Production of Modified Nucleosides in a Continuous Enzyme Membrane Reactor.

Nucleoside analogues are important compounds for the treatment of viral infections or cancers. While (chemo-)enzymatic synthesis is a valuable alternative to traditional chemical methods, the feasibility of such processes is lowered by the high production cost of the biocatalyst. As continuous enzyme membrane reactors (EMR) allow the use of biocatalysts until their full inactivation, they offer a valuable alternative to batch enzymatic reactions with freely dissolved enzymes. In EMRs, the enzymes are retained in the reactor by a suitable membrane. Immobilization on carrier materials, and the associated losses in enzyme activity, can thus be avoided. Therefore, we validated the applicability of EMRs for the synthesis of natural and dihalogenated nucleosides, using one-pot transglycosylation reactions. Over a period of 55 days, 2'-deoxyadenosine was produced continuously, with a product yield >90%. The dihalogenated nucleoside analogues 2,6-dichloropurine-2'-deoxyribonucleoside and 6-chloro-2-fluoro-2'-deoxyribonucleoside were also produced, with high conversion, but for shorter operation times, of 14 and 5.5 days, respectively. The EMR performed with specific productivities comparable to batch reactions. However, in the EMR, 220, 40, and 9 times more product per enzymatic unit was produced, for 2'-deoxyadenosine, 2,6-dichloropurine-2'-deoxyribonucleoside, and 6-chloro-2-fluoro-2'-deoxyribonucleoside, respectively. The application of the EMR using freely dissolved enzymes, facilitates a continuous process with integrated biocatalyst separation, which reduces the overall cost of the biocatalyst and enhances the downstream processing of nucleoside production.

Dielectrophoresis: An Approach to Increase Sensitivity, Reduce Response Time and to Suppress Nonspecific Binding in Biosensors?

The performance of receptor-based biosensors is often limited by either diffusion of the analyte causing unreasonable long assay times or a lack of specificity limiting the sensitivity due to the noise of nonspecific binding. Alternating current (AC) electrokinetics and its effect on biosensing is an increasing field of research dedicated to address this issue and can improve mass transfer of the analyte by electrothermal effects, electroosmosis, or dielectrophoresis (DEP). Accordingly, several works have shown improved sensitivity and lowered assay times by order of magnitude thanks to the improved mass transfer with these techniques. To realize high sensitivity in real samples with realistic sample matrix avoiding nonspecific binding is critical and the improved mass transfer should ideally be specific to the target analyte. In this paper we cover recent approaches to combine biosensors with DEP, which is the AC kinetic approach with the highest selectivity. We conclude that while associated with many challenges, for several applications the approach could be beneficial, especially if more work is dedicated to minimizing nonspecific bindings, for which DEP offers interesting perspectives.

Dielectrophoretic separation of blood cells.

Microfluidic dielectrophoretic (DEP) devices enable the label-free separation and isolation of cells based on differences in their electrophysiological properties. The technique can serve as a tool in clinical diagnostics and medical research as it facilitates the analysis of patient-specific blood composition and the detection and isolation of pathogenic cells like circulating tumor cells or malaria-infected erythrocytes. This review compares different microfluidic DEP devices to separate platelets, erythrocytes and leukocytes including their cellular subclasses. An overview and experimental setups of different microfluidic DEP devices for the separation, trapping and isolation or purification of blood cells are detailed with respect to their technical design, electrode configuration, sample preparation, applied voltage and frequency and created DEP field based and related to the separation efficiency. The technique holds the promise that results can quickly be attained in clinical and ambulant settings. In particular, point-of-care-testing scenarios are favored by the extensive miniaturization, which would be enabled by microelectronical integration of DEP devices.

Separation of Heterotrophic Microalgae Crypthecodinium cohnii by Dielectrophoresis.

Microalgae constitute an abundant source of poly-unsaturated fatty acids which are applied in various biotechnological fields such as pharmaceuticals and food supplement. Separating microalgae cells with respect to their lipid content would establish a relevant at-line analytical technique. The present study demonstrates an electrical approach for the separation of the lipid-producing microalgae Crypthecodinium cohnii using the effect of dielectrophoresis (DEP) in a microfluidic flow cell. Microalgae were cultivated for 8 days, while cell growth was characterized by optical density, dry cell weight, glucose concentration and lipid content via fluorescence microscopy. The size distribution of cells during cultivation was thoroughly investigated, since the DEP force scales with cell volume, but also depends on lipid content via cell electrophysiological constants. Thus, the challenge was to deconvolute one separation effect from the other, while the electrical cell constants of C. cohnii are not known yet. The DEP-dependent separation was realized by slanted top-bottom electrodes with the flowing cell suspension between them. Turning on the voltage deflected the cells from their initial path as determined by the streaming and thus changed their direction of flow. The separation efficiency of DEP was tested for various electrical field strengths and its performance was determined by quantitative analysis of optical and fluorescence videos. It could be shown for all size groups that the most lipid-containing cells were always subject to DEP separation and that the method is thus not only suitable for process analysis, but also for strain selection of the most productive cell lines.

An Approach to Ring Resonator Biosensing Assisted by Dielectrophoresis: Design, Simulation and Fabrication.

The combination of extreme miniaturization with a high sensitivity and the potential to be integrated in an array form on a chip has made silicon-based photonic microring resonators a very attractive research topic. As biosensors are approaching the nanoscale, analyte mass transfer and bonding kinetics have been ascribed as crucial factors that limit their performance. One solution may be a system that applies dielectrophoretic forces, in addition to microfluidics, to overcome the diffusion limits of conventional biosensors. Dielectrophoresis, which involves the migration of polarized dielectric particles in a non-uniform alternating electric field, has previously been successfully applied to achieve a 1000-fold improved detection efficiency in nanopore sensing and may significantly increase the sensitivity in microring resonator biosensing. In the current work, we designed microring resonators with integrated electrodes next to the sensor surface that may be used to explore the effect of dielectrophoresis. The chip design, including two different electrode configurations, electric field gradient simulations, and the fabrication process flow of a dielectrohoresis-enhanced microring resonator-based sensor, is presented in this paper. Finite element method (FEM) simulations calculated for both electrode configurations revealed ∇E2 values above 1017 V2m-3 around the sensing areas. This is comparable to electric field gradients previously reported for successful interactions with larger molecules, such as proteins and antibodies.

Separation, Characterization, and Handling of Microalgae by Dielectrophoresis.

Microalgae biotechnology has a high potential for sustainable bioproduction of diverse high-value biomolecules. Some of the main bottlenecks in cell-based bioproduction, and more specifically in microalgae-based bioproduction, are due to insufficient methods for rapid and efficient cell characterization, which contributes to having only a few industrially established microalgal species in commercial use. Dielectrophoresis-based microfluidic devices have been long established as promising tools for label-free handling, characterization, and separation of broad ranges of cells. The technique is based on differences in dielectric properties and sizes, which results in different degrees of cell movement under an applied inhomogeneous electrical field. The method has also earned interest for separating microalgae based on their intrinsic properties, since their dielectric properties may significantly change during bioproduction, in particular for lipid-producing species. Here, we provide a comprehensive review of dielectrophoresis-based microfluidic devices that are used for handling, characterization, and separation of microalgae. Additionally, we provide a perspective on related areas of research in cell-based bioproduction that can benefit from dielectrophoresis-based microdevices. This work provides key information that will be useful for microalgae researchers to decide whether dielectrophoresis and which method is most suitable for their particular application.

Comparison of time-gated surface-enhanced raman spectroscopy (TG-SERS) and classical SERS based monitoring of Escherichia coli cultivation samples.

The application of Raman spectroscopy as a monitoring technique for bioprocesses is severely limited by a large background signal originating from fluorescing compounds in the culture media. Here, we compare time-gated Raman (TG-Raman)-, continuous wave NIR-process Raman (NIR-Raman), and continuous wave micro-Raman (micro-Raman) approaches in combination with surface enhanced Raman spectroscopy (SERS) for their potential to overcome this limit. For that purpose, we monitored metabolite concentrations of Escherichia coli bioreactor cultivations in cell-free supernatant samples. We investigated concentration transients of glucose, acetate, AMP, and cAMP at alternating substrate availability, from deficiency to excess. Raman and SERS signals were compared to off-line metabolite analysis of carbohydrates, carboxylic acids, and nucleotides. Results demonstrate that SERS, in almost all cases, led to a higher number of identifiable signals and better resolved spectra. Spectra derived from the TG-Raman were comparable to those of micro-Raman resulting in well-discernable Raman peaks, which allowed for the identification of a higher number of compounds. In contrast, NIR-Raman provided a superior performance for the quantitative evaluation of analytes, both with and without SERS nanoparticles when using multivariate data analysis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1533-1542, 2018.

Prolonged Corrosion Stability of a Microchip Sensor Implant during In Vivo Exposure.

A microelectronic biosensor was subjected to in vivo exposure by implanting it in the vicinity of m. trapezii (Trapezius muscle) from cattle. The implant is intended for the continuous monitoring of glucose levels, and the study aimed at evaluating the biostability of exposed semiconductor surfaces. The sensor chip was a microelectromechanical system (MEMS) prepared using 0.25 µm complementary metal-oxide-semiconductor CMOS/BiCMOS technology. Sensing is based on the principle of affinity viscometry with a sensoric assay, which is separated by a semipermeable membrane from the tissue. Outer dimensions of the otherwise hermetically sealed biosensor system were 39 × 49 × 16 mm. The test system was implanted into cattle in a subcutaneous position without running it. After 17 months, the device was explanted and analyzed by comparing it with unexposed chips and systems. Investigations focused on the MEMS chip using SEM, TEM, and elemental analysis by EDX mapping. The sensor chip turned out to be uncorroded and no diminishing of the topmost passivation layer could be determined, which contrasts remarkably with previous results on CMOS biosensors. The negligible corrosive attack is understood to be a side effect of the semipermeable membrane separating the assay from the tissue. It is concluded that the separation has enabled a prolonged biostability of the chip, which will be of relevance for biosensor implants in general.

Micro-Electromechanical Affinity Sensor for the Monitoring of Glucose in Bioprocess Media.

An affinity-viscometry-based micro-sensor probe for continuous glucose monitoring was investigated with respect to its suitability for bioprocesses. The sensor operates with glucose and dextran competing as binding partner for concanavalin A, while the viscosity of the assay scales with glucose concentration. Changes in viscosity are determined with a micro-electromechanical system (MEMS) in the measurement cavity of the sensor probe. The study aimed to elucidate the interactions between the assay and a typical phosphate buffered bacterial cultivation medium. It turned out that contact with the medium resulted in a significant long-lasting drift of the assay's viscosity at zero glucose concentration. Adding glucose to the medium lowers the drift by a factor of eight. The cglc values measured off-line with the glucose sensor for monitoring of a bacterial cultivation were similar to the measurements with an enzymatic assay with a difference of less than ±0.15 g·L-1. We propose that lectin agglomeration, the electro-viscous effect, and constitutional changes of concanavalin A due to exchanges of the incorporated metal ions may account for the observed viscosity increase. The study has demonstrated the potential of the MEMS sensor to determine sensitive viscosity changes within very small sample volumes, which could be of interest for various biotechnological applications.

Continuously Operating Biosensor and Its Integration into a Hermetically Sealed Medical Implant.

An integration concept for an implantable biosensor for the continuous monitoring of blood sugar levels is presented. The system architecture is based on technical modules used in cardiovascular implants in order to minimize legal certification efforts for its perspective usage in medical applications. The sensor chip operates via the principle of affinity viscometry, which is realized by a fully embedded biomedical microelectromechanical systems (BioMEMS) prepared in 0.25-µm complementary metal⁻oxide⁻semiconductor (CMOS)/BiCMOS technology. Communication with a base station is established in the 402⁻405 MHz band used for medical implant communication services (MICS). The implant shall operate within the interstitial tissue, and the hermetical sealing of the electronic system against interaction with the body fluid is established using titanium housing. Only the sensor chip and the antenna are encapsulated in an epoxy header closely connected to the metallic housing. The study demonstrates that biosensor implants for the sensing of low-molecular-weight metabolites in the interstitial may successfully rely on components already established in cardiovascular implantology.