DEVELOPMENT OF A DEVICE PLATFORM FOR PREDICTIVE AND PROGNOSTIC POINT-OF-CARE TESTING USING THE EXAMPLE OF PATHOGEN IDENTIFICATION Reiner Götzen2, Frank Scherag1, Gerd Sulz3, Martin Schmidt4, Marcus Panning5, Hans Attig6, Thomas Brandstetter1 and Jürgen Rühe1 1 Department of Microsystems Engineering IMTEK, University of Freiburg, Germany 2 microTEC Gesellschaft fürMikrotechnologie mbH, Duisburg, Germany 3 Fraunhofer Institute for Physical Measurement Technique IPM, Freiburg, Germany 4 Micropelt GmbH, Freiburg, Germany 5 Institute for Medical Microbiology and Hygiene, University of Freiburg, Germany 6 QIAGEN GmbH, Hilden, Germany
ABSTRACT We present a device platform for point-of-care testing of whole blood samples. It enables the determination of the viral load of blood on a processor-controlled device platform. For the analysis process two disposable cartridges carrying the fluidic and a media module are placed together in one device platform. In order to verify the operational capability of the device the following sample viruses were selected for the project and hoarded in two bio banks: Cytomegalovirus (CMV), varicella-zoster virus (VZV), herpes simplex virus (HSV) 1 and 2 and Epstein-Barr virus (EBV). KEYWORDS: Point-of-care testing, whole blood analysis, multiparametric diagnostics INTRODUCTION The field of micro total analysis systems (μTAS) or lab‐on‐a‐chip devices (LOC) has recently extended into many new fields and applications [1]. Especially in the context of in-vitro and point-of-care (POC) diagnostics the role and significance of lab‐on‐chip systems increases strongly to the need for fast multiparametric diagnostic measurements. At the same time the devices have to be robust and simple to allow a point-of-care application at low cost [2, 3]. The ultimate goal for a POC genetic analysis device is to have sample-in-answer-out capabilities [4]. In the presented “Total Analysis System” electronic, mechanical, fluidic, pneumatic, hydraulic, optical and thermal functions have been integrated, where two disposable cartridges carry the fluidic and the media module. It enables the determination of the viral load of blood on a processor-controlled device, which is capable of blood-plasma separation, magnetic bead-based DNA separation and real-time PCR amplification in a completely automated manner. FUNCTIONAL PRINCIPLE From the clinical point of view it was prerequisite to generate a closed loop in which the reagents would not interfere at any step with the device platform. The combination of blood-plasma separation, DNA extraction and its optical analysis requires a complex interplay of different components, which all pose separate requirements onto the analysis process (Figure 1).
Fig. 1: Schematic depiction of all processes for the analysis of the virus loads in blood. Shown are the principle mechanisms and the consecutive preparation steps for the sample-in-answerout disposable device. Source: microTEC The design presented here consists of a media cartridge, which contains the required reagents for the bead-based DNA extraction, such as isopropanol, lysis buffer with magnetic beads, wash buffer 1 and 2, water and the elution buffer, as well as a waste chamber for the remaining byproducts. The fluidic module and the media cartridge are two separate 978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001
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17th International Conference on Miniaturized Systems for Chemistry and Life Sciences 27-31 October 2013, Freiburg, Germany
disposable products that get connected when introduced for use in the device (Fig. 2). The fluidic module interconnects the plasma separation with the DNA preparation and the PCR equipment (Fig. 3). Both are generated using the RMPD mask process. RMPD, Rapid Micro Product Development in which 3D structures are generated in a photo polymerization process. The RMPD mask method was used to manufacture the very fine structures with a high accuracy along the zdirection as well as sharp edges laterally. Thereby, the sharp-edged structures are essential for the use of the capillary effects. The materials used in RMPD were carefully tested for biocompatibility in the analysis process. For the analysis process, 350 µl of whole blood from an EDTA tube are introduced into the device via a Luer-Lock adapter into the blood-plasma separation chamber. In the following step the entire microfluidic module rotates whereby the rotational forces and the different densities of the components ensure that the cellular components and the serum are separated [5]. By applying a negative pressure to the empty waste chamber and opening the vent for the separation chamber, the serum flows via the so-called plasma source into the mixing chamber for bead-based DNA extraction. By opening the venting of the PCR chambers the water with the DNA sequences is transferred to the numerous 16 µl capacity PCR chambers by capillary forces via channels that have the same length. Prior to the opening of the venting a natural over pressure is received due to the pending liquid column on the PCR chambers. Inside of the PCR chambers are different lyophilized reagents for real-time PCR. The selected temperature in the 4 x 4 x 1 mm3 large PCR chambers is applied via a 1 mm thick aluminum plate that was integrated into the fluidic modules. The aluminum plate has a burnished black anodized surface and a special PCR compatible coating. The plate has been integrated during the RMPD mask method process. Underneath the aluminum plate, integrated into the device platform, sits a Peltier element, which controls and regulates the temperature of the aluminum plate and therefore of the fluid in the PCR chambers. During each temperature cycle light of a white LED is directed from the sides into the PCR chamber. The white light induces fluorescence which is recorded by a camera through an optical system with three different filters as images of the four PCR chambers. The three images are analyzed via image processing systems using appropriate algorithms. They provide the basis of the measurement of the virus load of whole blood for each chamber over the temperature cycles of the PCR analysis. The overall time from the blood sample to the test result was less than one hour.
Fig. 2: Left: cartridge for the storing the buffers and the fluidic module; right: platform with all the technical components, necessary for the analyzing process. Source: microTEC, IPM Fraunhofer
Fig. 3: Fluidic module in detail, illustrated are all inlets and outlets, chambers, which are included in the process for DNA extraction and PCR-on-chip; Source: microTEC
RESULTS AND DISCUSSION Rotational forces applied to the entire microfluidic module resulted in a clear blood-plasma separation line (Fig. 4). Depending on the percentage proportion of the serum (50-73 %) different radii for the line of serum and cellular components are received. DNA samples from Herpes viruses have been mixed with a BSA solution and the mixture was extracted with the disposable chip in order to check the function and extraction efficiency of the chip with the proposed platform. The DNA recovery by the extraction method was controlled by real-time PCR analysis. DNA extract samples show good amplification products with the chip based PCR system and only minor loss of target DNA during extraction (Figure 5). DNA data bases of Herpes viruses have been established and contain lyophilized plasma samples as well as extracted DNA which are frozen at ‐80°C.
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Fig. 4: Separation of the cellular parts and the plasma. Source: microTEC, IMMH
Fig. 5: DNA recovery by the extraction method of Herpes simplex virus intermixed with a BSA solution was controlled by real-time PCR analysis [A]. DNA extract samples show good amplification products with the chip based PCR [B]. CONCLUSION The presented multiparametric biomolecular analysis of whole blood in one disposable device is the result of several innovative microfluidic approaches. The functionality of the blood plasma separation, DNA extraction and PCR performance was proven. The samples from the Herpes DNA databases will also be used to prove the informative value, which is given by this sample-in-answer-out system. ACKNOWLEDGEMENTS The cooperation partners gratefully acknowledge for the financial support from the Federal Ministry of Education and Research (BMBF FKZ0315596F) as well as our associated partner QIAGEN (QIAGEN GmbH, Hilden, Germany).
REFERENCES 1. Arora, A., et al., Latest Developments in Micro Total Analysis Systems. Analytical Chemistry, 2010. 82(12): p. 4830-4847. 2. Olasagasti, F. and J.C.R. de Gordoa, Miniaturized technology for protein and nucleic acid point-of-care testing. Translational Research, 2012. 160(5): p. 332-345. 3. Schumacher, S., et al., Highly-integrated lab-on-chip system for point-of-care multiparameter analysis. Lab on a Chip, 2012. 12(3): p. 464-473. 4. Ahmad, F. and S.A. Hashsham, Miniaturized nucleic acid amplification systems for rapid and point-of-care diagnostics: A review. Analytica Chimica Acta, 2012. 733: p. 1-15. 5. Reiner Götzen Patent pending DE 10 2011 012 464 A1; Verfahren zur Erzeugung von freiem Blutplasma aus Vollblut in einem Fluidikmodul und dessen weitere Behandlung und kapillare Führung im Modul. CONTACT Reiner Götzen, microTEC- Gesellschaft für Mikrotechnologien mbH, Bismarckstrasse 142 b, 47057 Duisburg, mail:
[email protected] 469
