Time-Resolved Single-Step Protease Activity Quantification Using Nanoplasmonic Resonator Sensors

Abstract

Protease activity measurement has broad application in drug screening, diagnosis and disease staging, and molecular profiling. However, conventional immunopeptidemetric assays (IMPA) exhibit low fluorescence signal-to-noise ratios, preventing reliable measurements at lower concentrations in the clinically important pM∼nM range. Here, we demonstrated a highly sensitive measurement of protease activity using nanoplasmonic resonator (NPR). NPRs enhance Raman signals by 6.1×10 10 times in a highly reproducible manner, enabling fast detection of proteolytically active Prostate Specific Antigen (paPSA) activities in real-time, at sensitivity level at 6 pM (0.2 ng/ml) with a dynamic range of 3 orders of magnitude. Experiments on extracellular fluid (ECF) from the paPSA-positive cells demonstrate specific detection in a complex bio-fluid background. This method offers a fast, sensitive, accurate, and one-step approach to detect the proteases activities in very small sample volumes. KeywordsPlasmonic resonator; Surface Enhanced Raman Scattering; Sensing; Prostate Cancer; Protease Originally developed in 1928, Raman spectroscopy has been used extensively to characterize molecular properties 1 . Surface-Enhanced Raman Spectroscopy (SERS) increases the Raman signal significantly 2-5 through enhanced electromagnetic fields in close proximity to a surface. Additional enhancement can be obtained by utilizing molecular resonance Raman (RR) effect when the molecule was excited at its absorption band 6 . SERS measurements performed on dispersed metal nanoparticle aggregates, which is the mostly commonly used SERS substrate 7 , have demonstrated detection sensitivity up to single molecule level [8][9][10] . However, these measurement often suffer from poor reproducibility 11 . To improve the reproducibility, other methods including self-assembly of metallic colloidal nano-particles, 12 nanosphere lithography (NSL) and metal film over nanosphere (MFON) 13 The lack of specificity causes a high false-positive rate and often leads to costly prostate needle biopsies for diagnosis and post-biopsy complications as well as considerable anxiety. 22,28,29 Recent research has identified a family of highly specific peptides that can be cleaved by paPSA isoform in xenografts models 30 and human samples 31, 32 thus, measurement of paPSA protease activity from in vivo samples is possible and would be potentially valuable as a more specific screening agent for prostate cancer and in detection of recurrent disease. However, reported results based on immunopeptidemetric assays (IMPA) exhibit low fluorescence signal-to-noise ratios, preventing reliable measurements at lower concentrations in the clinically important range of 60-300 pM.31 , 32 In addition, there is usually a limited number of prostate cancer cells (<1000) isolated from fine needle biopsy or circulating cell capture. No method exists that can perform a paPSA protease activity assay on a small number of cells for clinical staging. Therefore, a key goal of this work is to devel...

Subcellular Resolution Mapping of Endogenous Cytokine Secretion by Nano-Plasmonic-Resonator Sensor Array

“…As we reported previously 16, 17 , the TNPR structure is 100 nm in diameter and consists of two 20 nm gold layers with an intervening 5 nm silicon dioxide thin film (Fig. 2a).…”

“…Plasmonic nanostructures provide a novel approach to concentrating electromagnetic energy at the nanoscale, and they can significantly enhance biosensing signals, such as for Surface Enhanced Raman Scattering (SERS) measurements 10 . Many novel nanostructures have been explored for ultrasensitive biosensor applications 11-15 with one powerful structure being a tunable-nano-plasmonic-resonator (TNPR) 16, 17 . TNPRs are multilayered nano-plasmonic structures that consist of feature sizes in the range of 100 nm that are assembled from alternating noble metal and dielectric films 16, 17 .…”

“…Many novel nanostructures have been explored for ultrasensitive biosensor applications 11-15 with one powerful structure being a tunable-nano-plasmonic-resonator (TNPR) 16, 17 . TNPRs are multilayered nano-plasmonic structures that consist of feature sizes in the range of 100 nm that are assembled from alternating noble metal and dielectric films 16, 17 . TNPRs provide unique advantages, such as extraordinary field enhancements, tunable resonance frequencies, and compatibility with a top-down mass production process which enables the fabrication of large numbers of uniform nano-plasmonic structures with controllable resonance spectra and electromagnetic field enhancements.…”

Cancer biomarker detection: recent achievements and challenges

“…Taking the analogical detection principle, a nanoplasmonic resonator (NPR) sensor was constructed by Zhang and coworkers for the measurement of protease activity. 202 The proteolytically active prostate-specific antigen (paPSA) cleaves the peptide substrate, leading to the release of the SERS molecule Rhodamine 19 and a subsequent decrease in the Raman scattering intensity in a dose-and time-dependent manner. The protease activity could be accurately quantified at a sensitivity level of 6 pM (0.2 ng mL À1 ) and a dynamic range of three orders of magnitude.…”

Shifts in plasmon resonance due to charging of a nanodisk array in argon plasma

“…At certain wavelengths, maximum coupling from electromagnetic waves to localized surface plasmons can be achieved and these are known as the localized surface plasmon resonances (LSPRs) of the structure. LSPRs of metal nanostructures depend on properties such as the size, 10,11 shape, 12-14 metal material, 15 surrounding dielectric material, [16][17][18][19][20][21][22] and the charge of the metal. [23][24][25][26][27][28][29][30] Active plasmonic devices can be realized by externally tuning the LSPR of nanostructures.…”