F. Lacharme1, C. Vandevyver2 and M.A.M. Gijs1 Institute of Microelectronics and Microsystems Research Commission EPFL-SNF Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland MAGNETIC BEADS RETENTION DEVICE FOR ON-CHIP SANDWICH IMMUNO-ASSAY Reporter : Yen-Po Lin ( 林林林 ) Date: December 30 , 2008
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F. Lacharme1, C. Vandevyver2 and M.A.M. Gijs1 Institute of Microelectronics and Microsystems Research Commission EPFL-SNF Ecole Polytechnique Fédérale.
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F. Lacharme1, C. Vandevyver2 and M.A.M. Gijs1 Institute of Microelectronics and Microsystems
Research Commission EPFL-SNF Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland
MAGNETIC BEADS RETENTION DEVICE FOR ON-CHIP SANDWICH IMMUNO-ASSAY
Enzyme-Linked Immuno-sorbent AssayWhat is magnetic micro-bead ? How can fix micro-bead?Examples of this topic
(A) Schematic representation of flow-based micro-immunoassay system. Paramagnetic particles from a dilute solution are collected near a rare earth magnet to form a packed bed within a capillary or channel. Reagents are introduced into the packed bed to perform standard immunoassays (either heterogeneous or sandwich assay), and the bed is imaged with an epifluorescence microscope with laser-induced excitation.
Mark A. Hayes, Nolan A. Polson, Allison N. Phayre Antonio A. Garcia
Schematic diagram of a generic micro-fluidic system for biochemical detection.
Analytical concept based on sandwich immunoassay and electrochemical detection.
Conceptual illustration of bio-sampling and immunoassay procedure: (a) injection of magnetic beads; (b) separation and (holding of beads: (c) flowing samples: (d) immobilization of target antigen; (e) flowing labeled antibody; @ electrochemicaldetection; and (g) washing out magnetic beads and ready for another immunoassay.
Jin-Woo Choi
Photograph of the fabricated bio-filter and immuno-sensor.
Photograph of the fabricated micro-fluidic biochemical detection system for magnetic bead-based immunoassay
Integrated micro-fluidic biochemical detection system has been successfully developed and fully tested for fast and low
Micro-fluidic biochemical analysis system that includes a surface-mounted bio-filter and immuno-sensor on a glass micro-fluidic motherboard.
Abstract
• Geometrical retention of self-assembled magnetic micro-beads in a structured micro-channel.
• Brought in a homogeneous magnetic field, a solution of 500 nm diameter magnetic micro-beads self-assembles in magnetic chains in the large sections of a periodically enlarge micro-channel
• Device is able to perform full on-chip direct and sandwich immuno-assays, in a total assay time of less than 30 min
Design
Micro-channel
Figure 1 Schematic layout of the 20um wide micro-channel showing the periodically enlarged sections of 30um
15 um
5 um
20um
30um
Fabrication 1
Pyrex a-si Photo-resist PDMS
Figure 2: (i) 2 μm thick a-Si deposition. (ii) pinning,exposure and development of the positive photoresist. (iii)DRIE of the a-Si protective mask and photoresist removal.(iv). DRIE of the a-Si to a depth of 8 μm. (iv) Removing of the a-Si protective mask in a KOH bath. (v) 2 mm PDMS cover with access holes placed on the microchip.
4 inch
Fabrication 2
Figure 4: Picture of the microchip (a) placed on its support (b) containing 2 permanent magnets (c) for the generation of the permanent homogeneous magnetic field.
Figure 3: SEM picture of the final micro-channel showing the periodically enlarged sections.
Principle 1
Figure 5: (a) Schematic diagram of the self-assembled magnetic beads in chains in the large cross-sections of the Micro-channel when applying a magnetic field H. (b)Schematic diagram of a flow applied through the magnetic chains exerting a dragging force which displaces the central part of the chains over a distance X.
μ0` : the magnetic permeability of vacuumM :magnetic moment of one beadRij :the distance between theMicro-beads with index i and jΘ: between the external magnetic field direction and the micro-bead center-to-center vector
Principle 2
Figure 6: Magnetic dipole energy of a chain of 60 Micro-beads (dashed line) and its magnetic dipolar force Fchain (solid line) as a function of X.
Fchain = - dEchain / dX
Figure 7: Optical image of the micro-channel with the self-assembled magnetic micro-bead chains.
Result 1 (Direct)
Figure 8: (a) Schematic drawing showing the formation of the fluorescent immuno-complexes during the direct immuno-assay on-chip. (b) Fluorescent microscopy image of the self-assembled magnetic micro-bead chains after the completion of the on-chip direct immuno-assay.
Figure 9: Fluorescence intensity profile along 800 μm of the micro-channel as derived from images like in figure 8b. The chains that are more upstream positioned in the micro-channel show the highest fluorescence intensity.
Result 2 (two-site)
Figure 10: (a) Schematic drawing showing the formation of the fluorescent immuno-complexes during the two-site immuno-assay on-chip.
Figure 11: Integrated fluorescence as a function of (a) the concentration of rabbit biotinylated IgG during the direct immuno-assay and (b) the mouse IgG concentration during the two-site immuno-assay.
3min
(i)rabbit biotinylated antibody anti-mouse IgG for 3 min,followed by a solution of (ii) purified mouse IgG, obtained from a cell culture, and (iii) a Cy3 fluorescent labeled antimouse IgG antibody solution during 5 min for the detection
Conclusion
• Micro-beads specifically capture and detect a low number of target analyte molecules (less than 105 ) in a very small sample volume of ~31 nL in an total assay time of less than 30 min.
• The magnetic chains gradually deplete the target antigen concentration from the flow• Our method permits to position a well-controlled and small amount of micro-beads in the micro-fluidic system• The way for fast, precise and extremely cheap detection of
specific molecules from a complex matrix
Reference[1]A. Manz, N. Graber, and H. M. Widmer, "Miniaturized Total Chemical-Analysis Systems -a Novel Concept for Chemical Sensing," Sens.Actuators A, vol. 10, pp. 244-248, 1990.[2] K. Sato, M. Tokeshi, H. Kimura, and T. Kitamori,"Determination of carcinoembryonic antigen inhuman sera by integrated bead bed immunoasay in a microchip for cancer diagnosis," Analytical Chemistry, vol. 73, pp. 1213-1218, 2001[3] M. A. M. Gijs, "Magnetic bead handling on-chip:new opportunities for analytical applications, ”Micro-fluidics and Nano-fluidics, vol. 1, pp. 22-40,2004.[4] M. A. Hayes, N. A. Polson, A. N. Phayre, and A.A. Garcia, "Flow-based micro-immunoassay," Anal.Chem., vol. 73, pp. 5896-5902, 2001.[5] J. W. Choi, K. W. Oh, J. H. Thomas, W. R.Heineman, H. B. Halsall, J. H. Nevin, A. J.Helmicki, H. T. Henderson, and C. H. Ahn, "An integrated micro-fluidic biochemical detectionsystem for protein analysis with magnetic bead based sampling capabilities," Lab on a Chip, vol. 2,pp. 27-30, 2002.[6] A. C. Siegel, S. S. Shevkoplyas, D. B. Weibel, D. A. Bruzewicz, A. W. Martinez, and G. M. Whitesides, "Cofabrication of electromagnets and microfluldic systems in poly(dimethylsiloxane),“ Angewandte Chemie-International Edition, vol. 45,pp. 6877-6882, 2006.
Reference
[9] M. Herrmann, "Microfluidic ELISA on nonpassivated PDMS chip using magnetic beadtransfer inside dual networks of channels," Lab Chip, pp. DOI: 10.1039/b707883h, 2007.[10] A. Rida, V. Fernandez, and M. A. M. Gijs, "Longrange transport of magnetic microbeads using simple planar coils placed in a uniform magnetostatic field," Applied Physics Letters, vol.83, pp. 2396-2398, 2003.[11] P. S. Doyle, J. Bibette, A. Bancaud, and J. L. Viovy, "Self-assembled magnetic matrices forDNA separation chips," Science, vol. 295, pp. 2237-2237, 2002.[12] L. E. Helseth, "Self-assembly of colloidal pyramids in magnetic fields," Langmuir, vol. 21, pp. 7276-7279, 2005.
[7] T. Deng, M. Prentiss, and G. M. Whitesides, "Fabrication of magnetic microfiltration systems using soft lithography," Applied Physics Letters, vol. 80, pp. 461-463, 2002.[8] A. Rida and M. A. M. Gijs, "Manipulation of selfassembled structures of magnetic beads for microfluidic mixing and assaying," Analytical Chemistry, vol. 76, pp. 6239-6246, 2004