Analysis Of Biomolecules Research Articles

Quantitative and Noninvasive Detection of SAH-Related MiRNA in Cerebrospinal Fluids In Vivo Using SERS Sensors Based on Acupuncture-Based Technology.

Quantitative analysis of microRNAs (miRNAs) in a noninvasive manner is of vital importance for disease diagnosis and prognosis evaluation. However, conventional strategies for realizing accurate, simple, and sensitive detection of target molecules are still a challenge, especially for miRNAs due to their low abundance and susceptibility in the complex biological environment. Here, a novel surface-enhanced Raman scattering (SERS) strategy was established for quantitative detection and monitoring of miRNA-21-5p (miR-21-5p) in living cells and in vivo cerebrospinal fluid (CSF) by applying hairpin DNA (hpDNA)-conjugated gold nanostars (GNSs) SERS probes combined with acupuncture-based technology. This strategy enabled ultrasensitive exploration toward miR-21-5p in a wide range from 1 fM to 100 pM in cell lysates. Moreover, SERS analysis facilitated the detection and long-term monitoring for in vivo miR-21-5p noninvasively. This developed strategy promises to offer a powerful method for the analysis of multiple biomolecules in single cells and living bodies.

Application Progress of Green Carbon Dots in Analysis and Detection

AbstractGreen chemistry is currently attracting more and more attention due to its eco‐friendly, and green carbon dots (g‐CDs) are creating a research boom. The g‐CDs have huge application prospects in analysis and detection due to their excellent optical properties, high biocompatibility, and low toxicity compared to other fluorescent materials. Based on the study of g‐CDs, the optical properties, synthesis methods, surface modification methods, and sensing mechanisms of g‐CDs are outlined, and the application in analysis and detection of environmental, food, biomolecules, and other organic small molecules are reviewed. At the same time, the challenges faced by g‐CDs are outlined. This review provides valuable references for further research and wider application of g‐CDs.

Modeling of the transient diffusion regime in fully porous particles—Application to the analysis of large biomolecules by ultra-high pressure liquid chromatography

The transient diffusion regime of large biomolecules such as monoclonal antibodies, double-stranded (ds) DNA (base pair number ∼ 100), and virus-like particles is modeled in a single fully porous particle utilized as size exclusion chromatography (SEC) packing materials in ultra-high pressure liquid chromatography (UHPLC). The expression of the time and space dependent concentration profiles is derived for a step concentration change. Four different UHPLC particles were considered in the calculations depending on the size of the analyte : 2.0 μm 125 Å Bridge-Ethylene-Hybrid (BEH) XBridgeTM particles for small molecules (size: 4 Å), 2.0 μm 125 Å and 250 Å BEH particles for monoclonal antibodies (size: 55 Å), 2.0 μm 250 Å and 2.5 μm 450 Å BEH particles for dsDNA (size: 160 Å), and 2.5 μm 900 Å BEH particles for virus-like particles (size: 500 Å). The accessible porosities and the hindrance diffusion factors of these analytes were determined from the physical reconstruction of the internal structure of a 2.0 μm 130 Å BEH particle and from the simulation of the analyte mobility in the reconstructed mesopores. The analysis of the transient diffusion profiles reveals that there is insufficient time for the largest analytes to fully equilibrate the BEH particles in UHPLC. The dynamic capacity of the BEH particles is estimated to decrease from more than 99% for small molecules to 96% for monoclonal antibodies, 74% for 100 base pair dsDNA, and to less than 2% for virus-like particles. A full but reduced column capacity relative to fully porous particles can be recovered for monoclonal antibodies by considering superficially porous particles with a core-to-particle diameter ratio of about 0.8. In contrast, this is not possible for either large dsDNA (base pair number ≥ 100) or virus-like particles because the shell thickness would become smaller than the mesopore size required. The presented results open up two research avenues in order to analyze such large biomolecules by UHPLC: prepare ultra-high pressure-resistant sub-3 μm silica-based particles with pore sizes much larger than 1000 Å and design proper nonporous macrostructures to load, trap, separate, and elute rapidly by gradient UHPLC.

Single-Image Super-Resolution Improvement of X-ray Single-Particle Diffraction Images Using a Convolutional Neural Network.

Femtosecond X-ray pulse lasers are promising probes for the elucidation of the multiconformational states of biomolecules because they enable snapshots of single biomolecules to be observed as coherent diffraction images. Multi-image processing using an X-ray free-electron laser has proven to be a successful structural analysis method for viruses. However, the performance of single-particle analysis (SPA) for flexible biomolecules with sizes ≤100 nm remains difficult. Owing to the multiconformational states of biomolecules and noisy character of diffraction images, diffraction image improvement by multi-image processing is often ineffective for such molecules. Herein, a single-image super-resolution (SR) model was constructed using an SR convolutional neural network (SRCNN). Data preparation was performed in silico to consider the actual observation situation with unknown molecular orientations and the fluctuation of molecular structure and incident X-ray intensity. It was demonstrated that the trained SRCNN model improved the single-particle diffraction image quality, corresponding to an observed image with an incident X-ray intensity (approximately three to seven times higher than the original X-ray intensity), while retaining the individuality of the diffraction images. The feasibility of SPA for flexible biomolecules with sizes ≤100 nm was dramatically increased by introducing the SRCNN improvement at the beginning of the various structural analysis schemes.

Real matrix-matched standards for quantitative bioimaging of cytosolic proteins in individual cells using metal nanoclusters as immunoprobes-label: A case study using laser ablation ICP-MS detection

The persistent lack of adequate matrix-matched reference materials still hinders the quantitative analysis of elements and biomolecules in biological samples by LA-ICP-MS. This fact is especially critical in cell cultures due to their complex matrix. In this work, we propose a novel matrix-matched calibration strategy, which fully mimics the matrix of cultured cells, by using the same cell line of the sample to create laboratory standards. As a model case, the quantitative imaging of two cytosolic proteins (MT2A and APOE) in individual HRPEsv cells was performed by LA-ICP-MS, both in cells subjected to inflammation with cytokine Interleukin-1α (IL-1α) and controls (CT). A single biomarker strategy using Au nanoclusters (AuNCs) as specific antibody labels was employed for the analysis of the selected proteins in individual cells by LA-ICP-MS. HRPEsv cells supplemented with suspensions containing nude AuNCs was employed to generate single-cell laboratory standards (HRPEsv cells@AuNCs). The preparation and characterization of the single-cell laboratory standards by both ICP-MS and LA-ICP-MS were optimized as well as the data treatment protocol required for obtaining the quantitative distribution of the proteins in individual cells. The mass of APOE and MT2A per cell in CT and IL1α−treated HRPEsv cells analysed by LA-ICP-MS using the proposed matrix-matched calibration were successfully corroborated with commercial ELISA kits. In addition, quantitative real time polymerase chain reaction (qPCR) analyses were performed to study the proteins gene expression.

PB1974: MULTIVARIATE SPECTRAL BIOTYPING OF EXTRAMEDULLARY MULTIPLE MYELOMA

Background: Multiple myeloma (MM) is the second most common hematological malignancy of the elderly. The bone marrow is infiltrated by malignant plasma cells. MM may progress into so-called extramedullary disease (EMD) - EMD occurs when a subclone of malignant plasma cells migrates out of the bone marrow and infiltrates soft tissues. Despite recent progress, pathogenesis of EMD still needs to be clarified. Aims: We focused on the analysis of low molecular weight molecules in peripheral blood of 20 MM and 20 EMD patients using MALDI-TOF mass spectrometry to create a tool for identification of MM and EMD, and discrimination of individual types. Methods: Matrix-Assisted Laser Desorption/Ionization Time-of Flight Mass Spectrometry (MALDI-TOF MS) has become an indispensable research tool, which is used for analysis of biomolecules and various organic molecules. Artificial Neural Networks (ANN) are components of artificial intelligence inspired by biological neural networks. Using ANN, we can model complex non-linear systems, as previously published. In our previous study, we recorded mass spectra of MM and healthy donor samples. ANN specifically predicted MM samples with high sensitivity, specificity and accuracy. The same approach as applied on MM and EMD. Results: The RStudio was used for statistical analysis, where the data were evaluated using Principal Component Analysis (PCA) and Partial least squares discriminant analysis (PSL-DA). Using MALDI-TOF MS, it was possible to distinguish between samples of MM patients and healthy donors, as well as MM and EMD patients. Informative patterns in mass spectra served as inputs for ANN that specifically distinguished between healthy donors and patients. Summary/Conclusion: We demonstrated that using MALDI-TOF MS coupled with ANN is a useful tool that can distinguish between healthy donors and MM/EMD patients. Thus, it can be used as a fast alternative to conventional analyses. This study was supported by grant of the Ministry of Health of the Czech Republic, NU21-03-00076.

Abstract 979: Analysis of metabolic and genetic changes in human colorectal cancer and colorectal metastatic tissue by combined NanoString technology and mass spectrometry imaging (MSI)

Abstract Introduction: The development of normal colon tissue to carcinoma can progress through several pathways, which exhibit different clinical, pathological and genetic features. Increasing evidence indicates that tumor development and metastases are not solely driven by genetic alterations but also the tumor microenvironment which is associated with changes in metabolic processes. While many studies show the importance of gene expression profiling in cancer progression, little is known about how this correlates with altered lipid metabolism at metastatic sites within the peritoneal cavity. NanoString nCounter provides specific and sensitive genetic profiling data identifying up to 800 genes simultaneously from small amounts of RNA. For biomolecule analysis, MSI is a powerful tool as it enables the rapid identification of phenotype-dependent MS signatures from tissues with little sample preparation. The aim of this study was to investigate the characteristics of secondary tumors at different metastatic sites compared to the primary tumors using a combination of immunohistochemistry, NanoString analysis and Desorption Electrospray Ionization (DESI) MSI. Methods: 12 fresh-frozen human colorectal adenocarcinoma and metastases samples were cut into 12μm sections. DESI-MSI (Xevo G2-XS, Waters) was performed in both polarities with 50μm spatial resolution. SCiLS Lab (Bruker) software were used for data analysis. Histologically-stained (H&E) adjacent sections were used for microscopy. For genomic analysis, RNA was extracted using AllPrep DNA/RNA micro kit from 20μm sections. Following this, RNA abundance was measured using Qubit fluorimeter. Lastly, mRNA hybridisation, detection and scanning was performed on a NanoString nCounter mRNA Gene Expression system and analysed using nSolver 4.0 software. Results: H&E staining allowed the visualisation of different tissue histopathologies. These differences are echoed in selected DESI-MSI ion images. DESI spectra from distinct regions of the tissue show differences in biomolecule expression especially for phospholipids (m/z 600-900). Ions such as m/z 812 correlated with the non-cancerous region whereas others such as m/z 861 were only observed in the cancerous regions of the tissue. Gene expression analysis showed concordance between primary and metastatic tumors such as in RAS/RAF genes. Others, such as IL-6 expression was seen to upregulated in the metastatic sites compared to the primary tumor. Multivariate analysis enabled identification of biomolecules representative of different tissue phenotypes, with the data showing putative MS biomarkers associated with genetic mutations. Conclusion: Results confirmed the advantage of the combined techniques to reveal spatial correlations between changes in lipid metabolism, tumor heterogeneity and gene expression. Citation Format: Yasmin Shanneik, Emrys Jones, Michal Smiga, Bipasha Chakrabarty, Omer Aziz, Steven Pringle, Kaye Williams, Adam McMahon. Analysis of metabolic and genetic changes in human colorectal cancer and colorectal metastatic tissue by combined NanoString technology and mass spectrometry imaging (MSI) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 979.

Simultaneous Multiplexed Imaging of Biomolecules in Transgenic Mouse Brain Tissues Using Mass Spectrometry Imaging: A Multi-omic Approach.

The importance of multi-omic-based approaches to better understand diverse pathological mechanisms including neurodegenerative diseases has emerged. Spatial information can be of great help in understanding how biomolecules interact pathologically and in elucidating target biomarkers for developing therapeutics. While various analytical methods have been attempted for imaging-based biomolecule analysis, a multi-omic approach to imaging remains challenging due to the different characteristics of biomolecules. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a powerful tool due to its sensitivity, chemical specificity, and high spatial resolution in visualizing chemical information in cells and tissues. In this paper, we suggest a new strategy to simultaneously obtain the spatial information of various kinds of biomolecules that includes both labeled and label-free approaches using ToF-SIMS. The enzyme-assisted labeling strategy for the targets of interest enables the sensitive and specific imaging of large molecules such as peptides, proteins, and mRNA, a task that has been, to date, difficult for any MS analysis. Together with the strength of the analytical performance of ToF-SIMS in the label-free tissue imaging of small biomolecules, the proposed strategy allows one to simultaneously obtain integrated information of spatial distribution of metabolites, lipids, peptides, proteins, and mRNA at a high resolution in a single measurement. As part of the suggested strategy, we present a sample preparation method suitable for MS imaging. Because a comprehensive method to examine the spatial distribution of multiple biomolecules in tissues has remained elusive, our strategy can be a useful tool to support the understanding of the interactions of biomolecules in tissues as well as pathological mechanisms.

Front Cover: The Institute of Biomolecules Max Mousseron: A Timely Move (Eur. J. Org. Chem. 21/2022)

The Front Cover shows an illustration of the new Balard Center which gathers on more than 25 000 square meters the two chemistry research centers of Montpellier, France: the Institute Charles-Gerhardt Montpellier (ICGM) and the Institute of Biomolecules Max Mousseron (IBMM). The blue Möbius ring, part of the IBMM logo, is here figuratively crossed by the DNA of the institute expressing the synthesis, analysis, and biological evaluation of biomolecules for therapeutic applications. An overview of the history of the Institute and the research currently being conducted by the various IBMM teams are presented in this issue. Cover design by Yanis Dufoix and Patrick Paris (Montpellier University). More information can be found in the Editorial by M. Smietana and P. Dumy.

Translational Detection of Indole by Complementary Cell-free Protein Synthesis Assay.

The information encoded in a single copy of DNA is processed into a plethora of protein molecules via the cascade of transcription and translation. Thus, the molecular process of gene expression can be considered an efficient biological amplifier from the viewpoint of synthetic biology. Cell-free protein synthesis (CFPS) enables the implementation of this amplification module for in vitro analysis of important biomolecules and avoids many of the problems associated with whole cell-based approaches. Here, we developed a method to analyze indole by using a combination of enzymatic conversion of indole and amino acid-dependent CFPS. In this method, indole molecules in the assay sample are used to generate tryptophan, which is incorporated into signal-generating proteins in the subsequent cell-free synthesis reaction. The activity of cell-free synthesized proteins was successfully used to estimate the indole concentration in the assay sample. In principle, the developed method could be extended to analyses of other important bioactive compounds.

Impact of structure and magnetic parameters of nanocrystalline cores on surface properties of molecularly imprinted nanoconjugates for analysis of biomolecules – A case of tyramine

In this paper, the investigations were carried out to reveal the complex nature of the nanocrystalline magnetic cores and to validate the hypothesis that magnetic molecularly imprinted polymer (MMIP) could be used as an effective sorbent during analysis of biomolecules but the adsorbate-sorbent process is independent from the amount of siloxane functionalized magnetite core (Fe3O4@SiO2-MPS). For that purpose, molecularly imprinted shell layer composed from poly(methacrylic acid-co-ethylene glycol dimethacrylate) was conjugated to various amounts of Fe3O4@SiO2-MPS. Magnetic measurements showed single-domain-type magnetic behavior for all materials. Thermal decomposition process revealed different enthalpies (110 to 279 J g−1) with the highest value for MMIP prepared from the lowest amount of Fe3O4@SiO2-MPS. The morphological and structural analyses proved a presence of single magnetite crystallite and elemental mapping confirmed core–shell structure. The binding capacities for MMIPs revealed similar values (0.897 ± 0.114 to 1.019 ± 0.129 µg g−1) and specificity (IF: 3.36 to 3.99). The MMIP sorbent was used to determine model biomolecule of tyramine in wine in the validated analytical method, proving the stability of sorbent. The lower tyramine levels were found in Chilean and Australian wines (0.42 ± 0.03 and 0.15 ± 0.01 mg L-1, respectively) and higher in French one (7.58 ± 0.15 mg L-1).

Chemical derivatization: recent advances in mass spectrometry imaging

<p indent="0mm">Recently, mass spectrometry imaging (MSI) has drawn more and more attention due to its continuous development and improvement as well as its vital role in the study of biological science. Many MSI-based strategies have been widely applied to the <italic>in situ</italic> qualitative and quantitative detection and the imaging of endogenous/exogenous molecules in tissues. Among them, matrix assisted laser desorption/ionization MSI (MALDI-MSI) and desorption electrospray ionization MSI (DESI-MSI) are generally regarded as the most commonly used two molecular imaging techniques. MSI, as a histology-based emerging molecular imaging technique, has significant advantages over other imaging techniques because it is a label-free technique which provides high sensitivity, high throughput, and molecular specificity derived from the use of mass spectrometers as detectors for a wide variety of ionized biomolecules <italic>in situ</italic> within a tissue section. But it still has obvious shortcomings in the <italic>in situ </italic>analysis of low-polarity and neutral biomolecules. The application of chemical derivatization technology in MSI is one of the most important approaches to overcome these deficiencies of MS molecular imaging. This review provides an overview of various chemical derivatization techniques used in MS analysis, such as esterification derivatization, acylation derivatization, addition reaction derivatization, substitution derivatization, oxidative derivatization, and other derivatizations, and highlights the application of these chemical derivatization methods in the <italic>in situ</italic> detection and imaging of biomolecules in tissues by MALDI-MSI and DESI-MSI. It can be predicted that with the advance progress of chemical derivatization technology, the application scope of MSI will be further expanded in the fields of spatial metabolomics, spatial lipidomics, spatial proteomics, and spatial glycomics.

Poly(diphenylamine) and its Nanohybrids for Chemicals and Biomolecules Analysis: A Review

Background:This is the first review on Poly(diphenylamine) and its nanohybrids which covers about 181 references demonstrating the brief discussion on the theoretical studies, chemical, electrochemical and other-phase preparation techniques, polymerization and oxidation-reduction (redox) mechanisms, physicochemical and electrochemical properties along with electrochemical sensors and spectroscopic applications on the detection of chemicals and biomolecules analysis applications.Objective:The main aim of this detailed report is merely to afford a survey of the literature existing on this multifunctional conducting organic polymer (poly(diphenylamine)) that provokes a pathway to innovations and discoveries in the near future claim its applications in multidisciplinary fields, especially in the detection of chemicals and bio-molecules applications.Methods:We discussed the overall studies on poly(diphenylamine) and its various nanohybrids, including copolymers, homopolymers, carbon-based, and metal/metal-oxide hybrids. The different synthesis methods of poly(diphenylamine) such as chemical/electrochemical/mechano-chemical polymerization in terms of morphology and electrical conductivity were briefly discussed.Conclusion:This review manuscript deliberates the various synthesis approaches and applications based on the multifunctional conducting polymer poly(diphenylamine) and its nanohybrids. This review provides an outlook and challenges ahead that ignites spotlight to innovations and discoveries in the near future claim its applications in multidisciplinary fields, particularly in electrochemical sensors and spectroscopic applications towards the detection of chemicals and bio-molecules.

Fabrication of Micro-Patterned Chip with Controlled Thickness for High-Throughput Cryogenic Electron Microscopy.

A major limitation for the efficient and high-throughput structure analysis of biomolecules using cryogenic electron microscopy (cryo-EM) is the difficulty of preparing cryo-EM samples with controlled ice thickness at the nanoscale. The silicon (Si)-based chip, which has a regular array of micro-holes with graphene oxide (GO) window patterned on a thickness-controlled silicon nitride (SixNy) film, has been developed by applying microelectromechanical system (MEMS) techniques. UV photolithography, chemical vapor deposition, wet and dry etching of the thin film, and drop-casting of 2D nanosheet materials were used for mass-production of the micro-patterned chips with GO windows. The depth of the micro-holes is regulated to control the ice thickness on-demand, depending on the size of the specimen for cryo-EM analysis. The favorable affinity of GO toward biomolecules concentrates the biomolecules of interest within the micro-hole during cryo-EM sample preparation. The micro-patterned chip with GO windows enables high-throughput cryo-EM imaging of various biological molecules, as well as inorganic nanomaterials.

Applications of nanopipettes in electrochemical analysis

AbstractNowadays, the application of glass nanopipettes in electrochemical analysis has attracted a lot of attention with the increasing demand for sensitive analysis and diagnostic tools for cellular biomolecules. The ultrasmall nanopipettes are becoming a bridge between the macro‐ and nanoworlds as they can transport very small volumes of liquids and materials as well as acting as electrochemical biosensors. In this minireview, we discuss the fabrication and characterization of nanopipettes and introduce our recent studies using nanopipettes for cell electroanalysis. The future application of nanopipettes has also been discussed.

A Cell-Anchored and Self-Calibrated DNA Nanoplatform for in Situ Imaging and Quantification of Endogenous MicroRNA in Live Cells: Introducing Two Controls to Normalize the Sensing Signals

A Cell-Anchored and Self-Calibrated DNA Nanoplatform for in Situ Imaging and Quantification of Endogenous MicroRNA in Live Cells: Introducing Two Controls to Normalize the Sensing Signals

Zinc ions negatively regulate proapoptotic signaling in cells expressing oncogenic mutant Ras.

Mutational activation of the Ras family of proto-oncogenes promotes cell survival and proliferation. Studies using cells cultured in vitro have shown that ectopic expression of constitutively active Ras suppresses apoptosis induced by serum deprivation. However, in some cellular contexts, constitutively active Ras exerts the opposite effects, including apoptosis of serum-starved embryonic fibroblasts. Such observations first came over two decades ago, but the molecular mechanisms by which mutant Ras increases the susceptibility of cells toserum deprivation leading to apoptosis are still not fully understood. To revisit this issue, I investigate the effects of serum depletion and mutant Ras expression on intracellular signaling and transcriptome of cells carrying an inducible allele of constitutively active mutant Hras (HrasG12V). I identify zinc ions (Zn2+) as a serum factor that suppresses proapoptotic signaling in cells expressing HrasG12V. Mechanistically, HrasG12V expression along with Zn2+ deficiency activates c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), which are required for caspase-3 activation involved in the induction of cell death. Transcriptome analyses suggest that HrasG12V induces the unfolded protein response (UPR). Further analyses of intracellular signaling biomolecules related to the UPR indicate that HrasG12V activates inositol-requiring protein 1 (IRE1), which synergizes with Zn2+ deficiency to activate JNK and p38 MAPK signaling. These results provide insights into a role of Zn2+ that counteracts proapoptotic signaling activated by mutationally activated Ras.

Label-Free Optical Analysis of Biomolecules in Solid-State Nanopores: Toward Single-Molecule Protein Sequencing.

Sequence identification of peptides and proteins is central to proteomics. Protein sequencing is mainly conducted by insensitive mass spectroscopy because proteins cannot be amplified, which hampers applications such as single-cell proteomics and precision medicine. The commercial success of portable nanopore sequencers for single DNA molecules has inspired extensive research and development of single-molecule techniques for protein sequencing. Among them, three challenges remain: (1) discrimination of the 20 amino acids as building blocks of proteins; (2) unfolding proteins; and (3) controlling the motion of proteins with nonuniformly charged sequences. In this context, the emergence of label-free optical analysis techniques for single amino acids and peptides by solid-state nanopores shows promise for addressing the first challenge. In this Perspective, we first discuss the current challenges of single-molecule fluorescence detection and nanopore resistive pulse sensing in a protein sequencing. Then, label-free optical methods are described to show how they address the single-amino-acid identification within single peptides. They include localized surface plasmon resonance detection and surface-enhanced Raman spectroscopy on plasmonic nanopores. Notably, we report new data to show the ability of plasmon-enhanced Raman scattering to record and discriminate the 20 amino acids at a single-molecule level. In addition, we discuss briefly the manipulation of molecule translocation and liquid flow in plasmonic nanopores for controlling molecule movement to allow high-resolution reading of protein sequences. We envision that a combination of Raman spectroscopy with plasmonic nanopores can succeed in single-molecule protein sequencing in a label-free way.

Mesoporous Ag–TiO2 based nanocage like structure as sensitive and recyclable low-cost SERS substrate for biosensing applications

We fabricated a porous Ag–TiO2 film with a nanocage (NC) like structure as a sensitive, recyclable, and low-cost SERS substrate using facile methods. The developed nanocage structure of TiO2 film was first coated on a glass plate by doctor-blade method, and then thin film of silver with an optimum thickness of 10 nm was deposited onto it using thermal evaporation method. The unique mesoporous cage-like structure of TiO2 film leads to the exceptional and non-uniform growth of Ag film. The developed Ag nanostructure is also porous, caps spherical in shape, and the average separation between two consecutive nanostructures is 10 nm. These closely decorated nanostructures of Ag produce hotspots by localising the incident electric field within the minimal volume. The unique nanocage structure of TiO2 film provides a larger effective surface area of adsorption to the analytes. Thus, the synergistic effects of Ag–TiO2 NC structure exhibit significant enhancement of 108 considering rhodamine-6g (R6G) molecule as a probe using a portable handheld Raman spectrometer. The amount of electromagnetic enhancement was further investigated by 3D FDTD simulation that matches well with the experiments. The recyclability of the developed SERS substrate was explored by placing the SERS substrate under UV illumination for 130 min. The enhanced photocatalytic activity of Ag–TiO2 NC SERS film degrades the dye molecules under UV radiation multiple times successively. Further, the Ag–TiO2 SERS substrate is highly sensitive to detect various concentrations of urea up to 1 mM covering the vital range of blood urea level. Thus, the optimised, low-cost, reusable SERS substrate can be used as level free biosensors for routine analysis of biomolecules in clinical applications.

High-Throughput Multiplexed Infrared Spectroscopy of Ion Mobility-Separated Species Using Hadamard Transform.

Coupling vibrational ion spectroscopy with high-resolution ion mobility separation offers a promising approach for detailed analysis of biomolecules in the gas phase. Improvements in the ion mobility technology have made it possible to separate isomers with minor structural differences, and their interrogation with a tunable infrared laser provides vibrational fingerprints for unambiguous database-enabled identification. Nevertheless, wide analytical application of this technique requires high-throughput approaches for acquisition of vibrational spectra of all species present in complex mixtures. In this work, we present a novel multiplexed approach and demonstrate its utility for cryogenic ion spectroscopy of peptides and glycans in mixtures. Since the method is based on Hadamard transform multiplexing, it yields infrared spectra with an increased signal-to-noise ratio compared to a conventional signal averaging approach.