Multivariate curve resolution -alternating least squares augmented with partial least squares baseline correction applied to mid-IR laser spectra resolves protein denaturation by reducing rotational ambiguity.

High spectral power density provided by advances in external cavity quantum cascade lasers (EC-QCL) have enabled increased transmission path lengths in mid-infrared (mid-IR) spectroscopy for more sensitive measurement of proteins in aqueous solutions. These extended path lengths also facilitate flow through measurements by avoiding congestion of the flow cell by protein aggregates. Despite the advantages presented by laser-based mid-IR spectroscopy of proteins, extraction of secondary structure information from spectra, especially in the presence of complex multi-component matrices with overlapping spectral features, remains an impediment that requires fine tuning of evaluation algorithms (e.g., band fitting, interpretation of second derivative spectra etc.). In this work, the use of multivariate curve resolution alternating least squares (MCR-ALS) for the analysis of a chemical de- and renaturation experiment has been demonstrated, since this technique offers the second-order advantage of extracting spectral signatures and concentration profiles even in the presence of unknown, uncalibrated constituents. Furthermore, we exhibit a partial least squares regression (PLSR) based subtraction of matrix component spectra prior to MCR-ALS as a method to obtain secondary structure information even in the absence of reference spectra. These approaches are showcased using the online reaction monitoring of the titration of ß-lactoglobulin (ß-LG) in water against the surfactants sodium dodecyl sulfate (SDS) and octaethylene glyol monododecyl ether (C12E8), using a commercially available laser-based IR spectrometer. Results for the automated PLSR correction plus MCR-ALS approach compare favorably to an MCR-ALS standalone approach using initial estimates as well as analysis of secondary structure using data processed with a manual baseline correction. The herein described chemometric approach suggests a way to simplify the challenge of handling complex matrices in protein structure analysis by isolating the background from the protein contributions, prior to analysis via other soft-modelling techniques. Consequently, the findings of this study indicate the suitability of online reaction monitoring through mid-IR spectroscopy combined with chemometric techniques as a potential tool in downstream quality control and process automation.

Bottom-Illuminated Photothermal Nanoscale Chemical Imaging with a Flat Silicon ATR in Air and Liquid.

We demonstrate a novel approach for bottom-illuminated atomic force microscopy and infrared spectroscopy (AFM-IR). Bottom-illuminated AFM-IR for measurements in liquids makes use of an attenuated total reflection setup where the developing evanescent wave is responsible for photothermal excitation of the sample of interest. Conventional bottom-illuminated measurements are conducted using high-refractive-index prisms. We showcase the advancement of instrumentation through the introduction of flat silicon substrates as replacements for prisms. We illustrate the feasibility of this technique for bottom-illuminated AFM-IR in both air and liquid. We also show how modern rapid prototyping technologies enable commercial AFM-IR instrumentation to accept these new substrates. This new approach paves the way for a wide range of experiments since virtually any established protocol for Si surface functionalization can be applied to this sample carrier. Furthermore, the low unit cost enables the rapid iteration of experiments.

A novel enzymatic method for isolation of plastic particles from human blood.

Microplastic particles have been detected in the human body. This study aimed to develop a blood digestion method that preserves microplastics during analysis. Acidic and alkaline reagents, commonly used for isolating plastic particles from organic materials, were tested on human blood samples and microplastics. Nitric acid, hydrochloric acid, potassium hydroxide, and sodium hydroxide were examined over time. Additionally, a pepsin-pancreatin combination was utilized for blood digestion. Light microscopy assessed digestion efficiency and particle count changes, while Raman microspectroscopy distinguished between plastic and cell debris. The acidic reagents were ineffective in removing the organic material, while alkaline reagents were effective without significant effects on microplastics. Blood digestion using pepsin and pancreatin demonstrated efficient digestion without negative consequences for the particles. While potassium hydroxide digestion is already established, novel use of the pepsin-pancreatin combination was introduced to digest human blood, indicating its potential for isolating plastic particles from tissue and human food.

AFM-IR for Nanoscale Chemical Characterization in Life Sciences: Recent Developments and Future Directions.

Despite the ubiquitous absorption of mid-infrared (IR) radiation by virtually all molecules that belong to the major biomolecules groups (proteins, lipids, carbohydrates, nucleic acids), the application of conventional IR microscopy to the life sciences remained somewhat limited, due to the restrictions on spatial resolution imposed by the diffraction limit (in the order of several micrometers). This issue is addressed by AFM-IR, a scanning probe-based technique that allows for chemical analysis at the nanoscale with resolutions down to 10 nm and thus has the potential to contribute to the investigation of nano and microscale biological processes. In this perspective, in addition to a concise description of the working principles and operating modes of AFM-IR, we present and evaluate the latest key applications of AFM-IR to the life sciences, summarizing what the technique has to offer to this field. Furthermore, we discuss the most relevant current limitations and point out potential future developments and areas for further application for fruitful interdisciplinary collaboration.

Chiral Monitoring Across Both Enantiomeric Excess and Concentration Space: Leveraging Quantum Cascade Lasers for Sensitive Vibrational Circular Dichroism Spectroscopy.

Recently, high-throughput quantum cascade laser-based vibrational circular dichroism (QCL-VCD) technology has reduced the measurement time for high-quality vibrational circular dichroism spectra from hours to a few minutes. This study evaluates QCL-VCD for chiral monitoring using flow-through measurement of a changing sample in a circulating loop. A balanced detection QCL-VCD system was applied to the enantiomeric pair R/S-1,1'-bi-2-naphthol in solution. Different mixtures of the two components were used to simulate a racemization process, collecting spectral data at a time resolution of 6 min, and over three concentration levels. The goal of this experimental setup was to evaluate QCL-VCD in terms of both molar and enantiomeric excess (EE) sensitivity at a time resolution relevant to chiral monitoring in chemical processes. Subsequent chemometric evaluation by partial least squares regression revealed a cross-validated prediction accuracy of 2.8% EE with a robust prediction also for the test data set (error = 3.5% EE). In addition, the data set was also treated with the least absolute shrinkage and selection operator (LASSO), which also achieved a robust prediction. Due to the operating principle of LASSO, the obtained coefficients constituted a few discrete spectral frequencies, which represent the most variance. This information can be used in the future for dedicated QCL-based instrument design, gaining a higher time resolution without sacrificing predictive capabilities.

Hydrolyzable tannins are incorporated into the endocarp during sclerification of the water caltrop Trapa natans.

The water caltrop (Trapa natans) develops unique woody fruits with unusually large seeds among aquatic plants. During fruit development, the inner fruit wall (endocarp) sclerifies and forms a protective layer for the seed. Endocarp sclerification also occurs in many land plants with large seeds; however, in T. natans, the processes of fruit formation, endocarp hardening, and seed storage take place entirely underwater. To identify potential chemical and structural adaptations for the aquatic environment, we investigated the cell-wall composition in the endocarp at a young developmental stage, as well as at fruit maturity. Our work shows that hydrolyzable tannins-specifically gallotannins-flood the endocarp tissue during secondary wall formation and are integrated into cell walls along with lignin during maturation. Within the secondary walls of mature tissue, we identified unusually strong spectroscopic features of ester linkages, suggesting that the gallotannins and their derivatives are cross-linked to other wall components via ester bonds, leading to unique cell-wall properties. The synthesis of large amounts of water-soluble, defensive aromatic metabolites during secondary wall formation might be a fast way to defend seeds within the insufficiently lignified endocarp of T. natans.

Beating thermal noise in a dynamic signal measurement by a nanofabricated cavity optomechanical sensor.

Thermal fluctuations often impose both fundamental and practical measurement limits on high-performance sensors, motivating the development of techniques that bypass the limitations imposed by thermal noise outside cryogenic environments. Here, we theoretically propose and experimentally demonstrate a measurement method that reduces the effective transducer temperature and improves the measurement precision of a dynamic impulse response signal. Thermal noise-limited, integrated cavity optomechanical atomic force microscopy probes are used in a photothermal-induced resonance measurement to demonstrate an effective temperature reduction by a factor of ≈25, i.e., from room temperature down as low as ≈12 K, without cryogens. The method improves the experimental measurement precision and throughput by >2×, approaching the theoretical limit of ≈3.5× improvement for our experimental conditions. The general applicability of this method to dynamic measurements leveraging thermal noise-limited harmonic transducers will have a broad impact across a variety of measurement platforms and scientific fields.

Dual-Beam Photothermal Spectroscopy Employing a Mach-Zehnder Interferometer and an External Cavity Quantum Cascade Laser for Detection of Water Traces in Organic Solvents.

We report on a mid-infrared (mid-IR) photothermal spectrometer for liquid-phase samples for the detection of water in organic solvents, such as ethanol or chloroform, and in complex mixtures, such as jet fuel. The spectrometer is based on a Mach-Zehnder interferometer (MZI) employing a He-Ne laser, a mini-flow cell with two embedded channels placed in the interferometer's arms, and a tunable external cavity quantum cascade laser (EC-QCL) for selective analyte excitation in a collinear arrangement. In this study, the bending vibration of water in the spectral range 1565-1725 cm-1 is targeted. The interferometer is locked to its quadrature point (QP) for most stable and automated operation. It provides a linear response with respect to the water content in the studied solvents and photothermal analyte spectra, which are in good agreement with FTIR absorbance spectra. The method is calibrated and validated against coulometric Karl Fischer (KF) titration, showing comparable performance and sensitivity. Limits of detection (LODs) for water detection in the single-digit ppm range were obtained for chloroform and jet fuel due to their low background absorption, whereas lower sensitivity has been observed for water detection in ethanol due to pronounced background absorption from the solvent. In contrast to KF titration, which requires toxic reagents and produces waste, the developed method works reagent-free. It can be applied in an online format in the chemical industry as well as for fuel quality control, being industrial applications where traces of water need to be accurately determined, preferably in real-time. It thus holds great promise as a green alternative to the offline KF titration method, which is the current standard method for this application.

Nanoscale chemical characterization of a post-consumer recycled polyolefin blend using tapping mode AFM-IR.

The routine analysis of polymer blends at the nanoscale is usually carried out using electron microscopy techniques such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM), which often require several sample preparation steps including staining with heavy metals and/or etching. Atomic force microscopy (AFM) is also commonly used, but provides no direct chemical information about the samples analyzed. AFM-IR, a recent technique which combines the AFM's nanoscale resolution with the chemical information provided by IR spectroscopy, is a valuable complement to the already established techniques. Resonance enhanced AFM-IR (contact mode) is the most commonly used measurement mode, due to its signal enhancement and relative ease of use. However, it has severe drawbacks when used in highly heterogenous samples with changing mechanical properties, such as polymer recyclates. In this work, we use the recently developed tapping mode AFM-IR to chemically image the distribution of rubber in a real-world commercially available polyethylene/polypropylene (PE/PP) recycled blend derived from municipal and household waste. Furthermore, the outstanding IR resolution of AFM-IR allowed for the detection of small PP droplets inside the PE phase. The presence of micro and nanoscale particles of other polymers in the blend was also established, and the polymers identified.

Quantum Cascade Laser-Based Vibrational Circular Dichroism Augmented by a Balanced Detection Scheme.

Vibrational circular dichroism (VCD) constitutes a powerful technique, enabling the determination of the absolute configuration of molecules without the need for specialized reagents. While delivering critical information, VCD signals commonly are several orders of magnitude weaker than classical absorbance signals, which so far necessitated long measurement times to achieve acceptable signal-to-noise ratios (SNRs) in VCD experiments. We present here an improved setup for the measurement of VCD in the range between 5.6 and 6.5 µm. Employing an external cavity quantum cascade laser (EC-QCL) as a high-power light source, we collected spectra with competitive noise levels in less than 5 min. The basis for this improvement was a balanced detection module combined with an optical path catered to VCD measurements. With the stabilization provided by the two-detector setup, noise originating from the laser source could be suppressed effectively. Noise level improvement up to a factor of 4 compared to the classical single detector EC-QCL-VCD could be reported. Compared to commercial Fourier transform infrared (FT-IR) instruments, the presented setup offers measurement time reductions of a factor of at least 6, with comparable noise levels. The applicability of the setup for qualitative and quantitative VCDs was proven. With the comparatively high temporal resolution provided, the monitoring of optically active processes will be possible in future applications.

High Throughput Nanoimaging of Thermal Conductivity and Interfacial Thermal Conductance.

Thermal properties of materials are often determined by measuring thermalization processes; however, such measurements at the nanoscale are challenging because they require high sensitivity concurrently with high temporal and spatial resolutions. Here, we develop an optomechanical cantilever probe and customize an atomic force microscope with low detection noise ≈1 fm/Hz1/2 over a wide (>100 MHz) bandwidth that measures thermalization dynamics with ≈10 ns temporal resolution, ≈35 nm spatial resolution, and high sensitivity. This setup enables fast nanoimaging of thermal conductivity (η) and interfacial thermal conductance (G) with measurement throughputs ≈6000× faster than conventional macroscale-resolution time-domain thermoreflectance acquiring the full sample thermalization. As a proof-of-principle demonstration, 100 × 100 pixel maps of η and G of a polymer particle are obtained in 200 s with a small relative uncertainty (<10%). This work paves the way to study fast thermal dynamics in materials and devices at the nanoscale.

Elemental mapping of fluorine by means of molecular laser induced breakdown spectroscopy.

The growing importance of fluoropolymers in high-tech applications and green technologies results in the rising need for their characterization. In contrast to conventional methods used for this task, laser-induced breakdown spectroscopy (LIBS) provides the advantage of a spatially resolved analysis. Nevertheless, the high excitation energy of fluorine results in low sensitivity of the atomic F(I) lines, which limits the feasibility of its LIBS-based analysis. This work presents a novel approach for quantitative mapping of fluorine in fluoropolymer samples. It bases on monitoring of molecular emission bands (CuF or CaF) arising from fluorine containing molecules. These species were generated during later stages of the LIBS plasma by a recombination of fluorine atoms originating from fluoropolymer sample with a molecule-forming partner (Cu or Ca) stemming from a surface coating. This approach enables F detection limits in the parts per million (µg g-1) range and elemental imaging using single shot measurements. The elements required for molecular formation are deposited on the sample surface prior to analysis. We evaluate two techniques - spray coating and sputter coating - with regards to their effects on sensitivity and spatial resolution in elemental mapping. Overall, both methods proved to be suitable for a spatially resolved analysis of fluorine: whereas sputter-coating of copper yielded a better sensitivity, spray coating of calcium provided a higher spatial resolution.

AFM investigation of APAC (antiplatelet and anticoagulant heparin proteoglycan).

Antiplatelet and anticoagulant drugs are classified antithrombotic agents with the purpose to reduce blood clot formation. For a successful treatment of many known complex cardiovascular diseases driven by platelet and/or coagulation activity, the need of more than one antithrombotic agent is inevitable. However, combining drugs with different mechanisms of action enhances risk of bleeding. Dual anticoagulant and antiplatelet (APAC), a novel semisynthetic antithrombotic molecule, provides both anticoagulant and antiplatelet properties in preclinical studies. APAC is entering clinical studies with this new exciting approach to manage cardiovascular diseases. For a better understanding of the biological function of APAC, comprehensive knowledge of its structure is essential. In this study, atomic force microscopy (AFM) was used to characterize APAC according to its structure and to investigate the molecular interaction of APAC with von Willebrand factor (VWF), since specific binding of APAC to VWF could reduce platelet accumulation at vascular injury sites. By the optimization of drop-casting experiments, we were able to determine the volume of an individual APAC molecule at around 600 nm3, and confirm that APAC forms multimers, especially dimers and trimers under the experimental conditions. By studying the drop-casting behavior of APAC and VWF individually, we depictured their interaction by using an indirect approach. Moreover, in vitro and in vivo conducted experiments in pigs supported the AFM results further. Finally, the successful adsorption of APAC to a flat gold surface was confirmed by using photothermal-induced resonance, whereby attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) served as a reference method.

Highly Biaxially Strained Silicene on Au(111).

Many of graphene's remarkable properties arise from its linear dispersion of the electronic states, forming a Dirac cone at the K points of the Brillouin zone. Silicene, the 2D allotrope of silicon, is also predicted to show a similar electronic band structure, with the addition of a tunable bandgap, induced by spin-orbit coupling. Because of these outstanding electronic properties, silicene is considered as a promising building block for next-generation electronic devices. Recently, it has been shown that silicene grown on Au(111) still possesses a Dirac cone, despite the interaction with the substrate. Here, to fully characterize the structure of this 2D material, we investigate the vibrational spectrum of a monolayer silicene grown on Au(111) by polarized Raman spectroscopy. To enable a detailed ex situ investigation, we passivated the silicene on Au(111) by encapsulating it under few layers hBN or graphene flakes. The observed spectrum is characterized by vibrational modes that are strongly red-shifted with respect to the ones expected for freestanding silicene. By comparing low-energy electron diffraction (LEED) patterns and Raman results with first-principles calculations, we show that the vibrational modes indicate a highly (>7%) biaxially strained silicene phase.

Polarimetric Balanced Detection: Background-Free Mid-IR Evanescent Field Laser Spectroscopy for Low-Noise, Long-term Stable Chemical Sensing.

In this work, we introduce polarimetric balanced detection as a new attenuated total reflection (ATR) infrared (IR) sensing scheme, leveraging unequal effective thicknesses achieved with laser light of different polarizations. We combined a monolithic widely tunable Vernier quantum cascade laser (QCL-XT) and a multibounce ATR IR spectroscopy setup for analysis of liquids in a process analytical setting. Polarimetric balanced detection enables simultaneous recording of background and sample spectra, significantly reducing long-term drifts. The root-mean-square noise could be improved by a factor of 10 in a long-term experiment, compared to conventional absorbance measurements obtained via the single-ended optical channel. The sensing performance of the device was further evaluated by on-site measurements of ethanol in water, leading to an improved limit of detection (LOD) achieved with polarimetric balanced detection. Sequential injection analysis was employed for automated injection of samples into a custom-built ATR flow cell mounted above a zinc sulfide multibounce ATR element. The QCL-XT posed to be suitable for mid-IR-based sensing in liquids due to its wide tunability. Polarimetric balanced detection proved to enhance the robustness and long-term stability of the sensing device, along with improving the LOD by a factor of 5. This demonstrates the potential for new polarimetric QCL-based ATR mid-IR sensing schemes for in-field measurements or process monitoring usually prone to a multitude of interferences.

High-Q dark hyperbolic phonon-polaritons in hexagonal boron nitride nanostructures.

The anisotropy of hexagonal boron nitride (hBN) gives rise to hyperbolic phonon-polaritons (HPhPs), notable for their volumetric frequency-dependent propagation and strong confinement. For frustum (truncated nanocone) structures, theory predicts five, high-order HPhPs, sets, but only one set was observed previously with far-field reflectance and scattering-type scanning near-field optical microscopy. In contrast, the photothermal induced resonance (PTIR) technique has recently permitted sampling of the full HPhP dispersion and observing such elusive predicted modes; however, the mechanism underlying PTIR sensitivity to these weakly-scattering modes, while critical to their understanding, has not yet been clarified. Here, by comparing conventional contact- and newly developed tapping-mode PTIR, we show that the PTIR sensitivity to those weakly-scattering, high-Q (up to ≈280) modes is, contrary to a previous hypothesis, unrelated to the probe operation (contact or tapping) and is instead linked to PTIR ability to detect tip-launched dark, volumetrically-confined polaritons, rather than nanostructure-launched HPhPs modes observed by other techniques. Furthermore, we show that in contrast with plasmons and surface phonon-polaritons, whose Q-factors and optical cross-sections are typically degraded by the proximity of other nanostructures, the high-Q HPhP resonances are preserved even in high-density hBN frustum arrays, which is useful in sensing and quantum emission applications.

Nanoscale Infrared Spectroscopy and Chemometrics Enable Detection of Intracellular Protein Distribution.

Determination of the intracellular location of proteins is one of the fundamental tasks of microbiology. Conventionally, label-based microscopy and super-resolution techniques are employed. In this work, we demonstrate a new technique that can determine intracellular protein distribution at nanometer spatial resolution. This method combines nanoscale spatial resolution chemical imaging using the photothermal-induced resonance (PTIR) technique with multivariate modeling to reveal the intracellular distribution of cell components. Here, we demonstrate its viability by imaging the distribution of major cellulases and xylanases in Trichoderma reesei using the colocation of a fluorescent label (enhanced yellow fluorescence protein, EYFP) with the target enzymes to calibrate the chemometric model. The obtained partial least squares model successfully shows the distribution of these proteins inside the cell and opens the door for further studies on protein secretion mechanisms using PTIR.

The Next Generation of IR Spectroscopy: EC-QCL-Based Mid-IR Transmission Spectroscopy of Proteins with Balanced Detection.

We report a mid-IR transmission setup for the analysis of the protein amide I and amide II band in aqueous solutions that achieves a limit of detection as low as 0.0025 mg mL-1 (outperforming our previous results and other state-of-the-art mid-IR-based techniques by almost an order of magnitude). This large improvement is made possible by combining the latest-generation external cavity-quantum cascade laser (EC-QCL) operated at room temperature with an optimized double-beam optical setup that adjusts the path length (26 µm) to ensure robust sample handling. For minimizing the noise introduced by the high-intensity laser light source, a thermoelectrically cooled mercury cadmium telluride balanced detection module was employed. In this way, noise levels better by a factor of up to 20 were achieved compared with single-channel measurements. Characteristic spectral features of proteins with different secondary structures were successfully identified at concentrations as low as 0.1 mg mL-1. Furthermore, a highly linear response was demonstrated for concentrations between 0.05 and 10 mg mL-1. The total acquisition time of the setup can be adapted to fulfill the required sensitivity of the protein measurements and to ensure maximum flexibility for future applications. The presented setup combines high sensitivity, large optical path lengths, and short measurement times and thus outperforms previous research type EC-QCL setups as well as commercially available instruments. This opens a wide range of future applications including protein-ligand interaction studies as well as qualitative and quantitative analyses of proteins in complex matrices such as those found in up- and downstream bioprocess monitoring and similar challenging applications which can not be readily met by conventional FT-IR spectroscopy.

Nanoscale Chemical Imaging of Individual, Chemotherapeutic Cytarabine-loaded Liposomal Nanocarriers.

Dosage of chemotherapeutic drugs is a tradeoff between efficacy and side-effects. Liposomes are nanocarriers that increase therapy efficacy and minimize side-effects by delivering otherwise difficult to administer therapeutics with improved efficiency and selectivity. Still, variabilities in liposome preparation require assessing drug encapsulation efficiency at the single liposome level, an information that, for non-fluorescent therapeutic cargos, is inaccessible due to the minute drug load per liposome. Photothermal induced resonance (PTIR) provides nanoscale compositional specificity, up to now, by leveraging an atomic force microscope (AFM) tip contacting the sample to transduce the sample's photothermal expansion. However, on soft samples (e.g. liposomes) PTIR effectiveness is reduced due to the likelihood of tip-induced sample damage and inefficient AFM transduction. Here, individual liposomes loaded with the chemotherapeutic drug cytarabine are deposited intact from suspension via nES-GEMMA (nano-electrospray gas-phase electrophoretic mobility molecular analysis) collection and characterized at the nanoscale with the chemically-sensitive PTIR method. A new tapping-mode PTIR imaging paradigm based on heterodyne detection is shown to be better adapted to measure soft samples, yielding cytarabine distribution in individual liposomes and enabling classification of empty and drug-loaded liposomes. The measurements highlight PTIR capability to detect ≈ 103 cytarabine molecules (≈ 1.7 zmol) label-free and non-destructively.

Revealing the Distribution of Metal Carboxylates in Oil Paint from the Micro- to Nanoscale.

Oil paints comprise pigments, drying oils, and additives that together confer desirable properties, but can react to form metal carboxylates (soaps) that may damage artworks over time. To obtain information on soap formation and aggregation, we introduce a new tapping-mode measurement paradigm for the photothermal induced resonance (PTIR) technique that enables nanoscale IR spectroscopy and imaging on highly heterogenous and rough paint thin sections. PTIR is used in combination with µ-computed tomography and IR microscopy to determine the distribution of metal carboxylates in a 23-year old oil paint of known formulation. Results show that heterogeneous agglomerates of Al-stearate and a Zn-carboxylate complex with Zn-stearate nano-aggregates in proximity are distributed randomly in the paint. The gradients of zinc carboxylates are unrelated to the Al-stearate distribution. These measurements open a new chemically sensitive nanoscale observation window on the distribution of metal soaps that can bring insights for understanding soap formation in oil paint.