Nanoparticle-Based Surface Localized Nucleic Acid Amplification for Rapid and Ultrasensitive Electrochemical Detection

Of various methods that have been developed for the diagnosis of diseases, molecular diagnostic approaches have proven to be of great significance and thus have been developed to a great extent. Of these approaches, nanoparticle-based systems for nucleic acid detection have gained much attention due to the remarkable features of the nanoparticle platform (large surface area, electrochemical catalysis, biocompatibility, etc.). These methods produced notable results for detection of disease-related genetic material, which suggested the potential to overcome the critical drawbacks of conventional PCR-based diagnosis. However, the acquisition of both high sensitivity and short detection runtime in a single method remain a challenge as methods with high sensitivity demand lengthy procedures while faster methods require higher concentrations of genetic material for detection. In response to this matter, we propose an innovative nanoparticle-based system composed of a nucleic acid amplification method paired with electrochemical assay for rapid and ultrasensitive detection of genetic biomarkers. Termed “nanoparticle surface localized amplification (nSLAM)”, the method uses DNA functionalized Fe₃O₄−Au core−shell nanoparticles as a platform for nucleic acid amplification and accumulation. The surface-anchored DNA act as primers that bind and amplify target DNA directly on the nanoparticle surfaces using conventional PCR procedures. The surface-accumulated amplicons are subsequently subjected to electrochemical detection, which induces significantly amplified current signals that identify the presence of the target material. The nanoparticles function as dispersible nanometer-sized electrodes that significantly improve the biochemical contact efficiency between the primers and target gene of interest, further enabling amplification and collection of extremely low concentrations of target material. Furthermore, multiple measurements of electrochemical detection induce rearrangement of the dispersible nanoparticles, substantially enhancing the current signals for both ultrasensitive and rapid detection of target genes. The detection performance was tested using the COVID-19 model, which produced a remarkable sensitivity of ~1 RNA copy/μL using 35 PCR cycles. This ultrasensitivity enabled the reduction of amplification cycles to as low as 4 cycles (~7 min runtime) using 1 fM COVID-19 cDNA, which demonstrated the system’s capacity for rapid detection. These results suggest the potential for the system to overcome critical drawbacks of conventional COVID-19 molecular diagnosis by speeding up the detection process to enable expeditious treatment-related measures taken by medical personnel during emergency situations. The nSLAM system was also tested using the estrogen receptor 1 (ESR1) gene model, a ctDNA biomarker from metastatic breast cancer, which resulted in a successful detection of as low as ~1 aM concentration using 40 PCR cycles and reduction to 10 PCR cycles (~18 min runtime) for 1 fM ESR1 detection. The detection results provide a solution to the crucial shortcomings of detecting ctDNA for liquid biopsy, as the short half-life and extremely low abundance of ctDNA in blood are supplemented by the rapid and ultrasensitive detection properties of the system. The system is a promising platform that can be used to detect cancer- and infectious disease-related genetic material in an ultrasensitive and rapid manner. The system also demonstrates great versatility that can be tailored to specific diseases by designing and substituting the primers used for amplification. We plan to further develop the system as a multiplex platform for simultaneous detection of various diseases.