Development of Nucleic Acid Detection Methods and Systems Using Nanobiosensors and Surface-enhanced Raman Spectroscopy for In Vitro Molecular Diagnostics
The development of new nucleic acid detection techniques for molecular diagnostics at the point-of-care (POC) and resource-limited settings has attracted great interest. Signal amplification-based nucleic acid detection can be an alternative to enzymatic amplification-based methods (e.g., PCR, i.e., polymerase chain reaction). Compared to enzymatic amplification, some advantages of signal amplification include simple detection workflow without the need of nucleic acid extraction and purification, being more resistant to contamination and inhibitors, etc. However, current signal amplification methods are usually not sufficiently sensitive or too complicated and require skill operators, thus affecting its translation to POC applications.
Advances in nanotechnology and nanomaterials offer new, versatile, and exciting platforms for POC diagnostics. Using nanobiosensors and surface-enhanced Raman scattering (SERS), we have been developing various novel nucleic acid detection methods for in vitro molecular diagnostics. Emphasis was placed on sensitivity and easy-to-use, two of the main requirements of POC molecular diagnostics. Two different and complementary strategies for nucleic acid detection including (1) a chip-based strategy and (2) a nanoparticle-based strategy were investigated. The chip-based strategy involves single-step multiplex detection using SERS-active Nanowave chips. The nanoparticle-based strategy involves sandwich hybridization using magnetic beads, target sequences, and SERS nanorattle nanoparticles.
First, we developed a method for fabrication of large area high enhancement SERS-active Nanowave chip. Using the method, wafer scale of SERS-active Nanowave chip with particularly high enhancement factor of ~108 were achieved. Based on the Nanowave chip platform, we developed molecular sentinel-on-chip assay and inverse molecular sentinel-on-chip assay for nucleic acid detection. To show the assays’ usefulness for molecular diagnostics, genetic biomarkers for respiratory infection and a specific DNA sequence of Dengue virus were used as test models.
Second, method for synthesis of ultrabright SERS nanorattles were developed. The nanorattles are suitable for sandwich hybridization-based nucleic acid detection due to its strong SERS signal and high stability. Using the synthesized nanorattles, we developed a sandwich hybridization assay to detect specific DNA sequence of malaria parasite P. falciparum and genetic biomarker of squamous cell carcinoma (SCC). For the malaria target DNA, detection limit of 3 pM using synthetic DNA was achieved. For the SCC biomarker, we could detect real biological samples by detecting real-time PCR products of RNA extracted from SCC cell lines. Sensitivity of 93% and specificity of 100% were achieved.
Third, we developed an integrated device for automation of the nanorattle sandwich assay. Using the device, we could directly detect malaria RNA in malaria-infected blood lysate. The detection process was quite simple by adding nanorattles pre-functionalized with reporter probes and magnetic beads pre-functionalized with capture probes to the blood lysate followed by several hour incubation and an automated washing by a second-generation lab-in-a-stick device. To our knowledge, this is the first time the SERS-based detection of pathogen nucleic acid in blood lysate without any nucleic acid extraction or enzymatic amplification was reported.
The results showed potential of our nanobiosensors, methods, and systems for nucleic acid-based molecular diagnostics. The nanorattle sandwich assay’s sensitivity and compatibility for automation make them suitable for POC applications. In combination with the lab-in-a-stick device, we can now directly detect malaria nucleic acid in blood lysate. Future works will be to improve the limit of detection so that the technique can be used for detection of bloodborne pathogens and genetic biomarkers at lower clinical-relevant concentrations.
Nucleic acid detection
Surface-enhanced Raman scattering
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