One-Shot Dual-Code Immunotargeting for Ultra-Sensitive Tumor Necrosis Factor-α Nanosensors by 3D Enhanced Dark-Field Super-Resolution Microscopy

Abstract

Tumor necrosis factor-α (TNF-α) is a significant mediator of autoimmune diseases and an inflammatory protein biomarker. A novel method for the immunotargeting of TNF-α has been developed using three-dimensional (3D) enhanced dark-field super-resolution microscopy (3D EDF-SRM) based on ultrasensitive dual-code plasmonic nanosensing. Dual-code EDF-based 3D SRM improved the localization precision and sensitivity with a least-cubic algorithm, which provides accurate position information for the immunotargeted site. A dual-view device and digital single-lens reflex (DSLR) camera were used for simultaneous dual confirmable quantitative and qualitative immunoscreening based on enhanced dark-field scattering images. Two different sizes of silver nanoparticles (40- and 80-nm AgNPs) were compared to enhance the scattering signal of the immunotargeted plasmonic nanoprobe for the 3D EDF-SRM system. The standard TNF-α was immunotargeted at a single-molecule level and was quantitatively analyzed by measuring the scattering signals of 80 nm AgNPs on an array chip with gold-nanostages (GNSs) with 100 nm spot diameters. The localization precision in the 80 nm AgNP immunotag on the GNS narrowed to ∼9.5 nm after applying the least-cubic algorithm. The developed nanosensor exhibited a detection limit of 65 zM (1.14 ag/mL; S/N = 3) with a wide dynamic detection range of 65 zM-2.08 pM (1.14 ag/mL-36.4 pg/mL; R = 0.9921). These values are 20-33 400 000 times lower than detection limits obtained using previous methods. In addition, a recovery greater than 98% was achieved by spiking standard TNF-α into human serum samples. This method should facilitate simultaneous improvements in immunotargeting precision and ultrahigh sensitive detection of various disease-related target protein molecules at a single-molecule level.

Plasmon-Amplified Endogenous Fluorescence Nanospectroscopic Sensor Based on Inherent Elastic Scattering for Ultratrace Ratiometric Detection of Capsaicinoids

“…A gold-nanopatterned chip was fabricated as per the previously reported procedure. 2 A glass wafer (WinWin Tech., Hwaseong, Korea) was the base of the spot and was washed with piranha solution (7:3 = H 2 SO 4 :/H 2 O 2 , v/v), after which poly(methyl methacrylate) (PMMA) was spin-coated on the surface of the glass wafer to a 150 nm thick layer. After exposure to electron beam lithography (Elionix E-beam system, 100 keV/100 pA), Au/Cr (20/5 nm thickness) was deposited and lifted off (Korea Advanced Nano Fab Center, Suwon, Korea) by dichloromethane.…”

“…A gold-nanopatterned chip was fabricated as per the previously reported procedure . A glass wafer (WinWin Tech., Hwaseong, Korea) was the base of the spot and was washed with piranha solution (7:3 = H 2 SO 4 :/H 2 O 2 , v/v), after which poly­(methyl methacrylate) (PMMA) was spin-coated on the surface of the glass wafer to a 150 nm thick layer.…”

“…Fluorescence probe-coupled, single-molecule imaging approaches have been universally employed for high precision detection and quantification. , Endogenous (native) fluorescence-based techniques have reversed the complexity of single-molecule imaging by reducing the use of additional labeled probes . However, the weak signal of endogenous fluorescence has been a formidable problem in quantitative single-molecule fluorescence imaging techniques.…”

Individual Plasmonic Nanoprobes for Biosensing and Bioimaging: Recent Advances and Perspectives

“…The modification of antibodies, which specifically bind to their corresponding antigens with high affinity on the surface of PNPs enables the development of immunoassays with high sensitivity and selectivity. [ 179 ] To detect cancer‐related protein biomarkers, including carcinoembryonic antigen (CEA), prostate‐specific antigen (PSA), alpha fetoprotein (AFP), and mutated protein, [ 180–183 ] a variety of immunoassays have been designed by tuning the morphology and surface chemistry of PNPs. For instance, Poon and coworkers developed a simple yet robust method for the quantification of CEA, PSA, and AFP by measuring single particle scattering intensity.…”

Dark-Field Microscopy: Recent Advances in Accurate Analysis and Emerging Applications

“…As a result, a single molecule could be measured by DFM . High-resolution imaging has also become an important branch of iDFM. , At the beginning of this review, the working principle of DFM is briefly discussed, and some explorations for new and effective scattering imaging modes are also summarized, which are important supplements to DFM.…”