We describe the fabrication of elliptical Au nanodisk arrays as a localized surface plasmon resonance (LSPR) sensing substrate for clinical immunoassay via thermal nanoimprint lithography (NIL) and enhancement in the sensitivity of the detection of the prostate-specific antigen (PSA) using the precipitation of 5-bromo-4-chloro-3-indolyl phosphate p-toluidine/nitro blue tetrazolium (BCIP/NBT), catalyzed by alkaline phosphatase. Au nanodisks were fabricated on glass through an unconventional tilted evaporation, which could preserve the thickness of imprinted resists and create an undercut beneficial to the subsequent lift-off process without any damage to pattern dimension and the glass while removing the residual polymers. To investigate the optically anisotropic property of the LSPR sensors, a probe light with linear polarization parallel to and perpendicular to the long axis of the elliptical nanodisk array was utilized, and their sensitivity to the bulk refractive index (RI) was measured as 327 and 167 nm/RIU, respectively. To our knowledge, this is the first application of enzyme-substrate reaction to sandwich immunoassay-based LSPR biosensors that previously suffered from a low sensitivity due to the short penetration depth of the plasmon field, especially when large-sized antibodies were used as bioreceptors. As a result, a large change in local refractive index because of the precipitation on the Au nanodisks amplified the wavelength shift of the LSPR peak in the vis-NIR spectrum, resulting in femtomolar detection limits, which was ∼10(5)-fold lower than the label-free detection without the enzyme precipitation. This method can be extended easily to the other clinical diagnostics with a high sensitivity.

A method to directly collect negatively charged nucleic acids, such as DNA and RNA, in the biosamples simply by applying an electric field in between the sample and collection buffer separated by the nanofilter membrane is proposed. The nanofilter membrane was made of low-stress silicon nitride with a thickness of 100 nm, and multiple pores were perforated in a highly arranged pattern using nanoimprint technology with a pore size of 200 nm and a pore density of 7.22 × 10 8 /cm 2 . The electrophoretic transport of hsa-mir-93-5p across the membrane was confirmed in pure microRNA (miRNA) mimic solution using quantitative reverse transcription-polymerase chain reactions (qRT-PCR). Consistency of the collected miRNA quantity, stability of the system during the experiment, and yield and purity of the prepared sample were discussed in detail to validate the effectiveness of the electrical protocol. Finally, in order to check the applicability of this method to clinical samples, liquid biopsy process was demonstrated by evaluating the miRNA levels in sera of hepatocellular carcinoma patients and healthy controls. This efficient system proposed a simple, physical idea in preparation of nucleic acid from biosamples, and demonstrated its compatibility to biological downstream applications such as qRT-PCR as the conventional nucleic acid extraction protocols.

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