Surface-Enhanced Raman Scattering-Based Immunoassay Technologies for Detection of Disease Biomarkers

Detection of biomarkers is of vital importance in disease detection, management, and monitoring of therapeutic efficacy. Extensive efforts have been devoted to the development of novel diagnostic methods that detect and quantify biomarkers with higher sensitivity and reliability, contributing to better disease diagnosis and prognosis. When it comes to such devastating diseases as cancer, these novel powerful methods allow for disease staging as well as detection of cancer at very early stages. Over the past decade, there have been some advances in the development of platforms for biomarker detection of diseases. The main focus has recently shifted to the development of simple and reliable diagnostic tests that are inexpensive, accurate, and can follow a patient’s disease progression and therapy response. The individualized approach in biomarker detection has been also emphasized with detection of multiple biomarkers in body fluids such as blood and urine. This review article covers the developments in Surface-Enhanced Raman Scattering (SERS) and related technologies with the primary focus on immunoassays. Limitations and advantages of the SERS-based immunoassay platform are discussed. The article thoroughly describes all components of the SERS immunoassay and highlights the superior capabilities of SERS readout strategy such as high sensitivity and simultaneous detection of a multitude of biomarkers. Finally, it introduces recently developed strategies for in vivo biomarker detection using SERS

Effect of spermidine on misfolding and interactions of alpha-synuclein.

Alpha-synuclein (α-Syn) is a 140 aa presynaptic protein which belongs to a group of natively unfolded proteins that are unstructured in aqueous solutions. The aggregation rate of α-Syn is accelerated in the presence of physiological levels of cellular polyamines. Here we applied single molecule AFM force spectroscopy to characterize the effect of spermidine on the very first stages of α-Syn aggregation--misfolding and assembly into dimers. Two α-Syn variants, the wild-type (WT) protein and A30P, were studied. The two protein molecules were covalently immobilized at the C-terminus, one at the AFM tip and the other on the substrate, and intermolecular interactions between the two molecules were measured by multiple approach-retraction cycles. At conditions close to physiological ones at which α-Syn misfolding is a rare event, the addition of spermidine leads to a dramatic increase in the propensity of the WT and mutant proteins to misfold. Importantly, misfolding is characterized by a set of conformations, and A30P changes the misfolding pattern as well as the strength of the intermolecular interactions. Together with the fact that spermidine facilitates late stages of α-Syn aggregation, our data demonstrate that spermidine promotes the very early stages of protein aggregation including α-Syn misfolding and dimerization. This finding suggests that increased levels of spermidine and potentially other polyamines can initiate the disease-related process of α-Syn

Direct Detection of α-Synuclein Dimerization Dynamics: Single-Molecule Fluorescence Analysis

AbstractThe aggregation of α-synuclein (α-Syn) is linked to Parkinson’s disease. The mechanism of early aggregation steps and the effect of pathogenic single-point mutations remain elusive. We report here a single-molecule fluorescence study of α-Syn dimerization and the effect of mutations. Specific interactions between tethered fluorophore-free α-Syn monomers on a substrate and fluorophore-labeled monomers diffusing freely in solution were observed using total internal reflection fluorescence microscopy. The results showed that wild-type (WT) α-Syn dimers adopt two types of dimers. The lifetimes of type 1 and type 2 dimers were determined to be 197 ± 3 ms and 3334 ± 145 ms, respectively. All three of the mutations used, A30P, E46K, and A53T, increased the lifetime of type 1 dimer and enhanced the relative population of type 2 dimer, with type 1 dimer constituting the major fraction. The kinetic stability of type 1 dimers (expressed in terms of lifetime) followed the order A30P (693 ± 14 ms) > E46K (292 ± 5 ms) > A53T (226 ± 6 ms) > WT (197 ± 3 ms). Type 2 dimers, which are more stable, had lifetimes in the range of several seconds. The strongest effect, observed for the A30P mutant, resulted in a lifetime 3.5 times higher than observed for the WT type 1 dimer. This mutation also doubled the relative fraction of type 2 dimer. These data show that single-point mutations promote dimerization, and they suggest that the structural heterogeneity of α-Syn dimers could lead to different aggregation pathways

Post-ER Stress Biogenesis of Golgi Is Governed by Giantin

Background: The Golgi apparatus undergoes disorganization in response to stress, but it is able to restore compact and perinuclear structure under recovery. This self-organization mechanism is significant for cellular homeostasis, but remains mostly elusive, as does the role of giantin, the largest Golgi matrix dimeric protein. Methods: In HeLa and different prostate cancer cells, we used the model of cellular stress induced by Brefeldin A (BFA). The conformational structure of giantin was assessed by proximity ligation assay and atomic force microscopy. The post-BFA distribution of Golgi resident enzymes was examined by 3D SIM high-resolution microscopy. Results: We detected that giantin is rather flexible than an extended coiled-coil dimer and BFA-induced Golgi disassembly was associated with giantin monomerization. A fusion of the nascent Golgi membranes after BFA washout is forced by giantin re-dimerization via disulfide bond in its luminal domain and assisted by Rab6a GTPase. GM130-GRASP65-dependent enzymes are able to reach the nascent Golgi membranes, while giantin-sensitive enzymes appeared at the Golgi after its complete recovery via direct interaction of their cytoplasmic tail with N-terminus of giantin. Conclusion: Post-stress recovery of Golgi is conducted by giantin dimer and Golgi proteins refill membranes according to their docking affiliation rather than their intra-Golgi location

Ultra-fast charging in aluminum-ion batteries: electric double layers on active anode

With the rapid iteration of portable electronics and electric vehicles, developing high-capacity batteries with ultra-fast charging capability has become a holy grail. Here we report rechargeable aluminum-ion batteries capable of reaching a high specific capacity of 200 mAh g−1. When liquid metal is further used to lower the energy barrier from the anode, fastest charging rate of 104 C (duration of 0.35 s to reach a full capacity) and 500% more specific capacity under high-rate conditions are achieved. Phase boundaries from the active anode are believed to encourage a high-flux charge transfer through the electric double layers. As a result, cationic layers inside the electric double layers responded with a swift change in molecular conformation, but anionic layers adopted a polymer-like configuration to facilitate the change in composition

Effect of Spermidine on Misfolding and Interactions of Alpha-Synuclein

Alpha-synuclein (α-Syn) is a 140 aa presynaptic protein which belongs to a group of natively unfolded proteins that are unstructured in aqueous solutions. The aggregation rate of α-Syn is accelerated in the presence of physiological levels of cellular polyamines. Here we applied single molecule AFM force spectroscopy to characterize the effect of spermidine on the very first stages of α-Syn aggregation – misfolding and assembly into dimers. Two α-Syn variants, the wild-type (WT) protein and A30P, were studied. The two protein molecules were covalently immobilized at the C-terminus, one at the AFM tip and the other on the substrate, and intermolecular interactions between the two molecules were measured by multiple approach-retraction cycles. At conditions close to physiological ones at which α-Syn misfolding is a rare event, the addition of spermidine leads to a dramatic increase in the propensity of the WT and mutant proteins to misfold. Importantly, misfolding is characterized by a set of conformations, and A30P changes the misfolding pattern as well as the strength of the intermolecular interactions. Together with the fact that spermidine facilitates late stages of α-Syn aggregation, our data demonstrate that spermidine promotes the very early stages of protein aggregation including α-Syn misfolding and dimerization. This finding suggests that increased levels of spermidine and potentially other polyamines can initiate the disease-related process of α-Syn

Modular Micro Raman Reader Instrument for Fast SERS-Based Detection of Biomarkers

Sensitive detection of biomarkers is very critical in the diagnosis, management, and monitoring of diseases. Recent efforts have suggested that bioassays using surface-enhanced Raman scattering as a signal read-out strategy possess certain unique beneficial features in terms of sensitivity and low limits of detection which set this method apart from its counterparts such as fluorescence, phosphorescence, and radiolabeling. Surface-enhanced Raman scattering (SERS) has also emerged as an ideal choice for the development of multiplexed bioassays. Such promising features have prompted the need for the development of SERS-based tools suitable for point-of-care applications. These tools must be easy to use, portable, and automated for the screening of many samples in clinical settings if diagnostic applications are considered. The availability of such tools will result in faster and more reliable detection of disease biomarkers, improving the accessibility of point-of-care diagnostics. In this paper, we describe a modular Raman reader instrument designed to create such a portable device suitable for screening a large number of samples with minimal operator assistance. The device’s hardware is mostly built with commercially available components using our unique design. Dedicated software was created to automatically run sample screening and analyze the data measured. The mRR is an imaging system specifically created to automate measurements, eliminating human bias while enhancing the rate of data collection and analysis ~2000 times. This paper presents both the design and capabilities of the custom-built modular Raman reader system (mRR) capable of automated and fast measurements of sandwich immunoassay samples on gold substrates using modified gold nanoparticles as Raman tags. The limit of detection (LOD) of the tested MUC4-specific iSERS assay was measured to be 0.41 µg/mL

DNA-Templated Silver Nanoclusters as Dual-Mode Sensitive Probes for Self-Powered Biosensor Fueled by Glucose

Nanomaterials have been extensively explored in developing sensors due to their unique properties, contributing to the development of reliable sensor designs with improved sensitivity and specificity. Herein, we propose the construction of a fluorescent/electrochemical dual-mode self-powered biosensor for advanced biosensing using DNA-templated silver nanoclusters (AgNCs@DNA). AgNC@DNA, due to its small size, exhibits advantageous characteristics as an optical probe. We investigated the sensing efficacy of AgNCs@DNA as a fluorescent probe for glucose detection. Fluorescence emitted by AgNCs@DNA served as the readout signal as a response to more H2O2 being generated by glucose oxidase for increasing glucose levels. The second readout signal of this dual-mode biosensor was utilized via the electrochemical route, where AgNCs served as charge mediators between the glucose oxidase (GOx) enzyme and carbon working electrode during the oxidation process of glucose catalyzed by GOx. The developed biosensor features low-level limits of detection (LODs), ~23 μM for optical and ~29 μM for electrochemical readout, which are much lower than the typical glucose concentrations found in body fluids, including blood, urine, tears, and sweat. The low LODs, simultaneous utilization of different readout strategies, and self-powered design demonstrated in this study open new prospects for developing next-generation biosensor devices