Dose-response curve of a microfluidic magnetic bead-based surface coverage sandwich assay

Magnetic micro-and nanoparticles ('magnetic beads') have been used to advantage in many microfluidic devices for sensitive antigen (Ag) detection. Today, assays that use as read-out of the signal the number count of immobilized beads on a surface for quantification of a sample's analyte concentration have been among the most sensitive and have allowed protein detection lower than the fg mL(-1) concentration range. Recently, we have proposed in this category a magnetic bead surface coverage assay (Tekin et al., 2013 [1]), in which 'large' (2.8 mm) antibody (Ab)-functionalized magnetic beads captured their Ag from a serum and these Ag-carrying beads were subsequently exposed to a surface pattern of fixed 'small' (1.0 mm) Ab-coated magnetic beads. When the system was exposed to a magnetic induction field, the magnet dipole attractive interactions between the two bead types were used as a handle to approach both bead surfaces and assist with Ag-Ab immunocomplex formation, while unspecific binding (in absence of an Ag) of a large bead was reduced by exploiting viscous drag flow. The dose-response curve of this type of assay had two remarkable features: (i) its ability to detect an output signal (i.e. bead number count) for very low Ag concentrations, and (ii) an output signal of the assay that was non-linear with respect to Ag concentration. We explain here the observed dose-response curves and show that the type of interactions and the concept of our assay are in favour of detecting the lowest analyte concentrations (where typically either zero or one Ag is carried per large bead), while higher concentrations are less efficiently detected. We propose a random walk process for the Ag-carrying bead over the magnetic landscape of small beads and this model description explains the enhanced overall capture probability of this assay and its particular non-linear dose response curves. Research Paper

Automated Analysis of Rare Biomarkers Using a Magnetic Bead Surface Coverage Assay Coupled with Chaotic Mixing on a Microfluidic Chip

Hüseyin Cumhur Tekin

Extremely sensitive protein detection is crucial for early diagnosis and monitoring of diseases. Present day’s limit of detection for an antibody-antigen recognition-based immunoassay in serum without having to use special target antigen amplification techniques, like immuno-linked polymerase chain reaction (PCR), is 10 fg/mL, a sensitivity achieved thanks to the use of relatively large sample volumes (40-100 μL). In this thesis, a new method is developed to analyze rare protein biomarkers in a serum sample using a magnetic bead surface coverage assay. The assay protocols are realized on a microfluidic chip having channels, valves, an integrated mixer, and a protein micro-array. The microfluidic chip provides automated analysis, minute sample consumption (5 μL), resulting in an unprecedented 2 fg/mL detection performance for Tumor Necrosis Factor-α (TNF-α) in serum. A first part of the thesis consists in fabricating protein micro-arrays using the surface of immobilized ‘small’ (1.0 μm size) superparamagnetic beads that are placed in microfluidic flow channels. The small beads are immobilized on a glass chip with pre-patterned aminopropyl- trietoxysilane (APTES) structures using an electrostatic self-assembly method. Different target antigens are captured from a flow passing over the micro-array inside the microfluidic channel, and are specifically bound on the small bead surfaces. The presence of target antigens is detected as a fluorescent signal by realizing a sandwich immunocomplex on the capture antibody-functionalized surfaces of the small beads. However, the fluorescent detection method is abandoned due to high fluorescence background levels, resulting in only a 0.25 ng/mL detection limit for mouse immunoglobulin in phosphate-buffered saline. In a second part of the thesis, a polydimethylsiloxane (PDMS) chip having microfluidic channels, valves and an integrated mixer is designed. A new active mixing strategy is performed in the chip using multiple source-sink microfluidic flows. To do so, four different pressure- controlled actuation chambers are arranged on top of the 5 μL volume of the mixing chamber. After microfluidic valves, realized using ‘microplumbing technology’, seal the mixing volume, a virtual source-sink pair is created by pressurizing one of the membranes and, at the same time, releasing the pressure of a neighboring one. This creates microfluidic flows from the squeezed region (source) to the released region (sink) where the PDMS membrane is turned into the initial state. Perfect mixing is 7000-fold faster than achievable with pure diffusion-based mixing. This excellent performance is possible thanks to the implementation of chaotic advection by inducing vortices inside the mixing chamber. A DNA isolation and purification protocol using magnetic beads in the mixing chamber is also performed on-chip, showing the enhanced DNA binding performance due to active mixing. Finally, a new protein detection method is realized in a microfluidic chip. ‘Large’ (2.8 μm size) functionalized superparamagnetic beads specifically capture target antigens from a serum matrix under active microfluidic mixing. Subsequently, the large beads loaded with the antigens gently expose antibody-functionalized small bead patterns on the glass detection chip. During the exposure, attractive magnetic bead dipole-dipole interactions improve the contact between the two bead types and help the antigen-antibody immunocomplex formation, while non- specific large bead adsorption is limited by exploiting viscous drag forces in the microfluidic channel on the detection chip. This efficient transport mimics a biological process, where leukocytes roll and slow down on blood vessel walls by selectin-mediated adhesion for rapid recognition of tissue molecules. Afterwards, the antigen concentration is detected by simply counting the surface pattern-bound large magnetic beads. The new technique allows detection of only a few hundreds of TNF-α molecules (600 yoctomole) spiked in the 5 μL serum sample. This performance, in terms of detectable number of molecules, is at least 20 times better than today’s most-sensitive PCR-free antibody-based methods. Furthermore, the method has the supplementary advantage of being rapid, resulting from the automated assay protocols and the extremely fast (1 minute) antigen capture.

EPFL2012

Attomolar protein detection using a magnetic bead surface coverage assay

Matteo Cornaglia, Martinus Gijs, Hüseyin Cumhur Tekin

We demonstrate a microfluidic method for ultra-sensitive protein detection in serum. First, ‘large’ (2.8 μm) antibody-functionalized magnetic beads specifically capture antigen from a serum matrix under active microfluidic mixing. Subsequently, the large beads loaded with the antigens are gently exposed to a surface pattern of fixed ‘small’ (1.0 μm) antibody-coated magnetic beads. During the exposure, attractive magnetic bead dipole–dipole interactions improve the contact between the two bead types and help the antigen-antibody immunocomplex formation, while non-specific large bead adsorption is limited by exploiting viscous drag forces in the microfluidic channel on the small-bead pattern. This efficient antigen-antibody recognition and binding mechanism mimics a biological process of selective recognition of tissue molecules, like is the case when leukocytes roll and slow down on blood vessel walls by selectin-mediated adhesion. After exposure of the large beads to the pattern of small beads, the antigen concentration is detected by simply counting the number of surface pattern-bound large magnetic beads. The new technique allows detection of proteins down to the sub-zeptomole range. In particular, we demonstrate detection of only 200 molecules of Tumor Necrosis Factor-α (TNF-α) in a serum sample volume of 5 μL, corresponding to a concentration of 60 attomolar or 1 fg/mL.

Royal Society of Chemistry2013

Magnetic Particle-Scanning for Ultrasensitive Immunodetection On-Chip

Matteo Cornaglia, Martinus Gijs, Thomas Lehnert, Hüseyin Cumhur Tekin, Raphaël Etienne Jean Trouillon

We describe the concept of magnetic particle-scanning for on-chip detection of biomolecules: a magnetic particle, carrying a low number of antigens (Ag's) (down to a single molecule), is transported by hydrodynamic forces and is subjected to successive stochastic reorientations in an engineered magnetic energy landscape. The latter consists of a pattern of substrate-bound small magnetic particles that are functionalized with antibodies (Ab's). Subsequationuent counting of the captured Ag-carrying particles provides the detection signal. The magnetic particle-scanning principle is investigated in a custom-built magneto-microfluidic chip and theoretically described by a random walk-based model, in which the trajectory of the contact point between an Ag-carrying particle and the small magnetic particle pattern is described by stochastic moves over the surface of the mobile particle, until this point coincides with the position of an Ag, resulting in the binding of the particle. This model explains the particular behavior of previously reported experimental dose-response curves obtained for two different ligand-receptor systems (biotin/streptavidin and TNF-alpha) over a wide range of concentrations. Our model shows that magnetic particle-scanning results in a very high probability of irrununocomplex formation for very low Ag concentrations, leading to an extremely low limit of detection, down to the single molecule-per-particle level. When compared to other types of magnetic particle-based surface coverage assays, our strategy was found to offer a wider dynamic range (>8 orders of magnitude), as the system does not saturate for concentrations as high as 10(11) Ag molecules in a 5 mu L drop. Furthermore, by emphasizing the importance of maximizing the encounter probability between the Ag and the Ab to improve sensitivity, our model also contributes to explaining the behavior of other particle-based heterogeneous immunoassays.