimal c... more In the field of bottom-up synthetic biology, lipid membranes are the scaffold to create minimal cells and mimic reactions and processes at or across the membrane. In this context, we employ here a versatile microfluidic platform that enables precise positioning of nanoliter droplets with user-specified lipid compositions and in a defined pattern. Adjacent droplets make contact and form a droplet interface bilayer to simulate cellular membranes. Translocation of molecules across membranes are tailored by the addition of alpha-hemolysin to selected droplets. Moreover, we developed a protocol to analyze the translocation of non-fluorescent molecules between droplets with mass spectrometry. Our method is capable of automated formation of one- and two-dimensional droplet networks, which we demonstrated by connecting droplets containing different compound and enzyme solutions to perform translocation experiments and a multistep enzymatic cascade reaction across the droplet network. Our pl...

Current Opinion in Biotechnology

Insert a brief two or three sentence description of the hardware and the design files.

Insert a brief two or three sentence description of the hardware and the design files.

2017 IEEE 30th International Conference on Micro Electro Mechanical Systems (MEMS), 2017

Droplet microfluidics affords researchers the ability to rapidly generate discretized conditions ... more Droplet microfluidics affords researchers the ability to rapidly generate discretized conditions at kHz rates cheaply and controllably. We leveraged a simple microwell array to enable a parallel process of droplet microarraying, droplet sample deposition through surfactant removal, and thermally actuated device disassembly for facile integration with MALDI mass spectrometry analysis.

<strong>HardwareX manuscript templates</strong> Word document (.docx) OpenDocument Te... more <strong>HardwareX manuscript templates</strong> Word document (.docx) OpenDocument Text (.odt) LaTeX (.tex, overleaf.com) HardwareX manuscripts are submitted at Editorial Manager. Additional instructions can be found in the Guide for Authors. If there is an error in the instructions or something is not clear, contact Co-Editor-In-Chief Todd Duncombe.

SSRN Electronic Journal, 2022

Nature Protocols, 2016

No two cells are the same. Cell-to-cell variation affects biological systems, from development 1 ... more No two cells are the same. Cell-to-cell variation affects biological systems, from development 1 to stem cell biology 2 to cancer 3,4. Unfortunately, conventional bulk measurements mask the biology occurring in each individual cell 5. Single-cell measurement techniques capture cell-to-cell variation, and microfluidic tools have a central role in bringing biochemical assays to single-cell resolution. The small device length scales and precision fluid control can maintain high local concentrations of single-cell lysates 6. In particular, RNA sequencing and genotyping on ten to thousands of single cells is possible, owing to microwell arrays and pneumatic microfluidic valves that control RT-PCR 1,7-11. These tools are making contributions such as elucidating genome diversity and identifying mutations that occur during gametogenesis 1. The study of events that occur at low frequency has also benefited 5. Isolation of rare circulating tumor cells with micropillar arrays 12,13 and RNA analysis in those cells offer the prospect of an enhanced cancer taxonomy for clinical medicine. Nevertheless, even with large strides in single-cell genomics and transcriptomics, nucleic acid expression levels do not always correlate with protein expression levels 14,15 , which drive cell fate. Consequently, advances in single-cell proteomic assays will complement advances in nucleic acid and imaging assays. For a more in-depth treatment of microfluidic single-cell analyses-including signaling dynamics 16 and lineage tracing using microscopy 17-we direct the reader to a recent review of these topics 6. Here we describe a single-cell resolution western blot assay with enhanced selectivity as compared with immunoassays 18,19 , which rely on only antibody binding for detection 20-23. Performing a protein sizing (electrophoretic mobility) stage with a subsequent immunoassay stage can identify protein isoforms and off-target signals that are not discernible with immunoassays alone. scWB performs thousands of concurrent single-cell western blots on one microdevice within a 4-6 h workflow. scWB has been applied to studies ranging from stem cell differentiation 18 to variations in cancer cell responses 19. Development of the protocol The scWB protocol presented here builds upon the efforts of our group to miniaturize western blotting assays for rapid, sensitive and selective quantitative analysis of protein expression 24. The scWB assay leverages microfabrication techniques to pattern microwells that accommodate single cells in a thin PAG 18. A key innovation is UV immobilization (blotting) of resolved proteins in the sieving gel 25 , thus maintaining high local protein concentrations for in-gel immunoprobing. Furthermore, photopatterning of hundreds to thousands of 1-mm-long pore-gradient microgels 26 extends scWB to analyses of wide molecular mass ranges 27. We have characterized cell lysis, PAGE and immunoprobing performance of scWB 19,28 , and critical insight from our findings is included throughout this protocol. scWB is designed to be an economical assay compatible with bench-top equipment commonly found in well-equipped life science laboratories and is adaptable to the needs of users seeking to directly assay proteins in single mammalian cells. Overview of the procedure The procedure begins with a step-by-step guide to scWB microdevice fabrication, including surface silanization of standard glass microscope slides and epoxy-based negative photoresist (SU-8) mold casting of microwells in a thin PAG layered on the glass microscope slide. Options are provided to create either a PAGE gel of uniform pore size, using chemical polymerization with an SU-8 silicon (Si) mold, or a PAGE gel of gradient pore size, using a grayscale photopatterning technique with an SU-8 glass mold. To facilitate in-gel immunoprobing of immobilized proteins with large antibodies, we also describe an alternative gel formulation that incorporates an acid-labile cross-linker chemistry 27. Once the microdevice has been fabricated, the scWB assay workflow comprises five stages (Fig. 1): sedimentation of cells, in-well chemical cell lysis and protein solubilization, PAGE of each cell lysate, photoactivated protein immobilization and in-gel immunoprobing.

Analytical Chemistry, 2016

High throughput, efficient, and readily adoptable analytical tools for the validation and selecti... more High throughput, efficient, and readily adoptable analytical tools for the validation and selection of reliable antibody reagents would impact the life sciences, clinical chemistry, and clinical medicine. To directly quantify antibody−antigen association and dissociation rate constants, k on and k off , in a single experiment, we introduce a microfluidic free-standing kinetic polyacrylamide gel electrophoresis (fsKPAGE) assay. Here, an antibody is immobilized in zones along the length of a single freestanding polyacrylamide gel lane of varying cross-sectional width. Fluorescently labeled antigen is electrophoresed through each immobilized antibody zone, with local cross-sectional area determining the local electric field strength and, thus, the local interaction time between immobilized antibody and electromigrating antigen. Upon crossing, the interaction yields immobilized immunocomplex. The k on is quantified by assessing the amount of immunocomplex formed at each interaction time. To quantify k off , immobilized zones of fluorescently labeled immunocomplex are subjected to a buffer dilution and monitored over time. We determine k on and k off for prostate-specific antigen (PSA) and make a comparison to gold-standard values. The fsKPAGE assay determines k on and k off in a single experiment of less than 20 min, using 45 ng of often limited antibody material and standard laboratory equipment. We see the fsKPAGE assay as forming the basis for rapid, quantitative antibody-screening tools.

This repository describes how to use HardwareX's approved file repositories. A file repositor... more This repository describes how to use HardwareX's approved file repositories. A file repository accompanies the submission of a manuscript using the Editorial Manager online interface and HardwareX's manuscript template. > Zenodo Instructions<br> > Open Science Framework Instructions<br> > Mendeley Data Instructions When submitting to HardwareX, the complete set of design files must be either uploaded to an approved online repository, uploaded at the time of submission on the online Editorial Manager submission interface as supplementary materials (CAD files, videos…), or included in the body of the manuscript (e.g. figures). The three external approved online repositories are Mendeley Data, the Open Science Framework, and Zenodo. The Open Science Framework and Zenodo repositories must be fully public at the time of submission. The Mendeley Data repository can be public or embargoed for up to one year during the review process. Once accepted the Mendeley ...

We describe a platform for high-throughput electrophoretic mobility shift assays (EMSAs) for iden... more We describe a platform for high-throughput electrophoretic mobility shift assays (EMSAs) for identification and characterization of molecular binding reactions. A photopatterned free-standing polyacrylamide gel array comprised of 8 mmscale polyacrylamide gel strips acts as a chassis for 96 concurrent EMSAs. The high-throughput EMSAs was employed to assess binding of the Vc2 cyclic-di-GMP riboswitch to its ligand. In optimizing the riboswitch EMSAs on the free-standing polyacrylamide gel array, three design considerations were made: minimizing sample injection dispersion, mitigating evaporation from the open free-standing polyacrylamide gel structures during electrophoresis, and controlling unit-to-unit variation across the large-format free-standing polyacrylamide gel array. Optimized electrophoretic mobility shift conditions allowed for 10% difference in mobility shift baseline resolution within 3 min. The powerful 96-plex EMSAs increased the throughput to ∼10 data/ min, notably mo...

2017 IEEE 30th International Conference on Micro Electro Mechanical Systems (MEMS), 2017

We present the self-assembly of fluorinated-Au nanoparticle films as a mass-producible fabricatio... more We present the self-assembly of fluorinated-Au nanoparticle films as a mass-producible fabrication methodology for generating nanostructure-initiator mass spectrometry substrates. The nanostructured surfaces enable the direct high sensitivity detection of peptides (20 fmol) and other small molecules using laser desorption ionization. Further, through a photolithographic liftoff technique we can realize micropatterned fluorinated-Au nanoparticle films. These micropatterns create a discrete wettability patterns, allowing us to passively fractionate hydrophobic molecules of interest from high-salt background environments for robust and predictable MS.

We report here for the first time a new open-microchannel architecture for rapid protein electrop... more We report here for the first time a new open-microchannel architecture for rapid protein electrophoresis. Owing to its open architecture, this technology is optimized for interfacing with automated robotic controllers and downstream processing (e.g., sample spotters, immunological probing, mass spectroscopy). We show the design, fabrication and initial feasibility testing of an open-microchannel array with favorable attributes for up-scaling into high-throughput electrophoretic separations. This multiplexed open-channel platform is uniquely well-suited for massively parallelized proteomics, a major unrealized goal from bioanalytical technology.

Author(s): Duncombe, Todd Andrew | Advisor(s): Herr, Amy E | Abstract: Seminal bioanalytical tech... more Author(s): Duncombe, Todd Andrew | Advisor(s): Herr, Amy E | Abstract: Seminal bioanalytical technologies for high-throughput analysis, such as flow cytometry and capillary electrophoresis, were leveraging microfluidic physical phenomena long before the advent of the term “microfluidics”. Transitioning from the initial solid-state micro-electronic fabrication approaches, microfluidic fabrication has moved towards polymer based technologies that are amenable to a rapid design, prototype, and test development cycle. In my dissertation, I took advantage of these features to create new tools for performing electrophoresis-based protein assays over a range of applications, including, rapid low-power electrophoretic immunoassays, open-microfluidic ‘soft-MEMS’ platform for high-throughput protein analysis, and spatially a temporally controlled separation media for enhanced single-cell western blotting assays.Rapid low-power electrophoretic immunoassay: To reduce the power requirements for ...

2012 Solid-State, Actuators, and Microsystems Workshop Technical Digest, May 23, 2012

We report a new open-channel architecture that enables the utility of microfluidics without sacri... more We report a new open-channel architecture that enables the utility of microfluidics without sacrificing accessibilityfor high performance protein electrophoresis. We utilize photo-patterned free-standing polyacrylamide gels as both an open-microchannel and a sieving matrix for protein sizing. The open format allows for easy downstream sample access for immunoblotting after rapid microfluidic protein separations are performed. For the purposes of multiplexing, the assay footprint is minimized to a single channel through the implementation of moving boundary electrophoresis protein separations.

We introduce the UV−vis spectra-activated droplet sorter (UVADS) for high-throughput label-free c... more We introduce the UV−vis spectra-activated droplet sorter (UVADS) for high-throughput label-free chemical identification and enzyme screening. In contrast to previous absorbancebased droplet sorters that relied on single-wavelength absorbance in the visible range, our platform collects full UV−vis spectra from 200 to 1050 nm at up to 2100 spectra per second. Our custombuilt open-source software application, "SpectraSorter," enables real-time data processing, analysis, visualization, and selection of droplets for sorting with any set of UV−vis spectral features. An optimized UV−vis detection region extended the absorbance path length for droplets and allowed for the direct protein quantification down to 10 μM of bovine serum albumin at 280 nm. UV−vis spectral data can distinguish a variety of different chemicals or spurious events (such as air bubbles) that are inaccessible at a single wavelength. The platform is used to measure ergothionase enzyme activity from monoclonal microcolonies isolated in droplets. In a label-free manner, we directly measure the ergothioneine substrate to thiourocanic acid product conversion while tracking the microcolony formation. UVADS represents an important new tool for high-throughput label-free in-droplet chemical analysis.

Communications Biology

In the field of bottom-up synthetic biology, lipid membranes are the scaffold to create minimal c... more In the field of bottom-up synthetic biology, lipid membranes are the scaffold to create minimal cells and mimic reactions and processes at or across the membrane. In this context, we employ here a versatile microfluidic platform that enables precise positioning of nanoliter droplets with user-specified lipid compositions and in a defined pattern. Adjacent droplets make contact and form a droplet interface bilayer to simulate cellular membranes. Translocation of molecules across membranes are tailored by the addition of alpha-hemolysin to selected droplets. Moreover, we developed a protocol to analyze the translocation of non-fluorescent molecules between droplets with mass spectrometry. Our method is capable of automated formation of one- and two-dimensional droplet networks, which we demonstrated by connecting droplets containing different compound and enzyme solutions to perform translocation experiments and a multistep enzymatic cascade reaction across the droplet network. Our pl...

Current Opinion in Biotechnology