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SERS-active microchannels on hydrophobic polydimethylsiloxane (PDMS) with both high reproducibility and sensitivity are fabricated. First, uniform and size controllable silver nanoparticles (Ag NPs) are prepared with the proposed two-step method, in which the nucleation and growth stages are carried out independently. The average size of Ag NPs is 65 nm, which is balanced for the stability of silver colloids and high enhancement of SERS at 633 nm excitation, with a relative standard deviation (RSD) of 7.80%. The uniform Ag NPs are evenly coated on hydrophobic PDMS and formed a dense monolayer Ag NPs film with a RSD of 7.07%. A SERS-active microchannel is fabricated by integrating the SERS-active substrate in a PDMS microchannel and shows excellent uniformity with a RSD of 4.6% along the flow direction. The SERS signals of a variety of lable free amino acid and proteins are detected with good signal-to-noise ratios on the SERS-active substrate. By comparing the SERS spectra detected on the substrate and in the microchannel during flow, the adsorption mode of PMP22-TM4 on the surface of Ag NPs are revealed. More important, the results indicate that PMP22-TM4 might denature while drying on the surface of Ag NPs, but maintain the native structure in the SERS-active microchannels. Therefore, measuring SERS in a microchannel under the condition of flow offers a way to study protein conformation while keeping it in the native state.

Highly uniform SERS-active microchannel on hydrophobic PDMS: a balance of high reproducibility and sensitivity for detection of proteins