Highly Sensitive Protein Concentration Assay over a Wide Range via Surface-Enhanced Raman Scattering of Coomassie Brilliant Blue

In the Bradford protein assay, protein concentrations are determined by the absorbance at 595 nm due to the binding of Coomassie brilliant blue G-250 (CBBG) to proteins. In a protein−CBBG liquid mixture, surface-enhanced Raman scattering (SERS) is sensitive to the amount of unbound CBBG molecules adsorbed on silver surfaces, and the bound CBBG amount is directly related to the target protein concentration. Accordingly, a novel method for detecting total protein concentration in a solution has been developed based on SERS of unbound CBBG with an internal standard of silicon. Two obvious advantages of the proposed protein assay over conventional Bradford protein assay are its much wider linear concentration range (10−5−10−9 g/mL) and 200 times lower limit of detection (1 ng/mL), which demonstrates its great potential in rapid, highly sensitive concentration determination of high and low-abundance proteins

Highly Sensitive and Selective Determination of Iodide and Thiocyanate Concentrations Using Surface-Enhanced Raman Scattering of Starch-Reduced Gold Nanoparticles

In this report, we propose a novel technique for the determination of the concentrations of iodide and thiocyanate by surface-enhanced Raman scattering (SERS) of starch-reduced gold nanoparticles. Starch-reduced gold nanoparticles show an intrinsic Raman peak at 2125 cm–1 due to the −CC– stretching mode of a synthesized byproduct. Because of the high adsorptivity of iodide on a gold surface, the intensity of the SERS peak at 2125 cm–1 decreases with an increase in the iodide concentration. Thiocyanate also strongly adsorbs on a gold surface, and a new peak appears at around 2100 cm–1, attributed to the −CN stretching vibration in a SERS spectrum of starch-reduced gold nanoparticles. These two peaks were successfully used to determine the iodide and thiocyanate concentrations separately, even in their mixture system. The detection limit of this technique for iodide is 0.01 μM with a measurement range of 0.01–2.0 μM, while the detection limit of this technique for thiocyanate is 0.05 μM with a measurement range of 0.05–50 μM. This technique is highly selective for iodide and thiocyanate ions without interference from other coexisting anions such as other halides, carbonate, and sulfate

Coupling Reaction-Based Ultrasensitive Detection of Phenolic Estrogens Using Surface-Enhanced Resonance Raman Scattering

Studies have shown that many adverse health effects are associated with human exposure to dietary or environmental estrogens. Therefore, the development of rapid and highly sensitive detection methods for estrogens is very important and necessary to maintain hormonal concentration below the safety limit. Herein, we demonstrate a simple and rapid approach to detect trace amounts of phenolic estrogen based on surface-enhanced resonance Raman scattering (SERRS). Because of a coupling reaction between diazonium ions and the phenolic estrogens, azo compounds are formed with strong SERRS activity, which allows phenolic estrogen recognition at subnanomolar levels in solution. The proposed protocol has multiplexing capability, because each SERRS fingerprint of the azo dyes specifically corresponds to the related estrogen. Moreover, it is universal and highly selective, not only for phenolic estrogens but also for other phenolic molecules, even in complex systems

Charge Transfer at the TiO₂/N3/Ag Interface Monitored by Surface-Enhanced Raman Spectroscopy

The interface of semiconductor–dye–metal system is a crucial issue for investigating dye-sensitized solar cells (DSSCs), where the electron transfer takes place. In this work, a series of assemblies of TiO₂/N3 (<i>cis</i>-bis­(isothiocyanato)­bis­(2,2′-bipyridyl-4,4′-dicarboxylato)­ruthenium­(II)) and TiO₂/N3/Ag have been fabricated, which were employed for the investigation of the adsorption configuration and conformational change of N3 molecules. We plot degree of charge transfer (CT) (ρ_CT) as a function of excitation wavelength of TiO₂/N3 and TiO₂/N3/Ag assemblies, which contributes to the understanding of the CT process in the series of N3 assemblies. According to the variation tendency of ρ_CT, when laser energy exceeds the CT energy threshold 2.071 eV, ρ_CT shows an obvious increasing trend with the increasing laser energy. In the case of TiO₂/N3/Ag assembly, when the laser energy exceeds the CT energy threshold 1.877 eV, ρ_CT becomes lager with the increase in the laser energy, until asymptotic behavior appears under higher laser energy. To explain the variation tendency of ρ_CT and the shift of CT energy threshold, we have proposed two models about the energy level scheme of TiO₂/N3 and TiO₂/N3/Ag assemblies. Furthermore, we investigated the influence of crystal structure of TiO₂ NPs on the CT process by the fabrication TiO₂/N3/Ag assemblies based on anatase and rutile TiO₂ NPs. It is noted that the TiO₂/N3/Ag assembly based on TiO₂ NPs calcinated at 450 °C with highest ρ_CT and lowest CT energy threshold is most in favor of CT process. Besides the specific chemical binding mode in the TiO₂/N3/Ag system, this study also found the relationship between the ρ_CT and the CT process, which is of considerable importance and relevance to solar energy conversion

Magnetic Silver Hybrid Nanoparticles for Surface-Enhanced Resonance Raman Spectroscopic Detection and Decontamination of Small Toxic Molecules

Magnetic hybrid assemblies of Ag and Fe₃O₄ nanoparticles with biocompatibly immobilized myoglobin (Mb) were designed to detect and capture toxic targets (NO₂^–, CN^–, and H₂O₂). Mb was covalently attached to chitosan-coated magnetic silver hybrid nanoparticles (M-Ag-C) <i>via</i> glutaraldehyde that serves as a linker for the amine groups of Mb and chitosan. As verified by surface-enhanced resonance Raman (SERR) spectroscopy, this immobilization strategy preserves the native structure of the bound Mb as well as the binding affinity for small molecules. On the basis of characteristic spectral markers, binding of NO₂^–, CN^–, and H₂O₂ could be monitored and quantified, demonstrating the high sensitivity of this approach with detection limits of 1 nM for nitrite, 0.2 μM for cyanide, and 10 nM for H₂O₂. Owing to the magnetic properties, these particles were collected by an external magnet to achieve an efficient decontamination of the solutions as demonstrated by SERR spectroscopy. Thus, the present approach combines the highly sensitive analytical potential of SERR spectroscopy with an easy approach for decontamination of aqueous solutions with potential applications in food and in environmental and medical safety control

In Situ Monitoring of Membrane Protein Electron Transfer via Surface-Enhanced Resonance Raman Spectroscopy

In situ analysis of membrane protein–ligand interactions under physiological conditions is of significance for both fundamental and applied science, but it is still a big challenge due to the limits in sensitivity and selectivity. Here, we demonstrate the potential of surface-enhanced resonance Raman spectroscopy (SERRS) for the investigation of membrane protein–protein interactions. Lipid biolayers are successfully coated on silver nanoparticles through electrostatic interactions, and a highly sensitive and biomimetic membrane platform is obtained in vitro. Self-assembly and immobilization of the reduced cytochrome b5 on the coated membrane are achieved and protein native biological functions are preserved. Owing to resonance effect, the Raman fingerprint of the immobilized cytochrome b5 redox center is selectively enhanced, allowing for in situ and real-time monitoring of the electron transfer process between cytochrome b5 and their partners, cytochrome c and myoglobin. This study provides a sensitive analytical approach for membrane proteins and paves the way for in situ exploration of their structural basis and functions

In Situ Raman Spectroscopy Reveals Cytochrome <i>c</i> Redox-Controlled Modulation of Mitochondrial Membrane Permeabilization That Triggers Apoptosis

The selective interaction of cytochrome c (Cyt c) with cardiolipin (CL) is involved in mitochondrial membrane permeabilization, an essential step for the release of apoptosis activators. The structural basis and modulatory mechanism are, however, poorly understood. Here, we report that Cyt c can induce CL peroxidation independent of reactive oxygen species, which is controlled by its redox states. The structural basis of the Cyt c–CL binding was unveiled by comprehensive spectroscopic investigation and mass spectrometry. The Cyt c-induced permeabilization and its effect on membrane collapse, pore formation, and budding are observed by confocal microscopy. Moreover, cytochrome c oxidase dysfunction is found to be associated with the initiation of Cyt c redox-controlled membrane permeabilization. These results verify the significance of a redox-dependent modulation mechanism at the early stage of apoptosis, which can be exploited for the design of cytochrome c oxidase-targeted apoptotic inducers in cancer therapy

In Situ and Real-Time Monitoring of Mitochondria–Endoplasmic Reticulum Crosstalk in Apoptosis via Surface-Enhanced Resonance Raman Spectroscopy

The crosstalk between mitochondria and endoplasmic reticula plays a crucial role in apoptotic pathways in which reactive oxygen species (ROS) produced by microsomal monooxygenase (MMO) are believed to accelerate cytochrome c release. Herein, we successfully demonstrate the potential of surface-enhanced resonance Raman spectroscopy (SERRS) for monitoring MMO-derived ROS formation and ROS-mediated cytochrome c release. Silver nanoparticles coated with nickel shells are used as both Raman signal enhancers and electron donors for cytochrome c. SERRS of cytochrome c is found to be sensitive to ROS, allowing for in situ probing of ROS formation with a cell death inducer. Label-free evaluation of ROS-induced apoptosis is achieved by SERRS-based monitoring of cytochrome c release in living cells. This study verifies the capability of SERRS for label-free, in situ, and real-time monitoring of the mitochondria–endoplasmic reticulum crosstalk in apoptosis and provides a novel strategy for the rational design and screening of ROS-inducing drugs for cancer treatment

Surface-Enhanced Raman Scattering for Direct Protein Function Investigation: Controlled Immobilization and Orientation

Surface-enhanced Raman spectroscopy (SERS) has exhibited great potential in protein identification and quantification. However, the poor spectral reproducibility, originating from random protein immobilization on SERS substrates, still makes it challenging for SERS to probe protein functions without any extrinsic Raman labels. Here, in our study, spacer molecules between proteins and SERS substrates are optimized for both biocompatible protein immobilization and Raman scattering enhancement. We have accordingly prepared iminodiacetic acid (IDA)-functionalized silver substrates, which are used for capturing His-tagged proteins via nickel–imidazole coordination. The controlled immobilization enables excellent SERS spectral reproducibility as evidenced by 6 polypeptides. Furthermore, the interactions between two model proteins, Erv1C (C-terminal domain of flavine adenine dinucleotide-dependent mitochondrial cytochrome c reductase Erv1) and AFP (alpha-fetoprotein), and their ligands Cyt c (cytochrome c) and ATRA (all-trans-retinoic acid) are examined, respectively. The results indicate that the IDA-functionalized silver substrates enable controlled protein immobilization and allow label-free protein function investigation by SERS. As a proof-of-concept study, the proposed functionalized SERS-active substrates combined with immobilized metal-affinity chromatography will be useful for mechanism studies on protein–ligand interactions, which is crucially important for understanding the structural basis of protein functional versatility and will contribute to the fields of drug design and biotechnology

Mitochondria-Specific Molecular Crosstalk between Ferroptosis and Apoptosis Revealed by In Situ Raman Spectroscopy

Ferroptosis and apoptosis are two types of regulated cell death that are closely associated with the pathophysiological processes of many diseases. The significance of ferroptosis–apoptosis crosstalk in cell fate determination has been reported, but the underlying molecular mechanisms are poorly understood. Herein mitochondria-mediated molecular crosstalk is explored. Based on a comprehensive spectroscopic investigation and mass spectrometry, cytochrome c-involved Fenton-like reactions and lipid peroxidation are revealed. More importantly, cytochrome c is found to induce ROS-independent and cardiolipin-specific lipid peroxidation depending on its redox state. In situ Raman spectroscopy unveiled that erastin can interrupt membrane permeability, specifically through cardiolipin, facilitating cytochrome c release from the mitochondria. Details of the erastin–cardiolipin interaction are determined using molecular dynamics simulations. This study provides novel insights into how molecular crosstalk occurs around mitochondrial membranes to trigger ferroptosis and apoptosis, with significant implications for the rational design of mitochondria-targeted cell death reducers in cancer therapy