Magnetic nanoparticle-enhanced SPR on gold nanoslits for ultra-sensitive, label-free detection of nucleic acid biomarkers

We have demonstrated a detection method for the ultra-sensitive detection of an mRNA biomarker. The method utilizes functionalized magnetic nanoparticles (MNPs) for signal enhancement in conjunction with surface plasmon resonance (SPR) on gold nanoslits. The approach for detection includes double hybridization at two different specific locations in two steps. First, the biomarker target molecule is captured with MNPs, and second, MNPs carrying the target molecule are introduced to the SPR chip to hybridize with probes immobilized on the gold nanoslits. In this work, MNPs were applied for a dual purpose: to isolate the target molecule from the sample matrix to prevent non-specific binding and to enhance the SPR response. Gold nanoslits that provide SPR sensing were fabricated by nanoimprinting lithography on polycarbonate (PC) film. The film was integrated with a microliter volume microfluidic chip to form the SPR detection chip. This detection method was used to detect mRNA heterogeneous nuclear ribonucleoproteins (hnRNP B1) in two cancer cell lines, CL1-0 and CL1-5. hnRNP B1 is an mRNA biomarker that is overexpressed in lung cancer tissue in the early stage of cancer and can be found in the serum and plasma of lung cancer patients. A synthetic target molecule and extracted total RNA from the cell lines were used as samples. Without amplification and labeling of the target molecule, the SPR results demonstrate a specific and sensitive method for the detection of hnRNP B1 mRNA in extracted RNA from the two selected cell lines. The method is capable of measuring down to 30 fM of the target molecule in a 7 mu l sample (corresponding to 1.26 x 10(5) molecules) without amplification and labeling of the target molecule.