Klaus Drese

Utilization of Micromixers for Extraction Processes

The aim of the investigation was to evaluate the extraction performance of a mixer settler set-up for miniplant technology using static micromixers as an alternative to conventional stirring apparatuses. A comprehensive experimental study was conducted at BASF AG to broaden the technology base for the “extraction” unit operation which is well established for miniplants in order to be utilized for microplant systems. The work proved that micromixers, or micromixer arrays, are highly efficient apparatuses for extraction purposes. The extraction efficiency was found to be a function of volume flow, which could be explained in light of the volume flow dependence of the mean droplet size and, hence, the specific surface area of the dispersions intermediately formed. At optimal flow conditions, one practical mixer settler stage was found to yield one theoretical plate for most systems investigated.

Amperometric Immunosensor for Carcinoembryonic Antigen in Colon Cancer Samples Based on Monolayers of Dendritic Bipodal Scaffolds

Detection of proteins that signal the presence or recurrence of cancer is a powerful therapeutic tool for effective early diagnosis and treatment. Carcinoembryonic antigen (CEA) has been extensively studied as a tumor marker in clinical diagnosis. We report on the development of an amperometric biosensor for the detection of CEA based on the immobilization of anti-CEA monoclonal antibody on a novel class of bipodal thiolated self-assembled monolayers containing reactive N-hydroxysuccinimide (NHS) ester end groups. The current variations showed a linear relationship with the concentration of CEA over the range of 0-200 ng/mL with a sensitivity of 3.8 nA x mL x ng(-1) and a detection limit of 0.2 ng/mL, which is well below the commonly accepted concentration threshold (5 ng/mL) used in clinical diagnosis. Real time and accelerated stability studies of the reporter antibody under various storage conditions demonstrated that the enzymatic activity and antibody affinity of the conjugate is retained for long periods of time in commercial stabilizing buffers such as StabilGuard Biomolecule Stabilizer, and a prediction of the stability trends was carried out using the kinetic and thermodynamic parameters obtained from the Arrhenius equation. The developed immunosensor as well as a commercially available enzyme-linked immunosorbent assay (ELISA) kit were successfully applied to the detection of CEA in serum samples obtained from colon cancer patients, and an excellent correlation of the levels of CEA measured was obtained. Ongoing work is looking at the incorporation of the developed biosensor into a platform for multiplexed simultaneous detection of several breast cancer related biomarkers.

Electrophoretic partitioning of proteins in two-phase microflows

This work reports on protein transport phenomena discovered in partitioning experiments with a novel setup for continuous-flow two-phase electrophoresis consisting of a microchannel in which a phase boundary is formed in flow direction. Proteins can be partitioned exploiting their affinity to different aqueous phases in two-phase systems. This separation process may be enhanced or extended by applying an electric field perpendicular to the phase boundary. In this context, microsystems offer new possibilities, as interfacial forces usually dominate over volume forces, thus allowing a superior control of the formation and arrangement of liquid/liquid phase boundaries. The two immiscible phases which are injected separately into the microchannel are taken from a polyethylene glycol (PEG)-dextran system. The side walls of the channel are partially made of gel material which serves as an ion conductor and decouples the channel from the electrodes, thus preventing bubble generation inside the separation channel. The experiments show that the electrophoretic transport of proteins between the laminated liquid phases is characterized by a strong asymmetry. When bovine serum albumin (BSA) is introduced into the PEG-rich phase, it can easily be transferred into the dextran-rich phase via an applied electric field of low strength or just by diffusion. In the reverse case, up to a certain field strength the transfer to the opposing phase is strongly inhibited. Only if the field strength is further increased will the BSA molecules leave the dextran-rich phase almost completely.

Design and testing of a packaged microfluidic cell for the multiplexed electrochemical detection of cancer markers

We present the rapid prototyping of electrochemical sensor arrays integrated to microfluidics towards the fabrication of integrated microsystems prototypes for point‐of‐care diagnostics. Rapid prototyping of microfluidics was realised by high‐precision milling of polycarbonate sheets, which offers flexibility and rapid turnover of the desired designs. On the other hand, the electrochemical sensor arrays were fabricated using standard photolithographic and metal (gold and silver) deposition technology in order to realise three‐electrode cells comprising gold counter and working electrodes as well as silver reference electrode. The integration of fluidic chips and electrode arrays was realised via a laser‐machined double‐sided adhesive gasket that allowed creating the microchannels necessary for sample and reagent delivery. We focused our attention on the reproducibility of the electrode array preparation for the multiplexed detection of tumour markers such as carcinoembryonic antigen and prostate‐specific antigen as well as genetic breast cancer markers such as estrogen receptor‐α, plasminogen activator urokinase receptor, epidermal growth factor receptor and erythroblastic leukemia viral oncogene homolog 2. We showed that by carefully controlling the electrode surface pre‐treatment and derivatisation via thiolated antibodies or short DNA probes that the detection of several key health parameters on a single chip was achievable with excellent reproducibility and high sensitivity.

Automated microsystem for electrochemical detection of cancer markers

The development of a fully automated microsystem housing an amperometric immunosensor is presented. The microfluidic cell integrates reagent storage and electrochemical immunodetection and was applied for the detection of breast cancer markers. The main advantage of this system is that no external fluidic storage is required and the instrumental setup is thus greatly simplified. The fluidics of the microsystem is computer controlled and requires minimal end‐user intervention. The analytical performance of the device was compared with a manually driven system and applied for the amperometric detection of the carcinoembryonic antigen (CEA) and cancer antigen 15‐3 (CA15‐3). This automation methodology greatly improves the analytical performance of the immunosensor in terms of accuracy and reproducibility as evidenced by a reduction of LOD observed for CEA and CA15‐3 with respect to a manually driven system. Finally, the automated microsystem was applied for the analysis of real patient serum samples, demonstrating excellent correlation with a commercial ELISA.

Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV) E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n = 28) from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection.

Microsystem for Isolation of Fetal DNA from Maternal Plasma by Preparative Size Separation

BACKGROUND Routine prenatal diagnosis of chromosomal anomalies is based on invasive procedures, which carry a risk of approximately 1%-2% for loss of pregnancy. An alternative to these inherently invasive techniques is to isolate fetal DNA circulating in the pregnant mothers plasma. Free fetal DNA circulates in maternal plasma primarily as fragments of lengths <500 bp, with a majority being <300 bp. Separating these fragments by size facilitates an increase in the ratio of fetal to maternal DNA. METHODS We describe our development of a microsystem for the enrichment and isolation of cell-free fetal DNA from maternal plasma. The first step involves a high-volume extraction from large samples of maternal plasma. The resulting 80-microL eluate is introduced into a polymeric microsystem within which DNA is trapped and preconcentrated. This step is followed by a transient isotachophoresis step in which the sample stacks within a neighboring channel for subsequent size separation and is recovered via an outlet at the end of the channel. RESULTS Recovered fractions of fetal DNA were concentrated 4-8 times over those in preconcentration samples. With plasma samples from pregnant women, we detected the fetal SRY gene (sex determining region Y) exclusively in the fragment fraction of <500 bp, whereas a LEP gene (leptin) fragment was detected in both the shorter and longer recovery fractions. CONCLUSIONS The microdevice we have described has the potential to open new perspectives in noninvasive prenatal diagnosis by facilitating the isolation of fetal DNA from maternal plasma in an integrated, inexpensive, and easy-to-use microsystem.

Fast preparation and testing methods using a microstructured modular reactor for parallel gas phase catalyst screening

Abstract Heterogeneous gas phase catalyst screening with catalysts applied on titer-plates is presented. It will be shown that titer-plates can as well be used for the sample preparation outside the reactor and for the catalyst testing in the reactor. For the catalyst preparation, the known wash-coating-impregnation procedure is applied. An alumina coated precursor is introduced into a manifold which utilizes the flow features of the separated wells for the impregnation of the precursor with solutions of the salts of a collection of transition metals. The impregnation liquid is flowing through the wells thus impregnating the surface of the porous γ-alumina layers within the microchannels. Less porous coatings are realized by the simultaneous gradient sputtering process developed on the basis of the standard sputtering process. Some details about the reactor development and characterization as well as problems which arose are reported. For the description of the reactor performance, a number of coated titer-plates were tested and these results are presented. After this so-called primary screening, a second evaluation phase normally follows. In this second phase, the kinetic properties of a single catalyst shall be studied. For this purpose, a model which describes the fluidic behavior of the reacting gas in microchannels was developed. Applying the model will give further insights into the underlying reaction kinetic of a single selected catalyst in the so-called secondary screening phase.

Fast nucleic acid amplification for integration in point‐of‐care applications

An ultrafast microfluidic PCR module (30 PCR cycles in 6 min) based on the oscillating fluid plug concept was developed. A robust amplification of native genomic DNA from whole blood samples could be achieved at operational conditions established from systematic investigations of key parameters including heat transfer and in particular flow velocities. Experimental data were augmented with results from computational fluid dynamics simulations. The reproducibility of the current system was substantially improved compared to previous concepts by integration of a closed reservoir instead of utilizing a vented channel end at ambient pressure rendering the devised module suitable for integration into complex sample‐to‐answer analysis platforms such as point‐of‐care applications.

Microsystem for field-amplified electrokinetic trapping preconcentration of DNA at poly(ethylene terephthalate) membranes.

In electrokinetic trapping (EKT), the electroosmotic velocity of a buffer solution in one area of a microfluidic device opposes the electrophoretic velocity of the analyte in a second area, resulting in transport of DNA to a location where the electrophoretic and electroosmotic velocities are equal and opposite and DNA concentrates at charged nanochannels. The method does not require an optical plug localization, a considerable advantage as compared to preconcentration techniques previously presented. In the work reported here, the trapping process is preceded by a field-amplification in the sample reservoir to reduce trapping time, as field-amplified EKT is shown to be an effective technique to preconcentrate samples from larger volumes. A theoretical model explaining the principle of field-amplified EKT considers different ionic strengths and cross-sectional areas in the microchip segments. The model is supported by experimental data using nucleic acids and fluorescein as sample analytes. An incorporated poly(ethylene terephthalate) (PET) membrane provides anion exclusion due to a negatively charged surface. A fluidic counter flow supports the trapping process in 100 nm pores due to anion exclusion. An analysis of Joule heating gives evidence that temperature gradient focusing effects are negligible and charge exclusion is responsible for trapping. The theoretical model developed and experimentally demonstrated can be exploited for the preconcentration of cell free fetal DNA circulating in maternal plasma and other rare nucleic acid species present in large sample volumes.