Patrick F. Wagler

DNA computing in microreactors

The goal of this research is to improve the modular stability and programmability of DNA-based computers and in a second step towards optical programmable DNA computing. The main focus here is on hydrodynamic stability. Clockable microreactors can be connected in various ways to solve combinatorial optimisation problems, such as Maximum Clique or 3-SAT. This work demonstrates by construction how one micro-reactor design can be programmed to solve any instance of Maximum Clique up to its given maximum size (N). It reports on an implementation of the architecture proposed previously. This contrasts with conventional DNA computing where the individual sequence of biochemical operations depends on the specific problem. In this pilot study we are tackling a graph for the Maximum Clique problem with N<EQ12, with a special emphasis for Nequals6. Furthermore, the design of the DNA solution space will be presented, which is symbolized by a set of bit-strings (words).

Microfabrication of a BioModule composed of microfluidics and digitally controlled microelectrodes for processing biomolecules

This work focuses on the development of an online programmable microfluidic bioprocessing unit (BioModule) using digital logic microelectrodes for rapid pipelined selection and transfer of deoxyribonucleic acid (DNA) molecules and other charged biopolymers. The design and construction technique for this hybrid programmable biopolymer processing device is presented along with the first proof of principle functionality. The electronically controlled collection, separation and channel transfer of the biomolecules is monitored by a sensitive fluorescence set-up. This hybrid reconfigurable architecture couples electronic and biomolecular information processing via a single module combination of fluidics and electronics and opens new fields of applications not only in DNA computing and molecular diagnostics but also in applications of combinatorial chemistry and lab-on-a-chip biotechnology to the drug discovery process. Fundamentals of the design and silicon–polydimethylsiloxane (PDMS)-based construction of these electronic microfluidic devices and their functions are described as well as the experimental results.

On demand nanoliter-scale microfluidic droplet generation, injection, and mixing using a passive microfluidic device

We here present and characterize a programmable nanoliter scale droplet-on-demand device that can be used separately or readily integrated into low cost single layer rapid prototyping microfluidic systems for a wide range of user applications. The passive microfluidic device allows external (off-the-shelf) electronically controlled pinch valves to program the delivery of nanoliter scale aqueous droplets from up to 9 different inputs to a central outlet channel. The inputs can be either continuous aqueous fluid streams or microliter scale aqueous plugs embedded in a carrier fluid, in which case the number of effective input solutions that can be employed in an experiment is no longer strongly constrained (100 s-1000 s). Both nanoliter droplet sequencing output and nanoliter-scale droplet mixing are reported with this device. Optimization of the geometry and pressure relationships in the device was achieved in several hardware iterations with the support of open source microfluidic simulation software and equivalent circuit models. The requisite modular control of pressure relationships within the device is accomplished using hydrodynamic barriers and matched resistance channels with three different channel heights, custom parallel reversible microfluidic I/O connections, low dead-volume pinch valves, and a simply adjustable array of external screw valves. Programmable sequences of droplet mixes or chains of droplets can be achieved with the device at low Hz frequencies, limited by device elasticity, and could be further enhanced by valve integration. The chip has already found use in the characterization of droplet bunching during export and the synthesis of a DNA library.

Hybrid poly(dimethylsiloxane)-silicon microreactors used for molecular computing

The goal of this research is to improve the modular stability and programmability of DNA-based computers and is a second step towards optical programmable DNA computing. The main focus here is on hydrodynamic stability. Clockable microreactors can be connected in various ways to solve combinatorial optimization problems, such as maximum clique or 3-SAT. This work demonstrates by construction how one microreactor design can be programmed to solve any instance of maximum clique up to its given maximum size (N). It reports on an implementation of the architecture proposed previously (McCaskill J S 2001 Biosystems 59 125?38). This contrasts with conventional DNA computing where the individual sequence of biochemical operations depends on the specific problem. In this pilot study we are tackling a graph for the maximum clique problem with N ? 12, with a special emphasis on N = 6. Furthermore, the design of the DNA solution space will be presented, which is symbolized by a set of bit-strings (words).

An Electronically Controlled Microfluidic Approach towards Artificial Cells

This work focuses on the application of on-line programmable microfluidic bioprocessing as a complementation vehicle towards the design of artificial cells. The electronically controlled collection, separation and channel transfer of the biomolecules are monitored by a sensitive fluorescence setup. Two different physical effects, electrophoresis and electroosmotic flow, are used to allow for a detailed micro-control of fluids in micro-reaction environments. A combination of these two basic electronically controlled input reaction chambers makes combinatorial fluidic networks and indefinitely sustained biochemical or chemical reaction networks feasible. Experimental data showing the power of this approach is presented. Not only does this processing power pave the way towards the development of artificial cells (using a technology to complement not yet established autonomous metabolic or replication capabilities) but it also opens up new processes for applications of combinatorial chemistry and lab-on-a-chip biotechnology to drug discovery and diagnosis.

Field programmable chemistry: Integrated chemical and electronic processing of informational molecules towards electronic chemical cells

The topic addressed is that of combining self-constructing chemical systems with electronic computation to form unconventional embedded computation systems performing complex nano-scale chemical tasks autonomously. The hybrid route to complex programmable chemistry, and ultimately to artificial cells based on novel chemistry, requires a solution of the two-way massively parallel coupling problem between digital electronics and chemical systems. We present a chemical microprocessor technology and show how it can provide a generic programmable platform for complex molecular processing tasks in Field Programmable Chemistry, including steps towards the grand challenge of constructing the first electronic chemical cells. Field programmable chemistry employs a massively parallel field of electrodes, under the control of latched voltages, which are used to modulate chemical activity. We implement such a field programmable chemistry which links to chemistry in rather generic, two-phase microfluidic channel networks that are separated into weakly coupled domains. Electric fields, produced by the high-density array of electrodes embedded in the channel floors, are used to control the transport of chemicals across the hydrodynamic barriers separating domains. In the absence of electric fields, separate microfluidic domains are essentially independent with only slow diffusional interchange of chemicals. Electronic chemical cells, based on chemical microprocessors, exploit a spatially resolved sandwich structure in which the electronic and chemical systems are locally coupled through homogeneous fine-grained actuation and sensor networks and play symmetric and complementary roles. We describe how these systems are fabricated, experimentally test their basic functionality, simulate their potential (e.g. for feed forward digital electrophoretic (FFDE) separation) and outline the application to building electronic chemical cells.

Molecular systems on-chip (MSoC) steps forward for programmable biosystems

This work describes online programmable microfluidic bioprocessing units using digital logic microelectrodes for rapid pipelined translocation of DNA molecules and other charged biopolymers as well as nanoparticles. Fundamentals of the design and fabrication technique both the silicon-PDMS and a polyimide-PDMS based construction (a new method based on conventional printed circuit board materials) of these electronic microfluidic devices and their functions are described as well as the experimental results along with the first proof of principle functionality. The electronically controlled collection, separation and channel transfer of the biomolecules and nanosized beads are monitored by a sen-sitive fluorescence setup and controlled by a custom-designed hardware for camera-control and feature selection. This hybrid reconfigurable architecture couples electronic and biomolecular information processing via a single module combination of fluidics and electronics and opens new fields of applications not only in DNA computing and molecular diagnostics but also in applications of combinatorial chemistry and lab-on-a-chip biotechnology to the drug discovery process.

Electronic pH switching of DNA triplex reactions

Electronic reversible switching of sequence-specific DNA interactions and reactions is an important operation for programming complex molecular and microscopic processes. While both quadruplex and triplex structures are suitable for moderate pH control (pH 5–7), we focus here on the large family of DNA sequences forming pH-sensitive triplex structures. These involve Hoogsteen and Watson–Crick base pairs in pyrimidine–purine–pyrimidine (Y:R:Y) motifs. We demonstrate electronically controlled local pH cycling, integrated into a microfluidic chip, which induces DNA hybridization switching in these triplex complexes. We also show that pH switching can be used to control rapid DNA ligation in double strand templated triplex structures using disulphide linkages. Switching between DNA complexes induced by pH is first characterized using capillary gel electrophoresis before employing local microelectrodes. Robust pH cycling is achieved over a moderate pH range (4–8), by voltage-biased gold microelectrodes immersed in quinhydrone redox systems, both in solution and immobilized on the surface, and is monitored via fluorescence ratio imaging with SNARF-4F. The resulting switching of DNA structures (reversible triplex to duplex, triplex-based DNA ligation) based on a hairpin template is spatially monitored by dye–quencher fluorescence. The integration of electronically controlled pH cycling into a microfluidic reactor allows both local patterning of pH and the maintenance of constant ionic strength over many cycles.

DNA-library assembly programmed by on-demand nano-liter droplets from a custom microfluidic chip

Nanoscale synthetic biology can benefit from programmable nanoliter-scale processing of DNA in microfluidic chips if they are interfaced effectively to biochemical arrays such as microwell plates. Whereas active microvalve chips require complex fabrication and operation, we show here how a passive and readily fabricated microchip can be employed for customizable nanoliter scale pipetting and reaction control involving DNA. This recently developed passive microfluidic device, supporting nanoliter scale combinatorial droplet generation and mixing, is here used to generate a DNA test library with one member per droplet exported to addressed locations on microwell plates. Standard DNA assembly techniques, such as Gibson assembly, compatible with isothermal on-chip operation, are employed and checked using off-chip PCR and assembly PCR. The control of output droplet sequences and mixing performance was verified using dyes and fluorescently labeled DNA solutions, both on-chip and in external capillary channels. Gel electrophoresis of products and DNA sequencing were employed to further verify controlled combination and functional enzymatic assembly. The scalability of the results to larger DNA libraries is also addressed by combinatorial input expansion using sequential injection plugs from a multiwell plate. Hence, the paper establishes a proof of principle of the production of functional combinatorial mixtures at the nanoliter scale for one sequence per well DNA libraries.

General-Purpose, Parallel and Reversible Microfluidic Interconnects

The packaging of microfluidic chips is a limiting factor in their ease of use and widespread application. A simple and versatile casting procedure to fabricate a parallel minimal dead-volume connector using polydimethylsiloxane elastomers without the application of any adhesive materials is demonstrated. The interconnections reversibly join standard-sized teflon, glass, or plastic tubing to modular microfluidic systems with minimal dead volume for biological and chemical applications. Parallel port connectors with 18 and 35 channels have been developed to illustrate the applicability of this novel universal fluidic interface to the large-scale integration of microfluidic (chip) devices, which will scale without problem to 100 parallel channels. The advantages of this novel interface are evaluated in relation to a comprehensive classification and analysis of other world to chip interconnection technologies.