Integrated Microfluidic Compact Disc Device with Potential Use in Both Centralized and Point-of-Care Laboratory Settings

Reducing the size of reaction volumes in immunodiagnostic procedures can reduce not only reagent cost but also analysis time. With reduced sample and reagent volumes and use of a flow-through mode, reactions reach completion rapidly, potentially decreasing turnaround time and making such systems suitable for point-of-care testing. However, decreased reaction volumes typically lead to other problems, such as imprecision of liquid metering, liquid evaporation, and problems related to the increased surface-to-volume ratio. To address these challenges, we have developed a new bioanalytical system composed of a disposable microfluidic compact disc (CD) device (Gyrolab Bioaffy™) that is intended for sandwich immunoassays and an instrument for automatic processing of CDs (Gyrolab™ Workstation). The Gyrolab Bioaffy CD (diameter, 120 mm) contains 104 parallel microstructures conveniently fitting into the CD, each covering a surface area of ∼2 × 15 mm. The CD is manufactured by injection molding using a cycloolefin polymer. The CD is covered with a lid in which appropriately placed holes enable liquid introduction by the robot into the microstructures; the lid protects liquids from evaporation once in the CD. Liquid movement and localization are achieved by a combination of capillary action, centrifugal force, and hydrophobic barriers within the microstructure. Each microstructure contains a 15-nL particle-based column coupled with streptavidin in which reactions take place (Fig. 11 ). The Gyrolab Bioaffy is designed to be an open system; i.e., it is compatible with various assays that use biotinylated reagents, which bind to the streptavidin column as the first layer of the sandwich. Thus the capture column in a microstructure can promptly be functionalized for different analytes by addition of selected biotinylated capture reagents in excess. Each microstructure contains integrated functions for several process steps, such as volume metering, and each microstructure can be accessed through an individual inlet or through a common channel connecting 8 microstructures. The workstation automatically handles the analytical process from addition of capture reagent to detection of reactions. It is equipped with a robotic station for liquid transfer from a microtiter plate to a CD, a CD spinner, and a detector for HeNe laser-induced fluorescence at 633 nm. Reagents and samples, kept in microtiter (MT) plates, are placed in a carousel in the instrument. The system is designed to handle maximally 5 CDs in a batch run. The instrument is also equipped with dedicated software to handle all process steps and evaluate the data. Fluorescence detection is carried out on column by rotating the CD while moving the detector in the radius direction. The fluorescent signal is integrated and represents the total response of the reaction. A few microliters of sample can be handled by the robotic system. For each data point, 200 nL of sample is analyzed. Wash liquids, reagents, and samples are sequentially introduced into the CD according to the method. All process steps, including the analyte capture step, are performed under flow conditions by spinning the CD at appropriate rotational speeds. More than 100 data points can be generated in 50 min. To avoid evaporation in MT plates, an aluminum foil that is penetrated by capillaries on aspiration of liquids is attached on top of MT plates. Commercially available antibodies specific for human analytes were selected for their assay performance in the system. Recombinant proteins were used to create calibration curves diluted in Gyros Standard Diluent (Gyros AB). Capture antibodies were labeled with biotin (cat. no. 21335; Pierce) and detection antibodies with ALEXA 647 (cat. no. A-20186; Molecular Probes) according to instructions from the manufacturers. Biotinylated capture reagents were used at a concentration of 100 mg/L, which is sufficient to saturate the streptavidin column, and detection reagents at 2–5 mg/L. Samples were prepared by mixing sample and equal volumes of Gyros Sample Diluent (Gyros AB). All quantitative assays are critically dependent on precise metering of sample volume. In the Gyrolab Bioaffy, volume definition is performed within the CD as part of the analysis procedure. When human EDTA plasma was subjected to volume definition within the CD microstructure, a 200-nL aliquoted sample volume was prepared with a CV of 0.75% (n = 560). The measuring range for a given analyte is dependent on the reagent properties and the binding capacity of the capture column. When working at low analyte concentrations, the analyte content of the 200-nL sample volume is enriched to a few nanoliters at the top of the capture column. When using high-quality immunoreagents, subpicomolar concentrations of analyte can be determined with a measuring range of 3–4 orders of magnitude of analyte concentrations before the column becomes saturated (data not shown). To illustrate the intra-CD precision of the Gyrolab Bioaffy, recombinant human interleukin-2 (hIL-2) was diluted to 50, 500, and 5000 ng/L. Each preparation was repeatedly analyzed for hIL-2 content in 3 separate CDs, each generating 104 data points per concentration. The CVs were 6.0%, 4.0%, and 4.2% for hIL-2 concentrations of 50, 500, and 5000 ng/L, respectively. To determine recovery in the Gyrolab Bioaffy, we added recombinant hIL-1β to 2 sets of human serum and plasma samples (citrate, EDTA, and heparin) at 2 concentrations (20 and 200 ng/L). The concentration of hIL-1β was measured with use of a set of immunoreagents specific for human hIL-1β. Mean recovery of hIL-1β was 74%–117% in serum, 102%–104% in citrate plasma, 95%–101% in EDTA plasma, and 88%–105% in heparin plasma. We developed a prototype CD containing microstructure functions for integrated separation of whole blood into plasma combined with an immunoassay for myoglobin as an illustrative example of how integration of functions for an intended process can be achieved with CD microfluidic technology. The process took place in 4 steps: First, 3 μL of fresh heparin blood was applied to a CD structure inlet that filled by capillary action. Plasma was then generated in the upper part of the microfluidic structure (not shown here) by a sedimentation spin at 1500 rpm for 180 s. Next, an overflow channel was activated and defined the plasma volume of 0.5 μL (CV <1%). Finally, plasma was transferred to the top of the capture column through an outlet channel by increasing the rotational frequency to 5000 rpm. The subsequent assay procedure was similar to the Gyrolab Bioaffy method described above. To test the prototype functions, fresh heparin blood samples were collected from 3 donors. Plasma was prepared within the CD and analyzed for myoglobin. In parallel, plasma was generated outside the CD by conventional means, introduced into the CD through a downstream inlet, and analyzed for myoglobin. The fluorescence intensities of the reactions representing the amount of myoglobin were compared for each sample set (Table 11 ). In summary, the results obtained for hIL-2 and hIL-1β regarding precision, dynamic range, and recovery in different matrices illustrate the performance of the Gyrolab Bioaffy. The features of the CD microfluidic technology can be adapted to meet specific needs and requirements for in vitro diagnostic purposes, for example, by incorporating functions for blood separation in point-of-care laboratory settings. Costs for reagents can be lowered 10- to 100-fold by reducing reagent consumption. Our experience to date suggests that existing assays developed for established in vitro diagnostic formats can be readily transferred into a CD microfluidic format. Gyrolab Bioaffy CD for sandwich immunoassay. Comparison of response from CD-based immunoassay of myoglobin in heparin plasma with separation of plasma from blood cells performed either inside or outside the CD. Comparison of response from CD-based immunoassay of myoglobin in heparin plasma with separation of plasma from blood cells performed either inside or outside the CD.