Open Sandwich FRET Immunoassay of Estrogen Receptor β in a PDMS Microfluidic Channel

Abstract

Immunoassay is a fast and cost-effective protein detection method that can be applied to clinical diagnostics and biological research. Enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and protein array chips are the most widely used immunoassay techniques for the detection of a target protein. In all of these methods, however, antibodies or antigens should be immobilized on a solid substrate. As a result, they require a long assay time for complete immunoreaction between antigens and antibodies, being limited by the diffusion kinetics. Moreover, they also require several cycles of consecutive binding and washing steps to separate free reagents from binding reagents. To overcome the problem of slow immunoreactions in conventional immunoassay techniques, we recently reported a fast and sensitive one-step immunoassay of estrogen receptor β (ERβ) using quantum dot (QD)-based fluorescence resonance energy transfer (FRET). To induce a strong FRET effect, QD was used as a fluorescence donor. FRET occurs when the electronic excitation energy of a donor chromophore is transferred to a nearby acceptor molecule via a dipole-dipole interaction between the donoracceptor pair. Conventional fluorescence dyes, which cause an appreciable overlap between the emission band of the donor and the absorption band of the acceptor, have generally been used as a donor-acceptor set. However, QD provides significant advantages over conventional fluorescence dyes, including brighter fluorescence, resistance to photobleaching, and narrow emission bands, which makes it suitable as a sensitive fluorescence donor. As a result, QD reaches a longer distance to the acceptor than conventional fluorescence dyes because of their superior optical properties. Furthermore, the QD-based FRET immunoassay of ERβ was faster (30-40 min of immunoassay time including incubation) than conventional immunoassay techniques because it does not need any solid-phase carriers and multiple washing steps for free reagent separations. In our previous work, the advantages of a QD-based FRET immunoassay in an open space were fully explored. Nonetheless, a faster reaction time and a smaller sample volume are still needed to apply this QD-based FRET immunoanalysis technique to clinical diagnostics. In the present work, a lab-on-a-chip technique, combined with the QD-based FRET immunoassay method, was used to develop a more sensitive and faster immunoanalysis technique. The lab-on-a-chip technique has several advantages over conventional bench-top techniques, such as minimal sample requirement, reduced reaction time, ease of use, improved product conversion, and reduced waste generation. In particular, the reaction time can be greatly reduced to less than a few minutes if a properly designed channel to obtain highly efficient mixing is adopted. Furthermore, only a small reaction volume is required in the lab-ona-chip analysis. An alligator-teeth-shaped poly(dimethylsiloxane) (PDMS) microfluidic channel was fabricated to obtain a fast immunoreaction between antibody and antigen under a flow condition. The detailed fabrication process for this PDMS channel has been reported elsewhere. ERβ is one of the most important breast cancer markers that are closely related with cell proliferations. The antiERβ monoclonal antibody (McAb, mouse ascites, IgM isotype; Sigma, St. Louis, MO, USA) was labeled on QD 565 using an antibody conjugation kit (Invitrogen, Eugene, OR, USA). In addition, the anti-ERβ polyclonal antibody (PcAb, rabbit immune serum, IgG; Biogenex, San Ramon, CA, USA) was labeled with AF 568 protein labeling kits (Invitrogen, Eugene, OR, USA). The formation of an open sandwich immunocomplex and its energy transfer process from QD-McAb to AF-PcAb are illustrated in Figure 1(a).