Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

University of Freiburg

For simultaneous measurement of glucose, lactate, glutamine, and glutamate a biosensor array is implemented in a micro flow-system thus giving a microsystem. The microsystem consists of a glass chip with the integrated biosensor array and a bottom part, which comprises a gold counter electrode, a 300 microm thick seal, and electrical interconnection lines. The flow device has a total internal volume of 2.1 or 6 microl when integrated with a mixer on chip. The biosensors with no crosstalking and high long term stability were produced by modifying the electrochemical transducers and utilizing photopatternable enzyme membranes. The use of appropriate miniaturization technology leads to mass producable devices for in vivo and ex vivo applications in whole blood and fermentation broth. Due to a novel glutaminase with an activity optimum in the neutral pH range direct and simultaneous monitoring of glutamine together with glucose, lactate, and glutamate could be performed.

Biosensor arrays for simultaneous measurement of glucose, lactate, glutamate, and glutamine.

Charge transfer mechanisms of several conducting polymers and polymer/CdSe nanocrystal composites (hybrid bulk heterojunction composites) were investigated by means of cyclic voltammetry. Potential application of these composites in hybrid light-emitting diodes was discussed. It was found that charge transfer is observed in most of the composites, used so far, but was relatively slow or incomplete. The PVPy/CdSe nanocrystal composite showed promising results, and is favorable for use in electroluminescent devices.

Charge transfer mechanism in hybrid bulk heterojunction composites.

In this letter, we present a low-temperature synthesis route revealing a new type of ultrasmall CdSe nanoparticle family with exceptional narrow blue emissions between 437 and 456 nm and full width at half-maxima below 20 nm. Transmission electron microscopy characterization shows the uniformity of the nanoparticles, which have a diameter of 1.6 nm. After surface modification, the spherical particles assemble into nanowires, demonstrating their potential as building blocks for the generation of highly ordered superstructures. They can also be used as single source precursors for the synthesis of CdSe nanocrystals.

Blue luminescence and superstructures from magic size clusters of CdSe.

The lack of sample pre-treatment concepts that are easily automatable, miniaturized and highly efficient for both small volumes and low target concentrations, is one of the key issues that block the road towards effective miniaturized diagnostic instruments. This paper presents a novel, highly efficient and simple method for low-molecular weight RNA extraction using electricity only. Cells are lysed by thermo-electric lysis and RNA is purified using a gel-electrophoretic purification step. The combination of the two steps in one integrated cartridge reduces the time frame between the two steps, thus protecting RNA from enzymatic degradation. A disposable chip solution is proposed using a novel dry film resist laminate technology that allows cheap, large-scale fabrication. The chip contains crucial microfluidic innovations that allow for a simple user interface, reproducible functioning and precise quantification. Phaseguides are invented that allow controlled spatial injection of gel, injection of sample and recovery of extracted RNA. A precise sample volume can be defined by integrating electrophoretic actuation electrodes in the microfluidic chamber. Electrolytic gas bubbles that are the result of constant-current actuation are driven out from the chip by the novel introduction of capillary bubble-expulsion techniques. The extraction approach and the functionality of the chip are demonstrated for Escherichia coli and Streptococcus thermophilus bacteria. Linear extraction behavior is obtained for transfer-messenger RNA down to one colony-forming unit per microlitre, or five colony-forming units per chip. The latter is an increase in extraction efficiency of a factor of 1000 with respect to the commercial extraction kit Ambion Ribopure. The chip shows particularly good performance for extraction of low-molecular weight RNA, thereby eliminating the need for large ribosomal RNA and DNA removal. RNA can be extracted in less than 11 min, being a speed-up of more than a factor of 20 with respect to commercial extraction kits. The presented solution may find broad acceptance and application in drug discovery and clinical diagnostics.

A microfluidic approach for high efficiency extraction of low molecular weight RNA.

Phaseguide technology gives complete control over filling and emptying of any type of microfluidic structures, independent of the chamber and channel geometry. The technique is based on a step-wise advancement of the liquid-air interface using the meniscus pinning effect. In this paper, the main effects and parameters underlying the phaseguiding principle are discussed and a demonstration is given of its potential for dead angle filling, spatially controlled phaseguide overflow and sequential phaseguide overflow, all accumulating in a passive valving approach. Phaseguides represent a new direction in microfluidic design thinking that will prove a leap forward towards more simple, flexible and reliable microfluidic systems.

Phaseguides: a paradigm shift in microfluidic priming and emptying.

Macro- to micro-volume concentration of viable bacteria is performed in a microfluidic chip. The enrichment principle is based on free flow electrophoresis and is demonstrated for Gram positive bacteria. Bacteria from a suspension flow are trapped on a gel interface that separates the trapping location from integrated actuation electrodes in order to enable non-destructive trapping. The microfluidic chip contains integrated electrolytic gas expulsion structures and phaseguides for gel and liquid handling. Trapping efficiency is systematically optimized to reach 25 times the initial concentration from a theoretical maximum of 30. Finally, enrichment from analytically relevant concentrations down to 3 × 10(2) colony forming units per millilitre is demonstrated with a trapping efficiency of 80% which represents the most important parameter in enrichment.

Enrichment of viable bacteria in a micro-volume by free-flow electrophoresis.

In this contribution, we present a system for efficient preconcentration of pathogens without affecting their viability. Development of miniaturized molecular diagnostic kits requires concentration of the sample, molecule extraction, amplification, and detection. In consequence of low analyte concentrations in real-world samples, preconcentration is a critical step within this workflow. Bacteria and viruses exhibit a negative surface charge and thus can be electrophoretically captured from a continuous flow. The concept of phaseguides was applied to define gel membranes, which enable effective and reversible collection of the target species. E. coli of the strains XL1-blue and K12 were used to evaluate the performance of the device. By suppression of the electroosmotic flow both strains were captured with efficiencies of up to 99%. At a continuous flow of 15 μl/min concentration factors of 50.17 ± 2.23 and 47.36 ± 1.72 were achieved in less than 27 min for XL1-blue and K12, respectively. These results indicate that free flow electrophoresis enables efficient concentration of bacteria and the presented device can contribute to rapid analyses of swab-derived samples.

Microfluidic concentration of bacteria by on-chip electrophoresis.

With a view to developing a rapid pathogen detection system utilizing isothermal nucleic acid amplification, the necessary micro-mixing step is innovatively implemented on a chip. Passive laminar flow mixing of two 6.5 μl batches differing in viscosity is performed within a microfluidic chamber. This is achieved with a novel chip space-saving phaseguide design which allows, for the first time, the complete integration of a passive mixing structure into a target chamber. Sequential filling of batches prior to mixing is demonstrated. Simulation predicts a reduction of diffusive mixing time from hours down to one minute. A simple and low-cost fabrication method is used which combines dry film resist technology and direct wafer bonding. Finally, an isothermal nucleic acid detection assay is successfully implemented where fluorescence results are measured directly from the chip after a one minute mixing sequence. In combination with our previous work, this opens up the way towards a fully integrated pathogen detection system in a lab-on-a-chip format.

A phaseguided passive batch microfluidic mixing chamber for isothermal amplification.

A microfluidic chip is presented for lysis and one-step RNA purification from bacteria. Bacteria are lysed by joule-heating followed by a gel electrophoresis step for clean-up and subsequent elution of small RNA. Bubble formation during electrophoresis at constant current is suppressed through the use of a silver chloride cathode and a silver anode. To prevent silver chloride sediment in the bulk solution, the anode was immersed in a saturated chloride solution. Salt bridges in the form of polyacrylamide gels are used that could be precisely patterned with the help of phaseguides. Bubble-free actuation could be performed for more than 20 min under a constant current. For longer actuation times, cathodic silver-chloride became depleted and a silver-chloride sediment formed in the anodic microchamber at increasing distance from the anode with time. The chip functioning was verified by extraction of transfer-messenger RNA from Escherichia coli and subsequent amplification using reverse transcription real-time PCR. Incorporation of salt bridges enables effective bubble free actuation of Ag/AgCl electrodes in a microfluidic chip. This opens up new possibilities in a surge towards fully integrated diagnostic cartridges that are miniaturized and disposable.

Bubble-free electrode actuation for micro-preparative scale electrophoresis of RNA.

Cell migration has been recognized as one hallmark of malignant tumor progression. By integrating the method of electrical cell-substrate impedance sensing (ECIS) with the Boyden chamber design, the state-of-the-art techniques provide kinetic information about cell migration and invasion processes in three-dimensional (3D) extracellular matrixes. However, the information related to the initial stage of cell migration with single-cell resolution, which plays a unique role in the metastasis-invasion cascade of cancer, is not yet available. In this paper, we present a microfluidic device integrated with ECIS for investigating single cancer cell migration in 3D matrixes. Using microfluidics techniques without the requirement of physical connections to off-chip pneumatics, the proposed sensor chip can efficiently capture single cells on microelectrode arrays for sequential on-chip 2D or 3D cell culture and impedance measurement. An on-chip single-cell migration assay was successfully demonstrated within several minutes. Migration of single metastatic MDA-MB-231 cells in their initial stage can be monitored in real time; it shows a rapid change in impedance magnitude of approximately 10 Ω/s, whereas no prominent impedance change is observed for less-metastasis MCF-7 cells. The proposed sensor chip, allowing for a rapid and selective detection of the migratory properties of cancer cells at the single-cell level, could be applied as a new tool for cancer research.

Microfluidic chip with integrated electrical cell-impedance sensing for monitoring single cancer cell migration in three-dimensional matrixes.

We present a novel, multiparametric microphysiometry system for the dynamic online monitoring of human cancer cell metabolism. The optically transparent, modular, hybrid microsystem is based on a glass chip and combines a cell cultivation chamber, microfluidics and metabolic monitoring with fully integrated chemo- and biosensors. pH and oxygen are measured in the cell culture area, and biosensors for lactate and glucose are connected downstream by microfluidics. The wafer-level fabrication features thin-film platinum and iridium oxide microelectrodes on a glass chip, microfluidics in an epoxy resist, a hybrid assembly and an on-chip reference electrode. The reliable analytical performance of the sensors in cell culture medium was demonstrated. The pH sensors exhibit a long-term stable, linear response. The oxygen sensors show a linear behaviour, which is also observed for low oxygen concentrations. Glucose and lactate measurements show a linear, long-term stable, selective and reversible behaviour in the desired range. T98G human brain cancer cells were cultivated and cell culture metabolism was measured on-chip. Stop/flow cycles were applied and extracellular acidification, respiration, glucose consumption and lactate production were quantified. Long-term metabolic rates were determined and all parameters could be measured in the outlet channel. A placement downstream of the cell cultivation area for biosensors was realised. A highly effective medium exchange and undiluted sampling from the cell culture chamber with low flow rates (2 μl min(-1)) and low volumes (15 μl per cycle) were achieved. The drug screening application was demonstrated by detecting alteration and recovery effects of cellular metabolism induced by the addition of substances to the medium.

Cell culture monitoring for drug screening and cancer research: a transparent, microfluidic, multi-sensor microsystem.

We present a flexible microsensor, based on a polymer substrate, for multiparametric, electrochemical in vivo monitoring. The sensor strip with a microelectrode array at the tip was designed for insertion into tissue, for fast and localized online monitoring of physiological parameters. The microsystem fabrication on a wafer-level is based on a polyimide substrate and includes the patterning of platinum microelectrodes as well as epoxy and dry-film-resist insulation in a cost-effective thin-film and laminate process. A stable, electrodeposited silver/silver chloride reference electrode on-chip and a perm-selective membrane as an efficient interference rejection scheme are integrated on a wafer-level. Amperometric, electrochemical, enzyme-based biosensors for the neurotransmitter L-glutamate and the energy metabolite L-lactate have been developed. Hydrogel membranes or direct cross-linking as stable concepts for the enzyme immobilization are shown. Sensor performance including high selectivity, tailoring of sensitivity and long-term stability is discussed. For glutamate, a high sensitivity of 2.16 nAmm(-2) µM(-1) was found. For lactate, a variation in sensitivity between 2.6 and 32 nAmm(-2)mM(-1) was achieved by different membrane compositions. The in vivo application in an animal model is demonstrated by glutamate measurements in the brain of rats. Local glutamate alterations in the micromolar range and in nanoliter-range volumes can be detected and quantified with high reproducibility and temporal resolution. A novel, versatile platform for the integration of various electrochemical sensors on a small, flexible sensor strip for a variety of in vivo applications is presented.

Polymer-based, flexible glutamate and lactate microsensors for in vivo applications.

Miniaturized electrochemical in vivo biosensors allow the measurement of fast extracellular dynamics of neurotransmitter and energy metabolism directly in the tissue. Enzyme-based amperometric biosensing is characterized by high specificity and precision as well as high spatial and temporal resolution. Aside from glucose monitoring, many systems have been introduced mainly for application in the central nervous system in animal models. We compare the microsensor principle with other methods applied in biomedical research to show advantages and drawbacks. Electrochemical sensor systems are easily miniaturized and fabricated by microtechnology processes. We review different microfabrication approaches for in vivo sensor platforms, ranging from simple modified wires and fibres to fully microfabricated systems on silicon, ceramic or polymer substrates. The various immobilization methods for the enzyme such as chemical cross-linking and entrapment in polymer membranes are discussed. The resulting sensor performance is compared in detail. We also examine different concepts to reject interfering substances by additional membranes, aspects of instrumentation and biocompatibility. Practical considerations are elaborated, and conclusions for future developments are presented. Graphical Abstract ᅟ.

Microfabricated, amperometric, enzyme-based biosensors for in vivo applications.

3D hepatic microtissues, unlike 2D cell cultures, retain many of the in-vivo-like functionalities even after long-term cultivation. Such 3D cultures are increasingly applied to investigate liver damage due to drug exposure in toxicology. However, there is a need for thorough metabolic characterization of these microtissues for mechanistic understanding of effects on culture behaviour. We measured metabolic parameters from single human HepaRG hepatocyte spheroids online and continuously with electrochemical microsensors. A microsensor platform for lactate and oxygen was integrated in a standard 96-well plate. Electrochemical microsensors for lactate and oxygen allow fast, precise and continuous long-term measurement of metabolic parameters directly in the microwell. The demonstrated capability to precisely detect small concentration changes by single spheroids is the key to access their metabolism. Lactate levels in the culture medium starting from 50µM with production rates of 5µMh(-1) were monitored and precisely quantified over three days. Parallel long-term oxygen measurements showed no oxygen depletion or hypoxic conditions in the microwell. Increased lactate production by spheroids upon suppression of the aerobic metabolism was observed. The dose-dependent decrease in lactate production caused by the addition of the hepatotoxic drug Bosentan was determined. We showed that in a toxicological application, metabolic monitoring yields quantitative, online information on cell viability, which complements and supports other methods such as microscopy. The demonstrated continuous access to 3D cell culture metabolism within a standard setup improves in vitro toxicology models in replacement strategies of animal experiments. Controlling the microenvironment of such organotypic cultures has impact in tissue engineering, cancer therapy and personalized medicine.

Accessing 3D microtissue metabolism: Lactate and oxygen monitoring in hepatocyte spheroids.

Multiplexed point-of-care testing (xPOCT), which is simultaneous on-site detection of different analytes from a single specimen, has recently gained increasing importance for clinical diagnostics, with emerging applications in resource-limited settings (such as in the developing world, in doctors' offices, or directly at home). Nevertheless, only single-analyte approaches are typically considered as the major paradigm in many reviews of point-of-care testing. Here, we comprehensively review the present diagnostic systems and techniques for xPOCT applications. Different multiplexing technologies (e.g., bead- or array-based systems) are considered along with their detection methods (e.g., electrochemical or optical). We also address the unmet needs and challenges of xPOCT. Finally, we critically summarize the in-field applicability and the future perspectives of the presented approaches.

Gerald A. Urban

Department of Microsystems Engineering,
Freiburg Materials Research Center

University of Freiburg

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

Gerald A. Urban

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Microsystems Engineering,Freiburg Materials Research Center

University of Freiburg

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

Department of Microsystems Engineering,
Freiburg Materials Research Center