Characterization of oxygen transfer in vertical microbubble columns for aerobic biotechnological processes.

This paper presents the applicability of a microtechnologically fabricated microbubble column as a screening tool for submerged aerobic cultivation. Bubbles in the range of a few hundred micrometers in diameter were generated at the bottom of an upright-positioned microdevice. The rising bubbles induced the circulation of the liquid and thus enhanced mixing by reducing the diffusion distances and preventing cells from sedimentation. Two differently sized nozzles (21 × 40 µm(2) and 53 × 40 µm(2) in cross-section) were tested. The gas flow rates were adjustable, and the resulting bubble sizes and gas holdups were investigated by image analysis. The microdevice features sensor elements for the real-time online monitoring of optical density and dissolved oxygen. The active aeration of the microdevice allowed for a flexible oxygen supply with mass transfer rates of up to 0.14 s(-1). Slightly higher oxygen mass transfer rates and a better degassing were found for the microbubble column equipped with the smaller nozzle. To validate the applicability of the microbubble column for aerobic submerged cultivation processes, batch cultivations of the model organism Saccharomyces cerevisiae were performed, and the specific growth rate, oxygen uptake rate, and yield coefficient were investigated.

An in-line photonic biosensor for monitoring of glucose concentrations.

This paper presents two PDMS photonic biosensor designs that can be used for continuous monitoring of glucose concentrations. The first design, the internally immobilized sensor, consists of a reactor chamber, micro-lenses and self-alignment structures for fiber optics positioning. This sensor design allows optical detection of glucose concentrations under continuous glucose flow conditions of 33 µL/h based on internal co-immobilization of glucose oxidase (GOX) and horseradish peroxidase (HRP) on the internal PDMS surface of the reactor chamber. For this design, two co-immobilization methods, the simple adsorption and the covalent binding (PEG) methods were tested. Experiments showed successful results when using the covalent binding (PEG) method, where glucose concentrations up to 5 mM with a coefficient of determination (R2) of 0.99 and a limit of detection of 0.26 mM are detectable. The second design is a modified version of the internally immobilized sensor, where a microbead chamber and a beads filling channel are integrated into the sensor. This modification enabled external co-immobilization of enzymes covalently onto functionalized silica microbeads and allows binding a huge amount of HRP and GOX enzymes on the microbeads surfaces which increases the interaction area between immobilized enzymes and the analyte. This has a positive effect on the amount and rate of chemical reactions taking place inside the chamber. The sensor was tested under continuous glucose flow conditions and was found to be able to detect glucose concentrations up to 10 mM with R2 of 0.98 and a limit of detection of 0.7 mM. Such results are very promising for the application in photonic LOC systems used for online analysis.

PDMS based photonic lab-on-a-chip for the selective optical detection of heavy metal ions.

The selective absorbance detection of mercury(II) (Hg(2+)) and lead(II) (Pb(2+)) ions using ferrocene-based colorimetric ligands and miniaturized multiple internal reflection (MIR) systems implemented in a low-cost photonic lab on a chip (PhLoC) is reported. The detection principle is based on the formation of selective stable complexes between the heavy metal ion and the corresponding ligand. This interaction modulates the ligand spectrum by giving rise to new absorbance bands or wavelength shifting of the existing ones. A comparative study for the detection of Hg(2+) was carried out with two MIR-based PhLoC systems showing optical path lengths (OPLs) of 0.64 cm and 1.42 cm as well as a standard cuvette (1.00 cm OPL). Acetonitrile solutions containing the corresponding ligand and increasing concentrations of the heavy metal ion were pumped inside the systems and the absorbance in the visible region of the spectra was recorded. The optical behaviour of all the tested systems followed the expected Beer-Lambert law. Thus, the best results were achieved with the one with the longest OPL, which showed a linear behaviour in a concentration range of 1 µM-90 µM Hg(2+), a sensitivity of 5.6 × 10(-3) A.U. µM(-1) and a LOD of 2.59 µM (0.49 ppm), this being 1.7 times lower than that recorded with a standard cuvette, and using a sample/reagent volume around 190 times smaller. This microsystem was also applied for the detection of Pb(2+) and a linear behaviour in a concentration range of 3-100 µM was obtained, and a sensitivity of 9.59 × 10(-4) A.U. µM(-1) and a LOD of 4.19 µM (0.868 ppm) were achieved. Such a simple analytical tool could be implemented in portable instruments for automatic in-field measurements and, considering the minute sample and reagent volume required, would enable the deployment of high throughput environmental analysis of these pollutants and other related hazardous species.

Dual photonic-electrochemical lab on a chip for online simultaneous absorbance and amperometric measurements.

A dual lab on a chip (DLOC) approach that enables simultaneous optical and electrochemical detection working in a continuous flow regime is presented. Both detection modes are integrated for the first time into a single detection volume and operate simultaneously with no evidence of cross-talk. The electrochemical cell was characterized amperometrically by measuring the current in ferrocyanide solutions at +0.4 V vs gold pseudoreference electrode, at a flow rate of 200 µL min(-1). The experimental results for ferrocyanide concentrations ranging from 0.005 to 2 mM were in good agreement with the values predicted by the Levich equation for a microelectrode inside a rectangular channel, with a sensitivity of 2.059 ± 0.004 µA mM(-1) and a limit of detection (LoD) of (2.303 ± 0.004) × 10(-3) mM. Besides, optical detection was evaluated by measuring the absorbance of ferricyanide solutions at 420 nm. The results obtained therein coincide with those predicted by the Beer-Lambert law for a range of ferricyanide concentrations from 0.005 to 0.3 mM and showed an estimated LoD of (0.553 ± 0.001) × 10(-3) mM. The DLOC was finally applied to the analysis of L-lactate via a bienzymatic reaction involving lactate oxidase (LOX) and horseradish peroxidase (HRP). Here, the consumption of the reagent of the reaction (ferrocyanide) was continuously monitored by amperometry whereas the product of the reaction (ferricyanide) was recorded by absorbance. The DLOC presented good performance in terms of sensitivity and limit of detection, comparable to other fluidic systems found in the literature. Additionally, the ability to simultaneously quantify enzymatic reagent consumption and product generation confers the DLOC a self-verifying capability which in turn enhances its robustness and reliability.

Development and integration of xerogel polymeric absorbance micro-filters into lab-on-chip systems.

This work reports on the implementation of different absorption micro-filters based on a dye-doped hybrid organic-inorganic xerogel polymeric material synthesized by the sol-gel process. Microstructures containing eight different filter widths were fabricated in polydimethylsiloxane (PDMS), bonded to glass substrates and filled with the corresponding dye doped polymeric material by a soft lithography approach. The filtering capacity as a function of dye concentration and filter width was studied and revealed a linear dependence with both parameters, as expected according to the Beer-Lambert law. Zero passband transmittance values and relatively sharp stopband regions were achieved with all the filters, also showing rejection levels between -6 dB and -55 dB. Finally, such filters were monolithically integrated into a disposable fluorescence-based photonic lab-on-a-chip (PhLoC) approach. Calibration curves carried out with a model fluorophore target analyte showed an over two-fold increase in sensitivity and a thirty-fold decrease of the limit of detection (LOD) compared with the values recorded using the same PhLoC system but without the polymeric filter structure. The results presented herein clearly indicate the feasibility of these xerogel-based absorbance filtering structures for being applied as low-cost optical components that can be easily incorporated into disposable fluorescence-based photonic lab on a chip systems.

Poly(dimethylsiloxane) photonic microbioreactors based on segmented waveguides for local absorbance measurement.

We present the development of microbioreactors (MBRs) based on poly(dimethylsiloxane) (PDMS) segmented waveguides (SWG) for local absorbance measurements. Two different MBRs were studied, either using symmetric or asymmetric SWG (being defined as MBR-S and MBR-A, respectively). Their optical and fluidic performances were numerically analyzed, showing robustness from an optical point of view and distinct fluid flow profile. The optical characterization was done in two steps. Initially, the experimental limit of detection (LOD) and the sensitivity were determined for two different analytes (fluorescein and methylorange). With both systems, a similar limit of detection for both analytes was obtained, being in the micromolar level. Their sensitivities were 20.2±0.3 (×10⁻³) A.U./µM and 5.5±0.2 (×10⁻³) A.U./µM for fluorescein and methylorange, respectively. Once validated its applicability for local absorbance measurements, a continuous cultivation of Saccharomyces cerevisiae was done to test the viability of the proposed systems for photonic MBRs. Concretely, the cell growth was locally monitored inside the MBR during 33 h. Spectral analysis showed that the determination of the culture parameters were wavelength dependant, with a growth rate of 0.39±0.02 h⁻¹ and a doubling time of 1.65±0.09 h at an optimal wavelength of 469.9±0.3 nm. Besides the easy and monolithic integration of the SWG into poly(dimethylsiloxane) microfluidic systems, the results presented here are very promising for the application in any disposable photonic lab-on-a-chip systems used for online analysis or photonic MBRs.

Selective functionalisation of PDMS-based photonic lab on a chip for biosensing.

A comparative study of different approaches for the selective immobilisation of biomolecules on the surface of poly(dimethylsiloxane) (PDMS) is reported. The motivation of this work is to set a robust and reliable protocol for the easy implementation of a biosensor device in a PDMS-based photonic lab-on-a-chip (PhLoC). A hollow prism configuration, previously reported for the colorimetric detection of analytes was chosen for this study. Here, the inner walls of the hollow prism were initially modified by direct adsorption of either polyethylene glycol (PEG) or polyvinyl alcohol (PVA) linear polymers as well as by carrying out a light chemical oxidation step. All these processes introduced hydroxyl groups on the PDMS surface to a different extent. The hydroxyl groups were further silanised using a silane containing an aldehyde end-group. The interaction between this group and a primary amine moiety enabled the selective covalent attachment of a biomolecule on the PDMS surface. A thorough structural characterisation of the resulting modified-PDMS substrates was carried out by contact angle measurements, X-ray photoelectron spectroscopic (XPS) analysis and atomic force microscopy (AFM) imaging. Using horseradish peroxidase as a model recognition element, different biosensor approaches based on each modification process were developed for the detection of hydrogen peroxide target analyte in a concentration range from 0.1 µM to 100 µM. The analytical performance was similar in all cases, a linear concentration range between 0.1 µM and 24.2 µM, a sensitivity of 0.02 a.u. µM(-1) and a limit of detection around 0.1 µM were achieved. However, important differences were observed in the reproducibility of the devices as well as in their operational stability, which was studied over a period of up to two months. Considering all these studies, the PVA-modified approach appeared to be the most suitable one for the simple fabrication of a biosensor device integrated in a PDMS PhLoC.

Application of an e-tongue to the analysis of monovarietal and blends of white wines.

This work presents a multiparametric system capable of characterizing and classifying white wines according to the grape variety and geographical origin. Besides, it quantifies specific parameters of interest for quality control in wine. The system, known as a hybrid electronic tongue, consists of an array of electrochemical microsensors-six ISFET based sensors, a conductivity sensor, a redox potential sensor and two amperometric electrodes, a gold microelectrode and a microelectrode for sensing electrochemical oxygen demand--and a miniaturized optofluidic system. The test sample set comprised eighteen Catalan monovarietal white wines from four different grape varieties, two Croatian monovarietal white wines and seven bi- and trivarietal mixtures prepared from the Catalan varieties. Different chemometric tools were used to characterize (i.e., Principal Component Analysis), classify (i.e., Soft Independent Modeling Class Analogy) and quantify (i.e., Partial-Least Squares) some parameters of interest. The results demonstrate the usefulness of the multisensor system for analysis of wine.

Monolithic PDMS passband filters for fluorescence detection.

We present the fabrication and characteristics of monolithically integrated ink dyed poly(dimethylsiloxane) (PDMS) filters for optical sensing in disposable lab-on-a-chip. This represents a migration of auxillary functions onto the disposable chip with the goal of producing truly portable systems. Filters made from commercially available ink (Pelikan) directly mixed into PDMS oligomer without the use of any additional solvents were patterned with standard soft lithography technologies. Furthermore, a fabrication process based on capillary forces is presented allowing PDMS coloration of arbitrary shapes. Different filters of varying thickness fabricated using red, green and blue ink in four different concentrations were characterized. The optimal performance was found with filter thicknesses of 250 microm and ink to PDMS ratios of 0.1 (mL ink : mL PDMS oligomer) resulting in a transmittance ranging from -15.1 dB to -12.3 dB in the stopband and from -4.0 dB to -2.5 dB in the passband. Additionally, we demonstrate the robustness of this approach as the ink dyed PDMS filters do not exhibit temporal ageing due to diffusion or autofluorescence. We also show that such filters can easily be integrated in fluorescence systems, with stopbands efficient enough to allow fluorescence measurements under non-optimal conditions (broadband excitation, 180 degrees configuration). Integrated ink dyed PDMS filters add robust optical functionalities to disposable microdevices at a low cost and will enable the use of these devices for a wide range of fluorescence and absorbance based biological and chemical analysis.

Cell screening using disposable photonic lab on a chip systems.

A low-cost photonic lab on a chip with three different working regimes for cell screening is presented. The proposed system is able to perform scattering, scattering + absorption, and absorption measurements without any modification. Opposite to the standard flow cytometers, in this proposed configuration, a single 30 ms scan allows to obtain information regarding the cell optical properties. An additional novelty is that the whole spectrum is obtained and analyzed, being then possible to determine for each regime which is the optimal working wavelength that would provide the best performance in terms of sensitivity and limit of detection (LOD). Experimental results have provided with an LOD of 54.9 +/- 0.7 cells (in the scattering regime using unlabeled cells), 53 +/- 1 cells (in the scattering + absorption regime using labeled cells), and 105 +/- 4 cells (in the absorption regime using labeled cells). Finally, the system has also been used for measuring the dead/live cell ratio, obtaining LODs between 7.6 +/- 0.4% and 6.7 +/- 0.3%, depending on the working regime used.

Hybrid electronic tongue based on optical and electrochemical microsensors for quality control of wine.

A multiparametric system able to classify red and white wines according to the grape varieties and for analysing some specific parameters is presented. The system, known as hybrid electronic tongue, consists of an array of electrochemical microsensors and a colorimetric optofluidic system. The array of electrochemical sensors is composed of six ISFETs based sensors, a conductivity sensor, a redox potential sensor and two amperometric electrodes, an Au microelectrode and a microelectrode for sensing electrochemical oxygen demand. The optofluidic system is entirely fabricated in polymer technology and comprises a hollow structure, air mirrors, microlenses and self-alignment structures. The data obtained from these sensors has been treated with multivariate advanced tools; Principal Component Analysis (PCA), for the patterning recognition and classification of wine samples, and Partial-Least Squares (PLS) regression, for quantification of several chemical and optical parameters of interest in wine quality. The results have demonstrated the utility of this system for distinguishing the samples according to the grape variety and year vintage and for quantifying several sample parameters of interest in wine quality control.

Development of 3D Force Sensors for Nanopositioning and Nanomeasuring Machine.

In this contribution, we report on different miniaturized bulk micro machined three-axes piezoresistive force sensors for nanopositioning and nanomeasuring machine (NPMM). Various boss membrane structures, such as one boss full/cross, five boss full/cross and swastika membranes, were used as a basic structure for the force sensors. All designs have 16 p-type diffused piezoresistors on the surface of the membrane. Sensitivities in x, y and z directions are measured. Simulated and measured stiffness ratio in horizontal to vertical direction is measured for each design. Effect of the length of the stylus on H:V stiffness ratio is studied. Minimum and maximum deflection and resonance frequency are measured for all designs. The sensors were placed in a nanopositioning and nanomeasuring machine and one point measurements were performed for all the designs. Lastly the application of the sensor is shown, where dimension of a cube is measured using the sensor.

A micromachined capillary electrophoresis chip with fully integrated electrodes for separation and electrochemical detection.

A novel analytical microsystem with fully integrated electrodes for electrophoresis and amperometrical detection is described. With respect to the lab-on-a-chip concept a capillary electrophoresis (CE) microsystem has been fabricated with a total of six gold electrodes for sample injection, separation and electrochemical detection using standard microfabrication technologies. The device is a ready-to-use system that does not need any extra mechanical apparatus for electrode insertion. The CE-chip has successfully been tested by measuring hydrogen peroxide, ascorbic acid and uric acid simultaneously. All three oxidizable species could be detected in less than 70 s. Glucose was detected by performing an enzymatic reaction along the separation channel. The microsystem showed a very good reproducibility.

Recombinant antibody fragments allow repeated measurements of C-reactive protein with a quartz crystal microbalance immunosensor.

C-reactive protein (CRP) is a serum marker highly upregulated in inflammation after bacterial infection. Robust, reliable and quick quantification of CRP would be a substitute for erythrocyte sedimentation rate (ESR) with superior diagnostic value. Quartz crystal microbalance (QCM) based sensors coated with specific antibodies and integrated into lab-on-chip systems are in development for rapid point of care quantification. In this study, we isolated three CRP specific single chain (sc)Fv antibody fragments using phage display from an antibody gene library. Their affinities ranged from 2.7 × 10(-8) to 1.0 × 10(-8) M when measured by surface plasmon resonance. ScFv antibody fragment LA13-IIE3 showed best affinity, high long-term stability and remarkable resistance to denaturation. This scFv antibody fragment was coupled to a QCM sensor. CRP quantification in up to 15 samples sequentially measured on the same sensor with intermitting regeneration by buffer was demonstrated.

Monolithically integrated biophotonic lab-on-a-chip for cell culture and simultaneous pH monitoring.

A poly(dimethylsiloxane) biophotonic lab-on-a-chip (bioPhLoC) containing two chambers, an incubation chamber and a monitoring chamber for cell retention/proliferation and pH monitoring, respectively, is presented. The bioPhLoC monolithically integrates a filter with 3 µm high size-exclusion microchannels, capable of efficiently trapping cells in the incubation chamber, as well as optical elements for real-time interrogation of both chambers. The integrated optical elements made possible both absorption and dispersion measurements, which were comparable to those made in a commercially available cuvette. The size-exclusion filter also showed good and stable trapping capacity when using yeast cells of variable size (between 5 and 8 µm diameter). For cell culture applications, vascular smooth muscle cells (VSMC), with sizes between 8 and 10 µm diameter, were used as a mammalian cell model. These cells were efficiently trapped in the incubation chamber, where they proliferated with a classical spindle-shaped morphology and a traditional hill-and-valley phenotype. During cell proliferation, pH changes in the culture medium due to cell metabolism were monitored in real time and with high precision in the monitoring chamber without interference of the measurement by cells and other (cell) debris.

Vertical microbubble column-A photonic lab-on-chip for cultivation and online analysis of yeast cell cultures.

This paper presents a vertically positioned microfluidic system made of poly(dimethylsiloxane) (PDMS) and glass, which can be applied as a microbubble column (µBC) for biotechnological screening in suspension. In this µBC, microbubbles are produced in a cultivation chamber through an integrated nozzle structure. Thus, homogeneous suspension of biomass is achieved in the cultivation chamber without requiring additional mixing elements. Moreover, blockage due to produced carbon dioxide by the microorganisms-a problem predominant in common, horizontally positioned microbioreactors (MBRs)-is avoided, as the gas bubbles are released by buoyancy at the upper part of the microsystem. The patterned PDMS layer is based on an optimized two-lithographic process. Since the naturally hydrophobic PDMS causes problems for the sufficient production of microbubbles, a method based on polyelectrolyte multilayers is applied in order to allow continuous hydrophilization of the already bonded PDMS-glass-system. The µBC comprises various microelements, including stabilization of temperature, control of continuous bubble formation, and two optical configurations for measurement of optical density with two different sensitivities. In addition, the simple and robust application and handling of the µBC is achieved via a custom-made modular plug-in adapter. To validate the scalability from laboratory scale to microscale, and thus to demonstrate the successful application of the µBC as a screening instrument, a batch cultivation of Saccharomyces cerevisiae is performed in the µBC and compared to shake flask cultivation. Monitoring of the biomass growth in the µBC with the integrated online analytics resulted in a specific growth rate of 0.32 h(-1), which is almost identical to the one achieved in the shake flask cultivation (0.31 h(-1)). Therefore, the validity of the µBC as an alternative screening tool compared to other conventional laboratory scale systems in bioprocess development is proven. In addition, vertically positioned microbioreactors show high potential in comparison to conventional screening tools, since they allow for high density of integrated online analytics and therefore minimize time and cost for screening and guarantee improved control and analysis of cultivation parameters.

Cell analysis using a multiple internal reflection photonic lab-on-a-chip.

Here we present a protocol for analyzing cell cultures using a photonic lab-on-a-chip (PhLoC). By using a broadband light source and a spectrometer, the spectrum of a given cell culture with an arbitrary population is acquired. The PhLoC can work in three different regimes: light scattering (using label-free cells), light scattering plus absorption (using stained cells) and, by subtraction of the two former regimes, absorption (without the scattering band). The acquisition time of the PhLoC is ∼30 ms. Hence, it can be used for rapid cell counting, dead/live ratio estimation or multiparametric measurements through the use of different dyes. The PhLoC, including microlenses, micromirrors and microfluidics, is simply fabricated in a single-mask process (by soft lithographic methods) using low-cost materials. Because of its low cost it can easily be implemented for point-of-care applications. From raw substrates to final results, this protocol can be completed in 29 h.

Polyelectrolyte multilayer surface functionalization of poly(dimethylsiloxane) (PDMS) for reduction of yeast cell adhesion in microfluidic devices.

Polyelectrolyte multilayers (PEMs) based on the combinations poly(diallyldimethylammonium chloride)∕poly(acrylic acid) (PDADMAC∕PAA) and poly(allylamine hydrochloride)∕PAA (PAH∕PAA) were adsorbed on poly(dimethylsiloxane) (PDMS) and tested for nonspecific surface attachment of hydrophobic yeast cells using a parallel plate flow chamber. A custom-made graft copolymer containing poly(ethylene glycol) (PEG) side chains (PAA-g-PEG) was additionally adsorbed on the PEMs as a terminal layer. A suitable PEM modification effectively decreased the adhesion strength of Saccharomyces cerevisiae DSM 2155 to the channel walls. However, a further decrease in initial cell attachment and adhesion strength was observed after adsorption of PAA-g-PEG copolymer onto PEMs from aqueous solution. The results demonstrate that a facile layer-by-layer surface functionalization from aqueous solutions can be successfully applied to reduce cell adhesion strength of S. cerevisiae by at least two orders of magnitude compared to bare PDMS. Therefore, this method is potentially suitable to promote planktonic growth inside capped PDMS-based microfluidic devices if the PEM deposition is completed by a dynamic flow-through process.

Microfluidic reactor for continuous cultivation of Saccharomyces cerevisiae.

A diffusion-based microreactor system operated with a reaction volume of 8 µL is presented and characterized to intensify the process understanding in microscale cultivations. Its potential as screening tool for biological processes is evaluated. The advantage of the designed microbioreactor is the use for the continuous cultivation mode by integrating online measurement technique for dissolved oxygen (DO) and optical density (OD). A further advantage is the broaden application for biological systems. The bioreactor geometry was chosen to achieve homogeneous flow during continuous process operation. The device consisted of a microstructured top layer made of poly(dimethylsiloxane) (PDMS), which was designed and fabricated using UV-depth and soft lithography assembled with a glass bottom. CFD simulation data used for geometry design were verified via microparticle-image-velocimetry (µPIV). In the used microreactor geometry no concentration gradients occurred along the entire reaction volume because of rapid diffusive mixing, the homogeneous medium flow inside the growth chamber of the microreactor could be realized. Undesirable bubble formation before and during operation was reduced by using degassed medium as well as moistened and moderate incident air flow above the gas permeable PDMS membrane. Because of this a passive oxygen supply of the culture medium in the device is ensured by diffusion through the PDMS membrane. The oxygen supply itself was monitored online via integrated DO sensors based on a fluorescent dye complex. An adequate overall volumetric oxygen transfer coefficient K(L)a as well as mechanical stability of the device were accomplished for a membrane thickness of 300 µm. Experimental investigations considering measurements of OD (online) and several metabolite concentrations (offline) in a modified Verduyn medium. The used model organism Saccharomyces cerevisiae DSM 2155 tended to strong reactor wall growth resembling a biofilm.