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 (林諺伯 )

Date: December 30 , 2008

Outline

IntroductionAbstractDesign FabricationPrincipleResultsConclusionReference

Introduction

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

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Reference

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