Bottom-Up Fabrication of Plasmonic Nanoantenna-Based High-throughput Multiplexing Biosensors for Ultrasensitive Detection of microRNAs Directly from Cancer Patients’ Plasma

Abstract

There is an unmet need in clinical point-of-care (POC) cancer diagnostics for early state disease detection, which would greatly increase patient survival rates. Currently available analytical techniques for early stage cancer diagnosis do not meet the requirements for POC of a clinical setting. They are unable to provide the high demand of multiplexing, high-throughput, and ultrasensitive detection of biomarkers directly from low volume patient samples (“liquid biopsy”). To overcome these current technological bottle-necks, herein we present, for the first time, a bottom-up fabrication strategy to develop plasmonic nanoantenna-based sensors that utilize the unique localized surface plasmon resonance (LSPR) properties of chemically synthesized gold nanostructures, gold triangular nanoprisms (Au TNPs), gold nanorods (Au NRs), and gold spherical nanoparticles (Au SNPs). Our Au TNPs, NRs, and SNPs display refractive index unit (RIU) sensitivities of 318, 225, and 135 nm/RIU respectively. Based on the RIU results, we developed plasmonic nanoantenna-based multiplexing and high-throughput biosensors for the ultrasensitive assay of microRNAs. MicroRNAs are directly linked with cancer development, progression, and metastasis, thus they hold promise as next generation biomarkers for cancer diagnosis and prognosis. The developed biosensors are capable of assaying five different types of microRNAs at an attomolar detection limit. These sets of microRNAs include both oncogenic and tumor suppressor microRNAs. To demonstrate the efficiency as a POC cancer diagnostic tool, we analyzed the plasma of 20-bladder cancer patients without any sample processing steps. Importantly, our liquid biopsy-based biosensing approach is capable of differentiating healthy from early (“non-metastatic”) and late (“metastatic”) stage cancer with a p value <0.0001. Further, receiver operating characteristic analysis shows that our biosensing approach is highly specific, with an area under the curve of 1.0. Additionally, our plasmonic nanoantenna-based biosensors are regenerative, allowing multiple measurements using the same biosensors, which is essential in low- and middle-income countries. Taken together, our multiplexing and high-throughput biosensors have the unmatched potential to advance POC diagnostics and meet global needs for early stage detection of cancer and other diseases (e.g., infectious, autoimmune, and neurogenerative diseases).

Multiplexed and High-Throughput Label-Free Detection of RNA/Spike Protein/IgG/IgM Biomarkers of SARS-CoV-2 Infection Utilizing Nanoplasmonic Biosensors

“…An example of the LSPR peak shift for SARS-CoV-2 N-836 is shown in Figure B. The magnitude of the LSPR shift is in agreement with our previous work on microRNA detection using the same sensing mechanism . This shift is from a combination of change in the local dielectric environment of Au TNPs and delocalization of their excitons within the -ssDNA/RNA duplex .…”

“…The magnitude of the LSPR shift is in agreement with our previous work on microRNA detection using the same sensing mechanism. 22 This shift is from a combination of change in the local dielectric environment of Au TNPs and delocalization of their excitons within the -ssDNA/RNA duplex. 38 LSPR peak shifts for the other five nucleic acid gene sequences are provided in the Supporting Information (Tables S2 and S4).…”

“…Glass coverslips containing nearly 95 Au TNPs were glued to the bottom of a no-bottom, 96-well plate using a previously published procedure. 22 Briefly, a small amount of glue was applied to the edges of the wells, followed by careful placement of the Au TNP-functionalized coverslip onto the glue area. Gentle pressure was applied to the coverslip for appropriate attachment.…”

“…To construct the biosensor, ∼42 nm edge-length Au TNPs were chemically attached onto silanized glass coverslips (see the Supporting Information), followed by performing a tape-cleaning procedure to remove nonprismatic nanostructures. Glass coverslips containing nearly 95 Au TNPs were glued to the bottom of a no-bottom, 96-well plate using a previously published procedure . Briefly, a small amount of glue was applied to the edges of the wells, followed by careful placement of the Au TNP-functionalized coverslip onto the glue area.…”

Selective Detection and Ultrasensitive Quantification of SARS-CoV-2 IgG Antibodies in Clinical Plasma Samples Using Epitope-Modified Nanoplasmonic Biosensing Platforms

“…High-throughput biosensing platforms were fabricated based on previously published procedures with modifications. 30 Au TNP-attached glass coverslips were glued to the bottom of a no-bottom 96-well plate by applying a small amount of glue around the edges of the wells on the plate and gently applying pressure on the coverslip to adhere to the well plate. The coverslip-attached plate was allowed to dry for at least an hour at room temperature.…”