This paper presents an optical concept for the read-out of a parallel, bead-based fluorescence immunoassay conducted on a lab-on-a-disk platform. The reusable part of the modular setup comprises a detection unit featuring a single LED as light source, two emission-filters, and a color CCD-camera as standard components together with a spinning drive as actuation unit. The miniaturized lab-on-a-disk is devised as a disposable. In the read-out process of the parallel assay, beads are first identified by the color of incorporated quantum dots (QDs). Next, the reaction-specific fluorescence signal is quantified with FluoSpheres-labeled detection anti-bodies. To enable a fast and automated read-out, suitable algorithms have been implemented in this work. Based on this concept, we successfully demonstrated a Hepatitis-A assay on our disk-based lab-on-a-chip

Brenner, T.

Ducrée, Jens

Ehlert, O.

Grumann, M.

Mittenbuhler, K.

Nann, T.

Pastewka, L.

Riegger, L.

Riegler, J.

Urban, G.

Zengerle, Roland

English

Irish Universities

Parallelization of chip-based fluorescence immuno-assays with quantum-dot labelled beads

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3A1.4 PARALLELIZATION OF CHIP-BASED FLUORESCENCE IMMUNO-ASSAYS WITH QUANTUM-DOT LABELLED BEADS M. Grumann‘, L. Riegger’, T. Nand,  J,  Riegler2, 0. Ehlert2, K. Mittenbiihle?, G. Urbad4,  L. Pastcwkal, T. Brenner’, R. Zengerle’, and J. Ducree’ ‘IMTEK - University of Freiburg, Laboratory for MEMS Applications, Georges-Kohler-Allee 106, D-79110 Freiburg, Germany ’University of Freiburg, FMF University Hospital Freiburg, Institute for Molecular Medicine and Cell Research 41MTEK ~ University of Freiburg, Laboratory for Sensors 3 ABSTRACT This paper presents a novel optical concept for the read- out of a parallel, bead-based fluorescence immunoassay conducted on a lab-on-a-disk platform. The reusable part of thc modular setup comprises a detection unit featuring a single LED as light source, two emission- filters, and a color CCD-camera as standard components together with a spinning drive as actuation unit. The miniaturized lab-on-a-disk is devised as a disposable. In the read-out process of the parallel assay, beads are first identified by the color of incorporated quantum dots (QDs). Next, the reaction-specific fluorescence signal is quantified with FluoSpheres-labeled detection anti- bodies. To enable a fast and automated read-out, suit- able algorithms have been implemented in this work. Based on this concept, we successfully demonstrated a Hepatitis-A assay on our disk-based lab-on-a-chip. Keywords: FIA, parallel assays, beads, quantum dots, centrifugal microfluidics INTRODUCTION The miniaturization of devices for clinical diagnostics towards point-of-care applications 1s subject of numer- ous approaches. Typically, such “lab-on-a-chip’’ or “micro total analysis” systems (pTAS) feature a full process integration, a high reproducibility, reduced consumption of sample and reagents as well as short times-to-result, and ease of handling [ 11-[6]. Besides the development of the analytical chips which are pref- erably disposable, detection devices have been developed which basically comprise an actuation and a read-out unit [7][8]. To further advance towards a comprehensive picture of the patient’s health state, especially in the field of emer- gency diagnostics, the parallel determination of a set of relevant biochemical markers in a given sample would drastically accelerate the benefit of these pTAS. As a special type of “lab-on-a-chip” systems, a “lab-on- a-disk” [SI-[ 121 exploits centrihgal forces generated by a spinning drive to pump liquids. On its way radially outwards, the sample is processed [ 13][ 141 and finally reaches a detection chamber. While the spinning drive, as the actuation unit, is a robust and reusable “macro” component, the “micro”-parts are here reduced to their functional essence, i.e. passive microstructures which can be fabricated in a very economic fashion, e.g. by industrial scale micromachining techniques such as hot- embossing or injection molding. The implicit advantages of centrifugal microfluidics like its robustness and reproducibility together with its cost efficiency recommend this platform for point-of-care applications, especially in the field of emergency as well as clinical diagnostics [15]-[17]. In our approach, a set of fluorescence immunoassays (FIAs) is run on a lab-on-a-disk with the use of color- tagged beads. Prior to loading them into the disk, the beads are fhctionalized with the relevant capture anti- bodies. In the automated read-out of the assay results using an image processing software, each bead is local- ized and spectrally identified in a first “tag-color” CCD image. A second fluorescence picture acquires com- pound fluorescence intensity of each bead. QUANTUM-DOTS AS OPTICAL BEAD-TAG Quantum-dot (QD) crystallites excel with their broad- band excitation and their narrow-band luminescence. Their spectrum can be tuned by their diameter ( ~ Q D  5 nm) during fabrication [ 18]-[20]. We use the large number of optically distinguishable QDs made of CdSe for bead identification in parallel assays (Fig. 1). To this end, the QDs are first suspended with poly- styrene (PS) beads (abead = 150 pm) in an organic sol- vent. The solvent swells the PS, thus incorporating roughly IO5 quantum-dots in the polymer matrix of the bead. By vacuum drying, the pores shrink to irreversi- bly capture the QDs. TRANSDUCERS’OS The 13th International Conference on Solid-state Sensors, Actuators and Microsystcms, Seoul, Korea, June 5-9,2005 0-7803-8952-2/05/$20.00 02005 IEEE. 1114 Authorized licensed use limited to: DUBLIN CITY UNIVERSITY. Downloaded on July 12,2010 at 13:16:33 UTC from IEEE Xplore.  Restrictions apply. 3A1.4 154 5W 55D MO 61(1 7W 7sO Wavelength /z (nm) Fig. 1 Quantum-dots as optical tag of the beads: Three types of quantum-dots (QD578, QDhlO, QD653) are incorporated in beads and their lu- minescence spectra are recorded while being commonly excited by an LED (beak = 475 run). To optically distinguish the bead-tag from the signal of the high-intensity FluoSpheresTM [24] as assay label two optical filters are required. A long-pass filter (LP 570) selects the luminescence of the QDs to record the bead-tag while a band-pass filter (BP 505- 540) exclusively transmits the signal of the fluorochrome as a measure of the assay result. PARALLEL FLUORESCENCE IMMUNOASSAY As an off-disk preparative step of the parallel bead- based FIA, each type o f  QD-encoded bead is first h n c -  tionalized with designated capture proteins (Fig. 2). Next, the QD-beads representing different capture anti- bodies are mixed, suspended in a buffer solution, and loaded into the inlet reservoir of the disk (material: Cyclic Olefin Copolymer, COC). The disk also com- prises a detection chamber and an outlet drain (Fig. 3 - A). The liquid is then transported by the centrifugal force F ,  into the detection chamber. Here, the outlet drain of the detection chamber establishes a geometrical barrier to thc beads as the depth of the chamber do,, is smaller than the bead diameter (i.e., do,, = 90 pm = 0.6 . dkad) (Fig. 3 -B). On the other hand, the depth of the detection chamber a,,, = 180 F]-m is chosen only slightly larger than dbead to aggregate a statistically distributed monolayer in the detection chamber. This configuration allows a direct optical access to all aggregated beads for a CCD camera mounted in a top view position of [211[221. The subsequent on-disk fluorescence immunoassay (FIA) is implemented by a custom-made spinning protocol Nt) controlling the flow rates and the incuba- tion times at high accuracy. After the delivery of the sample and washing, the FluoSpheresTM labelled, detection antibodies [24] specifically react with the previously formed antibody-antigen complex. A Functionalization with capture antibody B Sample containing target antigens C Detection antibody 3 Capture antibody Target antigen Detection antibody with fluorescent label Fig. 2 Concept of a fluorescence immunoassay (FIA) based on beads as solid-phase. (A) The beads are first functionalized with a certain capture protein and then loaded into the detection chamber on-disk. (B) Next, the sample con- taining the target antigens is transported into the detection chamber and the specific antigen- antibody complex is formed. (C) After wash- ing, the added detection antibodies which are labelled with fluorochromes specifically bind to these antigen-antibody complexes. The read-out procedure as the final step of the parallel FIA significantly deviates from the procedure of the conventional single-channeI assay (Fig. 3 ~ C). The fluorescence intensity has to be spatially resolved to allow the assignment to the type of bead in the subse- quent step. We first acquire an image of the bead-tag distribution (LP 570, Fig. 1) by illumination with a high-power LED (beak =475 nm) [23] and capture the image with a color CCD-camera [25]. With the same light source, the distribution of the assay-specific fluo- rescence intensity is recorded in a second image with a bandpass filter (BP 505-540, Fig. 1 ). READ-OUT PROCESSING ALGORITHM The post-processing of the first image locates the cen- ters and identifies the color-IDs of the QD-labelled beads using a Hough transform (Fig. 4) [XI. TRANSDUCERS’OS The 13th International Conference on Solid-state Sensors, Actuators and Microsystems, Seoul, Korea, June 5-9,2005 1115 Authorized licensed use limited to: DUBLIN CITY UNIVERSITY. Downloaded on July 12,2010 at 13:16:33 UTC from IEEE Xplore.  Restrictions apply. 3A1.4 The recognition of beads starts by taking the Euclidean distance between the vector of a pixel and reference vectors of a QD-type in RGB-space. This distance is compared to a threshold Ri and, if accepted, the current pixel is transformed to the Hough space of the bead centers for a given bead radius. Here, each pixel-value within the bead-radius of the current pixel is increased by one. The bead centers are then determined by finding the maxima in Hough space. Additional robustness is achieved by constraining the distance of neighbouring beads to more than the bead radius. The programmed routine lasts less than 15 s and displays a reliability better than 80 % . color C C D - ~ ~ ~ ~ ~ ~  ,center of rotation aggregation & pattern of 3 types of beads I do,, -v- -.- B I  I - * ~  0 L. c.ul---l dd!t C P,m aggregated beads I 1 emission filter Read-out of a bead-based fluorescence immu- noassay (FIA) on-disk. (A) Beads as solid phase of the HA are loaded into the detection chamber of the disk where they aggregate as a monolayer (B) in front of a geometrical bar- rier. (C) After the assay is conducted, the beads are excited by the light of an LED [23] with peak wavelength hlcak. The induced light (Am) of the fluorochrome [24] is spectrally fil- tered by the emission filter to suppress the re- flected light of the exciting LED and then di- rected towards a color CCD-camera [ 2 5 ]  as de- tector. Note that the photographs originally features distinguishable colors.' Fig. 3 A Color image of QD-luminescence Hough-space /"?, I$-) QDz Fig. 4 Concept of the image processing routine for a three-fold assay: (A) each pixcl of the QD- luminescence image is transferred as a vector into RGB-spacc and the Euclidean distance d to the reference vectors QDi (i = I ,  2, 3) of the QD-colors is measured. (B) For d < Ri, with R, as a color threshold, this pixel is assigned to QDi  and transformed to the Hough space 1261 of the bead centers for a given bead radius. (C) The bead centers are then determined by find- ing the maxima in Hough space. (D) From the fluorescence image, the assay results are meas- ured by averaging the fluorescencc signal within a concentric circle to suppress signal overlapping. RESULTS To demonstrate the feasibility of a parallel, disk-based FIA with the QD-encoded beads, a dilution series is run. It starts from standard human serum (Paul Ehrlich Insti- tute) containing a known (calibrated) concentration of Hepatitis-A antibodies (Fig. 5). The measured curve clearly allows to distinguish the state of long-term pro- tection by Hepatitis-A vaccination (10 international units per ml according to the WHO regulations). TRAN SDUC ERS'OS The 13th International Conference on Solid-state Sensors, Actuators and Microsystcms, Seoul, Korea, lune 5-9,2005 1116 Authorized licensed use limited to: DUBLIN CITY UNIVERSITY. Downloaded on July 12,2010 at 13:16:33 UTC from IEEE Xplore.  Restrictions apply. 3A1.4 ~ inslfficient I,/ long-term protection v by vaccination x 50 4- .- VJ 0.1 1 10 100 Concentration of Hepatitis A antibodies (IUlml) Fig. 5 Calibration curve of the Hepatitis A assay: the result clearly allows to determine the state of protection by Hepatitis-A vaccination accord- ing to the WHO regulation (limit at 10 intema- tional units (IU) per mi). CONCLUSION We realized a rugged optical setup assembled by con- ventional components which is suitable for parallelized fluorescence immuno-assays. Color encoding is imple- mented by incorporating quantum-dots into the beads while high-intensity FluoSpheresTM are coupled to the detection antibodies. We successfully demonstrated a Hepatitis-A assay on a miniaturized lab-on-a-disk platform. In contrast to complex high-end setups such as sequentially operating flow cytometers, our setup well complies with the technical and economic demands of diagnostic point-of-care applications. ACKNOWLEDGEMENTS The authors are grateful to the partial support by the Ministry of Science, Research and the Arts of the Ger- man federal state of Baden-Wurttemberg (contract 24- 720.43 1 - 1 -7/2) and the good cooperation with HSG- IMIT and Jobst-Technologies. REFERENCES [ I ]  Sia, S.; Linder, V.; Parviz, B.; Siegel, A.; Whitesi- des, G. Angew. Chem. Int. 2004,43,498-502. [2] Vilkner, T.; Janasek, D.; Manz, A. Anal. Chem. [3] Ducree, J.; Zengerle, R. FlowMap - MicroJuidics roadmap for the hJe sciences, 2004, Books on De- mand GmbH: Norderstedt, Germany, ISBN 3- 8334-0744- 1, tvww.microfl uidics-roadmap.com. [4] Reyes, D.; Iossifidis, D.; Auroux, P.; Manz, A. Anal. Chem., 2002,74,2623-2636. 2004,76,3373-3386. [ 5 ]  Auroux, P.; Reyes, D.; Iossifidis, D,; Manz, A. Anal. Chem., 2002, 74, 2637-2652. [6] Examples in point-of-care diagnostics: Careside Analyzer*, Careside Inc, USA; Biosite@ Biosite Inc., France; Pelikan SunTM of Pelikan Technolo- gies Inc., USA; Chempaq Analyzer, Chempaq Ais, Denmark; i-STAT Portable Clinical Analyzer, i- STAT Corp., USA. [7] Triage Meter Plus, Biosite Inc., USA, www.biosite.com. [ X I  Cardiac Reader, F. Hoffmann-La Roche Ltd., Switzerland, www.roche.ch. [9] Schembri, C.; Burd, T.; Kopf-Sill, A.; Shea, L.; and Braynin, B., Journal of Automatic Chemistty, [IO] Puckett, L.; Dikici, E.; Lai, S.; and Madou, M. Anal. Chem., 2004,76,7263-7268. (111 Ekstrand, G.; Holmquist, C.; Orleforn, A. E.;  Hellmann, E.; Larsson A.; and Andersson, P. Proc. pTAS2DOO conference, 2000,311-314. [I21 Ducree, J.; Brenner, T.; Grumann, M.; Bessler, W.; Stelzle, M.; Messner, S.; Nann, T.; Ruhe, J.; Moser, 1.; Zengerle, R. Bio-Disk - A Centrifugal Platform for Integrated Point-of care Diagnostics on Whole Blood, 2003, www.bio-disk.com. Brenner, T.; Haberle, S.; Zengerle, R.; and Ducrie, J. Proc. pTAS 2004 conference, 2004,566-568. Grumann, M.; Geipel, A.; Riegger, L.; Zengerle, R.; and DucrCe, J .  Lab on a Chip, 2005, accepted for publication. [ 151 LabCDTM, Tecan Group Ltd., Switzerland, www. tecan.com. [ 161 Gyrolab Bioaffy CD, Gyros AB, Sweden (formerly Pharmacia Inc., USA), www.gyros.com. [ 171 Piccolo, Abaxis Inc., USA, www.abaxis.com. [ 181 Nann, T.; Riegler, J. Chem. Eur. J. ,  2002, 8(20), [I91 Kucur, E.; Riegler, J.; Urban, G.; and Nann, T. J. [20] Nann, T.; Mulvaney, P. Angew. Cham. Int. Ed, [21] Grumann, M.; Schipppers, P.; Dobmeier, M,; Ha- berle, s.; Geipel, A.; Brenner, T.; Kuhn, c.; Frit- sche, M.; Zengerle, R.; and Ducree, J. Proc. [22] Grumann, M.; Dobmeier, M.; Schippers, P.; Bren- ner, T.; Zengerle, R.; and Ducree, J .  Lab on a chip, [23] Luxeon 111 Star, Lumileds Inc., USA, [24] F8774 (Axc = 505 nm, A,, = 515 nm), Molecular 1251 Axiocam MRc, Carl Zeiss AG, Germany, [26] Young, T. Y. Handbook ofpuffer# recognition and 1995,17 (3), 99-104. 479 14795. Chem. Phys,. 2003, 119(4), 2333-2337. 2004,43,5393-5396. IMECE (ASME), 2003,2003-41427. 2004,4,209-2 13. www.lumileds.com. Probes, USA, www.probes.com. www.zeiss.de. imageprocesssing, 1986, Academic Press. TRANSDUCERS05 The 13th International Confcrence on Solid-state Sensors, Actuators and Microsystems, Seoul, Korea, June 5-9,2005 1117 Authorized licensed use limited to: DUBLIN CITY UNIVERSITY. Downloaded on July 12,2010 at 13:16:33 UTC from IEEE Xplore.  Restrictions apply. 

Bio-Disk - A Centrifugal Platform for Integrated Point-of care Diagnostics on Whole Blood,

Examples in point-of-care diagnostics: Careside Analyzer*, Careside Inc, USA; Biosite@ Biosite Inc., France; Pelikan SunTM of Pelikan Technologies Inc., USA; Chempaq Analyzer, Chempaq Ais, Denmark; i-STAT Portable Clinical Analyzer,

Parallelization of chip-based fluorescence immuno-assays with quantum-dot labelled beads

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