Determination of Secondary Structure of Proteins by Nanoinfrared Spectroscopy.

Nanoscale infrared spectroscopy (AFMIR) is becoming an important tool for the analysis of biological sample, in particular protein assemblies, at the nanoscale level. While the amide I band is usually used to determine the secondary structure of proteins in Fourier transform infrared spectroscopy, no tool has been developed so far for AFMIR. The paper introduces a method for the study of secondary structure of protein based on a protein library of 38 well-characterized proteins. Ascending stepwise linear regression (ASLR) and partial least square (PLS) regression were used to correlate spectrum characteristic bands with the major secondary structures (α-helixes and ß-sheets). ASLR appears to provide better results than PLS. The secondary structure predictions are characterized by a root mean square standard error in a cross validation of 6.39% for α-helixes and 6.23% for ß-sheets.

Surface Heterogeneity in Amorphous Silica Nanoparticles Evidenced from Tapping AFM-IR Nanospectroscopy.

In this work, we propose to evaluate and validate an emerging spectroscopic space-resolved technique: atomic force microscopy coupled with infrared spectroscopy (AFM-IR) for inorganic materials in tapping mode at the nanoscale. For this aim, a preliminary investigation of sample preparation techniques was done and the stability of tapping AFM-IR spectra was evaluated on reference samples [poly(methyl methacrylate) and silica]. It was concluded that for a homogeneous polymer, it is possible to compare AFM-IR spectra with conventional Fourier-transform infrared (FTIR) spectra obtained in transmission. When an inorganic solid is considered, AFM-IR spectra are different from the global FTIR spectrum which indicates that the AFM-IR technique probes a volume which is not representative of global composition, that is, the external surface layer. Moreover, local infrared spectra recorded in the tapping mode of the external surface are significantly different depending on the analyzed regions of the same particle and between particles of the amorphous silica, implying surface heterogeneity. The AFM-IR technique allows surface description of amorphous inorganic materials at the nanoscale and opens new frontiers in the characterization of functional nanoscale and crystalline materials.

In-place molecular preservation of cellulose in 5,000-year-old archaeological textiles.

The understanding of fossilization mechanisms at the nanoscale remains extremely challenging despite its fundamental interest and its implications for paleontology, archaeology, geoscience, and environmental and material sciences. The mineralization mechanism by which cellulosic, keratinous, and silk tissues fossilize in the vicinity of archaeological metal artifacts offers the most exquisite preservation through a mechanism unexplored on the nanoscale. It is at the center of the vast majority of ancient textiles preserved under nonextreme conditions, known through extremely valuable fragments. Here we show the reconstruction of the nanoscale mechanism leading to the preservation of an exceptional collection of ancient cellulosic textiles recovered in the ancient Near East (4,000 to 5,000 years ago). We demonstrate that even the most mineralized fibers, which contain inorganic compounds throughout their histology, enclose preserved cellulosic remains in place. We evidence a process that combines the three steps of water transport of biocidal metal cations and soil solutes, degradation and loss of crystallinity of cellulosic polysaccharides, and silicification.

Quantification of drug loading in polymeric nanoparticles using AFM-IR technique: a novel method to map and evaluate drug distribution in drug nanocarriers.

Researchers are increasingly thinking smaller to solve some of the biggest challenges in nanomedicine: the control of drug encapsulation. Although recent years have witnessed a significant increase in the development and characterization of polymeric drug nanocarriers, several key features are still to be addressed: Where is the drug located within each nanoparticle (NP)? How much drug does each NP contain? Is the drug loading homogeneous on an individual NP basis? To answer these questions, individual NP characterization was achieved here by using atomic force microscopy-infrared spectroscopy (AFM-IR). A label-free quantification methodology was proposed to estimate with a nanoscale resolution the drug loadings of individual poly(lactic acid) (PLA) NPs loaded with an anticancer drug. First, a drug loading calibration curve was established using conventional IR microspectroscopy employing PLA/drug homogeneous films of well-known compositions. Then, single NPs were investigated by AFM-IR acquiring both IR mappings of PLA and drug as well as local IR spectra. Besides, drug location within single NPs was unravelled. The measured drug loadings were drastically different (0 to 21 wt%) from one NP to another, emphasizing the particular interest of this methodology in providing a simple quantification method for the quality control of nanomedicines.

Probing amyloid fibril secondary structures by infrared nanospectroscopy: experimental and theoretical considerations.

Amyloid fibrils are composed of aggregated peptides or proteins in a fibrillary structure with a higher ß-sheet content than their native structure. Attenuated total reflection Fourier transform infrared spectroscopy only provides bulk analysis of a sample therefore it is impossible to discriminate between different aggregated structures. To overcome this limitation, near-field techniques like AFM-IR have emerged in the last twenty years to allow infrared nanospectroscopy. This technique obtains IR spectra with a spatial resolution of ten nanometres, the size of isolated fibrils. Here, we present essential practical considerations to avoid misinterpretations and artefacts during these analyses. Effects of polarization of the incident IR laser, illumination configuration and coating of the AFM probes are discussed, including the advantages and drawbacks of their use. This approach will improve interpretation of AFM-IR spectra especially for the determination of secondary structures of species not accessible using classical ATR-FTIR.

Infrared and Raman chemical imaging and spectroscopy at the nanoscale.

The advent of nanotechnology, and the need to understand the chemical composition at the nanoscale, has stimulated the convergence of IR and Raman spectroscopy with scanning probe methods, resulting in new nanospectroscopy paradigms. Here we review two such methods, namely photothermal induced resonance (PTIR), also known as AFM-IR and tip-enhanced Raman spectroscopy (TERS). AFM-IR and TERS fundamentals will be reviewed in detail together with their recent crucial advances. The most recent applications, now spanning across materials science, nanotechnology, biology, medicine, geology, optics, catalysis, art conservation and other fields are also discussed. Even though AFM-IR and TERS have developed independently and have initially targeted different applications, rapid innovation in the last 5 years has pushed the performance of these, in principle spectroscopically complimentary, techniques well beyond initial expectations, thus opening new opportunities for their convergence. Therefore, subtle differences and complementarity will be highlighted together with emerging trends and opportunities.

Discrete Nanoscale Distribution of Hair Lipids Fails to Provide Humidity Resistance.

The subcellular distribution of lipids in human hair was investigated to better understand their role in water permeability. Unlike stratum corneum where lipids are organized under a precisely ordered continuous structure, the removal of free lipids in hair does not lead to an increase of water permeability. Esterified and CH2-enriched molecules were tracked at a 10 nm resolution by infrared nanospectroscopy (atomic force microscopy coupled to infrared spectroscopy, AFMIR). Free and bound lipids in the 25 nm thick intercellular spaces were directly detected for the first time, further substantiating the potential of AFMIR to study complex biomaterials. We observed that they were mostly found accumulated in some regions of the external cuticle layers, as "hotspots" in nonkeratinous portions of the internal cortex, and that they do not seem to modulate much the water exchanges due to their discrete distribution throughout the hair fiber.

Nanometric Chemical Speciation of Abnormal Deposits in Kidney Biopsy: Infrared-Nanospectroscopy Reveals Heterogeneities within Vancomycin Casts.

Infrared (IR) spectromicroscopy allows chemical mapping of a kidney biopsy. It is particularly interesting for chemical speciation of abnormal tubular deposits and calcification. In 2017, using IR spectromicroscopy, we described a new entity called vancomycin cast nephropathy. However, despite recent progresses, the IR microspectrometer spatial resolution is intrinsically limited by diffraction (a few micrometers). Combining atomic force microscopy and IR lasers (AFMIR) allows acquisition of infrared absorption spectra with a resolution and sensitivity in between 10 and 100 nm. Here we show that AFMIR can be used on standard paraffin embedded kidney biopsies. Vancomycin cast could be identified in a damaged tubule. Interestingly unlike standard IR spectromicroscopy, AFMIR revealed heterogeneity of the deposits and established that vancomycin coprecipitated with phosphate containing molecules. These findings highlight the high potential of this approach with nanometric spatial resolution which opens new perspectives for studies on drug-induced nephritis, nanocrystals, and local lipid or carbohydrates alterations.

Microstructure Characterization of Oceanic Polyethylene Debris.

Plastic pollution has become a worldwide concern. It was demonstrated that plastic breaks down to nanoscale particles in the environment, forming so-called nanoplastics. It is important to understand their ecological impact, but their structure is not elucidated. In this original work, we characterize the microstructure of oceanic polyethylene debris and compare it to the nonweathered objects. Cross sections are analyzed by several emergent mapping techniques. We highlight deep modifications of the debris within a layer a few hundred micrometers thick. The most intense modifications are macromolecule oxidation and a considerable decrease in the molecular weight. The adsorption of organic pollutants and trace metals is also confined to this outer layer. Fragmentation of the oxidized layer of the plastic debris is the most likely source of nanoplastics. Consequently the nanoplastic chemical nature differs greatly from plastics.

Characterization by Nano-Infrared Spectroscopy of Individual Aggregated Species of Amyloid Proteins.

Amyloid fibrils are composed of aggregated peptides or proteins in a fibrillar structure with a higher ß-sheet content than in their native structure. To characterize them, we used an innovative tool that coupled infrared spectroscopy with atomic force microscopy (AFM-IR). With this method, we show that we can detect different individual aggregated species from oligomers to fibrils and study their morphologies by AFM and their secondary structures based on their IR spectra. AFM-IR overcomes the weak spatial resolution of usual infrared spectroscopy and achieves a resolution of ten nanometers, the size of isolated fibrils. We characterized oligomers, amyloid fibrils of Aß42 and fibrils of α-synuclein. To our surprise, we figured out that the nature of some surfaces (ZnSe) used to study the samples induces destructuring of amyloid samples, leading to amorphous aggregates. We strongly suggest taking this into consideration in future experiments with amyloid fibrils. More importantly, we demonstrate the advantages of AFM-IR, with a high spatial resolution (≤ 10 nm) allowing spectrum recording on individual aggregated supramolecular entities selected thanks to the AFM images or on thin layers of proteins.

AFM-IR: Technology and Applications in Nanoscale Infrared Spectroscopy and Chemical Imaging.

Atomic force microscopy-based infrared spectroscopy (AFM-IR) is a rapidly emerging technique that provides chemical analysis and compositional mapping with spatial resolution far below conventional optical diffraction limits. AFM-IR works by using the tip of an AFM probe to locally detect thermal expansion in a sample resulting from absorption of infrared radiation. AFM-IR thus can provide the spatial resolution of AFM in combination with the chemical analysis and compositional imaging capabilities of infrared spectroscopy. This article briefly reviews the development and underlying technology of AFM-IR, including recent advances, and then surveys a wide range of applications and investigations using AFM-IR. AFM-IR applications that will be discussed include those in polymers, life sciences, photonics, solar cells, semiconductors, pharmaceuticals, and cultural heritage. In the Supporting Information , the authors provide a theoretical section that reviews the physics underlying the AFM-IR measurement and detection mechanisms.

How to unravel the chemical structure and component localization of individual drug-loaded polymeric nanoparticles by using tapping AFM-IR.

AFM-IR is a photothermal technique that combines AFM and infrared (IR) spectroscopy to unambiguously identify the chemical composition of a sample with tens of nanometer spatial resolution. So far, it has been successfully used in contact mode in a variety of applications. However, the contact mode is unsuitable for soft or loosely adhesive samples such as polymeric nanoparticles (NPs) of less than 200 nm of wide interest for biomedical applications. We describe here the theoretical basis of the innovative tapping AFMIR mode that can address novel challenges in imaging and chemical mapping. The new method enables gaining information not only on NP morphology and composition, but also reveals drug location and core-shell structures. Whereas up to now the locations of NP components could only be hypothesized, tapping AFM-IR allows accurately visualizing both the location of the NPs' shells and that of the incorporated drug, pipemidic acid. The preferential accumulation of the drug in the NPs' top layers was proved, despite its low concentration (<1 wt%). These studies pave the way towards the use of tapping AFM-IR as a powerful tool to control the quality of NP formulations based on individual NP detection and component quantification.

Chloroform induces outstanding crystallization of poly(hydroxybutyrate) (PHB) vesicles within bacteria.

Poly[(R)-3-hydroxyalkanoate]s or PHAs are aliphatic polyesters produced by numerous microorganisms. They are accumulated as energy and carbon reserve in the form of small intracellular vesicles. Poly[(R)-3-hydroxybutyrate] (PHB) is the most ubiquitous and simplest PHA. An atomic force microscope coupled with a tunable infrared laser (AFM-IR) was used to record highly spatially resolved infrared spectra of commercial purified PHB and native PHB within bacteria. For the first time, the crystallinity degree of native PHB within vesicle has been directly evaluated in situ without alteration due to the measure or extraction and purification steps of the polymer: native PHB is in crystalline state at 15% whereas crystallinity degree reaches 57% in commercial PHB. Chloroform addition on native PHB induces crystallization of the polymer within bacteria up to 60%. This possibility of probing and changing the physical state of polymer in situ could open alternative ways of production for PHB and others biopolymers. Graphical abstract An atomic force microscope coupled with a tunable infrared laser (AFM-IR) has been used to record local infrared spectra of biopolymer PHB within bacteria. Deconvolution of those spectra has allowed to determine in situ the crystallinity degree of native PHB.

Conducting polymer nanostructures for photocatalysis under visible light.

Visible-light-responsive photocatalysts can directly harvest energy from solar light, offering a desirable way to solve energy and environment issues. Here, we show that one-dimensional poly(diphenylbutadiyne) nanostructures synthesized by photopolymerization using a soft templating approach have high photocatalytic activity under visible light without the assistance of sacrificial reagents or precious metal co-catalysts. These polymer nanostructures are very stable even after repeated cycling. Transmission electron microscopy and nanoscale infrared characterizations reveal that the morphology and structure of the polymer nanostructures remain unchanged after many photocatalytic cycles. These stable and cheap polymer nanofibres are easy to process and can be reused without appreciable loss of activity. Our findings may help the development of semiconducting-based polymers for applications in self-cleaning surfaces, hydrogen generation and photovoltaics.

Radiolytic method as a novel approach for the synthesis of nanostructured conducting polypyrrole.

In this study, a novel and extremely facile method for the synthesis of conducting polypyrrole (PPy) was achieved in aqueous solution. This radiolytic method is totally free of template and environmentally friendly compared with traditional chemical methods. According to ultraviolet-visible (UV-vis) spectroscopy and Fourier transform infrared (FTIR) spectroscopy analysis, pyrrole (Py) monomers were polymerized into PPy thanks to their oxidation by HO(•) radicals produced by the radiolysis of water when exposed to γ irradiation. The morphology of PPy was characterized by cryo-transmission electron microscopy (cryo-TEM) in aqueous solution and by scanning electron microscopy (SEM) after deposition. In an original way, high-resolution atomic force microscopy, coupled with infrared nanospectroscopy, was used to probe the local chemical composition of PPy nanostructures. The results demonstrated that spherical and chaplet-like PPy nanostructures were formed by γ-radiolysis. Thermogravimetric analysis (TGA) and electronic conductivity measurements showed that radiosynthesized PPy had good thermal stability and an electrical conductivity higher than that of chemically synthesized PPy.

Minimising contributions from scattering in infrared spectra by means of an integrating sphere.

Mid-infrared spectra of biological matter such as tissues or microbial and eukaryotic cells measured in a transmission-type optical setup frequently show strongly distorted line shapes which arise from mixing of absorption and scattering contributions. Scattering-associated distorted line shapes may considerably complicate the analysis and interpretation of the infrared spectra and large efforts have been made to understand the mechanisms of scattering in biological matter and to compensate for spectral alterations caused by scattering. The goals of the present study were two-fold: firstly, to get a deeper understanding of the physics of scattering of biological systems and to explore how physical parameters of the scatterers such as shape, size and refractive index influence the line shape distortions observed. In this context, simulations based on the full Mie scattering formalism for spherical particles were found to be useful in explaining the characteristics of the Mie scatter-associated distortions and yielded a size criterion for the scattering particles similar to the well-known near field criterion. The second objective of the study was to investigate whether alternative optical setups allow minimisation of the effects of scattering. For this purpose, an optical system is proposed which is composed of an integrating sphere unit originally designed for diffuse reflection measurements, an off-axis DLaTGS detector to collect scattered and transmitted light components and a commercial Fourier transform infrared (FTIR) spectrometer. In the context of this study transmission type (tt-) FTIR spectra and spectra acquired by means of the integrating sphere setup (is-FTIR) were acquired from monodisperse poly(methyl) methacrylate (PMMA) microspheres of systematically varying sizes. The tt-FTIR spectral data of different PMMA particles confirmed earlier observations such as the presence of size-dependent oscillating spectral baselines, peak shifts, or derivative-like spectral line shapes. Such effects could be dramatically minimised when is-FTIR spectra were acquired by the integrating sphere unit. Utilisation of an integrating sphere is suggested as a convenient and easy-to use alternative to computer-based methods of scatter correction.

Detection of an estrogen derivative in two breast cancer cell lines using a single core multimodal probe for imaging (SCoMPI) imaged by a panel of luminescent and vibrational techniques.

3-Methoxy-17α-ethynylestradiol or mestranol is a prodrug for ethynylestradiol and the estrogen component of some oral contraceptive formulations. We demonstrate here that a single core multimodal probe for imaging - SCoMPI - can be efficiently grafted onto mestranol allowing its tracking in two breast cancer cell lines, MDA-MB-231 and MCF-7 fixed cells. Correlative imaging studies based on luminescence (synchrotron UV spectromicroscopy, wide field and confocal fluorescence microscopies) and vibrational (AFMIR, synchrotron FTIR spectromicroscopy, synchrotron-based multiple beam FTIR imaging, confocal Raman microspectroscopy) spectroscopies were consistent with one another and showed a Golgi apparatus distribution of the SCoMPI-mestranol conjugate in both cell lines.

Toward conformational identification of molecules in 2D and 3D self-assemblies on surfaces.

The design of supramolecular networks based on organic molecules deposited on surfaces, is highly attractive for various applications. One of the remaining challenges is the expansion of monolayers to well-ordered multilayers in order to enhance the functionality and complexity of self-assemblies. In this study, we present an assessment of molecular conformation from 2D to 3D supramolecular networks adsorbed onto a HOPG surface under ambient conditions utilizing a combination of scanning probe microscopies and atomic force microscopy- infrared (AFM-IR). We have observed that the infrared (IR) spectra of the designed molecules vary from layer to layer due to the modifications in the dihedral angle between the C=O group and the neighboring phenyl ring, especially in the case of a 3D supramolecular network consisting of multiple layers of molecules.

Macromolecular organic matter in samples of the asteroid (162173) Ryugu.

Samples of the carbonaceous asteroid (162173) Ryugu were collected and brought to Earth by the Hayabusa2 spacecraft. We investigated the macromolecular organic matter in Ryugu samples and found that it contains aromatic and aliphatic carbon, ketone, and carboxyl functional groups. The spectroscopic features of the organic matter are consistent with those in chemically primitive carbonaceous chondrite meteorites that experienced parent-body aqueous alteration (reactions with liquid water). The morphology of the organic carbon includes nanoglobules and diffuse carbon associated with phyllosilicate and carbonate minerals. Deuterium and/or nitrogen-15 enrichments indicate that the organic matter formed in a cold molecular cloud or the presolar nebula. The diversity of the organic matter indicates variable levels of aqueous alteration on Ryugu's parent body.

Revealing Lipid Body Formation and its Subcellular Reorganization in Oleaginous Microalgae Using Correlative Optical Microscopy and Infrared Nanospectroscopy.

The purpose of this work is to develop an integrated imaging approach to characterize without labeling at the sub-cellular level the formation of lipid body droplets (LBs) in microalgae undergoing nitrogen starvation. First conventional optical microscopy approaches, gas chromatography, and turbidimetry measurements allowed to monitor the biomass and the total lipid content in the oleaginous microalgae Parachlorella kesslerii during the starvation process. Then a local analysis of the LBs was proposed using an innovative infrared nanospectroscopy technique called atomic force microscopy-based infrared spectroscopy (AFM-IR). This label-free technique assessed the formation of LBs and allowed to look into the LB composition thanks to the acquisition of local infrared spectra. Last correlative measurements using fluorescence microscopy and AFM-IR were performed to investigate the subcellular reorganization of LB and the chloroplasts.