Stefanie Demming

Multiple internal reflection poly(dimethylsiloxane) systems for optical sensing

Compact poly(dimethylsiloxane)-based (PDMS) multiple internal reflection systems which comprise self-alignment systems, lenses, microfluidic channels and mirrors have been developed for highly sensitive absorbance measurements. With the proper definition of air mirrors at both sides of the sensing region, the optical path of the light from the LED has been meaningfully lengthened without a dramatic increase of the mean flow cell volume. By recursive positioning of such air mirrors, propagating multiple internal reflection (PMIR) systems have been designed, simulated and characterized. Experimental results confirm the ray-tracing predictions and allow the determining that there are some regions of the mean flow cell volume that do not contribute to the increase of the sensitivity. The tailoring of the sensing region, following the optical path, results in a similar limit of detection (110 nM) for fluorescein diluted in phosphate buffer. Finally, a ring configuration, labelled RMIR, has also been developed. With the addition of a third air mirror, the LOD can be decreased to 41 nM with the additional advantage of a substantial decrease of the length of the sensing region. These results confirm the validity of the proposed systems for high sensitivity measurements.

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 KLa 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.

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.

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 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.

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.

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.

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.

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.